"SOLID LIPID NANOPARTICLES OF ETOPOSIDE USING SOLVENT EMULSIFICATION DIFFUSION TECHNIQUE FOR PARENTERAL ADMINISTRATION"
Research Article ISSN: 2321-2969 Received: 20 April 2013, Accepted: 29 April 2013 Int. J. Pharm. Biosci. Technol. To cite this Article: Click here 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- email@example.com 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 polysorbate 80, 650 mg polyethylene glycol 300, Etoposide (Fig. 1), an epipodophyllotoxin and 30.5% (v/v) alcohol. Most of these anticancer molecule is found to be effective excipients are responsible for side effects such as against small cell lung carcinoma, germ cell pain, inflammation, tissue damage, necrosis at the tumors, hematologic and other types of site of injection, and substantial hemolysis. malignancies . Its prime mechanism of action is These issues have prompted the renewed interest inhibition of topoisomerase-II and activation of in the development of formulation which is oxidation reduction reactions to produce parenterally safe, robust and stable to dilutions derivatives that bind directly to DNA and cause DNA damage . 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 0.2–0.4 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.  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 Fig 1: Chemical Structure of Etoposide Fernandes et al Pg. 27 Int. J. Pharm. Biosci. Technol. Few of the approaches employed for parenteral Miscibility studies formulation of etoposide include long-/medium- The study involved the assessment of the miscible chain triglycerides-based lipid emulsion , nature of solid lipid Softisan®601 with the pegylated parenteral emulsion (PE) , submicro- parenterally acceptable surfactants. Miscibility of emulsion [8-9], phospholipid-based solid lipid and surfactants was ascertained visually microemulsion , liposomes ,solid lipid for phase separation in the ratio of 1:1. nanoparticles [2,11-12], poly(lactic-co-glycolic acid) (PLGA) and Polycaprolactone (PCL) Formulation development nanoparticles . Among these formulations, solid lipid nanoparticles (SLNs) have emerged as Methodology versatile systems for parenteral drug delivery Typically, a weighed amount of etoposide was which can provide sustained release of drug solubilized in lipid melt. Thereafter, the surfactant thereby reducing the frequency of drug dosing. and water saturated benzyl alcohol was Also, they can confer protection against incorporated, followed by warm water metabolizing enzymes; pH based degradation  (maintained at temperature above the melting and further, they have shown to enhance the point of the lipid). The mixture was stirred at 2500 efﬁcacy and residence time of the cytotoxic drugs rpm for 1 min to obtain o/w emulsion. Following with concomitant reduction in the side-effects which, the resultant emulsion was diluted with associated with them.  Therefore, the work warm 0.9% w/v saline and gradually cooled to discussed herein investigates the potential of solid room temperature to obtain solid lipid lipid nanoparticles (SLN) of etoposide for nanoparticles of etoposide under stirring. parenteral delivery. Further, the work entails the acute toxicity study of the novel lipid for Optimization parameters parenteral delivery. Effect of solvent concentration In this study, the concentration of the solvent; MATERIALS AND METHODS benzyl alcohol in the coarse dispersion was varied Materials from to 10-20% w/v and observed visually for increase in clarity of the dispersion on dilution. Etoposide was gift sample from Khandelwal Lab, India. Softisan®601 (mixture of glyceryl cocoate, Effect of dilution volume hydrogenated coconut oil and ceteareth-25) was a In this study, the coarse dispersion was diluted to generous gift sample from Sasol Germany GmbH. 10 ml, 25 ml, 50 ml, 75 ml, 100 ml and observed Polysorbate 80 (Tween® 80), benzyl alcohol, visually for increase in clarity of the dispersion on methanol, ethanol (99%), concentrated dilution. hydrochloric acid, disodium phosphate, potassium dihydrogen phosphate and sodium chloride, were Effect of stabilizer concentration purchased from s.d. Fine chemicals. Lutrol® F127 In this study, the stabilizer concentration was was procured from BASF, India. All other varied from 0.5 – 2 % w/v and observed visually chemicals were of AR grade. Double distilled water filtered through 0.45 µm membrane was for increase in clarity of the dispersion on dilution. prepared freshly whenever required. Characterization of the SLN dispersion Preformulation studies Particle size determination Apparent solubility studies Photon correlation spectroscopy (PCS) using laser The fixed amount (1g) of surfactant/lipid was light scattering is frequently used to determine weighed accurately and transferred to a small test particle size of colloidal system. A Beckman N4 tube. In this test tube, the drug was added to Plus submicron Particle Size Analyzer was assess the equilibrium solubility of the drug at the employed to monitor particle size of the SLN end of 24 h at ambient condition of 25 to 27°C. The dispersion. The instrument calculated the mean solubility was established by visual estimation of particle size and polydispersity from intensity, the samples for transparency and quantification assuming spherical particles. Light scattering was using UV-visible spectroscopy. Thereafter, the monitored at 90° scattering angle and temperature amount of surfactant/oil required to solubilize the of 25°C. Prior to analysis, the formulation was drug was determined. suitably diluted with double distilled water filtered through membrane filter of pore size 0.22 μm. The measurements were done in triplicate. Fernandes et al Pg. 28 Int. J. Pharm. Biosci. Technol. Drug content erythrocytes were removed by centrifugation. One hundred ml of resulting supernatant was SLN dispersion (equivalent to 20 mg of drug) was added to 2 ml of an ethanol/HCl mixture [(39 parts taken in a 10 ml volumetric flask, and suitably ethanol (99% v/v) + 1 part of HCl (37% w/v)]. This diluted to 10 ml with methanol. Following which, mixture dissolved all components and avoided the etoposide was extracted from SLN dispersion by precipitation of hemoglobin. The absorbance of subjecting the solution to sonication for 5 min. the mixture was determined at 398 nm by Then, 1 ml of this solution was suitably diluted to spectrometer monitoring against a blank sample. 50 ml using methanol. The drug content of the Control sample of 100% lysis (in water x 100) was resultant solution was determined in triplicate at employed as standard in the experiment. The λmax of 283 nm using developed UV-Vis percentage of hemolysis caused by the test spectrophotometric method. This procedure was sample was calculated by following equation: repeated for placebo, to evade any interference Hemolysis caused by sample (%) = (Absorbance from the excipients. Both the solutions were of the test sample-Absorbance at 100% lysis) x analyzed using water as blank. 100. Drug Encapsulation Efficiency Acute toxicity study For the quantitative determination of etoposide, 1 Toxicity status of excipients is a major issue for the ml of the SLN dispersion containing drug was use of a delivery system. In this research work, the subjected to centrifugation at the speed of 14000 lipid considered for the study has not been rpm for about 30 min. The supernatant obtained recommended for parenteral administration. after centrifugation was analyzed for drug content Hence, toxicity study was undertaken in using the similar procedure as mentioned for drug accordance to OECD guidelines to assess its safety content. Similar, procedure was repeated for SLN for parenteral route. The experimental protocol dispersions without drug. Entrapment efficiency was approved by the Institutional Animal Ethical was calculated using following equation; Committee. In accordance to the Organization for Economic Co-operation and Development (OECD) Winitial drug −Wfree drug 425 guidelines, the acute parenteral toxicity of Entrapment Efficiency = ------------------------ ×100 Softisan® 601 was determined as lethal dose (LD50). For acute parenteral toxicity studies, Winitial drug female Swiss albino mice weighing 20–25 g (10–12 where, “Winitial drug” is the mass of initial drug week) were used. Throughout the experiments, added, “Wfree drug” is the mass of free drug the animals were fed with a standard mice diet and analyzed in the supernatant after centrifugation. were provided with clean drinking water adlibitum. Animals were divided into six groups pH Measurement comprising of 5 animals each; The pH of formulation was measured before and Group I: Control, Group II: Dose 5mg/Kg, Group after autoclaving by Systronic Digital pH meter III: Dose 50 mg/Kg, Group IV: Dose 300 mg/Kg, 335, standardized using pH 4.0 and 7.0 standard Group V: Dose 500 mg/Kg, Group VI: Dose buffers. 2000mg/Kg In Vitro erythrocyte toxicity study The animals of Group I-VI were parenterally The erythrocyte toxicity assay was conducted as administered lipid emulsion at the respective described by Bock et al. . Fresh blood was dose. The animals were observed at regular collected in the vial containing ethylene-diamine- intervals on day of dosing and once daily tetraacetic acid (EDTA). Red blood cells (RBCs) thereafter for 15 days. Following observations were isolated by centrifugation (5,000 rpm for 5 pertaining to any gross change in the activity and min) and the RBCs were washed three times with behavioral pattern; presence of tremors, isotonic phosphate buffer pH 7.4 before diluting convulsions, salivations, diarrhea and lethargy with buffer to prepare erythrocyte stock was noted. Additionally, the food consumption, dispersion (three parts of centrifuged erythrocytes body weight and mortality were recorded. plus 11 parts buffer). The washing step was repeated in order to remove debris and serum RESULTS AND DISCUSSION protein. A 100 μl aliquot stock dispersion was For the formulation of SLNs, the selection of solid added per ml of test sample. The resulting solution lipid is the most critical aspect of the formulation. was incubated at 37°C for a period of 1 h. After The basis of selection of lipid was governed by the incubation under shaking, debris and intact solubility of the drug in the lipid melt. Besides this, Fernandes et al Pg. 29 Int. J. Pharm. Biosci. Technol. surfactants and cosurfactants, an integral aspect of reason for lower ideal solubility and hence poor this formulation, were investigated based on the aqueous solubility.  Furthermore, the aforesaid criterion. As depicted in the Fig. 2(a-b), etoposide molecule shows presence of –OH the drug was found to soluble to more extent in the groups which impart hydrophilic nature to some surfactant as compared to the lipid. This poor extent. Together, these contrasting reasons could solubility of etoposide could be ascribed to its possibly be the contributing factors to poor high melting point (240-250°C), an indicative solubility in lipid, whereas relatively better factor of strong crystal lattice energy; a possible solubility in surfactant. 25 120 Apparent solubility of etoposide Apparent solubility of etoposide a b 100 20 80 15 (mg/g) 60 (mg/ml) 10 40 5 20 0 0 Fig. 2. Apparent solubility profile of etoposide; 2(a) surfactants/cosolvents, 2(b) lipids For parenteral delivery, SLNs have proven to be solvent. Additionally, benzyl alcohol also versatile systems, they combine the advantages of plays a pivotal role in reducing the curvature of the systems such as emulsions, liposomes and the lipid particle thereby resulting in the reduction polymeric nanoparticles, with minimize of interfacial tension; consequently reduction in drawbacks. The prominent features of SLNs particle size of the dispersion. include the use of excipients of accepted status Generally, in the emulsification–diffusion (FDA-approved constituents), which can technique the emulsification rate governs particle immobilize the hydrophilic or hydrophobic drugs size. As clearly outlined for polymeric particles, in the solid matrix, sustain its release, prevent its similar theory is prevalent for SLNs generation; premature degradation and overall, reduce the higher the rate of emulsification there is a risk of acute and chronic toxicity. [15,18] proportional increase in exhaustive fragmentation In this investigation, the solvent-emulsion diffusion in the organic phase, resulting in small emulsion technique was adopted to encapsulate the droplets and consequently, smaller particle sizes sparingly soluble anticancer drug, etoposide in of SLNs. In addition to this, the organic/aqueous the lipid core. Herein, the particle was obtained phase ratio also appears to have an influence on by the rapid solvent diffusion from the droplets particle size, highlighting non-homogeneity in the into aqueous medium. The primary requirement emulsion at low phase ratios.  Similar study for this technique was to prepare a solvent-in- (Table 1.) was undertaken to study the influence of water emulsion with a partially water-miscible saturated benzyl alcohol on the emulsion droplet solvent, containing the lipid, as disperse phase. size. As anticipated, there was increase in the For this study, benzyl alcohol was chosen as the clarity of the emulsion with higher solvent for its parentally acceptability, organic/aqueous phase ratio indicative of preservative activity and its partial miscibility in decrease particle size. From toxicological water. Benzyl alcohol exhibits solubility of 1 part in standpoint, F6 was considered for further study. 13 parts of water at 25 °C, lower than this volume benzyl alcohol behaves like an immiscible Fernandes et al Pg. 30 Int. J. Pharm. Biosci. Technol. Table 1: Effect of solvent concentration Composition (% w/v) F1 F2 F3 F4 F5 F6 F7 Etoposide 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Softisan® 601 25 25 25 25 25 25 25 Tween20 35 35 35 35 35 35 35 Benzyl alcoholsata 10 12.5 13.75 15 17.5 18.75 20 0.9 % w/v saline 100 100 100 100 100 100 100 a Benzyl alcohol saturated with water Another important variable is the rate of diffusion w/v saline was chosen as dilution medium for this which is governed by the solubility of the organic study to facilitate compatibility with the parenteral solubility in dilution medium. Rapid diffusion of route of administration. Despite, the presence of the solvent results in lower particle size. As sodium and chloride ions, there was visible clarity depicted in Table 2. There was marked decrease of the dispersion. However, on standing for 6 h, in the particle size of the resultant dispersion with there was loss of transparency possibly arising increase in the volume of dilution medium due to the instability of the surfactants by the suggesting rapid and complete diffusion of benzyl electrolyte in the saline. alcohol in external aqueous medium. The 0.9% Table 2. Effect of dilution volume Composition (% w/v) F8 F9 F10 F11 F12 Etoposide 0.5 0.5 0.5 0.5 0.5 Softisan 601® 25 25 25 25 25 Tween 20 35 35 35 35 35 Benzyl Alcoholsat 18.75 18.75 18.75 18.75 18.75 0.9 % w/v saline 0.5 0.5 0.5 0.5 0.5 Final Dilution Volume (ml) 10 25 50 75 100 a Benzyl alcohol saturated with water To circumvent this issue of particle instability, the this particle size was noted at the end of 10 h use of stabilizer was considered. It is known that suggesting that lower particle size could be coating of nanocarriers with surfactants/stabilizers achieved if freeze dried powder of the developed does impart stability as well as improve the SLNs is reconstituted and immediately performance of the colloidal dispersion in administered by slow infusion. The pH of biological fluids. Herein, pluronic block formulation was found to be in an acceptable copolymer was selected as stabilizer. This class of range for intravenous administration and surfactant has shown to sensitize multidrug- conducive for etoposide stability (Table 3.) resistant cells by inhibiting drug efflux Furthermore, using this technique at the lipid load transporters, improve cellular uptake and confer of 2.5%, encapsulation of about 66% was achieved long circulations time by disguising the particles for sparingly soluble etoposide with appreciable as hydrophilic entity thereby deceiving the drug content (Table 3). monocyte phagocyte system of the body.  On Colloidal drug carrier systems serve to minimize incorporation of Lutrol F127, there was the side effects of drugs used for parenteral enhancement in stability for period exceeding 10 applications such as trauma arising from the h. The lack of stability could be attributed the destruction of corpuscles of blood or tissue cells at decrease in critical micellization concentration the site of injection. To corroborate this statement, (CMC) and temperature (CMT) in the presence of the hemolytic activity was done for estimating the salts with possible dehydrating effect in presence membrane damage caused by formulation in vivo. of benzyl alcohol. In comparison to double distilled water, the Nevertheless, the average diameter and the components of the formulation and formulation polydispersity index of SLNs prepared and itself exhibited considerably less hemolytic dispersed in 0.9% w/v saline at the end of 10 h activity (Table 4.) The study revealed that % was less than 300 nm. Although, at this particles haemolysis of SLNs dispersion is very low and can size, it is rapidly engulfed by the phagocytes, the be acceptable for the parenteral administration. presence of hydrophilic coating by Lutrol F127 could possible evade this opsonization. Further, Fernandes et al Pg. 31 Int. J. Pharm. Biosci. Technol. Table 3. Results of Characterization financial support for this project. 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Preparation of solid lipid nanoparticles by a solvent To receive bibliographic information in RIS emulsification–diffusion technique. format International Journal of Pharmaceutics. 2003; (For Reference Manager, ProCite, EndNote): 257 153–160. Send request to: firstname.lastname@example.org ___________________________________________ Fernandes et al Pg. 33