Supporting Information Nitric oxide releasing photoresponsive nanohybrids as excellent therapeutic agent for cervical cancer cell lines

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1 upporting Information itric oxide releasing photoresponsive nanohybrids as excellent therapeutic agent for cervical cancer cell lines Priya udhesh a, Kaviyarasan Tamilarasan a, Palaniappan Arumugam a and heela Berchmans* a Electrodics and Electrocatalysis Division, CIR-central Electrochemical Research Institute, Karaikudi, Tamilnadu IDIA * sheelaberchmans@yahoo.com Preparation of MBI GP g of AuCl 4 is dissolved in 10ml water and 600 µl 0.1M a is added to it. This solution was stirred for 15 minutes under inert condition g of MBI is dissolved in 20ml of water in presence of 0.1M a to get a clear solution. This solution was maintained in room temperature under inert conditions. After two hours, ice cold ab 4 (0.214 g) was added to the above solution. Colour of the solution changes from yellow orange to dark brown after two hours. Unreacted MBI was removed by dialysis for 24 hours. Figure. 1- IR spectra of free MBI a and MBI GP-b Calculating concentration of MBI-GP and ligands per GP

2 a) Calculating Au atoms in a nanoparticle V cluster = V atom =[ r cluster /r atom ] 3 = b) umber of surface atoms s =4πr 2 /πr 2 atom= Molecular weight of GP, Mw = Mgold x = Da c) Calculation of concentration of GP Mass of AuCl 4 = 0.015g Moles = 0.15/ = x 10-5 o. of Au atoms = x 10-4 x x = 2.29 x10 19 o of P = P = atom / =2.29 x / 16674= 1.37x10 15 Concentration of P= CP = P / Avg o = 2.27 x 10-9 Weight of MBI-GP per ml = Mwt of GPx Volx Conc.GP/1000 = 4 µg d) Calculating number of ligands from TGA Weight loss due to ligand = 22%of mg P = g Molecular weight of ligand= Moles= /195.2= x 10-7 o. of ligands= 1.555x 10-7 x x = 9.367x o. of moles of GP= mass/molecular wt of GP= x o of GP taken = x Ligands per nanoparticle = 9.36x /4.018 x = 2331 ligands per cluster Concentration of ligand = C P x no.ligands per cluster= 2.28x10-8 x 2300= 52µM e) Estimation of itric oxide release from 0.5 ml GP using griess assay release from GP was studied both in the presence and in the absence of light. Griess assay was carried out by taking GP solution in 2 eppendorfs and covered with a dialysis membrane. This eppendorfs are then immersed in Griess assay solution, so that released from GP only reacts with griess assay.ne is irradiated with visible light and other

3 is kept under dark condition. We can see red colour formation upon diazo coupling of the Griess reagent, which shows absorption at 546 nm. Linear regression equation from the standard plot, A= C(µM) Intensity of absorption due to release from GP in the presence of light = Concentration of released from 0.5 ml GP in presence of light = 22 µm Intensity of absorption due to release in the absence of light = Concentration of released in the absence of light = 5 µm Figure.2- Thermogravimetric analysis of free MBI and MBI GP

4 Absorption λ(nm) Figure.3-UV-Vis spectra pf GP after irradiation with visible light Figure. 4-TEM image after release

5 chematic representation showing mechanism of release itrite 2 2 itric oxide xyradical Assay Protocol Cytotoxicity of GP (Gold ano Particle) was assessed by MTT (3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Briefly cells/ml of ela cells were directly seeded in a 96 well tissue culture plate for 24 hours at 37 C in a humidified incubator supplied (-Biotek 203) with 5% C 2 (For attachment). After incubation, cells were incubated with various volume of (2µl to 20 µl) test sample and exposed to the light for 1 hour (fluorescent tubular lamp of 40W- Philips). It was further incubated for 24 hrs at 37 C in a humidified incubator supplied with 5% C 2. Cisplatin (10µg/ml) and Mercapto nitro imidazole (100µg/ml) was used as positive controls. After incubation the growth medium containing the test sample was removed and the cells

6 were gently washed once with Phosphate buffer saline (PB, p 7.4). The cytotoxicity was assessed by incubating the cells with MTT solution (1mg/ml) at 37 C for 3 hours. After incubation, hundred microlitre of DM was added to dissolve the formazan crystals (which gives purple colour if the cells are metabolically active). The absorbance was measured at 540 nm using pectramax M3 Multi-Mode Microplate Reader. The percentage of viability was calculated by using the following formula. % of Viability = Absorbance of Test Absorbance of the Control 100 TE: 1. DM was used as a Blank. 2. Growth medium Dulbecco s Modified Eagle Medium (DMEM) + 10% Fetal Bovine erum (FB) + 1X Antibiotic solution (10,000 units Penicillin and 10mg/ml streptomycin in 0.9% normal saline). Purchased from imedia, India.

7 Control MBI MBI-GP Figure. 5 - Cytotoxicity for cervical cancer (ela) cells

8 Control MBI MBI-GP Figure. 6- Cytotoxicity for cervical cancer (iha) cells

9 Control MBI MBI-GP Figure. 7- Cytotoxicity study of breast cancer (Mcf7 ) cells

10 Figure-8 Cytotoxicity study of lung cancer cell (A549) Cytotoxicity and apoptosis monitoring using oechst staining for analysing chromatin condensation by oechst staining, the cells were grown on 96-well plates and stained with 10mg/ml of oechst for 10 minutes and viewed under UV filter sets using ikon Epifluorescent microscope (TE2000E).