In addition, levels of activated caspase-3 and caspase-9 were significantly higher in cells treated with Photosan-II loaded in nanoparticles than free Photosan-II. Finally, treatment with nanoscale photosensitizers increased mouse survival and reduced tumor volume in mice to a greater extent compared with free photosensitizers. Overall, our data indicate that hollow nanoparticles IDO inhibitor containing photosensitizers more efficiently inhibit hepatoma cells than free photosensitizers, through induction of apoptosis, both in vivo and in vitro. Methods Cell lines The HepG2 human hepatoma cell line was purchased

from the cell center of the Xiangya School of Medicine of Central South University. Experimental animals Specific pathogen-free (SPF)-grade female BALB/c nude mice (26 to 30 days, 18 to 22 g) were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. Mice were housed in SPF-grade animal laboratory of the Second Xiangya Hospital of Central South University in a temperature and humidity controlled room with food and water ad libitum. All procedures were approved

by the Animal Ethical Committee of Second Xiangya Hospital of Central South University. Preparation ACP-196 of nanoscale photosensitizers Nanoscale photosensitizers were prepared using a one-step wet chemical-based synthesis at room temperature, as previously described [15]. Tetraethyl orthosilicate (TEOS, 99.99%), polyacrylic acid (PAA, M.W = 3,000) were purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). Anhydrous ethanol (99.7%) and ammonia (25% to 28%) were purchased from Sinopharm Chemical Reagent Co. Ltd (China) and Photosan-II (C34H38N4NaO5) obtained from Seehof Laboratorium F&E GmbH (Wesselburenerkoog, Germany). The resulted nanoscale photosensitizers (Photosan-II-loaded

also hollow silica nanospheres, 10 mg/L) showed good sphericity and narrow diameter distribution, ranging from 25 to 90 nm (mean value 37.8 nm). The encapsulation efficiency reached 95%. Cell culture and passaging Cryopreserved HepG2 human hepatoma cells were thawed and cultured in appropriate volume of 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM) purchased from Gibco (USA), at 37°C and 5% CO2. Cell growth was observed daily, and culture media were changed as needed. Cells grown to logarithmic phase were trypsinized and passaged. MTT assay Two hundred microliters of a 105 cells/mL suspension was seeded into a 96-well plate and cultured as described above. Photosensitizers used were either conventional Photosan or nanoscale Photosan.