Preparation Of Folic Acid-bovine Serum Albumin-cypate Nanaparticles For Targeted Photothermal Therapy On Bladder Cancer

Objective:Based on the light-thermal conversion characteristics, the cypate can be used to cure malignant tumor. In this work, the preparation and application of a new type of organic nano-stuctured materials were explored. We designed a kind of targeted drug delivery system, folic acid-bovine serum albumin-cypate (FA-BSA-Cypate) nanoparticles. Folic acid and cypate can be linked simultaneously into the different sites of bovine serum albumin.Using the high affinity of folate and folate receptor and the enhanced permeability and retention effect, cypate can be more effectively uptaked by bladder tumor cells. We investigated the photothermal effect of FA-BSA-Cypate nanoparticle on bladder cancer in vitro and in vivo experiments, as well as potential antitumor mechanisms. It is expected to provide the theoretical basis for the later clinical application.Methods:(1) The Cypate we synthesized was characterized by'H-NMR. The FA-BSA-Cypate nanoparticles were prepared by the dialysis method. The Preparation procedures were optimized with the particle size, entrapment efficiency, drug-loading. The morphology and size of Folate-BSA-Cypate nanoparticle were measured using transmission electron microscopy (TEM) and dynamic light scattering (DLS), The drug loading and encapsulation efficiency were measured using UV-VIS spectrophotometer. Temperature time curve was observed in different concentrations (0?1?10?100?g/ml) of nanoparticles. (2) Folate receptor alpha on 5637 cell was confirmed by immunofluorescence technique. In vitro 5637 cell absorption of FA-BSA-Cypate nanoparticles and BSA-Cypate nanoparticles were measured using a UV-VIS spectrometer. Furthermore, the uptake of FA-BSA-Cypate nanoparticles and BSA-Cypate nanoparticles are tested by confocal fluorescence microscope. (3) MTT Assay was used to estimate the in vitro cytotoxicity of FA-BSA-Cypate nanoparticles on 5637 cells. In addition, In different condition of nanoparticles concentration?laser energy density and irradiation time, MTT assay was used to investigate the photothermal cytotoxicity of FA-BSA-Cypate nanoparticles against 5637 cells under 808 nm photoirradiation. (4) We investigated the generation of singlet oxygen from cypate using 1,3-diphenylisobenzofuran (DPBF) as a probe which can be quenched in the presence of singlet oxygen. To observe the levels of reactive oxygen species on 5637 cells, dihydroethidium was utilized as a probe to moniter the generation of ROS upon photoirradiation. (5) Upon 808nm infared laser irradiation (1.5W/cm2,5min), The cells were divided into four groups:Control group, Laser group, BSA-Cypate+Laser group, FA-BSA-Cypate+Laser group. The number of apoptotic cells in experimental group and the control group was investigated with flow cytometry. Transwell assays were performed on the cells to detect the migration ability. The mRNA and protein expression levels of bcl-2? bax?Caspase-3?Survivin?Hsp70 and Hsp90 in each group were detected by qRT-PCR and western blotting. (6) The in vivo photothermal anti-tumor effect of FA-BSA-Cypate nanoparticles combined with near infared laser was investigated in mice bearing 5637 tumor.Results:FA-BSA-Cypate nanaparticles with a mean size of (101.2±9.3) nm was successfully achieved. The FA-BSA-Cypate had drug loading of (45.8±3.3)% and encapsulation efficiency of (91.5±4.2)%. Upon 808nm infared laser irradiation (1.5W/cm2,5min), the maximum temperature of FA-BSA-Cypate nanaparticles increased to 59? in a dose dependent manner. The expression of Folate receptor alpha on 5637 cell was confirmed. Cellular uptake assay indicated that FA-BSA-Cypate nanaparticles were folate-targeted nanoparticles for 5637 cell. The FA-BSA-Cypate nanaparticles of different concentrations had no obvious cytotoxicity to 5637 cells after 24h incubation. Upon laser irradiation,the cellular survival rates significantly decreased with the increased concentration of nanoparticles,energy density and irradiation time. The singlet oxygen production induced by FA-BSA-Cypate nanaparticles upon near-infared light irradiation also had cytotoxicity to 5637 cells. The Flow cytometry and Transwell assays indicated that high apoptosis rate and low migration ability of 5637 cell in FA-BSA-Cypate combined with Laser group. The mRNA and Western blot detection exhibited that the expressions of Caspase-3, Bax, Hsp70 and Hsp90 were significantly increased, while the expression of Survivin and Bcl-2 was obviously decreased in group of FA-BSA-Cypate nanaparticle combined with laser. The in vivo study showed the group of FA-BSA-Cypate nanaparticle combined with laser displayed strong anti-tumor efficacy on bladder carcinoma in nude miceConclusions:The FA-BSA-Cypate nanaparticles possess the characteristic of low-toxicity and active tumor targeting, Photothermal therapy induced by FA-BSA-Cypate nanaparticles have effective killing effect on 5637 cells and bladder cancer in nude mice with the mechanism of induction the apoptosis and inhibition of cell proliferation. The study also provide a theoretical basis of FA-BSA-Cypate nanaparticles in future clinical application of bladder cancer targeted photothermal therapy.