Selectively Estrogen Regulation And Platelets Blockade By Copper-Based Bioactive Nanovesicles For Metastasisinhibited Combination Triple-Negative Breast Cancer Therapy

Triple-negative breast cancer（TNBC）is a kind of breast cancer lacking estrogen receptor（ER）,progesterone receptor（PR）and human epidermal growth factor receptor（HER-2）.It has the characteristics of high malignant degree,easy recurrence,easy metastasis and poor prognosis,so there is no targeted therapy strategy for TNBC.Studies have shown that metastasis is the main cause of death in patients with triple negative breast cancer,and as a complex physiological process,estrogen and platelets play an important driving role in this process.Estrogen can not only increase the proliferation rate of tumor cells,but also increase the expression of matrix metalloproteinase 2（MMP-2）,degrade extracellular matrix and promote tumor cell metastasis.The degradation of tumor extracellular matrix can lead to the increase of platelet infiltration in the tumor,which makes more platelets activated by tumor cells.Activated platelets promote the epithelial-mesenchymal transformation of tumor cells by secreting TGF-β,which makes it more likely to metastasize.In addition,activated platelets can bind to tumor cells to help them achieve immune escape in blood circulation.Therefore,effectively down-regulating the level of estrogen in tumor tissue and blocking platelet activation will be a promising strategy for anti-triple negative breast cancer metastasis.Based on this,we developed a copper-based bioactive nano-vesicle（CPAD）,which can consume estrogen,cooperate with the production of nitric oxide（NO）to block platelet activation and cooperatively inhibit the proliferation and metastasis of TNBC.CPAD was prepared by loading cisplatin/arginine prodrug（Pt-Arg）into copper-based MOF（Cu-GA）and modified with hydrophilic shell（DSPE-PEG2000）.The designed CPAD can specifically release copper ions and prodrug Pt-Arg,copper ions to catalyze the oxidation of estrogen and reduce the level of in situ estrogen in tumor under acidic conditions,thus inhibiting TNBC metastasis through the ERK/CANP/MMP-2 pathway.At the same time,the released Pt-Arg prodrug produces free Pt and L-Arg under the activation of tumor tissue GSH,the Pt produces H₂O₂under the action of NOX enzyme,then oxidizes L-Arg to produce NO to prevent activated platelet-induced tumor EMT.Therefore,by virtue of local estrogen regulation and platelet activation blockade,CPAD made the inhibition rate of lung metastasis in 4T1breast cancer model as high as 88.4%.More importantly,the down-regulation of local estrogen,the production of NO and Pt-mediated chemotherapy improve the therapeutic effect of primary tumor,and the nanosystem has no significant toxicity to the body.Therefore,this study proposes a therapeutic strategy that cooperates with estrogen regulation and platelet activation blocking to inhibit the proliferation and metastasis of TNBC.The specific research contents are as follows:1、Preparation and characterization of CPAD nanoparticles.First,the prodrug Pt-Arg（PA）were prepared.¹H NMR and HPLC confirmed the successful synthesis of PA and its GSH responsiveness.After copper-based MOF（Cu-GA）nanoparticles were synthesized,PA was loaded into Cu-GA by physical stirring,and then DSPE-PEG2000was modified to obtain CPAD nanoparticles.The results of DLS and TEM showed that the hydrated particle size of CPAD was 200±10 nm,and the potential was 33.1 m V.Under the condition of p H 7.4,the stability of CPAD nanoparticles was good,while under the condition of p H 4.5,the release rate of Cu²⁺was 89.14%,and the release rate of Pt-Arg was as high as 95.43%.It shows that it has the ability to release drugs under the condition of tumor acid lysosome.Secondly,the estrogen consumption ability of copper ion was verified by detecting the production of H₂O₂,and the ability of prodrug Pt-Arg to produce NO was verified by NO detection kit,in which the yield of NO after the reaction of 10 m M Pt-Arg with 10 m M H₂O₂ was about 185±10μM.DLS results show that CPAD nanoparticles have good stability and dispersion.2、In vitro investigation of CPAD nanoparticle estrogen regulation and platelet blockade.In this study,triple negative breast cancer cell line 4T1 was used as a model.First of all,cell uptake experiments showed that the uptake of CPAD nanoparticles in4T1 cells was time-dependent,and the preparation could successfully achieve lysosome escape.Secondly,this study found that E2 upregulates the expression of MMP-2 through ERK/CANP signal pathway,while CPAD can significantly increase the content of intracellular Cu²⁺,successfully decrease the content of intracellular estrogen,and then down-regulate the protein levels of Capn4 and MMP-2.In addition,after the treatment of CPAD,a large number of H₂O₂ and NO were generated in the cells.Platelet aggregation assay,cell immunofluorescence assay and Western Blot assay showed that NO could significantly inhibit platelet activation and aggregation,thus blocking the deformation and EMT transformation of tumor cells.These results confirm that CPAD nanoparticles can effectively regulate estrogen and block platelet activation by producing copper ions and NO in vitro,thus inhibiting the expression of MMP-2 and the transformation of EMT in tumor cells.3、Study on anti-tumor and anti-metastatic activity of CPAD nanoparticles in vitro.First of all,the results of cell scratch test,transwell migration and invasion assay showed that the migration and invasion of 4T1 cells was increased after treat with platelets,and the ability of migration and invasion was further enhanced by the continued addition of E2,which was related to the promoting effect of platelets on EMT and the expression of MMP-2 induced by E2.CPAD nanoparticles could significantly inhibit the migration and invasion of 4T1 cells through estrogen regulation and platelet blockade.In addition,the results of cytotoxicity test,Calcein-AM/PI staining test and apoptosis test showed that the blank vector CD had partial inhibitory effect on cells,but the anti-tumor effect of CPAD nanoparticles was the most significant.The viability of cells treated with 50μg/m L CPAD was 27.1%,and the apoptosis rate was 53.7%.These results show that CPAD can not only effectively inhibit the invasion and metastasis of triple-negative breast cancer,but also exert its anti-tumor effect in vitro.4、Study on anti-tumor and anti-metastatic activity of CPAD nanoparticles in vivo.In this study,the animal model of 4T1 breast cancer in situ was established.Firstly,the aggregation of the preparation in the tumor site and the distribution of the main organs in mice were characterized by loading near infrared fluorescence dye IR783.Secondly,the anti-tumor properties of CPAD were evaluated by tumor volume,tumor tissue quality,H&E staining and TUNEL fluorescence staining.The results showed that CPAD nanoparticles had significant antigenic tumor effect.After treatment,the tumor volume increased slowly,the tumor quality decreased significantly,the nucleus shrank and the integrity was poor,and the tumor inhibition rate was as high as 80.9%.Then,the results of lung tissue weighing,lung metastasis count,Bouin’s and H&E staining showed that the weight of lung tissue after CPAD treatment was the same as that of normal mice,and there were almost no lung metastatic nodules,the metastasis inhibition rate was as high as 88.4%.Then,the anti-metastasis mechanism of CPAD in vivo was discussed by ICP-MS,Western Blot,immunohistochemistry and immunofluorescence experiments.It was found that the contents of Cu²⁺,ROS,NO and E-cadherin in tumor tissue increased significantly,while the indexes of E2,Capn4,MMP-2,p-selectin,TGF-βand N-cadherin decreased significantly,which confirmed the anti-metastasis mechanism of CPAD by triggering estrogen down-regulation and platelet blocking.Finally,we made a preliminary evaluation of the biosafety of the preparation,and the results showed that CPAD had good biosafety.In summary,the copper-based nano-active system loaded with cisplatin prodrug was synthesized to achieve effective anti-tumor and anti-metastasis therapy through chemotherapy,estrogen regulation and platelet activation blocking.Good biosafety shows that it has a certain clinical potential,which provides a new idea for the treatment of triple negative breast cancer.