To this mixture, 3mL absolute ethanol (EtOH, 99.99%) and sodium hydroxide (NaOH, 1M) mixture (in equal volume) were added and subjected to microwave assisted pyrolysis for 5min till color of the mixture turned to wine red. This mixture was separated by sucrose density gradient centrifugation (SDGC) using 50–100% gradient concentration of sucrose. Three distinct bands were removed carefully and their properties were studied. Bands are referred to B1, B2, and B3 for further discussions. Each fraction was subjected to repeated centrifugation steps to get rid of residual sucrose and pure C-dots were Inhibitors,research,lifescience,medical collected by spinning at 8385×g for 15min. On vacuum heating

for 8h, powdered form of black colored C-dots was obtained which was then used to make 100mg/mL stock solution and stored at −20°C. 2.4. Synthesis of Cipro@C-Dot Conjugate For the synthesis of the above conjugate, 0.5mL (1000μM) ciprofloxacin solution was added to 9.5mL (95mg/mL) C-dots and stirred for 3h at 30×g. Change in the optical properties of Cipro@C-dots conjugate was studied using UV-Vis Spectroscopy Inhibitors,research,lifescience,medical in the spectra window of 200–600nm with respect to pure C-dots. Further attachments of C-dots and ciprofloxacin were confirmed using Fourier transform infrared (FTIR)

and thermogravimetric analysis (TGA). Drug loading efficiency (DLE) of C-dots was calculated using the following equation (see Supplementary Material, Scheme 1a, available online Inhibitors,research,lifescience,medical at http://dx.doi.org/10.1155/2014/282193): DLE=Theoretical  amount  of  drug  loaded−Free  drugTheoretical  amount  of  drug  loaded×100. (1) 2.5. Antibiotic Release Studies 2mL of Cipro@C-dots Inhibitors,research,lifescience,medical conjugate was transferred to a fresh dialysis bag (MW cutoff 12–14kD, Pore size 2.4nm) and dialyzed against 1% phosphate buffer saline (PBS, pH 7.2) at 37°C. The antibiotic release at regular time intervals (0–48h) was

measured LGK-974 nmr spectrophotometrically at 277nm. Each time the reading appropriate volume of fresh phosphate buffer saline (PBS, pH 7.2) prewarmed and maintained at 37°C in an incubator was added to the dialysis chamber. 2.6. Cytotoxicity Studies Cytotoxic effect of the Cipro@C-dots conjugate was studied Inhibitors,research,lifescience,medical on most commonly used Terminal deoxynucleotidyl transferase Vero cells using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vero cells were seeded (3 × 105/mL) in 96 well plates and incubated at 37°C under 5% CO2 for 24h. After satisfactory growth of the cells, growth medium was replaced with the respective test solutions and incubated for 48h. Finally, C-dots or Cipro@C-dots solution was replaced with MTT (150μg/mL). Cells were incubated for 2h at 28 ± 2°C to initiate formation of formazan. After completion of the reaction, medium was replaced with 300μL of DMSO (Sigma, USA). This conjugate was agitated moderately to dissolve formazan crystals. Finally, the dissolved formazan in DMSO was transferred to fresh 96 well plates and read on microplate reader (Thermo, USA) at 570nm. 2.7.