Chapter 5shodhganga.inflibnet.ac.in/bitstream/10603/13755/13/13_chapter 5.pdf · Autoanalyzer Erba Chem 7, Germany Hematolyzer Sysmex, USA Optical Microscope DX31, Oylmpus, Japan

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.

Chapter 5 - Materials and methods

-83-

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


-84-

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.


-85-

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.


-86-

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.


-87-

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


-88-

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


-89-

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


-90-

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


-91-

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

×


-92-

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.


-93-

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


-94-

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


-95-

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.


-96-

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)


-97-

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.


-98-

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


-99-

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


-100-

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


-101-

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


-102-

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


-103-

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)






9. Group 9 ( 200 mg/kg, Paclitaxel loaded EL-SP3)

10. Group 10 (10 mg/kg, Paclitaxel loaded EL-SD3)






16. Group 16 (200mg/kg, paclitaxel loaded EL-SD3)

17. Group 17 (10 mg/kg, Marketed formulation)





-104-

Table 5.4: Repeated-dose 28 days subacute toxicity study frame.

S. No. Group Treatment

1. Group 1 Sham control

2. Group 2 Control















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


-105-

(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.


-106-

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.


-107-

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


-108-

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.

Documents

Chapter 5shodhganga.inflibnet.ac.in/bitstream/10603/13755/13/13_chapter 5.pdf · Autoanalyzer Erba Chem 7, Germany Hematolyzer Sysmex, USA Optical Microscope DX31, Oylmpus, Japan