Formulation development and in vitro evaluation of Valsartan loaded lipid nanoparticles

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1 Arunprasath.et al Volume (), 0, Page 667 International Journal of Medicine and Nanotechnology Access online at Original Article Formulation development and in vitro evaluation of Valsartan loaded lipid nanoparticles B Arunprasath, *, S A Sreenivas, K V Subrahmanyam, Janardhanan, Jeevitha,Shruthi,Sukanya Department of Pharmaceutics, Samskruti College of Pharmacy, Ghatkesar (M) Telangana, India Sri Dattha Institute of Pharmacy, Ibhrahimpatnam, Telangana, India. Article Received on: 9070 Accepted on: 9080 ABSTRACT In the present study three different types of lipids Dynasan, Dynasan 6 and Dynasan 8 were used at %, % and % concentrations to provide the solid lipid nanoparticles. The SLN were prepared by hot homogenization technique followed by ultrasonication. The process variables like homogenization time, sonication time and the formulation variables like polysorbate 80 concentration, soyalecithin concentration on particle size were optimized. The homogenization time was optimized to min and sonication time was optimized to min. The polysorbate 80 concentration was optimized to % and the soyalecithin concentration was optimized to %. After months of storage at different temperatures, the particle size of the SLN dispersions remains the same (< m), which emphasis the physical stability of these lipid particles. The entrapment efficiency and the drug release profile depend upon the concentration of the lipid employed. The in vitro drug release was found to be 96.8% at the end of hrs. The mathematical modeling again confirmed that the steady release of the drug is by following zero order kinetics with diffusional mechanism. The optimized SLN formulation F9 has got a drug content of 0.9mg/ml and the entrapment efficiency of 89.60%.The optimized formulation F9 showed good drug release of 9.8 during ex vivo permeation studies at the end of hrs. Carbopol 9 has been used to formulate the gels. The carbopol allows the drug to be easily dispersed in it due to its less hardness. The prepared gels were found to have good homogeneity, ph of 6.8 and good consistency of 7.mm and spreadability of.99 g.cm/sec. The aqueous dispersion as well as the hydrogel formulation described in this thesis was easy to manufacture and have shown good drug release and rheological properties and they may be potential for the topical delivery of Valsartan. Keywords: Dynasan, polysorbate 80, Carbopol 9, hydrogel, Valsartan. Corresponding Author B.Arunprasath * Department of Pharmaceutics, Samskruti College of Pharmacy, Ghatkesar (M) Telangana, India arunprasad0@gmail.com INTRODUCTION The main objective of the study was to assess the feasibility of formulating SLNs loading Valsartan by hot homogenization technique to prolong the drug release and reduce the toxicity or adverse effects and thereby improve the therapeutic efficiency and bioavailability of the drug. Further IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 66

2 Arunprasath.et al Volume (), 0, Page 667 goal of the study was to investigate the role of drug SLN in disease states. The investigation included the formulation and optimization of the physicochemical properties of the colloidal system Valsartan loaded SLNs such as particle size, zeta potential, and drug loading capacity, drug entrapment efficiency and in vitro and ex vivo drug release behavior. The following characters can also be achieved such as Production of particles in the size range of 80000nm, Controlled release and targeting of the drugs by Modification of dissolution rate of incorporated drugs, and thus enhancement of bioavailability for poorly water soluble molecules, which gives protection of chemically sensitive compounds in the gut and simultaneously absorption enhancement,improve the drug stability, Reduce the biotoxicity of the carrier, high drug payload., MATERIALS AND METHODS Valsartan was procured as a gift sample from Sasol, Witten, Germany Trimyristin (Dynansan ), Tripalmitin ( Dynasnan 6) and Tristearin (Dynansan 8) purchased from Hi Media Laboratories, Mumbai Experimental Methods Calibration Curve for Valsartan : Valsartan drug equivalent to 00mg was taken and dissolved in 00ml of 0.N NaOH solution. The solution was filtered and was further diluted to get 0,0,0,0 and 0 g/ml. The absorbance of solutions was determined at 0 nm using 0.NNaOH as blank. Formulation of SLN Hot Homogenization followed by sonication technique: Valsartan (0.%w/v), Lipid (Dynasan / Dynasan 6/ Dynasan 8) and Phosphatidyl choline were dissolved in 0ml of : mixture of cholorform and methanol. Organic solvents were completely removed and drug embedded lipid layer was melted by heating at 700C. An aqueous phase was prepared by dissolving the polysorbate 80 (%w/v) in double distilled water at 700C. Hot aqueous phase was added to the oil phase and homogenization was carried out at 8000rpm for min. Coarse hot oil in water nanoemulsion obtained was ultrasonicated for min. V SLN was obtained by allowing hot nanoemulsion to cool to room temperature. Optimization of Process Variables Effect of homogenization time on particle size: Homogenization was carried out for different time periods (min) and analysed for particle size. Effect of ultrasonication time on particle size: Sonication was carried out for 0,, and 0mins and measured for particle size. Optimization of Formulation Variables Effect of Polysorbate 80 Concentration: Various concentrations of polysorbate 80 (%, %, %) were prepared and analyzed for the particle size. Effect of Soyalecithin Content Various concentrations of Soyalecithin (%,.%, %) containing SLN were prepared and and the particle size was measured and examined for physical stability by storing at room temperature. Preparation of Optimized formulation Nine different optimized SLN formulations of Valsartan were prepared by homogenization followed by ultrasonication method. The concentrations of various ingredients taken for preparation of different SLN formulations. Measurement of particle size of SLN Average size and polydispersity index of different formulations of VSLN were measured by Photon Correlation Spectroscopy (PCS) using Malvern zetasizer at fixed angle of 90 at room temperature. The SLN dispersions were diluted :00 with the aqueous phase of the formulation to get a suitable Kilo counts per second (Kcps) Estimation of Drug content 0. ml of formulation was diluted to 0 ml with chloroform: methanol ( :) using standard volumetric flask. Final dilution was made with 0. N sodium hydroxide and the samples were analyzed spectrophotometrically at 0 nm. Entrapment efficiency ml of SLN equivalent to 0mg of the drug was taken and diluted with ml of 0.N NaOH. ml was taken from the solution and diluted to ml. The absorbance was measured UV spectrometerically at 0nm. Scanning Electron Microscopy (SEM) IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 67

3 Arunprasath.et al Volume (), 0, Page 667 The sample for the SEM analysis were prepared by applying a monolayer of the SLN dispersion on to one side of the electron microscope brass stub and the stubs were then coated with gold in an ion sputter (JSMT0A, JEOL, Japan). The pictures were taken with random scanning of the stub., IR Spectroscopy IR spectroscopic studies were carried out for Valsartan, Dynasan 8 and physical mixture of Valsartan and Dynasan 8 using Spectrum one, FTIR Spectrophotometer and these IR spectrum were compared to the assess the compatibility of the excipients with Valsartan. In Vitro Diffusion studies 6 In vitro diffusion studies were performed using Franz Diffusion Cells with a surface area of.cm. Dialysis membrane having pore size.nm, molecular weight cutoff between,000 and,000 was used. Membrane was soaked in doubledistilled water for h before mounting in a Franz diffusion cell. ml of ValsartanSLN formulation was kept in the donor compartment and the receptor was filled with a dialysis medium of 0 ml of Phosphate buffer ph 6.8. A temperature of 70C ± 0C was kept and a uniform distribution was maintained by using magnetic stirring through out the experiment. At fixed time intervals, ml of the sample was withdrawn from receiver compartment through side tube and the same volume of aliquots was replaced immediately to maintain sink condition. The withdrawn samples were suitably diluted and the absorbance of the solution was determined at specified wavelength of 0 nm. All experiments were done in triplicate. Ex vivo permeation study Franz diffusion cell with an effective diffusion area of. cm was used for in vitro permeation studies. The skin samples were mounted carefully on diffusion cells with the stratum corneum side up, donor solutions consisting of ml of SLN containing 0 mg Valsartan. The receiver compartment was filled with 0 ml phosphate buffer of ph 7. and its temperature was maintained at 7 ± 0.OC with magnetic stirring at 600 rpm throughout the experiment. For each experiment, ml of the receiver medium was withdrawn at predetermined time and then the volume was made up with the equal volume of fresh receiver medium. All samples were filtered a 0. µm pore size cellulose membrane filter and analyzed by UVVIS Spectrophotometer (Shimadzu). The drug release at hours was noted. Stability Studies All the formulations were subjected to a stability testing for three months at a temperature of C± C and C± C. It was analyzed for the particle size. 7 Formulation of SLN based Gel To the formed VSLN, % of carbopol 9 which was previously neutralized with Triethanolamine was slowly added and was stirred at 00 rpm using mechanical stirrer (Remi, India). After the formation of clear SLN based hydrogel, it was stored and was evaluated. 8, 9 Evaluation of hydrogel containing VSLN Rheological studies on the gels Brookefield SynchroLectric Viscometer ( Model LVE) with helipath stand was used for rheological studies. The sample (0 g) was placed in a beaker and was allowed to equilibrate for min before measuring the dial reading using a TC spindle at 0 to 00 rpm of angular velocity. At each speed, the corresponding dial reading on the viscometer was noted. The spindle 6 speed was successively lowered and the corresponding dial reading was noted. The measurements were carried in duplicate at ambient temperature. Direct multiplication of the dial readings with factors given in the Brookfield viscometer catalogue gave the viscosity in centipoises. Spreadability Test 0.g of the gel was placed within a circle of cm diameter premarked on the glass plate over which a second glass plate was paced. A weight of 00g was allowed to rest on the upper glass plate for minute. The increase in diameter due to spreading of the gel was noted. ph The ph of the various gel formulations was determined by using digital ph meter (ElicoSL 999Ap/09) IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 68

4 Arunprasath.et al Volume (), 0, Page 667 Consistency The measurement of consistency of the prepared gels was done by dropping a cone attached to a holding rod from affixed distance of 0cm in such way that it should fall on the center of the glass cup filled with the gel. The penetration was measured from the surface of the gel to the tip of the cone in side the gel. The distance traveled by cone was noted down after 0sec. Homogeneity All developed gels were tested for homogeneity by visual inspection after the gels have been set in the container. They were tested for their appearance of any aggregates. RESULTS AND DISCUSSION Standard Curve of Valsartan FTIR spectroscopy studies have been done in order to ensure that no chemical interaction between the drugs and the polymers had occurred. From the FTIR interpretation the following results were obtained. The FTIR of pure drug Valsartan show intense band at 96cm, 7 cm, 60 cm, 70 cm, 0 cm, cm corresponding to the functional groups methyl asymmetric/ symmetric stretch, aldehyde, amino, methyl asymmetric / asymmetric bend. The peaks obtained in the FTIR of the physical mixture of Valsartan and Dynasan 8 at 80 cm, 76 cm, 60 cm, 7 cm,80 cm. The pure Dynasan 8 shows intense bands at 97 cm, 89 cm, 77 cm, 7 cm,80 cm and the physical mixture of Valsartan and Dynasan 8 shows intense bands at 97 cm, 80 cm, 76 cm, 7 cm, 80 cm.from the above interpretation it was found that there was no major chemical interaction between the Valsartan and Dynasan 8 which were used in the formulation. The FTIR of pure drug Valsartan, Dynasan 8 and the corresponding physical mixtures. Effect of homogenization time on particle size Average particle size after homogenization is shown in table 6. As the homogenization time increased, the particle size was decreased and least particle size (. m was obtained at the end of min of homogenization with PI of 0.. There was no decrease in particle size with further increase in homogenization time. Thus the homogenization time was optimized to min. The PI too decreased with increase in homogenization time. During initial stages of homogenization, the concentration of larger particles is considerably high and there was less frequency of collision between the particles. As the number of shear applications increased, the larger particle was reduced and the number of smaller particles increases and reaches the ideal size. Further homogenization produces higher incidence of particle collision and causes increase in particle size by coalescence due to insufficient coverage of particles by surface by phospholipids. Table : Effect of homogenization time on particle size SL. Homogenization Average particle size ( m) No time (min).±0..0±0..±0..0±0..07±0. PI Table : Effect of Polysorbate 80 concentration on particle size of Valsartan SLN SL. No Concentration Particle PI Zetapotential of polysorbate 80 Size (%) 8.7 (nm) ± ± ± (mv) Table : Effect of Soyalecithin concentration on particle size SL. No Concentration of Soyalecithin (%) Particle size PI. 0.7 (nm) ± ± ± Effect of Sonication time on particle size Average particle size after sonication is shown in table 7. As the sonication time increased, the particle size was decreased and the least particle size (8.) was obtained at the end of min. A small increase in particle size was seen with further increase in sonication time. Thus the sonication IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 69

5 Arunprasath.et al Volume (), 0, Page 667 time was optimized to min. The PI was found to be decreased with increase in sonication time. Table : Formulation of Valsartan SLN SL Ingredients (%) Valsartan solid lipid nanoparticles No 6 7 Valsartan Dynasan Dynasan 6 Dynasan 8 Phosphotidyl choline Polysorbate 80 F F F F F F6 F7 F8 F Table : Assay and entrapment efficiency SL. NoFormulation Average drug F F F F F F6 F7 F8 content(mg/ml) Average entrapment efficiency (%) 8.±0.8 8.± ± ± ± ± ± ±0.99 Table 6: Measurement of Particle size and zeta potential of Optimized formulation SL No Formulation Particle Size (nm) Zeta potential (mv) F F F F F F6 F7 F8 F9 0.6 ± 0.. ± ± ± ± ± 0..7 ± ± ± Effect of Polysorbate 80 concentration on particle size of Valsartan SLN As the concentration of polysorbate 80 was increased, the particle size was decreased with optimized homogenization time and sonication time. The zeta potential was increased with increased polysorbate 80 concentrations. Even though a high zeta potential can provide an electric repulsion, Tween 80 also provide a steric stability for maintaining the stability of SLN. The PI was decreased with increase in concentration of the polysorbate 80 with optimized homogenization time and sonication time. Table 7: Average percentage of Drug release in phosphate buffer (ph 7.) from VSLN Percentage of drug Time (hrs) 6 release 8(%) F.7.± 0.98± 8.99±.± 0.60± 87.7± F F F F F6 F7 F8 F9 ± ± ± ±0..6 ± Fig : FTIR of Physical mixture of Valsartan and Dynasan 8 ±0. 7.6±.9±.±.0±.0± 96.8±. ± ±.8 Fig : Effect of homogenization time on particle size ± ± 0.99.± 0.09.±.07.± ± ±.±.9.± ± 0..±. 0.7± 0.98.± ±.8.± ± 0. 0.± ± 0.8.7± ± ± 0.9± ± ± 0.07.± ± ± ± 0..98± ± ± ± 0. 8.± ±.09 6.± 8.96± ± ± ± ± ± ±. 8.± 0.9 IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 70

6 Arunprasath.et al Volume (), 0, Page 667 Fig : Effect of polysorbate 80 concentration on polydispersity index Fig : Scanning Electron Microscopy of Valsartan SLN Fig : Comparison of In vitro drug release of VSLN and VSol Formulation of Valsartan SLN In the present study, we have developed an economical, simple and reproducible method for the preparation of SLN, i.e. homogenization followed by ultrasonication at above the melting point of the lipid. To disperse the Valsartan homogeneously in the lipid, solvent system chloroform/methanol (:) was used. Homogenization time was optimized to min. There was no decrease in particle size with further increase in homogenization time. To reduce the particle size below m, probe ultrasonicator was used. A min sonication was necessary to obtain solid lipid nanoparticles below m. Assay and Entrapment Efficiency Due to the lipophilic nature of the Valsartan, a high amount of Valsartan could be incorporated into the SLN. The assay results showed that the concentration of Valsartan ranges from 0.79 to 0.9 mg/ml for different optimized formulations and the entrapment efficiency varied from 8.% to 89.60%. The values of entrapment efficiency and assay range were given in table. In vitro Drug Release The cumulative drug release for nine different formulations of Valsartan was investigated for a period of hrs. Each sample was analyzed in triplicate. The release rate depends on the total concentration of drug in the formulation. The cumulative percentage drug release from formulation F to F9 were found to be 87.7%, 8.%, 8.0%, 80.%, 8.%, 88.%, 89.%, 8.% and 96.8% respectively. The prolonged release in the later stage can be attributed to the slow diffusion from the lipid matrix. This is probably caused by the release of the drug adsorbed on the nanoparticulate surface or precipitated from the superficial lipid matrix.the formulation F9 was regarded as a optimal preparation due to the fact that, it has highest drug release (96.8%) comparing to all the other formulations which inturn was due to the optimized homogenization and sonication time. In vitro Drug Release Kinetics The kinetic model of all the formulations have been given in the table. In order to describe the kinetics of the drug release from SLN preparation, mathematical modeling have been proposed, where zero order model describes the systems were independent of the concentration and the first order describes the systems were concentration dependent, while Higuchi model tells us about the mode of the release and a semi emperical equation was proposed to describe the drug release mechanism which is so called Korsmeyerpeppas law. The kinetic treatment reflected that the release data of the optimized batch (F9) showed R value of 0.9 and 0.9 for the zero order and first IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 7

7 Arunprasath.et al Volume (), 0, Page 667 order respectively, indicating that zero order and first order kinetics, fitted at these points showing statistically significant fit to this model. Furthermore Korsemeyerpeppas model described drug release kinetics in the most befitting manner, where the values of the diffusional exponent n obtained from the slopes of the fitted Korsemeyerpeppas model was found to be (n=.6) which is above, indicating that drug release follows supercase transport. The goodness of the fit was in the order of Zero order > First order > Higuchi > Korsmeyer peppas. Thus the results unequivocally point out the prevalence of diffusional mechanism with zero order release. Ex vivo Permeation Studies The formulation F9 had shown the maximum drug release through the membrane was further studied for drug release by using rat skin. The ex vivo skin permeation of VSLN were shown in table. Valsartan SLN exhibited 96.8% of cumulative percentage of drug permeation in h. The results of the drug permeation from F9 through the rat skin confirmed that Valsartan was released and permeated through the rat skin and hence could possibly permeate through the human skin. Effect of Stability Studies on Entrapment Efficiency After three months of storage at room temperature, the entrapment efficiencies were found to be lowered from 89.60% to 8.6%. Transitions of dispersed lipid from metastable forms to stable form might occur slowly on storage due to small particle size and the presence of emulsifier that may lead to drug expulsion from solid lipid nanoparticles. Therefore lowered entrapment efficiency observed on storage may be due to drug expulsion during lipid modification. Effect of Stability Studies on particle size Stability studies were carried out at 0C and at room temperature. After storing for months at room temperature and 0C, there is no much difference in the size of the particles (< m). This indicates that the VSLN dispersion is stable which is due to the stability of the lipid particles. Evaluation of Valsartan SLN enriched hydrogels Homogeneity, Spreadability, Consistency and ph Nine different formulations of hydrogels have been prepared by incorporating various SLN formulations into the gels. The homogeneity of all the formulations were good. Spreadbility is an important property of topical formulation from the patient compliance point of view. The diameter was found to be.90g.cm/sec for F9G. The formulations F9G showed good consistency of 7.mm. The ph of all the hydrogel formulations was found to be equivalent to the dermal ph. For the optimized formulation F9G ph was 6.8. Rheology profile of various VSLN enriched hydrogels Carbopol 9 has been used in order to increase the viscosity of the VSLN based hydrogel. As the angular velocity was increased from 000, the viscosity of the formulations decreased for all the formulations. At high shear rate, carbopol particles as a whole and polymer chain segments deform and align themselves in the direction of the flow and the gels exhibit pseudoplastic behaviour. The viscosity of the F9G formulation has decreased from 99 to 9 as the angular velocity increased. This showed that the gel is a shear thinning system. CONCLUSION The success of a new developed pharmaceutical formulation is related to the fact that it is able to deliver the active substance to the target organ at therapeutically relevant levels, with negligible discomfort and side effects, increasing the patient compliance to the therapeutics. Regarding this respect, the route of administration is of major relevance. Topical administration of active substances offers several attractions compared to traditional routes, e.g. it avoids the hepatic firstpass metabolism, it has the potential of longterm controlled release with avoidance of the topical peakthrough plasma profiles associated with frequent dosage regiments, it is easy to administer and it has the possibility for treatment withdrawal if that is the case. Nevertheless, topical administration route is the main target when the skin itself is the damaged organ. In the present study three different IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 7

8 Arunprasath.et al Volume (), 0, Page 667 types of lipids Dynasan, Dynasan 6 and Dynasan 8 were used at %, % and % concentrations to provide the solid lipid nanoparticles. The SLN were prepared by hot homogenization technique followed by ultrasonication. The process variables like homogenization time, sonication time and the formulation variables like polysorbate 80 concentration, soyalecithin concentration on particle size were optimized. The homogenization time was optimized to min and sonication time was optimized to min. The polysorbate 80 concentration was optimized to % and the soyalecithin concentration was optimized to %. After months of storage at different temperatures, the particle size of the SLN dispersions remains the same (< m), which emphasis the physical stability of these lipid particles. The entrapment efficiency and the drug release profile depend upon the concentration of the lipid employed. The in vitro drug release was found to be 96.8% at the end of hrs. The mathematical modeling again confirmed that the steady release of the drug is by following zero order kinetics with diffusional mechanism. The optimized SLN formulation F9 has got a drug content of 0.9mg/ml and the entrapment efficiency of 89.60%.The optimized formulation F9 showed good drug release of 9.8 during ex vivo permeation studies at the end of hrs. Carbopol 9 has been used to formulate the gels. The carbopol allows the drug to be easily dispersed in it due to its less hardness. The prepared gels were found to have good homogeneity, ph of 6.8 and good consistency of 7.mm and spreadability of.99 g.cm/sec. The aqueous dispersion as well as the hydrogel formulation described in this thesis was easy to manufacture and have shown good drug release and rheological properties and they may be potential for the topical delivery of Valsartan.. Westesen K, Bunjes H, Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J Controlled Release 997;8:.. Soukharev AR, Muller RH. Chemical stability of lipid excipients in SLN and shortterm stability. Pharmazie 006;6:0.. Schubert MA, MüllerGoyman CC. Solvent injection as a new approach for manufacturing lipid nanoparticles evaluation of the method and process parameters. Eur J Pharm Biopharm 00;:.. Jenning V, Thunemann AF, Gohla SH, Int J Pharm 000;99; Weiss J, Helgason T, McClements DJ, Kristbergsson K, Decker E, Awad T. Solid lipid nanoparticles as delivery systems for bioactive food components. Food Biophys 008;:6. 7. Muller RH, Lucks JS. Arzneistofftra ger aus festen Lipidteilchen, Feste Lipidnanospha ren (SLN). Eur Patent No. EP Müller RH, Runge SA. Solid lipid nanoparticles for controlled drug delivery. In: Benita S, editor. Submicron emulsion in drug targeting and delivery. Netherlands: Harwood Academic Publishers; 998. p Jores K, Mehnert W, Dreschler M, Bunjes H, Johann C, Karsten M. Investigations on the structure of solid lipid nanoparticles (SLN) and oilloaded solid lipid nanoparticles by photon correlation spectroscopy, fieldflow fractionation and transmission electron microscopy. J Control Release 00;9: 7. REFERENCES. Muller RH, Keck CM. Challenges and solutions for the delivery of biotech drugs a review of drug nanocrystal technology and lipid nanoparticles. J Biotech 00;:70. IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 7