Antitumor Effect Of Photothermal Therapy Sensitized With Nitric Oxide Activated By Near-infrared Light

Objectives:Firstly,AM（L-Arg-MNP）nanoparticles were synthesized by the functionalization of MELANin nanoparticles（MNP）with L-Arginine（Arg）.In order to improve the retention of AM nanoparticles in tumor tissues,hydrogel HF was coated on the outside of AM nanoparticles.HFAM nanogels were successfully prepared by using the thermosensitive hydrogel,which could transform phase at appropriate temperature.Secondly,it was verified that nano-gel HFAM can release NO under the action of near-infrared light,and the released NO can effectively sensitize PTT by inhibiting the autophagy mechanism of tumor cells,realizing the purpose of mild photothermal treatment of malignant tumors.Methods:1.Construction and characterization of HFAM nanogelA novel water-soluble melanin nanoparticles were prepared and modified by arginine functionalized acid to synthesize AM nanoparticles.Fourier transform infrared spectroscopy（FTIR）and DLS were used to verify the successful synthesis of AM nanoparticles by INFRARED spectroscopy and Zeta potential.The appearance and particle size of AM nanoparticles were investigated by TRANSMISSION electron microscopy（TEM）and DLS.Nano gel HFAM was prepared by physical mixing of AM nanoparticles with HF hydrogel.Single factor analysis was used to evaluate the effects of different concentrations of F127 on gel,and finally determine the optimal concentration of F127.The morphology of HFAM was observed by scanning electron microscope.Thermal imager and Griess reagent were used to evaluate the photothermal properties and NO release ability of HFAM.2.In vitro efficacy study of HFAM nanogelCCk-8 method was used to detect the cytotoxicity of HFAM on 4T1 cells and the inhibitory effect of HFAM on the proliferation of 4T1 cells under laser irradiation.The uptake of HFAM nanoparticles by 4T1 cells at different time points was investigated by flow cytometry to determine the optimal incubation time.Annexin V-FITC/PI double staining and flow cytometry were used to detect the apoptosis induced by HFAM under different conditions.The NO release ability of HFAM under different conditions was detected by Daf-FM DA fluorescence probe.Cyto-id autophagy kit and Westernblot were used to investigate the mechanism of HFAM+Laser’s mild and photothermal effects.3.In vivo efficacy study of HFAM nanogelThe mouse model of 4T1 subcutaneous breast cancer was established.PBS,HF+Laser,MNP+Laser and HFAM+Laser were used to study the photothermal effect and NO release performance of HFAM in mice.After treatment in different groups,tumor growth,mouse body weight and HE pathological section of mice 14 days later were observed to comprehensively evaluate the anti-tumor effect of HFAM+Laser in vivo.Results:1.Construction and characterization of HFAM nanogelCharacterized by infrared spectroscopy and elemental mapping,it was proved that the new nanoparticle AM was successfully synthesized.The prepared nanoparticles were uniformly dispersed and spherical under the transmission electron microscope,and the particles were measured by the particle size analyzer.The diameter was 10.1 nm,and the zeta potential was+6.28 m V.When the concentration of F127 was 18%,we obtained an ideal HFAM with excellent photothermal conversion ability and NO release ability.2.In vitro efficacy study of HFAM nanogelCCK-8 experiments showed that the HFAM did not show obvious cytotoxicity within the set concentration range.In the cell proliferation inhibition experiment,the inhibitory effect on the proliferation of 4T1 cells was shown as HFAM+Laser>HFAM.Calcein-AM/PI double staining assay for qualitative detection of cell apoptosis and Annexin V-FITC/PI double staining assay for quantitative detection of cell apoptosis both showed that HFAM+Laser group could induce apoptosis by inducing cell apoptosis.The cytostatic effect was achieved;flow cytometry showed that the uptake of HFAM by 4T1 cells increased with time and reached 99.98%after 4 h.The fluorescence intensity of the DAF-FM DA fluorescent probe in the HFAM+H₂O₂+Laser group was the highest.The results showed that the HFAM+H₂O₂+Laser group had the strongest NO release ability.The CYTO-ID autophagy kit and Western blot experiments proved that HFAM could enhance the sensitivity of 4T1 cells to photothermal therapy by inhibiting autophagy.3.In vivo efficacy study of HFAM nanogelCompared with the control group,the tumor volume in the HFAM+Laser group basically did not grow,and the bodyweight of the mice in each group did not decrease significantly.H&E staining can prove that HFAM has no obvious toxicity to the health of mice,and in the near-infrared Irradiation can effectively kill tumor cells.The above results indicated that HFAM+Laser successfully and efficiently inhibited the growth of the 4T1 tumor.Conclusion:In this paper,nano-gel HFAM was successfully prepared,which can enhance its retention in tumor sites and has good photothermal properties and NO release ability.In addition,nano-gel HFAM can trigger the release of NO under near-infrared light and enhance the sensitivity of cells to photothermal therapy by inhibiting autophagy,thus realizing the effect of mild photothermal therapy on malignant tumors.