15 - Chapter 6 PDF

6.
0 Preparation and Evaluation of Tenofovir nanoparticles
6.1 Introduction
Nanoparticle technology has evolved over the years in pharmaceutical drug

delivery. This technology has been used to targeting drug to different sites of the
body as per requirements of the disease. Well documented and extensive work
has been done in cancer therapy using nanoparticle technology. The major
problem involved in treatment of complex diseases is penetration of drug
through blood brain barrier. Nanoparticles have overcome this problem to a
great extent. Over the past decade nanotechnology has evolved for anti-
retroviral therapy. Published literature from Christy Anthony pillai (2006), Scott
Letendre (2008) and P.P. Koopmans (2009) to name a few have reported on the
need and effectiveness of nanoparticle technology in HIV therapy.
In the following chapter we have developed and optimised Tenofovir

nanoparticle formulation and established all parameters required for its
optimisation. Drug interaction has been studied using differential scanning
calorimetry and infrared spectroscopy as a part of Preformulation studies.
Attempt was made to prepare tenofovir nanoparticle formulation by various
techniques like nanoprecipitaion, double emulsion method and the steps were
taken to optimise formulation. Physio chemical characterisation of optimised
formulation like the size of the nanoparticle, zeta potential (which establishes
the surface charge), drug entrapment studies for establishing the amount of drug
present inside the nanoparticle, Surface morphology was done by scanning
electron microscopy. Stability studies were carried out to check the stability as
per standard guidelines. The topics are discussed under various subtopics below.
6.2 Materials and Methods
During the development and optimisation of Tenofovir nanoparticle formulation

various materials, reagents, instruments, chemicals are used and they are
mentioned in detail.
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6.0 Preparation and Evaluation of Tenofovir nanoparticles
6.2.1 Drug
Tenofovir disproxyl fumarate gift sample supplied by Matrix pharmaceuticals
limited Hyderabad.
6.2.2 Chemicals and reagents

Acetonitrile and Methanol (HPLC grade) supplied by Merck India, Water by
Millipore waters system, Eudragit RS 100 and RL 100 gift sample from Evonic
Mumbai, India, PLGA (50:50) polymer purchased from Boehringer Ingelheim
Germany, Polyvinyl alcohol (30000 to 70000) molecular weight from Sigma-
Aldrich, Mannitol is supplied by Himedia.
6.2.3 Instruments used

HPLC system (Waters 2695 separation module with millennium software),
Lichrocart analytical column 250 x 4.6mm, 5µ, pH meter (Systronics make),
UV spectrophotometer (2450 Shimadzu Kyoto, Japan), Infrared
spectrophotometer (Shimadzu FT-IR 8300), Differential scanning calorimeter
(DSC 60 Shimadzu Japan), ultra centrifuge (Sigma Aldrich 3k30), Analytical
balance (Sartorius), deep freezer (Sanyo), waters purification system (Milli-Q),
vortexer (Spinix USA), micropipette (eppendorf), particle size analyser(), high
speed homogeniser(Polytron PT 3100 Germany),lyophiliser().
6.3 Experimental methods
6.3.1 Pre formulation studies

Drug excipient compatibility studies performed using DSC-60 instrument. Alumina
was taken as reference. Temperature range is set from 350c to 8000c. Samples were
analysed with the above mentioned parameters and data collected using the collection
monitor software. Fourier transform infrared spectroscopy (FT-IR), the samples are
mixed with potassium bromate, triturated with help of mortar and pestal to ensure
uniform mixing of the sample. Pellets are prepared by taking the mixture into IR
holder and using a hydraulic press. It is ensured that thickness of pellet is uniform and
thin. Spectrum was recorded by Shimadzu IR solution 1.30 software. The FT-IR
range for preformulation studies were carried out at 4000cm-1 to 400 cm-1
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Differential Scanning Calorimetry (DSC) were performed for pure drug alone
and drug with excipients in the ratio of 1:1
Table 11: Samples analysed for FT-IR and DSC
S.No Sample
1. Tenofovir
2. Tenofovir+ PLGA 50:50 + Mannitol
3. Tenofovir+ Eudragit RL+ Mannitol
4. Tenofovir+ Eudragit RS+ Mannitol
6.3.2 Preparation of Nanoparticles
Preparation of nanoparticle was done by simple double emulsion method.

[Lamprecht et al (2000), Cristina et al (2002)] Tenofovir was dissolved in 0.5%
poly vinyl alcohol in a beaker and sonicated using ultra sonicator to ensure
complete dissolution of drug and vortexed to ensure uniform distribution in
solution. Accurately weighed amount of polymer (weights of polymer taken are
given in the table 12,13&14) was dissolved in two ml of dichloromethane in a
beaker and the solution obtained was clear with no particulate suspension
visible to naked eye. Primary emulsion was prepared in the following way.
Drug solution was added to polymer solution drop by drop and homogenised by
high speed homogeniser at 10000 RPM for 15 minutes. The time is counted
after complete addition of drug to polymer solution. The solution is probe
sonicated for 8 minutes (80w). Once the primary emulsion was formed,
secondary emulsion is prepared.
In another beaker, 0.5% Poly vinyl alcohol was homogenised with addition of
primary emulsion drop by drop at 10000 rpm for 15 minutes. The above
emulsion was probe sonicated 8 minutes (80w). The secondary emulsion was
kept for DCM evaporation for 4 hours at 370c and checked for odour of solvent
at regular intervals. The same procedure was followed for Eudragit-RS and
Eudragit-RL polymers.
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6.3.3 Separation of nanoparticles
After complete evaporation of DCM, the nanoparticles were centrifuged at 5000

rpm at 50c for 5 minutes to remove un entrapped drug and micro particles if
any. Sigma centrifuge (Germany) was used and further centrifugation was
carried out at 30000g for 30 minutes at 50c. The nanoparticles were dispersed in
Mannitol solution in 1:1 ratio (mannitol & Polymer). The sample solution was
kept at 00c for 2 hours and latter freezed at -700c overnight before proceeding
for lyophilisation.
6.3.4 Lyophilisation of Nanoparticles
Freeze drying of nanoparticles is method of choice to remove the water from

nanoparticles. Freeze drying of nanoparticles will help in overcoming stability
issues like aggregation of nanoparticles, drug leakage etc. After lyophilisation
of nanoparticles further steps like characterization of nanoparticles, particle
size, zeta potential, poly dispersity index and drug entrapment were carried out.
Table 12: Composition for TNF formulation and PLGA (50:50)

nanoparticles
Ingredient TNF- TNF- TNF- TNF- TNF- TNF-

04 05 06 25A 25B 25C
Tenofovir 8.0 8.0 8.0 8.0 8.0 8.0
(mg)
Poly lactic 10 20 40 60 60 60
glycolic acid
(PLGA) 50:50
(mg)
Poly vinyl -- -- -- 2.0 -- --
alcohol
(0.25%) (ml)
Poly vinyl 2.0 2.0 2.0 -- 2.0 --
alcohol (0.5%)
(ml)
Poly vinyl -- -- -- -- -- 2.0
alcohol (1.0%)
(ml)
Dichlorometha 2.0 2.0 2.0 2.0 2.0 2.0
ne (ml)
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Table 13: Composition for TNF formulation and Eudragit RL-100

nanoparticles

15 19 21 30A 30B 30C
Tenofovir 8.0 8.0 8.0 8.0 8.0 8.0
(mg)
Eudragit-RL 10 20 40 60 60 60
100 (mg)
Poly vinyl -- -- -- 2.0 -- --
alcohol
(0.25%) (ml)
Poly vinyl 2.0 2.0 2.0 -- 2.0 --
alcohol
(0.5%) (ml)
Poly vinyl -- -- -- -- -- 2.0
alcohol
(1.0%) (ml)
Dichlorometh 2.0 2.0 2.0 2.0 2.0 2.0
ane (ml)
Table 14: Composition for TNF formulation and Eudragit RS-100

nanoparticles

18 20 29 31A 31B 31C
Tenofovir 8.0 8.0 8.0 8.0 8.0 8.0
(mg)
Eudragit-RS 10 20 40 60 60 60
100 (mg)
Poly vinyl -- -- -- 2.0 -- --

alcohol
(0.25%) (ml)
Poly vinyl 2.0 2.0 2.0 -- 2.0 --
alcohol
(0.5%) (ml)
Poly vinyl -- -- -- -- -- 2.0
alcohol
(1.0%) (ml)
Dichlorometh 2.0 2.0 2.0 2.0 2.0 2.0
ane (ml)
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6.3.5 Nanoparticle characterization
Particle size analysis & Polydispersity index, Zeta potential, Drug entrapment,
Percentage yield, FE-SEM (Field Emission Scanning Electron Microscope),
FT-IR (Fourier transform infrared spectroscopy) and Differential Scanning
Calorimetry (DSC) were carried out. Zambaux et al (1998)
6.3.6 Particle size Analysis
The nanoparticle size was determined by dynamic light scattering (DLS)

technique. using Zeta sizer nano instrument from Malvern. The technique
measures the time dependent fluctuations in the intensity of scattered light
which occurs due to the particles in constant Brownian motion. This technique
provides the information about whole particulate system. Particle size of nano
formulation is very important. Parameters like biological fate, distribution in the
body and physiochemical parameters depend on particle size. Polydispersity
index (PDI) is the measure for width of size distribution. The value of PDI close
to zero indicates homogenous distribution of Nano particles and values close to
one indicates heterogeneous distribution. The Nano formulation is dispersed in
water as medium and temperature at 250c. Clear disposable zeta cells are used
for measurements were taken in triplicates and mean particle size reported.
Thirumala Govender et al (1999).
6.3.7 Zeta potential
It is described as charge of electric double layer created by ions of the liquid

which exists around each particle. (Malvern Webinar on particle size and zeta
potential available over the web) suggests this particle will attract positive ions
to the surface, they form a layer and this layer is called as “Stern layer”. If we
move further from the particles ions diffuse more freely around the particle.
This layer is called as the “Diffuse layer”. Somewhere within this diffuse layer
there is a band ring called as “Hydrodynamic plane of shear” also called as
“Slipping plane”. Ions outside the slipping plane will not move along with the
particle whereas ions within the plane move with it.
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There are 3 types of potential. First one is Surface potential (Surface of

particle), second one Stern potential (potential of stern layer) and third Zeta
potential (potential of slipping plane). So zeta potential is the potential at hydro
dynamic shear or also called as slipping plane. It depends not only on the
particle surface but also on the dispersant, it may be unrelated to the surface
potential, it can be affected by the small change in the pH or ionic charge.
Particles interact according to the magnitude of zeta potential not the surface
charge, Therefore it helps in knowing dispersion stability.
If all the particles have higher negative or positive zeta potential then there will
be dispersion stability due to repulsion. The dividing line between an aqueous
particle dispersion being stable and un stable is considered to be +30mV and -
30mV. Particles outside the above stated range are stable. The Nano
formulation is dispersed in water as medium and temperature at 250c. Clear
disposable zeta cells are used for measurements were taken in triplicates and
mean particle size reported.
6.3.8 Percentage yield
The percentage yield of the formulation of PLGA (50:50), Eudragit-RL 100 and
Eudragit-RS 100 formulation are done by using the formula
Percentage yield = Weight of freeze dried nanoparticles (mg) X 100
Weight of drug (mg) + Polymer (mg) + Mannitol (mg)
6.3.9 Percentage Entrapment Efficiency
It is defined as the amount of drug entrapped inside the nano formulation and is
assayed by HPLC assay method developed and validated as indicated in early
chapter. The sample was prepared in the following way. Amount equivalent to
10 mg of Tenofovir from its salt form was weighed and dissolved in 10 ml
volumetric flask. The solution was sonicated for 10 minutes and volume made
up to mark with mobile phase. The clear supernatant solution was centrifuged
for 5000 rpm for 5 minutes and injected into the system. Percentage entrapment
efficiency is calculated by using the formula, Jin-Wook Yoo et al (2011)
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EE (%) = Amount of drug in the prepared nanoparticle x 100

Amount of drug loaded in nanoparticle
6.3.10 Fourier Transform Infrared Spectroscopy (FT-IR) and Differential

Scanning Calorimetry (DSC)
Fourier Transform Spectroscopy (FT-IR) and DSC analysis of optimised batch

was carried out for Tenofovir formulation involving PLGA (50:50), Eudragit-
RS 100 and Eudragit-RL 100 polymers. The above mentioned parameters help
us in understanding the physical characteristic of drug and polymer interactions.
6.3.11 Field Emission Scanning Electron Spectroscopy (FE-SEM)
The surface morphology of optimised formulations was studied using Field

Emission Scanning Electron Spectroscopy (FE-SEM Hitachi SU 6600 Japan).
Dry nano formulation powder was taken on a dye with double side sticky tape
attached to it. The formulation is finely dispersed over the tape and gold coated
for 20 seconds. The die along with sample was loaded into the system and
photographed for its surface morphology.
6.3.12 Invitro Drug Release
Invitro release studies were carried out to nanoparticle formulation by Diffusion

method [T. Niwa, H. Takeuchi, T (1992), Einat et al (2009)] .0.1N HCl was
taken as dissolution media. Due to very high solubility of drug (13.9 mg/ml). 40
ml media was taken. Sigma membrane immersed in the dissolution media is
kept for overnight. This differentiates the donor and acceptor compartment.
Amount equivalent to one dose of drug was taken and placed in donor
compartment. A magnetic needle with 50 RPM was used and temperature is
maintained at 370c. Time points were 5, 10, 15, 30, 45, 60, 120, 240, 360, 480,
720, 960, 1200 and 1440 minutes. Sample were taken at the designated time and
replaced with fresh disso media maintained at the same temperature. Detection
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of drug release was done at UV wavelength of 260 nm. The experiments are
performed in duplicate and the drug concentration is converted into cumulative
drug release and results reported.
6.3.13 Release Kinetics
Release Kinetics is an important parameter for pharmaceutical dosage forms

and determining the release kinetics of our nano formulation is a proof of
adequacy of its design. To fulfil these criteria, cumulative release profiles of
optimised nano formulation are described using mathematical equation. These
models illustrate the drug release from nano formulation. In the present study
the best describing kinetic models like Krosmeyers-Peppas model (Mt/M∞ = kt
n), „n‟ is the diffusional exponent, „k‟ is kinetic constant, „Mt‟ is the amount
drug released at time t, „M∞‟ is amount released at time infinity. Higuchi (Q =
Kt1/2) model, Zero order model (percent release against time), First order (log
percent release against time). The criteria to choose the best model was
determined by coefficient of determination R2 to assess the best fit model.
Srinivas et al (2008).
6.3.14 Stability Studies
Stability is a very important criterion for formulation development. Stability

helps in assessment of shelf life. Different regulatory bodies like ICH have
guidelines and stringent norms of real time stability data that needs to be
established. This stability data gives information about shipping and storage of
samples. Stability studies were conducted for the optimised formulation as per
ICH guidelines. The optimised formulations were stored in glass vials at 40c +
20c. all three batches TNF-25, TNF-30 and TNF-31 were stored under the above
stated condition as well as at accelerated stability condition of 400 + 20c / 75% +
5% RH for a period of 15, 30,60,90 and 180 days. The samples were analysed
for change in physical appearance, entrapment efficiency, particle size and zeta
potential.
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6.4 Results and Discussion
6.4.1 Preformulation studies
As mentioned earlier Preformulation studies were carried out of Drug +PLGA

(50:50) polymer ( TNF-25 batch), Drug + Eudragit-RL 100 (TNF-30) and Drug
+ Eudragit-RS 100( TNF-31). Drug excipient reaction was studied by FT-IR
and DSC.
6.4.2 IR interpretation:
Presence of following functional groups in Tenofovir drug was confirmed by

the following functional groups, Carbonyl group at 1759 cm-1 and OH group at
3227 cm-1. PLGA polymer contain the following group OH at 3516 cm -1 and
carbonyl group at 1757 cm-1. In TNF-25 (PLGA 50:50) the spectrum of
polymer with drug showed that there is a physical interaction between carbonyl
group of drug and OH group of polymer due to hydrogen bond formation by
weak van der Waal‟s forces. Further release of drug invitro indicates only
physical interaction not chemical interaction.
Eudragit RL (TNF-30) the following functional group are found, Alkyl groups
below 3000cm-1, bend of alkyl CH3 and CH2 at 2359 cm-1 and 2332cm-1.
Carbonyl group at 1735cm-1 and 1726cm-1.In TNF-30 and TNF-31 IR
chromatograms indicates that drug entrapment is within the polymer by weak
forces of attraction like hydrogen bonding and van der Waal‟s forces.
6.4.3 DSC interpretation:
Thermogram of Tenofovir disproxil fumarate showed a sharp melting transition

endotherm ranging from 114.64°c to 120.52°c with a peak at 117.69°c. The
DSC Thermogram of formulation TNF 25 (drug + PLGA polymer) showed
small melting endotherm at 157.21°c followed by a sharp endotherm at
158.58°c indicating decomposition of drug. Thermogram of formulation TNF
30 (drug + Eudragit RS) showed a small melting endotherm at 157.29°c
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followed by an endotherm at 169.49°c indicating decomposition of drug. A

similar type of DSC Thermogram was observed for formulation TNF 31 with a
slight change in the peak position, 157.84°c followed by an endotherm at
168.25°c, with increased intensity. The absence of endothermic peak of the drug
at 117.69°c in the DSC Thermogram of the formulations i.e. TNF 25, TNF 30
and TNF 31 suggests that the drug existed in an amorphous or crystalline phase
as a molecular dispersion in the polymeric matrix.
Figure 17: FT-IR Spectrum of TNF-25 (Tenofovir + PLGA 50:50)
1755.28
75
2766.01
1637.62
511.15
2332.02
968.30
1197.83
2359.02
2875.96
%T 1255.70
628.81
881.50
682.82
50
1371.43
717.54
1330.93
2933.83
931.65
1458.23
25
1022.31
1089.82
3271.38
3750 3000 2250 1500 750

TNF-25 1/cm
Figure 18: FT-IR Spectrum of TNF-31 (Tenofovir + Eudragit-RS 100)
10
0
%
T
7
5
3730.45
412.78
2561.55
2332.02
476.43
2359.02
5
2677.29
509.22
0
2767.94
964.44
2993.62
1192.05
1149.61
879.57
1253.77
626.89
2
1732.13
5
1329.00
1375.29
715.61
929.72
2935.76
1458.23
0
1022.31
1087.89
3273.31
375 300 225 150 75

TNF-
0 0 0 0 0 1/c
31 m
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Figure 19: FT-IR Spectrum of TNF-30 (Tenofovir + Eudragit-RL100)
.942677
2332
5
.29
2767
.02
2359
%
509.
.02
22
966.
2989
T
881.37
.76
5
628.
50
81
.29 1251
.86 1188
0
1151
.84
.19
.54
715.
1332
1375
61
2935
931.
1732
.76
65
.13
1458
2
.23
5
1022
1087
.31
3282
3273
.89
.95
.31
0
375 300 225 150 75
TNF- 1/c
0 0 0 0 0
30 m
DSC Spectrum
Figure 20: DSC of TNF-25 (Tenofovir + PLGA 50:50)
TNF + TNF 25
DSC
mW
10.00
157.21 0x10
C
0.00
-10.00 0
117.69 x10
C
0
168.58 x10
C
-20.00
100.00 200.00 300.00
Temp [C]
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Figure 21: DSC of TNF-30 (Tenofovir + Eudragit-RL100)
TNF + TNF 30
DSC
mW
10.00
157.29 0x10
0.00 C
-10.00 117.69 0x10

C
0
169.49 x10
C
-20.00
100.00 200.00 300.00

Temp [C]
Figure 22: DSC of TNF-31 (Tenofovir + Eudragit-RS100)
TNF + TNF 31
DSC
mW
10.00
0
0.00 157.84 x10
C
-10.00 117.69 0x10

C 0
168.25 x10
C
100.00 200.00 300.00

Temp [C]
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6.4.4 Formulation Development
In the field of nano formulations PLGA (50:50) is most widely used bio
degradable polymer. The polymer is widely accepted by regulatory bodies
around the world for its safety was well known, no known major toxic effects
till now. Eudragit-RL 100 and Eudragit-RS 100 are selected for their stability at
varying pH. pH plays an important role in Tenofovir nano formulation
preparation by nano precipitation method Thirumala Govender et al (1999).
The problem with Tenofovir formulation was its high aqueous solubility of drug
which makes drug entrapment difficult as most of the drug remains in the
aqueous phase in this method both the drug and polymer is dissolved in organic
phase and then this was added to aqueous phase with constant stirring. The
organic layer was evaporated and then further steps of centrifugation at 20,000
rpm 50c were carried out. For aqueous phase we have chosen 2-[4-(2-Hydroxy
ethyl)1-piperazinyl] ethane sulphonic acid (HEPES buffer) 1mM strength and
pH adjusted to 7.0 by NaOH. The idea was to prevent the ionization of TNF
into aqueous solution so that more amount of drug is entrapped to the polymer.
pka of TNF is 3.75, so the pH 7.0 was chosen as trial. The particle size analysis
of the nanoparticles obtained by this method was high (884 nm), so double
emulsion method was tried out. The procedure for double emulsion method was
as mentioned earlier.
Most commonly used Poly vinyl alcohol was chosen as stabiliser. The ratio of
polymer with respect to drug was increased and this resulted in increase of drug
entrapment and drug yield. Varying concentrations of stabiliser did not make a
significant difference in nano particle characteristics like size, zeta potential or
drug entrapment etc. Homogenisation speed was optimised at 10000 rpm after
trials at lower and higher speeds. Probe sonication for reducing the particle size
was tried at varying amplitudes and the final optimised condition was kept at
80w/8 amps/ 8 mts.
Centrifugation speed was optimised at 30000g for 30 minutes at 40C. Higher

speeds made the re dispersion of nano formulation difficult and in few batches
re dispersion was not possible resulting in lump formation in the centrifuge
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tube. Lyophilisation to remove aqueous phase was done and it did not
contribute in any significant change in particle size or poly dispersity index and
zeta potential. The above method was good enough to have batch to batch
reproducibility and minimum variability with respect to final formulation
characterisation. Invitro release studies were carried out by widely reported
diffusion cell method with minor modifications to suit the developed
formulation and drug release was established for a period of 24 hours.
6.4.5 Characterisation of Nanoparticles
6.5 Physiochemical characterisation
6.5.1 Percentage yield

The percentage yield of Tenofovir nano formulation increased with increase in
polymer. Increase in polymer was necessary for increase in drug entrapment.
The final yield of all the three polymers (PLGA 50:50, Eudragit-RL, Eudragit-
RS) was comparable and reproducible. The final yield consists of drug, polymer
and cryoprotectant Mannitol.
Table 15: Percentage yield

Batch Number TNF-25 TNF-30 TNF-31
Polymer used in PLGA (50:50) Eudragit-RL 100 Eudragit-Rs 100

nano Polymer Polymer Polymer
formulation
Total yield 87.14% + 1.54 86.45% + 1.72 87.00% + 1.34
6.5.2 Percentage Encapsulation Efficiency
It is the measure of drug entrapped with polymer and is of great importance in

the final optimised formulation for drug delivery. Entrapment of drug is
dependent on factors like nature and types of polymers used. The encapsulation
efficiency for TNF-25 optimised formulation is found to be 13.45% + 1.20. In
case of TNF-30, Eudragit RL 100 the entrapment was established at 11.55% +
1.24. For TNF-31, Eudragit RS 100 the entrapment was established at 11.12% +
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1.32. There is no significant change in entrapment with change in Poly vinyl

alcohol which is used as stabiliser. The drug entrapment was increased with
change in drug polymer ratio from 1:1.25 to 1:7.5. This was attributed to
increase in surface area available to drug with increase in polymer.
Table 16: Tenofovir + PLGA (50:50) Polymer + Mannitol
Drug: Batch Particle Zeta

Polymer PVA No Size PDI Potential % EE
1:1.25 0.50% TNF-4 250.8 0.117 -21.3 1.72
1:2.5 0.50% TNF-5 226.2 0.143 -23.6 3.85
1:50 0.50% TNF-6 738 0.596 -28 11.9
1:7.5 0.50% TNF-25 231 0.348 -8.46 13.45
20
15
10
0
1 2 3 4
Table 17: Tenofovir + Eudragit-RL 100 + Mannitol
Drug: Particle Zeta

Polymer PVA Batch No Size PDI Potential % EE
1:1.25 0.50% TNF-15 845 1.000 30.4 0.99
1:2.5 0.50% TNF-26 290 0.252 49.6 9.45
1:50 0.50% TNF-28 284 0.314 51.4 10.26
1:7.5 0.50% TNF-30 325 0.351 47.7 11.55
20
15
10
0
1 2 3 4
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Table 18: Tenofovir + Eudragit-RS 100 + Mannitol
Drug: Particle Zeta

Polymer PVA Batch No Size PDI Potential % EE
1:1.25 0.50% TNF-18 291 0.418 34.5 1.75
1:2.5 0.50% TNF-27 290 0.316 49.4 8.87
1:50 0.50% TNF-29 333 0.352 40.5 9.98
1:7.5 0.50% TNF-31 187 0.209 30.9 11.12
14
12
10
8
6
4
2
0
1 2 3 4
6.5.3 Particle Size Analysis
Mean particle size and poly dispersity index is the measure of quality of nano
formulation. Particle size was determined by using dynamic light scattering
technique of Malvern zeta sizer. The average particle size of TNF-25
(Tenofovir + PLGA 50:50) batch is found to be 232 nm and poly dispersity
index was found to be 0.348. For Eudragit-RL 100 nano formulation the size
was found to be 326 nm and poly dispersity index 0.351. Eudragit-RS 100
particle size was found to be 187 nm and poly dispersity index a healthy 0.209.
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Figure 23: Particle size of TNF-25 Tenofovir + PLGA (50:50) Polymer
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Figure 24: Particle size of TNF-30 Tenofovir + Eudragit-RL 100 Polymer
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Figure 25: Particle size of TNF-31 Tenofovir + Eudragit-RS 100 Polymer
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6.5.4 Zeta potential
It is one of the important parameters for stability of nano formulation and the
negative charge indicates repulsion of particles thus preventing aggregation of
formulation and more stable during stability of formulation on storage. The
results show that zeta potential depends on particle size. The TNF-25 batch
containing PLGA (50:50) polymer has zeta potential -8.46 + 1.42 mV and TNF-
30 batch containing Eudragit-RL 100 polymer‟s has a zeta potential +47.7 +
1.34 mV, TNF-31 batch containing Eudragit-RS 100 polymer‟s zeta potential is
established at + 30.9 + 1.45 mV. TNF-25 negative charge is due to surfactants
adhering to the nanoparticle surface covering the carboxylic groups in the
polymer and presence of terminal carboxylic group in PLGA 50:50 polymer.
Eudragit polymer formulations have a very high value of zeta potential which is
well above the specified value of + 30 mV. This value of zeta potential
indicates the stability of formulation.
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Figure 26: Zeta Potential of TNF-25 Tenofovir + PLGA (50:50) Polymer
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Figure 27: Zeta Potential of TNF-30 Tenofovir + Eudragit-RL 100 Polymer
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Figure 28: Zeta Potential of TNF-31 Tenofovir + Eudragit-RS 100 Polymer
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6.5.5 Field Emission Scanning Electron Microscope

The surface morphology of optimised formulations was studied and the SEM
images indicate that the nanoparticles were spherical and no agglomeration of
particles is seen which supports the zeta potential data. The particles are
spherical in nature and seen against Mannitol in background.
Figure 29: SEM image of TNF-25 Tenofovir + PLGA (50:50) Polymer
Figure 30: SEM image of TNF-30 Tenofovir + Eudragit-RL 100 Polymer
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Figure 31: SEM image of TNF-31 Tenofovir + Eudragit-RS 100 Polymer
6.5.6 Fourier Transform Infrared (FT-IR) Spectroscopy
FT-IR and DSC analytical techniques give us the complete picture of drug
polymer interactions and also the influence it has on nanoparticles. Tenofovir
disproxyl fumarate displays the aliphatic CH stretching at 2985 cm-1and
aromatic CH stretching at 3051 cm-1. C=O stretching is spotted at 1759 cm-1.
The predominant group of NH stretching is seen at 3227 cm-1 and 3271 cm-
1
.C=O stretching is also seen at 1269 cm-1. The major group (NH) has not
interacted with polymer indicating the stability of the nano formulation as
evident in the spectrum.
Page 78
25
50
75
0
25
50
75
100
%T
%T
0
25
50
75
100
%T
3936.84
3880.91
60
70
80
90
0
25
50
75
0
25
50
75
100
%T
%T
%T
TNF
3863.55
3850.04
PLGA
TNF-25
3811.47
TNF
3750
3750
3797.96
3750
3730.45
TNF-28
3649.44
3750
3750
3750
3516.35
EUDRAGIT
3591.57
Polymer
3554.93
3510.56
3437.26 3271.38
3230.87 3227.02
3381.33
3282.95 3099.71
3273.31 3227.02 3051.49
2999.41 2985.91
3099.71
3000
3000
2955.04
3000
2933.83 2935.76
3051.49 2887.53 2875.96
2989.76 2985.91
3000
3000
3000
2935.76 2935.76 2748.65 2766.01 2748.65
2661.85 2683.07
2767.94 2748.65 2613.63
2677.29 2683.07 2546.12 2532.62
2503.69 2476.68
2532.62 2409.17
2359.02
2476.68 2357.09
2332.02
2359.02 2359.02 2258.72
2250
2250
2250
2332.02 2332.02
2114.05
2079.33
2250
2250
2250
1757.21 1755.28 1759.14
1681.98
1735.99 1759.14 1620.26 1637.62 1626.05
1732.13
1726.35 1681.98
1637.62 1506.46
1626.05 1454.38 1467.88
1458.23
1500
1500
1500
1506.46 1429.30 1421.58

1392.65 1371.43
1458.23 1467.88 1381.08
1500
1446.66
1500
1500
1330.93
1384.94 1375.29 1421.58 1278.85 1269.20
1255.70 1180.47
1332.86 1381.08 1197.83
1274.99 1269.20 1174.69 1155.40
1251.84 1126.47 1101.39
1242.20 1188.19 1180.47 1089.82
1151.54 1155.40 1089.82 1022.31 1033.88
1151.54
1087.89 1101.39 968.30 987.59
1024.24 1033.88 956.72 950.94
1022.31 931.65
989.52 966.37 987.59 862.21 881.50 895.00
958.65 931.65 950.94 829.42
850.64 895.00 750.33 717.54 788.91
Figure 32: TNF-25 Tenofovir Drug + Formulation + PLGA (50:50) Polymer
881.50
750
750
750
829.42 711.76 682.82 729.12

788.91 628.81 698.25
Figure 33: TNF-30 Tenofovir Drug + Formulation + Eudragit RL 100
750
750
715.61
750
729.12 570.95 653.89
628.81 698.25 511.15 599.88
653.89 567.09
1/cm
1/cm
1/cm
509.22 599.88 478.36

567.09
1/cm
1/cm
1/cm
478.36
Page 79
0
25
50
75
0
25
50
75
100
%T
%T
60
75
90
%T
TNF
TNF-28
3750
3750
3750
3585.79
Polymer
3549.14
EUDRAGIT RS
Polymer
mW
3437.26
DSC
0.00
10.00
-10.00
3390.97
3282.95
3273.31 3227.02
3099.71
3051.49
2989.76 2987.84 2985.91
3000
3000
3000
2935.76 2951.19 2935.76
0
C
45.38 x10
2767.94 2748.65
2677.29 2683.07
2532.62
0
2476.68
100.00
117.69 x10
2359.02 2359.02
2332.02 2330.09
2250
2250
2250
C
157.21 0x10
Temp [C]
1732.13 1734.06 1759.14
0
1681.98
C
1637.62 1626.05
168.58 x10
TNF + TNF 25+PLGA
200.00
1479.45 1506.46
1458.23 1467.88
1500
1500
1500
1446.66 1421.58
1375.29 1386.86
1332.86 1381.08
1251.84 1271.13 1269.20
1188.19 1242.20 1180.47
1151.54 1149.61 1155.40
1087.89 1101.39
1022.31 1026.16 1033.88
966.37 987.59 987.59
931.65 950.94
848.71 895.00
881.50 829.42
756.12 788.91
750
715.61
750
Figure 35: TNF-25 Tenofovir Drug + Formulation + PLGA (50:50)

750
Figure 34: TNF-31 Tenofovir Drug + Formulation + Eudragit RS 100
300.00
671.25 729.12
628.81 698.25
653.89
509.22 599.88
480.29 567.09
1/cm
1/cm
1/cm
478.36
Page 80
Figure 36: TNF-30 Tenofovir Drug + Formulation + Eudragit RL 100

Polymer
TNF + TNF 30 + RS 100

DSC
mW
10.00
0.00 0C
157.29 x10
-10.00 117.69 0x10

C
169.49 0x10
C
-20.00
-30.00
100.00 200.00 300.00

Temp [C]
Figure 37: TNF-31 Tenofovir Drug + Formulation + Eudragit RS 100

Polymer
TNF + TNF 31 + RL 100

DSC
mW
10.00
0.00 0C
157.84 x10
-10.00 117.69 0x10

C 0C
168.25 x10
-20.00
100.00 200.00 300.00

Temp [C]
Page 81
DSC is useful in the investigation of solid-state interactions. Thermograms were

generated for pure drug and different formulations. The thermal curve of
tenofovir showed an endothermic peak at 117.69°c corresponding to its melting
point/transition temperature displaying crystalline nature of the pure compound.
In the freeze dried nanoparticles the peak of tenofovir was disappeared
indicating possibility of conversion of tenofovir in to amorphous form. The
drug molecules might have entirely dispersed in the polymer matrix. As per
literature, decrease in crystallinity and amorphization of the drug exhibits more
solubility (Usha et al., 2008; Mutalik et al., 2008). However appearance of the
endothermic peaks at 168-169°c is due to the presence of cryoprotectant,
mannitol. This was confirmed by the native thermogram of mannitol.
6.5.7 Invitro Release Study
Invitro release studies give an insight about the formulation behaviour invivo.
The release pattern shows that the release is erosion kind of release. TNF-25
(PLGA 50:50 polymer) has faster release than TNF-30 & 31 (Eudragit RL 100
& RS 100). Tenofovir has a very high solubility in water (13.46 mg/ml), and it
goes into the solution at a rapid rate. In TNF-25 the drug release rate was high
from initial time point and at 60 minutes it was 85% + 2.7. TNF 30 the drug
release rate was 64% + 3.42 .TNF 31 batch the drug release rate was 59% +
3.29. This is due to the affinity of drug to the polymer which was due to
hydrogen bonding by van der Waals forces between the polymer and drug.
However it was observed that at TNF-25 drug released was 101% + 1.97 in four
hours and the concentration remained constant throughout indicating its
complete release from the polymer. In case of TNF 30 batches the drug release
was 100% + 1.48 in 8 hours more than double the time taken for PLGA
polymer formulation. TNF-31 batch drug release was 100% + 1.89 in 12 hours.
All the batches were tested for 24 hours and more and found out the drug
release was constant after 16 hours.
Page 82
Figure 38: Invitro drug release profile of TNF-25 (PLGA 50:50) polymer
120
R 100
D e 80
r l
60
u a
g s 40
e 20
0
0 500 1000 1500
Time in Minutes
Figure 39: Invitro drug release profile of TNF-30 (Eudragit RS 100)

polymer
120
R 100
e
D 80
l
r 60
e
u
a 40
g
s 20
e 0
0 500 1000 1500
Time in Minutes
Figure 40: Invitro drug release profile of TNF-30 (Eudragit RS 100)

polymer
120
R 100
D e 80
r l
60
u a
g s 40
e 20
0
0 500Time in Minutes
1000 1500
Page 83
6.5.8 Release Kinetics

The non curvilinear shape of the graph shows that the optimised batches have
followed Higuchi release kinetics. The obtained data was also tried out in other
mathematical models. The data suggest that drug entrapped outside the nano
particle released immediately followed by polymer breaking down and
releasing the entrapped drug.
Table 19: Release Kinetics data of optimised formulation
Nano particle Batch TNF-25 TNF-30 TNF-31

Zero order (R2) 0.2359 0.5241 0.7400
First order (R2) 0.7879 0.6280 0.6656
Higuchi model (R2) 0.8035 0.8713 0.8635
Krosmeyers-Peppas (n) 0.7951 0.7835 0.7889
Model fit Higuchi Higuchi Higuchi
6.5.9 Stability Studies
Stability studies were conducted as per the ICH guidelines. The results of
optimised nano formulation of TNF-25, TNF-30 and TNF-31 are performed at
accelerated stability conditions 400 + 20c / 75% + 5% RH and the results are
given in the following table no 21,22&23. Nano formulations of the above
batches did not show any agglomeration or flocculation of dry powder. The
percentage encapsulation efficiency was reduced in all the formulations with
time. Particle size was constant but slight fluctuation of zeta potential in TNF-
25 batch. Poly dispersity index was well within 0.5 value so the optimised
formulation was homogenous. In TNF-30 batch there was fluctuation in particle
size but it did not cross more than 400 nm. Zeta potential was well outside the
+30mV value which indicates the stability of formulation. Poly dispersity index
was well within the limit but showed a slight increase in 06 months data. In
TNF-31 batch, there was steady increase in particle size but till the last 06
months it has not crossed 400 nm which is good for oral formulation. Poly
dispersity index was also well within the limit with one value going above 0.5.
Zeta potential was outside+30mV but in 06 months the zeta potential has come
Page 84
down to + 19mv. which was a good value which indicates the stability of
formulation.
Table 20: Stability data of TNF-25 Tenofovir + PLGA (50:50) Polymer at

400 + 20c / 75% + 5% RH (n = 3)
Time % Encapsulation Particle PDI Zeta

(Months) Efficiency Size (nm) Potential
0.0 13.45 231.0 0.348 -08.46
0.5 13.44 224.2 0.120 -10.62
01 13.40 226.2 0.143 -23.60
02 13.39 250.8 0.117 -21.30
03 13.24 265.1 0.239 -18.20
06 13.19 267.9 0.244 -22.80
Figure 41: Log % Drug remaining against time plot of TNF-25 at 400 + 20c
/ 75% + 5% RH
2.002
R y = -0.0015x + 2
2 R² = 0.8903
e
L
m 1.998
o
a
g g 1.996
i
n 1.994
%
i 1.992
n
1.99
0 2 4 6 8
Time in Months
Table 21: Stability data of TNF-30 Tenofovir + Eudragit-RL 100 Polymer

at 400 + 20c / 75% + 5% RH (n = 3)

0.0 11.55 325 0.351 +47.70
0.5 11.52 284 0.314 +51.40
01 11.48 290 0.252 +49.46
02 11.47 275 0.396 +37.00
03 11.36 291 0.418 +34.50
06 11.35 371 0.613 +19.50
Page 85
/ 75% + 5% RH
2.002 y = -0.0013x + 1.9991

R R² = 0.8287
2
L e 1.998
o m
n 1.996
g a
g 1.994
i
1.992
% n
i 1.99
0 2 4 6 8
Time in Months
Table 22: Stability data of TNF-31 Tenofovir + Eudragit-RS 100 Polymer

at 400 + 20c / 75% + 5% RH (n = 3)

0.0 11.22 187 0.209 +30.90
0.5 11.22 241 0.130 +38.40
01 11.18 290 0.316 +49.40
02 11.10 291 0.418 +34.50
03 11.09 333 0.352 +40.50
06 11.05 371 0.613 +19.50
/ 75% + 5% RH
2.002 y = -0.0012x + 1.9995

R R² = 0.8345
2
e
1.998
L m
o a 1.996
g
g i 1.994
% n 1.992
i 0 2 4 6 8
n Time in Months
Page 86

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6.

0 Preparation and Evaluation of Tenofovir nanoparticles

Nanoparticle technology has evolved over the years in pharmaceutical drug

In the following chapter we have developed and optimised Tenofovir

6.2 Materials and Methods

During the development and optimisation of Tenofovir nanoparticle formulation

6.2.2 Chemicals and reagents

6.2.3 Instruments used

6.3 Experimental methods

6.3.1 Pre formulation studies

Table 11: Samples analysed for FT-IR and DSC

6.3.2 Preparation of Nanoparticles

Preparation of nanoparticle was done by simple double emulsion method.

6.3.3 Separation of nanoparticles

After complete evaporation of DCM, the nanoparticles were centrifuged at 5000

6.3.4 Lyophilisation of Nanoparticles

Freeze drying of nanoparticles is method of choice to remove the water from

Table 12: Composition for TNF formulation and PLGA (50:50)

Ingredient TNF- TNF- TNF- TNF- TNF- TNF-

Table 13: Composition for TNF formulation and Eudragit RL-100

Ingredient TNF- TNF- TNF- TNF- TNF- TNF-

Table 14: Composition for TNF formulation and Eudragit RS-100

Ingredient TNF- TNF- TNF- TNF- TNF- TNF-

Poly vinyl -- -- -- 2.0 -- --

6.3.5 Nanoparticle characterization

6.3.6 Particle size Analysis

The nanoparticle size was determined by dynamic light scattering (DLS)

6.3.7 Zeta potential

It is described as charge of electric double layer created by ions of the liquid

There are 3 types of potential. First one is Surface potential (Surface of

6.3.8 Percentage yield

6.3.9 Percentage Entrapment Efficiency

EE (%) = Amount of drug in the prepared nanoparticle x 100

6.3.10 Fourier Transform Infrared Spectroscopy (FT-IR) and Differential

Fourier Transform Spectroscopy (FT-IR) and DSC analysis of optimised batch

6.3.11 Field Emission Scanning Electron Spectroscopy (FE-SEM)

The surface morphology of optimised formulations was studied using Field

6.3.12 Invitro Drug Release

Invitro release studies were carried out to nanoparticle formulation by Diffusion

6.3.13 Release Kinetics

Release Kinetics is an important parameter for pharmaceutical dosage forms

6.3.14 Stability Studies

Stability is a very important criterion for formulation development. Stability

6.4 Results and Discussion

6.4.1 Preformulation studies

As mentioned earlier Preformulation studies were carried out of Drug +PLGA

Presence of following functional groups in Tenofovir drug was confirmed by

6.4.3 DSC interpretation:

Thermogram of Tenofovir disproxil fumarate showed a sharp melting transition

followed by an endotherm at 169.49°c indicating decomposition of drug. A

Figure 17: FT-IR Spectrum of TNF-25 (Tenofovir + PLGA 50:50)

3750 3000 2250 1500 750

Figure 18: FT-IR Spectrum of TNF-31 (Tenofovir + Eudragit-RS 100)

375 300 225 150 75

Figure 19: FT-IR Spectrum of TNF-30 (Tenofovir + Eudragit-RL100)

Figure 20: DSC of TNF-25 (Tenofovir + PLGA 50:50)

Figure 21: DSC of TNF-30 (Tenofovir + Eudragit-RL100)

-10.00 117.69 0x10

100.00 200.00 300.00

Figure 22: DSC of TNF-31 (Tenofovir + Eudragit-RS100)

-10.00 117.69 0x10

100.00 200.00 300.00

6.4.4 Formulation Development