Preparation Of Immune Adjuvant-Loading Phycocyanin Nanosystem And Its Application In Sono-Immune Combined Anti-Tumor Therapy

Cancer is a serious disease that endangers human life and health.Current clinical treatment options for cancer include surgical treatment（ST）,radiotherapy（RT）and chemotherapy（CT）.Each treatment has its advantages,but also has limitations,such as postoperative complications,drug resistance,damage to the human immune system,and systemic side effects.Moreover,once the tumor spreads,it becomes even more difficult to cure.Therefore,it is crucial to develop new therapeutic methods and strategies to kill tumor cells in situ as well as to suppress tumor metastasis and recurrencein the field of antitumor research.Nano-platform/nanocarrier based sonodynamic therapy-immunotherapy combination is a potential antitumor strategy that has shown a synergistic effect（"1+1>2"）in treatment.Therefore,it is particularly important to use multifunctional nano-systems with simple design,simple preparation process,safety and effectiveness to ensure and enhance the realization of the synergistic effect of sonodynamic immunotherapy.This study used a one-step cross-linking method to prepare phycocyanin nanoparticles（PCNPs）as a carrier to deliver imiquimote hydrochloride[R837（hy）]and as a nano-sonosensitizer to generate reactive oxygen species（ROS）to achievesonodynamic therapy.The pharmacologic properties,in vitro and in vivo properties,and antitumor effects of the obtained multifunctional nano-system[PCNP-R837（hy）]were evaluated as follows.1.PCNP-R837（hy）was successfully prepared by desolvation method.The characterization and properties of PCNP-R837（hy）were investigated through experiments such as transmission electron microscopy（TEM）observation of morphology,measurement of particle size and potential,examination of various spectral characteristics,and determination of encapsulation efficiency and drug loading.The in vitro stability of PCNP-R837（hy）was tested for fourteen days.The ugeneration ability of reactive oxygen species（ROS）such as ¹O₂ and.OH under ultrasound irradiation were investigated in vitro,and the potential of PCNP-R837（hy）application was preliminarily evaluated.The experimental results showed that the prepared nanoparticles have uniform spherical appearance,good dispersion,suitable particle size.They exhibited good stability in PBS and serum,and can effectively produce ROS（including ¹O₂ and·OH）under ultrasound irradiation,indicating that PCNP has a good role as a sonosenstizer.R837（hy）can be released significantly under acidic and ultrasonic conditions,indicating that R837（hy）was successfully loaded into PCNP.PCNP is an acidic and sonosensitive carrier,which has the potential to achieve tumor-targeted delivery and release of R837（hy）for sonodynamic-immune synergistic therapy.2.Mouse colon cancer cell CT26 and mouse breast cancer cell 4T1-LUC cells expressing luciferase were selected as study models to investigate the biocompatibility of PCNP in vitro and the in vitro anti-tumor effect of combined sonodynamic-immunotherapy based on PCNP-R837（hy）.MTT results showed that,PCNP/PCNP-R837（hy）has good biocompatibility,andbased on the sonodynamic-immunetherapy,it exhibited a strong killing effect on tumor cells in vitro.The study also examined the stimulating effect of PCNP-R837（hy）on murine bone marrow-derived Dendritic cells（DCs）and the induction of immunogenic cell death（ICD）in vitro.Flow cytometry results showed that compared with blank control group,PCNP-R837（hy）could significantly improve the maturity of DC cells,indicating that it could further promote antigen presentation and enhance immune response.Laser confocal images showed obvious CRT expression on the cell surface after ultrasound treatment based on PCNP-R837（hy）,indicating that ultrasound can induce immunogenic death of tumors and thus improve the level of immune response.3.To investigate the antitumor effect of PCNP-R837（hy）based sonodynamic-immunotherapy in vivo.（1）The study used a mouse colon cancer tumor to examine the biosafety of PCNP-R837（hy）in vivo by constructing and to investigate the mechanism of action of sonodynamic-immunetherapy based on PCNP-R837（hy）on DCs maturation and the mechanism of immune response.The experimental results showed that PCNP-R837（hy）had good biosafety in vivo,and the prepared nanoparticles still effectively promoted the maturation of DC in vivo under ultrasound irradiation,thus enhancing the efficiency of antigen presentation and improving the immune response.The flow cytometry demonstrated that Treg（CD4+Foxp3+T）levels were significantly increased after ultrasound treatment withoutanti-CTLA-4 antibody treatment.Therefore,it is speculated that the antitumor immune response induced by ICDs alone after ultrasound therapy may be compromised by Tregs recruited after ultrasound therapy.（2）By constructing a mouse colon cancer bilateral tumor model,the therapeutic effect of sonodynamic-immunotherapy based on PCNP-R837（hy）combined with cytotoxic T lymphocyte-associated antigen 4（CTLA-4）immune checkpoint blockade,as well as the mechanism of anti-tumor immune response were investigated.The experimental results showed that after sonodynamic-immunotherapy based on PCNP-R837（hy）,further combined with anti-CTLA-4 antibody therapy,the primary tumor could be effectively treated,the growth of distant tumor could be suprressed,and the CD8+T in distant tumors could be enhanced after treatment,which the proportion of of Treg cells in distant tumor was significantly reduced.Effectively inhibit the activity of immunosuppressive T cells,which can improve the anti-tumor immune response,play a role in killing tumors,and inhibit the growth of distant tumors.（3）A mouse model of breast cancer metastasis was constructed to investigate the therapeutic effect of PCNP-R837（hy）based sonodynamic-immunotherapy combined with CTLA-4 immune checkpoint blocking on tumor metastasis model and the mechanism of anti-tumor immune memory.The experimental results showed that the combination therapy could significantly inhibit tumor metastasis,prolong the survival time of mice to 50 days,and significantly inhibit tumor regrowth when the body was attacked by cancer cells again.From the perspective of in vivo immune indexes,the proportion of effector memory T cells（TEM CD3+CD8+CD62L-CD44+）in spleen of mice was significantly increased after treatment,and the proportion of several typical cytokines（TNF-α,IFN-γand IL-12p40）in serum of mice was up-regulated.indicating that PCNP-R837（hy）sonodynamic-immunotherapy combined with anti-CTLA-4 antibody treatment could induce strong immune memory and effectively prevent tumor recurrence.To sum up,this study prepared phycocyanin nanosystem loaded with immune adjuvant by one-step cross-linking method,and the nanosystem achieved two functions:snondynamic therapy based on sonosensitivity and immune adjuvant delivery based on nanoparticle carrier.It is a simple and effective multifunctional nanocarrier.Based on this system,the combination of sonodynamic and immune therapy has shown good therapeutic effect.It provides a new idea for the construction of simple nano delivery system,and explores a new way for the application of PC nanocarriers in sonodynamic-immunotherapy for anti-tumor treatment.