Preparation Of Manganese-Based Multifunctional Nanoparticles For Chemo-Photothermo-Immunotherapy In Triple-Negative Breast Cancer

Objective:Triple-negative breast cancer（TNBC）is defined as a“cold”tumor,which is characterized by a scarcity of T lymphocyte infiltration and an immunosuppressive tumor microenvironment（TME）,and is considered a poorly immunogenic tumor.These features greatly limit the immunotherapy effect of TNBC.Therefore in this study,bovine serum albumin（BSA）-templated MnO₂ nanoparticles（BSA/MnO₂ NPs）were prepared with the TME-response and photothermal ability,and then efficiently loaded doxorubicin（DOX,DOX-BSA/MnO₂ NPs）.The chemo-photothermal combining therapy-induced immunogenic cell death（ICD）with amelioration of the hypoxic TME would improve the immunogenicity of tumors and improve the immunosuppressive TME,which therefore can activate anti-tumor immune response.Accordingly,this work provides an alternative strategy for treating TNBC.Methods:（1）Biomineralization method was used to prepare DOX-BSA/MnO₂ NPs,which formulation and preparation process were further optimized and characterized.（2）The stability of nanoparticles under different conditions was investigated;The in vitro release experiment was conducted using the dialysis method;The photothermal effect and photothermal stability of nanoparticles at different concentrations and different laser power densities were investigated under 635 nm laser irradiation;The influence of nanoparticles on hemolysis was investigated.（3）The in vitro biocompatibility of BSA/MnO₂ NPs and anti-tumor effect of DOX-BSA/MnO₂ NPs were measured by MTT assay;In vitro cellular uptake of free DOX and DOX-BSA/MnO₂ NPs was observed by confocal laser scanning microscope and quantified by flow cytometry;The cell viability under varying laser irradiation time was investigated for evaluating the ability of photothermal ablation on killing tumor cells;The production capacity of calreticulin（CRT）,high mobility group box-1（HMGB-1）,adenosine triphosphate（ATP）by tumor cells induced by photothermal therapy alone,chemotherapy alone,and photothermal combined chemotherapy was evaluated.（4）The pharmacokinetic properties of free DOX solution compared with DOX-BSA/MnO₂ NPs were determined in rats after tail vein injection.The tissue distribution of DOX and DOX-BSA/MnO₂ NPs in tumor-bearing BALB/c mice was investigated.（5）The TNBC model in BALB/c mice were constructed,and a living body imaging system was used to investigate the distribution of nanoparticles in different organs and tissues after tail vein injection.To investigate the abscopal therapeutic effect on distant tumors,a bilateral tumor model of BALB/c mice was developed.The in vivo anti-tumor effect of photothermal therapy alone,chemotherapy alone,and photothermal combined chemotherapy was evaluated;Immunofluorescence analysis was performed to measure the effects of nanoparticles on the expression of hypoxia inducible factor-1α（HIF-1α）,CD4⁺,CD8⁺T lymphocytes and Foxp3（the marker of Tregs）;The polarization of tumor-associated macrophages（TAM）and expression of interleukin-10（IL-10）,tumor necrosis factor-α（TNF-α）were detected by flow cytometry assay;TUNEL fluorescent staining was performed to investigate the cell apoptosis of tumors induced by nanoparticles;HE staining was used to investigate the biocompatibility of nanoparticles to normal tissues.Results:（1）The optimized formulation and preparation process of DOX-BSA/MnO₂ NPs were obtained by a single factor analysis.Briefly,20 mg of KMnO₄ was fully dissolved in 5 m L of deionized water,and 5 m L of BSA solution（20 mg/m L）was then added dropwise into the KMnO₄ solution（4 mg/m L）in a water bath sonicator（200W）.Continuous ultrasonication of the mixture for 1 h yielded the BSA/MnO₂ NPs.Subsequently,1 m L of the obtained BSA/MnO₂ NPs was mixed with 1 m L of DOX solution（2 mg/m L）under stirring（450 r/min）for 7 h to obtain the DOX-BSA/MnO₂ NPs.The particle size,polydispersity index,zeta potential,encapsulation efficiency,loading efficiency of the obtained DOX-BSA/MnO₂ NPs were（32.50±2.31）nm,0.240±0.006,（-26.80±0.70）m V,（99.33±0.09）%,（23.88±0.28）%,respectively.The successful preparation of the DOX-BSA/MnO₂ NPs was confirmed by transmission electron microscope（TEM）,X-ray photoelectron spectroscopy（XPS）,ultraviolet-visible spectrum（UV-vis）,fourier transform infrared spectrum（FTIR）.The results of differential scanning calorimetry（DSC）and X-ray diffraction（XRD）analysis were both proving that DOX in nanoparticles is amorphous crystals.（2）Incubation of the DOX-BSA/MnO₂ NPs with purified water,saline,RPMI 1640 containing 10%FBS for 7 days at 4℃barely changed the particle size and PDI,indicating its good stability;What’s more,DOX-BSA/MnO₂ NP lyophilized powder can improve the stability;The result of hemolysis analysis showed that DOX-BSA/MnO₂ NPs had good blood biocompatibility and could be applicated for intravenous injection;Both BSA/MnO₂ NPs and DOX-BSA/MnO₂ NPs had good photothermal effect and photothermal stability to meet the requirements of photothermal therapy;The result of in vitro release experiment proved the TME-responsive and photothermal-responsive release properties of DOX-BSA/MnO₂NPs;The result of in vitro oxygen generation confirmed that the ability to improve tumor hypoxic microenvironment of DOX-BSA/MnO₂ NPs.（3）BSA/MnO₂ NPs presented good biocompatibility to cells;The cellular uptake of DOX-BSA/MnO₂ NPs showed obvious nuclear localization phenomenon and could improve in vitro anti-tumor effect;Compared with chemotherapy alone and photothermal therapy alone,the combination of DOX-BSA/MnO₂ NP-mediated chemotherapy and photothermal therapy could improve the anti-tumor effect and could greatly increase the exposure of CRT,the secretion of ATP and HMGB-1.（4）DOX-BSA/MnO₂ NPs could significantly influence the pharmacokinetic properties of DOX in vivo.The t_1/2 was prolonged by 3.88 times,the CL was decreased by 0.52 times,the AUC was increased by 1.02 times,the V was increased by 1.42 times,and the MRT was increased by 0.42 times.DOX-BSA/MnO₂ NPs could reduce the DOX distribution in heart which helps lower the toxicity of DOX.The results also showed that DOX-BSA/MnO₂ NPs had good ability of tumor targeting which may improve the anti-tumor efficacy.（5）The results of in vivo experiments presented that DOX-BSA/MnO₂ NPs could effectively improve hypoxic TME,reduce the expression level of HIF-1α,and then reduce the expression of Tregs,and induce the polarization of M2-type TAM to M1-type TAM,thereby promoting the secretion of TNF-αand inhibiting the secretion of IL-10.Therefore,the immunosuppressive TME could be improved.Moreover,DOX-BSA/MnO₂ NP-mediated chemo-photothermal therapy could effectively improve the infiltration of CD4⁺and CD8⁺T lymphocytes in the tumor site,which could greatly improve the immunogenicity of TNBC and result in an enhanced immunotherapy effect.Thus,the growth of primary and distant tumor could be effectively inhibited.Conclusion:Therefore in this study,DOX-BSA/MnO₂ NPs were successfully prepared with excellent biocompatibility and simplicity of the preparation,which was beneficial to industrialized production.DOX-BSA/MnO₂ NP-mediated chemo-photothermal therapy could not only induce the ICD of tumor cell,but also could effectively modified the immunosuppressive microenvironment by relieving the hypoxic TME.Thus,DOX-BSA/MnO₂NPs had the ability to favor antitumor immunity and improve the efficacy and safety of TNBC treatment.