Preparation and Evaluation of Biopolymeric Nanoparticles As Drug Delivery System in Effective

Supplementary Material

Preparation and evaluation of biopolymeric nanoparticles as drug delivery system in effective treatment of rheumatoid arthritis

Vijay Kumar1, Ankita Leekha1, Aakriti Tyagi1, Ankur Kaul2, Anil Kumar Mishra2, Anita Kamra Verma1*

1 Nano Biotech Lab, Department of Zoology, Kirori Mal College, University of Delhi, Delhi-110007

2Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumder Road, Delhi 110054, India.

*Correspondance:

Dr. Anita KamraVerma, Nano Biotech Lab,

Kirori Mal College, University of Delhi,

Delhi-110007.

Email: ,

Contact No: +91-9818921222

Radiotagging studies with 99mTc andBiodistribution studies

Briefly, to a premixed solution of stannous chloride dissolved in 0.1 N hydrochloric acid solution, 500 microlitres of 99mTc (74 Bq ml-1) added and the pH of the solution was adjusted to 6.5 using sodium bicarbonate solution. 0.5 ml of CHNPs were added to the above solution and incubated for various timepoints at room temperature and finally was filtered through Sephadex G25 column. Various parameters like concentration of stannous chloride, incubation time and pH of the solution werestudied to obtain high labeling efficiency. The labeling efficiency was estimated chromatographically using silica gel-coated sheets (ITLC-SG) strips (Pall Corporation, USA) as the stationary phase and 100% acetone as mobile phase. The radioactivity in each half was determined using gamma counter. The free pertechnetate with an Rf of 0.9–1.0, migrated to the top portion of the strip and the radiolabeled CHNP remained at the bottom.

Glutathione -S-transferase (GST) Assay

The reaction mixture was the composition of 9.8ml of phosphate buffer (pH 6.5), 100mM reduced glutathione (GSH) (100μl) and 100mM CDNB (100μl). To the final assay mixture of 200μl, 20μl of diluted serum and 180μl of reaction cocktail was added in each well. Increase in absorbance was measured at 340nm and the enzyme activity was determined using molar extinction coefficient of CDNB i.e. 9.6 mm−1cm−1.

Reduced Gluthathione (GSH) Assay

Briefly, 100μl of diluted serum was precipitated with 20μl of 5% trichloroacetic acid (TCA) in eppendrof tubes to remove the precipitated protein, which were then centrifuged at 1200 rpm for 5 minutes at room temperature and supernatant was collected. To the final assay mixture having volume 110μl, 45μl of collected serum (treated with TCA), 45μl of sodium phosphate buffer (0.2M, pH 8) and 20μl of dinitrobenzoic acid (DTNB-10mM) were added. The absorbance was read at 412nm after 10 minutes. The amount of reduced glutathione present was calculated using molar extinction coefficient of DTNB i.e. 13.6mm−1cm−1and was expressed as μmol/ml of the sample.

Glutathione Reductase Assay

Rat serum was diluted in GR cocktail containing 100mM phosphate buffer having 3.4mM EDTA pH 7.6, 30mM GSSG, 0.8mM β-NADPH and 1% BSA. GR activity was determined by monitoring the decrease in absorbance at 340 nm due to β-NADPH reduction, which directly correlates to GR activity. The activity of GR was expressed in terms of units/mg protein.

Figure S1: (A) FTIR spectra of MTX, chitosan and MTXCHNP, sTPP(B) FTIR spectra of DEX, chitosan and DEXCHNP

Figure S2: (A): Zero order plot of MTXCHNP at pH 7.4 and pH 5.8 (B): First order plot of MTXCHNP at pH 7.4 and pH 5.8 (C): Higuchi plot of MTXCHNP at pH 7.4 and pH 5.8 (D): KoresmeyerPeppas model of MTXCHNP at pH 7.4 and pH 5.8

Figure S3: (A): Zero order plot of DEXCHNP at pH 7.4 and pH 5.8 (B): First order plot of DEXCHNP at pH 7.4 and pH 5.8 (C): Higuchi plot of DEXCHNP at pH 7.4 and pH 5.8 (D): KoresmeyerPeppas model of DEXCHNP at pH 7.4 and pH 5.8

Figure S4: Serum Stability studies of CHNP.