Cary 50 bio uv vis spectrophotometer

R-0.6 and R-1.2 PβAE gel discs were cut in 1-cm discs, weighed and incubated in 1mg/ml riboflavin-DMSO solution at 37°C and allowed to react for various durations of time up to 48 hours. After 6, 12, 24 and 48 hours, gels were taken out, washed twice in DMSO to remove unreacted riboflavin followed by a single wash in anhydrous acetonitrile in order to leach out residual DMSO. The gel discs were then freeze-dried overnight to remove any residual solvents. The next day, each gel disc sample was placed in separate wells of a 12-well plate and read at 444 and 600 nm using Varian Cary 50 Bio UV-Vis spectrophotometer. Absorbance at 600 nm was subtracted from absorbance value at 444 nm as a baseline correction to obtain the absorbance only due to riboflavin conjugation and not the disc thickness/optical density.
Next, the riboflavin conjugated gel discs were degraded in PBS (35 mg gel/ ml of PBS) for gels to undergo hydrolysis and release pure riboflavin. The degradation products in PBS were analyzed once again under UV-Vis at 444 nm and riboflavin concentrations were calculated using standard calibration curve.

Analysis of PEG content in nanogels was performed using the barium-iodide assay [29 ]. To carry out the assay, two solutions were prepared. A) 60 mg/ml barium chloride in 1.2 N HCl (HCl stock diluted in DI water), B) potassium iodide and iodine in DI water with final concentration of 20 and 12.5 mg/ml respectively. Separately, 1 mg of nanogels was rapidly hydrolyzed in 500 µL of 5 N NaOH for 4 hours at 80o°C. Hydrolyzed nanogels were neutralized by addition of 500 µL of 5 N HCL. PEGMEMA4000 dissolved in DI water was used for standard calibration within the range of 0–10µg per well in a 96 well plate. 20 µL of the hydrolyzed nanogel solution was added to the wells. Sample and the calibration volumes were diluted to 170 µL with DI water. 40 µL of solution A was then added to each well followed by mixing. 40 µL of solution B after doing 1/5th dilution was added subsequently to all the wells and mixed. The reaction was allowed to develop for 10 minutes after which absorbance was recorded at 550 nm using Varian Cary 50 Bio UV-Vis spectrophotometer. Calibration curve of PEG (5000 MW) was used as a calibration standard.

It was important to assess that the released curcumin from CNGs was still active with its antioxidant potential intact as its phenolic groups were chemically altered during the nanoparticle synthesis. Therefore, trolox equivalent antioxidant capacity (TEAC) assay was performed in order to verify the antioxidant potential or radical scavenging property of released degradation products. TEAC is a colorimetric assay based on scavenging of 2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical anions (ABTS.-) in presence of any antioxidant. Briefly, 7 mM ABTS radical cation stock solution was prepared by mixing 1 ml of 8 mg/ml of ABTS solution with 1 ml of 1.32 mg/ml of potassium persulfate solution in DI water overnight. This concentrated ABTS radical stock solution was diluted in PBS to an absorbance of 0.4 AU at 734 nm to prepare working solution after baseline correction. Trolox, with known concentrations ranging from 0 to 0.27 mM, was used for calibration. The assay was carried out in a 96-well plate and 10 μl of the sample was added to each well, followed by 200 μl of ABTS radical working solution. Five minutes later, absorbance was read at 734 nm using Varian Cary 50 Bio UV-Vis spectrophotometer. A trolox calibration curve was used to determine equivalent trolox concentrations in the supernatant degradation products.