Chapter 5
Materials and Methods
5.1 Materials
Chemicals, drug and reagents were procured from the sources given below in
Table 5.1. All solvents used were of analytical grade.
Table 5.1: List of chemicals, drug, reagents and their sources.
Chemical Source
Paclitaxel Provided ex gratia by Dabur India, Ltd.
Ghaziabad (India)
Phosphatidylcholine (PC) Provided ex gratia by Life care
innovations Pvt. Ltd. Gurgaon, India
Methanol, Potassium dihydrogen
phosphate, Di-Sodium hydrogen phosphate,
Chloroform
S. D. Fine chemicals Ltd. Mumbai,
India
Acetonitrile Loba Chemie Pvt. Ltd. Mumbai, India
Span 80, Sodium Deoxycholate, Cellophane
membrane, MTT.
HIMEDIA Ltd. Mumbai, India
Chitosan Central Marine and Fisheries Research
Institute, Cochin
Ethanol, AR Bengal chemicals.
Sodium chloride E. Merck Ltd.
Paclitaxel marketed formulation Intaxel, Dabur Pharma, India.
Total Protein, cholesterol, triglycerides,
glucose, creatinine, SGOT, SGPT and Urea
estimation kit.
Erba diagnostic, Mannheim, Germany.
Fetal bovine serum , RPMI 1640 Media
Acridine Orange and Rhodamine 123
Sigma Aldrich Co. Ltd, USA.
Cytoselect 96-Well cell transformation
assay kit
Cell biolabs, USA.
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5.2 Instruments
Instruments used during the experimental work are listed in Table 5.2.
Table 5.2: List of instruments used.
Instrument Source
U.V. Spectrophotometer Beckman, California, USA (DU 640B)
HPLC Waters, 2489 UV/Visible Detector
Electronic weight balance Afoset, Mumbai (ER-182A)
Rotary evaporator Elma 570/H
Incubator Narang Scientific Works Pvt. Ltd., New
Delhi
5% CO2 Incubator Excella Eco-170, New Brunswick
Scientific, USA
Shaking incubator Remi equipments, Mumbai, India
Diffusion Cell Apparatus Fabricated by USIC Pbi. Uni. Patiala
Probe Sonicator VCX 505, Sonics USA
Transmission Electron Microscope Philips, 400T, TEM, New Brunswick,
Canada
Scanning Electron Microscope SEM, LEO43 SVP, Cambridge
Confocal Laser Scanning Microscope LSM 510, Carl Zeiss, Germany
Fluorescence Microscope Axio version, Carl Zeiss, Germany
Elisa Plate Reader APR4 Microplate Reader, Germany
Flow cytometer Beckton Dickinson, FACS Scan, USA
Autoanalyzer Erba Chem 7, Germany
Hematolyzer Sysmex, USA
Optical Microscope DX31, Oylmpus, Japan and B-Series,
Motic, Xiamen, China
24 and 96 Well Plates Corning Incorporated, Corning NY,
USA
Particle size and zeta potential analzser Beckman Coulter (Delsa NanoTM
,
Common)
Hemocytometer Marienfeld, Germany
Tewa meter (TM 210) Courage and khazaka electronic GmbH
Koln, Germany
Viscometer Brookfield Engineering Laboratories,
Inc, USA
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5.3 Preformulation Study
5.3.1 Physical appearance
The physical appearance of the drug was noted by visual observation. The drug
obtained was solid white or almost white, dried crystalline powder.
5.3.2 Ultraviolet absorption maxima (λmax)
The organic molecules in solution when exposed to light in the ultra-violet region
of the spectrum absorb light of particular wavelength, depending on the type of electronic
transition associated with the absorption. For determining the absorption maxima (λmax),
scanning was carried out in the UV range of 200-400 nm. A standard solution of
paclitaxel (100 g/mL) was prepared by dissolving 10 mg of the drug in 100 mL
methanol. This solution after dilution (10 g/mL) was scanned in the wavelength range
of 200-400 nm and the absorption maxima were determined spectrophotometrically.
5.3.3 Melting point determination
Melting Point of the paclitaxel was determined by taking small amount of drug in
a capillary tube closed at one end and placed in the melting point apparatus and the
temperature at which drug melts was noted in triplicate.
5.3.4 Infrared spectral assignment
Infrared spectrum of any compound or drug gives information about the
functional groups present on that particular compound. The infrared spectral assignment
of paclitaxel was carried out using FTIR spectrophotometer.
5.3.5 Solubility study
For the quantitative solubility study, a defined quantity of paclitaxel was taken in
each thoroughly cleaned and dried volumetric flask. Different investigative solvents were
added and volumetric flasks were tightly closed. After shaking for 24 h in the shaker
incubator, the mixture was filtered. The drug concentration in the supernatant solution
was determined spectrophotometerically.
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5.3.6 Partition coefficient
Paclitaxel (10 mg) was accurately weighed and taken in the stopper vials
containing 10 mL each of 2 immiscible phases, n-octanol and aqueous phase (distilled
water and PBS 7.4). The vials were placed in shaker incubator for 24 h. The phase
separation was done using a separating funnel and the aqueous phase was analyzed
spectrophotometrically for the amount of drug after suitable dilution. The partition
coefficient was calculated by using the formula:
P.C. = Co/Cw
Where, P.C. = Partition coefficient
Co = Concentration of drug in n-octanol phase
Cw = Concentration of drug in distilled water
5.3.7 Preparation of calibration curve
The calibration curve of paclitaxel was prepared in different media [Methanol, 1%
SLS in phosphate buffer saline (PBS 7.4) and Methanol; PBS (pH 7.4)]. Accurately
weighed paclitaxel (10 mg) was dissolved in 100 mL of media resulting in a stock
solution of 100 g/mL. From the stock solution, aliquots of 0.2 to 2.0 mL were
withdrawn in a series of 10 mL volumetric flasks and diluted to 10.0 mL. The resulting
aliquots had a concentration range of 2 to 20 g/mL. The absorbance of each aliquot was
measured spectrophotometrically at 229 nm. The data were processed using a Microsoft
Excel computer program and various statistical parameters were calculated.
5.3.8 HPLC analysis
5.3.8.1 Determination of retention time by HPLC
The drug molecules in optimized mobile phase showed the characteristic retention
time under standard set of conditions e.g. column, temperature and flow rate. The 0.002%
w/v solution of the paclitaxel in Acetonitrile: Distilled water (70:30), as mobile phase was
passed through the C-18 column at the flow rate of 1 mL/min for period of 15 min and
retention time was determined.
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5.3.8.2 Preparation of calibration curve of paclitaxel
Accurately weighed two mg of paclitaxel was dissolved in minimum quantity of
HPLC grade methanol. The volume was made up to 100 mL with the mobile phase
(acetonitrile: distilled water 70:30) resulting in a stock solution of 20 g/mL. From this
stock solution, aliquots of 0.2, 0.4, ……, 1.8, 2.0 mL were withdrawn in a series of 10.0
mL volumetric flasks and diluted to 10.0 mL with mobile phase. This gave a
concentration range of 0.2, 0.4, 1.8, 2.0 g/mL. These samples were subjected to HPLC
analysis by injecting 20 μL of sample into injection port. The area under the curve for
each peak obtained was plotted against concentration to make the calibration curve of
paclitaxel.
5.3.8.3 Preparation of calibration curve of paclitaxel in plasma
Blood sample was taken from mice and plasma was separated by centrifugation at
4000 rpm for 10 min. Equal volume of acetonitrile was added to the obtained plasma for
deproteinisation. After 15 min, the plasma was centrifuged at 4000 rpm for 10 min to
remove the precipitated proteins. Supernatant was passed through a 0.2µm filter.
Accurately weighed paclitaxel 20 mg was transferred to a 100 volumetric flask. Then
volume was made up to 100 (stock A; 200 µg/mL) with mobile phase. One mL of stock
A was taken into 10 mL volumetric flask and further diluted up to 10 mL with mobile
phase (stock B; 20 µg/mL). Aliquots of stock B were further diluted up to 10 mL to get
concentration of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 µg/mL. In each aliquot
100 µL of plasma was added. These samples were subjected to HPLC analysis by
injecting 20 µL of sample into injection port. The AUC for each peak obtained was
plotted against concentration to make the calibration curve of paclitaxel in plasma.
5.3.8.4 Validation study
5.3.8.4.1 Accuracy
The accuracy of the method was determined by standard addition method. In this
method, replicate analysis of three different concentration levels and analytical recovery
experiments were performed by adding known concentration of drug in three different
aliquots.
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5.3.8.4.2 Precision
Intermediate precision study was carried out by intraday and interday precision
method. Intraday precision means precision study carried out on the same day at different
time interval by the same solution. Interday precision means precision study carried out
on the different day with the same solutions under same experimental conditions.
Repeatability was determined by replicate analysis of six sets each of high, middle and
low quality control samples. Relative standard deviation (RSD) was calculated from the
ratios of standard deviation (SD) to the mean and expressed as percentage.
5.3.8.4.3 Limit of detection and quantification
The limit of detection was measured by determining the signal to noise ratio.
Determination of the signal to noise ratio is performed by comparing measured signals
from samples with low concentration of analyte with those of blank samples and
establishing minimum concentration at which the analyte can be reliable detected.
The limit of quantification is expressed as:
Limit of detection = 3.3 (SD/S)
Where SD is the standard deviation of the response and S is the slope of the calibration
curve of the analyte.
The limit of quantification is expressed as:
Limit of quantification = 10 (SD/S)
Where S.D is the standard response and S is the slope of the calibration curve of the
analyte.
5.4 Preparation of Elastic Liposomal Formulation
The elastic liposomes were prepared by conventional rotary evaporation-
sonication method as described by Cevc et al. (1997). Different batches of elastic
liposomes were prepared using surfactant and phospholipid in different ratios as shown in
Table 6.28 (Chapter 6.2). Accurately weighed amount of phospholipid, surfactant and
drug were taken in a clean, dry, round-bottom flask and this lipid mixture was dissolved
in a small quantity of chloroform. Chloroform was removed by rotary evaporation under
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reduced pressure at 40±1 C. Final traces of solvent were removed under vacuum
overnight. The deposited lipid film was hydrated with 7% ethanolic solution in PBS 7.4
at 60 rev/min for 1 h. The resulting vesicles were swollen for 2 h at room temperature to
get large multi lamellar vesicles (LMLVs). To prepare smaller vesicles, LMLVs were
probe sonicated for 10 min. The final lipid and drug concentrations in paclitaxel elastic
liposomal formulation were 5% w/v and 0.6% w/v, respectively.
The same method was used for preparing the conventional liposomal formulation
(phosphatidylcholine: cholesterol, 7:3) that was used as control for comparison purpose.
(Garg et al., 2008) The final lipid concentration in the conventional liposomal
formulation was also 5% w/v. Fluorescence marker Rhodamine 123 loaded elastic
liposomal formulation was prepared by the method similar to that described above, by
taking fluorescence marker in place of drug in first step.
5.5 Preparation of Elastic Liposomal in Situ Thermosensitive Hydrogel Formulation
Elastic liposomal in situ thermosensitive hydrogel formulation was prepared in
three steps. In first step, paclitaxel elastic liposomal formulation was prepared using
conventional rotary evaporation sonication method as described above in section 5.4. In
second step, chitosan-dibasic sodium phosphate based hydrogel (C-DSP) was prepared
and in third step, this hydrogel system was mixed with paclitaxel elastic liposomal
formulation to form elastic liposomal in situ thermosensitive hydrogel formulation.
Chitosan solution (2 % w/v) was prepared by dissolving 200 mg in 10 mL of 0.5% acetic
acid. Dibasic sodium phosphate solution (9% w/v) was prepared in distilled water. In a
glass vial, 10 mL chitosan solution was placed, magnetically stirred in an ice bath and
1mL of dibasic sodium phosphate was added drop wise into chitosan solution. After
mixing, the resultant solution was left for 4 h without stirring at room temperature to
degas and stored in refrigerator till further investigation. Elastic liposomal formulation
was added to the C-DSP solution under stirring to obtain elastic liposomal in situ
thermosensitive hydrogel formulation. The formulations were characterized for gelation
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time, gelation temperature and viscosity. On the basis of obtained results the
concentration of dibasic sodium phosphate was optimized for final preparation.
5.6 IN VITRO Characterization of Paclitaxel Elastic Liposomes
5.6.1 Vesicle shape and type by transmission electron microscopy (tem)
and phase contrast microscopy
Morphological characterization of the elastic liposomal vesicles was done by
using transmission electron microscopy (Philips, 400T, TEM, New Brunswick, Canada)
with an accelerating voltage of 100kV. The vesicles were diluted in a ratio of 1:10 with
distilled water and one drop of the dilution was subsequently taken and placed onto a
carbon-coated copper grid. The excess liquid was removed with filter paper and allowed
to stand for 10 min. The grid was then stained with 1% phosphotungastic acid (PTA) and
allowed to air dry for 5 min. The sample was viewed under transmission electron
microscope (TEM) and photomicrographs were taken (Guo et al., 2000).
Elastic liposomal vesicles without sonication were also visualized by using an
optical microscope. A thin film of elastic liposomes was spread on a slide and after
placing cover slip it was observed under the optical microscope and photomicrographs
were taken (Olympus DX 31, Japan).
5.6.2 Vesicles size and size distribution
The vesicle size and size distribution of elastic liposomal formulation before and
after sonication were determined by dynamic light scattering method (DLS), using
particle size analyzer (El Maghraby et al., 1999).
5.6.3 Zeta potential
As an indicator of the colloidal stability, surface charge of the elastic liposomes
was analyzed by Beckman Coulter Delsa NanoTM
, Zeta sizer. (El Maghraby et al., 2000).
5.6.4 Vesicle population
This is the important parameter for optimizing the composition and other process
variables of elastic liposomal formulation. Elastic liposomal formulation (without
sonication) was diluted five times with 0.9% NaCl solution and numbers of vesicles/mm3
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was counted by optical microscopy using haemocytometer. The elastic liposomes in 80
small squares were counted and no. of vesicles per cubic was calculated using the
following formula:
5.6.5 Turbidity measurement
Turbidity of elastic liposomal formulations was determined as per method
reported by Fang et al. (1997). Elastic liposome was diluted with distilled water to give a
total lipid concentration of 0.312 mM. After rapid mixing by bath sonication for 5 min
the turbidity was measured as absorbance at 400 nm with UV-visible spectrophotometer.
5.6.6 Drug entrapment efficiency
Drug entrapment efficiency was determined after separating unentrapped drug by
dialysis method for 4 h against the solution of 1% SLS in phosphate buffer saline (PBS
7.4) (Mura et al., 2007). Paclitaxel loaded elastic liposomes was placed into a cellulose
acetate dialysis bag (Cellophane membrane, MW cut-off 12000-14000, HIMEDIA, India)
immersed in 100 mL of 1% SLS in PBS 7.4 and magnetically stirred at 30 rpm. Samples
taken at different time intervals from the receiver solution were replaced with equal
volumes of fresh solvent and drug content was analyzed by HPLC assay. The experiment
was stopped when constant drug concentration values were obtained in subsequent
withdrawals from the receiver phase (taking into account the progressive dilution of the
medium). The dialyzed formulation was lysed using Triton-X 100 (10% v/v) and
subsequently analyzed for drug content using HPLC. The percent of entrapment
efficiency (EE %) was then calculated according to the following equation:
5.6.7 Drug content
To determine the drug content, 0.5 mL of the formulation was taken in a
volumetric flask and 4.5 mL of ethanol was added to dissolve it. From the resulting
solution, 1 mL was taken, diluted up to 5 mL with acetonitrile: water (7:3) and the drug
content was determined using HPLC assay.
Total no. of vesicles/mm3
Total no. of squares counted
Total no. of elastic liposomes counted= X Dilution factor X 4000
100% xdrugTotal
drugDiffuseddrugTotalEfficiencyEntrapment
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5.6.8 Elasticity measurement
For measurement of elasticity of vesicular membrane, elastic liposomal
formulations were extruded through the polycarbonate filter membrane having pore
diameter of 100 nm and diameter of 25 mm at 2.5 bar using a stainless steel pressure
holder with 200 mL capacity barrel. The amount of vesicle dispersion which was
extruded during 5 min was measured and vesicle size before and after filtration was also
monitored by dynamic light scattering method.. The elasticity of vesicle membrane was
calculated by using the following formula as shown in equation (Jain et al., 2005).
Where, E = elasticity of vesicle membrane;
J = amount of dispersion, which was extruded during 5 min;
rv = vesicles size (after extrusion);
rp = pore size of the barrier
Elasticity of vesicle membrane was further confirmed by SEM and TEM of the
treated filter. The pieces of filters were fixed in Karnovsky’s fixative overnight at 4C,
dehydrated with graded ethanol solutions (30, 50, 70, 90, 95 and 100%), coated with gold
and examined in a scanning electron microscope (SEM, LEO43 SVP, Cambridge).
Further these pieces were prepared for TEM by fixing at 4 oC in Karnovsky’s fixative
overnight, 1% osmium tetraoxide for 1 h and 0.2% ruthenium tetraoxide + 0.25%
K3Fe(CN)6 for 20 min. Subsequently, the samples were dehydrated with graded ethanol
solutions (30, 50, 70, 90, 95 and 100%) and embedded in Spurr’s resin. Ultra thin
sections were cut (Ultracut E. Reichert-Jung, Austria), collected on formvar coated grids
and examined by using transmission electron microscope (Philips) (van den Bergh et al.,
2001).
E = Jrv
rp
2
×
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5.7 Optimization Study
5.7.1 Optimization of paclitaxel into vesicles dispersion at saturated
concentration
To determine the maximum amount of paclitaxel that can be loaded in vesicle,
formulations were prepared with increasing concentrations of paclitaxel (1, 2, 4, 6, 8 and
10 mg/mL). Drug loaded formulations were examined over a period of 14 days for the
appearance of drug crystals, turbidity and entrapment efficiency.
5.7.2 Optimization of surfactant: phospholipid ratio
In this optimization study elastic liposomal formulations were prepared with
different types of surfactant at different ratio of phospholipids by using conventional
rotary evaporation sonication method as described in section 5.4. Formulations were
characterized for parameters like particle size, entrapment efficiency, vesicle population,
turbidity, elasticity, skin permeation and deposition study. The obtained results were used
for the optimization of types of surfactant and surfactant: phospholipid ratio.
5.7. 3 Optimization of hydration medium
The hydration medium for elastic liposomal formulation was optimized with the
objective to get maximum drug entrapment efficiency. For this purpose formulations
were prepared with optimized surfactant: phospholipid ratio and different hydration
medium were used. Formulations were characterized for drug entrapment efficiency and
shape. The obtained results were used for the optimization of hydration medium.
5.7.4 Optimization of sonication time
To get the required particle size of vesicular dispersion, sonication time was
optimized. Particle size reduction was carried out using ultra sonication technique. For
this purpose, 10 mL of elastic liposomal formulation was taken and formulation was
sonicated at 25 W at 40% out frequency for 5, 10, 15 and 20 min, and particle size was
determined by DLS method. At time period where particle size in 100-200 nm range was
obtained considered as optimized time period for preparation of formulation.
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5.8 Skin Permeation and Deposition Study
5.8.1 Preparation of animal skin
Albino rats (8-12 weeks old, weighing 100-200 g) were sacrificed by cervical
dislocation method. The hair of test animals were carefully trimmed short (< 2 mm) with
a pair of scissors and the abdominal skin was separated from the underlying connective
tissue using scalpel. The excised skin was placed on aluminum foil and the dermal side of
the skin was gently teased off and any adhering fat and/or subcutaneous tissue was gently
removed from the dermal side of the skin. The skin was then carefully checked by using
magnifying glass to ensure that samples were free from any surface irregularities such as
tiny holes or cervices in the portion that was to be used for skin permeation study. The
skin was washed with physiological buffer saline and stored under -20 oC till it was used.
The thickness of the skin was measured with vernier caliper.
5.8.2 Skin permeation study
The in vitro skin permeation of paclitaxel from different formulations was studied
using Franz glass diffusion cell maintained at 37±1 °C under non-occlusive condition.
The effective permeation area of the diffusion cell was 2.303 cm2. The receptor
compartment contained 22.5 mL of 1% SLS in PBS (7.4) and was constantly stirred at
100 rpm. Excised albino abdominal rat skin was mounted between the donor and the
receptor compartment. The elastic liposomal formulation (2.0 mL) was applied to the
epidermal surface of skin. Samples (2.0 mL) were withdrawn through the sampling port
of the diffusion cell at 1, 2, 4, 6, 14, 20 and 24 h time intervals and analyzed for drug
content by using HPLC assay. An equal volume of fresh receptor fluid maintained at
37±1 °C was replaced into the receptor compartment after each sampling.
5.8.3 Skin deposition study
Skin deposition study was carried out using same protocol as discussed above for
skin permeation study. At the end of the permeation experiment the surface of the skin
was washed five times with 50% ethanol to remove excess drug from the surface. The
washing protocol was verified and found to remove > 95% of the applied dose at zero
time. The skin was then cut into small pieces. The tissue was further homogenized with
50% ethanol and left for 24 h at room temperature. After shaking and centrifuging for 5
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min at 3000 rpm, the paclitaxel content in the supernatant was determined by HPLC
assay.
5.9 Vesicle Skin Interaction Study
5.9.1 Scanning electron microscopy
Albino rats, weighing 150-200 g were divided into five groups, each comprising
of three rats. The first group was untreated group, second group was treated with drug
solution, third, fourth group treated with optimized elastic liposomes (EL-SP3 and EL-
SD3) and fifth group treated with conventional liposome. The formulations were applied
non-occlusively to the abdominal side of the rat over an area of 1cm2. The treated rats
were caged and sacrificed after 6 and 24 h of treatment. The skin was removed
immediately and fixed in Karnvosky’s fixative overnight at 4 C, followed by 1% w/v
osmium tetroxide for 2 h, and finally in ruthenium tetraoxide 0.2% w/v and K3Fe(CN)6
0.25% w/v for 1h. After fixation, the samples were dehydrated in a range of ethanolic
solutions 70, 90, 95 and 100% v/v and coated with gold coater. The coated samples were
visualized under scanning electron microscope. All investigations were performed after
approval of the institutional Animal Ethics Committee of Department of Pharmaceutical
Sciences and Drug Research, Punjabi University, Patiala and in accordance with the
disciplinary principles and guidelines of CPCSEA.
5.9.2 Transmission electron microscopy
Transmission electron microscopy study was carried out using same protocol as
discussed above. The formulations were applied non-occlusively to the abdomen side of
the rat over an area of 1 cm2. The treated rats were caged and sacrificed after 6 and 24 h
of treatment. The skin was removed immediately and fixed at 4oC in Karnvosky’s
fixative overnight followed by 1% w/v osmium tetraoxide for 2 h and finally in ruthenium
tetraoxide 0.2 % w/v and K3Fe(CN)6 0.25% w/v for 1 h. The specimens were then
washed with phosphate buffer, dehydrated with graded acetone and then embedded in
araldite CY212 to make tissue blocks. Semi-thin (1 µm) as well as ultrathin sections (70-
80 nm) were cut by ultramicrotome (Ultracut E, Reichert, Austria). The sections were
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stained with uranyl acetate and lead acetate and examined under transmission electron
microscope (Philips, 400T).
5.9.3 FTIR and ATR-FTIR study
The vesicle-skin interaction of optimized elastic liposomal formulations was
evaluated by FTIR and ATR-FTIR and same protocol as discussed for transmission and
scanning electron microscopy was used. Treated skin samples were washed with
physiological saline solution and blotted dry. The FTIR and ATR-FTIR spectrum of the
skin was recorded in the range of 4000-400 cm-1
using FTIR spectrophotometer.
5.9.4 Biochemical Estimation
5.9.4.1 Influence of formulation treatment on excised rat skin cholesterol and triglyceride content
The influence of formulation treatment on excised rat skin cholesterol and
triglyceride content was studied. For this purpose rat skin was taken, epidermal sheet was
separated and treated with paclitaxel elastic liposomal formulations EL-SP3 and EL-SD3,
conventional liposomes, drug solution and normal saline. After 6 and 24 h of treatment
skin lipids were extracted by Folch method (Folch et al., 1957). In this method skin was
homogenized in methanol (10 mL) for 1 min. After that skin was again homogenized
with 20 mL chloroform for additional 2 min. The mixture was filtered and homogenized
the obtained residue in solution (30 mL) of chloroform: methanol (2:1) for 1 min. The
obtained solid was washed after filtration once each with chloroform (20 mL) and
methanol (10 mL). All obtained filtrates were combined and potassium chloride 0.88%
w/v was added with stirring equal to 25% of total volume of filtrate. After allowing it to
settle, the upper layer was aspirated and discarded. Solution of water: methanol (1:1)
equal to one fourth of remaining volume was added and then upper layer was discarded
after allowing the mixture to settle. Residue was collected and cholesterol, triglycerides
contents were determined by using their respective kit method. Standard procedure
provided along with commercial kit literature was used as described below.
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5.9.4.2 Estimation of cholesterol concentration
Cholesterol concentration in extracted residue was estimated by using cholesterol
estimation kit which employed modified Roeschalu’s method for estimation. The
principal of this method involves conversionn of cholesterol ester into cholesterol and
fatty acid in the presence of cholesterol esterase. Cholesterol thus formed is oxidized in
the presence of cholesterol oxidase to cholest-4-en-3-one and hydrogen peroxide.
Hydrogen peroxide then reacts with 4-aminoantipyrine and phenol in the presence of
peroxidase enzyme to form a quinoneimine dye.
Assay procedure
Pipette into tubes marked Blank Standard Test
Working reagent 1000 µL 1000 µL 1000 µL
Distilled water 20 µL -- --
Standard -- 20 µL --
Test -- -- 20 µL
Reagent 1: Cholesterol reagent
Cholesterol esterase >200 IU/L
Cholesterol oxidase >150 IU/L
Peroxidase >2000 IU/L
4-Aminoantipyrine 0.52 mmol/L
Sodium phenolate 20 mmol/L
Phosphate buffer 68 mmol/L
Lipid clearing agent --
Reagent 2: Cholesterol standard
All the components were mixed well and incubated for 10 min at 37ºC. The blank
was aspired following standard and tests. The absorbance of standard and each test tube
was read against reagent blank by using autoanalyzer (Erba Chem 7, Germany).
Cholesterol standard 200 mg/dL (5.14 mmol/L)
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Calculation
Cholesterol (mg/dL) = (Absorbance of Test/ Absorbance of Standard) x 200
Concentration of Standard = 200 mg/dL
5.9.4.3 Estimation of triglycerides content
Triglycerides were estimated by Erba triglycerides kit. Principle involves lipase
hydrolyses triglycerides sequentially to di and monoglycerides and finally to glycerol.
Glycerol kinase using ATP as P04 source converts glycerol liberated to Glycerol 3
phosphate. G3 oxidises (GPO) oxides, G3 phosphate formed to dihydroxyacetone
phosphate and hydrogen peroxide is formed. Peroxidase uses hydrogen peroxide formed
to oxidize 4-aminoantipyrine and DHBS (3,5,dichloro-2-hydroxy benzene sulphate) to
red coloured complex. The absorbance of coloured complex is measured at 520 nm or
with green filter which is proportional to triglycerides concentration.
Reagent composition
5x10 mL 5x20 mL 4x50 mL 20x50 mL
Triglycerides (Enzymes, chromogen) 5 5 4 20
Triglycerides (Buffer) 1 1 2 10
Triglycerides Standard (200mg/dl) 1 1 1 2
Assay procedure
Pipette in to test tube Blank Standard Test
Working reagent (mL) 1 1 1
Standard (mL) -- 0.01 --
Sample (mL) -- -- 0.01
All the samples were mixed well and incubated for 5 min at 37 oC. Absorbance of
each test and standard was read at 520 nm against reagent blank.
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5.9.4.4 Influence of formulation on viable rat skin cholesterol and
triglyceride
The influence of formulations treatment on viable rat skin cholesterol and
triglyceride was also determined. Five groups of albino rats (n=3) were taken and two
patches on dorsal side of each rat was prepared by shaving the hair with mechanical
clipper. One patch was left untreated and served as control. Animals were treated with
optimized paclitaxel elastic liposomal formulations EL-SP3 and EL-SD3, conventional
liposomes, drug solution and normal saline as control. Animals were sacrificed at 6 h and
24 h after treatment Skin was collected and epidermal sheet was separated. Similar
method as described in sections 5.9.4.2 and 5.9.4.3 was followed for the determination of
cholesterol (CHOL) and triglycerides (TGL) content. Percentage of cholesterol and
triglyceride extracted was calculated by using following formula =
5.10 Determination of Localization Index
5.10.1 Fluorescence and confocal laser scanning microscopy (CLSM)
Albino Rats, weighing 150-200 g were divided into four groups, each group
comprising of three rats. The first group served as control and received topical
application of 0.16 w/v solution of marker Rhodamine 123. The second, third and fourth
groups received conventional liposome and elastic liposomal formulations EL-SP3 and
EL-SD3 loaded with Rhodamine 123 (0.16 w/v) as fluorescence marker, respectively.
The formulations were applied topically on the abdominal side of the rat at a marked area
of 1 cm2. The animals were caged individually after application of formulation and were
sacrificed after application of 24 h. The skin was removed immediately, cut into pieces
and washed with PBS. The skin was blotted and fixed in carny’s fluid using procedure as
described by Garg et al. (2008). The sections were viewed under fluorescence and
confocal laser scanning microscope and photomicrographs were taken.
1 - Content remaining in skin afte treatmentX100
Content in normal skin
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5.11 In Vitro Characterization of In Situ Elastic Liposomal
Thermosensitive Hydrogel Formulation
5.11.1 Gelation time and temperature
The time required for the gelation of chitosan-DSP solution with and without
paclitaxel loaded elastic liposomal formulations was determined according to the
technique described by Gilbert et al. (1987). Thermo gelling C-DSP solution, either plain
or elastic liposomal formulation loaded was transferred to a glass vial and put in a water
bath at 25 C. The temperature of water bath was increased in increments of 3 ºC; left to
equilibrate for 15 min at each new setting. The samples were then examined for gelation,
which was said to have occurred when the meniscus would no longer move upon titling
the vial through 90º angle. The maximum temperature tested was 43 ºC.
5.11.2 pH determination
The pH of the thermosensitive hydrogel formulation was determined by using pH
meter.
5.11.3 Morphology
Scanning electron microscopy was used to obtain information on morphology of
thermosensitive hydrogel. Samples (5 mL, vial) of hydrogel were incubated in water bath
under the same conditions used for gelation time study. When the hydrogel was transited
into gel, the gel was frozen in liquid nitrogen and freeze dried for 24 h. Samples in
solution state was also frozen in liquid nitrogen and freeze dried for 24 h. The samples
were stubbed on aluminum stab by using double sided C tape and sputter coating with
gold under vacuum, and the surface was investigated with SEM. The microstructure of
the samples was imaged at different scale bar of 20, 40, 50 and 100µm.
5.11.4 Rheological characterization
The measurement of viscosity of freshly prepared C-DSP solution with and
without paclitaxel loaded elastic liposomal formulations was done by using Brookfield
viscometer. The sample was placed in a beaker and spindle was centered into the gel
placed in a beaker until the meniscus was in the middle of the immersion mark. After that
spindle was rotated direct multiplication of the dial readings (% torque) with appropriate
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factors for the spindle and speed in use as given in the Brookfield viscometer catalogue
gave the viscosity in centipoises (cP). Changes in viscosity as a function of time and
temperature were studied using a specified spindle/speed combination. The
measurements were carried in triplicate.
5.11.5 In vitro drug release through cellophane membrane
The in vitro drug release of paclitaxel from optimized elastic liposomal
formulations, hydrogel formulations and drug solution was studied using Franz glass
diffusion cell maintained at 37±1 °C. The effective permeation area of the diffusion cell
was 2.303 cm2. The receptor compartment contained 22.5 mL phosphate buffer saline
(pH 7.4 with 1% SLS) and was constantly stirred at 100 rpm. Cellophane membrane
(molecular weight cut off 12000-140000) was mounted between the donor and the
receptor compartment. Formulation was applied to the donor side of membrane. The
samples were withdrawn through the sampling port of the diffusion cell at 1, 2, 4, 6, 14,
20 and 24 h time intervals and drug content was analyzed by using HPLC assay. An
equal volume of fresh phosphate buffer (pH 7.4 with 1% SLS) maintained at 37±1 °C
was replaced into the receptor compartment after each sampling.
5.11.6 Stability study
The optimized formulations were tested for stability by storing them at 4 ±1oC
and at room temperature. Formulations were stored in amber colored glass vials at4 ±1oC
and room temperature for 90 days. After 15, 30, 60 and 90 days, they were evaluated for
the vesicle size and size distribution, zeta potential, entrapment efficiency and drug
content as per the method described earlier in section 5.6.6. .
5.12 Ex Vivo study
5.12.1 Intracellular uptake study
5.12.1.1 FACS assay and fluorescence microscopy
Cellular uptake of elastic liposomal formulations by A549 cells was studied by
using acridine orange (AO) fluorescence dye. In brief, 0.5 × 105 cells/well in 24 well
culture plates were allowed to grow overnight. Cells were incubated with AO loaded
elastic liposomal formulation and AO Cremophor EL solution for 24 h at 37±1 ºC. After
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that, cells were trypsinized, washed two times with PBS (pH 7.4) and transferred to
polystyrene tubes for FACS analysis (Beckton Dickinson, FACS Scan). Shift in
fluorescent peak of AO dye is represented in terms of Mean Fluorescent Intensity (MFI).
The numbers of positive cells for AO were represented in terms of percentage. Cells were
also examined under fluorescence microscope and photographed by Olympus Inverted
microscope (Olympus, Tokyo, Japan).
5.12.2 Hemolytic toxicity assay
Hemolytic toxicity was determined using red blood cell (RBC) lysis assay as per
method reported by Singhai et al. (1997). Tested samples of paclitaxel commercial
formulation (Intaxel) and elastic liposomal formulations were diluted with 0.9% normal
saline solution in the concentration range of 6000 to 6.0 µg/mL with dilution factor of
10X. Human blood was freshly drawn from the antecubital vein and centrifuged at 3000
rpm for 5-10 min and the supernatant was discarded. The red blood cell (RBC)
suspension was diluted with saline solution to a concentration of 5% w/v. The RBC
suspension (0.5 mL) was mixed with distilled water, which is considered to produce
100% hemolysis, normal saline that produces no hemolysis (hence acting as blank),
paclitaxel commercial and elastic liposomal formulations in concentration range of 6000
to 6 µg/mL. After incubation at 37 ºC for 1 h, the mixture was centrifuged at 3000 rpm
for 10 min to separate non-lysed RBC. The supernatant was taken and diluted with an
equal volume of PBS (pH 7.4) and absorbance was measured at 540 nm against
supernatant of normal saline. The percent hemolysis was determined for each sample
using the following equation by considering absorbance of distilled water as 100%
hemolysis.
100%100
Ab
AbHemolysis
Sample
5.12.3 Cytotoxicity assay
Small lung cancer cell line (A549) were propagated in RPMI 1640 medium
(Sigma, USA) containing 10% fetal calf serum at 37±1 oC under a 5% CO2 atmosphere.
Cytotoxicity of optimized elastic liposomal formulations was compared with Cremophor
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EL vehicle using MTT assay. 100 l of A549 cell suspension (5000 cells/well) was
seeded in 96-well flat bottom tissue culture plate. The elastic liposomal formulations and
Cremophor EL vehicle were added to the cell suspension, mixed thoroughly and
incubated at 371 oC for 24 and 48 h in 5% CO2 incubator. After 24 and 48 h, 20 µL of
MTT solution (2.5 mg/mL) was added and plate was incubated for 4 h. MTT crystals
formed were dissolved in 40 µL of DMSO and absorbance was measured at 540 nm
using ELISA plate reader (APR4 Microplate Reader, Germany). Similarly, cytotoxicity
of drug loaded elastic liposomal formulations were compared with that of Cremophor EL
based marketed formulation.
5.13 In Vivo Study
5.13.1 Toxicity study
The toxicity of optimized elastic liposomal formulations EL-SP3 and EL-SD3 in
comparison to marketed formulation was evaluated by single dose acute toxicity study
and repeated dose 28 days sub-acute toxicity study.
5.13.1.1 Single dose toxicity study
The single dose acute toxicity of the paclitaxel elastic liposomal formulations
(EL-SP3 and EL-SD3) and marketed formulation was evaluated in mice. Albino mice of
either sex (weight: 25-35 g; age: 14-20 weeks) received formulation by intra peritoneal
route starting at dose of 10 mg/kg to 200 mg/kg (Park et al., 2009). The animals were
observed for toxic symptoms continuously for 1 h after dosing. Finally, the number of
survivors was noted after 24 h and these animals were maintained for further 14 days
with observation made daily. The planning of animal groups is given in Table 5.3.
5.13.1.2 Repeated-dose 28 days subacute toxicity study
Albino mice of either sex (weight: 25-35 g; age: 14-20 weeks) were randomly
assigned into sixteen groups (n=6). Groups of six mice were housed together in cages
(males separated from females). The first group of animals acted as sham control and did
not receive any treatment. The second group that served as a control received PBS (pH
7.4). Remaining groups received EL-SP3 and EL-SD3 at a dose of 20, 40, 80, 120 and
160 mg/kg and marketed formulation (Intaxel) at a dose of 20 and 40 mg/kg in every 72 h
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for 28 days. All animals were supplied with Fiel Purina Chow(R)
and tap water during the
testing period. All animals were weighed and observed daily for physiological and
behavior changes. Animals were examined at the end of the test period for hematological
and biochemical parameters. The planning of animal groups is given in Table 5.4.
Table 5.3: Single dose acute toxicity study frame.
S. No. Group Treatment
1. Group 1 Sham control
2. Group 2 Control
3. Group 3 (10 mg/kg, Paclitaxel loaded EL-SP3)
4. Group 4 (20 mg/kg, Paclitaxel loaded EL-SP3)
5. Group 5 (40 mg/kg, Paclitaxel loaded EL-SP3)
6. Group 6 (80 mg/kg, Paclitaxel loaded EL-SP3)
7. Group 7 (120 mg/kg, Paclitaxel loaded EL-SP3)
8. Group 8 (160 mg/kg, Paclitaxel loaded EL-SP3)
9. Group 9 ( 200 mg/kg, Paclitaxel loaded EL-SP3)
10. Group 10 (10 mg/kg, Paclitaxel loaded EL-SD3)
11. Group 11 (20 mg/kg, Paclitaxel loaded EL-SD3)
12. Group 12 (40 mg/kg, Paclitaxel loaded EL-SD3)
13. Group 13 (80 mg/kg, Paclitaxel loaded EL-SD3)
14. Group 14 (120 mg/kg, Paclitaxel loaded EL-SD3)
15. Group 15 (160 mg/kg, Paclitaxel loaded EL-SD3)
16. Group 16 (200mg/kg, paclitaxel loaded EL-SD3)
17. Group 17 (10 mg/kg, Marketed formulation)
18. Group 18 (20 mg/kg, Marketed formulation)
19. Group 19 (40 mg/kg, Marketed formulation)
20. Group 20 (80 mg/kg, Marketed formulation)
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Table 5.4: Repeated-dose 28 days subacute toxicity study frame.
S. No. Group Treatment
1. Group 1 Sham control
2. Group 2 Control
3. Group 3 (20 mg/kg, Paclitaxel loaded EL-SP3)
4. Group 4 (40 mg/kg, Paclitaxel loaded EL-SP3)
5. Group 5 (80 mg/kg, Paclitaxel loaded EL-SP3)
6. Group 6 (120 mg/kg, Paclitaxel loaded EL-SP3)
7. Group 7 (160 mg/kg, Paclitaxel loaded EL-SP3)
8. Group 8 (20 mg/kg, Paclitaxel loaded EL-SD3)
9. Group 9 (40 mg/kg, Paclitaxel loaded EL-SD3)
10. Group 10 (80 mg/kg, Paclitaxel loaded EL-SD3)
11. Group 11 (120 mg/kg, Paclitaxel loaded EL-SD3)
12. Group 12 (160 mg/kg, Paclitaxel loaded EL-SD3)
13. Group 13 (10 mg/kg, Marketed formulation)
14. Group 14 (20 mg/kg, Marketed formulation)
15. Group 15 (40 mg/kg, Marketed formulation)
16. Group 16 (80 mg/kg, Marketed formulation)
5.13.1.3 Blood analysis for hematological and biochemical parameters
Blood samples for hematological and biochemical estimations in repeated dose 28
days sub-acute toxicity study were collected on 0 day and 28th
day period of time
intervals. Both plasma and serum were used for evaluating the different biochemical
parameters such as glucose, cholesterol, urea, SGPT, SGOT, triglycerides, total proteins,
ALP, creatinine and bilirubin content using standard Erba estimation kit using auto
analyzer (Erba, Chem 7, Germany). Standard procedure as specified in the kit literature
was followed. The blood samples were kept at room temperature for 30 min, allowed to
coagulate and then centrifuged at 4000 rpm for 10 min to separate serum. The blood
samples containing the blood and anticoagulant (EDTA) were centrifuged at 4000 rpm
for 10 min to separate the plasma. The blood in the sodium citrate tubes was also used for
red blood cell count (RBC), hemoglobin concentration (Hb), mean corpuscular volume
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(MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), platelets (Plt), total leukocytes count (TLC), polymorphs,
lymphocytes, monocytes and eosinophils count using hematolyzer (Sysmex, USA).
5.13.1.4 Relative organ weight and histopathology study
Animals were sacrificed at the end of 28 days by cervical dislocation and heart,
kidney, spleen and liver, brain, stomach, lung, pancreas were taken out and washed with
PBS (7.4). Finally organs were dried with a fitter paper and weights were taken. Organs
were preserved in 10% formalin for histopathological examination. Sections were fixed
and blocks were made using the procedure as reported (Jain et al., 2003a). The sections
were stained with eosin-hematoxylin to determine gross histopathology. Histological
sections were examined using optical microscope with photographic arrangement.
5.14 Evaluation of Anti-Cancer Activity
5.14.1 Evaluation of anti-cancer activity using soft agar colony formation
assay
The anti-cancer activity of paclitaxel elastic liposomal formulations and marketed
formulation was tested by soft agar colony formation using Cytoselect 96-well cell
transformation assay kit following the manufacturer’s procedure (CytoselectTM
96-well
cell transformation assay kit, Cell Biolabs, San Diego, CA, USA). In brief, the base agar
layer was prepared by transferring 50 µL of mixture of 1.2 % agar solution and 1%
RPMI-1640/10% FBS medium at equal volume to each well of a 96-well microplate. 75
µL of the mixture of 1.2% agar solution, 1 × RPMI-1640/10% FBS medium and A549
cell suspension (1×105
cells/well) of (1:1:1) were seeded in the 96-well microplate. 100
µL of different paclitaxel formulations were poured in the well and the cells were
incubated for 6-8 days at 37 °C under 5% CO2. Cell colony formation was examined
under light microscope. Further, 50 µL of agar solubilization solution was added to each
well and incubated for 1 h at 37 °C, cell lysis in 25 µL of 8X lysis buffer and 90 µL of
cyQuant working solution was added to each well and incubated for 10 min at room
temperature. Absorbance was measured using the 96-well flourometer using 485/520 nm
filter set.
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5.14.2 Evaluation of anti-cancer activity against ehrlich ascites carcinoma
model in mice
Albino mice (weight: 25-35 g; age: 14-20 weeks) were randomly assigned into
seven groups (n=6) and housed in polyacrylic cages with not more than six animals per
cage and maintained under standard laboratory conditions (temperature 25± 2ºC, 12
light/dark cycle). The animals were fed with a balanced animal feed (Ashirwad Animal
Feed Industries, Punjab, India) and had free access to normal drinking water. In order to
generate tumor, 15x106
ascitic cells were subcutaneously inoculated in to the right hind
limb (thigh) of all the animals left for five days. All groups except first group received 20
µL of Ehrlich ascites cell (EAC) suspension. Day of tumor implantation was assigned as
day ‘0’. On day 1, the animals were randomized and divided into seven groups (n = 6).
The first group served as control. The second group served as EAC control. Third, fourth,
fifth, sixth and seventh group received paclitaxel elastic liposomal formulation (EL-SP3.
EL-SD3, elastic liposomal in situ thermosensitive hydrogel formulations EL-SP3-C-
DSP3, EL-SD3-C-DSP3 and Cremophor EL based marketed formulation at dose of 5
mg/kg body weight. All animals were weighted and observed daily for toxic symptoms.
Treatment was started from day 6 of tumor implantation and continued for 20 days.
Treatment was given subcutaneously every third day. Tumor mass was measured from
the 10th
day of tumor induction. The measurement was carried out every 7th
day for a
period of 20 days. The volume of tumor mass was calculated using the formula
V= 4/3πr2
where r is the mean of r1 and r2 which are two independent radii of the tumor mass
(Kuttan et al., 1990).
Twenty four hour after the last dose blood was collected for hematological and
biochemical parameters estimation. Animals were then sacrificed by cervical dislocation
method and different organs collected for biodistribution study. Tumor mass was
removed, weighted and preserved in 10 % formalin and processed for histopathological
investigations as per method described in section 5.13.1.5.
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5.14.2.1 Estimation of hematological parameters
Collected blood was used for the estimation of complete hematological profile of
mice blood as per the method described in section 5.13.1.3
5.14.2.2 Estimation of biochemical method
Blood samples were collected from the animals and serum was separated for the
biochemical estimations of serum glutamic pyruvate transminase (SGPT), serum
glutamic oxalo acetate transminase (SGOT), cretanine and bilirubin as per the method
described in section 5.13.1.3.
5.14.2.3 Pharmacokinetic and Biodistribution study
EAC bearing mice divided in six groups with each group having 6 animals. The
first group served as control. Second, third, fourth, fifth and sixth group received
paclitaxel elastic liposomal formulation (EL-SP3. EL-SD3, elastic liposomal in situ
thermosensitive hydrogel formulations EL-SP3-C-DSP3, EL-SD3-C-DSP3 and
Cremophor EL based marketed formulation at dose of 5 mg/kg body weight by SC
administration. Blood samples were collected from mice at 0, 0.5, 1, 2, 4, 6, 12 and 24 h
time intervals and were centrifuged at 4000 rpm for 6 min to separate plasma. Plasma
concentration of drug was determined by HPLC assay. For biodistribtion study animals
were sacrificed at 6, 12 and 24 h time intervals and tumor, lungs, liver, kidney, heart and
spleen were collected. Simple one step method, protein precipitation with acetonitrile was
used for the extraction of paclitaxel. In this procedure, first tissue was homogenized in
normal saline in the ratio of 1:1 (v/v). Paclitaxel was extracted from plasma and tissue
homogenate samples by precipitation with acetonitrile in 1:1 and 1:2 ratios (v/v),
respectively. Samples were then vortexed for 1 min followed by centrifugation for 10 min
at 10000 rpm. The supernatant was transferred to vials from which 20 µL was injected
onto HPLC column after filtration for quantitative determination.
5.15 Skin Irritation Potential
5.15.1 Draize test
The irritancy of different formulations was determined in male albino rabbits (1.9-
2.0 kg) based on the method described by Draize et al. (1944). The animals were housed
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in an air-conditioned room (222.0 C) and hair of the back was trimmed short 24 h
before the beginning of the test. Three squares were drawn on each side of the back of
each rabbit. The squares were divided into seven groups each consisting of three. The
first group did not receive any treatment and acted as a sham control, second group
(control) received topical application of the PBS (7.4) and third group received 20% SLS
solution acting as a positive control. Fourth, fifth, sixth and seventh groups received
paclitaxel solution and paclitaxel elastic liposomal formulations EL-SP3 and EL-SD3 and
conventional liposome, respectively. At different time intervals of 0, 1, 24, 48 and 72 h
after application, the exposed area was scored for the erythema and oedema on grade of
0-4.
5.15.2 Transepidermal water loss measurement (TEWL)
The TEWL measurement was carried out using same protocol as discussed above
for skin irritation study. The rabbit was anaesthetized during the measurement. TEWL
measurement was carried out at 0, 24 and 48 h time intervals. The TEWL was measured
using Tewameter TM 210. The probe of Tewameter was kept perpendicular to the surface
of the skin and a stable reading of TEWL was reached in about 60s. The results were
expressed in g/hm2. The measurement was performed in an air conditioned room at
temperature of 22±0.5 ºC.
5.16 Statistical Analysis
Data are expressed as the mean ± standard deviation (SD) of obtained results. The
statistical analysis of data was performed using analysis of variance (ANOVA) (Sigma
stat 3.5, STATCON and Witzenhausen, Germany). A value of p < 0.05 was considered
statistically significant.