FORMULATION AND EVALUATION OF LIPOSOMES CONTAINING

ARTEMISININ
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,

BANGALORE.
BY
P. ASHWINI PAIB.Pharma.

Under The Guidance of

Dr. A. R. SHABARAYAM.Pharm.,MBA,Ph.D.

DEPARTMENT OF PHARMACEUTICS.
SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE 574143
2013-2015

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCESBANGALORE,

KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.

Name of the Candidate and

Address:

MS. P.ASHWINI PAI

1stYEAR M.PHARM,
DEPT. OF PHARMACEUTICS,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE-574143.
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, FARANGIPETE POST,
MANGALORE-574143.

2.

Name of the Institution:

3.

Course of Study and Subject:

MASTER OF PHARMACY
(PHARMACEUTICS)

4.

Date of Admission:

25-07-2013

5.

Title of the Project:

FORMULATION AND EVALUATION OF LIPOSOMES CONTAINING
ARTEMISININ

6.

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study:
Liposomes are microscopic lipid vesicles containing a polar hydrophilic group and a
non polar hydrophobic group, amphiphilic in nature. When the lipids are dispersed in
water, there is orientation of the amphiphile molecules into ordered structures in which
the hydrophobic regions are brought into proximity with each other while the polar
groups are juxtaposed between them and the water continuum. The monolayers formed
are organized back to back to form a bi-layered sheeth or lamella in which hydrophobic
regions are sandwiched between the polar groups and segregated from bulk water. The
lipids exhibit polymorphism and exist as hexagonal, cubic, and reverse hexagonal1.
Liposomes can be prepared by passive loading techniques like thin film hydration, ether
injection, sonication, membrane extrusion, reverse phase evaporation. Different types of
liposomes are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), large
unilamellar vesicles (LUVs). Polar drugs can be incorporated in the aqueous core and
non polar drugs can be incorporated in the lipid bilayer of the liposomes. The prepared
liposomes can be characterized by stability studies, trapped volume, transitional
behaviour, freeze fracture electron microscopy, scanning electron microscopy2.
Parasitic diseases are of immense global significance as around 30% of worlds
population experiences parasitic infections. Amongst various parasitic infections,
malaria is the most life threatening disease and accounts for 1 million to 2 million
deaths round the globe every year. The tropical countries such as India are more prone
to the malaria and around 2million cases are reported annually. In humans, malaria is
caused by four distinct species of parasites: Plasmodium vivax, Plasmodium falciparum,
Plasmodium malariae and Plasmodium ovale. Amongst these, the most severe malaria
is caused by blood-borne Apicomplexan parasite P. falciparum which is responsible for
almost all malaria related deaths. Existing treatments for malaria include a limited
number of clinically effective antimalarial agents. However, the clinical utility of the
most of the antimalarial agents is hampered due to problems such as poor oral
bioavailability and the emergence of drug-resistant parasite strains. Paradoxically, due to
lack of economic incentives, there are not many initiatives for the development of new
anti-malarial agents. On the other hand, despite numerous efforts and investigations,

there is no effective and promising malaria vaccine on the horizon till date. This
scenario has enforced the smart and effective utilization of the current antimalarial
agents with the help of novel drug delivery systems3.
Currently, the treatment of choice for uncomplicated and severe paludism is based on
administration of artemisinin-type compounds used alone or in association with other
drugs. Artemisinin, or qinghaosu, is a lactone sesquiterpene extracted from sweet
wormwood, or Artemisia annua. Active against all species of Plasmodium, artemisininis
a powerful blood schizontocide of rapid action compared with other antimalarials. It is
able to eliminate all asexual stages of Plasmodium spp. and to kill immature and
developing gametocytes, the sexual stages that are essential for transmission. At the
present time, because of its low oral bioavailability, artemisinin is replaced in the
therapeutic arsenal by its derivatives, including dihydroartemisinin, artesunate
(hemisuccinate of artemisinin), artemether (methylether of artemisinin) and arteether
(AE, ethylether of artemisinin, also called artemotil). The exact mechanism of action of
artemisinin and its derivatives is still a matter of debate. Generation of free radicals
leading to alkylation of proteins and inhibition of the parasite encoded sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) have been reported in the literature. In
humans, the daily dose of artemisinin derivatives is 23 mg of artemisinin derivatives
per kilogram of body weight. Artesunate and artemether belong to the Model List of
Essential Drugs retained by the World Health Organization. In-vitro, AE has greater
antimalarial activity than artemether and articulate. However, because of its high
lipophilicity (logP = 3.84), poor water solubility (17g/ml) and low stability in gastric
medium, AE is only available as oily formulations for intramuscular injection4.
Artemether is a potent schizonticide drug is practically insoluble in water, belongs to
BCS class II and however, this drug holds some important shortcomings, including (i)
short half-life usually between 3 h and 5 h; (ii) poor aqueous solubility, and thus low
oral bioavailability (40%); (iii) risk of degradation in acidic conditions; and (iv)
associated risk of toxicity. The marketed dosage forms available for Artemether are
tablet, powder filled capsule and intramuscular injection. The oral formulations of
Artemether are rapidly but incompletely absorbed, limiting its use in malaria, whereas
parenteral oily formulation leads to pain on injection and poor patient compliance5.

In order to overcome the above mentioned disadvantages, artemisinin loaded liposomes

have been proposed for present study. The aim of the present research is to prepare and
evaluate the liposomes loaded with artemisinin derivatives like artemether, artesunate
etc.
6.2 Review of literature Bhambere Deepak Sunil, Doijad Rajendra, Deshmukh Narendra, Manvi F.V. and
Kankate R6 has designed and characterized the liposomal drug delivery for zidovudin.
Liposomes have been loaded by zidovudine (AZT) as a model drug using thin film
hydration technique for targeted delivery of this drug. Four different formulations were
prepared with various concentrations of egg phosphatidylcholine (EPC) and
dipalmitoylphosphatidylcholine (DPPC). A series of tests have been carried out to
characterize the carrier vesicles in vitro, including loading parameters, drug release
kinetics, particle size distribution, SEM analysis. Liposomes having acceptable loading
parameters, released their drug content according to zero-order kinetics were selected
for in vivo tissue distribution study. The AZT-loaded liposomes showed preferential
drug targeting to liver followed by lungs, kidney and spleen. From the study they
concluded, AZT-loaded liposomes seem to be a promising delivery system for targeting
the drug to reticuloendothelial system (RES).
Ajay Kumar, Shital Badde, Ravindra Kamble, Varsh B. Pokharkar 7, has developed
and characterized liposomal drug delivery system for nimesulide. They prepared
Nimesulide loaded liposome which was prepared by two methods ethanol injection
method and rotary evaporation technique. The average particle size, percent drug
entrapment, drug release was found to be 270703 m, 4958 %, and 65.71 % in case of
ethanol injection method, while in case of rotary evaporator it was found to be 112
m, 6986% and 76.97 % respectively. The Zeta potential for nimesulide loaded
liposomes of ethanol injection method and rotary evaporator method were found to be
21.23 and 26.78 mV respectively.
N

Manjunatha,

G.

Prakash

Naidu,

Vasanthi

Sutrave,

Kalpesh

Patel,

MKSamantha8, prepared and evaluated liposomes of an antiviral drug by reverse phase

evaporation method using various ratios of phosphatidyl choline with cholesterol and

Cephalin (phosphatidyl ethanolamine) with cholesterol. Based on evaluation of

entrapment efficiency, the best formulations were subjected to physicochemical
studies .i.e., photo microscopy, invitro drug release and stability studies. The percentage
entrapped drug in soya lecithin liposomes of batch F17 and F21 was 60.51 and 59.69and
cephalin liposomes of F41 and F45 was 58.23 and 57.38 respectively. The formulations
F17, F21, F41 and F45 sustained the release and at the end of 12 hr the % drug release
was 73.89, 79.48, 76.78 and 79.77 % respectively. Short-term stability studies were
carried out for the selected formulations (F17, F21, F41 and F45) for a period of two
months at 4C 252C/ 605% RH and 302C/ 655% RH. The liposomes stored at
4C were found to be stable for duration of two months compared to other storage
conditions. Hence it was concluded that four formulations can be used for controlled
release of acyclovir
Rakesh P. Patel, Hardik Patel and Ashok H. Baria 9 has formulated and evaluated the
liposomes of ketaconazole.

Ketoconazole liposomes were prepared by thin film

hydration technique using soya lecithin, cholesterol and drug in different weight ratios.
The prepared liposomes were characterized for size, shape, entrapment efficiency, invitro drug release (by franz diffusion cell) and physical stability. The studies
demonstrated successful preparation of Ketoconazole liposomes and effect of soya
lecithin: cholesterol weight ratio on entrapment efficiency and on drug release.
Medha Joshi, Sulabha Pathakb, Shobhona Sharmab, Vandana Patravale 3has
formulated solid microemulsion preconcentrate (NanOsorb) and it significantly
improved the therapeutic efficacy of artemether demonstrating the utility of the novel
drug delivery strategies for antimalarial agents. In-vivo studies clearly demonstrated that
NanOsorb-ARM has significantly higher antimalarial activity as compared to the ARM
solution and marketed formulation (Larither). The acute and subacute toxicity studies
successfully established the safety of the NanOsorb in the animals.
NishaRaina, Amith K. Goyal, Pillai C. R., Goutham Rath

10

has developed and

characterized artemether loaded solid lipid nanoparticles. SLN was prepared using hot
melt method. The SLNs were evaluated for particle size, zeta potential, shape, zeta
potential, poly dispersity index (P.D.I), entrapment efficiency, in vitro drug release and

in vivo hemolysis. The antimalarial activity of ARM loaded SLNs and marketed
formulation was evaluated in Plasmodium berghei infected mice.SEM revealed the
optimized formulation was found to be smooth, spherical and non aggregated. Zeta
potential and P.D.I of optimized formulation was found to be -31.45mV and 0.025
respectively. SLN containing artemether showed lesser hemolysis but better antimalarial
activity comparable to the free drug and marketed formulation in preventive stage and
concluded that Artemether loaded solid lipid nanoparticles prepared by hot melt method
is a significant delivery system for targeting liver cells for treatment of malarial disease.
Sangeeta H. Rivankar11 has studied on the various excipients to be selected for NDDS
parenteral formulations. Excipients play a critical role in the creation of medicines,
helping to preserve the efficacy, safety and stability of active pharmaceutical ingredients
(APIs) and ensuring that they deliver their promised benefits to patients. A carefull
choice of excipients is crucial for development of effective and innovative
pharmaceuticals. Lipid based and polymer matrix based sustained release formulations
have gained significant interest in recent years for the parenterals delivery of active
ingredients because of their versatility. Designing polymer based sustained release
dosage formulation posses a number of challenges to formulators that include but are
not limited to excipient selection an understanding of the drug release mechanism,
manufacturing process considerations, and an understanding of the API characteristics
that may contribute to physical and chemical challenges.
Mansoori M.A, Jawade S., Agrawal S., Khan M.I. 12 has developed ketoprofen
(NSAID) liposomal gel for better anti-inflammatory activity and reduced adverse
effects. Liposome carriers, well known for their potential and topical drug delivery was
chosen to help transport ketoprofen molecule in skin layers. Ketoprofen was
encapsulated in liposomes for topical application. Ketoprofen liposomes were prepared
by thin film hydration technique using soya lecithin, cholesterol and drug in different
weight ratios. Carbopol 934 was used as a vehicle for topical drug delivery in the
concentration range 1%. In evaluation study, the effect of varying concentrations of
lipid on the properties of liposomes such as encapsulation efficiency, particle size,
invitro drug release and physical stability were studied. Phase transition study was
carried out to confirm the complete interaction of ketoprofen with bilayer structure of
liposome. Results of analysis revealed that the size of liposomes and entrapment
efficiency were dependent on the lipid concentration. Moreover, the release of the drug

was also modified and extended over a period of 8 h in all formulations. Drug lipid
interaction study showed no interaction between drug and lipid. Ketoprofen liposomal
gel together, the result indicates that the diclofenac liposomal gel is better than the
regular gel without liposome.
Lakshmi N Ramana, Swaminathan Sethuraman, Udaykumar Ranga and Uma M
Krishnan13 developed liposomal nanodelivery system for nevirapine. In the present
study, we have evaluated a liposome system for the delivery of nevirapine, a
hydrophobic non-nucleoside reverse transcriptase inhibitor. Liposomes were prepared
from egg phospholipids using thin film hydration. The parameters of the process were
optimized to obtain spherical liposomes below 200 nm with a narrow polydispersity.
The encapsulation efficiency of the liposomes was optimized at different ratios of egg
phospholipid to cholesterol as well as drug to total lipid. Thedata demonstrate that
encapsulation efficiency of 78.14% and 76.25% were obtained at egg phospholipid to
cholesterol ratio of 9:1 and drug to lipid ratio of 1:5, respectively. We further observed
that the size of the liposomes and the encapsulation efficiency of the drug increased
concomitantly with the increasing ratio of drug and lipid and that maximum stability
was observed at the physiological pH. Thermal analysis of the drug encapsulated
liposomes indicated the formation of a homogenous drug-lipid system. The magnitude
of drug release from the liposomes was examined under different experimental
conditions including in phosphate buffered saline (PBS), Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with 10% fetal bovine serum or in the presence of an
external stimulus such a slow frequency ultrasound. Within the first 20 minutes 40, 60
and 100% of the drug was released when placed in PBS, DMEM or when ultrasound
was applied, respectively and they concluded that nevirapine-loaded liposomal
formulations reported here could improve targeted delivery of the anti-retroviral drugs
to select compartments and cells and alleviate systemic toxic side effects as a
consequence.
Avinash Kumar Seth and Ambikanandan Misra 14 has Preparation and Evaluation of
Acyclovir Liposomes by Two Techniques. The aim of this study was to prepare
liposomes of acyclovir (ACY) by thin layer evaporation (TLE) and reverse phase
evaporation (REV) methods. Twenty-seven batches of liposomes from each method

were prepared using technique of three variables at three levels (33) factorial design.
Drug/Lipid (molar ratio), hydration volume and hydration time were considered three
independent variables in TLE method while that of Drug/Lipid (molar ratio), organic
phase volume and aqueous phase volume in REV method. Liposomes, obtained by TLE
method (TLEs) and REV method (REVs) were evaluated for geometric mean diameter,
and percent drug entrapment (PDE). REVs of 3.5(2.3) m with 77.2% and of 3.4(2.2)
m with 71.1% drug entrapment was obtained with Drug/PC/CHOL (in molar ratio) of
1:4:0.5and 1:2:0.5 respectively while TLEs of 4.3(1.7) m with 79.5% drug entrapment
was obtained with Drug/PC/CHOL (in molar ratio) of 1:20:10. In vitro studies were
conducted to compare drug diffusion pattern across the human cadaver skin (HCS) of
promising batches of TLEs and REVs.
6.3 OBJECTIVE OF THE STUDY
The present investigation is aimed at
(A) Preparation of liposomes using phospholipids and cholesterol.
(B) Investigation of the above preparation with respect to 1. Particle size and shape analysis and its distribution
2. Percentage drug entrapment.
3. Determination of Zeta potential.
4. In vitro drug release studies and comparison within the formulation.
5. Stability study of the formulations as per ICH guidelines.
MATERIALS AND METHOD
Materialsa) Drug - Artemisinin type of compounds.
b) Phospholipids ECP, DSPC, SPC etc and Cholesterol
All other chemicals will be of analytical grade.
Method-

a) Lipsomes will be prepared by thin film hydration technique using rotary flash
evaporator.
Phospholipids are dissolved in mixture of organic solvents. The lipids are
deposited as stacks of film from the organic solvent on the wall of the round
bottom flask by process of rotary evaporation under reduced pressure. Upon
7.

hydration of lipids by addition of aqueous buffer containing the drug, lipids tend
to swell and peel off from the wall of round bottom flask results in formation of
multilamellar vesicle. Mechanical energy is required to cause swelling of lipids
and dispersion of lipid film by simple hand shaking technique. The MLVs
formed will be sonicated either by using probe sonicator or bath sonicator.

7.1 Source of Data:

a) Review of literature fromJournals such as : International Journal of Pharma Professionals Research.
International journal of pharma tech research.
Scholar research library.
International journal of pharm tech research.
International journal of pharm. research and development.
South African Medical Journal.
International journal of pharmacy and pharmaceutical sciences.
Journal of Innovative trends in pharmaceutical sciences.
Scholar research library.
Asian journal of pharmaceutical and clinical research.
International journal of pharmacy and pharmaceutical sciences.
Indian journal of pharmaceutical education and research.
International Quarterly Research Journal of Chemical Sciences.
( Rasayan journal)
American Association of Pharmaceutical Scientists journal.
International Journal of Drug Delivery.
Internet websites
www.pubmed.com

 www.sciencedirect.com
www.jgate-helinet.informindia.co.in
7.2 Method of Collection of Data:
1. The data related to properties of the drug and liposomes will be collected from drug
information centre, various standard books, journals and other sources like research
literature data bases such as science direct etc. and laboratory experiment.
2. Formulation of liposomes by thin film hydration technique using rotary flash
evaporator.
3. Evaluation of formulated liposome will be done as follows,

Morphology of liposomes using TEM or SEM.

Percentage drug entrapment.

Determination of particle size, shape, Zeta potential using Malvern nano zeta

sizer.
In vitro drug release studies and comparison within the formulation.
Stability study of the formulations as per ICH guidelines.

7.3 Does the study require any investigations or interventions to be conducted on patients
or other humans or animals? If so, please describe briefly.
Not Applicable

7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Not Applicable
LIST OF REFERENCES :
1. Kirby CJ, Gregoriadis G. Liposomes. In: Collins G. Encyclopedia of pharmaceutical
controlled drug delivery. New York: John Wiley and Sons, Inc; 1999. 461-89.
2. Sawarbrik J, Boylan JC. Liposomes as pharmaceutical dosage forms. Encyclopedia
of pharmaceutical technology. New York: Marcel Dekker, Inc; 1994:1-31.

3. Joshi M, Pathak S, Sharma S, Patravale V. Solid microemulsion preconcentrate

(NanOsorb) of artemether for effective treatment of malaria. Int. J. of
Pharmaceutics, 2008;362:172178.
4. Memvanga PB, Preat V. Formulation design and in vivo antimalarial evaluation of
lipid-based drug delivery systems for oral delivery of b-arteether. Eur J of
Pharmaceutics and Biopharmaceutics, 2012;82:112119.
5. Raina N, Amith K. Goyal, Pillai C R. Development and characterization of
8.

artemether loaded solid lipid nanoparticles. Ind. J of Pharmaceutical Edu And Res.,
2013 April-June;47(2):123-128.
6. Bhambere DS, Doijad R, Deshmukh N, Manvi FV, Kankate R. Liposomal drug
delivery system for zidovudine: design and characterization. Int J Drug Dev & Res.,
2010 Jan-March;2(1):8-14.
7. Ajay K,Shital B, Ravindra K, Varsha BP. Development and characterization of
liposomal drug delivery system for nimesulide. Int J Pharma and Pharma Sci.
2010;2(4):87-89.
8. Manjunatha N, Naidu GP, Sutrave V, Patel K, Samanta MK. Preparation and
Evaluation of Liposomes of an Antiviral Drug. Ind J Novel Drug Delivery. 2009
Oct-Dec;1(1):25-31.
9. Patel RP, Patel H, Baria AH. Formulation and Evaluation of Liposome of
Ketoconazole. Int. J of Drug Delivery Tech., 2009;1(1):16-23.
10. Raina N, Goyal KA, Pillai CR, Rath G. Development and characterization of
artemether loaded solid lipid nanoparticles. Ind J Pharm Edu Res., 2013; 47(2):123128.
11. Rivankar SH. Excipient selection for NDDS parenteral formulations. Pharma Times,
2013;45(3):33-40.
12. Mansoori MA, Jawade S, Agrawal S, Khan MI.
ketoprofen

liposomal

gel.

Journal

of

Formulation development of

Pharmaceutics

and

Cosmetology

2012;2(10):23-29.
13. Ramana LN, Sethuraman S, Ranga U, Krishnan UM. Development of a liposomal

nanodelivery system for nevirapine. J. Of Biomedical Sci., 2010;17(57):1-9.

14. Seth AK, Misra A. Preparation and Evaluation of Acyclovir Liposomes by Two
Techniques: A Comparative Study. Iranian J Of Pharm Sci., 2007;6(2):75-81.
15. Kulkarni PR, Yadav JD, Vaidya KA. Liposomes: A Novel Drug Delivery System.Int
J Curr Pharm Res., 2013;(2):10-18.
16. Bhatia A, Rajiv K, Om PK. Tamoxifen in topical liposomes: Development,
characterization and in-vitro evaluation. J Pharm Pharmaceut Sci.,2004;7(2):252-9.
17. Abhijit A. Date B, Medha D. Joshi A, Vandana BP. Parasitic diseases: Liposomes
and polymeric nanoparticles versus lipid nanoparticles. Advanced Drug Delivery
Reviews, 2007;59:505521.
18. Durn N, Priscyla DM, Teixeira Z, Durn M, Costa FTM, Brocchi M. State of the
Art

of

Nanobiotechnology Applications

Nanoscience,2009;5:396-408.

in

Neglected

Diseases.

Current

9.

Signature of the candidate

(P. ASHWINI PAI)

10.

Remarks of the Guide

The work, which is assigned to

P. ASHWINI PAI is under my guidance.

11.

11.1 Name and Designation of the

Guide

Dr. A. R. SHABARAYA M.Pharm.,MBA,Ph.D.

HOD
Department of Pharmaceutics,
Srinivas College of Pharmacy,
Valachil, Mangalore- 574143.

11.2 Signature

11.3 Name and Designation of the

Co-Guide

-------------

11.4 Signature

-------------

11.5 Head of the Department

Dr. A. R. SHABARAYA M.Pharm., MBA,PhD.

Principal and Director,
Srinivas College of Pharmacy,
Valachil, Mangalore- 574143.

11.6 Signature
12.

12.1 Remarks of the Principal

12.2 Signature

Recommended and forwarded for favourable

consideration.