Anti-Breast Cancer Study Of PDT/PTT Combined Immunotherapy Based On Multifunctional Nanoparticles

Objective In recent years,breast cancer has an increasing impact on women’s health.Its incidence is on the rise,and the age of onset tends to be younger,so women pay more and more attention to breast cancer.Despite existing treatments such as surgical treatment,chemotherapy treatment,radiotherapy treatment,traditional Chinese-medicine treatment have played an important role in the treatment of breast cancer,there are also large trauma,easy to relapse and metastasis,systemic toxic side effects and other shortcomings.In addition,as new therapies for breast cancer,such as immunotherapy,photothermal therapy and photodynamic therapy,they have achieved good clinical results due to their advantages of high therapeutic efficiency,less invasiveness,less side effects,and good therapeutic specificity.However,in clinical practice and exploratory research,these new therapies also have inherent defects.The tumor cannot be completely eliminated to achieve the best therapeutic effect.For example,immunotherapy has the disadvantages of limited tissue and tumor penetration,long half-life,immunogenicity and high cost.PTT may lead to uneven heat distribution within the tumor,and the hypoxic microenvironment within the tumor also limits the effect of PDT,so it is still difficult to eradicate the tumor by a single treatment method,especially deep tumor.In addition,the pathogenesis of breast cancer is also very complex,and the tumor also has the characteristics of diversity and heterogeneity,so a single treatment can not meet the clinical needs.Therefore,based on the existence of the above problems,a new type of Fe₃O₄ carrier was prepared,loaded with the photothermal agent indocyanine green（ICG）and surfaced with a cyclic tripeptide of arginine-glycine-aspartic acid（cRGD）multifunctional ICG@SANPs-cRGD nanoparticles.It is used for PDT/PTT combined immunotherapy in the treatment of breast cancer,hoping to explore a safe and efficient combined treatment strategy to comprehensively and accurately inhibit or eliminate cancer foci.Methods（1）Fe₃O₄ nanoparticles with magnetic properties were synthesized by high temperature decomposition,and coated with oleic acid to obtain SANPs.Then,ICG was coated on the surface of SANPs to obtain ICG@SANPs.Finally,cRGD was coupled and modified to the surface of ICG@SANPs to prepare into the required ICG@SANPs-cRGD nanoparticles.Transmission electron microscopy（TEM）was used to observe the dispersion and particle size distribution of nanoparticles.the size and hydrated particle size of the magnetic nanoparticles were determined by dynamic light scattering;Zeta potential was detected by nano-particle potential analyzer;Determination of near-infrared absorption spectrum of nanoparticles by UV-Vis-NIR spectrophotometr;vibrating sample magnetometer determination and magnetic resonance imaging system to detect the magnetic properties and imaging capabilities of ICG@SANPs-RGD nanoparticles;hemolysis experiments to verify its biological safety.（2）Subsequently,a series of cell experiments were conducted to evaluate the in vitro anti-tumor ability of ICG@SANPs-cRGD PDT/PTT.The cellular internalization of ICG@SANPs-cRGD nanoparticles was observed by CLSM;The photothermal conversion ability of ICG@SANPs-cRGD nanoparticles was evaluated by infrared thermal imager.The in vitro cytotoxicity and the effect on cell proliferation were detected by CCK-8 kit;the effects of magnetic nanoparticles on the proliferation,invasion and migration of 4T1 cells were observed by plate cloning and transwell chamber invasion and migration experiments;the production of reactive oxygen species was detected by the fluorescent probe DCFH-DA;the effect of NIR+ICG@SANPs-cRGD nanoparticles on the dead and alive state of 4T1 cells was detected by Calcein-AM/PI kit;Annexin V-FITC/PI kit was used to detect the apoptosis of4T1 cells after NIR+ICG@SANPs-cRGD treatment.（3）Finally,the therapeutic effect and immune activation mechanism of PDT/PTT combined immunotherapy based on multifunctional nanoparticles were evaluated in vivo.The photothermal conversion ability of ICG@SANPs-cRGD nanoparticles in vivo was evaluated by infrared thermal imager;the tumor enrichment and MRI imaging ability were observed by small animal fluorescence imaging system and MRI imaging system;The therapeutic effect of PDT/PTT combined with immune checkpoint blocker Anti-PD-L1 based on ICG@SANPs-cRGD nanoparticles was observed in 4T1tumor-bearing mice.The biosafety of the combined treatment strategy was observed by staining,blood routine and blood biochemical indicators.H&E staining and TUNEL staining were used to evaluate the morphological changes and apoptosis of tumor cells in mice after combined treatment.Bax apoptotic protein,antigen Ki67,ROS production in tumor tissue,hypoxia-inducible factor 1α,infiltration of cytotoxic T cells,and expression of calreticulin CRT were determined by immunofluorescence technique and immunohistochemical（IHC）staining;the secretion of various immune-related cytokines in serum was measured by ELISA.Results（1）ICG@SANPs-cRGD nanoparticles were synthesized by pyrolysis method.The TEM images showed that the nanoparticles were dispersive and had no aggregation phenomenon;their particle sizes were uniformly distributed in DLS.Zeta results showed that cRGD was successfully modified,UV/vis-NIR showed that it has a wide absorbance in the near-infrared region,VSM and MRI verified that it is paramagnetic and can be used for MRI imaging,and hemolysis test showed that it had good biocompatibility.（2）In vitro therapeutic effect studies have shown that ICG@SANPs-cRGD nanoparticles can be maximized by cell uptake.The near-infrared photothermal performance test showed that the ICG@SANPs-RGD nanoparticles have good photothermal conversion ability.The detection of CCK-8 kit showed that ICG@SANPs-cRGD nanoparticles had no obvious cytotoxicity to normal cell lines,but the toxicity to 4T1 breast cancer was higher.Under NIR irradiation,ICG@SANPs-cRGD nanoparticles enhanced the effect of PDT/PTT and significantly inhibited the proliferation,cloning,invasion and migration of 4T1 cells;DCFH-DA assay showed that under NIR irradiation,ICG@SANPs-cRGD nanoparticles produced a large amount of cytotoxic ROS in 4T1 cells and induced immunogenic cell death;the results of calcein-AM/PI double staining and flow cytometry analysis showed that the number of cell death and apoptosis rate of 4T1 increased under the PDT/PTT enhanced by ICG@SANPs-cRGD nanoparticles.（3）The results of in vivo combined treatment effect study and immune activation mechanism study found that ICG@SANPs-cRGD nanoparticles have good photothermal conversion performance in vivo,which can enhance the effect of PTT.Fluorescence imaging showed that ICG@SANPs-cRGD nanoparticles can be targeted and enriched at tumor sites,the results of the MRI imaging system proved that the magnetic nanoparticles have the ability of T1-weighted imaging;the detection of H&E staining,blood routine and blood biochemical indicators proved that the magnetic nanoparticles have good biosafety in the process of tumor treatment in vivo.The therapeutic effects of the three mouse tumor models demonstrated that ICG@SANPs-cRGD enhanced PDT/PTT combined with Anti-PD-L1 therapy could not only destroy the primary tumor,but also identify,inhibit and eliminate distant metastases.In the study of the mechanism of anti-tumor immune activation in vivo,we found that after combined treatment,cells in the mouse tissues underwent necrosis and apoptosis,the expression of Bax protein increased,the expression of Ki-67 antigen decreased,and the production of ROS in vivo enhanced ICD,hypoxia-inducible factor HIF-1αexpression was decreased,cytotoxic T cells CD3⁺and CD8⁺tumor infiltration increased,calreticulin CRT exposure increased,proinflammatory factor secretion increased,and immunosuppressive factors were downregulated.Conclusion The as-synthesized ICG@SANPs-RGD in this experiment are small in diameter,dispersed and uniform in size;they have good paramagnetic properties,biocompatibility.Under the irradiation of NIR in vitro,ICG@SANPs-cRGD nanoparticles prepared in this study have the ability to enhance photodynamic and photothermal therapeutic effects,effectively inhibited the proliferation activity and the ability of invasion and migration of breast cancer cells and promoted the apoptosis of cancer cells by ROS cytotoxicity,which has a good ability to kill tumor cells in vitro.The ICG@SANPs-cRGD nanoparticles achieved Fluorescence imaging/MRI dual-modality imaging,enhanced the therapeutic effect of PDT/PTT+Anti-PD-L1,and realized the integration of tumor multimodality therapy and diagnosis and treatment,which is a promising candidate for breast cancer patients.Combination therapy provides theoretical basis and research ideas.