Preparation of Nano-Polymer Containing Active Ingredient of Arsenic for Photochemotherapy and Its Activation of Anti-Glioblastoma Immunity

OBJECTIVE To investigate the in vitro antitumor activity and in vivo targeting of nanopolymers co-loaded with arsenic trioxide (ATO), an active ingredient of traditional Chinese medicine arsenic, and photosensitizer (IR780) to activate the patient's own immune system in the treatment of brain glioma in synergistically with chemotherapy and phototherapy.

METHODS PLGA/AI containing ATO and IR780 was prepared by volatilization with multiple emulsion solvent. The particle size, potential and polydispersity index(PDI) were determined by dynamic light scatterometer (DLS) at different feed ratios. Transmission electron microscopy (TEM) was used to detect the morphology of PLGA/AI. Fluorescence spectrophotometer was used to investigate the spectroscopic characteristics. UV-vis spectrophotometer was used to determine the drug loading and encapsulation rate of IR780. Under laser irradiation, the physiological release of ATO was measured by dialysis bag method. The uptake of PLGA/AI in GL261 cells was photographed by confocal microscopy (CLSM). The cell uptake mechanism of PLGA/AI was investigated by flow cytometry (FCM). 3D tumor spheroid model was constructed to simulate the deep penetration of PLGA/AI in solid tumors. The synergistic anti-tumor effects of PLGA/AI in vitro were studied by MTT assay and dying staining. DCFH-DA staining and FCM determination of the co-expression of CD80 CD86 co-stimulatory molecules verified the immune activation induced by PLGA/AI in vitro photochemotherapy. The in vivo targeting of PLGA/AI and the tissue distribution of the main organs were investigated by means of in vivo imaging of small animals after tail vein injection.

RESULTS The optimal ratio of ATO to IR780 was 1 ∶ 10, its particle size was (134.11±2.19) nm, Zeta potential was (-7.02±0.649) mV, PDI was 0.254±0.059, and it was a uniform spherical shape under TEM. The fluorescence spectra showed that IR780 was successfully loaded onto PLGA, and the drug loading and encapsulation rate of IR780 in PLGA/AI were (2.53±0.02)% and (71.26±0.38)% respectively. The results of in vitro release experiments showed that ATO could be released in response to laser light by IR780-mediated photo-dynamics. Cell uptake experiments showed that the nano-polymer could effectively enter tumor cells under clathrin-mediated endocytosis. In vitro investigation of cell viability, ROS detection and dendritic cell maturation experiment showed that it could effectively kill tumor cells by inducing a large amount of ROS to generate and activate immune cells. In vivo targeting and biological distribution study confirmed that PLGA/AI could effectively penetrate BBB into tumor sites.

CONCLUSION The active ingredients of traditional Chinese medicine arsenic, ATO and IR780, use PLGA as carriers to form nano-polymers through the volatilization of multiple emulsion solvents, which can cross the BBB and effectively accumulate at the tumor site. Based on "strengthening the healthy qi and eliminating pathogenic factors" and combined with chemotherapy and photo-immune activation, it has the potential for long-term anti-glioblastoma treatment.