Declaration

SELECTION OF THE BEST VESICULAR CARRIER SYSTEM
FOR ENHANCED TRANSDERMAL DRUG DELIVERY: A

COMPARATIVE STUDY
A Thesis submitted to the

Jawaharlal Nehru Technological University Anantapur
For the award of
DOCTOR OF PHILOSOPHY
in
PHARMACEUTICAL SCIENCES
By
K. SRIKANTH
[Reg. No. 0900PH1309]
Under the guidance of
Dr. V. RAMA MOHAN GUPTA & Dr. DEVANNA NAYAKANTI
RESEARCH AND DEVELOPMENT

JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR
ANANTHAPURAMU – 515002, A.P., INDIA
JULY-2014
DECLARATION
I declare that the thesis entitled “SELECTION OF THE BEST VESICULAR
CARRIER SYSTEM FOR ENHANCED TRANSDERMAL DRUG DELIVERY: A
COMPARATIVE STUDY” has been prepared by me under the guidance of Dr. V.
RAMA MOHAN GUPTA, Professor & Principal, Pulla Reddy Institute of
Pharmacy, Medak, Hyderabad and Dr. N. Devanna, Director, JNTUA-OTRI,
Ananthapuramu. No part of this thesis has formed the basis for the award of end
degree or fellowship previously.
K.SRIKANTH
Dept of Pharmaceutical sciences,
Jawaharlal Nehru Technological University,Anantapur,
Ananthapuramu.
Dt:
CERTIFICATE
We certify that K. Srikanth has preparaed his thesis entitled “SELECTION
OF THE BEST VESICULAR CARRIER SYSTEM FOR ENHANCED TRANSDERMAL
DRUG DELIVERY: A COMPARATIVE STUDY” for the award of Ph.D degree of the
Jawaharlal Nehru Technological University Anatapur, Ananthapuramu under our
guidance. He has carried out the work at the Dept of Pharmaceutics, Pulla Reddy
Institute of Pharmacy, Medak, Hyderabad, A.P, (INDIA.).

Signature of the co-guide Signature of the co-guide
Dr. N. Devanna Dr. V. Rama Mohan Gupta
Director, Professor & Principal
JNTUA-OTRI, Pulla Reddy Institute ofPharmacy,
Ananthapuramu Medak, Hyderabad

ACKNOWLEDGEMENT
It would not have been possible to do research work and to write my
doctoral thesis without the help and support of my god Saibaba. I don’t have words
to express my deep sense of gratitude towards my God Sai Baba for his blessings.
I have only option to make him happy is that following his words. I want to be a
dust particle under his feet forever.
At the outset, I would like to thank my supervisor Dr. V. Rama Mohan
Gupta, Principal, Pulla Reddy Institute of Pharmacy for his quick evaluation and
decision on my PhD application. In fact, it was a big surprise to receive his positive
reply within a few minutes after my request for the PhD position, grateful for his
confidence and trust on me. It is my privilege to have his encouragement and
galvanizing my spirit in accomplishing this project successfully. His valuable and
precious suggestions, criticisms have been guiding force throughout the process of
my work. The sincerity, righteousness and virtuousness which he has inculcated in
me will take me a long way in my life. During this period, I had a great freedom to
plan and execute my ideas in research without any pressure. This made me to
identify my own strength and drawbacks and particularly boosted my self-
confidence. It is a great pleasure working under his supervision and I would ever be
indebted to him for the unstinted support exleished to me during my Ph.D.
I will forever be thankful to my co-guide Dr. N. Devanna, Director, OTRI,
Anathapur for his timely help and sincere support during my research period. He
was and remains my best role model as a scientist, mentor, and teacher. His helping
hand is a source in crossing the barriers associated in completion of synopsis,
thesis, etc.
I take this opportunity to sincerely acknowledge Mr. CH. Sathi Reddy,
Chairman, Pulla Reddy Institute of Pharmacy, Medak and Mr. Aditya, Director,
Pulla Reddy Institute of Pharmacy, Medak for providing facilities to carry out
my research work at Pulla Reddy Institute of Pharmacy which buttressed me to
perform my research work comfortably.
I oblige my deep sense of gratitude to Mr. V.S. Prakash Rao,
Administrative Officer, Pulla Reddy Institute of Pharmacy, Medak for his
supportive suggestions and motivations from the commencement of my project
work. He shared his experiences which he had faced in his long tenure and offered
valuable suggestions to become ideal person.
I express my sincere gratitude to my colleagues Mr. Ashwaq Hussain, Mr.
Subhas Sahoo, Mrs. Srivani Voviliveni, Ms. Nikunja B.Pati, Mrs. Parimala devi,
and others who have been behind me and for their timely help during my Ph.D and
bench works.
I wish to thank Kremoint Pharma Pvt. Ltd, Thane, Maharastra and Lipoid
AG, Switzerland for providing gift samples of drugs and polymers.
I extend my sincere thanks to all the supporting staff members of my
college Mr. Srinivas, Mr. Omkar Rao, Mr. Siddaiah, Mrs. Sangeetha for their
involvement and friendly attitude during my work.
I would like to pay high regards to my Mother, Father and Brother for
their sincere encouragement and inspiration throughout my research work and
lifting me uphill this phase of life. I owe everything to them. Words fail me to
express my appreciation to my mother and father for their support, generous care.
A simple thanks doesn’t seem sufficient but it is said with appreciation and respect
to my brother who has been instrumental for the good shape of my thesis because
of his help and suggestions in formatting the entire thesis. During the inevitable
ups and downs of conducting my research he often reminded me life’s true
priorities by what could be the influence of a loving God Sai baba. A journey is
easier when you travel together. My wife always stood beside me during the happy
and hard moments to push me and motivate me. I am much indebted to my wife for
her love, care, support and creating a pleasant atmosphere at home during my
entire research period.
Besides this, my sincere thanks also goes to several people who have
knowingly and unknowingly helped me in the successful completion of this project.
K. SRIKANTH
ABSTRACT
The present research work aimed to select the best vesicular carrier
system among niosomes, ethosomes and transferosomes (primary objective) for
administration of drugs across the skin to produce systemic effect. Nystatin, a
polyene antifungal agent, was used as model drug in the present study. The
secondary objective of the study is enhancement of bioavailability of the
nystatin and to decrease the adverse effects associated with higher doses of the
same by administering through skin.
Niosomal and transferosomal suspensions of nystatin prepared by using
thin film hydration Technique whereas ethosomal suspensions of nystatin was
prepared method reported by Touitou et al. Totally 24 vesicular suspensions
(eight formulations from each type of vesicular systems) were prepared.
Niosomal suspensions were prepared using two different non-ionic
surfactants i.e span-80 and span-60. Each surfactant used at four different
concentrations (eight niosomal suspensions) was prepared. Similarly, eight
transferosomal and eight ethosomal suspensions were prepared.
Transferosomal suspensions were prepared using two different edge activators
i.e. span-60 and span-80 at four different concentrations whereas in
preparation of ethosomal suspensions ethanol and lecithin concentrations has
been changed for preparing the same (each at four different concentrations).
The prepared niosomal, ethosomal and transferosomal suspensions (24
formulations) evaluated for different parameters like vesicles morphology (size,
shape and surface texture), entrapment efficiency and in vitro drug release. The
drug release data fitted in different mathematical models such as Zero order,
First order, Higuchi, Hixon-crowel, Korsmeyer-peppas to find out the order and
mechanism of drug release from all formulations.
Based on the size, entrapment efficiency and invitro drug release results,
one optimized vesicular suspension from each type of vesicular formulation
was selected.
Elasticity and surface charge (Zeta potential) of the vesicles present in
three optimized vesicular suspensions was studied.
After performing the zetapotential and elasticity studies, using the
optimized niosomal, ethosomal and transferosomal suspensions niosomal,
ethosomal and transfersosmal gels having concentration of 0.01%w/w was
prepared respectively. Along with three vesicular nystatin gels, conventional
nystatin gel having same concentration was also prepared with pure nystatin
sample.
The prepared gels were characterized for in vitro and ex vivo drug release,
skin retention, stability studies. Finally in vivo studies also carried out to find
out the systemic availability of nystatin from individual gels in rabbits.
In vitro, ex vivo drug release data showed a good correlation between
each other. The study revealed that ethosomes are effective for permeation of
nystatin across the dialysis membrane and skin than other two vesicles.
Transdermal flux, permeation coefficient and enhancement ratio was
calculated from ex vivo drug release data of all three vesicular carrier gels. The
data showed higher efficiency to the ethosomes than other two vesicular gels.
Skin retention study was performed to find out the quantity of nystatin that
reached systemic circulation and retained in and on the skin after ex vivo drug
release study. The skin retention study also showed that ethosomes are
efficient than other two vesicular carriers i.e. transferosomes and niosomes.
In vivo study was also carried out in rabbits to find out the efficiency of
all vesicular gels and conventional gel in production of systemic effect. Highest
Cmax and AUC was exhibited by the ethosomes than other two vesicular and
conventional gels which stating that ethosomes are effective carriers to produce
systemic effect than other two carriers.
Our findings contribute to the evidence base for enhancing the
permeability of nystatin across the skin and to produce the systemic anti
fungal activity. (Secondary objective)
Key Words: transdermal drug delivery, systemic effect, localized effect,
niosomes, ethosomes, transferosomes, gels.

PREFACE
This Ph.D. work I have started by the grace of my god Sai baba and is
desire of my father. This thesis contains my five years research work which I
have carried out at Pulla Reddy Institute of Pharmacy, Medak. It was my
brother who made me a real person. He is the person who ignited passion in
me towards research. Without this foundation, I probably would not have dared to
study an emergent technology which, at the point I started this work, still had a very
unclear future. I would have never reached the point of finishing my dissertation
without the help and support of others.
These five years have been a challenging trip, with both ups and downs.
Fortunately, I was not alone on this road, but accompanied by an extended
team of experts, always willing to coach, sponsor, help, and motivate me. For
this, I would like to kindly thank them.
This thesis is divided into eight chapters. Chapter one focuses on the
general aspects of Drug Delivery and the need for various approaches to Novel
Drug Delivery Systems. It also provides an overview of transdermal drug
delivery that envisages briefly about vesicular carriers in drug delivery, through
skin. Chapter two includes a careful and systematic presentation of published
literature that has been reviewed to develop and evaluate the system designed
in the present investigation. Chapter three outlines the aim & objective of the
present investigation, preformulation studies of drug and various polymers
with their characterization. It deals with brief profiles of the drug, polymers,
Materials and methods adopted in the preparation and characterization of the

microspheres in the present work. Chapter four outlines the results of all
investigations obtained in an orderly manner. Chapter Five deals with
discussions of all results obtained in the investigation with their probable
implications. Chapter Six deals with summary and conclusion drawn from the
present work. Chapter seven gives list of Bibliography/References. Chapter
eight gives list of Publications & Conferences.

TABLE OF CONTENTS
Declaration
Certificate
Acknowledgements
Abstract
Preface
List of Tables
List of Figures
List of Abbreviations
List of Appendices
Chapter No. Name of the Chapter Page No.
1. Introduction
2. Literature Survey
3. Theoretical Analysis
4. Experimental Investigations
5. Experimental Results
6. Discussion of Results
7. Summary, Conclusion and Recommendations
8. References/Bibliography
Index
LIST OF TABLES
Table No Title of the table Pg.no
Chapter -1
1.1 Transdermal drugs approved by the US FDA
Chapter -3
3.5.1 List of chemicals
3.5.2 List of equipments
Chapter -5
5.1 Solubility of nystatin in Phosphate buffer saline solution pH 7.4
Standard curve of nystatin in Phosphate buffer
5.2
saline solution pH 7.4
5.3 Composition of niosomal formulations
5.4 Composition of ethosomal formulations
5.5 Composition of transferosomal formulations
Vesicles size & Entrapment efficiency of
5.6
all niosomal formulations
Vesicles size & Entrapment efficiency of
5.7
all ethosomal formulations
Vesicles size & Entrapment efficiency of all
5.8
transferosomal formulations
Summary of in vitro drug release from all niosomal
5.9
formulations across the dialysis membrane
5.10 In vitro drug release from niosomal formulation FN1
Summary of release kinetics data of all nystatin
5.18
Niosomal Formulations
Summary of in vitro nystatin release from all ethosomal
5.19
5.20 In vitro drug release from ethosomal formulation FE1
Summary of Release kinetics data of all
5.28
nystatin ethosomal suspension
Summary of in vitro nystatin release from all transferosomal
5.29
5.30 In vitro drug release from transferosomal formulation FT1
Summary of Release kinetics data of all nystatin
5.38
transferosomal suspension
Elasticity measurements of three optimized vesicular
5.39
formulations
5.40 Zeta potential of three optimized vesicular formulations
Composition of conventional, niosomal, ethosomal
5.41
&transferosomal gels
In vitro drug release from conventional gel across
5.42
the dialysis membrane
In vitro drug release from niosomal gel across
5.43
In vitro drug release from ethosomal gel across
5.44
In vitro drug release from transferosomal gel across
5.45
5.46 Ex vivo drug release from conventional gel across the rat skin
5.47 Ex vivo drug release from niosomal gel across the rat skin
5.48 Ex vivo drug release from ethosomal gel across the rat skin
5.49 Ex vivo drug release from transferosomal gel across the rat skin
Nystatin retention from all gels during ex vivo drug
5.50
release study in and on skin
5.51 Stability studies of optimized niosomal formulation gel (FN3)
5.52 Stability studies of optimized ethosomal formulation gel (FE6)
Stability studies of optimized transferosomal formulation gel
5.53
(FT7)
Analysis of ex vivo drug release study of three
5.54
optimized vesicular formulations
5.55 Calibration curve of the nystatin by HPLC
5.56 In vivo study of the conventional gel in rabbits
5.57 In vivo study of the niosomal gel in rabbits
5.58 In vivo study of the ethosomal gel in rabbits
5.59 In vivo study of the transferosomal gel in rabbits
Summary of the nystatin concentrations in blood plasma at
5.60
different time intervals (In vivo study)
5.61 Bioavailability studies of nystatin from all gels
LIST OF FIGURES
Figure
Title of the figure Pg.no
No
Chapter -1
1.1 Different routes of drug administration
1.2 Market segmentation the global drug delivery market in 2007
1.3 Topical and transdermal drug delivery systems
1.4 Number of Transdermal drugs approved in each year.
1.5 Structure of the skin
1.6 Pathways of drug absorption through the skin
1.7 Chemical structure of typical chemical penetration enhancers
1.8 Basic design of electroporation drug delivery device
1.9 Basic principle of iontophoresis.
1.10 Basic principle of phonophoresis
1.11 Basic design of microneedle delivery devices
1.12 Enhancement of transdermal permeation by pressure wave
1.13 various pharmaceutical carriers
1.14 Structure of typical vesicle
1.15 structure of conventional liposome
1.16 Chemical structure of Phosphatidylcholine (PC)
1.17 Structure of emulsomes
Proposed mechanism for penetration of molecule from
1.18 ethosomal system across the lipid domain of stratum
corneum
1.19 structure of sphingolipids
1.20 Structure of transferosomes
1.21 Mechanism of transferosomes permeation across the skin
1.22 Non-ionic surfactant vesicle / Niosome
Chapter -5
5.1 Absorption spectrum of Nystatin between 200-400nm
5.2 FTIR graph of Nystatin
5.3 Standard curve of nystatin in PBS pH 7.4
5.4 SEM images of the nystatin loaded niosomes
In vitro drug release from niosomal preparations
5.5
prepared using span-60
In vitro drug release from niosomal preparations
5.6
prepared using span-80
5.7 Release kinetics of niosomal suspension FN1
5.15 SEM Photographs of ethosomes
Effect of ethanol concentration on drug release from
5.16
ethosomes
Effect of lecithin concentration on drug release from
5.17
ethosomes.
5.18 Effect of cholesterol on drug release from ethosomes
5.19 Release kinetics of ethosomal formulation FE1
5.27 SEM photographs of transferosomes
In vitro drug release from transferosomal formulations
5.28
prepared using span - 60
n vitro drug release from transferosomal formulations
5.29
5.30 Release kinetics of transferosomal formulation FT1
Zeta potential measurement of nystatin loaded niosomes
5.38
of an optimized formulation (FN3)
Zeta potential measurement of nystatin loaded ethosomes
5.39
of an optimized formulation (FE6)
Zeta potential measurement of nystatin loaded
5.40 transferosomes
of an optimized formulation (FT7)
In vitro drug release from all gels across the dialysis
5.41
membrane
5.42 Ex vivo drug release from all gels across the rat skin
calculation of flux at steady state and other parameters from
5.43
ex vivo drug release data
5.44 Calibration curve of nystatin by HPLC
5.45 In vivo drug release from all gels
5.46 HPLC chromatogram of nystatin (300ng/ml)
Conventional gel HPLC chromatograms of 0th hour blood
5.53
sample in Rabbit -1
5.54
sample in Rabbit -2
Conventional gel HPLC chromatograms of 1st hour blood
5.55
sample in Rabbit -1
Conventional gel HPLC chromatograms of1st hour blood
5.56
sample in Rabbit -2
Conventional gel HPLC chromatograms of 3rd hour blood
5.57
sample in Rabbit -1
Conventional gel HPLC chromatograms of 3rd hour blood
5.58
sample in Rabbit -2
5.59
sample in Rabbit -1
5.60
sample in Rabbit -2
5.61
sample in Rabbit -1
5.62
sample in Rabbit -2
5.63
sample in Rabbit -1
5.64
sample in Rabbit -2
5.65
sample in Rabbit -1
5.66
sample in Rabbit -2
5.67
sample in Rabbit -1
5.68
sample in Rabbit -2
Niosomal gel HPLC chromatograms of 0th hour blood sample
5.69
in Rabbit -1
5.70
in Rabbit -2
Niosomal gel HPLC chromatograms of 1st hour blood sample
5.71
in Rabbit -1
Niosomalgel HPLC chromatograms of1st hour blood sample in
5.72
Rabbit -2
Niosomal gel HPLC chromatograms of 3rd hour blood sample
5.73
in Rabbit -1
Niosomalgel HPLC chromatograms of 3rd hour blood sample
5.74
in Rabbit -2
Niosomalgel HPLC chromatograms of 5th hour blood sample in
5.75
Rabbit -1
5.76
in Rabbit -2
5.77
in Rabbit -1
5.78
in Rabbit -2
5.79
in Rabbit -1
5.80
in Rabbit -2
5.81
in Rabbit -1
Niosomalgel HPLC chromatograms of 11th hour blood sample
5.82
in Rabbit -2
Niosomalgel HPLC chromatograms of 24th hour blood sample
5.83
in Rabbit -1
5.84
in Rabbit -2
Ethosomal gel HPLC chromatograms of 0th hour blood sample
5.85
in Rabbit -1
Ethosomall gel HPLC chromatograms of 0th hour blood sample
5.86
in Rabbit -2
Ethosomal gel HPLC chromatograms of 1st hour blood sample
5.87
in Rabbit -1
Ethosomal gel HPLC chromatograms of1st hour blood sample
5.88
in Rabbit -2
Ethosomal gel HPLC chromatograms of 3rd hour blood
5.89
sample in Rabbit -1
Ethosomal gel HPLC chromatograms of 3rd hour blood
5.90
sample in Rabbit -2
5.91
in Rabbit -1
5.92
in Rabbit -2
5.93
in Rabbit -1
5.94
in Rabbit -2
5.95
in Rabbit -1
Ethosomalgel HPLC chromatograms of 9th hour blood sample
5.96
in Rabbit -2
Ethosomal gel HPLC chromatograms of 11th hour blood
5.97
sample in Rabbit -1
5.98
sample in Rabbit -2
5.99
sample in Rabbit -1
5.100
sample in Rabbit -2
Transferosomal gel HPLC chromatograms of 0th hour blood
5.101
sample in Rabbit -1
5.102
sample in Rabbit -2
Transferosomal gel HPLC chromatograms of 1st hour blood
5.103
sample in Rabbit -1
Transferosomal gel HPLC chromatograms of1st hour blood
5.104
sample in Rabbit -2
Transferosomal gel HPLC chromatograms of 3rd hour blood
5.105
sample in Rabbit -1
Transferosomal HPLC chromatograms of 3rd hour blood
5.106
sample in Rabbit -2
5.107
sample in Rabbit -1
5.108
sample in Rabbit -2
5.109
sample in Rabbit -1
5.110
sample in Rabbit -2
5.111
sample in Rabbit -1
5.112
sample in Rabbit -2
5.113
sample in Rabbit -1
5.114
sample in Rabbit -2
5.115
sample in Rabbit -1
5.116
sample in Rabbit -2
LIST OF ABBREVIATIONS
µg microgram
µm micrometer
nm nanometer
ng nanogram
0
C degree centigrade.
hrs hours
min minutes
ml milliliter
v/v volume by volume
w/w weight by weight
w/v weight by volume
v/w olume by weight
% percentage
FN1-FN8 Nystatin niosomal suspensions
FE1-FE8 Nystatin ethosomal suspensions
FT1-FT8 Nystatin transferosomal suspensions
UV ultra violet
LUVs large unilamellar vesicles
SUVs small unilamellar vesicles.
MLVs multi lamellar vesicles
SEM Scanning electron microscope
HPLC High pressure liquid chromatography
n Release exponent
CPCSEA Committee for the purpose of supervision of
experiments on animals
ICH International conference on harmonization
SD Standard deviation
LIST OF APPENDICES
Appendix - 1 Publication - 1
Appendix - 4 Conference - 1
Appendix - 5 Conference - 2
Appendix - 6 Approval Letter from CPCSEA

Chapter - 1
INTRODUCTION
Chapter - 1: Introduction
Table of contents
S.No Name of the subtitle Page No

1.1 Advantages of Transdermal drug delivery system
1.2 Disadvantages of Transdermal drug delivery system
1.3 Physiology of the skin
1.4 Pathways of drug absorption through the skin
General method of drug absorption from skin
1.5 to produce systemic effect
1.6 Vesicular Carrier Systems
1. INTRODUCTION
Route of administration is also one of the important influential factors, to
be considered, to produce desired therapeutic effect and patient compliance.
Different routes are available to administer the drugs which includes IM, IV,
SC, Nasal, Oral, transdermal, topical, rectal, etc.

Figure 1.1: Different routes of drug administration
Though different routes are available, the Oral route is an ancient, highly
successful, comfortable and economical route for administration of drugs. Its
success rate is limited by different reasons includes first pas metabolism,
gastric irritation, nausea, vomiting, unpleasant taste and odour of some drugs,
etc. Third highly successful route of administration is transdermal route from
ancient days. Even in current days, the analysis of the global market scenario
of the drug delivery market revealing the same1.

Figure 1.2: Market segmentation the global drug delivery market in 2007
Transdermal drug delivery systems were defined as self–contained,
discrete dosage forms which when applied to the intact skin, deliver the drug
(s) through the skin at controlled rate to the systemic circulation 2.
There is much difference between the transdermal and topical drug
delivery systems. In transdermal drug delivery systems the drug reaches to the
deeper layers of the skin and from there into the systemic circulation and
produces the systemic effect. The concentration of the drug in the plasma is
significant.
Eg. NitroDur transdermal patch, Fentanyl transdermal patch.
Topical drug delivery can be defined as the application of a drug
containing formulation to the skin to directly treat cutaneous disorders with
the intent of containing the pharmacological or other effect of the drug to the
surface of skin or within3.

In topical drug delivery systems, the drug will not reach the deeper layers
of the skin but remains upper layers of the skin and produces the localized
effect. Concentration of the drug in plasma is insignificant.
Eg. Lidocain patch.
Figure 1.3: Topical and transdermal drug delivery systems
The process of administration of drugs to the skin is using over the
millennia. They used to administer the drugs to produce localized effect and
the medication is used to administer in the form of ointments, gels, creams,
pates and poultices, etc. these dosage formsare incapable to produce systemic
effect. Administration of drugs through skin to produce systemic effect is a
relatively new phenomenon and is introduced to overcome the drawbacks
associated with oral administration and IV routes 4.
The first adhesive transdermal delivery system (TDDS) patch was
approved by the Food and Drug Administration in 1979 (scopolamine patch for
motion sickness). Nitroglycerine patches were approved in 1981. This method

of delivery became widely recognized when nicotine patches for smoking
cessation were introduced in 1991. Transdermal drug delivery systems growth
between the 2003-2007 was more than tripled. Today, there are 19
transdermal delivery systems available in the market and the drug is
administering by different techniques such as usage of the permeation
enhancers, iontophoresis, etc5.
Figure 1.4: Number of Transdermal drugs approved in each year.

Table 1.1: Transdermal drugs approved by the US FDA.
Approval
Drug Indication Product Name Marketing company
year
Novartis Consumer
Transderm-
1979 Scopolamine Motion sickness Health (Parsippany,
Scop
NJ)
Transderm- Novartis (East

1981 Nitroglycerin Angina pectoris
Nitro Hannover, NJ)
Boehringer Ingelheim
1984 Clonidine Hypertension Catapres-TTS
(Ridgefield, CT)
Menopausal Novartis (East

1986 Estradiol Estraderm
symptoms Hannover, NJ)
Janssen
1990 Fentanyl Chronic pain Duragesic Pharmaceutica
(Titusville, NJ)
GlaxoSmithKline
Nicoderm, (Philadelphia, PA),
1991 Nicotine Smoking cessation Habitrol, Novartis Consumer
ProStep Health (Parsippany,
NJ) Elan (Gainesville)
Testosterone Alza, Mountain View,

1993 Testosterone Testoderm
deficiency CA
Approval
year
Lidocaine/
Local dermal Iomed (Salt Lake
1995 epinephrine Iontocaine
analgesia City, UT)
(iontophoresis)
Estradiol/ Menopausal Novartis (East

1998 Combipatch
norethidrone symptoms Hannover, NJ)
Endo
Post-herpetic
1999 Lidocaine Lidoderm Pharmaceuticals
neuralgia pain
(Chadds Ford, PA)
Ortho-McNeil
Ethinylestradiol/
2001 Contraception Ortho Evra Pharmaceutical
norelgestromin
(Raritan, NJ)
Bayer Healthcare
Estradiol/ Menopausal
2003 Climara Pro Pharmaceuticals
levonorgestrel symptoms
(Wayne, NJ)
2003 Oxybutynin Overactive bladder Oxytrol Watson Pharma
Lidocaine Local dermal Echo Therapeutics

2004 SonoPrep
(ultrasound) anesthesia (Franklin, MA)
Endo
Lidocaine/ Local dermal
2005 Synera Pharmaceuticals
tetracaine analgesia
(Chadds Ford, PA)
Approval
year
Fentanyl HCl Acute postoperative Alza, Mountain View,

2006 Ionsys
(iontophoresis) pain CA
Attention deficit
2006 Methylphenidate hyperactivity Daytrana Shire (Wayne, PA)
disorder
Major depressive Bristol-Myers Squibb

2006 Selegiline Emsam
disorder (Princeton, NJ)
Schwarz Pharma
2007 Rotigotine Parkinson’s disease Neupro
(Mequon, WI)
Novartis (East
2007 Rivastigmine Dementia Exelon
Hannover, NJ)
1.1. Advantages of Transdermal drug delivery system
 Avoidance of first pass metabolism of drugs.

 Transdermal medication delivers a steady infusion of a drug over a prolonged
period of time. Adverse effects or therapeutic failures frequently associated with
intermittent dosing can also be avoided.
 The simplified medication regimen leads to improved patient compliance and
reduced the side effects, inter and intra-patient variability.
 No interference with gastric and intestinal fluids.
 Maintains stable or constant and controlled blood levels for longer period of
time.
 Comparable characteristics with interavenous infusion.
 It increases the therapeutic value of many drugs via avoiding specific problems
associated with the drug like GI irritation, lower absorption, decomposition due
to ‘hepatic first pass’ effect.
 This route is suitable for the administration of drugs having very short half life,
narrow therapeutic window and poor oral availability.
 Improved patient compliance and comfort via non-invasive, painless and simple
application.
 Flexibility of terminating the drug administration by simply removing the patch
from the skin.
 Self administration is possible in these systems.
1.2. Disadvantages of Transdermal drug delivery system

 The possibility of local irritation may develop at the site of application. Many
problems like Erythema, itching, and local edema can be caused by the drug,
the adhesive, or other excipients in the patch formulation.
 Drugs having large molecular size make absorption difficult. So drug molecule
should ideally be below 800-1000 daltons.
 Many drugs with a hydrophilic structure having a low penetration through the
skin and slowly to be of therapeutic benefit. Drugs with a lipophillic character,
however, are better suited for transdermal delivery.
 The barrier function of the skin changes from one site to another on the same
person, from person to person and with age.
 Transdermal drug delivery system cannot achieve high drug levels in
blood/plasma.
1.3. Physiology of the skin
The skin6 also knows as the cutaneous membrane covers the external
surface of the body and is the largest organ of the body in the both surface
area and weight. In adults, the skin covers an area of about 2 square meters
(22 square feet) and weight 4.5-5 kg (10-11 lb), about 7% of total body weight.
It ranges in thickness from 0.5 mm on the eyelids to 4.0 mm on the heels. Over
most of the body it is 1-2 mm thick. Anatomically, the skin has many
histological layers but in general, it is described in terms of three major tissue
layers. Figure shows the structure of the skin.
The three layers of the skin are as follows.
1. The epidermis
2. The dermis and
3. The hypodermis.
The superficial, thinner portion, which is composed of epithelial tissue, is
the epidermis. The deeper, thicker connective tissue portion is the dermis.
While the epidermis is a vascular, the dermis is vascular. For this reason, if
you cut the epidermis there is no bleeding, but if you cut the dermis there is
bleeding.
Deep to the dermis, but not part of the skin, is the subcutaneous layer.
Also called the hypodermis, this layer consists of areolar and adipose tissues,
Fibers that extend from the dermis anchor the skin to underlying fascia, the
connective tissue around muscles and bones. The subcutaneous layer serves
as a storage depot for fat and contains large blood vessels that supply the skin.
This region (and sometimes the dermis) also contains nerve endings called
pacinian (lamellated) corpuscles that are sensitive to pressure.
1.3.1. Epidermis
The epidermis is composed of keratinized stratified squamous epithelium.
It contains four principal types of cells. They are
1. Keratinocytes
2. Melanocytes
3. Langerhans cells and
4. Merkel cells.
About 90% of epidermal cells are Keratinocytes, which are arranged in
four or five layers and produce the protein keratin. Recall from chapter four
that keratin is a tough, fibrous protein that helps protect the skin and
underlying tissues from abrasions, heat, microbes, and chemicals.
Keratinocytes also produce lamellar granules, which release a water-repellent
sealant that decreases water entry and loss and inhibits the entry of foreign
materials.
About 8% of the epidermal cells are melanocytes, which develop from the
ectoderm of a developing embryo and produce the pigment melanin. Their long,
slender projections extend between the keratinocytes and transfer
melaningranules to them. Melanin is a yellow-red or brown-black pigment that
contributes to skin color and absorbs damaging ultraviolet (UV) light. Once
inside keratinocytes, the melanin granules cluster to from a protective veil over
the nucleus, on the side towards the skin surface. In this way they shield the
nuclear DNA from damage by UV light. Although their melanin granules
effectively protect keratinocytes, melanocytes themselves are particularly
susceptible to damage by UV light.
Langerhans cells also called epidermal dendritic cells, arise from red
bone marrow and migrate to the epidermis ,where they constitute a small
fraction of the epidermal cells.they participate in immune responses mounted
against microbes that invade the skin ,and are easily damaged by UV light.
Their role in the immune response is to help other cells of the immune system
recognize an invading microbe and destroy it.
Merkel cells are the least numerous are the epidermal cells. They are
located in the deepest layer of the epidermis, where they contact the flattened
process of a sensory neuron, a structure called a merkel (tactile) disc .Merkel
cells and their associated merkel discs detect touch sensations.
Several distinct layers of keratinocytes in various stages of development form
the epidermis. In most regions of the body the epidermis has four strata or
layers –stratum basale, stratum spinosum, stratum granulosum and a thin
stratum corneum. This is called thin skin. Where exposure to friction is
greatest, such as in the fingertrips, palms and soles, the epidermis has five
layers - stratum basale, stratum spinosum, stratum granulosum, stratum
lucidem, and a thick stratum corneum. This is called thin skin.
1.3.1.1. Stratum basale
The deepest layer of the epidermis is the stratum basle composed of a
single row of cuboidal or columnar keratinocytes.Some cells in this layer are
stem cells that undergo cell division to continually produce new keratinocytes.
The nuclei of keratinocytes in the stratum basale are large, and their cytoplasm
contains many ribosomes,a small Golgi complex, a few mitochondria , and
some rough endo plasmic reticulum.the cytoskeleton within keratinocytes of
the stratum basale includes scatterd intermeadiare filaments, called keratin
intermediate filaments (tonofilaments) . The keratin intermediate filament
forms the tough protein keratin and its more superficial epidermal layers.
Keratin protects the deeper layers from injury. Keratin intermediate filaments
attach to desmosomes, which bind cells of the stratum basale to rach other
and to the cell of the adjacent stratum spinosum, and to hemidesmosomes,
which bind the keratinocytes to the basement membrane posisioned between

the epidermis and the dermis. Melanocytes and merkel cells with their
associated merkel discs are scatterd among the keratinocytes of the basal layer
.The stratum basale is also known as the stratum germinativum to indicate its
role in forming new cells.
1.3.1.2. Stratum spinosum
Superficial to the stratum basale there is stratum spinosum. This
stratum mainly consists of numerous keratinocytes arranged in 8-10 layers.
Cells in the more superficial layers become somewhat flattened.The
keratinocytes in the stratum spinosum, which are produced by the stem cells
in the basale layer, have the same organells as cells of the stratum basale and
some retained their ability to devide. The keratinocytes of these layers produce
coarser bundles of keratin in intermediate filaments than those of the basal
layer. All though they are rounded and larger in living tissue ,cells of the
stratum spinosum shrink and pull apart when prepared for microscopic
examination so that they appeare to be coverd with thornlike spins. At each
spinelike projection, bundles of keratin intermediate filaments insert in to
desmosomes, which tightly join the cells to one another. This arrangement
provides both strength and flexibility to the skin. Langerhans cells and
projections of melonocytes are also presents in the stratum spinosum.
1.3.1.3. Stratum granulosum

At about the middle of the epidermis, the stratum granulosum consists
of three to five layers of flattened keratinocytes that are undergoing apoptosis.
The nuclei and other organelles of these cells begin to degenerate as they move
further from their source of nutrition. Even though keratin intermediate
filaments or no longer being produced by these cells, they become more
apparent because the organelles in the cells are regressing. A distinctive future
of cells in this layer is the presence of darkly staining granules of a protein
called keratohyalin, which assembles keratin intermediate filaments into
keratin. Also present in the keratinocytes are membrane-enclosed lamellar
granules, which fuse with the plasma membrane and release a lipid-rich
secretion. This secretion is deposited in the spaces between cells of the stratum
granulosum, stratum lucidum, and stratum corneum. The lipid-rich secretion
act as a water –repellent sealant,retardingloss and entry of water and entry of
foreign materials. As their nuclei break down during apoptosis, the
keratinocytes of the stratum granulosum can do longer carry on vi-tal
metabolic reactions, and they die. Thus, the stratum granulosum marks the
transition between the deeper, metabolically active strata and the dead cells of
the more superficial strata.
1.3.1.4. Stratum Lucidum
The stratum lucidum is present only in the thick skin of areas such as
the fingertips, palms, and soles. It consists of four to six layers of flattened
clear, dead keratinocytes that contain large amount of keratin and thickened
plasma membranes. This probably provides an additional level of toughness in
this resign of thick skin.
1.3.1.5. Stratum Corneum
The stratum corneum consists on average of 25 to 30 layers of flsttened
dead keratinocytes, but can range in thickness from a few cells in thin skin to
50 or more cell layers in thick skin. The cells are extremely thin, flat, plasma
membrane –enclosed packages of keratin that no longer contain a nucleus or
any internal organelles. They are the final product of the differentiation process
of the keratinocytes. The cells within each layer overlap one another like the
scales on the skin of a snake. Neighboring layers of cells also form strong
connections with one another. The plasma membranes of adjacent cells are
arranged in complex, wavy folds that fit together like pieces of a jigsaw puzzle
to hold the layers together. In this outer stratum of the epidermis, cells are
continuously shed and replaced by cells from the deeper strata. Its multiple
layers from injury and microbial invasion. Constant exposure of skin to friction
stimulates increased cell production that results in the formation of a callus,
an abnormal thickening of the stratum corneum.
1.3.2. Dermis
The second, deeper part of the skin, the dermis, is composed of dense
irregular connective tissue containing collagen and elastic fibers. This woven
network of fibers has great tensile strength. The dermis also has the ability to
stretch and recoil easily. It is much thicker than the epidermis, and this
thickness varies from region to region in the body, reaching is greatest

thickness on the palms and soles. Leather, which we use for belts, shoes,
baseball gloves, and basketballs, is the dried and treated dermis of other
animals. The few cells present in the dermis include predominantly fibroblasts,
with some macrophages, and a few adipocytes near its boundary with the
subcutaneous layer. Blood vessels, nerves, glands, and hair follicles are
embedded in the dermal layer. The dermis is essential to the survival of the
epidermis, and these adjacent layers form many important structural and
functional relations. Based on its tissue structure, the dermis can be divided
into a thin superficial papillary region and a thick deeper reticular region.
The papillary region makes up one-fifth of the thickness of the total
layer. It consists of thin collagen and fine elastic fibers. Its surface area is
greatly increased by dermal papillae, small, nipple-shaped structure that
project into the undersurface of the epidermis. All dermal papillae contain
capillary loops. Some also contain tactile receptors called meissner corpuscles (
MIS-ner) or corpuscles of touch , nerve endings that are sensitive to touch. Still
other dermal papillae also contain free nerve endings, dendrites that lack any
apparent structural specialization. Different free nerve endings initiate signals
that give rise to sensations of warmth, coolness, pain, tickling, and itching.
The reticular region, which is attached to the subcutaneous layer,
consists of bundles of thick collagen fibers, scattered fibroblasts, and various
wandering cells. Some adipose cells can be present in the deepest part of the
layer, along with some coarse elastic fibers. The collagen fibers in the reticular
region are arranged in a netlike manner and have a more regular arrangement
than those in the papillary region. The more regular orientation of the large
collagen fibers helps the skin resist stretching. Blood vessels, nerves, hair
follicles, sebaceous (oil) glands, and sudoriferous (sweat) glands occupy the
spaces between fibers.
The combination of collagen and elastic fibers in the reticular region
provides the skin with strength, extensibility, the ability to stretch, and
elasticity, the ability to return to original shape after stretching. The
extensibility of skin can be readily seen around joints and in pregnancy and
obesity.
The surfaces of the palms, fingers, soles, and toes have a series of ridges
and grooves. They appear either as straight lines or as a pattern of loops and
whorls, as on the tips of the digits. These epidermal ridges are produced during
the third month of fetal development as downward projections of the epidermis
into the dermis between the dermal papillae of the papillary region. The
epidermal ridges create a strong bond between the epidermis and dermis in a
region of high mechanical stress. The epidermis ridges also increase the
surface area of the epidermis and thus increase the grip of the hand or foot by
increasing friction. Finally, the epidermal ridges greatly increase surface area,
which increase the number of meissner corpuscles and thus increases tactile
sensitivity. Because the ducts of sweat glands open on the tops of the
epidermal ridges as sweat pores, the sweat and ridges form fingerprints on
touching a smooth object. The epidermal ridges pattern is in part genetically
determined and is unique for each individual. Even identical twins have
different patterns. Normally, the ridge pattern does not change during life,
except to enlarge, and thus can serve as the basis for identification. The study
of the pattern of epidermal ridges is called dermatoglyphics.
In addition to forming epidermal ridges, the complex papillary surface of
the dermis has other functional properties. The dermal papillae greatly
increase the surface contact between the dermis and epidermis. This increased
dermal contact surface, with its extensive network of small blood vessels,
serves as an important source of nutrition for the overlying epidermis.
Molecules diffuse from the small blood capillaries in the dermal papillae to the
cells of the stratum basale, allowing the basal epithelial stem cells to divide and
the keratinocytes to grow and develop. As keratinocytes push toward the
surface and away from the dermal blood source, they are no longer able to
obtain the nutrition they require, leading to the eventual breakdown of their
organelles.
The dermal papillae fit together with the complementary epidermal ridge
to form an extremely strong junction between the two layers. This jigsaw
puzzle-like connection strengthens the skin against shearing forces that
attempt to separate the epidermis from the dermis.

Figure 1.5: Structure of the skin
1.4. Pathways of drug absorption through the skin
Several investigations were carried out to determine the penetration
pathways of the topically applied active principles into/through the skin to
produce both localized and/or systemic effects. The investigations proposed
different pathways through which drugs can be absorbed into and/or across
the skin depending on the physicochemical properties of the drug. Both
hydrophilic and lipophilic drugs are absorbed from different routes of the skin.
The barrier properties of the stratum corneum will not permeate drugs through
the skin but due to the presence of various absorption routes facilitates the
permeation of drugs and allows the drugs to reach the systemic circulation.
The different absorption routes proposed by the researchers are as follows7:
1. Transfollicular route
2. Transcellular route
3. Intercellular route
1.4.1. Transfollicular route
This route is the shortest pathway for drugs to reach the systemic
circulation that provides a large area for diffusion of drugs. Various oil glands,
hair follicles, sweat glands and pores openings present on the outer surface of
the skin via their ducts. These ducts acts continuous channels for
transportation of the drugs across the stratum corneum but extent of drug
transportation across the skin will be influenced by various factors like
secretions from the glands, amount of secretion and content etc. However
transappendageal route occupies only 0.1% of total skin surface and therefore
contributes minimum.
1.4.2. Transcellular route
In transcellular route drug transportation will takes place from the
corneocytes which consists of highly hydrated keratin. This corneocytes will
create hydrophilic pathways for transportaion of drugs. Around the corneocytes
there will be lipids which connect these cells. Therefore drugs require
partitioning and diffusion steps for permeation into the systemic circulation.
This is the widely using route for transportation of various drugs. In this route
the drugs will pass through the matrix (cytoplasm) of the cells and is suitable
route for hydrophilic drugs. The highly hydrated keratin provide aqueous
pathway to the hydrophilic drugs. A number of partitioning and diffusion steps
are require to pass the drug through the cell matrix.
1.4.3. Intercellular route
In intercellular route, the drugs diffuse through the lipid matrix present
between the cells. The barrier property of this route is due to the tortuous
structure formed by corneocytes and the drug has to pass through the
alternating lipid and aqueous domain by partitioning into the lipid bilayer and
diffusing to the inner side. It has been found that water has to travel 50 times
more by this route so; it is suitable mainly for uncharged lipophilic drugs.
Figure 1.6: Pathways of drug absorption through the skin

1.5. General method of drug absorption from skin to produce systemic
effect
To produce desired therapeutic effect, the drug concentration in the
plasma should lie in the therapeutic window. i.e. between minimum effective
concentration (MEC) and maximum safe concentration (MSC). All drugs of
desired concentration will not reach the therapeutic window when applied on
the skin by means of conventional dosage forms such as ointments, creams,
gels, etc because of various reasons like barrier properties of the skin, high
molecular weight, high melting point, less lipophilicity of the drug etc. To
overcome these difficulties and to achieve of desired concentration in the
plasma different researchers introduced different technologies. The different
technologies introduced by the researchers for administration of drugs across
the skin to produce systemic effect are as follows.
 Chemical potential adjustment
 Ion pairs and complex coacervates
 Eutectic systems
 Hydration
 Chemical penetration enhancers
 Electrophoresis
 Iontophoresis
 Ultrasound ( Phonophoresis, Sonophoresis)
 Laser radiation and photochemical waves
 Radio frequency
 Magnetophoresis
 Temperature (“thermophoresis”)
 Microneedle based devices
 Skin puncture and perforation
 Needleless injection
 Suction ablation
 Application of Pressure
 Skin stretching
 Skin abrasion
 Pharmaceutical carrier systems, etc
1.5.1. Chemical potential adjustment
The maximum skin penetration rate is obtained when a drug is at its
highest thermodynamic activity as is the case in a supersaturated. The
diffusion of paraben from saturated solutions in eleven different solvents
through a silicone membrane was determined. Due to the different solubility of
the parabens in the various solvents, the concentration varied over two orders
of magnitude. However, paraben flux was the same from all solvents, as the
thermodynamic activity remained constant because saturated conditions were
maintained throughout the experiment. Supersaturated solutions can occur
due to evaporation of solvent or by mixing of cosolvents. Clinically, the most
common mechanism is evaporation of solvent from the warm skin surface,
which probably occurs, in many topically applied formulations. In addition, if

water is imbibed from the skin into the vehicle and acts as an antisolvent, the
thermodynamic activity of the permeant would increase .

8-11
1.5.2. Ion pairs and complex coacervates:
Charged drug molecules do not readily partition into or permeate
through human skin. Formation of lipophilic ionpairs has been investigated to
increase stratum corneum penetration of charged species. This strategy
involves adding an oppositely charged species to the charged drug, forming an
ion-pair in which the charges are neutralised so that the complex can partition
into and permeate through the stratum corneum. The ion-pair then dissociates
in the aqueous viable epidermis releasing the parent charged drug, which can
diffuse within the epidermal and dermal tissues12.
1.5.3. Eutectic systems
The melting points of a drug influences solubility and hence skin
penetration. According to regular solution theory, the lower the melting point,
the greater the solubility of a material in a given solvent, including skin lipids.
The melting point of a drug delivery system can be lowered by formation of a
eutectic mixture: a mixture of two components which, at a certain ratio, inhibit
the crystalline process of each other, such that the melting point of the two
components in the mixture is less than that of each component alone. A
number of eutectic systems containing a penetration enhancer as the second
components have been reported, for example: Ibuprofen with terpenes 13, and
methyl nicotinate14, propranolol with fatty acids15 and lignocaine with
menthol16.
1.5.4. Hydration:
Water is the most widely used and safest method to increase skin
penetration of both hydrophilic17 and lipophilic permeants18. The water content
of the stratum corneum is around 15 to 20% of the dry weight but can vary
according to humidity of the external environment. Additional water within the
stratum corneum could alter permeant solubility and thereby modify
partitioning from the vehicle into the membrane. In addition, increased skin
hydration may swell and open the structure of the stratum corneum leading to
an increase in penetration, although this has yet to be demonstrated
experimentally. For example, Scheuplein and Blank showed that the diffusion
coefficients of alcohols in hydrated skin were ten times that observed in dry
skin19. Hydration can be increased by occlusion with plastic films; paraffins,
oils, waxes as components of ointments and water-in-oil emulsions that
prevent transepidermal water loss; and oil-in-water emulsions that donate
water. Of these, occlusive films of plastic or oily vehicle have the most profound
effect on hydration and penetration rate20. A commercial example of this is the
use of an occlusive dressing to enhance skin penetration of lignocaine and
prilocane from EMLA cream in order to provide sufficient local anaesthesia
within about 1 hour. Also drug delivery from many transdermal patches
benefits from occlusion.
1.5.5. Chemical penetration enhancers (CPEs)
The use of CPEs over the other techniques has certain advantages,
including design flexibility of the patch and ease of patch application over a
large area (>10 cm2)21. An ideal penetration enhancer should reversibly reduce
the barrier resistance of the SC without damaging the skin cells. According to
Finnin et al.22 : ideal penetration enhancers should possess the following
properties:
 Pharmacologically inert
 Nontoxic, nonirritating, and non-allergenic
 Rapid onset of action; predictable and suitable duration of action for the
drug used
 Reversible effect of the CPE on the barrier property of SC
 Chemically and physically compatible with the delivery system
 Readily incorporated into the delivery system
 Inexpensive and cosmetically acceptable
Because the skin provides such a formidable barrier to the delivery of most
drugs, a broad range of different chemical additives have been tested to
enhance transdermal penetration during the last two decades. Much of the
cited literature is found in patents 23 as well as pharmaceutical science
literature24. Even though many chemical entities have been identified, only a
few were introduced in the market due to several limitations, which include
their economic feasibility and the toxic effects on skin, which make them
undesirable for developing transdermal patches. Penetration enhancers may
act by one or more of three main mechanisms 25:
 Disruption of the highly ordered structure of stratum corneum lipid.
 Interaction with intercellular protein.

 Improved partition of the drug, coenhancer or solvent into the stratum
corneum.
1.5.5.1. Sulphoxides and similar chemicals
Dimethyl sulphoxides (DMSO) is one of the earliest and most widely
studied penetration enhancers. It is a powerful aportic solvent which hydrogen
bonds with itself rather than with water. It is colourless, odourless and is
hydroscopic and is often used in many areas of pharmaceutical sciences as a
“universal solvent”. DMSO alone has been applied topically to treat systemic
inflammation. DMSO works rapidly as a penetration enhancer - spillage of the
material onto the skin can be tasted in the mouth within a second. Although
DMSO is an excellent accelerant, it does create problems. The effect of the
enhancer is concentration-dependent and generally cosolvents containing >
60% DMSO are needed for optimum enhancement efficacy. However, at these
relative high concentrations, DMSO can cause erythema and wheal of the
stratum corneum. Denaturing of some skin proteins results in erythema,
scaling, contact uticaria, stinging and burning sensation 26. Since DMSO is
problematic for use as a penetration enhancer, researchers have investigated a
similar chemically-related material as a accelerant. Dimethylacetamide (DMAC)
and dimethylformamide (DMF) are similarly powerful aportic solvents.
However, Southwell and Barry, showing a 12-fold increase in the flux of
caffeine permeating across a DMF-treated human skin, concluded that the
enhancer caused irreversible membrane damage27. DMF irreversibly damages
human skin membranes but has been found in vivo to promote the
bioavailability of betamethasone-17-benzoate as measured by vasoconstrictor
assay28, 29
. DMSO may also extract lipids, making the horny layer more
permeable by forming aqueous channels.
It has been postulated that DMSO denatures the intercellular structural
proteins of the stratum corneum, or promotes lipid fluidity by disruption of the
ordered structure of the lipid chains. In addition, DMSO may alter the physical
structure of the skin by elution of lipid, lipoprotein and nucleoprotein
structures of the stratum corneum. Decylmethylsulfoxide (DCMS) is thought to
promote permeation enhancement as a result of protein-DCMS interaction
creating aqueous channels, in addition to lipid interactions.
1.5.5.2. Alkanes
Long chain alkanes (C7-C16) have been shown to enhance skin
permeability by non-destructive alteration of the stratum corneum barrier 30.
These findings were confirmed in studies in which nonane was investigated as
an enhancer31, although there must be some destructive solubilisation and
biochemical extraction caused by these lipophilic solvents.
1.5.5.3. Azone
Azone (1-dodecylazacycloheptan-2-one or laurocapran) was the first
molecule specifically designed as a skin penetration enhancer. Azone is a
colourless, odourless liquid with a melting point of -7 ºC and it possesses a
smooth, oily but yet non-greasy feel. Azone is a highly lipophilic material with a
log p octanol / water of around 6.2 and it is soluble in and compatible with
most organic solvents including alcohol and propylene glycol. Azone enhances
the skin transport of a wide variety of drugs including steroids, antibiotics and
antiviral agents. Azone is most effective at low concentrations, being employed
typically between 0.1- 5% but more often between 1- 3% 32. Azone partitions
into a bilayer lipid to disrupt their packing arrangement but integration into
the lipid is unlikely to be homogeneous. Azone provokes dynamic structural
disorder of the intercellular lamellar lipid structure throughout the stratum
corneum and the creation of fluid domains involving the intercellular lipids,
which was suggested by 2H NMR assay. Another mechanism was also
proposed based on the alteration of the lateral bonding within stratum corneum
lipid lamellae33. Azone/PG increase penetration through the stratum corneum
by affecting both the hydrophilic and lipophilic routes of penetration. Azone
increases the fluidity of the lipid layer, while PG increases the water content of
the proteinaceous region and helps azone partition into the aqueous region. A
combination of these two helps the penetration of hydrophilic drugs greatly 34.
1.5.5.4. Pyrrolidones
Pyrrolidones have been used as permeation enhancers for numerous
molecules including hydrophilic (e.g. mannitol and 5-flurouracil) and lipophilic
(progesterone and hydrocortisone) permeants. N-methyl-2- pyrolidone was
employed with limited success as a penetration enhancer for captopril when
formulated in a matrix-type transdermal patch 35. The pyrrolidones partition
well into human stratum corneum within the tissue and they may act by
altering the solvent nature of the membrane. Pyrrolidones have been used to
generate reservoirs within the skin membrane. Such a reservoir effect offers a
potential for sustained release of a permeant from the stratum corneum over
extended time periods36.
1.5.5.5. Urea
Urea promotes transdermal permeation by facilitating hydration of the
stratum corneum and by the formation of hydrophilic diffusion channels within
the barrier. As urea itself possesses only marginal penetration enhancing
activity, attempts have been made to synthesis analogues containing more
potent enhancing moieties. Thus Wong and co-workers synthesised cyclic urea
analogues and found them to be as potent as Azone for promoting
indomethacin across shed snake skin and hairless mouse skin Cyclic urea
permeation enhancers are biodegradable and non-toxic molecules consisting of
a polar parent moiety and a long chain alkyl ester group. As a result,
enhancement mechanism may be a consequence of both hydrophilic activity
and lipid disruption mechanism37.
1.5.5.6. Fatty acids and Esters
Percutaneous drug absorption has been increased by a wide variety of
fatty acids and their esters, the most popular of which is oleic acid. A general
trend has been seen that unsaturated fatty acids are more effective in
enhancing percutaneous absorption of drugs than their saturated
counterparts. It is of interest to note that many penetration enhancers such as
azone contain saturated or unsaturated hydrocarbon chains and some
structure - activity relationships have been drawn from the extensive studies of
Aungst who employed a range of fatty acids, acids, alcohols, sulphoxides,

surfactants and amides as enhancers for naloxone 38, 39
. Shin et al40 studied
various penetration enhancers like glycols (diethylene glycol and tetraethylene
glycol), fatty acids (lauric acid, myristic acid and capric acid) and nonic
surfactant (polyoxyethylene-2-oleyl ether, polyoxy ethylene-2-stearly ether) on
the release of triprolidone. Lauric acid in Propylene glycol enhanced the
delivery of highly lipophilic antiestrogen 41. Oleic acid greatly increased the flux
of many drugs such as increasing the flux of salicylic acid 28-fold and 5-
flurouracil flux 56-fold through human skin membrane in vitro42. The enhancer
interacts with and modifies the lipid domains of the stratum corneum as would
be expected for a long chain fatty acid with cis- configuration.
1.5.5.7. Alcohols, fatty alcohols and glycols
Alcohols may influence transdermal penetration by a number of
mechanisms. The alkyl chain length of the alkanols (fatty alcohols) is an
important parameter in the promotion of permeation enhancement.
Augmentation appears to increase as the number of carbon units increases, up
to a limiting value43. In addition, lower molecular weight alkanols are thought
to act as solvents, enhancing the solubility of drugs in the matrix of the
stratum corneum. Disruption of the stratum corneum integrity through
extraction of biochemicals by the more hydrophobic alcohols almost certainly
also contributes to enhanced mass transfer through this tissue 44. Ethanol is
the most commonly used alcohol as a transdermal penetration enhancer.
Ethanol acts as a penetration enhancer by extracting large amounts of stratum
corneum lipids. It also increases the number of free sulphydryl groups of

keratin in the stratum corneum proteins. Usually, pretreatment of skin with
ethanol increases the permeation of hydrophilic compounds, while it decreases
that of hydrophobic ones45. The molecular complexity of different glycol
molecules is a determinant of their efficacy as permeation enhancers. Solubility
of the drug in the delivery vehicle is markedly influenced by the number of
ethylene oxide functional groups on the enhancer molecule; this solubility
modification may either enhance or retard transdermal flux depending on the
specific drug and delivery environment. The activity of propylene glycol (PG) is
thought to result from solvation of α keratin within the stratum corneum; the
occupation of proteinaceous hydrogen bonding sites reducing drug-tissue
binding and thus promoting permeation46. PG is widely used as a vehicle for
penetration enhancers and shows synergistic action when used with, for
example, oleic acid.
1.5.5.8. Surfactants
Many surfactants are capable of interacting with the stratum corneum to
increase the absorption of drugs and other active compounds from products
applied to the skin. Skin penetration measurements are valuable in quantifying
these effects and observing the influence of surfactant chemistry and
concentration. A surfactant interacts with skin by depositing onto the stratum
corneum, thereby disorganizing its structure. Then surfactant can solubilise or
remove lipids or water-soluble constituents in or on the surface of the stratum
corneum. Finally it can be transported into and through the stratum corneum.
This last effect is related to the surfactant and stratum corneum protein
interaction and epidermal keratin denaturation 47. In general, anionic
surfactants are more effective than cationic and nonionic surfactants in
enhancing skin penetration of target molecules. Some anionic surfactants
interact strongly with both keratin and lipids, whereas the cationic surfactants
interact with the keratin fibrils of the cornified cells and result in a disrupted
cell-lipid matrix. Nonionic surfactants enhance absorption by inducing
fluidization of the stratum corneum lipids.Scheuplein and Ross reported that
the capacity of the statum corneum to retain significant quantities of
membrane-bound water is reduced in the presence of sodium dodecanoate and
sodium dodecyl sulfate48. This effect is readily reversible upon removal of the
agents. These investigations proposed that anionic surfactants alter the
permeability of the skin by acting on the helical filaments of the stratum
corneum, thereby resulting in the uncoiling and extension of _-keratin
filaments to produce _- keratin. Then they cause an expansion of the
membrane, which increases permeability. However, more recent findings
suggest that impairment of the skin’s barrier properties is unlikely to result
from changes in protein conformation alone. Based on differential scanning
calorimetry results, sodium lauryl sulfate (SLS) disrupted both the lipid and
the protein components. The amount of surfactant that penetrates the skin
after the disruption of the skin barrier depends on the monomer activity and
the critical micelle concentration (CMC). Above the CMC, the added surfactant
exists as micelles in the solution and micelles are too large to penetrate the
skin. The extent of barrier disruption and penetration enhancement of a

surfactant is also strongly dependent on surfactant structure, especially alkyl
chain length. In general, studies have shown that surfactants having 12
carbons in their alkyl chain cause more disruption to the skin barrier and
allow drugs to penetrate more readily than those that have more or less than
12 carbons. The explanation for this optimum of 12 carbons is not known yet.
1.5.5.9. Essential oil, terpenes and terpenoids
Terpenes are found in essential oils, and are compounds comprising of
only carbon, hydrogen and oxygen atoms, but which are not aromatic.
Numerous terpenes have long been used as medicines as well as flavoring and
fragrance agents. The essential oils of eucalyptus, chenopodium and ylang-
ylang have been found to be effective penetration enhancers for 5-flouorouracil
transversing human skin in vivo49. Cornwell et al50 investigated the effect of 12
sesquiterpenes on the permeation of 5-flurouracil in human skin. Pretreatment
of epidermal membranes with sesquiterpene oil or using solid sesquiterpenes
saturated in dimethyl isosorbide increased the absorption of 5- flurouracil. L-
menthol has been used to facilitate in vitro permeation of morphine
hydrochloride through hairless rat skin as well as diffusion of imipramine
hydrochloride across rat skin and hydrocortisone through hairless mouse
skin51. One mechanism by which this agent operates is to modify the solvent
nature of the stratum corneum, thus improving drug partitioning into the
tissue. Many terpenes permeate human skin well and large amounts of terpene
have been found in the epidermis after application from a matrix-type patch.
Terpenes may also modify drug diffusivity through the membrane. During
steady state permeation experiments using terpenes as penetration enhancers,
the lag time for permeation was usually reduced, indicating some increase in
drug diffusivity through the membrane following terpene treatment.
1.5.5.10. Cyclodextrins
Cyclodextrins are biocompatible substances that can form inclusion
complexes with lipophilic drugs with a resultant increase in their solubility,
particularly in aqueous solutions. However, cyclodextrins alone were
determined be to less effective as penetration enhancers than when combined
with fatty acids and propylene glycol52.
1.5.5.11. Oxazolidinones
Oxazolidinones are a new class of chemical agents which have the
potential for use in many cosmetic and personal care product formulations.
This is due to their ability to localize co-administered drug in skin layers,
resulting in low systemic permeation. The structural features of these
permeation enhancers are closely related to sphingosine and ceramide lipids
which are naturally found in the upper skin layers. Oxazolidinones such as 4-
decyloxazolidin-2-one has been reported to localize the delivery of many active
ingredients such as retinoic acid and diclofenac sodium in skin layers. This
compound has a higher molecular weight and lipophilicity than other solvent-
type enhancers, physical characteristics that may be beneficial in terms of a
reduction in local toxicity because of the lack of effective absorption of these
enhancers into the lower skin layers where irritation is likely to be occur.
Figure 1.7: Chemical structure of typical chemical penetration enhancers
1.5.6. Electroporation
The use of electropermeabilization, as a method of enhancing diffusion
across biological barriers, dates back as far as 100 years 53. Electroporation
involves the application of high voltage pulses to induce skin perturbation.
High voltages (≥100 V) and short treatment durations (milliseconds) are most
frequently employed. Other electrical parameters that affect delivery include
pulse properties such as waveform, rate and number. The increase in skin
permeability is suggested to be caused by the generation of transient pores
during electroporation. The technology has been successfully used to enhance
the skin permeability of molecules with differing lipophilicity and size (i.e. small
molecules, proteins, peptides and oligonucleotides) including
biopharmaceuticals with a molecular weight greater that 7kDA, the current
limit for iontophoresis. Genetronics Inc (San Diego, California) have developed
a prototype electroporation transdermal device, which has been tested with
various compounds with a view to achieving gene delivery, improving drug
delivery and aiding the application of cosmetics. Other transdermal devices
based on electroporation have been proposed by various groups however, more
clinical information on the safety and efficacy of the technique is required to
assess the future commercial prospects.
Figure 1.8: Basic design of electroporation drug delivery device
1.5.7. Iontophoresis
This method involves enhancing the permeation of a topically applied
therapeutic agent by the application of a low level electric current either
directly to the skin or indirectly via the dosage form 54. Increase in drug
permeation as a result of this methodology can be attributed to either one or a
combination of the following mechanisms; electrorepulsion (for charged
solutes), electro osmosis (for uncharged solutes) and electropertubation (for
both charged and uncharged). Parameters that affect design of an iontophoretic

skin delivery system include; electrode type, current intensity, pH of the
system, competitive ion effect and permeant type. The launch of
commercialised systems of this technology has either occurred or is currently
under investigation by various companies. Extensive literature exists on the
many types of drugs investigated using iontophoretic delivery and the reader is
referred to the following extensive reviews. The PhoresorTM device (Iomed Inc.),
was the first iontophoretic system to be approved by the FDA in the late 70’s as
a physical medicine therapeutic device. In order to enhance patient compliance
the use of patient- friendly, portable and efficient iontophoretic systems have
been under intense development over the years. Such improved systems
include the Vyteris and E-TRANS iontophoretic devices. Previous work has also
reported that the combined use of iontophoresis and electroporation is much
more effective than either technique used alone in the delivery of molecules
across the skin. The limitations of ionotophoretic systems include the
regulatory limits on the amount of current that can be used in humans
(currently set at 0.5 mA cm-2) and the irreversible damage such currents could
do to the barrier properties of the skin. In addition, iontophoresis has failed to
significantly improve the transdermal delivery of macromolecules of >7000
Da55.
Figure 1.9: Basic principle of iontophoresis. A current passed between the
active electrode and the indifferent electrode repelling drug away from
the active electrode and into the skin.
1.5.8. Ultrasound (sonophoresis and phonophoresis)
Ultrasound involves the use of ultrasonic energy to enhance the
transdermal delivery of solutes either simultaneously or via pre-treatment and
is frequently referred to as sonophoresis or phonophoresis. The proposed
mechanism behind the increase in skin permeability is attributed to the
formation of gaseous cavities within the intercellular lipids on exposure to
ultrasound resulting in disruption of the SC 56. Ultrasound parameters such as
treatment duration, intensity and frequency are all known to affect
percutaneous absorption, with the latter being the most important. Although
frequencies between 20 kHz-16 MHz have been reported to enhance skin
permeation, frequencies at the lower end of this range (< 100 kHz) are believed
to have a more significant effect on transdermal drug delivery with the delivery
of macromolecules of molecular weight up to 48 kDa being reported. The

SonoPrep ® device (Sontra Medical Corporation) uses low frequency ultrasound
(55 kHz) for an average duration of 15 s to enhance skin permeability. This
battery operated hand held device consists of a control unit, ultrasonic horn
with control panel a disposable coupling medium cartridge, and a return
electrode. The ability of the SonoPrep® device to reduce the time of onset of
action associated with the dermal delivery of local anaesthetic from EMLA
cream was recently reported. In the study by Kost et al.(37), skin treatment by
ultrasound for an average time of 9 s resulted in the attainment of dermal
anaesthesia within 5 min, which was comparable to the 60 min required in for
non-treated skin. The use of other small, lightweight novel ultrasound
transducers to enhance the in vitro skin transport of insulin has also been
reported by a range of workers57.
Figure 1.10: Basic principle of phonophoresis. Ultrasound pulses are

passed through the probe into the skin fluidizing the lipid bilayer by the
formation of bubbles caused by cavitation.
1.5.9. Laser radiation and photomechanical waves
Lasers have been used in the clinical therapies for decades, therefore
their effects on biological membranes are well documented. Lasers are
frequently used for the treatment of dermatological conditions such as acne
and to confer ‘facial rejuvenation’ where the laser radiation destroys the target
cells over a short frame of time (~300 ns). Such direct and controlled exposure
of the skin to laser radiation results in ablation of the SC without significant
damage to the underlying epidermis. Removal of the SC via this method has
been shown to enhance the delivery of lipophilic and hydrophilic drugs. The
extent of barrier disruption by laser radiation is known to be controlled by
parameters such wavelength, pulse length, pulse energy, pulse number and
pulse repetition rate. A hand-held portable laser device has been developed by
Norwood Abbey Ltd (Victoria, Australia). In a study involving human
volunteers, the Norwood Abbey laser device was found to reduce the onset of
action of lidocaine to 3-5 min, whilst 60 min was required to attain a similar
effect in the control group. The Norwood Abbey system has been approved by
the US and Australian regulatory bodies for the administration of a topically
applied anaesthetic. Pressure waves (PW), which can be generated by intense
laser radiation, without incurring direct ablative effects on the skin have also
been recently found to increase the permeability of the skin. It is thought that
PW form a continuous or hydrophilic pathway across the skin due to expansion
of the lacunae domains in the SC. Important parameters affecting delivery such
as peak pressure, rise time and duration has been demonstrated. The use of
PW may also serve as a means of avoiding problems associated with direct
laser radiation. Permeants that have been successfully delivered in vivo include
insulin58, 40 kDa dextran and 20 nm latex particles. A design concept for a
transdermal drug delivery patch based on the use of PW has been proposed by
Doukas & Kollias.
1.5.10. Radio-frequency
Radio-frequency involves the exposure of skin to high frequency
alternating current (~ 100 kHz) resulting in the formation of heat-induced
microchannels in the membrane similar to when laser radiation is employed.
The rate of drug delivery is controlled by the number and depth of the
microchannels formed by the device, which is dependent on the properties of
the microelectrodes used in the device. The Viaderm device (Transpharma Ltd)
is a hand held electronic device consisting of a microprojection array (100
microelectrodes/cm2) and a drug patch. The microneedle array is attached to
the electronic device and placed in contact with the skin to facilitate the
formation of the microchannels. Treatment duration takes less than a second,
with a feedback mechanism incorporated within the electronic control
providing a signal when the microchannels have been created, so as to ensure
reproducibility of action. The drug patch is then placed on the treated area.
Experiments in rats have shown the device to enhance the delivery of
granisetron HCL, with blood plasma levels recorded after 12 h rising to 30
times higher levels than that recorded for untreated skin after 24 h. A similar
enhancement in diclofenac skin permeation was also observed in the same

study. The device is reported not to cause any damage to skin with the radio-
frequency-induced microchannels remaining open for less than 24 h. The skin
delivery of drugs such as testosterone and human growth hormone by this
device is also currently in progress.
1.5.11. Magnetophoresis
This method involves the application of a magnetic field which acts as an
external driving force to enhance the diffusion of a diamagnetic solute across
the skin. Skin exposure to a magnetic field might also induce structural
alterations that could contribute to an increase in permeability. In vitro studies
by Murthy showed a magnetically induced enhancement in benzoic acid flux,
which was observed to increase with the strength of the applied magnetic field.
Other in vitro studies using a magnet attached to transdermal patches
containing terbutaline sulphate (TS), demonstrated an enhancement in
permeant flux which was comparable to that attained when 4% isopropyl
myristate was used as a chemical enhancer. In the same paper the effect of
magnetophoresis on the permeation of TS was investigated in vivo using guinea
pigs. The preconvulsive time (PCT) of guinea pigs for those subjected to
magnetophoretic treatment was found to last for 36 h which was similar to that
observed after application of a patch containing 4% IPM. This was in contrast
to the response elicited by the control (patch without enhancer), when the
increase in PCT was observed for only 12 h. In human subjects, the level of TS
in the blood was higher but, not significantly different to that observed with the
patch containing 4% IPM. The fact that this technique can only be used with
diamagnetic materials will serve as a limiting factor in its applicability and
probably explains the relative lack of interest in the method.
1.5.12. Temperature (“thermophoresis”)
The skin surface temperature is usually maintained at 32ºC in humans
by a range of homeostatic controls. The effect of elevated temperature (non-
physiological) on percutaneous absorption was initially reported by Blank et
al., (54). Recently, there has been a surge in the interest of using
thermoregulation as means of improving the delivery profile of topical
medicaments. Previous in vitro studies59 have demonstrated a 2-3 fold increase
in flux for every 7-8°C rise in skin surface temperature. The increased
permeation following heat treatment has been attributed to an increase in drug
diffusivity in the vehicle and an increase in drug diffusivity in the skin due to
increased lipid fluidity. Vasodilation of the subcutaneous blood vessels as a
homeostatic response to a rise in skin temperature also plays an important role
in enhancing the transdermal delivery of topically applied compounds. The in
vivo delivery of nitroglycerin, testosterone, lidocaine, tetracaine and fentanyl
from transdermal patches with attached heating devices was shown to increase
as a result of the elevated temperature at the site of delivery. However, the
effect of temperature on the delivery of penetrants over 500 Da has not been
reported. The controlled heat-aided drug delivery patch (CHADD) (Zars Inc, Salt
Lake City, Utah) consists of a patch containing a series of holes at the top
surface which regulate the flow of oxygen in to the patch. The patch generates
heat chemically in a powder filled pouch by an oxidative process regulated by

the rate of flow of oxygen through the holes in to the patch. The CHADD
technology was used in the delivery of a local anaesthetic system (lidocaine and
tetracaine) from a patch (S-Caine®) and found to enhance the depth and
duration of the anaesthetic action in human volunteers when the results
obtained in active and placebo groups were compared. Zars Inc together with
Johnson and Johnson, recently submitted an investigational new drug (IND)
application to the FDA for Titragesia™ (a combination of CHADD disks and
Duragesic Patches, the latter contains fentanyl for treatment of acute pain).
Kuleza & Dvoretzky also describe a heat delivery patch or exothermic pad for
promoting the delivery of substances into the skin, subcutaneous tissues,
joints, muscles and blood stream, which may be of use in the application of
drug and cosmetic treatments. All the studies described above employed an
upper limit skin surface temperature of 40 - 42 oC, which can be tolerated for
a long period (> 1 h). In heat-patch systems where patient exposure to heat is ≤
24 h, such an upper limit may be necessary for regulatory compliance. In
addition, the issue of drug stability may also need to be addressed when
elevated temperatures are used. Thermopertubation refers to the use of
extreme temperatures to reduce the skin barrier. Such perturbation has been
reported in response to using high temperatures over a short duration (30ms),
with little or no discomfort, using a novel patch system 60. These investigators
developed a polydimethylsiloxane (PDMS) patch for non intrusive transdermal
glucose sensing via thermal micro-ablation. Ablation was achieved by
microheaters incorporated within the patch. The heat pulse is regulated by

means of a resistive heater, which ensures that the ablation is limited within
the superficial of dead layers of the skin. Average temperatures of 130 oC are
required for ablation to occur within 33 ms after which SC evaporation results.
Other heat assisted transdermal delivery devices under development include
the PassPort ® patch (Althea therapeutics) which ablates the SC via a similar
manner as the above described PDMS patch. The exposure of skin to low
(freezing) temperatures has been reported to decrease its barrier function but
has however not been exploited as means of enhancing skin absorption. The
final group of active enhancement methods entails the use of a physical or
mechanical means to breach or bypass the SC barrier.
1.5.13. Microneedle based devices
One of the first patents ever filed for a drug delivery device for the
percutaneous administration of drugs was based on this method 61. The device
as described in the patent consists of a drug reservoir and a plurality of
projections extending from the reservoir. These microneedles of length 50-110
μm will penetrate the SC and epidermis to deliver the drug from the reservoir.
The reservoir may contain drug, solution of drug, gel or solid particulates and
the various embodiments of the invention include the use of a membrane to
separate the drug from the skin and control release of the drug from its
reservoir. As a result of the current advancement in microfabrication
technology in the past ten years, cost effective means of developing devices in
this area are now becoming increasingly common. A recent commercialisation
of microneedle technology is the Macroflux® microprojection array developed

by ALZA Corporation. The macroflux® patch can either be used in combination
with a drug reservoir or by dry coating the drug on the microprojection array;
the latter being better for intracutaneous immunization. The lengths of the
microneedles have been estimated to be around 50-200 μm and therefore are
not believed to reach the nerve endings in the dermo-epidermal junction. The
microprojections/ microneedles (either solid or hollow) create channels in the
skin, hence allowing the unhindered movement of any topically applied drug.
Clinical evaluations report minimal associated discomfort and skin irritation
and erythema ratings associated with such systems are reportedly low. This
technology serves as an important and exciting advance in transdermal
technology due to the ability of the technique to deliver medicaments with
extremes of physicochemical properties (including vaccines, small molecular
weight drugs and large hydrophilic biopharmaceuticals) Yuzhakov et al.,
describes, the production of an intracutaneous microneedle array and provides
an account of its use (microfabrication technology). Various embodiments of
this invention can include a microneedle array as part of a closed loop system
‘smart patch’ to control drug delivery based on feedback information from
analysis of body fluids. Dual purpose hollow microneedle systems for
transdermal delivery and extraction which can be coupled with electrotransport
methods are also described by Trautman et al., Down et al., Allen et al.,62 These
mechanical microdevices which interface with electronics in order to achieve a
programmed or controlled drug release are referred to as
microelectromechanical systems (MEMS) devices.

Figure 1.11: Basic design of microneedle delivery devices. Needles of
approximately with or without centre hollow channels are placed onto the
skin surface so that they penetrate the stratum corneum and epidermis
without reaching the nerve endings present in the upper dermis.
1.5.14. Skin puncture and perforation
These devices are similar to the microneedle devices produced by
microfabrication technology. They include the use of needle-like structures or
blades, which disrupt the skin barrier by creating holes and cuts as a result of
a defined movement when in contact with the skin. Godshall and Anderson
(81), described a method and apparatus for disruption of the epidermis in a
reproducible manner. The apparatus consists of a plurality of microprotrusions
of a length insufficient for penetration beyond the epidermis. The
microprotrusions cut into the outer layers of the skin by movement of the
device in a direction parallel to the skin surface. After disruption of the skin,
passive (solution, patch, gel, ointment etc) or active (iontophoresis,
electroporation etc) delivery methods can then be utilised. Descriptions of other

devices based on a similar mode of action have been described by Godshall 63,
Kamen64, Jang65and Lin et al66.
1.5.15. Needleless injection
Needleless injection is reported to involve a pain free method of
administering drugs to the skin. This method therefore avoids the issues of
safety, pain and fear associated with the use of hypodermic needles.
Transdermal delivery is achieved by firing the liquid or solid particles at
supersonic speeds through the outer layers of the skin using a suitable energy
source. Over the years there have been numerous examples of both liquid (Ped-
O-Jet®, Iject® Biojector2000® Medi-jector® and Intraject®) and powder
(PMEDTM device formerly known as powderject® injector) systems. The latter
device has been reported to deliver successfully testosterone, lidocaine
hydrochloride and macromolecules such as calcitonin and insulin 66,67.
Problems facing needless injection systems include the high developmental cost
of both the device and dosage form and the inability, unlike some of the other
techniques described previously, to programme or control drug delivery in
order to compensate for inter-subject differences in skin permeability. In
addition, the long-term effect of bombarding the skin with drug particles at
high speed is not known thus, such systems may not be suitable for the
regular administration of drugs. It may however be very useful in the
administration of medicaments which do not require frequent dosing e.g.
vaccines.
1.5.16.Suction ablation
Formation of a suction blister, involves the application of a vacuum or
negative pressure to remove the epidermis, whilst leaving the basal membrane
intact. The cellpatch® (Epiport Pain Relief, Sweden) is a commercially available
product based on this mechanism. It comprises a suction cup, epidermatome
(to form a blister) and device (which contains morphine solution) to be attached
to the skin this method which avoids dermal invasivity there by avoiding pain
and bleeding is also referred to as skin erosion. Such devices have also been
shown to induce hyperaemia in the underlying dermis in in vivo studies which
was detected via laser Doppler flowmetry and confirmed via microscopy, and is
thought to further contribute to the enhancement of dextran and morphine
seen with this method. The disadvantages associated with the suction method
include the prolonged length of time required to achieve a blister (2.5 h),
although this can be reduced to 15-70 min by warming the skin to 38 oC. In
addition, whilst there is no risk of systemic infection compared to the use of
intravenous catheters, the potential for epidermal infections associated with
the suction method cannot be ignored even though the effects might be less
serious.
1.5.17.Application of Pressure
The application of modest pressures (i.e. 25 kPa) has been shown to
provide a potentially noninvasive and simple method of enhancing skin
permeability of molecules such as caffeine 68. These workers attributed the
increase in transcutaneous flux to either an improved transapendageal route or

an increased partition of the compound into the SC when pressure was
applied. This method may also work due to the increased solubility of caffeine
in the stratum corneum caused by the increase in pressure.
Figure1.12: Enhancement of transdermal permeation by pressure wave
1.5.18. Skin stretching
These devices hold the skin under tension in either a unidirectional or
multidirectional manner69,70. The authors claim that a tension of about 0.01 to
10 mP results in the reversible formation of micropathways. The efficiency of
the stretching process was demonstrated by monitoring the delivery of a
decapeptide (1 kDa) across the skin of hairless guinea pigs using a
microprotrusion array. The results of the study showed that the bi-directional
stretching of skin after microprotrusion piercing, allowed the pathways to stay
open (i.e. delayed closure) hence facilitating drug permeation to a greater extent
(27.9 ± 3.3 μg/cm2 h) than in the control group (9.8 ± 0.8 μg/cm2 h), where
the skin was not placed under tension after microneedle treatment. However,
increased skin permeation in the absence of microneedle pre-treatment was
found not to occur. Other methods involving the use of skin stretching with
subsequent use of delivery devices based on electrotransport, pressure,
osmotic and passive mechanisms have also been suggested but the value of
skin stretching alone without the benefit of a secondary active delivery device
remains to be seen.
1.5.19.Skin abrasion
These techniques, many of which are based on techniques employed by
dermatologists in the treatment of acne and skin blemishes (e.g.
microdermabrasion), involve the direct removal or disruption of the upper
layers of the skin to enhance the permeation of topically applied compounds.
The delivery potential of skin abrasion techniques are not restricted by the
physicochemical properties of the drug and previous work has illustrated that
such methods enhance and control the delivery of a hydrophilic permeant,
vitamin C vaccines and biopharmaceuticals. One current method is performed
using a stream of aluminium oxide crystals and motor driven fraises Sage &
Bock also describe a method of pre-treating the skin prior to transdermal drug
delivery which consists of a plurality of microabraders of length 50-200 μm.
The device is rubbed against the area of interest, to abrade the site, in order to
enhance delivery or extraction. The microabraders/microprotrusions terminate
as blunt tips and therefore do not penetrate the SC. The device functions by
removing a portion of the SC without substantially piercing the remaining
layer. Some of these methods are claimed to offer advantages such as minimal
patient discomfort, increased patient compliance, ease of use and less risk of
infection compared to their more “invasive” predecessors such as ablation and

the use of hypodermic needles/cannulas to deliver medicaments across the
skin.
1.5.20.Pharmaceutical carrier systems
Different types of pharmaceutical carriers are present. They are –
particulate, polymeric, macromolecular, and cellular carrier. Particulate type
carrier also known as a colloidal carrier system, includes lipid particles (low
and high density lipoprotein-LDL and HDL, respectively), microspheres,
nanoparticles, polymeric micelles and vesicular like liposomes, niosomes
pharmacosomes, virosomes, etc. 48- 51

these carriers were introduced for
different purposes and all will not have same qualities.
Figure1.13: various pharmaceutical carriers
1.6. Vesicular Carrier Systems
Since two decades the interest on the vesicles has been increased
tremendously because of the potential advantages of vesicles. Huge research
work is going on the vesicles to achieve different objectives. They become

vehicle of choice for drug delivery to researchers and are using these vesicles in
the areas like immunology, membrane biology, diagnostic techniques, genetic
engineering, etc and are using in the transport and targeting of active
pharmaceutical ingredients.
The vesicular systems are highly ordered assemblies of one or several
concentric lipid bilayers formed, when certain amphiphilic building blocks are
confronted with water. Vesicles can be formed from a diverse range of
amphiphilic building blocks. The terms such as synthetic bilayers allude to
the non-biological origin of such vesiculogenes. Biologic origin of these
vesicles was first reported in 1965 by Bingham71, and was given the name
Bingham bodies.
Figure1.14: Structure of typical vesicle
The different advantages of the vesicular carrier systems are as
follows.
1. Lipid vesicles have evolved successfully, as vehicles for controlled delivery.

2. Vesicles can be administered from different routs such as oral, IM, nasal,
transdermal, vaginal, rectal, ocular, etc. and possible to produce both the
localized and/or systemic effects.
3. Enahnaces the bioavailability of the drugs by increasing the mean residence
time in biological environment and/or by enhancing the solubility.
4. Reduces the adverse effects associated with the high doses of the drugs by
increasing the bioavalability of the active principles.
5. Enhances the stability of the drugs by avoiding the exposure of the drugs to the
susceptible environments.
6. Drug targeting can be possible by means of these vesicles.
7. Both hydrophilic and lipophilic drugs can incorporate in these vesicles.
As these vesicles have potential applications, the interest on these
vesicles have been increased and developed different vesicles for different
purposes. The different vesicles include liposomes, niosomes, ethosomes,
transferosomes, pharmacosomes, etc.
1.6.1. LIPOSOMES
Liposomes are simple microscopic lipid vesicles ranging from 20
nanometers to several micrometers in size and in these vesicles the lipid bilayer
encloses the aqueous environment. Both hydrophilic and lipophilic drugs can
be successfully entrapped72 in these vesicles. Hydrophilic and lipophilic drugs
entrapped in the central aqueous environment and within the lipid bilayers
respectively[6]. Liposomes can be used for delivery of drugs through oral,
topical, ocular, vaginal, etc routes.

Figure 1.15: structure of conventional liposome
Phospholipid and cholesterol are the main ingredients for manufacturing
of liposomes. Phosphatidylcholine (PC), a natural phospholipid, is most
commonly used for preparation of liposomes. One can also use other
phospholipids such as phosphatidyl ethanolamine (PE), phosphatidyl serine
(PS), phosphatidyl inositol (PI) and phosphatidyl glycerol (PG) for
manufacturing of liposomes. Cholesterol is used for different purposes such as
fluidity buffer and also provides rigidity to the lipid bilayer.
Figure 1.16: Chemical structure of Phosphatidylcholine (PC)
Liposomes, as particulate carriers, naturally can target cells of the

mononuclear phagocytic system(MPS), particularly macrophages to treat a
number of diseases such as infectious diseases, cancer and atherosclerosis
and inflammatory diseases. To achieve targeting of liposomes to monocytes,
macrophages and dendritic cells, the physicochemical properties of liposomes
has been modified by addition of surface ligands such as proteins, peptides,
antibodies, polysaccharides, glycolipids, glycoproteins and lectins. Doijad
Rajindra C et al prepared zidovudine, loaded liposomes for targeting to liver
followed by lungs, kidney and spleen against human immunodeficiency virus
(HIV)73. Though this liposomes have wide advantages, its success rate
decreased due to its bigger size, impermeable to deeper layers of the skin,
fusion, leakage, etc.
The proposed mechanisms for drug delivery across the skin through
liposomes are intact vesicular skin permeation, the penetration enhancing
effect, the adsorption effect and the penetration of liposomes through the
transappendageal route.
1.6.2. EMULSOMES
Emulsomes are lipid vesicles designed for poorly soluble drugs to
administer them through the parenteral route 74. The internal core of
emulsomes is prepared with fats and triglycerides and is stabilized in form of
o/w emulsion by adding high concentration of lecithin. These will posses both
liposomes and emulsions characteristics. Entrapment efficiency of lipophilic
drugs can be enhanced by the process of solidification or semisolidification of
internal oily core which also controls the release of drugs from vesicles. As
liposomes, in these vesicles also one can encapsulate hydrophilic drugs in
aqueous compartments of surrounding phospholipid layers. The solvent-
free and surfactant-free emulsome technologies were developed to increase the
encapsulation capacity of water insoluble antifungal and anticancer drugs
which showed improved preclinical efficacy for oral route. Vyas et al developed
zidovudine emulsomes for sustained and targeted drug delivery to liver for the
treatment of life-threatening viral infections like hepatitis, HIV and Epstein-
Barr virus infection.
Figure 1.17: Structure of emulsomes
1.6.3. ETHOSOMES
These lipid vesicles developed to deliver the drugs having poor
penetration through the skin. They are soft in nature having size range from
tens of nano meters to microns. These vesicles contains more concentration of
ethanol (20-50%) which brings soft malleable nature to the vesicles and also
solubilizes the inter cellular lipid and enhances the penetration of the drugs
through the skin75. Ethanol induces surface negative net charge to the vesicles
which decreases the size of the vesicles. Concentration of ethanol in vesicles is
inversely proportional to the size of the vesicles. Ethosomes has been used as
carrier for delivery of many drugs through the skin successfully to produce
both localized and/or systemic effects. The drugs includes antifungal agents
(fluconazole) antiviral agents (lamivudine, acyclovir, stavudine, and zidovudine),
NSAIDS (aceclofenac, diclofenac), antibiotics (cannabidol, erythromycin), etc. in
all the studies ethosomes has shown enhanced therapeutic effect when
compared with conventional methods.
Exact mechanism of drug permeation across the skin through ethosomes
is not proposed. It is assumed that a combination of two processes contribute
to the enhancing effect. The two processes are solubilisation of stratum
corneum lipid and on vesicles fluidity as well as dynamic interaction between
ethosomes and the stratum corneum.

Figure 1.18: Proposed mechanism for penetration of molecule from
ethosomal system across the lipid domain of stratum corneum
1.6.4. ENZYMOSOMES
These are developed to deliver the therapeutic proteins like enzymes and
are developed by complexing the enzymes with the lipids. Superoxide
Dismutase, a therapeutic agent for oxidative stress related diseases like
rheumatoid arthritis and ischaemia/reperfusion situations, loaded
enzymosomes have been developed for long circulation time in the blood, in
order to accumulate at inflammed target sites, while maintaining enzymatic
activity in its intact form.
1.6.5. SPHINGOSOME
These are introduced to overcome the stability problems associated with
liposomes and are called as sphingosomes due to the presence of the

sphigolipids in place of phospholipids in liposomes. Phospholipids readily
undergo chemical degradation such as oxidation, hydrolysis due to the
presence of ester linkage. In sphingolipids either an ether or amide linkage will
present which are resistant to chemical degradation and brings stability to the
sphingolipids than the phospholipids. Sphingosomes are efficient carriers
because of high stability, for targeting of the drug to the site of action and are
being biodegradable, innocuous nature and also identical to biological
membrane.
Sphingosomes offer selective passive targeting to tumour tissues and
flexibly bind with site-specific ligands to achieve active targeting. These are
much more stable to acid hydrolysis and have better drug retention
characteristics. Sphingosomal products e.g., Marqibo(TM) (vincristine loaded
sphingosomes) are loaded with active, cell cycle-specific anticancer agents that
are benefited from increased targeting and long duration of drug exposure at
the tumor site. Vinorelbine and topotecan are also selected for sphingosomal
formulation specifically for their ability to benefit from this novel
encapsulation.
Figure 1.19: structure of sphingolipids

1.6.6. TRANSFEROSOMES
Transferosome was introduced by GregorCevc in the year 1991. The
word “Transferosome” means “carrying body” and is derived from Latin word
“transferre” means “to carry across” and the Greek word “soma” means
“body”. The unique nature of these vesicles is that they have deformable
nature. Due to its deformable nature these vesicles penetrate through the
pores having less size than the vesicles and deliver the drugs across the skin
without making puncture of the skin and with less loss.
Figure 1.20: Structure of transferosomes
These are used as carriers for many drugs such as hoprmones,
proteins, peptides and various drug molecules. These vesicles are also used
successfully to deliver the active principles, such as insulin, interferons,
which are highly unstable in GIT, through the skin. Large molecular weight
substances such as proteins are difficult to deliver through skin due to their
large m.wt which can also delivered successfully through the skin by these
vesicles. In a study it was shown that these vesicles delivered diclofenac

sodium 10times more than the commercial hydrogel across the skin and
shown longer effect76. Anti-HIV agents like zidovudine, indinavir, etc also
enclosed in transferosomes and characterized for delivery of drugs across the
skin which shown higher concentration of drug in the systemic circulation.
Two mechanisms have been proposed to explain the drug permeation
across the skin by means of transferosomes. First, vesicles can act as drug
carrier systems, where by intact vesicles enter the saturation stratum
corneum carrying vesicle bound drug molecules into the skin. Second vesicles
can acts as penetration enhancers, whereby vesicle bilayers enter the stratum
corneum and subsequently modify the inter cellular lipid lamellae.
Figure1.21: Mechanism of transferosomes permeation across the
skin
1.6.7. PHARMACOSOMES
Pharmacosomes are novel vesicular drug delivery systems having unique
advantages over other drug delivery systems 77. Pharmacosomes are amphiphilic
lipoidal colloidal dispersions of drugs, covalently bound to lipids with potential
to improve bioavailability of poorly water soluble as well as poorly lipophilic
drugs. Any drug possessing a free carboxyl group or an active hydrogen atom(-
OH, -NH2) can be esterified (with or without a spacer group) to the hydroxyl
group of a lipid molecule, thus generating an amphiphilic prodrug. An
amphiphilic prodrug is then converted to pharmacosomes upon dilution with
water. The prodrug conjoins hydrophilic and lipophilic properties (thereby
acquiring amphiphilic characteristics), reduces interfacial tension, and at
higher concentration exhibits mesomorphic behavior. Because of decrease in
interfacial tension, the contact area increases, therefore bioavailability also
increases. As the drug is covalently conjugated with lipids, loss due to leakage
of drug does not occur. Hence, provides maximum entrapment efficiency. The
three main components for the preparation of pharmacosomes are drug,
solvent and lipid. Drug should contain active hydrogen atom (-COOH, OH,
NH2), can be esterified with lipid and form amphiphilic complexes, which
facilitate membrane transfer. The solvent should have high purity, volatility
and intermediate polarity (between the polarity of phospholipid and drug) for
the preparation of pharmacosomes. The most commonly used lipid for the
preparation of pharmacosomesis phosphatidylcholine. The pharmacosomes can
be prepared by hand-shaking and ether-injection methods. These have been
prepared for various non-steroidal anti-inflammatory drugs, proteins,

cardiovascular and antineoplastic drugs. Drug release from pharmacosomes is
by hydrolysis (including enzymatic) unlike liposomes the release of drug is by
diffusion through bilayer, desorption from the surface or degradation of
liposomes.
1.6.8. VIROSOMES
Virosomes are reconstituted viral envelopes that are composed of a lipid
bilayer in which inserted viral glycoproteins can be derived from different
enveloped viruses. Virosomes are described as liposomes with influenza virus
hemagglutinin (HA) and neuraminidase (NA) spikes on their surface.
Virosomes closely mimic the intact virus except that they do not contain virus
replication machineries. They retain the cell entry and membrane fusion
characteristics of the virus derived from. The two pathways by which
reconstituted vesicles are able to enter the cells and deliver their contents into
the cytoplasm are plasma membrane fusion(Sendai virus) and acid-induced
fusion from within endosomes (Influenza virus). As a result, foreign
substances encapsulated within the lumen of virosomes are effectively
delivered to the cytosol of target cells. Virosomes can be used in vaccination
for the efficient induction of antibody responses against the virus they are
derived from78.
1.6.9. NON-IONIC SURFACTANT VESICLES/NIOSOMES
First, in 1979, Handjanivila et al reported the niosomes will form upon
hydration of a mixture of cholesterol and single alkyl chain. These are also said
to be non-ionic surfactant vesicles. Since 1979, different non-ionic surfactants

such as polyglycerol alkyl ethers, glucosyldialkyl ethers, crown ethers,
polyoxyethylene alkyl ethers, ester linked surfactants, steroid linked
surfactant, brij and a series of spans, tweens, etc have to used to manufacture
the niosomes79. These have microscopic lamellar structure of size range 10-
1000nm consisting of spherical, uni or multilamellar and polyhedral vesicles in
aqueous media. cationic, anionic and ampholytic surfactants also used to
prepare niosomes but as they causes skin irritation, non ionic surfactants are
preferred which causes no irritation. These niosomes have many advantages
over lipid vesicles with respect to stability, manufacturing and storage. These
will not require special condition for manufacturing and storage because the
non ionic surfactants are less prone to oxidation comparatively lipids which are
used in preparation of liposomes and other lipid vesicles. These have shown
better patient compliance and better therapeutic effect in comparison to oily
formulations. Niosomes are osmotically active and stable and whose shape,
size, composition and fluidity of niosomes can be controlled as and when
required.
Figure 1.22: Non-ionic surfactant vesicle / Niosome
1.6.10. BILOSOMES
Bilosomes are the novel innovative drug delivery carriers consist of
deoxycholic acid incorporated into the membrane of niosomes. As
conventional vesicles (liposomes and niosomes) can cause dissolution and
undergo enzymatic degradation in gastro intestinal tract but incorporation of
bile salts (commonly used penetration enhancers) in niosomal formulation
could stabilize the membrane against the detrimental effects of bile acids in GI
tract. These bile salt stabilized vesicles are known as bilosomes. These are
highly biocompatible and have been found to improve the therapeutic efficacy
of drugs due to their stability in gastro intestinal tract. Bilosomes have been
found to increase the bioavailability of drugs as they can readily absorbed
through small intestine to the portal circulation (hepatocirculation). Through
this circulation they approach to liver and release the drug, so found to be an
effective tool in drug targeting to liver. Shukla et al showed that HBsAg loaded
bilosomes produced both systemic as well as mucosal antibody responses
upon oral administration. For extended humoral, cell-mediated and mucosal
immune responses, additional coating carrier system provided better
protection against disease for longer period of time. Optimum mannan coating
was found to stabilize the vesicles in gastrointestinal environment and also act
as a targeting ligand for mannose receptors expressed on macrophages and
dendritic cells80.
1.6.11. AQUASOMES
Aquasomes firstly developed by Kossovsky, are one of the most recently
developed delivery system for bioactive molecules 81. Aquasomes are three
layered structures (i.e. core, coating and drug) that are self-assembled
through non covalent bonds, ionic bonds and vander waals forces. They
consist of tin oxide, nanocrystalline carbon ceramic (diamonds) or brushite
(calcium phosphate dihydrate) core coated with oligomeric film to which
biochemically active molecules are adsorbed by copolymerization, diffusion or
adsorption with or without modification. The solid core provides the structural
stability, while the carbohydrate coating protects against dehydration and
stabilizes the biochemically active molecules. Aquasomes are spherical 60-
300nm size particles called Âbodies of waterÊ. Their water like properties
protects and preserves fragile biological molecules. Mechanism of action of
aquasomes is controlled by their surface chemistry, which deliver contents
through combination of specific targeting, molecular shielding and slow and
sustained release process. Due to their size and structural stability, these
avoid clearance by reticuloendothelial system and degradation by other
environmental changes. Aquasomes can be used as red blood cell substitutes
for the release of oxygen by haemoglobin. Aquasomes can be used as vaccines
for delivery of viral antigen, for targeted intracellular gene therapy, for delivery
of insulin and enzymes like DNAase and pigments/dyes.

Chapter - 2
LITERATURE REVIEW
Chapter - 2: Literature review
Table of contents

2.1 Literature on niosomes
2.2 Literature on ethosomes
2.3 Literature on transferosomes
2. LITERATURE REVIEW
Baillie et al82 (1985), have studied niosomes prepared by liposomal technology
and has concluded that niosomes are promising vehicles for drug delivery to
target organs, it was also concluded that these are less toxic than the vesicles
prepared from ionic surfactants.
Rogerson et al83 (1988), have studied the distribution of doxorubicin in mice
following the administration of niosomes and concluded that half life of the
drug were prolonged compared with the free drug solution profile and also
concluded that metabolism and maintenance of anti tumour effects are altered
when administered with the niosomes.
Chandra prakash et al84 (1990), have studied the pharmacokinetics of
surfactant vesicles entrapped methotrexate in tumour bearing mice and has
concluded that entrapment efficiency was increased with increase in the
lipophilicity of the surfactant and also concluded that its pharmacokinetics in
mice was markedly different in comparison to unentrapped drug.
Raja Naresh et al85 (1993), have studied the anti-inflammatory action of
niosomes encapsulated diclofenac sodium in rats and has concluded that
niosomal encapsulated diclofenac sodium shows higher anti-inflammatory
action than the diclofenac sodium when administered intra-peritoneally and
transdermally.
Uduppa et al86 (1993), have formulated and evaluated the methotrexate
niosomes and has concluded niosomes produce sustained activity and
niosomes are useful carrier for delivery of the methotrexate.

Malhotra et al87 (1994), have studied the properties and preparation of
niosomes for the delivery of bioactive agents and various drugs.
Partha Sarathi et al88 (1994), have studied the anti-cancer activity of vincristine
sulphate encapsulated niosomes in mice and has concluded that anti-cancer
activity increased with reduced toxicity.
Partha Sarathi et al89(1994), have formulated and evaluated the vincristine
encapsulated niosomes and have concluded that transmembrane pH gradient
drug uptake process was most satisfactory drug release from niosomes slowed
down and also stability of the system was enhanced.
Naresh et al90 (1996), have studied the antitumor activity of niosomes
encapsulated bleomycin and have concluded that these shows better
antitumor activity due to better delivery of bleomycin to the tumor site.
Raja naresh et al91 (1996), have studied the kinetics and tissue distribution of
niosomal bleomycin in tumour bearing mice and have concluded plasma
concentration of bleomycin significantly increased and removal of it from the
plasma decreased much when it administered niosomally than the free drug.
Arun et al92 (1998), have studied the effect of niosomes on the kinetics of
ketorlac tromethamine and have concluded that niosomes increases the
plasma level and biological half life of the drug significantly than the plain
drug.
Erdogan et al93 (1998), have studied the stability of niosomes and liposomes
encapsulated with iopromide and have concluded that niosomes have greater
stability than the liposomes.

Sheena et al94 (1998), have studied the antitumour efficiency of niosomal
entrapped withaferin-A and plain withaferin-A in mice and have concluded
that antitumor efficiency of niosomal encapsulated formulation is superior
than plain formulation.
Namdeo et al95 (1999), have prepared and evaluated the niosomes encapsulated
indomethacin and have concluded that niosomal encapsulated indomethacin
shown superior anti-inflammatory activity than the plain indomethacin
formulation.
Devi et al96 (2000), have studied the niosomal entrapped sumatriptan
succinate for nasal administration.
Ruckmani et al97 (2000), have prepared the niosomes encapsulated cytarabin
hydrochloride by film hydration method and evaluated for encapsulation,
storage and In vitro drug release and have concluded that drug release profile
was increased significantly and stability profile was good over long period.
Fang et al (2001)98, have studied the In vitro skin permeation from various
proniosomal gel formulations across the excited rat skin and have concluded
that the permeation of estradiol was increased after entrapping it in niosomes.
Ravichandran et al99 (2001), have prepared and evaluated the In vitro release
of diclofenac sodium from niosomes and have concluded that niosomal
formulations are stable and the drug release was extended for long period.
Ruckmani et al100 (2001), have studied the cytarabin (1-beta-D-
arabiofuranosylcytosine) entrapped niosomes and concluded that the

antitumor activity of cytarabin in mice was enhanced after encapsulation in
niosomes.
Alia sagar Shashiwala et al101 (2002), have studied the topical niosomal
encapsulated nimesulide gel and concluded that drug release was prolonged,
retention of the drug is increased, and permeation across the skin and anti
inflammatory activity have increased after entrapping in niosomes when
compared with plain nimesulide gel.
Balasubramaniam et al102 (2002), have prepared and evaluated niosomal
encapsulated daunorubicin hydrochloride formulation and concluded that
niosomal formulation shows greater /superior anti-tumor activity than the free
drug treatment.
Dutta Shivani et al103 (2002), have studied the niosomal delivery of plumbagin
ester and have concluded that it shows better anti fertility activity also reduces
toxicity of plumbagin ester.
Joseph et al104 (2002), have studied antitumor activity of etoposide
encapsulated niosomes against daltons lymphoma cells in Swiss albino mice
and have concluded that niosomes have the potential to produce significant
antitumor effect than the free drug and also increases the percentage of tumor
cell inhibition.
Manconi et al105 (2002), have prepared and evaluated the properties of trenitoin
loaded niosomes and have concluded that entrapment efficiency depends upon
the structure of the vesicle and In vitro drug release from niosomal formulation
is faster than from trenitoin solution.

Roux et al106 (2002), have studied the pH sensitive niosomal and liposomal
formulations bearing alkylated N-isopropylacrylamide copolymers with respect
to vesicle-polymer interaction, pH-responsiveness and stability in human
serum and have concluded that both formulations were released their contents
under mild acidic conditions, the niosomes lost their pH-sensitivity after
incubation in serum whereas liposomes maintained their ability to respond to
pH only when complexed with copolymer containing a high proportion of
hydrophobic anchor and also concluded that the interactions that take place
between the polymer and the vesicles strongly depends on the vesicle nature.
Satturwar et al107 (2002), was formulated and evaluated the ketoconazole
niosomes and have concluded that niosomes have the potential to reduce the
therapeutic dose of ketoconazole improving the performance.
Varshasoz et al108 (2003), have prepared insulin entrapped niosomes by film
hydration method and found that vesicles containing span 60 showed the
highest protection of insulin against proteolytic enzymes and good stability in
the presence of sodium desoxycholate & storage temperatures.
Aggarwal et al 109
(2004), have studied the topical niosomal preparation of
acetazolamide by ether injection method, lipid hydration method and reverse
phase evaporation methods and have concluded that drug entrapment
efficiency and percent of permeation vary with the charged and neutral
niosomes and also shown that topical niosomal formulation shows greater
physiological activity than the oral preparation of acetazolamide.

Dufes et al110 (2004), have studied the glucose targeted niosomes deliver
vasoactive intestinal peptide to the brain and have concluded that glucose
bearing vesicles are a novel tool to deliver drug across the blood brain barrier.
Mullaicharam et al111 (2004), have studied the organ distribution pattern of
niosomes encapsulated rifampicin compared with that of free rifampicin
solution in albino rats to find out the potential delivery system for the site
specific deliver of rifampicin and also to find out the most effective route of
administration in intravenous and intra tracheal routes and have concluded
that best route of administration is intra-tracheal administration and there is
significant difference in the distribution of rifampicin administered in the form
of solution and administered after entrapping in niosomes.
Aggarwal et al112 (2005), have prepared mucoadhesive niosomal ophthalmic
drug delivery system of timolol maleate and compared with the timolol maleate
solution in terms of in-vitro drug release and intra-occular pressure lowering
pharmacodynamic effect, finally concluded that niosomal formulation shown
significantly better results than the timolol maleate solution.
Alsarra et al113 (2005), have studied the uptake of atenolol from GIT tract
using the niosomes as drug carriers / permeation enhancers and have
concluded that atenolol entrapped in niosomes shows greater absorption. This
is evidenced by showing the greater permeation through an intestinal sac.
Guinedi et al114 (2005), have studied the bioavailability and corneal penetration
of the acetazolamide by loading the drug in niosomes and have concluded that
niosomes decreases the intra-ocular pressure significantly than the free drug
solution.
Huang et al115 (2005), have prepared cationic niosomes of sorbiton
monostearates by film hydration method and evaluated for their effect on
delivery of antisense oligonucleotides and the formulation results showed
positive results on cellular uptake of antisense oligonucleotides.
Jain et al116 (2005), have studied niosomes as carriers adjuvant system for
topical immunization and have concluded that niosomal immunization is more
effective when compared other topical immunizations.
Nasseri et al117 (2005), have studied the effect of cholesterol and temperature
on the elastic properties of niosomal membranes and have concluded that
interaction between the cholesterol and sorbitan monosterates should increase
the rigidity of the membranes.
Ning et al118 (2005), have prepared and evaluated the vaginal administering
niosomal clotrimazole gel and have concluded that it shows greater antifungal
activity than the plain clotrimazole gel.
Jain et al119 (2006), have studied the niosomal system for deliver of
rifampicin to lymphatic and have concluded that rifampicin encapsulated in
niosomes can successfully be used for the treatment of tuberculosis along
lymphatic system.
Manconi et al120 (2006), have studied the influence of thermodynamic activity
and niosome composition, size, lamellarity and charge on the transdermal
delivery of trenitoin and have concluded that small, negatively charged

niosomal formulations, which are saturated with trenitoin have shown to give
higher cutaneous drug retention than liposomes and marketed formulation.
Manivannan Rangasamy et al121 (2008) developed Acyclovir entrapped
niosomes by hand shaking and ether injection process with different ratios of
(1:1, 1;2 and 1:3) cholesterol (CHOL) and Span-80 (Non-ionic surfactant). They
found that the size range of niosomes prepared using ether injection method is
less than the niosomes prepared using hand shaking method. They also found
that the order of encapsulation efficiency increases when span-80
concentration was increased and also found In-vitro release of acyclovir more
from formulation prepared with CHOL: Span-80 (1:1).
Naresh Ahuja et al 122

(2008) developed Lansoprazole entrapped niosomes by
reverse phase evaporation method using a homogenizer and characterized for
its size rang, entrapment efficiency and in-vitro release of drug. They concluded
that they will reduce the systemic toxicity of drugs.
V. Sankar et al123 (2009) developed carboplatin niosomes for enhanced delivery
to cancer cells by thin film hydration technique using (cholesterol: surfactant)
in the micromolar ratio of 30:100 and with/without the addition of charge
inducing agents and Pluronic F 68. The In vitro cytotoxicity results of the
formulations reveal that the Tween 80 formulation with Pluronic shows the
highest level of cytotoxicity (90%) when compared with drug in solution. Also,
the stability studies of the formulations reveal that the formulations were
stable in refrigerated condition.

Gupta Naveen et al124 (2010) developed and evaluated Niosomes, a synthetic
microscopic vesicles consisting of an aqueous concentration enclosed in a
bilayer consisting of cholesterol and one or more nonionic surfactants to
improve the low corneal penetration and bioavailability characteristic shown by
conventional ophthalmic vehicles. They concluded that Niosomes formed from
span 60 and cholesterol in the ratio 200:100 (in mol) is a promising approach
to improve the bioavailability of ofloxacin even for an extended period of time
which showed good physicochemical properties, good stability and controlled
drug release pattern, thereby improving the bioavailability of the drug.
Y. Anand Kumar et al125 (2010) developed and optimized niosomal formulation
of aceclofenac in order to improve its bioavailability. They studied the effect of
the varying composition of non ionic surfactant and cholesterol on the
properties such as encapsulation efficiency, particle size and drug release.
They concluded that the bioavailability of aceclofenac can increase by this
technology.
Kandasamy Ruckmani et al126 (2010) developed Zidovudine entrapped
niosomes and characterized process-related variables like hydration and
sonication time, rotation speed of evaporation flask, and the effects of charge-
inducing agent and centrifugation on zidovudine entrapment and release from
niosomes ans stated that niosomes can be formulated by proper adjustment of
process parameters to enhance zidovudine entrapment and sustainability of
release. These improvements in zidovudine formulation may be useful in
developing a more effective AIDS therapy.

N. Paval rani et al127 (2010) prepared niosomes of rifampicin and gatifloxacin by
lipid hydration technique using rotary flash evaporator and evaluated for size,
entrppment efficiency and bactericidal activity. The prepared rifampicin and
gatifloxacin niosomes showed a vesicle size in the range of 100-300nm, the
entrapment efficiency were 73% and 70% respectively. The invitro release study
showed that 98.98% and 97.74% of release of rifampicin and gatifloxacin
niosomes respectively. The bactericidal activities of the niosomal formulation
were studied by BACTEC radiometric method using the resistant strains (RF
8554) and sensitive strains (H37Rv) of Mycobacterium tuberculosis which
showed greater inhibition and reduced growth index.
Saggam Srinivas et al128 (2010) developed aceclofenac niosomes and evaluated
for the effect of varying composition of non ionic surfactant and cholesterol on
the properties such as entrapment efficiency, particle size and drug release.
Release of the drug was modified and extended over a period of 72 h in all
formulations. The mechanism of dug release was found to be non fickian
(anomalous) diffusion governed by Peppas’ model.
D. Akhilesh et al129 (2011) possibility of manufacturing proniosomes and using
proniosome-based niosomes as drug carriers by preparing the Gliclazide
Loaded Maltodextrin Based Proniosomes. The study revealed that Proniosomes
are very promising as drug carriers.
Chawda Himmat Singh et al130 (2011) prepared niosome-encapsulated drug
delivery for anti-inflammatory drugs like nimesulide using different Non-ionic
surfactants like span 20, 40, 60 and cholesterol was used in different molar
ratios by lipid film hydration method and ether injection technique and
evaluated the vesicle size, encapsulation efficiency, In vitro release and physical
stability of the niosomes. Finding of all this investigation conclusively
demonstrate prolongation of drug release at a constant and controlled rate
after niosomal encapsulation of nimesulide.
P.U.Mohamed Firthouse et al131 (2011) formulated miconazole entrapped
Niosomes by varying the cholesterol and surfactant ratios as 1:0.5, 1:1,1:1.5 by
Thin Film Hydration Technique and each formulation was evaluated for
percentage of drug entrapment and for their cumulative drug release. They
found that formulation with 1:1 CHOL: SA ratio has shown higher entrapment
efficiency and drug release was found up to 24 hours.
M. Abraham Lingan et al132 (2011) formulated topical gel containing clobetasol
propionate niosomes to prolong the duration of action and prevent its side
effects and evaluated the same. The niosomes were prepared using by varying
the concentration ratios between various non-ionic surfactants (Span 40, 60,
80) and cholesterol by three methods such as thin film hydration method,
ether injection method and hand shaking methods. The results suggested that
the niosomal delivery of clobetasol propionate in carbopol gel base acts as a
suitable topical drug delivery system to prolong the duration of action.
Vyas Jigar et al133 (2011) developed erythromycin entrapped niosomes by thin
film hydration technique and various process parameters were optimized by
partial factorial design and concluded demonstrates prolongation of drug
release, an increase in amount of drug retention into skin and improved

permeation across the skin after encapsulation of Erythromycin into niosomal
topical gel.
V. satyavathi et al134 (2012) developed niosomal in situ gel ocular delivery
system of brimonidine tartrate to enhance bioavailability and evaluated the
same and concluded that the niosomal preparations are one of the tool to
enhance the bioavailability.
Pardakhty A et al135 (2012) developed different positively charged niosomal
formulations containing sorbitan esters, cholesterol and cetyl trimethyl
ammonium bromide by film hydration method for the entrapment of autoclaved
Leishmania major (ALM). Size distribution pattern and stability of niosomes
were investigated by laser light scattering method and ALM encapsulation per
cent was measured by the bicinchoninic acid method. Finally, the selected
formulation was used for the induction of the immune response against
cutaneous leishmaniasis in BALB/c mice. The percentage of ALM entrapped in
all formulations varied between 14.88% and 36.65%. In contrast to ALM, In
vivo studies showed that the niosomes containing ALM have a moderate effect
in the prevention of cutaneous leishmaniasis in BALB/c mice.
Disha Mehtani et al136 (2012) have employed the concept of mixed solvency, to
investigate the various solubilizers, to increase the solubility of surfactants and
the intended drug in diethyl ether, formulate niosomal gel for transdermal use
and perform its evaluation and concluded that this is one of the useful
technology for development of particulate carriers.

Rajesh Z. Mujoriya et al137 (2012) developed ketoprofen niosomes using various
concentrations of Surfactant (Span 60) Cholesterol and Dicetyl phosphate
(DCP) by thin film hydration method and evaluated all formulations. They
concluded based on the results that the optimized niosomal formulation
showed a sustained action of about 16 hrs was subjected to In vivo studies
(anti-inflammatory activity). This formulation was found to be more effective in
reducing edema after 2 hrs as compared to the free drug.
Guiling et al (2012)138 have prepared tracolimus loaded ethosomes to
characterize a novel ethosomal carrier for tacrolimus, an immunosuppressant
treating atopic dermatitis (AD), and to investigate inhibition action upon
allergic reactions of mice aiming at improving pharmacological effect for
tacrolimus in that commercial tacrolimus ointment with poor penetration
capability exhibited weak impact on AD compared with common glucocorticoid.
The results shown that the ethosomes showed lower vesicle size and higher
encapsulation efficiency (EE) as compared with traditional liposomes with
cholesterol. In addition, thequantity of tacrolimus remaining in the epidermis
at the end of the 24-h experiment was statistically significantly greater from
the ethosomal delivery system than from commercial ointment which indicates
the greater penetration capability of the ethosomes into the deep strata of the
skin. Finally concluded the ethosomal tacrolimus delivery systems may be a
promising candidate for topical delivery of tacrolimus in treatment of AD.
Manish K. Chourasia et al (2011) 139 developed ketoprofen loaded ethosomes
with an aim to study the potential applications of ethosomes as carrier for

improved transdermal drug delivery and evaluated the same. In the study they
revealed that there is improved drug permeation through the skin from
ethosomes than the hydroethonolic solution. The study also revealed that the
drug concentration in plasma increased with ethosomes rather than the
hydroethonilic solution.
Limsuwan et al140 (2012) developed ethosomes containing mycophenolic acid
with an aim to enhance the permeation of drug across the skin upon topical
administration and characterized for the same and theu substiated the same.
Godin et al141 (2004) have developed bacitricin loaded ethosomes with an aim to
investigate the dermal and intracellular delivery of bacitracin, a model
polypeptide antibiotic, from ethosomes and characterized. The evaluation
substantiated that the antibiotic peptide was delivered into deep skin layers
through intercorneocyte lipid domain of stratum corneum (SC). Occlusion had
no effect on the permeation profile of the drug from ethosomes in in vitro
experiments. Efficient delivery of antibiotics to deep skin strata from ethosomal
applications could be highly beneficial, reducing possible side effects and other
drawbacks associated with systemic treatment. Further the researchers
revealed, ethosomal delivery systems could be considered for the treatment of a
number of dermal infections, requiring intracellular delivery of antibiotics,
whereby the drug must bypass two barriers: the SC and the cell membrane.
Vaibhav Dubey et al142 (2007) have prepared melatonin loaded ethonolic
liposomes and characterized for physicochemical properties and transdermal
drug delivery. The results shown that a better skin tolerability of ethosomal
suspension on rabbit skin suggested that ethosomes may offer a suitable
approach for transdermal delivery of melatonin.
Vaibhav Dubey et al143 (2007) have developed ethosomes containg methotrexate
(MTX), an anti-psoriatic, anti-neoplastic, highly hydrosoluble agent, having
limited transdermal permeation, to evaluate the transdermal potential of novel
vesicular carrier, ethosomes and characterized for the same. The results shown
that ethosomes are an efficient carrier for dermal and transdermal delivery of
MTX.
Maria manconi et al144 (2011) have developed ethosomes containing diclofenac
to study the potentiality of carriers in permeation of drugs across the skin and
characterized for the same. The results the superior ability of the PEVs to
enhance ex vivo drug transport of both hydrophilic and lipophilic diclofenac
forms.
Vijay kumar et al145 (2010) have prepared diclofenac potassium enclosed
ethosomes with an aim to evaluate the transdermal potential of novel vesicular
carrier, ethosomes, having diclofenac potassium, a potent, water soluble non‐
steroidal anti‐inflammatory drug with lesser transdermal permeation and the
results are compared with the efficiency of marketed preparation. The
pharmacodynamic studies showed enhanced anti‐inflammatory activity of
ethosomal gel than the marketed gel formulation and also confirmed that the
ethosomes are an efficient carrier for dermal and transdermal delivery of
diclofenac potassium.
Ashoniya et al146 (2011) developed ethosomes containing ketoconozole and
evaluated. The researchers concluded based on the results that the ethosomes
are prominent tools for transdermal delivery of drugs.
Xingyan Liu et al147 (2011) have prepared ligustrazine loaded ethosomes and in
vitro and in vivo evaluation was carried out and the results are compared with
conventional ligustrazine administration. The comparison concluded confirmed
that ligustrazine ethosome patches could promote better drug absorption and
increase bioavailability. This study also demonstrated that the transdermal
action of the ligustrazine ethosome patch was comparatively good.
Elisabetta Esposito et al148 (2004) have developed ethosomes and liposomes
containing azelaic acid and preformulation study was carried out and the
results are compared with each other. The researchers are concluded that the
release rate was more rapid from ethosomal systems than from liposomal
systems. They also concluded that, ethosomes produced by the highest ethanol
concentration released azelaic acid more rapidly, and the same trend was
found using viscous forms.
Verma149 (2011) has developed ethosomal systems containing ascorbic acid to
observe the effect of penetration enhancer in ethosomal formulations and the
study shown that ethosomal preparation containing penetration enhancer
shown better permeation efficiency than the ethosomal preparation alone.
Jadupati Malakar et al150 (2012) have developed transferosomal gel containing
insulin by reverse phase evaporation method for painless insulin delivery to
treat insulin dependent diabetes mellitus. The prepared formulations were

evaluated and optimized one formulation and in vivo study was carried out
with optimized formulation. The in vivo study of optimized transferosomal gel in
alloxan-induced diabetic rat has demonstrated prolonged hypoglycemic effect
in diabetic rats over 24 h after transdermal administration.
Donatella Paolino et al151 (2012) have prepared ultradeformable vesicles, by the
extrusion technique, containing asiaticoside, a natural compound obtained
from Centella asiatica and evaluated for the potentiality of ultradeformable
vesicles as a possible topical delivery system. The results shown that
ultradeformable vesicles provided the greatest in vitro skin permeation of
asiaticoside showing a 10-fold increase with respect to the free drug solution
voured and increase in in vivo collagen biosynthesis and also suggested that
ultradeformable vesicles are suitable carriers for the pharmaceutical and
cosmetic application of the natural agent asiaticoside.
Gregor Cevc et al152 (2002) have developed ultradeformable lipid vesicles to
study the stability of various aggregates in the form of lipid bilayer vesicles was
tested by three different methods before and after crossing different semi-
permeable barriers. The researchers concluded that the ultradeformable
vesicles penetrate the skin intact, that is, without permanent disintegration.
Eun kyung Oha et al153 (2011) have aimed to develop 5-amaino levulinic acid
(5-ALA) loaded ultradeformable liposomes (UDL) with different surface charges,
and to investigate their physicochemical characteristics and capability for the
skin penetration and retention of 5-ALA for topical photodynamic therapy
(PDT). The effects of surface charges of UDL on in vitro permeation of 5-ALA

and in vivo accumulation of 5-ALA_induced PpIX in viable skin were
determined and then compared with conventional neutral liposomes
(nLiposome). All UDL showed smaller particle size and better deformability
than nLiposome. However, entrapment efficiency of 5-ALA was similar to each
vesicle. Among vesicles, the cationic UDL (cUDL) demonstrated higher stability
and permeability, and could deliver 5-ALA into deep skin tissue by topical
application.
Jorge Montanari et al154 (2010) have developed with an aim independent of
artificial power sources, self administered sunlight triggered photodynamic
therapyncould be a suitable alternative treatment for cutaneous leishmaniasis,
that avoids the need for injectables and the toxic side effects of pentavalent
antimonials and they have determined the in vitro leishmanicidal activity of
sunlight triggered photodynamic ultradeformable liposomes (UDL).
B. Geusens et al155 (2009) have developed Ultradeformable cationic liposomes
for delivery of small interfering RNA (siRNA) into human primary melanocytes
and also to block the expression of a specific Myosin Va exon F containing
isoform that is physiologically involved in melanosome transport in human
melanocytes and characterized for the same. The best results were obtained
with UCLs which are promising for future in vivo experiments.
Gregor Cevc et al156 (2001) have developed and evaluated the diclofenac loaded
ultrdeformable liposomes and proved that the these vesicles are prominent tool
for topical and transdermal delivery of active principles.

Ebtessam A. Essa et al157 (2003) have developed a neutral lipophilic compound
(estradiol) enclosed transferesomes and studied the effect of electroporation on
human epidermal penetration of a model neutral lipophilic compound
(estradiol) from saturated aqueous solution and when encapsulated in
ultradeformable liposomes and Total amount penetrated and skin deposition
were compared with values obtained from passive diffusion. The results shown
that the effect of electrical pulsing on liposome size was investigated. The
action of phosphatidylcholine on skin that was structurally altered by such
pulses was determined. Electroporation did not affect liposome size. Skin
pulsing considerably increased estradiol penetration and skin deposition from
solution, relative to passive delivery, with subsequent partial recovery of skin
resistance to molecular penetration. Surprisingly, with liposomes,
electroporation did not markedly affect estradiol skin penetration. Importantly,
liposomal phosphatidylcholine applied during or after pulsing accelerated skin
barrier repair, i.e. provided an anti-enhancer or retardant effect.
Gregor Cevc et al158 (2003) have developed novel formulations of the
halogenated corticosteroid, triamcinolone-acetonide, based on ultradeformable
mixed lipid vesicles, Transfersomes, and their performance was tested in vivo
using radioactive label measurements, to study the drug biodistribution, and
murine ear edema, to determine the drug bioactivity. Sparse use of drug-loaded
TransfersomesR on the skin ensures an almost exclusive delivery of
triamcinolone-acetonide into the organ, thus arguably increasing the treatment

safety. The researchers concluded based on the results transferosomes are
efficient carriers in transportation of drugs across the skin.
P. Loan Honeywell-Nguyen et al159 (2003) developed and several aspects of
elastic vesicle transport into human skin were investigated in vivo. Surfactant
based elastic vesicles were applied onto human skin in vivo and subsequently a
series of tape-strippings were performed, which were visualised by freeze
fracture electron microscopy. Factors of investigation for non-occlusive
treatment were the duration of application and the volume of application. In
addition, occlusive vs. non-occlusive application was studied. The results have
shown a fast penetration of intact elastic vesicles into the stratum corneum
after non-occlusive treatment, frequently via channel-like regions. A higher
volume of application resulted in an increase in the presence of vesicle material
found in the deeper layers of the stratum corneum. Micrographs after occlusive
treatment revealed very few intact vesicles in the deeper layers of the stratum
corneum, but the presence of lipid plaques was frequently observed. They have
also proposed a hypothesis that the channel-like regions represent
imperfections within the intercellular lipid lamellae in areas with highly
undulating cornified envelopes.
Gregor Cevc et al160 (1998) developed transferosomes containing insulin with an
aim to deliver the insulin through intact skin and evaluated for the same. The
researchers concluded that this process can be nearly as efficient as an
injection needle, as seen from the results of experiments in mice and humans
with the insulin-carrying vesicles and also said that the carrier-mediated
transcutaneous insulin delivery is unlikely to involve shunts, lesions or other
types of skin damage. Rather than this, insulin is inferred to be transported
into the body between the intact skin cells with a bio-efficiency of at least 50%
of the s.c. dose action.
Ramesh R. Boinpally et al161 (2003) developed lecithin vesicles containing
diclofenac, anti-inflammatory agent, to treat rheumatic diseases and the
permeation of diclofenac across human cadaver epidermis in-vitro from four
lecithin vesicle formulations and a few marketed semi-solid preparations and
they concluded that the lecithin vesicles of diclofenac appear to be
advantageous for the topical delivery of diclofenac based on the results.
Yuka Hiruta et al162 (2006) have incorporated Beta-sitosterol 3-β-D-glucoside
(Sit-G), an absorption enhancer, into ultra-deformable vesicles containing
bleomycin to attenuate drug toxicity in human keratinocytes and evaluated.
Based on the results researchers concluded that the presence of Sit-G
increased drug entrapment and improved in vitro stability of ultradeformable
vesicles. They also concluded that the Ultra-deformable formulation contained
Sit-G maintained flexibility for penetration through the skin, increased
entrapment efficiency of bleomycin and stability in vitro, and significantly
increased distribution of bleomycin in epidermis and dermis compared with
those without Sit-G.
P. Loan Honeywell-Nguyen et al163 (2003) have developed pergolide from L-595–
PEG-8-L elastic vesicle formulations and studied the in vitro transport of the
same across the human skin using flow-through Franz diffusion cells to
elucidate the transport mechanism. The findings show that there was a strong
correlation between the drug incorporation to saturated levels and the drug
transport, both of which were influenced by the pH of the drug–vesicular
system. The optimal pH was found to be 5.0, giving the highest drug
incorporation as well as the highest drug transport. Non-occlusive co-treatment
with elastic vesicles improved the skin delivery of pergolide compared to the
non-occlusive buffer control by more than 2-fold. Occlusion reduces the
actions of elastic vesicles, but could increase the pergolide transport since
water is a good penetration enhancer for this particular drug. Based on the
results obtained, a mechanism of action for the elastic vesicles was proposed.
Ajay Kumar et al164 (2012) have made review on transferosomes and they
suggested that Transferosomes are novel vesicular systems that are several
times more elastic than other vesicular systems. They also given information
like regulatory aspects of excipients used in preparation, summary of clinical
investigations performed, marketed preparations available, research reports
and patent reports related to transfersomes.
Tomonobu Uchino et al165 (2013) developed vesicular carrier system enclosed
ketoprofen to study the skin permeation of ketoprofen with vesicles and
characterized for the same and also studied the effect of elasticity of the vesicle
in permeation of the enclosed drug. The researchers concluded based on the
results that the Surfactant-based vesicles enhance ketoprofen transport across
human skin, while no enhancement of ketoprofen was observed when loaded in
elastic liposomes.
Sureewan Duangjit et al166 (2011) have developed lipid vesicles viz, liposomes
and transferosomes containing meloxicum with an aim to study potential use
of liposome and transfersome vesicles in the transdermal drug delivery of
meloxicam (MX) and evaluated for the same. The results suggested that MX-
loaded transfersomes can be potentially used as a transdermal drug delivery
system rather than the liposomes.
Alpana Ram et al167 (2012) developed transferosomes containing ibuprofen, an
anti-inflammatory agent and evaluated the ability of the vesicles in permeation
of the same. The study revealed that transfersomes are a promising prolonged
delivery system for Ibuprofen and have reasonably good stability characteristics
and the researchers concluded that transferosomes are prominent tool for
transportation of ibuprofen across the skin.

Chapter - 3
THEORITICAL ANALYSIS
Chapter - 3: Theoritical Analysis
Table of contents

3.1 Drug and excipient profiles
3.2 Objective of the study
3.3 Schematic presentation of the research work
3.4 parameters to be characterized
3.5 Materials
3. THEORETICAL ANALYSIS
3.1. DRUG AND EXCIPIENTS PROFILE
3.1.1. NYSTATIN
Nystatin168 is a polyene antifungal drug to which many molds and
yeasts are sensitive, including Candida spp. Nystatin has some toxicity
associated with it when given intravenously, but it is not absorbed across
intact skin or mucous membranes. It is considered a relatively safe drug
for treating oral or gastrointestinal fungal infections.
Chemical Name: (1S,3R,4R,7R,9R,11R,15S,16R,17R,18S,19E,21E, 25E,
27E, 29E, 31E, 33R, 35S, 36R, 37S)-33-[(3-amino-3, 6-dideoxy-β-L-
mannopyranosyl)oxy]-1, 3, 4, 7, 9, 11, 17,37-octahydroxy-15, 16,18-
trimethyl-13-oxo-14, 39 dioxabicyclo [33.3.1] nonatriaconta-19,
21,25,27,29,31-hexaene-36-carboxylic acid.
Chemical structure:
Molecular formula: C47H75NO17
Molecular weight: 926.09
Colour : Yellow to light tan powder

Odour: cereallike odor
Solubility: very slightly soluble in water (4mg/ml at 28oC & 360mg/L at
24oC), and slightly to sparingly soluble in alcohol.
Pharmacology:
Pharmacokinetics:
a) Bioavailability: 0% on oral ingestion
b) Metabolism: None (not extensively absorbed).
c) Half-life: Dependent upon GI transit time
d) Excretion: Faecal- 100 %.
Pharmacodynamics:
Nystatin is an antibiotic which is both fungistatic and fungicidal in vitro
against a wide variety of yeasts and yeast-like fungi, including Candida
albicans, C. parapsilosis, C. tropicalis, C. guilliermondi, C. pseudotropicalis, C.
krusei, Torulopsis glabrata, Tricophyton rubrum, T. mentagrophytes. Nystatin
acts by binding to sterols in the cell membrane of susceptible species resulting
in a change in membrane permeability and the subsequent leakage of
intracellular components. On repeated subculturing with increasing levels of
nystatin, Candida albicans does not develop resistance to nystatin. Generally,
resistance to nystatin does not develop during therapy. However, other species
of Candida (C. tropicalis, C. guilliermondi, C. krusei, and C. stellatoides) become
quite resistant on treatment with nystatin and simultaneously become cross
resistant to amphotericin as well. This resistance is lost when the antibiotic is

removed. Nystatin exhibits no appreciable activity against bacteria, protozoa, or
viruses.
Mechanism of action
Nystatin binds to ergosterol, a major component of the fungal cell
membrane. When present in sufficient concentrations, it forms pores in the
membrane that lead to K+ leakage, acidification, and death of the fungus. [7]
Ergosterol, is unique to fungi, so the drug does not have such catastrophic
effects on animals or plants. However, many of the systemic/toxic effects of
Nystatin are attributable to its effect on human cells via binding to mammalian
sterols, namely cholesterol. This is the effect that accounts for the
nephrotoxicity observed when high serum levels of Nystatin are achieved.
Uses:
1. It is used to treat vaginal, Cutaneous, mucosal and esophageal Candida
infections.
2. Oral nystatin is often used as a preventative treatment in people who are
at risk for fungal infections, such as AIDS patients with a low CD4 +
count and patients receiving chemotherapy.
3. It has been investigated for use in patients after liver transplantation
but fluconazole was found to be much more effective for preventing
colonization, invasive infection and death.
4. It is also used prophylactically in Very Low Birth Weight (<1500 g)
infants to prevent invasive fungal infections.

5. Liposomal Nystatinis not currently available but investigational use has
shown greater in vitro activity than colloidal formulations of
Amphotericin B.
6. It offers an intriguing possibilty for difficult to treat systemic infections,
such as invasive apergillosis, or infections that demonstrate resistance to
Amphotericin B. Additionally, liposomal Nystatin appears to cause fewer
cases of and less severe nephrotoxicity than observed with Amphotericin
B. However, at the time of this writing, plans for additional development
and investigation of liposomal nystatin appear in flux.
7. Cryptococcus is also sensitive to nystatin.
8. It is also used in cellular biology as an inhibitor of the lipid raft-caveolae
endocytosis pathway on mammalian cells, at concentrations around
3 µg/mL.
Adverse effects
The oral suspension form produces a number of adverse effects including
but not limited to
1. Diarrhea
2. Abdominal pain
3. Rarely, tachycardia, bronchospasm, facial swelling, muscle aches
Both the oral suspension and the topical form can cause
1. Hypersensitivity Reactions, including Stevens Johnsons Syndrome in
some cases
2. Rash, itching, burning.

3.1.2. CHOLSTEROL
Empirical Formula : C27H46O
Chemical structure :
Molecular Weight : 386.67
Synonyms : Cholesterin, cholesterolum.
Chemical Name : Cholest-5en-3β-ol.
Description : Cholesterol occurs as white or faintly yellow, almost
less, pearly leaflets needles, powder or granules, on prolonged exposure to light
and air cholesterol acquires a yellow to tan colour.
Boiling point : 360°C
Density : 1.052 g/cm3
Melting Point : 147-150°C.
Solubility : Chloroform 1 in 4.5; Ether 1 in 2.8 and
practically insoluble in water.

Applications : Emollient, Emulsifying agent. Cholesterol
is used in cosmetics and topical pharmaceuticals and at concentrations of
0.30%w/w imparts water absorbing power to an ointment and also has
emollient activity.
Method of manufacture:
The commercial material is normally obtained from the spinal cord of
cattle by extraction with petroleum ethers, but it may also be obtained from
wool fat. Purification is normally accomplished by repeated bromination.
Cholesterol may also be produced by entirely synthetic means.Cholesterol
produced from animal organs will always contain cholestanol and other
saturated sterols.
Safety:
Cholesterol is generally regarded as an essentially nontoxic and
nonirritant material at the levels employed as an excipient(18).It has, however,
exhibited experimental teratogenic and reproductive effects, and mutation data
have been reported. Cholesterol is often derived from animal sources and this
must be done in accordance with the regulations for human consumption. The
risk of bovine spongiform encephalopathy (BSE) contamination has caused
some concern over the use of animal-derived cholesterol in pharmaceutical
products. However, synthetic methods of cholesterol manufacture have been
developed.
Incompatibilities: Cholesterol is precipitated by digitonin.
Handling Precautions:
Observe normal precautions appropriate to the circumstances and
quantity of material handled. Rubber or plastic gloves, eye protection and a
respirator are recommended.
Regulatory status:
It is included in the FDA Inactive Ingredients Guide (injections,
ophthalmic, topical, and vaginal preparations). It is included in nonparenteral
medicines licensed in the UK andin the Canadian List of Acceptable Non-
medicinal Ingredients.
3.1.3. SPAN – 60
Empirical Formula : C24H46O6.
Chemical structure :
Molecular Weight : 431
Synonyms : Ablunol S-60, 6 - Octa deconoate
Arlaces60, Sorbitan Stearate, Spans 60.
Functional category: Emulsifying agent; nonionic surfactant; solubilizing
agent; wetting and dispersing/suspending agent.
Applications: In pharmaceutical formulation or technology Sorbitan
monoesters are a series of mixtures of partial esters of sorbitol and its mono-
and dianhydrides with fatty acids. Sorbitan diesters are a series of mixtures of
partial esters of sorbitol and its monoanhydride with fatty acids. Sorbitan
esters are widely used in cosmetics, food products, and pharmaceutical
formulations as lipophilic nonionic surfactants. They are mainly used in
pharmaceutical formulations as emulsifying agents in the preparation of
creams, emulsions, and ointments for topical application. When used alone,
sorbitan esters produce stable water-in-oil emulsions and microemulsions but
are frequently used in combination with varying proportions of a polysorbate to
produce water-in-oil or oil-in-water emulsions or creams of varying
consistencies.Sorbitan monolaurate, sorbitan monopalmitate and sorbitan

trioleate have also been used at concentrations of 0.01–0.05% w/v in the
preparation of an emulsion for intramuscular administration.
Description : Cream Solid
Typical properties
Acid value : 5-10
Flash point : >149°C
HLB value : 4.7
Hydroxyl value : 235-260
Iodine number : ≤1
Melting point : 53-57
Moisture content : ≤1
Saponification value : 147-157
Solubility: sorbitan esters are generally soluble or dispersible in oils; they are
also soluble in most organic solvents. In water, although insoluble, they are
generally dispersible.
Surface tension of 1% aqueous solution: 46mN/m
Stability and storage conditions: Gradual soap formation occurs with strong
acids or bases; sorbitan esters are stable in weak acids or bases. Sorbitan
esters should be stored in a well-closed container in a cool, dry place.
Safety: Sorbitan esters are widely used in cosmetics, food products, and oral
and topical pharmaceutical formulations and are generally regarded as
nontoxic and nonirritant materials. However, there have been occasional
reports of hypersensitive skin reactions following the topical application of

products containing sorbitan esters. When heated to decomposition, the
sorbitan esters emit acrid smoke and irritating fumes. The WHO has set an
estimated acceptable daily intake of sorbitan monopalmitate, monostearate,
and tristearate, and of sorbitan monolaurate and monooleate at up to 25mg/kg
body-weight calculated as total sorbitan esters.
LD50 (rat, oral): 31g/kg. Very mildly toxic by ingestion.
Handling precautions: Observe normal precautions appropriate to the
circumstances and quantity of material handled. Eye protection and gloves are
recommended.
Regulatory status: Certain sorbitan esters are accepted as food additives in
the UK. Sorbitan esters are included in the FDA Inactive Ingredients Guide
(inhalations; IM injections; ophthalmic, oral, topical, and vaginal preparations).
Sorbitan esters are used in nonparenteral medicines licensed in the UK.
Sorbitan esters are included in the Canadian List of Acceptable Non-medicinal
Ingredients.
3.1.4. SPAN – 80
Chemical formula : C24H44O6
Chemical structure:
Synonyms : Sorbitan oleate; Sorbitan (Z)-mono-9-
octadecenoate
Molecular weight : 428
Physical properties
Density : 0.986
Refractive Index : 1.48
Flash point : > 149o C
Stability : Stable, Combustible, Incompatible with
strong oxidizing Agents
Description : Liquid
Colour : Yellow
Odour : Fatty acid-like
Viscosity : Dynamic (25 °C) 1000 mPa*s
Boiling point : > 100 °C
Vapour pressure (20 °C) : < 1.4 hPa

Solubility : In water (20 °C), insoluble ethylacetate (20
°C), soluble acetone (20 °C).
HLB : 4.3
Storage : Store in dark. Kept under seal.
Handling : Prevention of sunlight, drench, heating
and bump, treat lightly and forbid mixed package and transport with
deleterious goods or other contaminated cargo.
Applications : Use as a w/o emulsifier or as an o/w
emulsifier for use in cosmetic formulations, oil field chemicals, plastics,
household products, coatings and textiles.

3.1.5. PHOSPHOLIPON-90H
Synonym : Hydrogenated phosphatidylcholine
Composition : Phosphatidylcholine (hydrogenated)
[g/100 g] n.l.t. 90.0
Structure formula :
Molecular formula : C43H95NO8P
Molecular weight : Average M. Wt 784.6 g/mol
Phase transition temperature : Approx. 55°C (in hydrated form)
Purity : Lysophosphatidylcholine [g/100 g]
NMT 4.0
Non-polar lipids : Triglycerides [g/100 g] NMT 2.0
Typical fatty acid composition : Σ (C16:0, C18:0) [g/100 g]n.l.t. 98
Iodine value : NMT 1

Water : [g/100 g] NMT 2.0
Residual solvents
Ethanol : [g/100 g] NMT 0.5
Physical and chemical Properties
Colour : White to whitish
Consistency : Powder
Odor : Odorless
Solubility (5% solution):
Ethanol : Soluble at 50 °C
Propylene glycol : Soluble 55 °C
Water : Dispersible 55 °C
Beeswax : Soluble 80 °C
MCT : Soluble 90 °C
Macadamia nut oil : Soluble 97 °C
Bulk density : 400–500 kg/m3
pH : 6 ± 1 at 10 g/l (20°C)
Minimum ignition energy : 3 mJ < MIE < 10 mJ
Specific resistance : 4, 32 x 10-11 Ωm
Bacteriological Data:
Total aerobic microbial count (TAMC)[/g]: NMT 100
Total combined yeasts & moulds count (TYMC) [/g]: NMT 10
E. Coli [/g] : Negative
Staphylococcus aureus [/g] : Negative

Pseudomonas aeruginosa [/g] : Negative
Packaging : 5 kg and 25 kg standard packaging
in double PE bag/aluminium foil bag
Storage
Stored in closed containers at +5 ± 3 °C. To avoid a negative impact on
the product quality by humidity, a cooled product unit must not be opened
without prior conditioning to ambient temperatures. Close opened containers
immediately.
Applications:
- Preparation of liposomes and emulsions for pharmaceuticals and
cosmetics
- Skin protectant
3.2. Objective of the study
From ancient days the search for new things, which will improve
comfortability, is going till today and will be there in future also to achieve
more and more comfortability and also there will not be end for the same.
Similarly there is lot of change has been taken place in the field of
pharmaceutical sciences also, when compared the current generation with
ancient days. Since beginning researchers trying to introduce new alternative
drug molecules and/or techniques to achieve better therapeutic effect, to
increase patient compliance, etc.
Generally researchers will have two options viz either introduction of new
molecules or using of existing molecules in better way to achieve the desired
objectives. The first one involves huge money, time, etc hence it is difficult to
follow the first option. The other option is modifying the existing drug
molecules to meet the objectives which less difficult rather the first option and
is preferable option. With the same intention researchers were adopted
different techniques to achieve the desired objectives. The different techniques
include modification of existing drug molecule, change in the route of
administration, change in the dosage form, change in drug delivery systems,
etc.
The modified drug delivery systems are classified under the class of new
drug delivery systems (NDDS). The ideal NDDS should fulfill two important
requirements. They are 1. They should capable to deliver the drug at required
concentration for required period of time. 2. These should deliver the drug to
the site of action. No NDDS were introduced till today which can were fulfill the
two requirements. To achieve the same different carrier systems were
introduced by the researchers which include cellular carrier, particulate,
macromolecular and polymeric carriers. Particulate type carrier includes lipid
particles (low and high density lipoprotein-LDL and HDL, respectively),
microspheres, nanoparticles, polymeric micelles and vesicular carriers like
liposomes, niosomes pharmacosomes, virosomes, etc.
Last three decades onwards the interest on the vesicular carrier systems
tremendously increased and huge research is carrying out as these carriers
has shown solution for many challenges like enhancing the solubility,
controlled drug release, targeting the abnormal sites, permeation of drugs
across the biological membranes, etc. These vesicular carrier systems are
highly ordered assemblies of one or several concentric lipid bilayers formed
when certain amphiphilic building blocks are confronted with water. Vesicles
can be manufactured using a diverse range of amphiphilic
molecules.Researchers developed variety of vesicles includes liposomes,
niosomes, ethosomes, transferosomes, aquasomes, pharmacosomes, etc for
different purposes. Here the primary aim of the work is to select the best
vesicular carrier system, among niosomes, ethosomes and transferosomes,
which shows enhanced transdermal delivery of active principles. The secondary
objective the research work is to enclose the nystatin, polyene antifungal
antibiotic, in the best vesicular carrier system and to characterize the same.
3.2.1. Particular goals were:
 Development of niosomal suspension using two different non-ionic
surfactants viz. span-60 and span-80 at different concentrations.
 Characterization of prepared niosomal suspensions and selection of best
formulation.
 Preparation of nystatin niosomal gel with optimized formulation and
carrying out ex vivo drug release study, stability, etc.
 Development of ethosomal suspension using different concentrations of
ethanol and lipid.
 Characterization of prepared ethosomal suspensions and selection of
best formulation.
 Preparation of nystatin ethosomal gel with optimized formulation and
carrying out ex vivo drug release study, stability, etc.
 Development of transferosomal suspension using different
concentrations of edge activators and lipid.
 Characterization of prepared transferosomal suspensions and selection
of best formulation.
 Preparation of nystatin transferosomal gel with optimized formulation
and carrying out ex vivo drug release study, stability, etc.
 Carrying out of in vivo study with optimized niosomal, ethosomal and
transferosomal gel and to determine the pharmacokinetic parameters.

 Concluding the best vesicle for enhanced transdermal drug delivery
based on the in vivo data.

3.3. SCHEMATIC PRESENTATION OF THE RESEARCH WORK
Preformulation studies
Niosomal formulations Ethosomal formulations Transferosomal formulations
Characterization Characterization Characterization

1. Vesicles morphology
2. Entrapment efficiency
3. In vitro drug release (Dialysis membrane)
4. Release kinetics
Selection of the Selection of the Selection of the

best formulation best formulation best formulation
1. Elasticity measurements.
2. Zetapotential
Preparation of gel Preparation of gel Preparation of gel

2. Ex vivo drug release (Rat skin)
3. Skin retention study
4. Stability study
5. In vivo performance
Conclusion of the best vesicular system

3.4. PARAMETERS TO BE CHARACTERIZED
1. Vesicles morphology (size, shape and surface appearance)
2. Entrapment efficiency
i. Dialysis method
ii. Ultra centrifugation method
4. Release kinetics
a. Zero order
b. First order
c. Higuchi model
d. Hixon-crowel model
e. Korsmeyer-peppas model
5. Elasticity measurements.
6. Zetapotential
7. Invitro drug release (Dialysis membrane)
8. Ex vivo drug release (Rat skin)
a. Transdermal flux.
b. Permeability co-efficient.
c. Enhancement ratio.
9. Drug retention study
10. Stability studies.
11. In vivo performance.
a. Pharmacokinetic parameters like Cmax, Tmax, AUC, etc

3.5. Materials
Table 3.5.1: List of chemicals
Sr.No. Chemical Name Grade Manufacturer
1 Phospolipon-90H HPLC Merck Ltd, Mumbai.
2 Ethanol HPLC Merck Ltd., Mumbai.
3 Hydrochloric acid AR Merck Ltd, Mumbai.
4 Dichloromethane AR Merck Ltd, Mumbai.
5 Span 80 AR Merck Ltd, Mumbai.
6 Span 60 AR Merck Ltd, Mumbai.
7 Cholesterol AR Merck Ltd, Mumbai.
8 Heavy liquid paraffin AR Merck Ltd., Mumbai.
9 Diethyl ether AR Merck Ltd., Mumbai.
10 Sodium bicarbonate AR Merck Ltd, Mumbai.
11 Ortho phosphoric acid HPLC Merck Ltd., Mumbai.
12 Anesthetic ether AR Sigma Aldrich, Mumbai
13 EDTA AR Merck Ltd., Mumbai.
14 Carbopol -971 AR Merck Ltd., Mumbai.
15 HPMC AR Merck Ltd., Mumbai.
16 Propylene glycol AR Merck Ltd., Mumbai.
17 Triethanolamine AR Merck Ltd., Mumbai.
18 Methanol HPLC Merck Ltd., Mumbai.

Table 3.5.2: LIST OF EQUIPMENTS
S. No Name of the Equipment Manufacturer
1 Digital Electronic balance Metler, Japan.
2 Roto vacuum evaporator Kshitij Innovations.
3 Magnetic stirrers Remi.
4 Refrigerated centrifuge Remi.
5 Magnetic beads Sunsil.
6 Dialysis membrane Himedia
7 Hot Air Oven Minicon
8 UV Spectrophotometer PG Instruments
9 HPLC Waters
10 Vacuum pump Kent Pvt Ltd
11 Filtration assembly Kshitij Innovations
12 Micro pippets Micro tech
13 Bath sonicator Kshitij Innovations

Chapter - 4
EXPERIMENTAL
INVESTIGATIONS
Chapter - 4: Experimental Investigations
Table of contents
S.No Title of the content Page No

4.1 Preformulation Studies
4.2 Standard curve of nystatin in phosphate buffer saline pH 7.4
4.3 Formulation of nystatin loaded niosomes
4.4 Formulation of nystatin loaded ethosomes
4.5 Preparation of nystatin loaded transferosomes.
4.6 Characterizations
4. EXPERIMENTAL INVESTIGATIONS
4.1. Preformulation Studies
4.1.1. Physico-chemical characterization of API
Preformulation study is the fundamental step in the development of new
formulations which involves characterization of different physic-chemical
parameters which were directly influence on the development of formulations.
In this study various physico-chemical and mechanical properties of drug and
excipients to be characterized so as to develop an effective dosage from. A
thorough understanding of the properties may ultimately provide a rational for
formulation design.
4.1.2. Identification of drug sample
The Nystatin drug, received as gift sample, was characterized and
confirmed by studying its physicochemical parameters like appearance,
solubility, absorption maximum and FTIR study. During the identification
process the drug sample was used without further purification.
4.1.3. Description
The sample of drug sample was studied for organoleptic characters such as
appearance, colour, odour and solubility. The drug sample was appeared as
yellow coloured powder with cereal like odor.
The solubility study and determination of absorption maximum (λ max) of
the drug sample was studied in phosphate buffer saline pH 7.4. The study was
carried out as follows.
4.1.4. Preparation of phosphate buffer saline pH 7.4

Accurately, 1.564gm of sodium hydroxide and 6.804gm potassium
dihydrogen phosphate was weighed and are solubilized in small volume of
distilled water. After complete solubilization of both the ingredients, the volume
was adjusted to 1000ml with distilled water. The resulting solution pH was
measured and adjusted to pH 7.4, if necessary.
4.1.5. Determination of absorption maximum (λmax) of drug sample
A stock solution of drug sample was prepared by dissolving 20mg of drug
sample in 100ml of phosphate buffer saline, pH.7.4. From the above stock
solution, 2µg/ml solution was prepared by serial dilution. The prepared
solution absorbance was recorded between the wavelengths 200-400nm by UV-
Visible spectrophotometer. From the absorbance data, wavelength at which
maximum absorbance was exhibited by the drug sample was observed. The
obesreved wavelength was considered as absorption maximum and was found
at 306nm. The absorption spectrum of given drug sample was given in figure
5.1.
4.1.6. Solubility
The solubility of drug sample was determined by adding excess amount
(100mg) of drug in the deaerated phosphate buffer saline (PBS) pH 7.4 and
kept aside for 12 hrs to acieve equilibrium with occasional shaking at room
temperature. From the above super saturated solution, 10ml of supernatant
liquid was collected and centrifuged at 3000RPM for 10min. After
centrifugation, 1ml of supernatant was transferred into the 100ml volumetric
flask and volume adjusted to 100ml with PBS pH7.4. The flask was shaken
well and the resulting solution absorbance was measured using UV-Visible
Spectrophotometer at 306nm. Based on the absorbance, the solubility of the
drug sample in PBS pH7.4 was calculated and tabulated in table 5.1.
4.1.7. FTIR study of the drug sample
FTIR spectrum of gift sample was determined to find out the
characteristic peaks for lactone carbonyl, N-H, C-OH, O-H bond stretching and
Carboxylate ion. The FTIR spectra of drug sample has given in figure 5.2.
Based on the results of the solubility, absorption maximum and FTIR
studies, the drug sample was confirmed as Nystatin.
4.2. Standard curve of nystatin in phosphate buffer saline pH 7.4
A stock solution of nystatin was prepared by dissolving 20 mg of drug in
100 ml of phosphate buffer saline pH 7.4. From the above stock solution,
solutions having concentrations of 2, 4, 6, 8 and 10µg/ml were prepared by
serial dilutions. The resultant solutions absorbances were recorded by UV –
Visible spectrophotometer at 306 nm. The absorbances of the solutions
tabulated in table 5.2 and the tandard curve has given in figure 5.3.
4.3. Formulation of nystatin loaded niosomes
Niosomal suspensions were prepared by the thin-film hydration method.
Accurately weighed quantities of drug, surfactant (Span – 60 & Span - 80), and
Cholesterol were dissolved in chloroform in a round-bottom flask. The
chloroform was evaporated at 60°C under reduced pressure using a rotary
flash evaporator (Buchi type). After chloroform evaporation, the flask was kept
under vacuum overnight to remove residual solvent. The thin film was
hydrated with 10 ml of phosphate buffer (PBS), pH 7.4, and the flask was kept
rotating at 60°C. Formulations were sonicated three times in a bath-sonicator
for 15 min with 5-min interval between successive times. Composition of all
niosomal suspensions was given in table 5.3.
4.4. Formulation of nystatin loaded ethosomes
Ethosomal suspensions were developed according to the method reported
by Touitou. Phospholipon and drug were dissolved in ethanol and the mixture
was heated to 30±1ºC. Double distilled water (consisting of dissolved
cholesterol) was heated to 30ºC±1ºC and was added slowly as a fine stream to
the lipid mixture with constant stirring (Mechanical stirrer, Remi equipment,
Mumbai) at 700rpm. The entire process was carried out in a closed vessel to
prevent the evaporation of ethanol at a temperature of 30ºC±1ºC. The stirring
was continued for 5 more minutes after addition of aqueous solution to lipid
mixture which results in the formation of ethosomal suspension. Composition
of all ethosomal suspensions was given in table 5.4.
4.5. Preparation of nystatin loaded transferosomes.
All transferosomal suspensions contanining nystatin were prepared by
the thin-film hydration method. Accurately weighed quantities of drug,
surfactant (Span – 60 & Span - 80), and lecithin were dissolved in chloroform
in a round-bottom flask. The chloroform was evaporated at 40°C under
reduced pressure using a rotary flash evaporator (Buchi type). After chloroform
evaporation, the flask was kept under vacuum overnight to remove residual
solvent. The thin film was hydrated with 10 ml of phosphate buffer saline
(PBS), pH 7.4, and the flask was kept rotating at 40°C. All the formulations
were sonicated three times in a bath-sonicator for 15 min with 5-min interval
between successive times. Composition of all transferosomal suspensions was
tabulated in table 5.5.
4.6. CHARACTERIZATION
4.6.1. Vesicle Morphology
Morphology (size, shape and surface) of the vesicles present in all
vesicular suspensions were characterized. The shape and surface of the
vesicles was observed by means of Scanning Electron Microscopy (SEM) and
the size of the vesicles was measured using optical microscope. The SEM
photographs of niosomes, ethosomes and transferosomes are given figures 5.4,
5.15 and 5.27 respectively. The vesicles present in all niosomal, ethosomal and
transferosomal formulations were tabulated in tables 5.6, 5.7 and 5.8
respectively.
4.6.2. Entrapment Efficiency
Entrapment efficiency of all vesicular suspensions was determined by
separating the unentrapped drug. Un-entrapped drug was separated out by
ultracentrifugation method.
Vesicular suspension was suitably diluted with Phosphate buffer saline
solution and centrifuged at 12000rpm for 20min at 8 oC. The supernatant liquid
was collected and analyzed for concentration of an un-entrapped drug by UV
spectrophotometer (PG instruments) at 306nm. Entrapment efficiency of all
vesicular suspensions was determined by using formulas given below. The

results were confirmed by dialysis method also. Entrapment efficiency of all
vesicular suspensions was tabulated in tables 5.6, 5.7 and 5.8.
4.6.3. In vitro Release Studies
In vitro drug release from vesicular suspensions was studied by
exhaustive dialysis method and was carried out in an artificial diffusion cell.
Two side open ended glass tube was taken and one side has been closed
with Dialysis membrane (HiMedia Laboratories Pvt. Ltd., M.Wt cut off: 12000).
The fabricated tube was used as donor compartment, in which 1ml of vesicular
suspension was taken and the entire set up placed in receptor compartment
(Beaker, 250ml capacity) containing 100 ml phosphate buffer saline. The
dialysis was carried out at 50 rpm at 37±0.5°C for 12hrs. Every hour 2ml of
diffusion fluid from receptor compartment was withdrawn and same volume of
fresh diffusion fluid was replaced to maintain sinc conditions. The withdrawn
diffusion fluids were suitably diluted and analyzed using UV spectrophotometer
at 306nm. The in vitro drug releases from all niosomal, ethosomal and
transferosomal formulations was tabulated in tables 5.9-5.17, 5.19-5.27 and
5.29-5.37 respectively and the same was graphically presented in figutes 5.5-
5.6, 5.16-5.18 and 5.28-5.29 respectively.

Specifications of the in vitro drug release study
Equipment : Artificial diffusion cell
Semipermeable membrane : Dialysis membrane (M.Wt cut off: 12000)
Diffusion medium : Phosphate buffer saline (PBS) pH 7.4
Volume of sampling fluid : 2ml
Temperature : 37±0.5°C
RPM : 50
Absorption maximum : 306nm
4.6.4. Kinetic modelling of drug release:
The order and mechanism of Nystatin release from all vesicular
suspensions was determined by fitting the in vitro drug release data in the
following mathematical models. The drug release kinetics was evaluated by
using the linear regression method.
1. Zero-order kinetics (cumulative % release vs time),
2. First-order kinetics (log % drug remaining vs time),
3. Higuchi kinetics (cumulative % drug release vs. square root of time),
4. Korsmeyer - Peppas (log cumulative % drug release vs log time) and
5. Hixson-Crowel models (cubic root of drug remaining vs time).
The r2 and n values are calculated for the linear curves obtained by
regression analysis of the above plots. The release pattern from all vesicular
suspensions was concluded based on the r 2 and n – values. The r2 and n –
values of all niosomal, ethosomal and transferosomal suspensions was
tabulated in tables 5.18, 5.28 and 5.38 respectively. Release kinetics of

niosomal, ethosomal and transferosomal suspensions are graphically presented
in figures 5.7-5.14, 5.19-5.26 and 5.30-5.37 respectively.
Based on the vesicle size, entrapment efficiency and in vitro drug release
profile from all vesicular suspensions, one vesicular suspension was selected
as an optimized formulation from each type vesicular carrier systems.
Elasticity of the vesicles membranes and surface charge of the vesicles
present in the three optimized suspensions (each from one type vesicular
suspension) was characterized as they are important factors which influence
on the drug permeability across the semipermeable membrane and physical
stability of the vesicles respectively.
4.6.5. Elasticity measurement
Three optimized vesicular suspensions were allowed to pass through the
filter paper having pore size of 50nm under vacuum conditions for 5min. After
5min, the volume of suspension permeated and the vesicles size, present in the
permeated suspension, was measured. Then elasticity of the vesicles was
calculated using the following formula. Elasticity study results of all three
optimized suspensions were tabulated in table 5.39.
Where, E – Elasticity of vesicle membrane; J – Volume of suspension
extruded in 5 min; rv - Vesicle size after extrusion and rp - Pore size of the
barrier.
4.6.6. Zeta potential
Zeta potential of the optimized formulations was measured by
instrument zetasizer nano ZS using DTS software (Malvern Instrument Limited,
UK) using M3-PALS technology. Zeta potential study results of three optimized
vesicular suspensions were tabulated in table 5.40.
4.6.7. Preparation of conventional and vesicular gels
Using raw Nystatin and optimized vesicular suspensions, four gel was
prepared having concentration of 0.01%w/w. for preparation of gels, carbopol-
971 and HPMC was used as gelling agents and are used at a ratio of 2:1. (Total
concentration of gelling agents was maintained as 1.5%w/w). Propylene glycol
was used as co-solvent and as dispersion medium for nystatin. The
performance of the gel, viscosity and physical properties was enhanced by
adding HPMC to carbopol. Neutralization and pH of the gel was adjusted by
adding triethanolamine (quantity sufficient).
Half quantity of the double distilled water was heated to 90 0C and HPMC
was added gradually to the above water with continuous stirring. The
remaining half quantity of water was cooled to 5 0C and this water added to the
HPMC dispersion with continuous stirring. Required quantity of Carbopol
which was passed through sieve no. 40 was weighed and added to the HPMC
dispersion while stirring. The resulting gel pH was adjusted by adding
triethanolamine. Required quantity of Nystatin was dispersed in propylene
glycol and transferred in to the mixture of carbopol and HPMC with continuous
stirring. Composition of all three formulations was given in table 5.41.

4.6.8. In vitro drug permeation from niosomal, ethosomal,
transferosomal and conventional gels
In vitro drug release from vesicular and conventional gels was studied by
exhaustive dialysis method and was carried out in an artificial diffusion cell.
Two side open ended glass tube was taken and one side has been closed
with Dialysis membrane (HiMedia Laboratories Pvt. Ltd., M.Wt cut off: 12000).
The fabricated tube was used as donor compartment, in which 1gm of gel was
taken and the entire set up placed in receptor compartment (Beaker, 250ml
capacity) containing 100 ml phosphate buffer saline. The dialysis was carried
out at 50 rpm at 37±0.5°C for 12hrs. Every hour 2ml of diffusion fluid from
receptor compartment was withdrawn and same volume of fresh diffusion fluid
was replaced to maintain sinc conditions. The withdrawn diffusion fluids were
suitably diluted and analyzed using UV spectrophotometer at 306nm. The in
vitro drug release from all conventional, niosomal, ethosomal and
transferosomal gels was tabulated in tables 5.42. 5.43, 5.44 and 5.45
respectively and the same were graphically presented in figutes 5.41.
Specifications of the in vitro drug release study
Semipermeable membrane : Dialysis membrane (M.Wt cut off: 12000)
RPM : 50
4.6.9. Ex vivo drug permeation from the conventional, niosomal,
ethosomal and transferosomal gels
Ex vivo drug permeation studies of all gels were carried out using an
artificial diffusion cell consisting of effective diffusional area of 1.32 Sq.cm and
100 ml of PBS pH 7.4 was placed in receiver compartment as diffusion
medium. Between donor and receiver compartments rat abdominal skin was
mounted and is used as semipermeable membrane.
Dehaired skin of anaesthetized male albino rats weighing between
200 -250 gms was separated from the adhering tissues. Circular section of
suitable diameter was cut to place in the diffusion cell.
The subcutaneous tissue from the skin was removed surgically and the
fat content adhered to the skin was removed by washing with isopropyl alcohol
followed by distilled water.
The prepared abdominal rat skin was arranged between donor and
receptor compartments in such way that the stratum corneum, barrier of the
skin, was faced towards the donor compartment and the dermal side faced
towards the receiver compartment side.
In donor compartment, 1gm of gel was taken and the receiver
compartment was filled with 100ml of diffusion medium, Phosphate buffer
saline pH7.4 and a magnetic bead was placed in the PBS to bring the agitation.
The diffusion was carried out at 50 rpm at 37°C for 10hrs. Every hour 2ml of
diffusion fluid from receptor compartment was withdrawn and same volume of
fresh diffusion fluid was replaced to maintain sinc conditions. The withdrawn
diffusion fluids were suitably diluted and analyzed using UV spectrophotometer
at 306nm. The ex vivo drug release from all conventional, niosomal, ethosomal
and transferosomal gels was tabulated in tables 5.46. 5.47, 5.48 and 5.49
respectively and the same were graphically presented in figutes 5.42.
Specifications of ex vivo drug release study
Semipermeable membrane : Rat Skin
RPM : 50
4.6.10. Transdermal flux
Amount of material flowing through a unit cross-sectional barrier in unit
time is said to be flux.
Mathematically it is expressed as,
Where,
J is the flux
Q is the quantity of compound traversing the membrane in time t
A is the area of exposed membrane in cm2

Flux of drug from all gels was calculated and compared with each other.
4.6.11. Data analysis of the ex vivo drug release
Cumulative amount of nystatin permeated (μg/cm 2) through the
membrane vs time (min) plot was constructed and parameters like transdermal
flux (Jss, μg/cm2/min) at steady state, permeation coefficient (Kp, cm 2/min),
etc was calculated. Slope of the linear portion of the cumulative amount of
nystatin permeated vs time was considered as transdermal flux. Permeation
coefficient (Kp) calculated by dividing the flux with the initial concentration of
the drug taken in the donor compartment and enhancement ratio (Er), extent
of increase in permeation of drug by vesicular gels than the conventional gel,
was calculated by dividing the nystatin permeation from vesicular gels by
concentration of nystatin permeated from conventional gel in a fixed time.
4.6.12. Drug retention study:
This is an important parameter for characterization of transdermal
drug delivery products which states the amount of drug reaches to the
systemic circulation and amount of drug retained in and on the skin. This
study will be carried out after completion of the ex vivo drug release study.
After completion of 12hrs ex vivo diffusion studies, the skin placed
between donor and receptor compartment was collected carefully and the gel
present on the skin was scrapped off precisely and preserved for further
studies.
The skin was sliced as very small thin pieces using surgical stainless
steel scalpel. The pieces were transferred into a 250ml beaker containing
100ml of PBS pH7.4. The beaker was kept under a mechanical stirrer and
stirring was initiated at a speed of 1000rpm and the same was continued for
24hrs. The entire process was carried out at a temperature of 37±1 0 C. After
24hrs, 10ml of solution collected from the beaker and filtered. The filtered
solution was centrifuged at 3000rpm for 30min and supernatant fluid was
collected and nystatin concentration was quantified using UV-Visible
spectrophotometer at 306nm.
The resultant nystatin concentration indicates the amount of the drug
retained in the skin. From the above data, amount of nystatin retained on the
skin was calculated by subtracting the amount of nystatin permeated (receptor
fluid concentration during ex vivo study) and retained within the skin. This was
confirmed by analyzing the collected overlying gel. i.e. gel collected from skin
surface169. The data of skin retention study was tabulated in table 5.50.
4.6.13. Stability Studies
In the present study, the stability of the vesicles was charecterized as per
ICH guidelines. Three optimized formulations preserved at refrigerated
temperature (4-8±1°C) and room temperature (25±2°C) for 180days. After 90

and 180days, shape, size and % entrapment efficiency of vesicles were
measured. The results were compared with the initial size, shape and %
entrapment efficiency of all three samples and are tabulated in tables 5.51-
5.53.
4.6.14. In vivo study of the gels in rabbits
An approved reliable reversed-phase high-performance liquid
chromatographic method170 was used for in vivo study. Four groups, consisting
of two rabbits in each group, were taken. Each group was treated with 1gm of
gels and is labled properly.
At different time points, given below, blood sapmle was collected from ear
vein of the rabbits and are analysed by HPLC for concentration of nystatin in
each sample. Nystatin is extracted by 1:2 (v/v) liquid-liquid extraction with
methanol. Separation is achieved by HPLC after direct injection on a
muBondapak C18 analytical column with a mobile phase composed of 10 mM
sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile adjusted
to pH 6. Detection is by ultraviolet absorbance at 305 nm. Quantitation is
based on the sum of the peak area concentration of the two major isomers of
nystatin, which elute at 7.5-8.5 and 9.5-10.5 min. The assay was linear over
the concentration range of 0.05 to 50µg/ml. in vivo study results of
conventional, niosomal, ethosomal and transferosomal gels are tabulated in
tables 5.56, 5.57, 5.58 and 5.59 respectively. Summary of the nystatin
concentrations in blood plasma at different time intervals was tabulated in
table 5.60 and pharmacokinetic parameters are tabulated in table 5.61. HPLC
chromatograms of conventional, niosomal, ethosomal and transferosomal gels
in rabbits 1&2 are given in figures 5.53-5.68, 5.69-5.84, 5.85-5.100 and 5.101-
5.116 respectively.
Chapter - 5
EXPERIMENTAL RESULTS
5. EXPERIMENTAL RESULTS
Table 5.1: Solubility of nystatin in Phosphate buffer saline solution pH 7.4
Corresponding Actual Actual Actual

concentration concentration, concentration, concentration,
mcg/ml mcg/ml mcg/100ml mg/100ml Solubility
(corresponding (Actual of
concentration, concentration, (Actual nystatin,
mcg/ml)Xdil.Factor mcg/ml) X concentration, mg/100ml
S.No Absorbance y=0.045x+0.006 (100) 100 mcg/100ml)/1000 of PBS
1 0.163 3.4889 348.8889 34888.89 34.8889 34.89
2 0.149 3.1778 317.7778 31777.78 31.7778 31.78
3 0.158 3.3778 337.7778 33777.78 33.7778 33.78
Average solubility 33.48
Std. deviation 1.58
Table 5.2: Standard curve of nystatin in Phosphate buffer saline
solution pH 7.4
S.No Concentration, (mcg/ml) Absorbance (nm)
1 0 0.000
2 2 0.102
3 4 0.192
4 6 0.275
5 8 0.364
6 10 0.460
Table 5.3: Composition of niosomal formulations
Composition, mg
Formulation
S.No Non-ionic Surfactant
code Nystatin Cholesterol
Span-60 Span-80
1 FN1 100 50 -- 100
2 FN2 100 100 -- 100
3 FN3 100 150 -- 100
4 FN4 100 200 -- 100
5 FN5 100 -- 50 100
6 FN6 100 -- 100 100
7 FN7 100 -- 150 100
8 FN8 100 -- 200 100
Table 5.4: Composition of ethosomal formulations
Formulation Nystatin, Lecithin, Ethanol, Cholesterol,

S.No
code mg mg ml mg
1 FE1 20 100 15 --
2 FE2 20 100 30 --
3 FE3 20 100 45 --
4 FE4 20 100 60 --
5 FE5 20 200 45 --
6 FE6 20 300 45 --
7 FE7 20 400 45 --
8 FE8 20 300 45 20
Table 5.5: Composition of transferosomal formulations
Formulation Nystatin, Lecithin, Edge activator, mg

S.No
code mg mg Span-60 Span-80
1 FT1 100 960 40 --
2 FT2 100 920 80 --

3 FT3 100 880 120 --
4 FT4 100 840 160 --

5 FT5 100 960 -- 40
6 FT6 100 920 -- 80

7 FT7 100 880 -- 120
8 FT8 100 840 -- 160
Table 5.6: Vesicles size & Entrapment efficiency of all niosomal formulations
S.No Formulation Vesicle % Entrapment efficiency

code size, nm Centrifugation method Dialysis method
1 FN1 363±2.3 59.4±3.2 62.7±2.9
2 FN2 326±1.5 63.7±2.6 61.5±1.7
3 FN3 281±1.7 72.5±1.9 73.7±2.3
4 FN4 278±1.4 72.8±2.4 73.6±1.8
5 FN5 431±1.2 51.2±2.2 47.9±1.1
6 FN6 366±2.1 56.6±1.6 58.8±2.1
7 FN7 298±1.9 59.3±2.1 63.2±3.3
8 FN8 289±1.2 61.9±2.6 58.5±1.9
Table 5.7: Vesicles size & Entrapment efficiency of all ethosomal formulations
Formulation Vesicle % Entrapment efficiency

S.No
1 FE1 463.2 42.6±1.6 44.2±1.5
2 FE2 416.1 64.8±1.9 62.5±2.3
3 FE3 371.7 78.7±2.1 78.3±2.7
4 FE4 291.2 61.5±1.1 64.8±3.3
5 FE5 438.2 81.3±2.4 80.7±1.6
6 FE6 412.5 88.4±1.4 87.5±2.1
7 FE7 403.3 86.8±1.7 83.9±2.8
8 FE8 391.6 87.6±2.2 85.3±2.4
Table 5.8: Vesicles size & Entrapment efficiency of all transferosomal formulations
Formulation Vesicle % Entrapment efficiency

S.No
344.2
1 FT1 56.2±2.1 52.1±2.1
317.6
2 FT2 58.7±1.6 57.4±2.6
271.3
3 FT3 61.3±1.9 60.5±2.8
266.7
4 FT4 57.4±2.2 58.8±2.5
384.4
5 FT5 62.4±2.6 59.9±2.7
369.9
6 FT6 65.3±1.7 62.2±1.9
374.1
7 FT7 69.4±1.9 70.6±1.8
331.6
8 FT8 66.1±2.3 61.3±2.2
Table 5.9: Summary of in vitro drug release from all niosomal formulations across the dialysis membrane
% nystatin release from all niosomal formulations

S.No Time, Hrs FN1 FN2 FN3 FN4 FN5 FN6 FN7 FN8
1 0 0 0 0 0 0 0 0 0
2 1 9.09 7.22 6.48 4.81 11.33 6.89 6.58 5.49
3 2 17.23 14.94 13.13 9.6 21.96 14.48 15 11.12
4 3 28.77 22.06 19.38 17.39 34.42 27.39 21.95 19.64
5 4 35.19 30.76 26.48 24.82 43.27 36.36 31.76 28.52
6 5 43.11 38.56 33.87 28.72 51.34 44.66 42.14 35.02
7 6 51.54 45.45 39.09 35.03 60.37 52.08 48.55 41.68
8 7 58.47 51.54 47.04 43.55 75.06 72.21 54.75 52.51
9 8 66.22 61.21 54.34 50.89 84.81 77.97 63.77 61.82
Table 5.10: In vitro drug release from niosomal formulation FN1

amt. cumulative
in amt in amt.
TIME 5ml 200ml. release log log cub rt
(min) t √t logt Abs conc (µg) (µg) (µg) CPR CPRU CPR CPRU (CPR)
0 0.000 0.000 0 0 0 0.0 0.00 100.00 2.00 0.0
60 7.746 1.778 0.030 0.54 2.7 108 108.0 9.09 90.91 0.96 1.96 2.1
120 10.954 2.079 0.051 1.01 5.05 202 204.7 17.23 82.77 1.24 1.92 2.6
180 13.416 2.255 0.081 1.67 8.35 334 341.8 28.77 71.23 1.46 1.85 3.1
240 15.492 2.380 0.096 2.01 10.05 402 418.1 35.19 64.81 1.55 1.81 3.3
300 17.321 2.477 0.115 2.43 12.15 486 512.2 43.11 56.89 1.63 1.76 3.5
360 18.974 2.556 0.135 2.87 14.35 574 612.3 51.54 48.46 1.71 1.69 3.7
420 20.494 2.623 0.150 3.21 16.05 642 694.7 58.47 41.53 1.77 1.62 3.9
480 21.909 2.681 0.168 3.59 17.95 718 786.7 66.22 33.78 1.82 1.53 4.0

amt. cumulative
in amt in amt.
0 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.027 0.46 2.3 92 92.0 7.22 92.78 0.86 1.97 1.9
120 10.954 2.079 0.048 0.94 4.7 188 190.3 14.94 85.06 1.17 1.93 2.5
180 13.416 2.255 0.068 1.37 6.85 274 281.0 22.06 77.94 1.34 1.89 2.8
240 15.492 2.380 0.091 1.89 9.45 378 391.9 30.76 69.24 1.49 1.84 3.1
300 17.321 2.477 0.111 2.34 11.7 468 491.3 38.56 61.44 1.59 1.79 3.4
360 18.974 2.556 0.128 2.72 13.6 544 579.0 45.45 54.55 1.66 1.74 3.6
420 20.494 2.623 0.143 3.04 15.2 608 656.6 51.54 48.46 1.71 1.69 3.7
480 21.909 2.681 0.167 3.58 17.9 716 779.8 61.21 38.79 1.79 1.59 3.9

cumulative
amt. amt in amt.
TIME in 5ml 200ml. release log log cub rt
(min)t √t logt Abs conc (µg) (µg) (µg) CPR CPRU CPR CPRU (CPR)
0 0.000 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.027 0.47 2.35 94 94.0 6.48 93.52 0.81 1.97 1.9
120 10.954 2.079 0.048 0.94 4.7 188 190.4 13.13 86.87 1.12 1.94 2.4
180 13.416 2.255 0.068 1.37 6.85 274 281.1 19.38 80.62 1.29 1.91 2.7
240 15.492 2.380 0.089 1.85 9.25 370 383.9 26.48 73.52 1.42 1.87 3.0
300 17.321 2.477 0.111 2.34 11.7 468 491.2 33.87 66.13 1.53 1.82 3.2
360 18.974 2.556 0.126 2.66 13.3 532 566.9 39.09 60.91 1.59 1.78 3.4
420 20.494 2.623 0.149 3.17 15.85 634 682.2 47.04 52.96 1.67 1.72 3.6
480 21.909 2.681 0.169 3.62 18.1 724 788.0 54.34 45.66 1.74 1.66 3.8

cumulative
amt. amt in amt.
0 0.000 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.022 0.35 1.75 70 70.0 4.81 95.19 0.68 1.98 1.7
120 10.954 2.079 0.037 0.69 3.45 138 139.8 9.60 90.40 0.98 1.96 2.1
180 13.416 2.255 0.062 1.24 6.2 248 253.2 17.39 82.61 1.24 1.92 2.6
240 15.492 2.380 0.085 1.75 8.75 350 361.4 24.82 75.18 1.39 1.88 2.9
300 17.321 2.477 0.096 1.99 9.95 398 418.2 28.72 71.28 1.46 1.85 3.1
360 18.974 2.556 0.114 2.4 12 480 510.1 35.03 64.97 1.54 1.81 3.3
420 20.494 2.623 0.139 2.96 14.8 592 634.1 43.55 56.45 1.64 1.75 3.5
480 21.909 2.681 0.160 3.42 17.1 684 740.9 50.89 49.11 1.71 1.69 3.7

cumulative
amt. amt in amt.
0 0.000 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.032 0.58 2.9 116 116.0 11.33 88.67 1.05 1.95 2.2
120 10.954 2.079 0.056 1.11 5.55 222 224.9 21.96 78.04 1.34 1.89 2.8
180 13.416 2.255 0.083 1.72 8.6 344 352.5 34.42 65.58 1.54 1.82 3.3
240 15.492 2.380 0.102 2.13 10.65 426 443.1 43.27 56.73 1.64 1.75 3.5
300 17.321 2.477 0.118 2.49 12.45 498 525.7 51.34 48.66 1.71 1.69 3.7
360 18.974 2.556 0.136 2.89 14.45 578 618.2 60.37 39.63 1.78 1.60 3.9
420 20.494 2.623 0.167 3.57 17.85 714 768.6 75.06 24.94 1.88 1.40 4.2
480 21.909 2.681 0.185 3.98 19.9 796 868.5 84.81 15.19 1.93 1.18 4.4

cumulative
amt. amt in amt.
0 0.000 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.024 0.39 1.95 78 78.0 6.89 93.11 0.84 1.97 1.9
120 10.954 2.079 0.042 0.81 4.05 162 164.0 14.48 85.52 1.16 1.93 2.4
180 13.416 2.255 0.074 1.52 7.6 304 310.0 27.39 72.61 1.44 1.86 3.0
240 15.492 2.380 0.096 1.99 9.95 398 411.6 36.36 63.64 1.56 1.80 3.3
300 17.321 2.477 0.114 2.41 12.05 482 505.6 44.66 55.34 1.65 1.74 3.5
360 18.974 2.556 0.131 2.77 13.85 554 589.6 52.08 47.92 1.72 1.68 3.7
420 20.494 2.623 0.179 3.84 19.2 768 817.5 72.21 27.79 1.86 1.44 4.2
480 21.909 2.681 0.189 4.07 20.35 814 882.7 77.97 22.03 1.89 1.34 4.3
amt. amt in cumulative

TIME in 5ml 200ml. amt. log log cub rt
(min) t √t logt Abs conc (µg) (µg) release CPR CPRU CPR CPRU (CPR)
(µg)
0 0.000 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.024 0.39 1.95 78 78.0 6.58 93.42 0.82 1.97 1.9
120 10.954 2.079 0.046 0.88 4.4 176 178.0 15.00 85.00 1.18 1.93 2.5
180 13.416 2.255 0.063 1.27 6.35 254 260.4 21.95 78.05 1.34 1.89 2.8
240 15.492 2.380 0.088 1.82 9.1 364 376.7 31.76 68.24 1.50 1.83 3.2
300 17.321 2.477 0.114 2.39 11.95 478 499.8 42.14 57.86 1.62 1.76 3.5
360 18.974 2.556 0.128 2.71 13.55 542 575.8 48.55 51.45 1.69 1.71 3.6
420 20.494 2.623 0.141 3.01 15.05 602 649.3 54.75 45.25 1.74 1.66 3.8
480 21.909 2.681 0.162 3.47 17.35 694 756.4 63.77 36.23 1.80 1.56 4.0
cumulative
amt. amt in amt.
0 0.000 0.000 0.000 0 0 0 0.0 0.00 100.00 #NUM! 2.00 0.0
60 7.746 1.778 0.021 0.34 1.7 68 68.0 5.49 94.51 0.74 1.98 1.8
120 10.954 2.079 0.037 0.68 3.4 136 137.7 11.12 88.88 1.05 1.95 2.2
180 13.416 2.255 0.060 1.19 5.95 238 243.1 19.64 80.36 1.29 1.91 2.7
240 15.492 2.380 0.083 1.71 8.55 342 353.1 28.52 71.48 1.46 1.85 3.1
300 17.321 2.477 0.099 2.07 10.35 414 433.6 35.02 64.98 1.54 1.81 3.3
360 18.974 2.556 0.115 2.43 12.15 486 516.0 41.68 58.32 1.62 1.77 3.5
420 20.494 2.623 0.143 3.04 15.2 608 650.1 52.51 47.49 1.72 1.68 3.7
480 21.909 2.681 0.165 3.54 17.7 708 765.3 61.82 38.18 1.79 1.58 4.0
Table 5.18: Summary of release kinetics data of all nystatin Niosomal Formulations
Formulation Zero First Hixon

Order Order Higuchi Korsmeyer Peppas crowel Mechanism of
S.No code R² R² R² R² n R² drug release
1 FN1 0.996 0.987 0.996 0.9 0.113 0.776 Fickian diffusion
Table 5.19: Summary of in vitro nystatin release from all ethosomal formulations across
Cumulative amount of nystatin permeated from ethosomal suspensions

S.No TIME, Min FE1 FE2 FE3 FE4 FE5 FE6 FE7 FE8
1 15 4.46 6.48 5.97 19.84 8.49 5.09 3.69 5.48
2 30 6.38 13.65 13.02 21.66 16.57 10.35 5.68 10.10
3 60 10.91 15.79 17.09 27.89 23.00 15.20 10.12 14.77
4 120 15.47 19.65 21.83 33.37 26.43 22.70 17.01 19.59
5 180 18.91 24.31 25.73 38.74 33.58 26.77 23.29 23.44
6 240 26.61 27.95 29.03 44.81 36.98 34.50 27.90 31.32
7 300 31.80 32.23 35.29 48.82 41.03 39.14 34.04 35.05
8 360 36.10 37.33 40.33 53.35 44.38 45.51 40.48 39.38
9 420 40.68 40.46 44.91 56.95 48.25 54.55 47.79 44.56
10 480 43.88 45.17 49.66 62.86 51.29 62.43 54.47 49.32
11 540 46.65 47.46 53.44 65.89 55.84 67.44 60.41 55.73
12 600 51.79 51.15 57.00 69.44 59.19 72.83 68.13 60.83
13 660 56.98 56.72 61.73 75.46 64.91 77.15 72.01 64.83
14 720 61.29 62.04 63.58 78.11 73.64 79.61 78.49 69.25
Table 5.20:In vitro drug release from ethosomal formulation FE1
cumulative
amt. in amt in amt.
TIME Conc, 2ml, 200ml, release log log cub rt
(min) t √t logt Abs (µg) (µg) (µg) (µg) CPR CPRU CPR CPRU (CPR)
15 3.873 1.176 0.015 0.19 0.38 38 38.0 4.46 95.54 0.65 1.98 1.6
30 5.477 1.477 0.018 0.27 0.54 54 54.4 6.38 93.62 0.80 1.97 1.9
60 7.746 1.778 0.027 0.46 0.92 92 92.9 10.91 89.09 1.04 1.95 2.2
120 10.954 2.079 0.035 0.65 1.3 130 131.8 15.47 84.53 1.19 1.93 2.5
180 13.416 2.255 0.042 0.79 1.58 158 161.1 18.91 81.09 1.28 1.91 2.7
240 15.492 2.380 0.056 1.11 2.22 222 226.7 26.61 73.39 1.43 1.87 3.0
300 17.321 2.477 0.065 1.32 2.64 264 270.9 31.80 68.20 1.50 1.83 3.2
360 18.974 2.556 0.073 1.49 2.98 298 307.6 36.10 63.90 1.56 1.81 3.3
420 20.494 2.623 0.081 1.67 3.34 334 346.6 40.68 59.32 1.61 1.77 3.4
480 21.909 2.681 0.087 1.79 3.58 358 373.9 43.88 56.12 1.64 1.75 3.5
540 23.238 2.732 0.091 1.89 3.78 378 397.5 46.65 53.35 1.67 1.73 3.6
600 24.495 2.778 0.100 2.09 4.18 418 441.3 51.79 48.21 1.71 1.68 3.7
660 25.690 2.820 0.109 2.29 4.58 458 485.4 56.98 43.02 1.76 1.63 3.8
720 26.833 2.857 0.117 2.46 4.92 492 522.2 61.29 38.71 1.79 1.59 3.9
Table 5.21: In vitro drug release from ethosomal formulation FE2
cumulative
amt. amt in amt.
15 3.873 1.176 0.025 0.42 0.84 84 84 6.48 93.52 1.97 1.86
30 5.477 1.477 0.046 0.88 1.76 176 176.84 13.65 86.35 1.13 1.94 2.39
60 7.746 1.778 0.051 1.01 2.02 202 204.6 15.79 84.21 1.20 1.93 2.51
120 10.954 2.079 0.062 1.25 2.5 250 254.62 19.65 80.35 1.29 1.91 2.70
180 13.416 2.255 0.075 1.54 3.08 308 315.12 24.31 75.69 1.39 1.88 2.90
240 15.492 2.380 0.085 1.76 3.52 352 362.2 27.95 72.05 1.45 1.86 3.03
300 17.321 2.477 0.097 2.02 4.04 404 417.72 32.23 67.77 1.51 1.83 3.18
360 18.974 2.556 0.111 2.33 4.66 466 483.76 37.33 62.67 1.57 1.80 3.34
420 20.494 2.623 0.119 2.51 5.02 502 524.42 40.46 59.54 1.61 1.77 3.43
480 21.909 2.681 0.132 2.79 5.58 558 585.44 45.17 54.83 1.65 1.74 3.56
540 23.238 2.732 0.137 2.91 5.82 582 615.02 47.46 52.54 1.68 1.72 3.62
600 24.495 2.778 0.146 3.12 6.24 624 662.84 51.15 48.85 1.71 1.69 3.71
660 25.690 2.820 0.161 3.45 6.9 690 735.08 56.72 43.28 1.75 1.64 3.84
cumulative
amt. in amt in amt.
15 3.873 1.176 0.027 0.47 0.94 94 94 5.97 94.03 1.97 1.81
30 5.477 1.477 0.052 1.02 2.04 204 204.94 13.02 86.98 1.11 1.94 2.35
60 7.746 1.778 0.066 1.33 2.66 266 268.98 17.09 82.91 1.23 1.92 2.58
120 10.954 2.079 0.082 1.69 3.38 338 343.64 21.83 78.17 1.34 1.89 2.79
180 13.416 2.255 0.095 1.98 3.96 396 405.02 25.73 74.27 1.41 1.87 2.95
240 15.492 2.380 0.106 2.22 4.44 444 456.98 29.03 70.97 1.46 1.85 3.07
300 17.321 2.477 0.127 2.69 5.38 538 555.42 35.29 64.71 1.55 1.81 3.28
360 18.974 2.556 0.144 3.06 6.12 612 634.8 40.33 59.67 1.61 1.78 3.43
420 20.494 2.623 0.159 3.39 6.78 678 706.92 44.91 55.09 1.65 1.74 3.55
480 21.909 2.681 0.174 3.73 7.46 746 781.7 49.66 50.34 1.70 1.70 3.68
540 23.238 2.732 0.186 3.99 7.98 798 841.16 53.44 46.56 1.73 1.67 3.77
600 24.495 2.778 0.196 4.23 8.46 846 897.14 57.00 43.00 1.76 1.63 3.85
660 25.690 2.820 0.211 4.56 9.12 912 971.6 61.73 38.27 1.79 1.58 3.95
720 26.833 2.857 0.216 4.66 9.32 932 1000.72 63.58 36.42 1.80 1.56 3.99
Table 5.23:In vitro drug release from ethosomal formulation FE4
cumulative
amt. amt in amt.
15 3.873 1.176 0.061 1.22 2.44 244 244 19.84 80.16 1.30 1.90 2.71
30 5.477 1.477 0.065 1.32 2.64 264 266.44 21.66 78.34 1.34 1.89 2.79
60 7.746 1.778 0.082 1.69 3.38 338 343.08 27.89 72.11 1.45 1.86 3.03
120 10.954 2.079 0.096 2.01 4.02 402 410.46 33.37 66.63 1.52 1.82 3.22
180 13.416 2.255 0.110 2.32 4.64 464 476.48 38.74 61.26 1.59 1.79 3.38
240 15.492 2.380 0.126 2.67 5.34 534 551.12 44.81 55.19 1.65 1.74 3.55
300 17.321 2.477 0.136 2.89 5.78 578 600.46 48.82 51.18 1.69 1.71 3.65
360 18.974 2.556 0.147 3.14 6.28 628 656.24 53.35 46.65 1.73 1.67 3.76
420 20.494 2.623 0.156 3.33 6.66 666 700.52 56.95 43.05 1.76 1.63 3.85
480 21.909 2.681 0.171 3.66 7.32 732 773.18 62.86 37.14 1.80 1.57 3.98
540 23.238 2.732 0.177 3.81 7.62 762 810.5 65.89 34.11 1.82 1.53 4.04
600 24.495 2.778 0.186 3.99 7.98 798 854.12 69.44 30.56 1.84 1.49 4.11
660 25.690 2.820 0.200 4.32 8.64 864 928.1 75.46 24.54 1.88 1.39 4.23
720 26.833 2.857 0.206 4.44 8.88 888 960.74 78.11 21.89 1.89 1.34 4.27
cumulative
amt. in amt in amt.
15 3.873 1.176 0.037 0.69 1.38 138 138 8.49 91.51 0.93 1.96 2.04
30 5.477 1.477 0.066 1.34 2.68 268 269.38 16.57 83.43 1.22 1.92 2.55
60 7.746 1.778 0.089 1.85 3.7 370 374.06 23.00 77.00 1.36 1.89 2.84
120 10.954 2.079 0.101 2.11 4.22 422 429.76 26.43 73.57 1.42 1.87 2.98
180 13.416 2.255 0.126 2.67 5.34 534 545.98 33.58 66.42 1.53 1.82 3.23
240 15.492 2.380 0.137 2.92 5.84 584 601.32 36.98 63.02 1.57 1.80 3.33
300 17.321 2.477 0.151 3.22 6.44 644 667.16 41.03 58.97 1.61 1.77 3.45
360 18.974 2.556 0.162 3.46 6.92 692 721.6 44.38 55.62 1.65 1.75 3.54
420 20.494 2.623 0.174 3.74 7.48 748 784.52 48.25 51.75 1.68 1.71 3.64
480 21.909 2.681 0.184 3.95 7.9 790 834 51.29 48.71 1.71 1.69 3.72
540 23.238 2.732 0.199 4.28 8.56 856 907.9 55.84 44.16 1.75 1.65 3.82
600 24.495 2.778 0.209 4.51 9.02 902 962.46 59.19 40.81 1.77 1.61 3.90
660 25.690 2.820 0.228 4.93 9.86 986 1055.48 64.91 35.09 1.81 1.55 4.02
720 26.833 2.857 0.258 5.59 11.18 1118 1197.34 73.64 26.36 1.87 1.42 4.19
cumulative
amt. in amt in amt.
15 3.873 1.176 0.026 0.45 0.9 90 90 5.09 94.91 0.71 1.98 1.72
30 5.477 1.477 0.047 0.91 1.82 182 182.9 10.35 89.65 1.01 1.95 2.18
60 7.746 1.778 0.066 1.33 2.66 266 268.72 15.20 84.80 1.18 1.93 2.48
120 10.954 2.079 0.095 1.98 3.96 396 401.38 22.70 77.30 1.36 1.89 2.83
180 13.416 2.255 0.110 2.32 4.64 464 473.34 26.77 73.23 1.43 1.86 2.99
240 15.492 2.380 0.140 2.98 5.96 596 609.98 34.50 65.50 1.54 1.82 3.26
300 17.321 2.477 0.157 3.36 6.72 672 691.94 39.14 60.86 1.59 1.78 3.40
360 18.974 2.556 0.181 3.89 7.78 778 804.66 45.51 54.49 1.66 1.74 3.57
420 20.494 2.623 0.215 4.65 9.3 930 964.44 54.55 45.45 1.74 1.66 3.79
480 21.909 2.681 0.245 5.3 10.6 1060 1103.74 62.43 37.57 1.80 1.57 3.97
540 23.238 2.732 0.262 5.69 11.38 1138 1192.34 67.44 32.56 1.83 1.51 4.07
600 24.495 2.778 0.281 6.11 12.22 1222 1287.72 72.83 27.17 1.86 1.43 4.18
660 25.690 2.820 0.295 6.43 12.86 1286 1363.94 77.15 22.85 1.89 1.36 4.26
720 26.833 2.857 0.303 6.61 13.22 1322 1407.42 79.61 20.39 1.90 1.31 4.30
cumulative
amt. in amt in amt.
15 3.873 1.176 0.020 0.32 0.64 64 64 3.69 96.31 0.57 1.98 1.54
30 5.477 1.477 0.028 0.49 0.98 98 98.64 5.68 94.32 0.75 1.97 1.78
60 7.746 1.778 0.045 0.87 1.74 174 175.62 10.12 89.88 1.01 1.95 2.16
120 10.954 2.079 0.072 1.46 2.92 292 295.36 17.01 82.99 1.23 1.92 2.57
180 13.416 2.255 0.096 1.99 3.98 398 404.28 23.29 76.71 1.37 1.88 2.86
240 15.492 2.380 0.113 2.37 4.74 474 484.26 27.90 72.10 1.45 1.86 3.03
300 17.321 2.477 0.136 2.88 5.76 576 591 34.04 65.96 1.53 1.82 3.24
360 18.974 2.556 0.159 3.41 6.82 682 702.76 40.48 59.52 1.61 1.77 3.43
420 20.494 2.623 0.186 4.01 8.02 802 829.58 47.79 52.21 1.68 1.72 3.63
480 21.909 2.681 0.211 4.55 9.1 910 945.6 54.47 45.53 1.74 1.66 3.79
540 23.238 2.732 0.232 5.02 10.04 1004 1048.7 60.41 39.59 1.78 1.60 3.92
600 24.495 2.778 0.260 5.64 11.28 1128 1182.74 68.13 31.87 1.83 1.50 4.08
660 25.690 2.820 0.272 5.92 11.84 1184 1250.02 72.01 27.99 1.86 1.45 4.16
720 26.833 2.857 0.296 6.44 12.88 1288 1362.5 78.49 21.51 1.89 1.33 4.28
cumulative
amt. in amt in amt.
15 3.873 1.176 0.028 0.48 0.96 96 96 5.48 94.52 0.74 1.98 1.76
30 5.477 1.477 0.046 0.88 1.76 176 176.96 10.10 89.90 1.00 1.95 2.16
60 7.746 1.778 0.064 1.28 2.56 256 258.72 14.77 85.23 1.17 1.93 2.45
120 10.954 2.079 0.082 1.69 3.38 338 343.28 19.59 80.41 1.29 1.91 2.70
180 13.416 2.255 0.096 2.01 4.02 402 410.66 23.44 76.56 1.37 1.88 2.86
240 15.492 2.380 0.127 2.68 5.36 536 548.68 31.32 68.68 1.50 1.84 3.15
300 17.321 2.477 0.140 2.98 5.96 596 614.04 35.05 64.95 1.54 1.81 3.27
360 18.974 2.556 0.156 3.33 6.66 666 690 39.38 60.62 1.60 1.78 3.40
420 20.494 2.623 0.175 3.75 7.5 750 780.66 44.56 55.44 1.65 1.74 3.55
480 21.909 2.681 0.192 4.13 8.26 826 864.16 49.32 50.68 1.69 1.70 3.67
540 23.238 2.732 0.215 4.65 9.3 930 976.42 55.73 44.27 1.75 1.65 3.82
600 24.495 2.778 0.233 5.05 10.1 1010 1065.72 60.83 39.17 1.78 1.59 3.93
660 25.690 2.820 0.247 5.35 10.7 1070 1135.82 64.83 35.17 1.81 1.55 4.02
720 26.833 2.857 0.263 5.71 11.42 1142 1213.24 69.25 30.75 1.84 1.49 4.11
Table 5.28: Summary of Release kinetics data of all nystatin ethosomal suspension
Formulation Zero First Hixon- Korsmeyer-

order order Higuchi crowel peppas Mechanism of
S.No Code R2 R2 R2 R2 R2 n drug release
1 FE1 0.992 0.992 0.979 0.924 0.994 0.684 Non-Fickian diffusion
4 FE4 0.988 0.988 0.989 0.948 0.973 0.367 Fickian diffusion
Table 5.29: Summary of in vitro nystatin release from all transferosomal formulations across the dialysis membrane
In released from all transferosomal formulations

S.No Time, Min FT1 FT2 FT3 FT4 FT5 FT6 FT7 FT8
1 15 1.7 1.0 3.1 1.0 1.8 7.7 19.7 5.3

2 30 3.2 2.9 6.8 5.5 5.2 15.3 24.1 10.6
3 60 5.8 5.5 10.8 11.4 10.0 21.5 28.9 15.7

4 120 9.7 9.7 14.8 12.9 16.0 28.3 35.9 20.3
5 180 14.8 14.3 19.9 16.7 20.6 32.4 39.9 25.8

6 240 19.0 19.4 24.4 21.3 24.9 38.0 44.9 30.4
7 300 23.4 23.2 27.6 25.3 28.9 43.9 49.5 34.2

8 360 27.1 26.7 32.1 30.0 33.6 48.5 53.5 39.5
9 420 31.8 30.8 37.0 32.9 40.1 53.4 56.6 45.4

10 480 36.6 35.9 41.3 35.4 44.4 56.7 60.3 49.5
11 540 41.9 39.9 45.1 40.9 48.3 59.6 66.3 55.6
12 600 45.1 43.2 49.0 46.8 54.3 63.2 69.2 61.6

13 660 48.5 45.8 51.8 51.2 60.0 65.5 72.3 66.9
14 720 51.1 54.2 58.3 55.3 65.2 69.2 75.2 72.1
Table 5.30: In vitro drug release from transferosomal formulation FT1
cumulative
amt. cub
TIME amt. in amt in release log log rt
(min) t √t logt Abs conc 2ml(µg) 200ml.(µg) (µg) CPR CPRU CPR CPRU (CPR)
15 3.873 1.176 0.018 0.27 0.54 54 54 4.98 95.02 0.70 1.98 1.71
30 5.477 1.477 0.034 0.62 1.24 124 124.54 11.49 88.51 1.06 1.95 2.26
60 7.746 1.778 0.056 1.1 2.2 220 221.78 20.46 79.54 1.31 1.90 2.74
120 10.954 2.079 0.078 1.6 3.2 320 323.98 29.89 70.11 1.48 1.85 3.10
180 13.416 2.255 0.086 1.77 3.54 354 361.18 33.32 66.68 1.52 1.82 3.22
240 15.492 2.380 0.093 1.93 3.86 386 396.72 36.60 63.40 1.56 1.80 3.32
300 17.321 2.477 0.099 2.07 4.14 414 428.58 39.54 60.46 1.60 1.78 3.41
360 18.974 2.556 0.103 2.16 4.32 432 450.72 41.58 58.42 1.62 1.77 3.46
420 20.494 2.623 0.106 2.23 4.46 446 469.04 43.27 56.73 1.64 1.75 3.51
480 21.909 2.681 0.109 2.29 4.58 458 485.5 44.79 55.21 1.65 1.74 3.55
540 23.238 2.732 0.110 2.31 4.62 462 494.08 45.58 54.42 1.66 1.74 3.57
600 24.495 2.778 0.111 2.34 4.68 468 504.7 46.56 53.44 1.67 1.73 3.60
660 25.690 2.820 0.115 2.42 4.84 484 525.38 48.47 51.53 1.69 1.71 3.65
720 26.833 2.857 0.120 2.54 5.08 508 554.22 51.13 48.87 1.71 1.69 3.71
cumulative
amt. cub
15 3.873 1.176 0.026 0.44 0.88 88 88 7.57 92.43 0.88 1.97 1.96
30 5.477 1.477 0.036 0.67 1.34 134 134.88 11.61 88.39 1.06 1.95 2.26
60 7.746 1.778 0.052 1.02 2.04 204 206.22 17.75 82.25 1.25 1.92 2.61
120 10.954 2.079 0.061 1.23 2.46 246 250.26 21.54 78.46 1.33 1.89 2.78
180 13.416 2.255 0.092 1.91 3.82 382 388.72 33.45 66.55 1.52 1.82 3.22
240 15.492 2.380 0.105 2.19 4.38 438 448.54 38.60 61.40 1.59 1.79 3.38
300 17.321 2.477 0.117 2.46 4.92 492 506.92 43.62 56.38 1.64 1.75 3.52
360 18.974 2.556 0.119 2.52 5.04 504 523.84 45.08 54.92 1.65 1.74 3.56
420 20.494 2.623 0.123 2.59 5.18 518 542.88 46.72 53.28 1.67 1.73 3.60
480 21.909 2.681 0.124 2.62 5.24 524 554.06 47.68 52.32 1.68 1.72 3.63
540 23.238 2.732 0.124 2.63 5.26 526 561.3 48.30 51.70 1.68 1.71 3.64
600 24.495 2.778 0.125 2.64 5.28 528 568.56 48.93 51.07 1.69 1.71 3.66
660 25.690 2.820 0.126 2.66 5.32 532 577.84 49.73 50.27 1.70 1.70 3.68
720 26.833 2.857 0.140 2.98 5.96 596 647.16 55.69 44.31 1.75 1.65 3.82
cumulative
amt. cub
15 3.873 1.176 0.025 0.43 0.86 86 86 7.06 92.94 0.85 1.97 1.92
30 5.477 1.477 0.035 0.64 1.28 128 128.86 10.58 89.42 1.02 1.95 2.20
60 7.746 1.778 0.051 1.01 2.02 202 204.14 16.76 83.24 1.22 1.92 2.56
120 10.954 2.079 0.062 1.24 2.48 248 252.16 20.70 79.30 1.32 1.90 2.75
180 13.416 2.255 0.110 2.32 4.64 464 470.64 38.64 61.36 1.59 1.79 3.38
240 15.492 2.380 0.114 2.4 4.8 480 491.28 40.33 59.67 1.61 1.78 3.43
300 17.321 2.477 0.124 2.62 5.24 524 540.08 44.34 55.66 1.65 1.75 3.54
360 18.974 2.556 0.130 2.76 5.52 552 573.32 47.07 52.93 1.67 1.72 3.61
420 20.494 2.623 0.133 2.83 5.66 566 592.84 48.67 51.33 1.69 1.71 3.65
480 21.909 2.681 0.135 2.86 5.72 572 604.5 49.63 50.37 1.70 1.70 3.67
540 23.238 2.732 0.137 2.92 5.84 584 622.22 51.09 48.91 1.71 1.69 3.71
600 24.495 2.778 0.142 3.02 6.04 604 648.06 53.21 46.79 1.73 1.67 3.76
660 25.690 2.820 0.149 3.17 6.34 634 684.1 56.17 43.83 1.75 1.64 3.83
720 26.833 2.857 0.153 3.26 6.52 652 708.44 58.16 41.84 1.76 1.62 3.87
cumulative cub
TIME amt. in amt in amt. release log log rt
15 3.873 1.176 0.017 0.24 0.48 48 48 4.13 95.87 0.62 1.98 1.60
30 5.477 1.477 0.023 0.38 0.76 76 76.48 6.58 93.42 0.82 1.97 1.87
60 7.746 1.778 0.036 0.66 1.32 132 133.24 11.47 88.53 1.06 1.95 2.25
120 10.954 2.079 0.039 0.74 1.48 148 150.56 12.96 87.04 1.11 1.94 2.35
180 13.416 2.255 0.061 1.22 2.44 244 248.04 21.35 78.65 1.33 1.90 2.77
240 15.492 2.380 0.069 1.4 2.8 280 286.48 24.65 75.35 1.39 1.88 2.91
300 17.321 2.477 0.070 1.42 2.84 284 293.28 25.24 74.76 1.40 1.87 2.93
360 18.974 2.556 0.082 1.69 3.38 338 350.12 30.13 69.87 1.48 1.84 3.11
420 20.494 2.623 0.089 1.84 3.68 368 383.5 33.00 67.00 1.52 1.83 3.21
480 21.909 2.681 0.095 1.97 3.94 394 413.18 35.56 64.44 1.55 1.81 3.29
540 23.238 2.732 0.108 2.26 4.52 452 475.12 40.89 59.11 1.61 1.77 3.45
600 24.495 2.778 0.116 2.44 4.88 488 515.64 44.38 55.62 1.65 1.75 3.54
660 25.690 2.820 0.122 2.58 5.16 516 548.52 47.20 52.80 1.67 1.72 3.61
720 26.833 2.857 0.125 2.64 5.28 528 565.68 48.68 51.32 1.69 1.71 3.65
cumulative
amt. cub
15 3.873 1.176 0.011 0.11 0.22 22 22 1.80 98.20 0.25 1.99 1.22
30 5.477 1.477 0.030 0.54 1.08 108 108.22 8.84 91.16 0.95 1.96 2.07
60 7.746 1.778 0.049 0.95 1.9 190 191.3 15.63 84.37 1.19 1.93 2.50
120 10.954 2.079 0.073 1.48 2.96 296 299.2 24.44 75.56 1.39 1.88 2.90
180 13.416 2.255 0.087 1.81 3.62 362 368.16 30.08 69.92 1.48 1.84 3.11
240 15.492 2.380 0.093 1.93 3.86 386 395.78 32.33 67.67 1.51 1.83 3.19
300 17.321 2.477 0.096 1.99 3.98 398 411.64 33.63 66.37 1.53 1.82 3.23
360 18.974 2.556 0.109 2.28 4.56 456 473.62 38.69 61.31 1.59 1.79 3.38
420 20.494 2.623 0.111 2.34 4.68 468 490.18 40.05 59.95 1.60 1.78 3.42
480 21.909 2.681 0.117 2.46 4.92 492 518.86 42.39 57.61 1.63 1.76 3.49
540 23.238 2.732 0.140 2.98 5.96 596 627.78 51.29 48.71 1.71 1.69 3.72
600 24.495 2.778 0.147 3.13 6.26 626 663.74 54.23 45.77 1.73 1.66 3.79
660 25.690 2.820 0.163 3.48 6.96 696 740 60.46 39.54 1.78 1.60 3.92
720 26.833 2.857 0.174 3.74 7.48 748 798.96 65.27 34.73 1.81 1.54 4.03

cumulative
amt. cub
15 3.873 1.176 0.030 0.53 1.06 106 106 8.31 91.69 0.92 1.96 2.03
30 5.477 1.477 0.050 0.97 1.94 194 195.06 15.29 84.71 1.18 1.93 2.48
60 7.746 1.778 0.067 1.36 2.72 272 275 21.55 78.45 1.33 1.89 2.78
120 10.954 2.079 0.083 1.72 3.44 344 349.72 27.41 72.59 1.44 1.86 3.02
180 13.416 2.255 0.097 2.02 4.04 404 413.16 32.38 67.62 1.51 1.83 3.19
240 15.492 2.380 0.112 2.36 4.72 472 485.2 38.03 61.97 1.58 1.79 3.36
300 17.321 2.477 0.128 2.71 5.42 542 559.92 43.88 56.12 1.64 1.75 3.53
360 18.974 2.556 0.140 2.98 5.96 596 619.34 48.54 51.46 1.69 1.71 3.65
420 20.494 2.623 0.153 3.26 6.52 652 681.3 53.39 46.61 1.73 1.67 3.77
480 21.909 2.681 0.161 3.44 6.88 688 723.82 56.73 43.27 1.75 1.64 3.84
540 23.238 2.732 0.168 3.59 7.18 718 760.7 59.62 40.38 1.78 1.61 3.91
600 24.495 2.778 0.176 3.78 7.56 756 805.88 63.16 36.84 1.80 1.57 3.98
660 25.690 2.820 0.181 3.89 7.78 778 835.44 65.47 34.53 1.82 1.54 4.03
720 26.833 2.857 0.190 4.09 8.18 818 883.22 69.22 30.78 1.84 1.49 4.11
TIME amt. in amt in cumulative log log cub

(min) t √t logt Abs conc 2ml(µg) 200ml.(µg) amt. CPR CPRU CPR CPRU rt
release
(µg) (CPR)
15 3.873 1.176 0.062 1.24 2.48 248 248 17.71 82.29 1.25 1.92 2.61
30 5.477 1.477 0.082 1.68 3.36 336 338.48 24.18 75.82 1.38 1.88 2.89
60 7.746 1.778 0.105 2.21 4.42 442 447.84 31.99 68.01 1.50 1.83 3.17
120 10.954 2.079 0.126 2.66 5.32 532 542.26 38.73 61.27 1.59 1.79 3.38
180 13.416 2.255 0.146 3.1 6.2 620 635.58 45.40 54.60 1.66 1.74 3.57
240 15.492 2.380 0.167 3.58 7.16 716 737.78 52.70 47.30 1.72 1.67 3.75
300 17.321 2.477 0.186 3.99 7.98 798 826.94 59.07 40.93 1.77 1.61 3.89
360 18.974 2.556 0.203 4.38 8.76 876 912.92 65.21 34.79 1.81 1.54 4.03
420 20.494 2.623 0.210 4.54 9.08 908 953.68 68.12 31.88 1.83 1.50 4.08
480 21.909 2.681 0.215 4.64 9.28 928 982.76 70.20 29.80 1.85 1.47 4.13
540 23.238 2.732 0.217 4.69 9.38 938 1002.04 71.57 28.43 1.85 1.45 4.15
600 24.495 2.778 0.220 4.75 9.5 950 1023.42 73.10 26.90 1.86 1.43 4.18
660 25.690 2.820 0.222 4.81 9.62 962 1044.92 74.64 25.36 1.87 1.40 4.21
720 26.833 2.857 0.224 4.84 9.68 968 1060.54 75.75 24.25 1.88 1.38 4.23
cumulative cub
TIME amt. in amt in amt. log log rt
(min) t √t logt Abs conc 2ml(µg) 200ml.(µg) release CPR CPRU CPR CPRU (CPR)
(µg)
15 3.873 1.176 0.021 0.34 0.68 68 68 5.34 94.66 0.73 1.98 1.75
30 5.477 1.477 0.036 0.67 1.34 134 134.68 10.57 89.43 1.02 1.95 2.19
60 7.746 1.778 0.051 0.99 1.98 198 200.02 15.70 84.30 1.20 1.93 2.50
120 10.954 2.079 0.063 1.27 2.54 254 258 20.25 79.75 1.31 1.90 2.73
180 13.416 2.255 0.078 1.61 3.22 322 328.54 25.79 74.21 1.41 1.87 2.95
240 15.492 2.380 0.091 1.89 3.78 378 387.76 30.44 69.56 1.48 1.84 3.12
300 17.321 2.477 0.101 2.11 4.22 422 435.54 34.19 65.81 1.53 1.82 3.25
360 18.974 2.556 0.115 2.43 4.86 486 503.76 39.54 60.46 1.60 1.78 3.41
420 20.494 2.623 0.131 2.78 5.56 556 578.62 45.42 54.58 1.66 1.74 3.57
480 21.909 2.681 0.141 3.01 6.02 602 630.18 49.46 50.54 1.69 1.70 3.67
540 23.238 2.732 0.158 3.37 6.74 674 708.2 55.59 44.41 1.74 1.65 3.82
600 24.495 2.778 0.173 3.72 7.44 744 784.94 61.61 38.39 1.79 1.58 3.95
660 25.690 2.820 0.187 4.02 8.04 804 852.38 66.91 33.09 1.83 1.52 4.06
720 26.833 2.857 0.200 4.31 8.62 862 918.42 72.09 27.91 1.86 1.45 4.16
Table 5.38: Summary of Release kinetics data of all nystatin transferosomal suspension
Zero First Hixon- Korsmeyer-

Formulation Higuchi Mechanism of
S.No order order crowel peppas
Code drug release
R2 R2 R2 R2 R2 n
1 FT1 0.862 0.915 0.862 0.738 0.66 0.055 Fickian diffusion
Fickian diffusion
2 FT2 0.88 0.916 0.88 0.811 0.765 0.056
Fickian diffusion
3 FT3 0.886 0.932 0.886 0.81 0.763 0.061
Fickian diffusion
4 FT4 0.99 0.991 0.99 0.934 0.873 0.074
Fickian diffusion
5 FT5 0.974 0.965 0.974 0.841 0.7 0.082
Fickian diffusion
6 FT6 0.981 0.998 0.981 0.911 0.85 0.059
Fickian diffusion
7 FT7 0.999 0.989 0.999 0.981 0.96 0.078
Fickian diffusion
8 FT8 0.998 0.962 0.998 0.96 0.896 0.074
Table 5.39: Elasticity measurements of three optimized vesicular formulations
Volume of
liquid Vesicle Pore
Name of the permeated size size of
vesicular after filter Elasticity,
carrier Formulation in 5min, extrusion, paper,
systems code ml nm (rv) µm (rp) (rv/rp) (rv/rp)2 nm.ml/µm Average ±SD
1.9 241 50 4.82 23.2324 44.1
Niosomes FN3 1.8 248 50 4.96 24.6016 44.3 45.2 1.7
2.1 237 50 4.74 22.4676 47.2
2.7 276 50 5.52 30.4704 82.3
2.5 269 50 5.38 28.9444 72.4
Ethosomes FE6 2.8 274 50 5.48 30.0304 84.1 79.6 6.3
3.3 292 50 5.84 34.1056 112.5
Transferosome 3.2 288 50 5.76 33.1776 106.2
s FT7 3.5 293 50 5.86 34.3396 120.2 113.0 7.0
Table 5.40: Zeta potential of three optimized vesicular formulations
Name of the vesicular carrier systems

Niosomes Ethosomes, Transferosomes,
S.No FN3 FE6 FT7
Zeta potential, mV 24.2 -38.1 -20
Table 5.41: Composition of conventional, niosomal, ethosomal &transferosomal gels
Ingredients Conventional gel Niosomal gel Ethosomal gel Transferosomal gel
Nystatin 0.01%w/w 0.01%w/w 0.01%w/w 0.01%w/w

Carbopol -971 1%w/w 1%w/w 1%w/w 1%w/w
HPMC 0.5% w/w 0.5% w/w 0.5% w/w 0.5% w/w
Propylene glycol 2% 2% 2% 2%
Triethanolamine q.s q.s q.s q.s
Double distilled
100% w/w 100% w/w 100% w/w 100% w/w
water q.s
Table 5.42: In vitro drug release from conventional gel across the dialysis membrane
cumulative
cumulative amt
TIME amt. in amt in amt.
√t logt Abs conc CPR permeated/
(min) t 2ml(µg) 100ml.(µg) release
Unit area
(µg)
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.00
60 7.746 1.778 0.007 0.021 0.042 2.1 2.1 2.10 1.59
120 10.954 2.079 0.007 0.032 0.064 3.2 3.2 3.24 2.46
180 13.416 2.255 0.008 0.037 0.074 3.7 3.8 3.81 2.88
240 15.492 2.380 0.008 0.047 0.094 4.7 4.9 4.88 3.70
300 17.321 2.477 0.008 0.055 0.11 5.5 5.8 5.77 4.37
360 18.974 2.556 0.009 0.06 0.12 6 6.4 6.38 4.84
420 20.494 2.623 0.009 0.062 0.124 6.2 6.7 6.70 5.08
480 21.909 2.681 0.009 0.064 0.128 6.4 7.0 7.03 5.32
540 23.238 2.732 0.009 0.067 0.134 6.7 7.5 7.46 5.65
600 24.495 2.778 0.009 0.067 0.134 6.7 7.6 7.59 5.75
Table 5.43: In vitro drug release from niosomal gel across the dialysis membrane
cumulative cumulative amt

√t logt Abs conc CPR
(min) t 2ml(µg) 100ml.(µg) release permeated/
(µg) Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.00
60 7.746 1.778 0.007 0.012 0.024 1.2 1.2 1.20 0.91
120 10.954 2.079 0.008 0.043 0.086 4.3 4.3 4.32 3.28
180 13.416 2.255 0.011 0.1 0.2 10 10.1 10.11 7.66
240 15.492 2.380 0.012 0.13 0.26 13 13.3 13.31 10.08
300 17.321 2.477 0.016 0.22 0.44 22 22.6 22.57 17.10
360 18.974 2.556 0.020 0.3 0.6 30 31.0 31.01 23.49
420 20.494 2.623 0.024 0.39 0.78 39 40.6 40.61 30.77
480 21.909 2.681 0.025 0.43 0.86 43 45.4 45.39 34.39
540 23.238 2.732 0.026 0.45 0.9 45 48.3 48.25 36.55
600 24.495 2.778 0.027 0.46 0.92 46 50.2 50.15 37.99
Table 5.44: In vitro drug release from ethosomal gel across the dialysis membrane

(µg) Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.0
60 7.746 1.778 0.017 0.25 0.5 25 25.0 25.00 18.9
120 10.954 2.079 0.024 0.41 0.82 41 41.5 41.50 31.4
180 13.416 2.255 0.029 0.51 1.02 51 52.3 52.32 39.6
240 15.492 2.380 0.030 0.53 1.06 53 55.3 55.34 41.9
300 17.321 2.477 0.030 0.53 1.06 53 56.4 56.40 42.7
360 18.974 2.556 0.030 0.54 1.08 54 58.5 58.46 44.3
420 20.494 2.623 0.033 0.59 1.18 59 64.5 64.54 48.9
480 21.909 2.681 0.033 0.59 1.18 59 65.7 65.72 49.8
540 23.238 2.732 0.034 0.63 1.26 63 70.9 70.90 53.7
600 24.495 2.778 0.035 0.64 1.28 64 73.2 73.16 55.4
Table 5.45: In vitro drug release from transferosomal gel across the dialysis membrane

(µg) Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.0
60 7.746 1.778 0.015 0.2 0.4 20 20.0 20.00 15.2
120 10.954 2.079 0.020 0.32 0.64 32 32.4 32.40 24.5
180 13.416 2.255 0.025 0.42 0.84 42 43.0 43.04 32.6
240 15.492 2.380 0.028 0.49 0.98 49 50.9 50.88 38.5
300 17.321 2.477 0.029 0.52 1.04 52 54.9 54.86 41.6
360 18.974 2.556 0.031 0.55 1.1 55 58.9 58.90 44.6
420 20.494 2.623 0.031 0.55 1.1 55 60.0 60.00 45.5
480 21.909 2.681 0.031 0.55 1.1 55 61.1 61.10 46.3
540 23.238 2.732 0.031 0.56 1.12 56 63.2 63.20 47.9
600 24.495 2.778 0.031 0.56 1.12 56 64.3 64.32 48.7
Table 5.46: Ex vivo drug release from conventional gel across the rat skin
cumulative amt
cumulative
TIME amt. in amt in
√t logt Abs conc amt. release CPR
(min) t 2ml(µg) 100ml.(µg) permeated
(µg)
/Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.00
60 7.746 1.778 0.007 0.018 0.036 1.8 1.8 1.80 1.36
120 10.954 2.079 0.007 0.029 0.058 2.9 2.9 2.94 2.22
180 13.416 2.255 0.008 0.037 0.074 3.7 3.8 3.79 2.87
240 15.492 2.380 0.008 0.046 0.092 4.6 4.8 4.77 3.61
300 17.321 2.477 0.008 0.055 0.11 5.5 5.8 5.76 4.36
360 18.974 2.556 0.009 0.062 0.124 6.2 6.6 6.57 4.98
420 20.494 2.623 0.009 0.064 0.128 6.4 6.9 6.89 5.22
480 21.909 2.681 0.009 0.066 0.132 6.6 7.2 7.22 5.47
540 23.238 2.732 0.009 0.067 0.134 6.7 7.5 7.45 5.65
600 24.495 2.778 0.009 0.068 0.136 6.8 7.7 7.69 5.82
Table 5.47: Ex vivo drug release from niosomal gel across the rat skin
cumulative amt
cumulative
TIME amt. in amt in
(µg)
/Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.00
60 7.746 1.778 0.006 0.01 0.02 1 1.0 1.00 0.76
120 10.954 2.079 0.008 0.04 0.08 4 4.0 4.02 3.05
180 13.416 2.255 0.010 0.09 0.18 9 9.1 9.10 6.89
240 15.492 2.380 0.012 0.13 0.26 13 13.3 13.28 10.06
300 17.321 2.477 0.015 0.21 0.42 21 21.5 21.54 16.32
360 18.974 2.556 0.019 0.29 0.58 29 30.0 29.96 22.70
420 20.494 2.623 0.024 0.39 0.78 39 40.5 40.54 30.71
480 21.909 2.681 0.025 0.42 0.84 42 44.3 44.32 33.58
540 23.238 2.732 0.026 0.44 0.88 44 47.2 47.16 35.73
600 24.495 2.778 0.026 0.44 0.88 44 48.0 48.04 36.39
Table 5.48: Ex vivo drug release from ethosomal gel across the rat skin
cumulative amt
cumulative
TIME amt. in amt in
(µg)
/Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.0
60 7.746 1.778 0.017 0.24 0.48 24 24.0 24.00 18.2
120 10.954 2.079 0.025 0.42 0.84 42 42.5 42.48 32.2
180 13.416 2.255 0.028 0.49 0.98 49 50.3 50.32 38.1
240 15.492 2.380 0.029 0.52 1.04 52 54.3 54.30 41.1
300 17.321 2.477 0.029 0.52 1.04 52 55.3 55.34 41.9
360 18.974 2.556 0.030 0.54 1.08 54 58.4 58.38 44.2
420 20.494 2.623 0.032 0.58 1.16 58 63.5 63.46 48.1
480 21.909 2.681 0.032 0.58 1.16 58 64.6 64.62 49.0
540 23.238 2.732 0.034 0.62 1.24 62 69.8 69.78 52.9
600 24.495 2.778 0.034 0.62 1.24 62 71.0 71.02 53.8
Table 5.49: Ex vivo drug release from transferosomal gel across the rat skin
cumulative amt
cumulative
TIME amt. in amt in
(µg)
/Unit area
0 0.000 0.000 0.000 0 0 0 0.0 0.00 0.0
60 7.746 1.778 0.015 0.19 0.38 19 19.0 19.00 14.4
120 10.954 2.079 0.020 0.31 0.62 31 31.4 31.38 23.8
180 13.416 2.255 0.025 0.42 0.84 42 43.0 43.00 32.6
240 15.492 2.380 0.028 0.48 0.96 48 49.8 49.84 37.8
300 17.321 2.477 0.029 0.51 1.02 51 53.8 53.80 40.8
360 18.974 2.556 0.030 0.53 1.06 53 56.8 56.82 43.0
420 20.494 2.623 0.030 0.53 1.06 53 57.9 57.88 43.8
480 21.909 2.681 0.031 0.55 1.1 55 60.9 60.94 46.2
540 23.238 2.732 0.031 0.55 1.1 55 62.0 62.04 47.0
600 24.495 2.778 0.031 0.55 1.1 55 63.1 63.14 47.8
Table 5.50: Nystatin retention from all gels during ex vivo drug release study in and on skin
Concentrations, µg/ml
Area
Conventional gel Niosomal gel Ethosomal gel Transferosomal gel
Drug permeated
4.3 28.6 16.3 21.9
in to the skin layers
Drug permeated in to the
5.8 36.4 53.8 47.8
receptor fluid
drug retained on the skin 89.9 35 29.9 30.3

Table 5.51: Stability studies of optimized niosomal formulation gel (FN3)
S.No Refrigerated temperature Room temperature

Formulation
FN3 Vesicles Vesicle size Vesicles Vesicle
% EE % EE
shape (nm) shape size (nm)
1 Freshly prepared Spherical 281±1.7 72.5±1.9 Spherical 281±1.7 72.5±1.9
2 After 3 month Spherical 282±1.2 71.9±1.6 Spherical 288±1.4 70.2±1.7
3 After 6 month Spherical 285±1.3 71.3±1.2 Spherical 290±2.7 67±2.3
Table 5.52: Stability studies of optimized ethosomal formulation gel (FE6)
Refrigerated temperature Room temperature

Formulation
S.No
FE6 Vesicles Vesicle Vesicles Vesicle
% EE % EE
shape size (nm) shape size (nm)
1 Freshly prepared Spherical 403.3 88.4 Spherical 403.3 88.4
2 After 3 month Spherical 414.5 85.2 Spherical 416.8 84.2
3 After 6 month Spherical 438.1 82.8 Spherical 485.2 71.1
Table 5.53: Stability studies of optimized transferosomal formulation gel (FT7)

Refrigerated temperature Room temperature
Formulation
S.No
FT7 Vesicle Vesicle
Vesicles shape % EE Vesicles shape % EE
size (nm) size (nm)
Freshly Approximately Approximately

1 374.1 70 374.1 70
prepared Spherical shape Spherical shape
After 3 Approximately Approximately

381.6 68.4 391.9 65.7
month Spherical shape Spherical shape
After 6 Approximately Approximately

2 419.3 62.8 446.2 59.4
month Spherical shape Spherical shape
Table 5.54: Analysis of ex vivo drug release study of three optimized
vesicular formulations
Kp,
S.No Nystatin gels Slope Area Jss sq.cm/hr Er
1 Conventional gel 0.948 1.32 0.72 0.007
2 Niosomal gel 2.297 1.32 1.74 0.017 2.4
3 Transferosomal gel 10.71 1.32 8.11 0.081 11.3
4 Ethosomal gel 12.83 1.32 9.72 0.097 13.5
Table 5.55: Calibration curve of the nystatin by HPLC
S.No Concentration, ng/ml Area of the peak

1 0 0
2 300 214265
3 600 428531
4 900 642797
5 1200 857063
6 1500 1071328
7 1800 1285594
8 2100 1499860
Table 5.56: In vivo study of the conventional gel in rabbits
Rabbit -I Rabbit-II Concentration,

Time, Avearge
Hrs Isomer -a Isomer-b Total area Isomer -a Isomer-b Total area area ng/ml
0 0 0 0 0 0 0 0 0
1 7755 5170 12925 7158 4772 11930 12427 17.40
3 9404 6269 15673 8680 5787 14467 15070 21.10
5 9092 6061 15153 8392 5595 13987 14570 20.40
7 9003 6002 15004 8310 5540 13850 14427 20.20
9 8869 5913 14781 8187 5458 13644 14213 19.90
11 8334 5556 13890 7693 5129 12822 13356 18.70
24 6373 4249 10622 5883 3922 9805 10213 14.30
Table 5.57: In vivo study of the niosomal gel in rabbits
Rabbit -I Rabbit-II Avearge Concentration,

Time,
0 0 0 0 0 0 0 0 0
1 39531 26354 65885 36490 24327 60817 63351 88.70
3 194670 129780 324450 179695 119797 299492 311971 436.81
5 196274 130850 327124 181176 120784 301961 314542 440.41
7 149702 99801 249503 138186 92124 230310 239906 335.91
9 148498 98999 247497 137075 91384 228459 237978 333.21
11 53614 35743 89357 49490 32994 82484 85921 120.30
24 25225 16817 42042 23285 15523 38808 40425 56.60
Table 5.58: In vivo study of the ethosomal gel in rabbits
Rabbit -I Rabbit-II Avearge Concentration,

Time,
0 0 0 0 0 0 0 0 0
1 132008 88006 220014 121854 81236 203090 211552 296.21
3 306133 204088 510221 282584 188389 470973 490597 686.92
5 573759 382506 956266 529624 353083 882707 919486 1287.44
7 757466 504977 1262443 699199 466133 1165332 1213887 1699.65
9 783983 522656 1306639 723677 482451 1206128 1256383 1759.15
11 761120 507413 1268534 702573 468382 1170954 1219744 1707.85
24 144665 96444 241109 133537 89025 222562 231836 324.61
Table 5.59: In vivo study of the transferosomal gel in rabbits
Rabbit -I Rabbit-II Concentration,

Time, Avearge
0 0 0 0 0 0 0 0 0
1 87486 58324 145809 80756 53837 134593 140201 196.31
3 221143 147429 368572 204132 136088 340220 354396 496.21
5 483421 322281 805702 446235 297490 743725 774714 1084.73
7 636777 424518 1061296 587795 391863 979658 1020477 1428.84
9 663696 442464 1106160 612643 408428 1021071 1063616 1489.24
11 645468 430312 1075780 595817 397211 993028 1034404 1448.34
24 88377 58918 147295 81579 54386 135965 141630 198.31
Table 5.60: Summary of the nystatin concentrations in blood plasma at
different time intervals (In vivo study)
Concentration of nystatin, ng/ml

Time,Hrs Conventional gel Niosomal gel Transferosomal gel Ethosomal gel
0 0 0 0 0
1 17.4 88.7 196.3 296.2
3 21.1 436.8 496.2 686.9
5 20.4 440.4 1084.7 1287.4
7 20.2 335.9 1428.8 1699.6
9 19.9 332.2 1489.2 1759.1
11 18.7 120.3 1448.3 1707.8
24 14.3 56.6 198.3 324.6
Table 5.61: Bioavailability studies of nystatin from all gels
Formulations Cmax, ng/ml Tmax, Hrs AUC, ngh/ml
Conventional gel 21.9 3 589.21
Niosomal gel 442.8 5 1092.7
Transferosomal gel 1471.6 9 2136.3
Ethosomal gel 1759.1 9 2449.2
Figure 5.1: Absorption spectrum of Nystatin between 200-400nm

Figure 5.2: FTIR graph of Nystatin
FTIR showed the characteristic peak for NH, OH stretch – 3350-3331cm-1, Lactone carbonyl stretch-
1701cm-1, Carboxylate ion –1566-1546cm-1, C-OH stretch-1068cm-1 (Fig.2)
Figure 5.3: Standard curve of nystatin in PBS pH 7.4
Figure 5.4: SEM images of the nystatin loaded niosomes
Figure 5.5: In vitro drug release from niosomal preparations prepared
using span-60
Figure 5.6: In vitro drug release from niosomal preparations prepared

using span-80
Figure 5.7: Release kinetics of niosomal suspension FN1
Figure 5.15: SEM Photographs of ethosomes
Figure 5.16: Effect of ethanol concentration on drug release from
ethosomes
Figure 5.17: Effect of lecithin concentration on drug release from

ethosomes.
Figure 5.18: Effect of cholesterol on drug release from ethosomes
Figure 5.19: Release kinetics of ethosomal formulation FE1
Figure 5.27: SEM photographs of transferosomes
Figure 5.28: In vitro drug release from transferosomal formulations
Figure 5.29: In vitro drug release from transferosomal formulations

Figure 5.30: Release kinetics of transferosomal formulation FT1
Figure 5.38: Zeta potential measurement of nystatin loaded niosomes of
an optimized formulation (FN3)
Figure 5.39: Zeta potential measurement of nystatin loaded ethosomes of
an optimized formulation (FE6)
Figure 5.40: Zeta potential measurement of nystatin loaded
transferosomes of an optimized formulation (FT7)
Figure 5.41: In vitro drug release from all gels across the dialysis
membrane
Figure 5.42: Ex vivo drug release from all gels across the rat skin
Figure 5.43: calculation of flux at steady state and other parameters from ex vivo drug release data
Figure 5.44: Calibration curve of nystatin by HPLC
Figure 5.45: In vivo drug release from all gels

Figure 5.46: HPLC chromatogram of nystatin (300ng/ml)
Figure 5.53: Conventional gel HPLC chromatograms of 0th hour blood sample in
Rabbit -1
Figure 5.54: Conventional gel HPLC chromatograms of 0 th hour blood sample in Rabbit -2
Figure 5.55: Conventional gel HPLC chromatograms of 1 st hour blood sample in Rabbit -1
Figure 5.56: Conventional gel HPLC chromatograms of 1 st hour blood sample in Rabbit -2
Figure 5.57: Conventional gel HPLC chromatograms of 3 rd hour blood sample in Rabbit -1
Figure 5.58: Conventional gel HPLC chromatograms of 3 rd hour blood sample in Rabbit -2
Figure 5.69: Niosomal gel HPLC chromatogram of nystatin at 0 th hr blood sample in rabbit - 1
Figure 5.71: Niosomal gel HPLC chromatogram of nystatin at 1 st hr blood sample in rabbit - 1
Figure 5.72: Niosomal gel HPLC chromatogram of nystatin at 1 st hr blood sample in rabbit - 2
Figure 5.73: Niosomal gel HPLC chromatogram of nystatin at 3 rd hr blood sample in rabbit - 1
Figure 5.74: Niosomal gel HPLC chromatogram of nystatin at 3 rd hr blood sample in rabbit - 2
Figure 5.85: Ethosomal gel HPLC chromatogram of nystatin at 0 th hr blood sample in rabbit - 1
Figure 5.87: Ethosomal gel HPLC chromatogram of nystatin at 1 st hr blood sample in rabbit - 1
Figure 5.88: Ethosomal gel HPLC chromatogram of nystatin at 1 st hr blood sample in rabbit - 2
Figure 5.89: Ethosomal gel HPLC chromatogram of nystatin at 3 rd hr blood sample in rabbit - 1
Figure 5.90: Ethosomal gel HPLC chromatogram of nystatin at 3 rd hr blood sample in rabbit - 2
Figure 5.101: Transferosomal gel HPLC chromatogram of nystatin at 0 th hr blood sample in rabbit - 1
Figure 5.103: Transferosomal gel HPLC chromatogram of nystatin at 1 st hr blood sample in rabbit - 1
Figure 5.104: Transferosomal gel HPLC chromatogram of nystatin at 1 st hr blood sample in rabbit - 2
Figure 5.105: Transferosomal gel HPLC chromatogram of nystatin at 3 rd hr blood sample in rabbit - 1
Figure 5.106: Transferosomal gel HPLC chromatogram of nystatin at 3 rd hr blood sample in rabbit - 2
Chapter - 6
DISCUSSION ON RESULTS
Chapter - 6: DISCUSSION ON RESULTS
Table of contents

6.1 Morphology of vesicles (size, shape and texture)
6.2 Entrapment efficiency
6.3 In vitro drug release
6.4 Release kinetics
6.5 Zeta potential
6.6 Elasticity measurement
6.7 In vitro and ex vivo drug release
6.8 Skin retention study
6.9 Stability study
6.10 In vivo study
6. DISCUSSION ON RESULTS
6.1. Morphology of vesicles (size, shape and texture)
The shape and size of the vesicles present in all formulations was
determined by using optical microscope. All vesicles appeared spherical shape.
But, transfersomes appeared not spherical as much as niosomes and
ethosomes. The surface of the vesicles was determined by SEM analysis.
Niosomes and transferosomes appeared with rough surface and ethosomes
appeared with smooth surface.
The average size of niosomes was found in the range of 278±1.4 to
431±1.2nm. The size of vesicles was decreased with increase in the
concentration of surfactant. This may due to decrease in surface tension with
increase in the concentration of surfactant.
The average size of ethosomes present in all formulations was found in
the range of 291.2 – 463.2nm. The size of the ethosomes was inversely
proportional to the concentration of ethanol. Vesicles size was decreased with
an increase in the concentration of ethanol from 15- 60%. This is may be due
to the modification in net charge of the system by the ethanol and some degree
of stearic stabilization.
The average size of the transferosomes present in all formulations was
found in the range of 266.7 – 374.1nm. The size of the vesicles in
transferosomal formulations prepared using span – 60 is less than the vesicles
present in transferosomal formulations prepared using span – 80. This is

because the size of the vesicles depends on the liposphilicty of the edge
activators. i.e. the size of the vesicles is influencing by their HLB values. The
particle size is inversely proportional to the HLB value of the surfactant. The
reason behind to this concept is that there is increase in surface free energy
with increasing the lipophilicity of surfactants. The surface free energy will
increase due to the interaction of the edge activators with the lipid head groups
present in the membrane which leads increase in packing density of the layer.
This increased surface free energy causes fusion between lipid bilayers and
also increase in size of the vesicles.
The size of the vesicles deceased with increase in the concentration of the
surfactant which may be due to the decrease in surface tension with increase
in concentration of surfactant. Among all formulations the vesicles size was
found asfollows Ethosomes > Transferosomes > Niosomes
6.2. Entrapment efficiency
Entrapment efficiency in niosomal formulation was observed that
the percentage entrapment efficiency increased with increase in surfactant
concentration. Entrapment efficiency is more in case of span-60 niosomes
rather than the niosomes prepared using span-80. This may be due to that the
Span-80 has the lowest transition temperature (Tc = -12ºC) and span-60 has
high transition temperature (Tc =53ºC).
In ethosomal formulations the Entrapment efficiency of Nystatin was
increased with an increase in ethanol concentration up to 45% and on further
increasing the concentration of ethanol, entrapment efficiency was decreased.

Increased entrapment of Nystatin with an increase in concentration of ethanol
owing to increase in fluidity of membranes and increased solubility of Nystatin
in ethanol. On increasing the concentration of ethanol further, the entrapment
efficiency was decreased. This may due to the leakage of the drug from bilayer
of the vesicles.
EE was also increased with an increase in the concentration of lipid to
certain extent. But after specific concentration, the percent EE was decreased.
This may be due to the saturation effect on increase in solubility of Nystatin in
lipid.
Formulations prepared with span – 80 have shown more entrapment
efficiency than the formulations prepared using span – 60. The influential
factor on the entrapment efficiency of nystatin in the transferosomes may be
the lipophilicity of the edge activators (span – 60 and span – 80).
Span – 80 is more lipophilic than the span – 60 than the span – 80. The
HLB values also exhibiting the same that the span – 60 (HLB value: 4.7) is less
liposphilic than the span – 80 (HLB value: 4.3). As nystatin is lipophilic in
nature, it is more soluble in the span – 80 than the span – 60. Hence the
nystatin has entrapped more in the lipid bilayers of the span – 80 than the
lipid bilayers of the span – 60 in transferosomes.
All the transferosomal formulations prepared using span – 80 and span –
60 showed increased entrapment efficiency with increased concentration of
edge activators, initially. Later on further increasing the concentration of edge
activators leads to decrease in the entrapment efficiency. This is may be due to

that, at initial the edge activators (monomers) may be associated with lipid
bilayers of the transferosomes and on increasing the concentration of edge
activators the association of monomers with lipid bilayers may increased which
increases the partitioning of the drug and increases in the entrapment
efficiency. On further increasing the concentration of the edge activators, the
association of edge activators with lipid bilayers may be saturated and some
amount of edge activators left in bulk solution. The unassociated monomers
might begin formation of micelles in lipid bilayers of the transferosomes which
results in the formation of pores in transferosomes and finally bilayers
conversion will takes place to mixed micelles. These mixed micelles are not able
to accommodate more quantity of the drug and are not capable to penetrate the
deeper layers of the skin because of their structural features.
6.3. In vitro drug release
In vitro diffusion studies of all formulations were carried out in fabricated
diffusion cell. The drug release from niosomes decreases with increase in the
concentration of surfactant and also from the data we shall conclude that
niosomal formulations prepared using Span 60 yielded a lower rate of drug
release compared to Span 80 niosomes. This can be explained by the fact that
niosomes exhibit an alkyl chain length-dependent release. The higher the chain
length, the lower the release rate. The nystatin release from FN4 formulation is
highly sustained than that of other niosomal preparations.
In ethosomal formulations, the release of the drug from vesicles was
increased as the concentration of ethanol increases. This may be due to

increased fluidity of the bilayers of the vesicles with increased concentration of
ethanol. However drug release from vesicles was decreased as the
concentration of lecithin increased above 300mg, due to decrease in the bilayer
malleability and deformability and increased rigidity of vesicles. Effect of
incorporation of cholesterol on drug release was also studied and was found
that decreased drug release with addition of cholesterol. This may be due to
the increased rigidity of the vesicle bilayer.
In vitro drug release from all transferosomal formulations showed
increased drug release with increase concentration of edge activators at lower
concentrations. On further increase in the concentration of edge activators the
drug release was decreased. This was observed in all formulations prepared
using both edge activators. This may be due to fact that the transferosomes
lipid bilayers are more ordered and less leaky at low concentration of edge
activators which results in less drug release. Whereas on increasing the
concentration of edge activators slowly, the drug release has increased. This
may be due to the increased deformability of the bilayers. On further increasing
the concentration of edge activators, the vesicle may lose their structure and
also may be due to the formation of mixed micelles which results in decrease in
drug release.
6.4. Release kinetics
All ethosomal, niosomal and transferosomal formulations in vitro drug
release data fitted in various release kinetic models and they showed that all
formulations following zero order drug release. The r 2 values of higuchi and
hixon crowel models showed that drug release from all formulations following
diffusion rather than the dissolution. This was further confirmed by fitting the
in vitro drug release data in Korsmeyer-Peppas model. The r 2 values of
Korsmeyer-Peppas model also showed the same. The n values of Korsmeyer-
Peppas model (n>0.5) showed that niosomes and transferosomal formulations
following fickian diffusion.
But the n-value in ethosomal formulation was found between 0.48-0.95
except FT4 formulation (0.37). this indicates that the ethosomal formulations
following non-fickian diffusion and zero order release.
Based on the entrapment efficiency, in vitro drug release and release
mechanism, one optimized formulation i.e. FN3, FE6 and FT7 from all
niosomal, ethosomal and transferosomal suspension was selected respectively.
The selected optimized suspensions were subjected to zeta potential and
elasticity measurements.
6.5. Zeta potential
The selected optimized formulations FN3, FE6 and FT7 was shown 24.2,
-38.1 and -20.0 mV respectively which substantiating that the ethosomal
formulations are more stable than other two vesicles. i.e. niosomes and
transferosomes. The stability patterns of all the vesicles are as follows based on
the zeta potential.
Ethosomes > Niosomes > Transferosomes
6.6. Elasticity measurement

The elasticity of the lipid/surfactant bilayers of the niosomes, ethosomes
and transferosomes present in three optimized vesicular suspensions was
measured and was found to be 45.2±1.7, 79.6±6.9, 113.0±7.0 nm.ml/µm
respectively. The results stated that the transferosomes more elastic than
others two vesicles followed by ethosomes. Least elasticity was exhibited by the
niosomes.
6.7. In vitro and ex vivo drug release
The nystatin release from skin was lesser than the dialysis membrane
which may be due to the complexicity of skin membrane. But, there is a good
correlation between the in vitro and ex vivo release of nystatin from all gels.
The ex vivo drug release data was analyzed for transdermal flux,
permeation co-efficient and enhancement ratio. The transdermal flux at steady
state for transferosomal, ethosomal, niosomal and conventional gel was found
to be 8.11, 9.72, 1.74 and 0.72 Jss, μg/cm2/min respectively. The permeation
co-efficient of transferosomal, ethosomal, niosomal and conventional gel was
found to be 0.081, 0.097, 0.017 and 0.007 sq.cm/hr respectively. The extent of
drug permeation from vesicular gels was compared with the drug permeation
from conventional gel was measured by calculating the enhancement ratio. The
enhancement ratio of niosomal, transferosomal and ethosomal gels was found
to be 2.4, 11.3 and 13.5 respectively. The effectiveness of the vesicles based on
the above data concluded as follows for administration of drugs across the
skin.
Ethosomal gel > Transferosomal gel > Niosomal gel > Conventional gel
6.8. Skin retention study
The skin retention study revealed that more amount of drug retained on
the skin in case of conventional gel and highest amount of nystatin permeated
from ethosomal gel. In ethosomal gel, less amount of drug retained on the skin
as well as within the skin. This may be due to the optimized flexibility of the
vesicle lipid bilayers. In case of transferosomes less quantity of the nystatin
retained on the skin and less quantity of the drug permeated into the systemic
circulation when compared with ethosomes but more amount of drug retained
within the skin in case of transferosomes than the ethosomes. This may be due
to the fact that the excessive flexibility of transferosomes. Due to high flexibility
of the lipid bilayers of transferosomes, they penetrated easily into the skin than
the ethosomes and may undergo rapture within the skin while penetrating into
the deeper layers of the skin. This may be the reason for accumulating more
quantity of the nystatin with in the skin in transferosomes than the ethosomes.
This was also confirmed in elasticity measurements of the vesicles. In niosomal
gel, neither nystatin permeated into the systemic circulation nor into the skin.
But, when compared with conventional gel, niosomal gel permeated more
quantity of the nystatin into the systemic circulation.The study revealed that
the extent of drug release in to the systemic circulation is as follows
Ethosomal gel > Transferosomal gel > Niosomal gel > Conventional gel
6.9. Stability study
All prepared gels were subject stability studies at room and refrigerated
temperatures for 6 months. The study was shown that there is no change in
the vesicles shape in all gels after 3 rd and 6th months also at both temperatures.
The change in the size and entrapment efficiency of the vesicles in all gels was
negligible after 6th month when compared with initial size and entrapment
efficiency at refrigerated temperature. But the variation in the size and
entrapment efficiency of vesicles in all gels at room temperature was more
when compared with the change that has been taken place at refrigerated
temperature.
If on comparison of the change that has taken place in size and
entrapment efficiency of the vesicles, within the gels, the change was as
follows.
Transferosomes > Ethosomes > Niosomes
The reason may be for this is their bilayer flexibility. The study was
shown that the stability is inversely proportional to the flexibility of the
bilayers.
6.10. In vivo study
The in vivo study and HPLC analysis revealed that the ethosomes are
prominent vesicular carrier system for enhanced transdermal delivery of
nystatin as it has shown highest C max (1759ng.ml) and AUC 2449.2 ngh/ml
than other gels. The study was shown that the in vivo performance of all gels
was as follows.
Ethosomal gel > Transferosomal gel > Niosomal gel > Conventional gel.
Chapter - 7
SUMMARY, CONCLUSION AND
RECOMMENDATIONS
7. SUMMARY, CONCLUSION AND RECOMMENDATIONS
The present research work aimed to select the best vesicular carrier
system among niosomes, ethosomes and transferosomes (primary objective) for
administration of drugs across the skin to produce systemic effect. Nystatin, a
polyene antifungal agent, was used as model drug in the present study. The
secondary objective of the study is enhancement of bioavailability of the
nystatin and to decrease the adverse effects associated with higher doses of the
same by administering through skin.
Niosomal and transferosomal suspensions of nystatin prepared by using
thin film hydration Technique whereas ethosomal suspensions of nystatin was
prepared method reported by Touitou et al. Totally 24 vesicular suspensions
(eight formulations from each type of vesicular systems) were prepared.
Niosomal suspensions were prepared using two different non-ionic
surfactants i.e span-80 and span-60. Each surfactant used at four different
concentrations (eight niosomal suspensions) was prepared. Similarly, eight
transferosomal and eight ethosomal suspensions were prepared.
Transferosomal suspensions were prepared using two different edge activators
i.e. span-60 and span-80 at four different concentrations whereas in
preparation of ethosomal suspensions ethanol and lecithin concentrations has
been changed for preparing the same (each at four different concentrations).
The prepared niosomal, ethosomal and transferosomal suspensions (24
formulations) evaluated for different parameters like vesicles morphology (size,

shape and surface texture), entrapment efficiency and in vitro drug release. The
drug release data fitted in different mathematical models such as Zero order,
First order, Higuchi, Hixon-crowel, Korsmeyer-peppas to find out the order and
mechanism of drug release from all formulations.
Based on the size, entrapment efficiency and invitro drug release results,
one optimized vesicular suspension from each type of vesicular formulation
was selected.
Elasticity and surface charge (Zeta potential) of the vesicles present in
three optimized vesicular suspensions was studied.
After performing the zetapotential and elasticity studies, using the
optimized niosomal, ethosomal and transferosomal suspensions niosomal,
ethosomal and transfersosmal gels having concentration of 0.01%w/w was
prepared respectively. Along with three vesicular nystatin gels, conventional
nystatin gel having same concentration was also prepared with pure nystatin
sample.
The prepared gels were characterized for in vitro and ex vivo drug release,
skin retention, stability studies. Finally in vivo studies also carried out to find
out the systemic availability of nystatin from individual gels in rabbits.
In vitro, ex vivo drug release data showed a good correlation between
each other. The study revealed that ethosomes are effective for permeation of
nystatin across the dialysis membrane and skin than other two vesicles.
Transdermal flux, permeation coefficient and enhancement ratio was
calculated from ex vivo drug release data of all three vesicular carrier gels. The
data showed higher efficiency to the ethosomes than other two vesicular gels.
Skin retention study was performed to find out the quantity of nystatin that
reached systemic circulation and retained in and on the skin after ex vivo drug
release study. The skin retention study also showed that ethosomes are
efficient than other two vesicular carriers i.e. transferosomes and niosomes.
In vivo study was also carried out in rabbits to find out the efficiency of
all vesicular gels and conventional gel in production of systemic effect. Highest
Cmax and AUC was exhibited by the ethosomes than other two vesicular and
conventional gels which stating that ethosomes are effective carriers to produce
systemic effect than other two carriers.
Our findings contribute to the evidence base for enhancing the
permeability of nystatin across the skin and to produce the systemic anti
fungal activity. (Secondary objective)
The present study demonstrates the utility of ethosomes as a tool for
administration of drugs across the skin to produce the systemic effect and
further studies such as clinical trials are required to conclude the same.
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62
INDEX
INDEX
A Epidermis
Application of Pressure Ethosomes
Aquasomes Eutectic systems
B Ex vivo drug release
Bilosomes F
Bingam bodies First order
C H
Chemical penetration enhancers Higuchi
Chemical potential adjustment Hixon-crowel
Cholesterol HPLC
D Hydration
Dermis I
Dialysis membrane In vitro drug release
Dialysis method In vivo study
Dialysis method Intercellular route
E Ion pairs and complex coacervates
Elasticity of the vesicles Iontophoresis
Electrophoresis K
Enhancement ratio Keratinocytes
Entrapment efficiency Korsmeyer-Peppas
Enzymosomes L
Langerhans cells
Laser radiation and photochemical S
waves Skin
Liposomes Skin abrasion
M Skin puncture and perforation
Magnetophoresis Skin stretching
Melanocytes Span-60
Merkel cells Span-80
Microneedle based devices Sphingosomes
Morphology Stability studies
N Stratum basale
Needleless injection Stratum corneum
Niosomes Stratum granulosum
Nystatin Stratum lucidem
P Stratum spinosum
Permeability co-efficient. Suction ablation
Pharmaceutical carrier systems T
Pharmacosomes Temperature (“thermophoresis”)
Phosphate buffer saline pH 7.4 Thin film hydration technique
Phospolipon-90H Topical drug delivery systems
R Transcellular route
Radio frequency Transdermal drug delivery systems
Routes of administration Transdermal flux.

Transferosomes V
Transfollicular route Vesicular Carrier Systems
U Virosomes
Ultra centrifugation method Z
Ultrasound ( Phonophoresis, Zero order
Sonophoresis) Zeta potential
UV-Visible spectrophotometer
LIST OF PUBLICATIONS &
CONFERENCES
LIST OF PUBLICATIONS & CONFERENCES

1. K. Srikanth, V. Rama Mohan Gupta, N. Devanna. Formulation and Evaluation
of Nystatin loaded Niosomes. International Journal of Pharmaceutical Sciences
and Research, 4(5), 2013, 2015 – 2020. (ISSN No: 0975 – 8232).
2. K. Srikanth, V. Rama Mohan Gupta, N. Devanna. Ethanolic Vesicles of
Nystatin for enhanced Transdermal Drug Delivery: Invitro & Exvivo Evaluation.
Indo-American Journal of Pharmaceutical Research, 3(9), 2013, 7316 – 7324.
(ISSN No: 2231 - 6876).
3. K. Srikanth, V. Rama Mohan Gupta, Sundaraj Manvi, N. Devanna. Particulate
Carrier Systems: A Review. International Research Journal of Pharmacy. 3(11),
2012, 22 -26. (ISSN No: 2230 - 8407).
4. Presented a Poster entitled “Formulation and Evaluation of Nystatin loaded
Niosomes” in an International Conference ‘Indo-American Pharmaceutical
Regulatory Symposium’ held at JNTUH, Hyderabad in 2012.
5. Gave an Oral Presentation entitled “Formulation and Evaluation of Nystatin
loaded Transferosomes” in AICTE sponsored a two days National Level
Conference “INNOVATE – 13” at Pulla Reddy Institute of Pharmacy, Medak in
2013.

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SELECTION OF THE BEST VESICULAR CARRIER SYSTEM

FOR ENHANCED TRANSDERMAL DRUG DELIVERY: A

A Thesis submitted to the

Under the guidance of

Dr. V. RAMA MOHAN GUPTA & Dr. DEVANNA NAYAKANTI

RESEARCH AND DEVELOPMENT

I declare that the thesis entitled “SELECTION OF THE BEST VESICULAR

CARRIER SYSTEM FOR ENHANCED TRANSDERMAL DRUG DELIVERY: A

COMPARATIVE STUDY” has been prepared by me under the guidance of Dr. V.

RAMA MOHAN GUPTA, Professor & Principal, Pulla Reddy Institute of

Pharmacy, Medak, Hyderabad and Dr. N. Devanna, Director, JNTUA-OTRI,

degree or fellowship previously.

Dept of Pharmaceutical sciences,

Jawaharlal Nehru Technological University,Anantapur,

We certify that K. Srikanth has preparaed his thesis entitled “SELECTION

OF THE BEST VESICULAR CARRIER SYSTEM FOR ENHANCED TRANSDERMAL

Jawaharlal Nehru Technological University Anatapur, Ananthapuramu under our

Institute of Pharmacy, Medak, Hyderabad, A.P, (INDIA.).

Dr. N. Devanna Dr. V. Rama Mohan Gupta

Director, Professor & Principal

JNTUA-OTRI, Pulla Reddy Institute ofPharmacy,

Ananthapuramu Medak, Hyderabad

dust particle under his feet forever.

At the outset, I would like to thank my supervisor Dr. V. Rama Mohan

confidence and trust on me. It is my privilege to have his encouragement and

galvanizing my spirit in accomplishing this project successfully. His valuable and

my work. The sincerity, righteousness and virtuousness which he has inculcated in

identify my own strength and drawbacks and particularly boosted my self-

indebted to him for the unstinted support exleished to me during my Ph.D.

I will forever be thankful to my co-guide Dr. N. Devanna, Director, OTRI,

hand is a source in crossing the barriers associated in completion of synopsis,

my research work at Pulla Reddy Institute of Pharmacy which buttressed me to

perform my research work comfortably.

I oblige my deep sense of gratitude to Mr. V.S. Prakash Rao,

Administrative Officer, Pulla Reddy Institute of Pharmacy, Medak for his

supportive suggestions and motivations from the commencement of my project

valuable suggestions to become ideal person.

I express my sincere gratitude to my colleagues Mr. Ashwaq Hussain, Mr.

AG, Switzerland for providing gift samples of drugs and polymers.

I extend my sincere thanks to all the supporting staff members of my

involvement and friendly attitude during my work.

their sincere encouragement and inspiration throughout my research work and

entire research period.

knowingly and unknowingly helped me in the successful completion of this project.

system among niosomes, ethosomes and transferosomes (primary objective) for

administration of drugs across the skin to produce systemic effect. Nystatin, a

secondary objective of the study is enhancement of bioavailability of the

same by administering through skin.

Niosomal and transferosomal suspensions of nystatin prepared by using

thin film hydration Technique whereas ethosomal suspensions of nystatin was

prepared method reported by Touitou et al. Totally 24 vesicular suspensions

(eight formulations from each type of vesicular systems) were prepared.

Niosomal suspensions were prepared using two different non-ionic

concentrations (eight niosomal suspensions) was prepared. Similarly, eight

transferosomal and eight ethosomal suspensions were prepared.

Transferosomal suspensions were prepared using two different edge activators

i.e. span-60 and span-80 at four different concentrations whereas in

preparation of ethosomal suspensions ethanol and lecithin concentrations has

The prepared niosomal, ethosomal and transferosomal suspensions (24

formulations) evaluated for different parameters like vesicles morphology (size,

mechanism of drug release from all formulations.

one optimized vesicular suspension from each type of vesicular formulation

Elasticity and surface charge (Zeta potential) of the vesicles present in

three optimized vesicular suspensions was studied.