Construction Of Biomimetic Nanosystem For Targeted Delivery Of Gamabufotalin And Their Combined Therapy For TNBC

Breast cancer is the most common malignancy among women in China.Triple-negative breast cancers(TNBC)refers to a subtype of breast cancer in which estrogen receptor,progesterone receptor,and human epidermal growth factor receptor-2 are all negative.Its incidence accounts for 15%to 20%of breast cancer,and has the characteristics of strong invasiveness,high degree of malignancy,easy recurrence and metastasis.At present,the comprehensive treatment mode based on surgery,chemotherapy,radiotherapy,endocrine therapy and targeted drug therapy has shown a good effect on early breast cancer and effectively prolong the total survival,but there is a lack of effective treatment for metastatic breast cancer,especially TNBC,and the prognosis is poor.Therefore,exploring the combined treatment strategy to reduce the recurrence and metastasis rate and improve the survival rate of patients has extremely important theoretical significance and extensive application value for TNBC.Traditional medicine has great potential in preventing recurrence and metastasis of TNBC.Gamabufotalin(CS-6),as the main active ingredient of Bufo bufonis,has the functions of anti-tumor and anti-inflammation,which has been preliminarily used in TNBC therapy.However,the low efficiency of tumortargeted delivery and cardiotoxicity greatly limits the clinical effect of CS-6.In this paper,we focus on the core problem of how to achieve accurate targeting therapy of TNBC,and construct biomimetic nanodrug of CS-6 combined with chemotherapy and photodynamic therapy,which can greatly improve the toxicity and efficiency of tumor targeted delivery of CS-6 to achieve the purpose of efficient targeted therapy of TNBC in vitro and in vivo to carry out the following work:(1)Construction and anti-TNBC study of the sequentially-targeted biomimetic nanodrug system based on CS-6 regulating DOX "back door effect".Doxorubicin(DOX)is widely used in clinical treatment as one of the standard treatments for TNBC.However,the long-term use of these drugs can enhance tumor metastasis and drug resistance by up-regulation the expression of cyclooxygenase-2(COX-2)to induce the "back door effect" of tumor.In this work,we proposed a therapeutic strategy of CS-6 and DOX combined chemotherapy based on the function of CS-6 in inhibiting the expression of COX2 and killing tumor,which can effectively reduce the drug dose and inhibit tumor growth,invasion,and metastasis.Thus,we developed a biomimetic nanodrug system(denoted as GTDC@M-R NPs)based on erythrocyte membrane(M)camouflaged graphene oxide quantum dots(GOQDs,G)for TNBC therapy.The TAT(T)and RGD(R)peptides were used to endow targeting accumulation ability of CS-6(C)and DOX(D)in tumor tissue.The characterization results show that the average size of GTDC@M-R NPs is about 70 nm,showing a classical coreshell structure,in which the core contains multiple GTDC NPs.In addition,the erythrocyte membrane protein was not damaged in the process of synthesis,which retained its ability to escape the host immune system and prolong blood circulation.The DOX and CS-6 drug model proved that GTDC@M-R NPs has a good drug-carrying capacity,and the loading rate of DOX and CS-6 are 42.2%and 38.9%,respectively.The drug in GTDC@M-R NPs released less drug in a neutral environment but released quickly in an acidic environment.The target cells(MDA-MB-231)proved that GTDC@M-R NPs promoted cells and nucleus targeting capability by recognizing the integrin αvβ3 protein of RGD peptide and the importin of TAT peptide,enhancing drug effect on tumor cells.The uptake mechanism proved that GTDC@M-R NPs can be taken up by cells through clathrin and caveolin-mediated endocytosis,then escaped from lysosomal to achieve the function of nuclear targeting.The cytotoxicity experiment proved that the combination of DOX(0.5 μM)and CS-6(0.05 μM)had synergistic effect when the concentration ratio was 10:1.GTDC@M-R NPs can induce cell cycle arrest and mitochondrial apoptosis mediated by p53 and BAX/Bcl-2 signal pathway,which could effectively inhibit the proliferation and promote apoptosis of TNBC cells.The wound healing and molecular biology experiments proved that GTDC@M-R NPs can inhibit the expression of COX-2,VEGF,MMP-9,and the activation of epithelial mesenchymal transformation(EMT)to prevent tumor invasion and metastasis.The blood circulation and tumor targeting experiments proved that the blood circulation time and accumulation in tumor sites of GTDC@M-R NPs increased 3-fold and 2-fold compared to naked GTDC NPs,respectively.In vivo assay proved that GTDC@M-R NPs has a better therapeutic effect of TNBC than a single therapy strategy(86.4%)and effectively prolong the survival cycle.Moreover,comparing with the control,the average number of lung metastatic nodules in tumor-bearing mice reduced 84.5%.Taken together,our results proved that the developed GTDC@M-R NPs can inhibit the growth and suppress metastasis of TNBC.However,the deficiency is that when the nano-drugs gather in the tumor focus,most of them accumulate in the periphery of the tumor tissue and can not enter the depths of the tumor tissue to exert their efficacy.Therefore,improving the permeability of nanodrugs in tumor tissue is of great significance to improve the bioavailability of drugs in solid tumors and improve the therapeutic efficacy of tumor therapy.In our next work,we will use sequential drug delivery strategy to solve the problem of poor permeability of nanodrugs by dilating blood vessels and changing particle size.(2)Construction and combination with photodynamic therapy for TNBC study of the high penetration CS-6 biomimetic nanodrug system based on“sequential drug delivery”strategy.In this study,we use capsaicin as a vasodilator to effectively improve the anoxic microenvironment of tumor and promote the penetration of nanodrugs in the tumor.Hence,we propose a sequential nanodrug delivery strategy based on capsaicin as a prodrug,which can enhance the combined therapy of CS-6 and PDT to effectively inhibit TNBC invasion and metastasis.We used hybrid membrane(HM)camouflaged PLGA to prepare capsaicinloaded biomimetic nano-prodrug delivery system(HM-PLGA@Cap NP s)and CS6&Ce6-co-loaded biomimetic nanodrug delivery system(HM-PLGA@GC&CS-6 NPs).The different characterization methods confirmed that the average size of the biomimetic nanodrug was about 122 nm,showing a classical core-shell structure,and successfully wrapped in the hybrid membrane.In addition,the membrane protein of the hybrid membrane was successfully retained during the synthesis,which provided a guarantee for escape from the host immune system,prolongation of blood circulation and homologous targeting of tumor.The HMPLGA@GC&CS-6 NPs model proved that pH and laser co-stimulation can realize the particle size conversion of the biomimetic nanodrug to release small-size nanodrug GC&CS-6 NPs(～15 nm)and drugs.The target cells(MDA-MB-231)and immune cells(RAW264.7)proved that HM-PLGA@GC&CS-6 NPs can effectively target homologous tumor cells and escape host immune cells by using the homologous targeting effect and immune recognition ability of hybrid membrane.The tumor 3D sphere model proved that the GC&CS-6 NPs released by HM-PLGA@GC&CS-6 NPs under co-stimulation can effectively enhance the penetration ability in tumor.The cytotoxicity test proved that HMPLGA@GC&CS-6 NPs can induce cell cycle arrest and mitochondrial apoptosis to inhibit the growth and promote apoptosis of tumor cells.The Transwell and Western blotting test proved that HM-PLGA@GC&CS-6 NPs can effectively inhibit the expression of hypoxia inducible factor-1α(HIF-1α),COX-2,and MMP-9 to prevent tumor invasion and metastasis induced by PDT.The endothelial cells(HUVECs)confirmed that HM-PLGA@Cap NPs can effectively enhance the influx of Ca2+and promote the release of nitric oxide(NO)to achieve the purpose of vasodilation.The immunofluorescence experiments proved that HM-PLGA@Cap NPs pretreatment can effectively dilate vessels and significantly reduce the expression of HIF-1α in tumor to improving tumor hypoxic microenvironment.Tumor targeting and penetration experiments in vivo proved that hybrid membrane camouflage can significantly increase the accumulation of HM-PLGA@GC&CS-6 NPs.In addition,HM-PLGA@Cap NPs pretreatment can significantly increase the penetration of HM-PLGA@GC&CS-6 NPs in the tumor.In vivo antitumor results demonstrated that the sequential administration strategy not only effectively inhibits tumor growth(77.5%),but also significantly reduces the expression of HIF-1α,COX-2,and MMP-9 in tumor tissues to block the metastasis of TNBC.Taken together,the study proved a high efficiency of the sequential treatment strategy by combining the vasodilator of capsaicin and tumor-infiltrating biomimetic nanodrugs for TNBC therapy.