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					Research Article                                                                                      ISSN: 2321-2969
Received: 20 April 2013, Accepted: 29 April 2013                                       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

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                      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-

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

                                                                 polysorbate 80, 650 mg polyethylene glycol 300,
Etoposide (Fig. 1), an epipodophyllotoxin
                                                                 and 30.5% (v/v) alcohol.[4] 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.[5]
malignancies [1]. 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 [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 0.2–0.4 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. [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                              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 [6],
                                                       nature of solid lipid Softisan®601 with the
pegylated parenteral emulsion (PE) [7], submicro-
                                                       parenterally acceptable surfactants. Miscibility of
emulsion         [8-9],       phospholipid-based
                                                       solid lipid and surfactants was ascertained visually
microemulsion [3], liposomes [10],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 [13]. 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 [14]
                                                       (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. [15] 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;
                                                       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
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.
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. [16]. 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. [17] 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.


                                                                                Apparent solubility of etoposide
Apparent solubility of etoposide

                                                                        a                                                                           b
                                   100                                                                             20
                                    80                                                                             15


                                     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.[19] 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. [20] 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
 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
    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.[21] 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. [22] 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.[23]
                                                              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. The authors are
                                                         also thankful to Sasol Germany GmbH and BASF
 Tests                              Observations         for providing the gift samples of lipid, and
 Particle Size (nm)                    233.4             surfactant.
 Polydispersity index                  1.408
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Fernandes et al                                                                                     Pg. 33

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Description: Key words: Etoposide, Solid lipid nanoparticles, Parenteral, Acute toxicity