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
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.
E-mail address- firstname.lastname@example.org
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. 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)
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
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
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
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,
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
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.
Apparent solubility of etoposide
Apparent solubility of etoposide
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
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. 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. 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.
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