Nanotherapeutic System Based On Cancer Cell Membrane With High Expression Of RGD And Its In Vitro Effects Targeting To Triple Negative Breast Cancer Through PDT And Chemotherapy

ObjectiveBreast cancer is one of the malignant tumors that seriously threaten the life and health of women,and its incidence has taken women’s first place as increasing every year.Triple negative breast cancer(TNBC)is a special type of breast cancer which accounting for about 15%among all types and featured by high local recurrence,tendency to metastasis,and insufficient of specific therapeutic targets.Currently,there has been no effective treatment method.Photodynamic therapy(PDT)combined with chemotherapy exibits obvious synergistic effects on TNBC,and therefore the investigations to explore appropriate strategies for combining PDT and chemotherapy have very important significance in clinic.In this study,poly(β-amino ester contained disulfide bond)(ss PBAE)and poly(lactic acid-co-glycolic acid)(PLGA)are used as the carrier materials to prepare nanoparticles for co-loading chlorin e6(Ce6),one of the second generation photosensitizers,and doxorubicin(DOX),a first-line chemotherapeutic drug,thus obtain hydrophobic ss PBAE/PLGA/Ce6/DOX(PPCD)nanocores.The cell membrane with high expression of RGD named as RGD-MEM is extracted and then coated on the surfaces of PPCD nanocores to construct a biomimetic nanotherapeutic system,RGD-MEM@ss PBAE/PLGA/Ce6/DOX(RMPPCD)nanoparticles.By using the homologous and active targeting property of RMPPCD nanoparticles,the synergistic effects of PDT combined with chemotherapy can be achieved in TNBC treatment.ContentsThis research was mainly divided into two parts as follow.In the first part,RMPPCD nanoparticles were prepared and characterized.First,p CDH-p-RGD-Ecadherin recombinant plasmid(p CDH-p-RGD-Ecad)was constructed and used to transfect MDA-MB-231 cells to obtain a high RGD-expressed cell line(RGD-231).Second,the RGD-Ecad expression by RGD-231 cells was evaluated at the m RNA and protein levels,and then RGD-MEM was extracted from these cells.Third,RMPPCD nanoparticles were prepared and characterized,and their drug release behaviors were further evaluated in vitro.In the second part,the targeting capability and therapeutic effects of RMPPCD nanoparticles were investigated in MDA-MB-231 cells.First,the cellular uptake and intracellular localization of RMPPCD nanoparticles were detected,and their PDT efficiency was further evaluated.Second,the inducting effects of RMPPCD nanoparticles on the cell apoptosis and the cell cycle arrest were detected in MDA-MB-231 cells,and furthermore their cytotoxicity was also measured.Third,the tumor-permeation ability of RMPPCD nanoparticles was assessed in 3D tumor sphere of MDA-MB-231 cells.Methods1.Preparation and characterization of RMPPCD nanoparticle.First,p CDH-p-RGD-Ecad was constructed and then used to transfect MDA-MB-231 cells to obtain a high RGD-expressed cell line(RGD-231)by using the lentiviral vector.The expression of RGD-Ecad by RGD-231 cells was verified at the m RNA level using the q RT-PCR and gel electrophoresis and at the protein level using Western blotting.RGD-MEM was extracted from RGD-231 cells and the total protein components were further analyzed by sliver stainning.According to a previous method we reported,ss PBAE was synthesized via two step chemical reactions and characterized by the ~1H NMR spectrum.ss PBAE and PLGA were used as the carrier materials to prepare nanoparticles for co-loading Ce6 and DOX,named as PPCD nanocores,by an ultrasonic emulsifying-solvent evaporation method.RGD-MEM was extracted and coated on the surfaces of PPCD nanocores to prepare RMPPCD nanoparticles using a co-extrusion method.Furthermore,MPPCD nanoparticles were also prepared as the control by the same method as above by using the cell membrane extracted from MDA-MB-231 cells.The sizes,size distributions,surface charge properties,and in vitro stabilities of PPCD nanocores and RMPPCD nanoparticles were evaluated using the dynamic laser scattering method and Zeta potential analyzer,moreover their morphology were observed by a transmission electron microscope(TEM).The existence of RGD-Ecad in RMPPCD nanoparticles was verified by Western blotting.The optical properties of RMPPCD nanoparticles were investigated by the ultraviolet-visible and fluorescence spectroscopy.The loading and encapsulation efficiencies of DOX and Ce6 in RMPPCD nanoparticles were detected using the high performance liquid chromotography(HPLC).The in vitro release behaviors of DOX from RMPPCD nanoparticles were investigated using the dynamic dialysis method.2.Evaluation of in vitro effects of RMPPCD nanoparticles.The cellular internalization and intracellular location of RMPPCD nanoparticles in MDA-MB-231cells and human umbilical vein endothelial cells(HUVECs)were observed using the confocal microscopy.Furthermore,the uptakes of RMPPCD nanoparticles in these cells were quantitatively analyzed using the flow cytometry.The PDT efficiency of RMPPCD nanoparticles was assessed in MDA-MB-231 cells by detecting the intracellular generation of reactive oxygen species(ROS)using the DCFH-DA fluorescence probe method.The inducting effect of RMPPCD nanoparticles on the cell apoptosis and their function mechanisms were investigated in MDA-MB-231cells through detecting the intracellular protein expressions of caspase 9,caspase 3and Bcl-2 using Western blotting.The arresting effect of RMPPCD nanoparticles on the cell cycle was assessed in MDA-MB-231 cells by the PI staining method.The cytotoxicity of RMPPCD nanoparticles to MDA-MB-231 cells was evaluated using the living/dead cell staining method and the MTT assay.The 3D tumor sphere was constructed from MDA-MB-231 cells,and the tumor-permeation ability of RMPPCD nanoparticles was further evaluated in vitro.In all above experiments,the in vitro effects of free Ce6,PPCD nanocores and MPPCD nanoparticles were also investigated at the same time for the comparison.And therefore,the homologous and active effects of RMPPCD nanoparticles targeting to MDA-MB-231 cells and the synergistic killing effects in vitro could be evaluated through comprehensively analyzing these experimental results.Results1.The RGD-231 cell line with high expression of RGD on cell membrane was successfully constructed.The results of q RT-PCR,gel electrophoresis,Western blotting and sliver staining verified the high expression of RGD on RGD-231 cells at the m RNA and protein levels.ss PBAE was successfully synthesized and the characteristic proton peaks in the ~1H NMR spectrum confirmed its chemical structure.PPCD nanocores were prepared and RGD-MEM extracted from RGD-231 cells was then coated on their surfaces to obtain RMPPCD nanoparticles.The size and zeta potential of RMPPCD nanoparticles were 167.8 nm and?24.9 m V,respectively.RMPPCD nanoparticles showed a good stability in vitro and a distinct membrane envelop on their surface under the observation of TEM.The results of Western blotting confirmed the existence of RGD-Ecad in the outer layer of RMPPCD nanoparticles,demonstrating that the biological activity of RGD-MEM could be retained during the preparation period.The ultraviolet-visible absorbance and fluorescence emission spectra both verified the co-loading of Ce6 and DOX and the retention of their optical properties in RMPPCD nanoparticles.The loading efficiency and encapsulation efficiency of Ce6 detected by the HPLC were 3.9%and 78%,and the loading efficiency and encapsulation efficiency of DOX is 0.403%and 80.6%,respectively.The results of dynamic dialysis method showed that the release of DOX from RMPPCD nanoparticles displayed p H and redox dual-responsive behaviours in vitro.2.The cell experimental results of RMPPCD nanoparticles compared with free Ce6,PPCD nanocores and MPPCD nanoparticles were as follows.The confocal images revealed that RMPPCD nanoparticles were mainly localized in the cytoplasm of MDA-MB-231 cells and HUVECs.The results of flow cytometry demonstrated that RMPPCD nanoparticles had the highest cellular uptake in these cells.RMPPCD nanoparticles triggered the generation of the largest amount of ROS in MDA-MB-231cells,indicating that they efficiently carried Ce6 into the cells and thus further had the strongest PDT efficiency under laser irradiation.The results of Western blotting showed that RMPPCD nanoparticles induced the apoptosis of MDA-MB-231 cells through the activation of mitochondrial apoptosis pathway.The results of PI staining revealed that RMPPCD nanoparticles arrested the cell cycle of MDA-MB-231 cells at G2/M stage.The results of living/dead cell staining and MTT assay both confirmed the strongest cytotoxicity of RMPPCD nanoparticles in MDA-MB-231 cells.RMPPCD nanoparticles displayed the strongest penetrating ability in the 3D tumor sphere.All of the above experimental results confirmed that RMPPCD nanoparticles had the homologous and active effects targeting to MDA-MB-231 cells,the intracellular drug release capability,and synergistic killing effects on MDA-MB-231cells by combining PDT and chemotherapy.ConclusionsIn this study,we constructed a biomimetic nano therapeutic system containing photosensitizer Ce6 and chemotherapy drug DOX,which was named as RMPPCD nanoparticles.In vitro characterizations showed that RMPPCD nanoparticles had a typical“core-shell”structure,good stability,and significant p H and redox dual-responsive drug release behaviour.The results of cell experiments showed that RMPPCD nanoparticles had excellent TNBC-targeting property,efficient intracellular drug release ability,and significant synergistic effects of PDT and chemotherapy against TNBC.Altogether,our study provides a new carrier for TNBC-targeted drug delivery and a novel strategy for TNBC combination treatment in clinic.