2.1 Materials.
Medical grade lactic acid was purchased from Malladi Drugs & Pharmaceuticals Ltd, Chennai. Boc-glycine was purchased from across organics. Curcumin (98% by HPLC) was obtained from Pharmaceutical Research labs,. Bovine Serum Albumin (BSA), Stannous Octoate ( Sn(otc)2), Poly (propylene glycol), TBTU, Trimethylamine, solvents Methanol, Dimethyl Formamide and all other remaining used chemicals were purchased from Sigma Alrich and Merck Chemicals.
2.2 Preparation of Poly (lactic acid)
100 ml of the Lactic acid, 1% Stannous Octoate (Sn(Oct)2) catalyst and 300 ml of toluene were added into 1000 ml three necked round bottom flask fitted with Dean Starck apparatus, mechanical stirrer and nitrogen gas supply unit setup and then heated the mixture at 160oC. The reaction was continued 14 hours under Nitrogen atmosphere @ 160oC and concurrently removed water from the mixture. After completion of the Polymerization reaction, the solvents and Catalyst were removed by distillation. The synthesized polymer was washed with 50 ml methanol in many times and dried with vacuum oven at 65o Celsius.
2.3 Preparation of PPG polymer with Boc- Glycine End groups.
The Boc-Glycine (1.0 gm) was dissolved in DMF (20mL). To that solution TBTU (2.91 gm and trimethylamine (1.19mL) was added. The mixture was stirred for 30 min at room temperature. Then PPG (1.0 gm) was added and stirred for 24 h at room temperature. Then the reaction mixture as quenched with crushed ice and filtered and dried in vacuum oven.
2.4 Preparation of Poly (lactic acid) – Poly(propylene glycol) with Boc-Glycine end group Copolymer
PLA-PPG with Boc Glycine21 polymer was synthesized by using a condensation polymerization technique. 5 gm of PLA and 5gm of PPG with Boc Glycine were added to 1000 ml three necked round bottom flask fitted with Nitrogen gas supplied and stirrer set up. 50 ml of Toluene added to the flask and stirred for the mixture at the duration of 15 minutes. Then the catalyst Stannous Octoate 1% added into the mixture and stirred for 10 minutes. Then the reactants steadily increased the temperature at 160oCelsius and continued the reaction upto 14 hours. After completion of the Polymerization reaction, the solvents and Catalyst were removed by distillation method. The synthesized polymer was washed with 50 ml methanol in many times and dried with vacuum oven at 65o Celsius.
2.5 Anti-inflammatory activity studies
BSA denaturation technique
The synthesized polymer (PLA-PPG-Boc- Glycine) and standard diclofenac sodium were screened for anti-inflammatory activity by using the inhibition of albumin denaturation technique with slight modification. The standard drug and polymer were dissolved in minimum quantity of Dimethyl formamide (DMF) and diluted with phosphate buffer (0.2 M, PH 7.4). The final concentration of DMF in all solution was less than 2.5%. Test Solution (4 mL) containing different concentrations of the drug was blended with 1 mL of 1 mM Bovine serum albumin solution in phosphate buffer and incubated at 37°C in an incubator for 15 min. Denaturation was induced by keeping the reaction mixture at 70°C in a water bath for 30 min. After cooling, the turbidity was measured at 660 nm. Percentage of Inhibition of denaturation was measured from control where no drug was added. The percentage inhibition of denaturation quantity was determined by using the following formula.
% of Inhibition = (Abs. Control Sample – Abs. Test Sample / Abs. Control Sample) X100
2.6 Drug releasing study
Preparation of drug-polymer matrix
50 mg of polymer was dispersed in 1 mL of methanol. To that mixture, Curcumin22–24 (20 mg/0.2 mL of chloroform) added drop wise. The mixture was stirred for 24 hours. The maximum quantity of supernatant was removed using micropipette and absorbance at 432 nm was recorded. From the absorbance of known Curcumin solution, the loaded drug quantity was calculated. (Drug Loading Percentage: 13.28% of Curcumin, Drug Loading Efficiency: 75.76%)
Kinetic studies of Curcumin release in Vitro
The drug release26–30 study was performed in Vitro. The drug (Curcumin) release from PLA-PPG-Boc Glycine with respect to time was investigated at RT in phosphate buffer (pH: 5.5, 6.8 and 7.4). 10 mg/10mL polymer-drug matrix of prepared material was placed in a dialysis membrane tube with a 12000 Da molecular cut off (Himedia, Dialysis membrane-110). This tube was dipped in a beaker containing 50 mL of Phosphate Buffer Solution. At time 0,5,10,15, 20 and 25 hour intervals, 2 mL of the solution was taken from the release medium and replaced with fresh PBS buffer. The released quantity of Curcumin was determined at various time intervals by measuring its absorbance of release medium by using a spectrophotometer at 432 nm.
2.7 Stability study of PLA-PPG-Boc-Glycine
The stability study was performed for the polymer is as followed by banuppriya et al. 25. The polymer was dissolved in minimum quantity of Dimethyl Formamide made up of using phosphate buffer (pH = 7.4, 5mL) at 1 m concentration. From the solution, 200 µL of the sample was diluted in 2 mL of phosphate buffer. The synthesized polymer solution was incubated at 37°C for 10 min. The absorbance (270 nm) was recorded which is considered as an initial time for degradation. The sample solutions were again kept at 37 0C and the experiment was repeated up to 3 days at a different time intervals. The stability of the polymer was determined from the decrease in absorbance by using UV Visible Spectrophotometer.
2.8 Characteristics
Infrared (IR) was carried out using Shimadzu Tracer 100 instrument. UV spectrum was carried out by using a UV –Vis spectrometer Labman 1900S Model. Thermo gravimetric analysis was carried out using a TA Q500HI-RES. Sample was scanned to a temperature range of 600oC at the rate of 10oC with a supplying of Nitrogen atmosphere. Nuclear Magnetic Resonance (NMR) study was carried out using DMSO as solvent in Bruker Advance 500 MHz model instrument.