Research Article

Received: 20 April 2013, Accepted: 29 April 2013

ISSN: 2321-2969

Int. J. Pharm. Biosci. Technol.

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International Journal of Pharma Bioscience and Technology; Volume 1, Issue 1, May 2013, Pg 27-33

Journal home page: www.ijpbst.com

SOLID LIPID NANOPARTICLES OF ETOPOSIDE USING SOLVENT EMULSIFICATION DIFFUSION TECHNIQUE FOR PARENTERAL ADMINISTRATION
Clara B. Fernandes, Sagar Mandawgade, Vandana B. Patravale *
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai 400019, Maharashtra, India. Corresponding Author* E-mail address- vbp_muict@yahoo.co.in

ABSTRACT: In this investigation, solid lipid nanoparticles were formulated for parenteral administration of etoposide. For this purpose, solvent emulsification diffusion technique in a saline medium was employed. The influence of process variables such as solvent concentration, dilution volume and stabilizer concentration was studied. The optimized formulation was characterized for parameters such as particle size, polydispersity index, zeta potential, drug content, entrapment efficiency and pH measurement. The in vitro erythrocyte toxicity study revealed the parenteral acceptability of the developed formulation. Additionally, acute toxicity study established the safety of the lipid for parenteral administration. Overall, the results suggest the potential use of developed formulation for parenteral delivery of etoposide. Key words: Etoposide, Solid lipid nanoparticles, Parenteral, Acute toxicity INTRODUCTION Etoposide (Fig. 1), an epipodophyllotoxin anticancer molecule is found to be effective against small cell lung carcinoma, germ cell tumors, hematologic and other types of malignancies [1]. Its prime mechanism of action is inhibition of topoisomerase-II and activation of oxidation reduction reactions to produce derivatives that bind directly to DNA and cause DNA damage [2]. Although effective, the usefulness of etoposide therapy is limited by its low solubility in water, chemical instability in aqueous solutions, and severe side effects such as hypotension, anaphylaxis, and bronchospasm. The parenteral administration of etoposide involves dilution of etoposide formulation in the infusion fluid to the concentration of 0.20.4 mg/mL of etoposide and slow infusion over a period of 30 /60 min. This low concentration and slow administration is essential to avoid the risk of precipitation and hypotension, respectively. [3] Besides this, the commercially available parenteral formulation comprises of 20 mg/ml etoposide in nonaqueous vehicle including 2 mg citric acid, 30 mg benzyl alcohol, 80 mg Fernandes et al polysorbate 80, 650 mg polyethylene glycol 300, and 30.5% (v/v) alcohol.[4] Most of these excipients are responsible for side effects such as pain, inflammation, tissue damage, necrosis at the site of injection, and substantial hemolysis.[5] These issues have prompted the renewed interest in the development of formulation which is parenterally safe, robust and stable to dilutions

Fig 1: Chemical Structure of Etoposide

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Int. J. Pharm. Biosci. Technol. Few of the approaches employed for parenteral formulation of etoposide include long-/mediumchain triglycerides-based lipid emulsion [6], pegylated parenteral emulsion (PE) [7], submicroemulsion [8-9], phospholipid-based microemulsion [3], liposomes [10],solid lipid nanoparticles [2,11-12], poly(lactic-co-glycolic acid) (PLGA) and Polycaprolactone (PCL) nanoparticles [13]. Among these formulations, solid lipid nanoparticles (SLNs) have emerged as versatile systems for parenteral drug delivery which can provide sustained release of drug thereby reducing the frequency of drug dosing. Also, they can confer protection against metabolizing enzymes; pH based degradation [14] and further, they have shown to enhance the efcacy and residence time of the cytotoxic drugs with concomitant reduction in the side-effects associated with them. [15] Therefore, the work discussed herein investigates the potential of solid lipid nanoparticles (SLN) of etoposide for parenteral delivery. Further, the work entails the acute toxicity study of the novel lipid for parenteral delivery. Miscibility studies The study involved the assessment of the miscible nature of solid lipid Softisan601 with the parenterally acceptable surfactants. Miscibility of solid lipid and surfactants was ascertained visually for phase separation in the ratio of 1:1. Formulation development Methodology Typically, a weighed amount of etoposide was solubilized in lipid melt. Thereafter, the surfactant and water saturated benzyl alcohol was incorporated, followed by warm water (maintained at temperature above the melting point of the lipid). The mixture was stirred at 2500 rpm for 1 min to obtain o/w emulsion. Following which, the resultant emulsion was diluted with warm 0.9% w/v saline and gradually cooled to room temperature to obtain solid lipid nanoparticles of etoposide under stirring. Optimization parameters Effect of solvent concentration MATERIALS AND METHODS Materials Etoposide was gift sample from Khandelwal Lab, India. Softisan601 (mixture of glyceryl cocoate, hydrogenated coconut oil and ceteareth-25) was a generous gift sample from Sasol Germany GmbH. Polysorbate 80 (Tween 80), benzyl alcohol, methanol, ethanol (99%), concentrated hydrochloric acid, disodium phosphate, potassium dihydrogen phosphate and sodium chloride, were purchased from s.d. Fine chemicals. Lutrol F127 was procured from BASF, India. All other chemicals were of AR grade. Double distilled water filtered through 0.45 m membrane was prepared freshly whenever required. Preformulation studies Apparent solubility studies The fixed amount (1g) of surfactant/lipid was weighed accurately and transferred to a small test tube. In this test tube, the drug was added to assess the equilibrium solubility of the drug at the end of 24 h at ambient condition of 25 to 27C. The solubility was established by visual estimation of the samples for transparency and quantification using UV-visible spectroscopy. Thereafter, the amount of surfactant/oil required to solubilize the drug was determined. In this study, the concentration of the solvent; benzyl alcohol in the coarse dispersion was varied from to 10-20% w/v and observed visually for increase in clarity of the dispersion on dilution. Effect of dilution volume In this study, the coarse dispersion was diluted to 10 ml, 25 ml, 50 ml, 75 ml, 100 ml and observed visually for increase in clarity of the dispersion on dilution. Effect of stabilizer concentration In this study, the stabilizer concentration was varied from 0.5 2 % w/v and observed visually for increase in clarity of the dispersion on dilution. Characterization of the SLN dispersion Particle size determination Photon correlation spectroscopy (PCS) using laser light scattering is frequently used to determine particle size of colloidal system. A Beckman N4 Plus submicron Particle Size Analyzer was employed to monitor particle size of the SLN dispersion. The instrument calculated the mean particle size and polydispersity from intensity, assuming spherical particles. Light scattering was monitored at 90 scattering angle and temperature of 25C. Prior to analysis, the formulation was suitably diluted with double distilled water filtered through membrane filter of pore size 0.22 m. The measurements were done in triplicate. Pg. 28

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Int. J. Pharm. Biosci. Technol. Drug content SLN dispersion (equivalent to 20 mg of drug) was taken in a 10 ml volumetric flask, and suitably diluted to 10 ml with methanol. Following which, etoposide was extracted from SLN dispersion by subjecting the solution to sonication for 5 min. Then, 1 ml of this solution was suitably diluted to 50 ml using methanol. The drug content of the resultant solution was determined in triplicate at max of 283 nm using developed UV-Vis spectrophotometric method. This procedure was repeated for placebo, to evade any interference from the excipients. Both the solutions were analyzed using water as blank. Drug Encapsulation Efficiency For the quantitative determination of etoposide, 1 ml of the SLN dispersion containing drug was subjected to centrifugation at the speed of 14000 rpm for about 30 min. The supernatant obtained after centrifugation was analyzed for drug content using the similar procedure as mentioned for drug content. Similar, procedure was repeated for SLN dispersions without drug. Entrapment efficiency was calculated using following equation; Winitial drug Wfree drug Entrapment Efficiency = ------------------------ 100 Winitial drug where, Winitial drug is the mass of initial drug added, Wfree drug is the mass of free drug analyzed in the supernatant after centrifugation. pH Measurement The pH of formulation was measured before and after autoclaving by Systronic Digital pH meter 335, standardized using pH 4.0 and 7.0 standard buffers. In Vitro erythrocyte toxicity study The erythrocyte toxicity assay was conducted as described by Bock et al. [16]. Fresh blood was collected in the vial containing ethylene-diaminetetraacetic acid (EDTA). Red blood cells (RBCs) were isolated by centrifugation (5,000 rpm for 5 min) and the RBCs were washed three times with isotonic phosphate buffer pH 7.4 before diluting with buffer to prepare erythrocyte stock dispersion (three parts of centrifuged erythrocytes plus 11 parts buffer). The washing step was repeated in order to remove debris and serum protein. A 100 l aliquot stock dispersion was added per ml of test sample. The resulting solution was incubated at 37C for a period of 1 h. After incubation under shaking, debris and intact Fernandes et al erythrocytes were removed by centrifugation. One hundred ml of resulting supernatant was added to 2 ml of an ethanol/HCl mixture [(39 parts ethanol (99% v/v) + 1 part of HCl (37% w/v)]. This mixture dissolved all components and avoided the precipitation of hemoglobin. The absorbance of the mixture was determined at 398 nm by spectrometer monitoring against a blank sample. Control sample of 100% lysis (in water x 100) was employed as standard in the experiment. The percentage of hemolysis caused by the test sample was calculated by following equation: Hemolysis caused by sample (%) = (Absorbance of the test sample-Absorbance at 100% lysis) x 100. Acute toxicity study Toxicity status of excipients is a major issue for the use of a delivery system. In this research work, the lipid considered for the study has not been recommended for parenteral administration. Hence, toxicity study was undertaken in accordance to OECD guidelines to assess its safety for parenteral route. The experimental protocol was approved by the Institutional Animal Ethical Committee. In accordance to the Organization for Economic Co-operation and Development (OECD) 425 guidelines, the acute parenteral toxicity of Softisan 601 was determined as lethal dose (LD50). For acute parenteral toxicity studies, female Swiss albino mice weighing 2025 g (1012 week) were used. Throughout the experiments, the animals were fed with a standard mice diet and were provided with clean drinking water adlibitum. Animals were divided into six groups comprising of 5 animals each; Group I: Control, Group II: Dose 5mg/Kg, Group III: Dose 50 mg/Kg, Group IV: Dose 300 mg/Kg, Group V: Dose 500 mg/Kg, Group VI: Dose 2000mg/Kg The animals of Group I-VI were parenterally administered lipid emulsion at the respective dose. The animals were observed at regular intervals on day of dosing and once daily thereafter for 15 days. Following observations pertaining to any gross change in the activity and behavioral pattern; presence of tremors, convulsions, salivations, diarrhea and lethargy was noted. Additionally, the food consumption, body weight and mortality were recorded. RESULTS AND DISCUSSION For the formulation of SLNs, the selection of solid lipid is the most critical aspect of the formulation. The basis of selection of lipid was governed by the solubility of the drug in the lipid melt. Besides this, Pg. 29

Int. J. Pharm. Biosci. Technol. surfactants and cosurfactants, an integral aspect of this formulation, were investigated based on the aforesaid criterion. As depicted in the Fig. 2(a-b), the drug was found to soluble to more extent in the surfactant as compared to the lipid. This poor solubility of etoposide could be ascribed to its high melting point (240-250C), an indicative factor of strong crystal lattice energy; a possible reason for lower ideal solubility and hence poor aqueous solubility. [17] Furthermore, the etoposide molecule shows presence of OH groups which impart hydrophilic nature to some extent. Together, these contrasting reasons could possibly be the contributing factors to poor solubility in lipid, whereas relatively better solubility in surfactant.

Apparent solubility of etoposide (mg/ml)

Apparent solubility of etoposide (mg/g)

120

25

a
100 80 60 40 20 0

b
20 15 10 5 0

Fig. 2. Apparent solubility profile of etoposide; 2(a) surfactants/cosolvents, 2(b) lipids For parenteral delivery, SLNs have proven to be versatile systems, they combine the advantages of the systems such as emulsions, liposomes and polymeric nanoparticles, with minimize drawbacks. The prominent features of SLNs include the use of excipients of accepted status (FDA-approved constituents), which can immobilize the hydrophilic or hydrophobic drugs in the solid matrix, sustain its release, prevent its premature degradation and overall, reduce the risk of acute and chronic toxicity. [15,18] In this investigation, the solvent-emulsion diffusion technique was adopted to encapsulate the sparingly soluble anticancer drug, etoposide in the lipid core. Herein, the particle was obtained by the rapid solvent diffusion from the droplets into aqueous medium. The primary requirement for this technique was to prepare a solvent-inwater emulsion with a partially water-miscible solvent, containing the lipid, as disperse phase. For this study, benzyl alcohol was chosen as the solvent for its parentally acceptability, preservative activity and its partial miscibility in water. Benzyl alcohol exhibits solubility of 1 part in 13 parts of water at 25 C, lower than this volume benzyl alcohol behaves like an immiscible solvent.[19] Additionally, benzyl alcohol also plays a pivotal role in reducing the curvature of the lipid particle thereby resulting in the reduction of interfacial tension; consequently reduction in particle size of the dispersion. Generally, in the emulsificationdiffusion technique the emulsification rate governs particle size. As clearly outlined for polymeric particles, similar theory is prevalent for SLNs generation; higher the rate of emulsification there is a proportional increase in exhaustive fragmentation in the organic phase, resulting in small emulsion droplets and consequently, smaller particle sizes of SLNs. In addition to this, the organic/aqueous phase ratio also appears to have an influence on particle size, highlighting non-homogeneity in the emulsion at low phase ratios. [20] Similar study (Table 1.) was undertaken to study the influence of saturated benzyl alcohol on the emulsion droplet size. As anticipated, there was increase in the clarity of the emulsion with higher organic/aqueous phase ratio indicative of decrease particle size. From toxicological standpoint, F6 was considered for further study.

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Int. J. Pharm. Biosci. Technol. Table 1: Effect of solvent concentration Composition (% w/v) Etoposide Softisan 601 Tween20 Benzyl alcoholsata 0.9 % w/v saline
a

F1 0.5 25 35 10 100

F2 0.5 25 35 12.5 100

F3 0.5 25 35 13.75 100

F4 0.5 25 35 15 100

F5 0.5 25 35 17.5 100

F6 0.5 25 35 18.75 100

F7 0.5 25 35 20 100

Benzyl alcohol saturated with water w/v saline was chosen as dilution medium for this study to facilitate compatibility with the parenteral route of administration. Despite, the presence of sodium and chloride ions, there was visible clarity of the dispersion. However, on standing for 6 h, there was loss of transparency possibly arising due to the instability of the surfactants by the electrolyte in the saline.

Another important variable is the rate of diffusion which is governed by the solubility of the organic solubility in dilution medium. Rapid diffusion of the solvent results in lower particle size. As depicted in Table 2. There was marked decrease in the particle size of the resultant dispersion with increase in the volume of dilution medium suggesting rapid and complete diffusion of benzyl alcohol in external aqueous medium. The 0.9% Table 2. Effect of dilution volume Composition (% w/v) Etoposide Softisan 601 Tween 20 Benzyl Alcoholsat 0.9 % w/v saline Final Dilution Volume (ml)
a

F8 0.5 25 35 18.75 0.5 10

F9 0.5 25 35 18.75 0.5 25

F10 0.5 25 35 18.75 0.5 50

F11 0.5 25 35 18.75 0.5 75

F12 0.5 25 35 18.75 0.5 100

Benzyl alcohol saturated with water this particle size was noted at the end of 10 h suggesting that lower particle size could be achieved if freeze dried powder of the developed SLNs is reconstituted and immediately administered by slow infusion. The pH of formulation was found to be in an acceptable range for intravenous administration and conducive for etoposide stability (Table 3.) Furthermore, using this technique at the lipid load of 2.5%, encapsulation of about 66% was achieved for sparingly soluble etoposide with appreciable drug content (Table 3). Colloidal drug carrier systems serve to minimize the side effects of drugs used for parenteral applications such as trauma arising from the destruction of corpuscles of blood or tissue cells at the site of injection. To corroborate this statement, the hemolytic activity was done for estimating the membrane damage caused by formulation in vivo. In comparison to double distilled water, the components of the formulation and formulation itself exhibited considerably less hemolytic activity (Table 4.) The study revealed that % haemolysis of SLNs dispersion is very low and can be acceptable for the parenteral administration.

To circumvent this issue of particle instability, the use of stabilizer was considered. It is known that coating of nanocarriers with surfactants/stabilizers does impart stability as well as improve the performance of the colloidal dispersion in biological fluids.[21] Herein, pluronic block copolymer was selected as stabilizer. This class of surfactant has shown to sensitize multidrugresistant cells by inhibiting drug efflux transporters, improve cellular uptake and confer long circulations time by disguising the particles as hydrophilic entity thereby deceiving the monocyte phagocyte system of the body. [22] On incorporation of Lutrol F127, there was enhancement in stability for period exceeding 10 h. The lack of stability could be attributed the decrease in critical micellization concentration (CMC) and temperature (CMT) in the presence of salts with possible dehydrating effect in presence of benzyl alcohol.[23] Nevertheless, the average diameter and the polydispersity index of SLNs prepared and dispersed in 0.9% w/v saline at the end of 10 h was less than 300 nm. Although, at this particles size, it is rapidly engulfed by the phagocytes, the presence of hydrophilic coating by Lutrol F127 could possible evade this opsonization. Further, Fernandes et al

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Int. J. Pharm. Biosci. Technol. Table 3. Results of Characterization Tests Particle Size (nm) Polydispersity index pH Drug Content (%) Drug Encapsulation Efficiency (%) Observations 233.4 1.408 5.23 98.5 66 financial support for this project. The authors are also thankful to Sasol Germany GmbH and BASF for providing the gift samples of lipid, and surfactant. REFERENCES 1. Zhanga F, Koha GY, Hollingsworth J, Russob P S, Stoutc RW, Liua Z. Reformulation of etoposide with solubility-enhancing rubusoside. International Journal of Pharmaceutics. 2012; 434 (1-2): 453-459. 2. Reddy L H, Adhikari J S, Dwarakanath B S, Sharma RK, Murthy RR. Tumoricidal effects of etoposide incorporated into solid lipid nanoparticles after intraperitoneal administration in daltons lymphoma bearing mice. AAPS J. 2006; 8 (2): Article 29. 3. Jain J, Fernandes C, Patravale V. 2010. Formulation development of parenteral phospholipid-based microemulsion of etoposide. AAPS PharmSciTech. 11(2):826-31. 4. (2000) Physicians desk reference, 54th edition. Medical Economics Company, Inc. Montvale, New Jersey, pp. 888889. 5. Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharmaceutical Research. 2004; 21:201230. 6. Dong W, Zhang L, Niu Y, Fan D, Wu X, Tang X, Cai C. 2013 A stable and practical etoposidecontaining intravenous long-/medium-chain triglycerides-based lipid emulsion formulation:pharmacokinetics, biodistribution, toxicity, and antitumor efficacy. Expert Opinion on Drug Delivery. 2013 7. Reddy PR, Venkateswarlu V. Pharmacokinetics and tissue distribution of etoposide delivered in long circulating parenteral emulsion. Journal of Drug Targeting. 2005; 13(10):543-53. 8. Tian LL, Tang X, He HB, Wang J Preparation and in vitro and in vivo evaluation of etoposide submicro-emulsion for intravenous injection. Yao Xue Xue Bao. 2007; 42(8):892-897. 9. Chen H, Shi S, Zhao M, Zhang L, He H, Tang X. A lyophilized etoposide submicron emulsion with a high drug loading for intravenous injection: preparation, evaluation, and pharmacokinetics in rats. Drug Development and Industrial Pharmacy.2010; 36(12):14441453.

Table 4. Comparative haemolysis after 1 hour incubation period Component Tween 20 Benzyl alcohol 2.5 % Lutrol F127 Solution SLN dispersion Water % haemolysis after 1 h of incubation 14 5.13 1.3 3.6 100

Reiterating that, the lipid used for this study has not been reported for parenteral administration. It was imperative that the parenteral acceptability of this lipid had to be established. For this purpose, the acute toxicity study of the plain lipid was undertaken. Since to evade the contributing effect of solvents, lipid was injected as emulsion in the tail vein of mice and behavior as well as mortality was observed during the period of 15 days. The various doses recommended for the study included 5 mg/kg, 50 mg/kg, 300 mg/kg, 500 mg/kg and 2000 mg/kg. With an exception of dose 2000 mg/ kg, all the dose levels had negligible effect on the mice, which showed normal behavior and survived the fifteen day observation period. At dose 2000 mg/ kg, the mortality of two mice was observed on 3rd day indicating that 2000 mg/ kg was toxic dose of the lipid. For the proposed application, the dose to be administered in human was found to be less than 5 mg/kg stating that the lipid was safe for parenteral administration in developed dosage form. CONCLUSIONS The solvent emulsification diffusion technique was successfully employed for fabrication of SLNs of etoposide. However, with this technique the desired stability was not achieved. Thus, the use of freeze-drying would be considered as a favorable option to exploit the use of this system. The in vitro erythrocyte toxicity study and acute toxicity study demonstrated the safety and acceptability of the formulation for parenteral administration. ACKNOWLEDGEMENTS The authors are grateful to the University Grant Commission (New Delhi, India) for providing

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Int. J. Pharm. Biosci. Technol. 10. Sistla A, Smith DJ, Kobrinsky NL, Kumar K. Pharmacokinetics and tissue distribution of liposomal etoposide in rats. Drug deliv. 2009;16(8):423-429. 11. Reddy LH, Sharma RK, Chuttani K, Mishra AK, Murthy RR. Etoposide-incorporated tripalmitin nanoparticles with different surface charge: formulation, characterization, radiolabeling, and biodistribution studies. AAPS J. 2004; 6(3):e23. 12. Harivardhan RL, Sharma RK, Chuttani K, Mishra AK, Murthy RS Influence of administration route on tumor uptake and biodistribution of etoposide loaded solid lipid nanoparticles in Dalton's lymphoma tumor bearing mice. Journal of Controlled Release. 2005; 105(3):185-198. 13. Snehalatha M, Venugopal K, Saha RN, Babbar AK, Sharma RK. Etoposide loaded PLGA and PCL nanoparticles II: biodistribution and pharmacokinetics after radiolabeling with Tc99m. Drug delivery. 2008; 15(5):277-287. 14. Patel PA, Patravale VB. AmbiOnp: solid lipid nanoparticles of amphotericin B for oral administration. Journal of Biomedical Nanotechnology. 2011; 7 (5):632-639. 15. Joshi MD, Muller RH. Lipid nanoparticles for parenteral delivery of actives. European Journal of Pharmaceutics and Biopharmaceutics. 2009; 71, 161172. 16. Bock TK, Muller BW. A novel assay to determine the hemolytic activity of drugs incorporated in colloidal carriers systems. Pharmaceutical Research. 1994; 11: 589591. 17. Yalkowsky S. Solubility and solubilization of nonelectrolytes. Techniques of Solubilization of Drugs, Dekker, New York, 1981; 2-15. 18. Martinsa S, Costa-Lima S, Carneiro T, Cordeiro-da-Silv A, Souto EB, Ferreir DC. Solid lipid nanoparticles as intracellular drug transporters: An investigation of the uptake mechanism and pathway. International Journal of Pharmaceutics. 2012; 430, 216 227. 19. Trotta M, Debernardi F, Caputo O. Preparation of solid lipid nanoparticles by a solvent emulsificationdiffusion technique. International Journal of Pharmaceutics. 2003; 257 153160. 20. Mora-Huertas CE, Fessi H, Elaissari A. Influence of process and formulation parameters on the formation of submicron particles by solvent displacement and emulsificationdiffusion methods Critical comparison. Advances in Colloid and Interface Science. 2011; 163, 90122. 21. Yordanov G, Skrobanska R, Evangelatov A. Colloidal formulations of etoposide based on poly(butyl cyanoacrylate) nanoparticles: Preparation, physicochemical properties and cytotoxicity Colloids and Surfaces B: Biointerfaces. 2013; 101, 215 222. 22. Kabanov AV, Alakhov VY. Pluronic block copolymers in drug delivery: from micellar nanocontainers to biological response modifiers. Critical Reviews in Therapeutic Drug Carrier Systems. 2002; 19(1):1-72. 23. Alexandridis P, Hatton TA. Poly(ethylene oxide)-poly(propylene oxide )-poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids Surf A Physicochem Eng Asp. 1995; 96, 1- 46. ___________________________________________ How to cite this article APA style Fernandes, C.B., Mandawgade, S., & Patravale, V. B. (2013). Solid lipid nanoparticles of etoposide using solvent emulsification diffusion technique for parenteral administration. International Journal of Pharma Bioscience and Technology, 1(1), 2733. Elsevier Harvard style Fernandes, C.B., Mandawgade, S., Patravale, V.B., 2013. Solid lipid nanoparticles of etoposide using solvent emulsification diffusion technique for parenteral administration. Int. J. Pharm. Biosci. Technol. 1, 2733. Vancouver Style Fernandes CB, Mandawgade S, Patravale VB. Solid lipid nanoparticles of etoposide using solvent emulsification diffusion technique for parenteral administration. Int. J. Pharm. Biosci. Technol. 2013; 1(1):2733. To receive bibliographic information in RIS format (For Reference Manager, ProCite, EndNote): Send request to: ijpbst@yahoo.com ___________________________________________

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