6.1. MATERIALS 6.1.1. List of Materials Used S.No...

Materials & Methods

Dept. of Pharmaceutics, J.S.S. College of Pharmacy, Udhagamandalam (J. S. S. University, Mysore) T.N. 58

6.1. MATERIALS

6.1.1. List of Materials Used

S.No. Chemicals/Materials Manufacturer/Suppliers

1. Curcumin Sami Labs Ltd., Bangalore

2. Betulinic acid Sigma Aldrich, Bangalore

3. Lenalidomide Apicore Pharmaceutical Pvt. Ltd., Gujrat

4.. Chitosan (CS) (specifications:

molecular weight: 100,000-

300,000 Da, deacetylation degree

>80%)

Sigma Aldrich, Bangalore

5. Sodium alginate(molecular weight

100 kDa and mannuronic to

guluronic acid ratio of 60:40)

CDH Labs Ltd., India

6. Gelatin B Sterling Biotech Ltd., Ooty

7. κ-Carragennan Sigma Aldrich, Bangalore

8. Xanthan gum, bovine serum

albumin and gelatin A

Affy Pharma Pvt. Ltd., Baddi, Himanchal

Pradesh

9. Mango gum, badam gum, ispagal

and fenugreek seed

Local market, Udhagamandalam,

Tamilnadu

10. Potassium-dihydrogen

orthophosphate AR

S.D. Fine Chem Ltd., Mumbai

11. Sodium hydroxide pellets AR Qualigens Pvt. Ltd., Mumbai

12. Concentrated hydrochloric acid

LR

Qualigens Pvt. Ltd., Mumbai

Materials & Methods


13. Methanol (HPLC grade) E. Merck (India) Ltd., Mumbai

14. Acetonitrile (HPLC grade) Rankem Ltd., New Delhi

15. Potassium chloride AR

Qualigens Pvt. Ltd., Mumbai

16. Brij 78 (M.P. 44-46 ºC) Sigma Aldrich, Bangalore

17. Calcium chloride AR Merck Pvt. Ltd., Mumbai

18. Sodium chloride AR Qualigens Pvt. Ltd., Mumbai

19. Sodium hydrogen carbonate AR S.D. Fine-Chem Ltd., Mumbai

20. Triethylamine (HPLC) Merck Pvt. Ltd., Mumbai

21. Orthophosphoric acid LR S.D. Fine-Chem ltd., Mumbai

22. Tween 80 LR Rankem Ltd., New Delhi

23. White leghorn chicken eggs Poultry Research Centre, Kerala.

Materials & Methods


6.1.2. List of Equipment Used

S.No. Equipment Manufacturer/Suppliers

1. pH meter pH/ /ion/MV

meter

ion 510 bench pH, Eutech Instruments, Mumbai

2. Blade stirrer with speed

regulator

Remi Electrotechnik Ltd., Vasai, India

3. UV-VIS

spectrophotometer

PharmaSpec 1700, Shimadzu, Japan

4. Digital oven HICON, Grover Enterprises, Delhi

5. Digital electronic balance Sartorius BT 224 S, Bangalore

6. Differential scanning

calorimeter

Water Q 200, Bangalore

7. FTIR spectrophotometer Shimadzu FTIR 84000 S, Japan

8. UFLC Shimadzu LC-20 AD, SPD-M201 230V, Japan

9. Zeta sizer Malvern, Model No. 3000 HF, Malvern (U.K.)

10. Deep freezer Labline, Mumbai

11. Freeze dryer SKL-12 N, Esquire Biotech, Chennai

12. Incubator Jindal Scientific Instruments, Delhi

13. Orbitory shaker IKA KS 4000 i control, Chennai

14. Single pan electronic

balance

K- Roy Instruments, Varanasi

15. Aseptic cabinet S.M Scientific Instrument (P) ltd., Delhi

16. Autoclave

HICON, Grover Enterprises, Delhi

17. Scanning electron

microscopy (SEM)

SEM, JFC 1200 fine coater, Japan

18. Transmission electron

microscopy (TEM)

TEM, Philips CM-10, USA

Materials & Methods


19. X-Ray diffractometer Bruker, AXS/8, Berlin, Germany

20. Elemental analyzer Perkin Elmer, 2400 Series, CHNSO Analyser,UK

21. Atomic absorption

spectroscopy

Shimadzu AA-6300, Serial No-A305245, Japan

22. Mass spectrometry Bruker Daltonik, Bremen, Germany

23. NMR spectroscopy Bruker AC-300 Spectrometer, Germany

24. GC/MS analyser Agilent Technologies, Palo Alto, USA

25. Stability humidity cabnit Grover Enterprises, New Delhi

Materials & Methods


6. 2. METHODS

6.2.1. Preformulation Studies

6.2.1.1. Selection of curcumin, betulinic acid and lenalidomide

The curcumin, betulinic acid and lenalidomide were selected on the basis of

docking studies on PKC-beta II/VEGF-A/HIF-1 alfa receptors.

6.2.1.1.1. Docking and molecular modelling studies

The molecular modelling studies were carried out on Windows 7 workstation

and Linux Fedora 7.0 workstation using Glide, version 5.7, Schrodinger suit 2011,

LLC on a Maestro graphical user interface.

6.2.1.1.2. Ligand Preparation

The structures of the ligands were generated in the CDX format converted

mol2 format and subjected to LigPrep module of Maestro in the Schrödinger suite of

tools. They were converted from 2D to 3D structures by including stereochemical,

ionization, tautomeric variations, as well as energy minimization and optimized for

their geometry, desalted and corrected for their chiralities and missing hydrogen

atoms. The bonds orders of these ligands were fixed and the charged groups were

neutralized. The ionization and tautomeric states were generated between pH of 6.8 to

7.2 using Epik module. In the final stage of LigPrep, compounds were minimized

using Optimized Potentials for Liquid Simulations-2005 (OPLS-2005) force field in

impact package of Schrodinger until a root mean square deviation of 1.8Ǻ was

achieved by algorithm and conjugate gradient method. A single low energy ring

confirmation per ligand was generated and the optimized ligands were used for

docking analysis127.

6.2.1.1.3. Protein Preparation

The crystal structure of protein PKC beta-II, VEGF-A, HIF-1 alfa (PDB ID:

2I0E, 1YWN, 1YCI) was downloaded from the RCSB Protein Data Bank (PDB) and

refined for their bond orders, formal charges and missing hydrogen atoms, topologies,

incomplete and missing residues and terminal amide groups. The water molecules

beyond 5 Å of the hetero atom were removed. The possible ionization states were

Materials & Methods


generated for the heteroatom present in the protein structure and the most stable state

was chosen. The hydrogen bonds were assigned and orientations of the retained water

molecules were corrected. Finally, a restrained minimization of the protein structure

was carried out using OPLS 2005 force field to reorient side-chain hydroxyl groups

and alleviate potential steric clashes. The minimization is restrained to the input

protein coordinates by a predefined root mean square deviation (RMSD) tolerance of

0.3 Å.

6.2.1.1.4. Receptor grid generation

The ligand ANP (phosphoaminophosphonic acid adenylate ester) was retained

in the crystal structure of the prepared protein which was used for the receptor grid

construction. The binding box dimensions (within which the centroid of a docked

pose is confined) of the protein was set to 14 Å x 14 Å x14 Å.

6.2.1.1.5. Validation of the docking programme

The accuracy of the docking procedure was determined by finding how

closely the lowest energy pose (binding conformation) of the co-crystallized ligand

predicted by the object scoring function, Glide score (G Score), resembles an

experimental binding mode as determined by X-ray crystallography. Extra precision

Glide docking procedure was validated by removing the co-crystallized ligand from

the binding site of the protein and redocking the ligand with its binding site. The

hydrogen bonding interactions and the root mean square deviation (RMSD) between

the predicted conformation and the observed X-ray crystallographic conformation

were used for analyzing the results.

6.2.1.1.6. Glide Ligand docking

The glide docking of the curcumin, betulinic acid and lenelidomide were

carried out using the previously prepared receptor grid and the ligand molecules. The

favorable interactions between ligand molecules and the receptor were scored using

Glide ligand docking program. All the docking calculations were performed using

extra precision (XP) mode and OPLS-2005 force field. The above docking process

was run in a flexible docking mode which automatically generates conformations for

each input ligand. The ligand poses generated were passed through a series of

Materials & Methods


hierarchal filters that evaluate the ligand’s interaction with the receptor. The initial

filter test the spatial fit of the ligand to the defined active site, and examines the

complementary of the ligand-receptor interactions using grid-based method patterned

after the empirical ChemScore function. This algorithm recognizes favorable

hydrophobic, hydrogen-bonding and metal-ligation interactions, and penalizes steric

clashes. Poses that pass these initial screens enter the final stage of the algorithm,

which involves evaluation and minimization of a grid approximation OPLS

nonbonded ligand-receptor interaction energy. Finally, the minimized poses were re-

scored using GlideScore scoring function. GlideScore is based on ChemScore, but

includes a steric-clash term, adds buried polar terms to penalize electrostatic

mismatches, and has modifications to other terms:

GScore = 0.065*vdW + 0.130*Coul + Lipo + Hbond + Metal + BuryP + RotB + Site

(vdW: Vander Waals energy, Coul: Coulomb energy, Lipo: Lipophilic term, Hbond:

Hydrogen-bonding term, Metal: Metal-binding term, BuryP: Penalty for buried polar

groups, RotB: Penalty for freezing rotatable bonds, Site: Polar interactions in the

active site).

6.2.1.2. Selection of polymers

Chitosan (C) was selected as cationic polymer and sodium alginate (SA),

bovine serum albumin (BSA), κ-carragennan (CA) and gelatin A (GA) were selected

as anionic polymers. The characterized plant based mucilages and gum was also

selected as anionic polymer in combination with chitosan for the preparation of

nanoreservoir system. For optimization of concentration of selected polymers,

mucilages and gum were subjected for following studies such as opalascent

suspension formation, effect of pH on opalascent suspension formation and swelling

index.

Materials & Methods


6.2.1.3. Isolation and purification of water soluble fractions of mucilages and

gum

6.2.1.3.1 Extraction and purification of mucilage from fenugreek and isphagula seed

The seeds were collected and washed with water to remove the dirt and debris.

The seeds (250g) were soaked in double distilled water (500 ml) overnight and then

heated at 50°C for 2h. The solution was filtered through muslin cloth and to the

filtrate equi-volume ratio of 90% alcohol was added. The obtained precipitate was

filtered and dried in a hot air oven at 45ºC to obtain ≈150g of powder. The obtained

powder was re-dissolved in 100 ml of water, filtered and centrifuged for 10 min at

3000 rpm. The supernatant clear solution was collected, evaporated and dried. This

process of purification was repeated thrice. The purified solid mass was dried under

reduced pressure at 40°C, grounded and passed through sieve no. 80 and stored in an

airtight container.

6.2.1.3.2. Extraction and purification of mango gum

Crude plant exudates were collected in season of March to June. Mango gum

was obtained from the incised trunk of Mangifera indica. The Mango gum resin

(250g) was extracted by distilled water 500 ml on a water bath maintaining at 40 – 50

°C for 45 min with intermittent stirring, extraneous materials were removed by

straining through a several folds of muslin cloth. The gum was then precipitated by

using 90% v/v alcohol followed by centrifugation at 3000 rpm. The extracted gum

was filtered and water was evaporated in oven at 45 °C. The obtained powder was re-

dissolved in 100 ml of water, filtered and centrifuged for 10 min at 3000 rpm. The

supernatant clear solution was collected, evaporated and dried. This process of

purification was repeated thrice. The purified solid mass was dried under reduced

pressure at 40°C, grounded and passed through sieve no. 80 and stored in an airtight

glass container128.

Materials & Methods


6.2.1.4. Physico-chemical and structural characterization of mucilages and gum

4.2.1.4.1. Physicochemical studies of isolated mucilages and gum

The obtained purified samples of mucilages and gum were subjectd to various

biochemical tests for carbohydrates, proteins, tannins, alkaloids, saponins, phenol and

flavonoids as per official methods129.

6.2.1.4.2. Structural elucidation

6.2.1.4.2.1. X-ray diffraction analysis (X-RD)

In X-ray studies, an automatic x-ray diffractometer equipped with a PW R30

x-ray generator was used. The dry sample powder was pressed into pellets and X-ray

diffraction spectra were recorded using nickel-filtered Cu kα1 radiation having a

wavelength of 1.5106 Å, operating at 35 kW and 20 mA. X-ray diffractograms were

obtained at a scanning rate of 1 degree (2θ) per minute.

6.2.1.4.2.2. Elemental composition analysis

Elemental compositions of C, H and N were analyzed by using an elemental

analyzer. Accurately weighed 0.5 mg sample was heated to 1150 ºC seperately and

the corresponding element was determined by using a thermal conductivity detector.

6.2.1.4.2.3. Atomic absorbtion spectroscpic (AAS) analysis

The AAS instrumental parameters for the estimation of major, trace and heavy

metals were set which include the lamp current, wavelength, slit width, lamp mode,

fuel flow rate, support gas flow rate, flame type and burner height were optimized and

are given in Table 6.1. Deionized water was used for all the dilutions. All the plastic

materials and glass wares were cleaned by soaking in dilute nitric acid solution for 24

h and rinsed with distilled water followed by deionized water prior to use. The

calibration curves for the analyte ions were drawn after setting various parameters.

All measurements were performed in triplicate (n=3) and the standard deviation (SD)

was recorded.

Materials & Methods


Table 6.1. System sutability parameters for estimation of major, trace and heavy

metals Instrument Parameter

Elements Ca Na K Cu Pb As Hg Cd Al

Lamp current (mA)

10 12 10 6 10 12 4 8 10

Wavelength (nm)

422.7 589.0 768.0 324.8 283.3 193.7 243.7 228.8 309.3

Slit weidth (nm)

0.7 0.2 0.7 0.7 0.7 0.7 0.7 0.7 0.7

Lamp mode BGC-D2

Non-BCG

Non-BCG

BGC-D2

BGC-D2

BGC-D2

BGC-D2 BGC-D2

BGC-D2

Fuel gas flow rate (L/min)

2.0 1.8 2.0 1.8 2.0 2.0 Nil 1.8 7.0

Support gas flow rate (L/min)

15 15 15 15 15 15 Nil 15 15

Flame type Air-C2H2

Air-C2H2

Air-C2H2

Air-C2H2

Air-C2H2

HVG (Air-C2H2)

HVG cold Vaporizer technique

Air-C2H2

N2O-C2H2

Burner height (mm)

7 7 7 7 7 7 Nil 7 11

Linearity range (ppm)

0.5-2.5 2-10 1-5 0.5-2.5 2-10 0.010-0.050

0.010-0.050

0.10-0.50

5-25

Regression eqution

Y=0.0137X-0.0015

Y=0.0921X+0.0261

Y=0.0142X+0.0028

Y=0.0712X-0.0064

Y=0.0021X+0.00092

Y=0.0238X+0.2108

Y=0.010X+0.0038

Y=0.000067X-0.00006

Y=0.0007X+0.0009

Correlation coefficient

0.9999 0.9989 0.9989 0.9999 0.9994 0.9945 0.9989 0.9998 0.9994

6.2.1.4.2.4. Fourier Transform Infrared spectroscopy (FTIR)

The FT-IR spectrum of sample was recorded on FT-IR spectrophotometer.

The dry sample (10 mg) was ground into fine powder using mortar and pastel then

pressed using potassium bromide (KBr 100 mg) disc technique under a hydraulic

press at 10,000 psi. Each KBr disc was scanned at 4 mm/s a resolution of 2 cm over a

wave number region of 4000 – 400 cm-1.

Materials & Methods


6.2.1.4.2.5. Mass Spectroscopic analysis (MS)

The average molecular weight of sample was determined by matrix assisted

laser desorption/ionization-time of flight (Reflex II MALDI-TOF instrument) analysis

in the negative mode. For the ionization, a nitrogen laser (337 nm, 3 ns pulse width, 3

Hz) was used. Sample (5mg) was dissolved in D2O solvent. 0.5mL of the sample was

applied to the target followed by the addition of 1 mL of matrix solution (2,5-

dihydroxybenzoic acid), and dried under a gentle stream of air. All spectra were

measured in the reflector mode using external calibration.

6.2.1.4.2.6. 1D and 2D NMR spectroscopy

NMR spectra of 1H and 13C of sample was recorded in an NMR spectroscopy.

100 mg of sample was dissolved in D2O and chemical shifts were reported in ppm

relative to an internal standard TMS (tetramethylsilane) for 1H NMR and for 13C

spectra. NMR spectrum was obtained at a base frequency of 400MHz, with 16

transitions and delay time 1.5s using D2O as solvent (samples in tubes of 0.5 cm id).

The spectral width was 200 ppm, chemical shifts were expressed in δ (ppm) relative

to the resonance of internal TMS.

2D NMR spectrum was applied using double-quantum filtered correlated

spectroscopy (DQF COSY), hetero nuclear single-quantum coherence (HMQC) with

a Bruker NMR spectrometer.

6.2.1.4.2.7. Acid-base hydrolysis

The pH of the sample solution (300 mg/ml) was adjusted to 2.0 by adding

0.5M sulfuric acid. The solution was heated to 100 ºC for 24 h. After cooling, the pH

of solution was adjusted to 10 by the addition of 0.5M NaOH and heated to 100 ºC for

24 h. After cooling the solution was neutralized with barium carbonate and filtered.

The solution was then dialyzed for 24 h against de-ionized water; freeze dried130. The

obtained hydrolysed dry samples were subjected for FTIR and Gas chromatography-

/mass spectrometry (GC-MS) studies.

6.2.1.4.2.7.1.

For freezed dried hydrolysed fraction of mucilages and gum were subjected

for FT-IR (method described in section 6.2.1.4.2.4) and GC-MS analysis was

FTIR and GC-MS analysis of hydrolysed fraction

Materials & Methods


performed using HP-5 capillary column (0.25 mm x 30 m) linked to model TD800

Finningam ion trap mass spectrometer (MS) operated at 70 eV. The columns were

programmed from 50-220 ºC at 40 ºC min-1.

6.2.1.4.2.8. Determination of surface charge and pH

1% w/v solution of sample was prepared in distilled water and determined

surface charge by potentiometric analysis using potentiometer and pH of same

solution (1% w/v) was determined by using digital pH meter. All measurements were

performed in triplicate (n=3) and the standard deviation (SD) was recorded.

6.2.1.4.2.9. In vitro Cell viability assay

Cytotoxicity of the sample was determined by the MTT (3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using L929 mouse

fibroblast cell line. 1 x 105cells/ml were plated in each well of a 96 well microtitre

plate in 100µl of medium and incubated at 37°C in CO2 incubator. In the first six

wells cells were left without any treatment as a positive control. Stock solutions of the

samples of different concentrations (0.05mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml and

5mg/ml) were made in 1% DMSO and diluted with minimal essential medium

(MEM) to a final concentration of 50, 500, 1000, 2000, 5000 µg/ml in the plate. Once

the confluent monlolayer was ready, concentrations of the test samples of 50μl was

added to each well in triplicate and incubated at 37 oC in a CO2 incubator for 72 h.

After incubation 50 µl of MTT dye (2mg/ml) was added to each well and again

incubated at 37 oC for another 4 h. Formed formazan was washed and dissolved using

50 µl of isopropanol and optical density was recorded with a microtitre plate reader at

550 nm131. All measurements were performed in triplicate (n=3) and the standard

deviation (SD) was recorded. Cell viability was calculated by the formula:

Where Isample is the absorbance of samples treated well and Icontrol is the absorbance of

contro wells without samples.

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Materials & Methods


6.2.1.5. Compatibility studies

The compatibility studies of curcumin, betulinic acid and lenalidomide in

combination with different polymers, mucilages and gum in 1:1 ratio (physical

mixture) were carried out by FT-IR and by DSC studies.

6.2.1.5.1. Fourier transform infrared spectroscopy (FTIR)

FT-IR spectra of curcumin, betulinic acid and lenalidomide alone were

recorded and their physical mixtures with polymers, mucilages and gum were

recorded using FT-IR spectrophotometer (method described in section 6.2.1.4.2.4).

6.2.1.5.2. Differential scanning calorimetry (DSC)

Differential scanning calorimetric analysis was used to characterize the

thermal behavior of the drug, polymers, mucilages, gum and their physical mixtures.

Sample was crimped in standard aluminium pans and heated from 20 to 400 ºC at a

heating rate of 10 ºC/min under constant purging of dry nitrogen at 30ml/min. An

empty pan, sealed in the same way as the sample, was used as a reference. DSC

thermograms were obtained using an automatic thermal analyzer system.

Temperature calibration was performed using Indium calibration reference standard.

6.2.1.6. Analytical method development

6.2.1.6.1 Calibration curve of curcumin

The stock solution (100µg/ml) of curcumin was prepared using acetonitrile.

From stock solution aliquots of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 ml were

pipetted out in 10 ml volumetric flask to obtain the concentration of 2000-16000

ng/ml and analysed at 418 nm by ultra fine liquid chromatography (UFLC) method.

Calibration curve data was subjected to linear regression analysis and obtained the

intercept, slope and regression equation. All measurements were performed in

triplicate (n=3) and the standard deviation (SD) was recorded.

6.2.1.6.2. Optimized liquid chromatographic conditions

UFLC analysis was carried out by gradient elution using Hibar C18 (250 x 4.6

mm, 5µ) column with a mobile phase of acetonitrile–phosphate buffer pH 6.5 (buffer

Materials & Methods


strength-25mM) in 85:15 ratio at a flow-rate of 1.0 ml/min. An aliquot of 20 µl using

Rheodyne 7725 injector of each sample was injected onto the UFLC column and the

eluate was monitored at 418 nm with retention time of 3.2 min. The procedure was

repeated three times for each sample.

6.2.1.6.3. Calibration curve of betulinic acid

The stock solution (10µg/ml) of betulinic acid was prepared in methanol.

From stock solution aliquots of 0.5, 2, 4, 6 and 8 ml were pipetted out in 10 ml

volumetric flask to obtain the concentration of 500-10,000 ng/ml. and analysed at 210

nm by UFLC method. Calibration curve data was subjected to linear regression

analysis and obtained the intercept, slope and regression equation. All measurements

were performed in triplicate (n=3) and the standard deviation (SD) was recorded.



mm, 5µ) column with a mobile phase of acetonitrile–phosphate buffer pH 3.0 (buffer

strength-25mM) in 80:20 ratio at a flow-rate of 1.2 ml/min. An aliquot of 20 µl using




6.2.1.6.5. Calibration curve of lenalidomide

The stock solution of Lenalidomide was prepared in acetonitrile. From stock

solution of 100 µg/ml aliquots of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 ml were

pipetted out in 10 ml volumetric flask to obtain the concentration of 2000-16000

ng/ml. and analysed at 220 nm by UFLC method. Calibration curve data was

subjected to linear regression analysis and obtained the intercept, slope and regression

equation. All measurements were performed in triplicate (n=3) and the standard

deviation (SD) was recorded.



mm, 5µ) column with a mobile phase of acetonitrile-water pH 4.0 (buffer strength-

Materials & Methods


25mM) in 90:10 ratio at a flow-rate of 1.0 ml/min. An aliquot of 20 µl using




6.2.1.6.7. Method Validation

Validation of the method was carried out after the development of the UFLC

methods. Validation parameters tested were- Selectivity/ Specificity, Sensitivity,

Linearity, Accuracy, Ruggedness and Robustness.

6.2.1.6.7.1. Selectivity/ Specificity

A method is said to be specific when it produces a response only for a single

analyte. Method selectivity is the ability of the method to produce a response for the

analyte in the presence of other interferences. In order to prove that the method chosen

was specific and selective the following sets of samples were processed and injected

into the UFLC using the extraction procedure. The % interference was calculated using

formula;

6.2.1.6.7.2. Sensitivity

It is expressed as limit of quantitation. It is the lowest amount of analyte in a

sample matrix that can be determined.

6.2.1.6.7.3. Linearity

Linearity and range of the methods were analyzed by preparing calibration

curves using different concentrations of the standard solution. The calibration curve

was plotted using response factor and concentration of the standard solutions. Linearity

was established over the range of 2000-16000 ng/ml using the weighted least square

regression analysis.

Materials & Methods


6.2.1.6.7.4. Accuracy

Accuracy of the method was determined by recovery experiments. The

recovery of the method was determined at single level by adding a known quantity of

curcumin, betulinic acid and lenalidomide separately to the known suspension of

nanoformulation and mixtures were reanalysed. The avarage recovery obtained from

each sample was reported as % nominal of the analyzed concentration which is

calculated using formula;

6.2.1.6.7.5. Ruggedness

Ruggedness of the method was studied by changing the experimental

conditions such as operators, instruments, source of reagents, solvents and column of

similar type. Chromatographic parameters such as retention time, asymmetric factor,

capacity factor and selectivity factors were evaluated.

6.2.1.6.7.6. Robustness

Robustness of the method was studied by injecting the standard solutions with

slight variations in the optimized conditions, ± 1% in the ratio of acetonitrile in the

mobile phase and ± 0.1 ml of the flow rate.

6.2.1.7. Solubility studies of curcumin/betulinic acid/lenalidomide

An excess amount of drug (50 mg) was added to 25 ml stoppered conical flask

containing 10 ml of simulated tear fluid (STF) pH 7.4 with different concentrations of

tween 80 and brij 78 (0.1%, 0.2%, 0.3%, 0.4%, and 0.5%) and without tween 80 and

brij 78 separately placed in rotary shaker at 200 rpm at 37± 0.5°C. At the end of 24

and 48 h the samples were collected, filtered and diluted (100 times) which were

analyzed for saturation solubility by UFLC. All measurements were performed in

triplicate (n=3) and the standard deviation (SD) was recorded132.

Materials & Methods


6.2.1.8. Preliminary parameters for nanoparticle preparation

6.2.1.8.1. Formation of opalescent suspension and effect of pH

The different concentration ranges of polymers, mucilages and gum (0.01-

1.0%w/v) were subjected in suitable media (distilled water and 0.1% acetic acid in

case of chitosan) at room temperature. Opalescent suspension formation was

determined by visual examination of the suspension under light alternatively against

white and black backgrounds. pH of the solution also plays important role in

opalascent suspension formation. The selected ranges of polymers, mucilages and

gum (0.02-01.0% w/v) was subjected to the different pH range (4.5-7.5) to study the

effects of pH on opalascent suspension formation. pH of different solutions were

manipulated by addition of 0.1N hydrochloric acid/0.1N sodium hydraoxide and pH

was measured by pH meter. The effect of pH on opalascent suspension was examined

under light and dark background. All measurements were performed in triplicate

(n=3) and the standard deviation (SD) was recorded133.

6.2.1.8.2. Determination of swelling index

A modified fabricated apparatus described by Sharma et al134 (Fig. 6.1) was

used to measure water uptake and swelling index (SI). Weighed quantity of sample

was subjected to the graduated arm A (internal diameter 10 mm) and the swelling

medium (distilled water and 0.1% acetic acid in case of chitosan) were poured in

graduated arm B (internal diameter 10 mm) to a level corresponding to the height of

the powder pile in arm A. The level of swelling medium was maintained constant

during the entire experiment. The increase in the volume (cm3) of sample was

recorded at different time intervals upto 48 h and SI was calculated by the following

formula;

Materials & Methods


Figure 6.1. Swelling index apparatus

6.2.2. Formulation studies

6.2.2.1. Preparation of nanoparticles

The nanoparticles were prepared by modified coacervation method135 using

design of experiment of 23 factorial design.

Chitosan polymer was dissolved in 0.1% w/v of acetic acid; volume adjusted

using double distilled water and pH 5.5 with 0.1M NaOH. Sodium alginate solution

was prepared by dissolving in double distilled water and pH adjusted 5.5 using 0.1N

hydrochloric acid. The chitosan and sodium alginate solutions were filtered under

vacuum. Prepared drug solution in tween 80/brij 78 added in chitosan solution with

constant stirring and resultant solution was added dropwise with the help of syringe in

sodium alginate solution. The solution was stirred at 3000 rpm for 2 h and formed

nanoparticles were collected by centrifugation. Before freeze drying 0.5% w/v of

mannitol was added as a lyoprotectant. Freeze dried nanoparticles were stored in

desiccator under vacuum.

The same procedure was followed for other anionic polymers, mucilages and

gum (bovine serum albumin, gelatin A, κ-carragennan fenugreek mucilage, ispagol

mucilage, mango gum) in combination with chitosan. The general method of

preparation of nanoparticles is shown in flowchart.

Materials & Methods


Flowchart for general method of preparation and sterlization of nanoparticles

6.2.2.2. Experimental design

Use of experimental design allows for testing a large number of factors

simultaneously and precludes the use of a huge number of independent variables. 23

factorial statistical design was used to optimized the formulation parameters and

evaluated the main effects. Experimental design was validated by using polynomial

equation;22

Y= B0+B1X1+ B2X2 + B3X3+B12X1X2+ B13X1X3+ B23X2X3+B123X1X2X3

Where, Y is the measured response associated with each factor level combination,

B0 is intercept, B1 to B3 are regression coefficients, X1, X2, X3 denotes the

concentration of cationic, anionic polymers and drug. The dependent variables were

particle size, zeta potential, morphology and encapsulation efficiency.

Materials & Methods


6.2.2.3. Selection of Optimized Formulation Optimized formulation was selected on the basis of small particle size, higher

zeta potential (in between 30-35 mV), spherical in shape and higher entrapment

efficiency.

6.2.2.4. Statistical analysis of responses by Design Expert

Design Expert 8 was used to analyse the effect of each variable on the

designated response. Surface responce charts were made for the analysis of each

response coefficient for its statistical significance. Quantitative and qualitative

contribution of each variable on each of the response was analyzed. The significant

response polynomial equations generated by Design Expert were used to validate the

statistical design136. Response surface plots were generated to visualize simultaneous

effect of each variable on each response parameter.

6.2.3. Evaluation of prepared nanoparticles

6.2.3.1. Percent recovery of nanoparticle

Freeze-dried nanoparticles weighed accurately, and nanoparticles recovery

(%) were calculated using formula;137

6.2.3.2. Particle size and zeta potential

Nanoparticles size distribution and zeta potential were determined using

zetasizer photon correlation spectroscopy. The size distribution analysis was

performed at a scattering angle of 90° and at a temperature of 25°C using samples

appropriately diluted with filtered water (0.2 μm filter), whereas zeta potential was

measured using a disposable zeta cuvette. For each sample, the mean diameter/zeta

potential ± standard deviation of six determinations was calculated applying

multimodal analysis.

Materials & Methods


6.2.3.3. Surface morphology

6.2.3.3.1 Scanning electron microscopy (SEM)

The surface morphology of nanoparticles was recorded by using SEM.

Appropriate samples were mounted on an aluminum stub with double-sided adhesive

tape. The tape was first firmly attached to the stub and the sample powder was

scattered carefully over its surface. The stub with the specimen was then sputter

coated with a thin layer of gold to make the specimens conductive. The processed

specimen was subjected to SEM analysis.

6.2.3.3.2. Transmission electron microscopic (TEM)

TEM was used to study the surface morphology of nanoparticles. Samples of

the nanoparticles suspension (5-10 μl) were dropped onto formvar-coated copper

grids. After complete drying, the samples were stained using 2% w/v phosphotungstic

acid. Digital Micrograph and soft imaging viewer software were used to perform the

image capture.

6.2.3.4. Determination of encapsulation efficiency

The encapsulation efficiency of nanoparticles was determined by the

separation of drug-loaded nanoparticles from the aqueous medium containing free

drug by cooling centrifugation at 18,000 rpm for 30 min. The amount of free drug in

the supernatant was measured by UFLC. The encapsulation efficiency of the

nanoparticles was determined in triplicate and calculated as follows;138

6.2.3.5. In-vitro release study

Lyophilized drug loaded nanoparticles were redispersed in 5 ml of ultrapure

water and quantity equivalent to 2 mg of the drug placed in dialysis membrane bag

with a molecular cut-off of 5 kDa, tied and placed in 280 ml of simulated tear fluid

(STF), pH 7.4. The entire system was kept at 37 ± 0.5 ºC with continuous magnetic

stirring (25 rpm). At appropriate time intervals (0, 0.30, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16,

18, 20, 22, 24 h), 2 ml of the release medium was removed and 2 ml fresh medium

was added into the system to maintain sink conditions. The amount of drug in the

Materials & Methods


release medium was evaluated by UFLC method. The cumulative % drug release

calculated and drug release data were calculated to study the possible mechanism of

drug release of nanoparticles. All measurements were performed in triplicate (n=3)

and the SD was calculated.

6.2.3.6. Stability of prepared nanoparticles

Freeze dried samples of nanoparticles were stored at room temperature for

stability studies. The nanoparticles were rehydrated with ultrapure water at every 3

months interval up to 9 months and evaluated for any change in particle size, zeta

potential and entrapment efficiency139.

6.2.4. Formulation of nanogel

Under aseptic condition the carbopol (carbopol C974, 0.1 % w/v) was soaked

in double distilled water for 5 h and benzalkonium chloride 0.02% w/v was added by

uniform mixing on magnetic stirrer. The prepared carbopol gel was sterilized by

autoclaving at 121°C for 20 min. The selected ideal batch of sterile freeze dried

nanoparticles in 1:1 ratio (equivalent to 5 mg drug) was incorporated to carbopol by

uniform mixing on magnetic stirrer for 2 h at room temperature. The gel was formed

at pH 6.8 by the addition of 0.1N triethylamine. The prepared nanogel was stored in

sterilized container and evaluated for clarity, pH, homogeneity, viscosity, ex-vivo

permeation study using goat cornea, sterility studies, in-vitro, in-vivo ocular irritation

studies, in-vitro tube formation studies and in-vitro, in-vivo anti-proliferation

studies140.

The optimized batches of curcumin, betulinic acid and lenalidomide

nanoparticles were incoporated into carbopol gel to formulate following batches of

nanogels (B1, B2, B3, B4, B5 with curcumin nanoparticles, B6, B7, B8, B9, B10 with

betulinic acid nanoparticles, B11, B12, B13, 14, 15 with lenalidomide nanoparticles).

6.2.5. Evaluation of nanogel

6.2.5.1. Clarity

The clarity of the nanogel formulations were determined by visual

examination under light alternatively against white and black backgrounds.

Materials & Methods


6.2.5.2. pH

The 1% w/v solution was prepared by double distilled water and pH of that

diluted suspension was determined by using digital pH meter. All measurements were

performed in triplicate (n=3) and the standard deviation (SD) was recorded.

6.2.5.3. Viscosity

Viscosity of nanogel was determined by using Brookfield’s viscometer

attached with S 34 spindle. In the small volume adaptor the nanogel was filled and the

angular velocity was increased gradually from 10, 20, 50 and 100 rpm. The hierarchy

of the angular velocity was reversed. The average of three readings was taken to

calculate the viscosity of the nanogels.

6.2.5.4. Ex-vivo corneal permeation studies using goat’s cornea

Goat cornea was used for the study permeation across the corneal membrane.

Whole eyeballs of goat were procured from a slaughter house, Ooty and transported to

laboratory in cold condition in normal saline maintained at 4ºC. The corneas were

carefully removed along with a 5–6 mm of surrounding scleral tissue and washed with

cold saline. The washed corneas were kept in cold freshly prepared solution of STF of

pH 7.4. The study was carried out by using Franz‐diffusion cell in such a way that

corneum side was contineously remained in an intimate contact with formulation in

the donor compartment. The receptor compartment was filled with STF, pH 7.4 at 34

± 0.5 ºC. The receptor medium was stirred on a magnetic stirrer. 2 ml samples were

withdrawn at different time intervals (0, 0.3, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20,

22, 24 h) and replenished with an equal volume of STF (pH 7.4). The percent drug

released was plotted against time to get release kinetics. All measurements were

performed in triplicate (n=3) and the SD was calculated141.

6.2.5.4. Stability study

The stability studies were carrierd out by ICH stability guideline at 25 °C/60%

RH, 30°C/65% RH, 40°C/75% RH. Samples were withdrawn at 0, 30, 90 and 180

days and were evaluated for the physical appearance, viscosity and for in-vitro drug

release. The logarithms of percent drug remaining were calculated and plotted against

time in days. The slope of straight line was determined and the degradation rate

Materials & Methods


constant was calculated with equation, slope=K/2.303, where K is a degradation rate

constant142.

6.2.5.5. Sterility study

The sterility testing was performed according to Indian Pharmacopoeia (IP)

2007 method on four different media namely, fluid thioglycolate media (FTM),

nutrient broth medium (NB), soyabean casein digests medium (SCDM) and sorbitol-

dextrose broth (SDB) to investigate the pesence or absence of aerobic bacteria,

anaerobic bacteria and fungi. FTM media was incubated at 37 ± 0.5°C under aerobic

condition, NB at 30 ± 2.5°C under anaerobic condition in a bacteriological incubator

while SCDM, SDB was incubated at 25 ± 0.5°C under aerobic condition in a fungal

incubator for 7 days. The experiment was performed in triplicate143.

6.2.5.6. In-vitro Ocular irritation studies Hen's egg test on the Chorio-Allantoic

Membrane assay (HET-CAM Test)

White Leghorn chicken eggs were obtained from the Poultry Research Centre,

Kerala. White Leghorn chicken eggs were selected for the study because they have no

hereditary defects and yields very consistent and reproducible results. Eggs were

incubated in the incubator for 9-10 days at 37 ºC. On day 9, eggs were tested with

candling light to ensure that all were viable. On day 10, the air cell was marked with a

pencil and removed the shell by tapered forceps. The membrane was carefully

moistened with 0.9 % NaCl solution at 37 ºC. The eggs were replaced in the incubator

until ready for assay. The moistening solution (0.9 % NaCl) was poured off from the

opened egg and the membrane was carefully removed without injuring any underlying

blood vessels. 0.1 N NaOH and 0.9 % NaCl were used as negative and solvent control

respectively. The CAM of each egg was applied directly with 0.3 ml of the

solvent/negative control and with different concentration of placebo (without drug

loaded) nanoparticles (50-2000 μg/ml) and placebo nanogels (50-2000μg ≈1 -1.5 mg

of nanogel). Two eggs for controls and three for test substances were used for each

assay. The reactions of hemorrhage, coagulation and lysis on the CAM were observed

over a period of 6 h. The time for each reaction was recorded in 5 min and an

irritation score (IS) was calculated according to the formula given below;144

Materials & Methods


H= hemorrhage; L = vessel lysis; C = coagulation; Sec = 5 min. After treating for 6h

the main reaction (hemorrhage, lysis or coagulation) was scored as follows: 0 = no

reaction; 1 = slight reaction; 2 = moderate reaction; 3 = severe reaction.

Each test was done in triplicate and the mean score of the three eggs was determined.

At the end of each assay the embryos were killed quickly by placing the eggs into a

freezer at −20 ºC.

6.2.5.7. Haemocompatibility studies

Blood samples of healthy human volunteers were obtained from blood bank of

government hospital, Ooty in evacuated siliconized glass tube containing sodium

citrate. Red blood cells were separated by centrifugation at 1500 rpm for 10 min and

then washed 3 times with phosphate buffer saline pH 7.4. Stock solution of

erythrocytes in PBS was prepared such that the cell count was 1×108 cells/ml. Equal

volumes of RBC suspension and nanoparticles dispersion were suspended in a

microcentrifuge tube such that the final concentrations of nanoparticle dispersion and

nanogel were 50- 200 μg/ml and incubated seperately at 37 ºC for 1 h. 1% Triton X

and PBS were used as positive and negative controls respectively. After 1 h the tubes

were centrifuged at 1500 rpm for 10 min and the hemoglobin released in the

supernatant was detected by UV absorbance at 540 nm. All measurements were

performed in triplicate (n=3) and the SD was calculated. The percent haemolysis was

calculated by the formula145;

Where Abssample is the absorbance of supernatant of erythrocyte and nanoparticles

suspension. Abs0% is the absorbance of supernatant of erythrocyte and PBS

suspension. Abs100% is the absorbance of supernatant of erythrocyte and Triton X

suspension.

6.2.5.8. In-vitro anti-proliferative studies

In-vitro anti-proliferation studies were performed by HET-CAM model using

concentrations from 50μg/ml to 200μg/ml of sterile drug, drug loaded nanoparticles

and nanogels. Watmann filter paper was cut into discs of diameter 3 mm sterilized by

autoclaving and dried properly. At the end of 9th day full growth of blood vessels

Materials & Methods


taken place and different concentration of sterile pure drug, drug loaded nanoparticles

and nanogels were added into the discs and placed on growing blood vessels, reseal

by parafilm and kept in incubator for 24 h. At the end of 24 h observed for the

reduction/inhibition in blood vessels development146.

6.2.5.9. In-vitro tube formation studies

The rabbit ocular endothelial cells were isolated and cultured in medium in

gelatin coated flasks. The cells from passages 4 to 7 were used for the angiogenesis

studies. Three dimensional collagen gels containing endothelial cells were prepared.

After gelation at 37 °C for 30 min the gels were overlaid with basal medium

supplemented with test substances at indicated concentrations 50-200 μg/ml. Gels

were examined and the tube length was determined. All experiments were terminated

at 48 h147.

6.2.6. In-vivo studies

The in-vivo studies were performed by the OECD 405 test guideline.

Experimental animals were obtained from central animal house J.S.S. College of

Pharmacy, Ooty and treated as prescribed in the publication guide for the care and the

use of laboratory animales (NIH publication No. 92-93, revised 1985). All procedures

using animals were reviewed and approved by the Institutional Animal Ethics

Committee of the J.S.S. College of Pharmacy (JSSCP/IAEC/PH.D/PH-

CEUTICS/01/2012-13). The animals were housed singly in standard cages, in a light

controlled room (12 h dark/12 h light cycle) at 19 ± 1°C and 50 ± 5% relative

humidity, with no restriction of food or water. During experimentation, the rabbits

were placed in restraining boxes in a way that they could move their head and eye

freely.

6.2.6.1. Acute eye irritation studies

Acute eye irritation potential of each placebo nanogels and nanoparticles were

tested on New Zealand albino rabbits. The irritation test was performed according to

the Organization for Economic Cooperation and Development (OECD) test guideline

405. Nanogels (20- 30μl) and nanoparticles (18.6-25.8μl) in the concentration range

of 50-200 μg/ml without drug were placed in the conjunctival sac of the eye of each

Materials & Methods


animal after gently pulling the lower lid away from the eyeball. The lid was then

gently held together for about 1 s to limit loss of the material. The left eye, which was

untreated, served as a negative control. The eyes were examined for the sign of

redness, lacrimation and inflammation at 1, 24, 48, 72 h. If there was no evidence of

irritation at 72 h, the study was terminated148. After 10 days of washout period the

other batches of nanogels and nanoparticles were instilled at same concentration and

observed for 72 h for above mentioned parameters.

6.2.6.2. In-vivo corneal anti-proliferations studies

In-vivo anti-proliferation studies were performed using rabbit model by alkali burn

method149. 54 New Zealand white rabbits weighing 2.0–2.5 kg each were divided into

6 groups.

Each group contains 3 animals-

Group 1 - Negative Control group (Untreated)

Group 2 - Curcumin, betulinic acid and lenalidomide drug solution

Group 3 - Placebo nanoparticles embedded in carbopol gel

Group 4 -Curcumin/ betulinic acid/ lenalidomide loaded nanoparticles

(Nanosuspension)

Group 5 - Curcumin/ betulinic acid/ lenalidomide loaded gel

Group 6 - Nanogel formulations of curcumin, betulinic acid and lenalidomide

Dose was calculated on the basis of standard drug (equivalent to 2 mg of drug).

6.2.6.2.1. Rabbit model of corneal neovasculrization

General anesthesia was induced with an intramuscular injection of 50 mg/kg

ketamine hydrochloride and 10 mg/kg xylazine. Central corneal alkali wound was

produced in right eyes of 54 rabbits by applying a 5-mm round filter paper, soaked in

1 N NaOH, for 60 s and left eyes was left as negative control. On the first day after

saturation, the following drugs and formulations according to groups were instilled

into the right eye once per day and in left eyes 0.9 %w/v of saline solution were

instilled. One the second day onwards eyes were examined for reducation in

neovasculrization and inflammation. At the end of 10 days of the treatment the eyes

Materials & Methods


were subjected for the histopathological studies. Same procedures were adapated for

betulinic acid and lenalidomide batches as above the method described.

6.2.6.2.2. Histopathological Examination After ten days, the eyes were enucleated, dissected and immediately double fixed in

4% glutraldehyde buffer, then 1.3% osmium tetraoxide in phosphate buffer (pH 7.3).

Retinal specimens were processed and embedded in araldite Cy212. Semi-thin

sections were stained with toluidine blue (TB). Slides were examined by Olympus

light microscope and photographed by Olympus camera.

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