6.0 Preparation and Evaluation of Tenofovir nanoparticles

Transcription

1 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 antiretroviral therapy. Published literature from Christy Anthony pillai (26), Scott Letendre (28) and P.P. Koopmans (29) 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. Page 53

2 6.2.1 Drug Tenofovir disproxyl fumarate gift sample supplied by Matrix pharmaceuticals limited Hyderabad Chemicals and reagents Acetonitrile and Methanol (HPLC grade) supplied by Merck India, Water by Millipore waters system, Eudragit RS 1 and RL 1 gift sample from Evonic Mumbai, India, PLGA (5:5) polymer purchased from Boehringer Ingelheim Germany, Polyvinyl alcohol (3 to 7) molecular weight from Sigma- Aldrich, Mannitol is supplied by Himedia Instruments used HPLC system (Waters 2695 separation module with millennium software), Lichrocart analytical column 25 x 4.6mm, 5µ, ph meter (Systronics make), UV spectrophotometer (245 Shimadzu Kyoto, Japan), Infrared spectrophotometer (Shimadzu FT-IR 83), Differential scanning calorimeter (DSC 6 Shimadzu Japan), ultra centrifuge (Sigma Aldrich 3k3), Analytical balance (Sartorius), deep freezer (Sanyo), waters purification system (Milli-Q), vortexer (Spinix USA), micropipette (eppendorf), particle size analyser(), high speed homogeniser(polytron PT 31 Germany),lyophiliser(). 6.3 Experimental methods Pre formulation studies Drug excipient compatibility studies performed using DSC-6 instrument. Alumina was taken as reference. Temperature range is set from 35 c to 8 c. 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 1.3 software. The FT-IR range for preformulation studies were carried out at 4cm -1 to 4 cm -1 Page 54

3 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 5:5 + Mannitol 3. Tenofovir+ Eudragit RL+ Mannitol 4. Tenofovir+ Eudragit RS+ Mannitol Preparation of Nanoparticles Preparation of nanoparticle was done by simple double emulsion method. [Lamprecht et al (2), Cristina et al (22)] Tenofovir was dissolved in.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 1 RPM for 15 minutes. The time is counted after complete addition of drug to polymer solution. The solution is probe sonicated for 8 minutes (8w). Once the primary emulsion was formed, secondary emulsion is prepared. In another beaker,.5% Poly vinyl alcohol was homogenised with addition of primary emulsion drop by drop at 1 rpm for 15 minutes. The above emulsion was probe sonicated 8 minutes (8w). The secondary emulsion was kept for DCM evaporation for 4 hours at 37 c and checked for odour of solvent at regular intervals. The same procedure was followed for Eudragit-RS and Eudragit-RL polymers. Page 55

4 6.3.3 Separation of nanoparticles After complete evaporation of DCM, the nanoparticles were centrifuged at 5 rpm at 5 c 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 3g for 3 minutes at 5 c. The nanoparticles were dispersed in Mannitol solution in 1:1 ratio (mannitol & Polymer). The sample solution was kept at c for 2 hours and latter freezed at -7 c overnight before proceeding for lyophilisation 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 (5:5) nanoparticles Ingredient Tenofovir (mg) Poly lactic glycolic acid (PLGA) 5:5 (mg) Poly vinyl alcohol (.25%) (ml) Poly vinyl alcohol (.5%) (ml) Poly vinyl alcohol (1.%) (ml) Dichlorometha ne (ml) A 25B 25C Page 56

5 Table 13: Composition for TNF formulation and Eudragit RL-1 nanoparticles Ingredient Tenofovir (mg) Eudragit-RL 1 (mg) Poly vinyl alcohol (.25%) (ml) Poly vinyl alcohol (.5%) (ml) Poly vinyl alcohol (1.%) (ml) Dichlorometh ane (ml) A 3B 3C Table 14: Composition for TNF formulation and Eudragit RS-1 nanoparticles Ingredient Tenofovir (mg) Eudragit-RS 1 (mg) Poly vinyl alcohol (.25%) (ml) Poly vinyl alcohol (.5%) (ml) Poly vinyl alcohol (1.%) (ml) Dichlorometh ane (ml) A 31B 31C Page 57

6 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) 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 25 c. Clear disposable zeta cells are used for measurements were taken in triplicates and mean particle size reported. Thirumala Govender et al (1999) 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. Page 58

7 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 +3mV and - 3mV. Particles outside the above stated range are stable. The Nano formulation is dispersed in water as medium and temperature at 25 c. Clear disposable zeta cells are used for measurements were taken in triplicates and mean particle size reported Percentage yield The percentage yield of the formulation of PLGA (5:5), Eudragit-RL 1 and Eudragit-RS 1 formulation are done by using the formula Percentage yield = Weight of freeze dried nanoparticles (mg) X 1 Weight of drug (mg) + Polymer (mg) + Mannitol (mg) 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 1 mg of Tenofovir from its salt form was weighed and dissolved in 1 ml volumetric flask. The solution was sonicated for 1 minutes and volume made up to mark with mobile phase. The clear supernatant solution was centrifuged for 5 rpm for 5 minutes and injected into the system. Percentage entrapment efficiency is calculated by using the formula, Jin-Wook Yoo et al (211) Page 59

8 EE (%) = Amount of drug in the prepared nanoparticle x 1 Amount of drug loaded in nanoparticle 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 (5:5), Eudragit- RS 1 and Eudragit-RL 1 polymers. The above mentioned parameters help us in understanding the physical characteristic of drug and polymer interactions 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 66 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 2 seconds. The die along with sample was loaded into the system and photographed for its surface morphology Invitro Drug Release Invitro release studies were carried out to nanoparticle formulation by Diffusion method [T. Niwa, H. Takeuchi, T (1992), Einat et al (29)]..1N HCl was taken as dissolution media. Due to very high solubility of drug (13.9 mg/ml). 4 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 5 RPM was used and temperature is maintained at 37 c. Time points were 5, 1, 15, 3, 45, 6, 12, 24, 36, 48, 72, 96, 12 and 144 minutes. Sample were taken at the designated time and replaced with fresh disso media maintained at the same temperature. Detection Page 6

9 of drug release was done at UV wavelength of 26 nm. The experiments are performed in duplicate and the drug concentration is converted into cumulative drug release and results reported 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 (M t /M = kt n), n is the diffusional exponent, k is kinetic constant, M t is the amount drug released at time t, M is amount released at time infinity. Higuchi (Q = Kt 1/2 ) 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 R 2 to assess the best fit model. Srinivas et al (28) 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 4 c + 2 c. all three batches 25, 3 and 31 were stored under the above stated condition as well as at accelerated stability condition of c / 75% + 5% RH for a period of 15, 3,6,9 and 18 days. The samples were analysed for change in physical appearance, entrapment efficiency, particle size and zeta potential. Page 61

10 6.4 Results and Discussion Preformulation studies As mentioned earlier Preformulation studies were carried out of Drug +PLGA (5:5) polymer ( 25 batch), Drug + Eudragit-RL 1 (3) and Drug + Eudragit-RS 1( 31). Drug excipient reaction was studied by FT-IR and DSC 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 25 (PLGA 5:5) 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 (3) the following functional group are found, Alkyl groups below 3cm -1, bend of alkyl CH 3 and CH 2 at 2359 cm -1 and 2332cm -1. Carbonyl group at 1735cm -1 and 1726cm -1.In 3 and 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 DSC interpretation: Thermogram of Tenofovir disproxil fumarate showed a sharp melting transition endotherm ranging from c to c with a peak at c. The DSC Thermogram of formulation TNF 25 (drug + PLGA polymer) showed small melting endotherm at c followed by a sharp endotherm at c indicating decomposition of drug. Thermogram of formulation TNF 3 (drug + Eudragit RS) showed a small melting endotherm at c Page 62

11 followed by an endotherm at 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, c followed by an endotherm at c, with increased intensity. The absence of endothermic peak of the drug at c in the DSC Thermogram of the formulations i.e. TNF 25, TNF 3 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 25 (Tenofovir + PLGA 5:5) 75 %T /cm Figure 18: FT-IR Spectrum of 31 (Tenofovir + Eudragit-RS 1) 1 % T /c 31 m Page 63

12 Figure 19: FT-IR Spectrum of 3 (Tenofovir + Eudragit-RL1) 7 5 % T /c m DSC Spectrum Figure 2: DSC of 25 (Tenofovir + PLGA 5:5) DSC mw 1. TNF + TNF x C x C x C Temp [C] Page 64

13 Figure 21: DSC of 3 (Tenofovir + Eudragit-RL1) DSC mw 1. TNF + TNF xc xc xc Temp [C] Figure 22: DSC of 31 (Tenofovir + Eudragit-RS1) DSC mw 1. TNF + TNF x C x C x C Temp [C] Page 65

14 6.4.4 Formulation Development In the field of nano formulations PLGA (5:5) 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 1 and Eudragit-RS 1 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 2, rpm 5 c 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. 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. 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 1 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 8w/8 amps/ 8 mts. Centrifugation speed was optimised at 3g for 3 minutes at 4 C. 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 Page 66

15 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 Characterisation of Nanoparticles 6.5 Physiochemical characterisation 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 5:5, Eudragit-RL, Eudragit- RS) was comparable and reproducible. The final yield consists of drug, polymer and cryoprotectant Mannitol. Table 15: Percentage yield Batch Number Polymer used in nano formulation PLGA (5:5) Polymer Eudragit-RL 1 Polymer Eudragit-Rs 1 Polymer Total yield 87.14% % % 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 25 optimised formulation is found to be 13.45% In case of 3, Eudragit RL 1 the entrapment was established at 11.55% For 31, Eudragit RS 1 the entrapment was established at 11.12% + Page 67

16 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 (5:5) Polymer + Mannitol Drug: Polymer PVA Batch No Particle Size PDI Zeta Potential % EE 1:1.25.5% :2.5.5% :5.5% :7.5.5% Table 17: Tenofovir + Eudragit-RL 1 + Mannitol Drug: Polymer PVA Batch No Particle Size PDI Zeta Potential % EE 1:1.25.5% :2.5.5% :5.5% :7.5.5% Page 68

17 Table 18: Tenofovir + Eudragit-RS 1 + Mannitol Drug: Polymer PVA Batch No Particle Size PDI Zeta Potential % EE 1:1.25.5% :2.5.5% :5.5% :7.5.5% 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 25 (Tenofovir + PLGA 5:5) batch is found to be 232 nm and poly dispersity index was found to be.348. For Eudragit-RL 1 nano formulation the size was found to be 326 nm and poly dispersity index.351. Eudragit-RS 1 particle size was found to be 187 nm and poly dispersity index a healthy.29. Page 69

18 Figure 23: Particle size of 25 Tenofovir + PLGA (5:5) Polymer Page 7

19 Figure 24: Particle size of 3 Tenofovir + Eudragit-RL 1 Polymer Page 71

20 Figure 25: Particle size of 31 Tenofovir + Eudragit-RS 1 Polymer Page 72

21 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 25 batch containing PLGA (5:5) polymer has zeta potential mv and 3 batch containing Eudragit-RL 1 polymer s has a zeta potential mv, 31 batch containing Eudragit-RS 1 polymer s zeta potential is established at mv. 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 5:5 polymer. Eudragit polymer formulations have a very high value of zeta potential which is well above the specified value of + 3 mv. This value of zeta potential indicates the stability of formulation. Page 73

22 Figure 26: Zeta Potential of 25 Tenofovir + PLGA (5:5) Polymer Page 74

23 Figure 27: Zeta Potential of 3 Tenofovir + Eudragit-RL 1 Polymer Page 75

24 Figure 28: Zeta Potential of 31 Tenofovir + Eudragit-RS 1 Polymer Page 76

25 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 25 Tenofovir + PLGA (5:5) Polymer Figure 3: SEM image of 3 Tenofovir + Eudragit-RL 1 Polymer Page 77

26 Figure 31: SEM image of 31 Tenofovir + Eudragit-RS 1 Polymer 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 -1 and aromatic CH stretching at 351 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

27 Figure 32: 25 Tenofovir Drug + Formulation + PLGA (5:5) Polymer Figure 33: 3 Tenofovir Drug + Formulation + Eudragit RL %T TNF /cm 75 %T %T /cm PLGA /cm Polymer 1 %T TNF /cm 75 %T /cm %T EUDRAGIT /cm Page 79

28 Figure 34: 31 Tenofovir Drug + Formulation + Eudragit RS 1 Polymer 1 %T TNF /cm %T EUDRAGIT RS /cm 75 %T /cm Figure 35: 25 Tenofovir Drug + Formulation + PLGA (5:5) Polymer DSC mw 1. TNF + TNF 25+PLG x C x C x C x C Temp [C] Page 8

29 Figure 36: 3 Tenofovir Drug + Formulation + Eudragit RL 1 Polymer DSC mw 1. TNF + TNF 3 + RS xc xc xc Temp [C] Figure 37: 31 Tenofovir Drug + Formulation + Eudragit RS 1 Polymer DSC mw 1. TNF + TNF 31 + RL xc xc xc Temp [C] Page 81

30 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 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., 28; Mutalik et al., 28). However appearance of the endothermic peaks at c is due to the presence of cryoprotectant, mannitol. This was confirmed by the native thermogram of mannitol 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. 25 (PLGA 5:5 polymer) has faster release than 3 & 31 (Eudragit RL 1 & RS 1). Tenofovir has a very high solubility in water (13.46 mg/ml), and it goes into the solution at a rapid rate. In 25 the drug release rate was high from initial time point and at 6 minutes it was 85% TNF 3 the drug release rate was 64% TNF 31 batch the drug release rate was 59% 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 25 drug released was 11% in four hours and the concentration remained constant throughout indicating its complete release from the polymer. In case of TNF 3 batches the drug release was 1% in 8 hours more than double the time taken for PLGA polymer formulation. 31 batch drug release was 1% 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

31 Figure 38: Invitro drug release profile of 25 (PLGA 5:5) polymer 12 D r u g R e l a s e Time in Minutes Figure 39: Invitro drug release profile of 3 (Eudragit RS 1) polymer D r u g R e l e a s e Time in Minutes Figure 4: Invitro drug release profile of 3 (Eudragit RS 1) polymer 12 D r u g R e l a s e Time in Minutes 1 15 Page 83

32 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 Zero order (R 2 ) First order (R 2 ) Higuchi model (R 2 ) Krosmeyers-Peppas (n) Model fit Higuchi Higuchi Higuchi Stability Studies Stability studies were conducted as per the ICH guidelines. The results of optimised nano formulation of 25, 3 and 31 are performed at accelerated stability conditions c / 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 25 batch. Poly dispersity index was well within.5 value so the optimised formulation was homogenous. In 3 batch there was fluctuation in particle size but it did not cross more than 4 nm. Zeta potential was well outside the +3mV value which indicates the stability of formulation. Poly dispersity index was well within the limit but showed a slight increase in 6 months data. In 31 batch, there was steady increase in particle size but till the last 6 months it has not crossed 4 nm which is good for oral formulation. Poly dispersity index was also well within the limit with one value going above.5. Zeta potential was outside+3mv but in 6 months the zeta potential has come Page 84

33 down to + 19mv. which was a good value which indicates the stability of formulation. Table 2: Stability data of 25 Tenofovir + PLGA (5:5) Polymer at c / 75% + 5% RH (n = 3) Time (Months) % Encapsulation Efficiency Particle Size (nm) PDI Zeta Potential Figure 41: Log % Drug remaining against time plot of 25 at c / 75% + 5% RH L o g % R e m a i n i n g y = -.15x + 2 R² = Time in Months Table 21: Stability data of 3 Tenofovir + Eudragit-RL 1 Polymer at c / 75% + 5% RH (n = 3) Time (Months) % Encapsulation Efficiency Particle Size (nm) PDI Zeta Potential Page 85

34 Figure 42: Log % Drug remaining against time plot of 3 at c / 75% + 5% RH L o g % R e m a i n i n g y = -.13x R² = Time in Months Table 22: Stability data of 31 Tenofovir + Eudragit-RS 1 Polymer at c / 75% + 5% RH (n = 3) Time (Months) % Encapsulation Efficiency Particle Size (nm) PDI Zeta Potential Figure 43: Log % Drug remaining against time plot of 31 at c / 75% + 5% RH L o g % R e m a i n i n g y = -.12x R² = Time in Months Page 86