The Study Of Photothermal Therapy Induced By Cypate/Liposome And Its Synergistic Effect With Immunotherapy

Photothermal therapy(PTT) has been the hot spot rapidly, owing to their unique advantages such as localized treatment,high efficacy,low side effect and so on. The principle of PTT is to utilizes near-infrared(NIR) light-absorbing agents enriched in the tumor site with irradiation, using its photothermal conversion effect to generate heat, to achieve local temperature elevation for destruction of cancer cells. The near-infrared(NIR) light-absorbing agents by Nano drug delivery system will selectively targeted to tumor irradiation with through the EPR effect and they can reduce the systemic toxicity, significantly improved the anti-tumor effect. In recent years, immunotherapy as a novel anti-tumor technology has attracted tremendous interest in tumor ablation. Immunotherapy main purpose is to activate the body’s immune function, enhance the ability of the immune system to recognize the tumor cells. However, immune therapy for the treatment of solid tumors is limited only as an aid in combination with other anti-tumor technologies, generating synergistic anti-tumor effect. According to a new study, PTT can not only cause irreversible damage to the tumor, but also co-stimulate the body’s immune function. Therefore applied PTT and IT can achieve significant antitumor action.In this paper, we first designed the compound preparation contained liposome loading organic photothermal agents Cypate, immunogen ADM-BSA and molecular bridge HA-ADM,consisting of anti-tumor agents, which can generating an obvious antitumor effect combined photothermal therapy and immunotherapy. These investigations are shown as follows:Firstly, this chapter is an overall introduction of photothermal therapy, immunotherapy and laser immunotherapy in the field of anti-tumor research, and we explained the background and content of our research.Then,we were prepared Cy-Liposome by solvent injection method and investigate their physical properties, chemical properties and pharmacodynamic properties in vitro or vivo. Experimental results show that, the average diameter of the prepared spherical Cy-Liposome was(111.9 ± 5.94) nm, with the encapsulation efficiency of(98.11 ± 1.13)%; Compared with free Cypate in vitro, the warming effect and singlet oxygen production of Cy-Liposome was significantly improved, and liposome has good physical stability. In vitro, compared with the free Cypate, the cellular uptake of Cy-Liposome is higher and it locates in acidic organelles lysosomes; the liposomes inhibited the growth of 4T1 cells in vitro significantly with the IC50 value decreasing 89.99 %(P < 0.01). In vivo, the tissue distribution of Cy-Liposome in the tumor-bearing mice showed that liposome entrapped at the tumor site after the enrichment of a 57.7 % increase, and the liposome still stranded in tumor at 96 h. The results of thermal imaging showed that when the tumor-bearing mice were treated with Cy-Liposome at the concentration of 3.0 mg·kg-1, the temperature achieved in the tumor site was increasing by 20 ℃,which would generare thermal ablation, which together result in effective tumor ablation in vivo without re-growth.At the same time, with the background of early study in lab, the experiment based on polyclonal antibody preparation technology and ligand binding theory. In order to obtain the desired immunogen, glutaraldehyde cross-linked hapten synthesis of exogenous ADM-BSA, purified immunogen through dialysis, by UV and FTIR spectrophotometry to identify them, and calculate ADM and BSA coupling rate is 12.8 by BCA quantitative method. We investigate the immunogenicity of ADM-BSA in the healthy mice and tumor-bearing mice serum after immunized subcutaneously by measuring antibodies anti-ADM through ELISA. The results show immunogen ADM-BSA has good immunogenicity, can stimulate the immune system to produce specific antibodies. Meanwhile, we synthesis molecular bridge HA-ADM by ceramide reaction, and the results showed that compared with ADM, HA-ADM inhibited the growth of 4T1 cells in vitro significantly with the IC50 value decreasing 86.7 %(P < 0.01),and the cellular uptake of HA-ADM is higher,which indicate that HA-ADM can target to tumor cells obviously. At the same time, cell ELISA results show that HA-AD has a bridging role, so that formed on the surface of tumor cells "CD44-HA-ADM- ADM anti-antibody" complex, which can enhance tumor cells recognize the role of the immune system. In vivo, the immunotherapy based on immunogen ADM-BSA and molecular bridge HA-ADM has some inhibition of tumor growth, but the inhibitory percentages was 25.55%, so the therapeutic effect is limited.In the end, based on the above research, we explore a complex anti-tumor agents consisting of Cy-Liposome, immunogen ADM-BSA and molecular bridge HA-ADM, and investigate its antitumor activity. The results in vivo showed that immunotherapy combined with PTT can generate synergistic anti-tumor effect, significantly reducing the recurrence rate of the tumor, decreased by 60%. In addition, PTT may also co-stimulatory immune function through cytokine ELISA kit results show that photothermal therapy combined with immunotherapy can significantly improve the content of IL-12, TNF-α in tumor-bearing mice spleen.This paper constructs the complex preparation combined targeting photothermal therapy with immunotherapy, and we study on their use in the treatment of cancer and provide a foundation of laser immunotherapy in cancer therapy field.