| The incidence rate of breast cancer is the highest and the mortality rate is high in China.The main treatment methods are surgery,radiotherapy,chemotherapy,endocrine therapy or molecular targeted therapy.However,surgery is invasive;radiotherapy is radioactive;drugs can not be visualized,and high-dose or long-term administration will increase systemic toxicity or drug resistance;targeted therapy has an off-target effect and is expensive.Therefore,exploring safe and effective treatment strategies have become the main directions for breast cancer.Photodynamic therapy(PDT)is a novel type of tumor therapy with low toxicity,non-radiation,non-invasive and high spatial selectivity.Laser activated photosensitizer can transfer energy to oxygen to produce cytotoxic singlet oxygen,which can kill tumor cells.However,most photosensitizers,such as Ce6,have poor water solubility,low tumor targeting ability and non-visibility,which affect the therapeutic effect of PDT.In addition,and the oxygen content in tumor microenvironment is limited,so PDT alone is not significant.Immunotherapy provides a novel method for the treatment of breast cancer,especially triple negative breast cancer(TNBC)with high malignancy,easy metastasis and recurrence.However,the response of TNBC to immune checkpoint blocking therapy is limited and the objective remission rate is low.Therefore,it is necessary to combine with other therapies to further improve the efficacy.Photothermal therapy(PTT)in nanomedicine is a safe and non-radiation local treatment method.It can induce the release of tumor related antigens and cytokines,enhance the recognition of tumor cells by immune cells,and can cooperate with PDT or immunotherapy.However,immune antibodies may be denatured and inactivated under high temperature,which brings challenges to PTT cooperative immunotherapy.At present,JQ1,a small molecule inhibitor,has shown potential as a type of immunosuppressant.JQ1 can prevent the transcription of TNBC related oncogene c-MYC and induce apoptosis,and directly down regulate the expression of PD-L1 by affecting the transcription of c-MYC.In addition,JQ1 has relatively good structural stability and thermal stability,which can play an important role in the photothermal immunotherapy.However,JQ1 has poor water solubility and low tumor targeting ability,which affects its further application in TNBC immunotherapy.The surface of multifunctional nano-carriers can be loaded with drugs or inhibitors to improve the concentration of drugs in tumor;some nanomaterials also have therapeutic or imaging effects to realize single or combined treatment under the guidance of imaging,which is very valuable for the accurate diagnosis and treatment or combined treatments of breast cancer.However,the complex synthesis steps or potential safety risks of nanomaterials have become the main limitations for clinical transformation.Biomimetic nanomaterials,polydopamine melanin nanoparticles,have been widely concerned because of their good biocompatibility,safety and degradability,and are taken as drug carriers,treatment(PTT)and magnetic resonance/photoacoustic imaging(MRI/PAI)agents.Therefore,it is of great significance to further explore the application of melanin nanoparticles in drug carriers,synergistic PDT or immunotherapy.The purpose of this study:1.To explore the ability of melanin nanoparticles to protect,improve the effect and enhance PDT of photosensitizer Ce6;to evaluate the synergistic PDT and PTT effect of breast cancer by PDMN-Ce6 guided by imaging.2.To determine whether melanin nanoparticles can improve the tumor targeting ability of JQ1 and the efficacy of TNBC immunotherapy,and prevent tumor metastasis and recurrence.The main research contents are as follows:1.Preparation of biomimetic melanin nanoparticlesMethods:dopamine polymerization method is used to synthesize PDMN in vitro.Chemical analysis,MRI,PAI technology,MTT,blood biochemical analysis and pathological staining are used to identify the PDMN.Experimental results:the particle size of PDMN is about 100 nm.PDMN can be slowly degraded in simulated tumor acidic environment.PDMN has photothermal stability,the temperature rise is dependent on the concentration and the laser power,which can be used for PTT.PDMN has MRI and PAI performance.When the cell incubation concentration is up to 8mg/m L,the cell viability is not affected.The results of biochemical analysis and H&E staining show that PDMN also has good biocompatibility in vivo.Conclusions:the multifunctional biomimetic material PDMN has been successfully prepared which has stable performance,good biocompatibility and degradability,and can be used in PTT or imaging research.2.Photosensitizers-loaded melanin nanoparticles for bimodal imaging-guided combined therapyMethods:Ce6 is loaded on the surface of PDMN byπ-πstacking(PDMN-Ce6).The surface properties,phototherapeutic ability,cell phagocytosis level,biocompatibility and combined therapy of PDMN-Ce6 are analyzed by chemical analysis,PDT/PTT performance analysis,flow cytometry analysis and MTT method.The animal model of subcutaneous tumor of breast cancer is established.The distribution and combination therapy in vivo of PDMN-Ce6 are guided by MRI/PAI.The biocompatibility of PDMN-Ce6 is verified by pathological staining,blood index analysis and hemolysis test.Results:PDMN is successfully loaded with photosensitizer Ce6,and its loading efficiency is 35.2 wt%.Compared with free Ce6,breast cancer cells show a higher uptake rate to PDMN-Ce6,which is related to energy dependent and actin driven endocytosis.Compared with single PDT or PTT,PDMN-Ce6 mediated phototherapy has stronger cell therapeutic effect,and the cell survival rate is only 11%.In addition,in vivo experiments show that the tumor accumulation of PDMN-Ce6 is2.8-fold higher than that of free Ce6 after intravenous injection for 24 h,under the guidance of fluorescence optical imaging mediated by Ce6,or MRI/PAI mediated by PDMN.Compared with the control group,PDMN-Ce6 does not cause hemolysis,important blood indexes changes,or normal tissue damage.The synergistic phototherapy mediated by PDMN-Ce6 can significantly inhibit tumor growth,cause tumor necrosis and inhibit tumor angiogenesis.Conclusions:PDMN can successfully load photosensitizer Ce6,protect Ce6,improve its water solubility and tumor targeting,and realize the combined treatment under the guidance of bimodal imaging,which will provide novel ideas for the integration of diagnosis and treatment for breast cancer.3.JQ1-loaded melanin nanoparticles inhibit c-MYC/PD-L1 to enhance photothermal immunotherapy for triple negative breast cancerMethods:PDMN is modified with small molecule JQ1(PDMN-JQ1)byπ-πstacking.Chemical analysis,PTT performance analysis,flow cytometry,western blot and MTT are used to test the basic properties,photothermal stability,biodegradability,target protein expression,cell apoptosis and therapeutic effect of PDMN-JQ1.The animal model of TNBC subcutaneous tumor is established,and the tumor therapeutic effect is analyzed.The lymphocyte expression is analyzed by flow cytometry and the important cytokines expression is analyzed by ELISA.After treatment,the animal model of distant tumor is established,and the growth of tumor and the expression of memory lymphocyte are detected.Results:PDMN is successfully loaded with JQ1,which exhibits a solid sphere with an average diameter of about 100 nm,and its loading efficiency is 23.3 wt%.It shows good biodegradability and can be degraded slowly.JQ1is released slowly as the carrier degradating.About 40%of JQ1 is detected after degradation for 24 h,and about 70%is detected after degradation for 14 d.PDMN-JQ1has good photothermal stability and biocompatibility.It can down regulate the expression of c-MYC and PD-L1,and induce apoptosis.Under the combined treatment,tumor cell proliferation is significantly inhibited.Compared with JQ1 or PDMN alone,c-MYC targeted therapy,PTT and immunomodulatory therapy mediated by PDMN-JQ1 can inhibit the growth of subcutaneous tumor,down regulate the expression of PD-L1,activate the expression of CD3~+T cells and CD8~+T lymphocytes,and stimulate the secretion of cytokines TNF-α,IFN-γand IL-2.Through the analysis of distant tumor animal model,PDMN-JQ1 can produce strong immune memory effect and inhibit the growth of distant tumor.Conclusions:a simple and multifunctional nano-platform PDMN-JQ1 is successfully prepared.PDMN-JQ1 can significantly increase the tumor concentration of JQ1,achieve triple synergistic therapy,significantly enhance the activation of cytotoxic T lymphocytes,and effectively inhibit the growth of TNBC subcutaneous tumor and distant tumor.Moreover,PDMN-JQ1 can induce strong immune memory effect by continuously releasing JQ1,which can effectively prevent tumor metastasis and recurrence. |
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