Multifunctional Nanosystems Based On Marine Polysaccharide And Biomimetic Modification With Cancer Cell Membrane For Combination Treatment Of Triple Negative Breast Cancer

Background and Objective:Triple negative breast cancer(TNBC)is a specific subtype of breast cancer and lack of estrogen receptor and progesterone receptor and human epidermal growth factor receptor 2.It has prominent clinical characteristics such as strong invasion,high metastasis and easy recurrence,resulting in a greatly shortened survival of patients.Single chemotherapy,immunotherapy and the emerging photodynamic therapy(PDT)have their own limitations,leading to the general therapeutic effect.Consideration of TNBC molecular heterogeneity,combination therapy of multiple methods is considered to be the most promising strategy.Nanocarrier technology provides the possibility to exert synergistic antitumor effects of combination therapy better.They can not only co-load different drugs,but also enhance their tumor enrichment through passive and active targeting effects.In this investigation,two nanosystems with multiple functions will be designed by taking advantages of good biological properties and high biosafety of natural polysaccharides and cell membranes,and to realize tumor-targeted delivery of drugs with different mechanisms as well as acquired synergistic effects against TNBC.Methods:Part Ⅰ: Multifunctional nanosystem based on marine polysaccharide for TNBC therapy via combining chemotherapy and COX-2 inhibitor.A marine sulfated polysaccharide,propylene glycol alginate sodium sulfate(PSS)that has multiple pharmacological activities was modified with benzaldehyde and then chemically linked to a chemotherapeutic drug doxorubincin(DOX)through a p H-cleavable benzoic imine bond,thus obtaining a polysaccharide prodrug PSS-DOX.And its structure was confirmed.Through one-step precipitation processing,PSS-DOX with amphiphilic property and celecoxib(CXB),a selective cyclooxygenase-2(COX-2)inhibitor with strong hydrophobic property,self-assembled to form PSS@DC nanoparticles(NPs).The morphology,size and surface potential of the NPs were observed by transmission electron microscopy(TEM)and size-Zeta potential analyzer.Differential thermal scanning(DSC)and X-ray powder diffraction(XRD)were applied to analyze drug existence forms in NPs.Loading contents and encapsulation efficiencies of drugs were determined by high performance liquid chromatography(HPLC)method.In vitro drug release behavior of NPs was assessed by using a dynamic dialysis method.The cell uptake ability and intracellular localization of PSS@DC NPs in 4T1 cells were observed by a confocal microscopy and flow cytometry.MTT assay was used to compare the cytotoxicity of NPs with free drugs.Cell apoptosis-inducing and cycle-arresting were evaluated by flow cytometry.In addition,migration,invasion and adhesion assays were applied to investigate the inhibitory effect of NPs on the movement of 4T1 cells.An orthotopic TNBC mouse model was constructed using luciferase labeled 4T1(4T1-Luc)cells.The tissue distribution and tumor accumulation of PSS@DC NPs were investigated by IVIS in vivo imaging system.Synergistic antitumor efficacy of NPs was investigated in 4T1-Luc tumor-bearing mice through detecting tumor size and body weight changes during the whole treatment process and observing lung metastasis after treatment.Furthermore,organs and tumors were treated with hematoxylin and eosin(H&E)staining and immunohistochemical staining,to explore antitumor mechanisms of PSS@DC NPs.Part Ⅱ: Multifunctional nanosystem based on biomimetic modification with homologous cancer cell membrane for TNBC treatment via combinaing photodynamic therapy and melittin.Poly(β-amino ester)(ssPBAE)was synthesized by a two-step method and its structure was characterized.ssPBAE and poly(lactic-co-glycolic acid)(PLGA)were used as carrier to prepare NPs with loading of photosensitizer temoporfin(m THPC)(TPP NPs)by emulsification method.Homologous cancer cell membrane was extracted from 4T1 tumor-bearing mice and then fused with melittin(Mel)in the presence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DSPE)to obtain Mel-DSPE-Mem(MDM).Hemolysis experiment was used to evaluate the shielding effect of MDM on the hemolytic toxicity of Mel.Next,MDM was coated on the surfaces of TPP NPs under sonication to prepare MDM@TPP NPs.The morphology,particle size and Zeta potential were investigated.Total protein staining was applied to evaluate the retention of cell membrane-associated proteins on MDM@TPP NPs.Ultraviolet spectrophotometry was used to determine the loading content of m THPC in NPs.A specific fluorescence probe(SOSG)for singlet oxygen,was applied to assess photodynamic performance of the NPs after 652 nm laser irradiation.The uptake and distribution of MDM@TPP NPs in 4T1 cells were investigated by laser confocal microscopy and flow cytometry,and the permeability of NPs was evaluated by tumor spheres.The intracellular ROS production level of the NPs combined with652 nm laser irradiation was detected by reactive oxygen species probe DCFH-DA.The synergistic killing effect of PDT/Mel on 4T1 cells was investigated by MTT assay and Live/Dead cell staining.Flow cytometry and fluorescence techniques were used to investigate the synergistic induction effect on apoptosis and its mechanism.Immunofluorescence technique was applied to assess their immunogenic effect in4T1 cells with combination treatment.The activation of immune cells processed with tumor antigens was evaluated using flow cytometry.An IVIS imaging system was used to investigate the tissue distribution and tumor accumulation of MDM@TPP in4T1-Luc tumor-bearing mice,to evaluate the homologous tumor-targeting effect.The in vivo photodynamic efficacy of NPs was assessed of the ROS generation by using SOSG.A bilateral back tumor model of TNBC was established to investigate synergistic anitumor effects of MDM@TPP NP-mediated combination treatment on the distal tumor and lung metastases of the original tumor as well as the immune-activated effect in vivo.The synergistic anti-tumor effect of MDM@TPP NPs combined with PDT/Mel was evaluated by histomopathological and immunohistochemical analysis of the main organs and tumors.Results:Part Ⅰ: Spectral data demonstrated that PSS-DOX was synthesized successfully.PSS@DC NPs had “core-shell” nanostructure.Their average size was 126.5±5.2 nm and their Zeta potential was –34.9 m V.DSC and XRD curves indicated that drugs existed in NPs as amorphous states.Encapsulation efficiencies of CXB and DOX in NPs were 86.9% and 84.8%,and their loading contents were 66% and 6.2%correspondingly.DOX exhibited obvious p H-responsive release behavior.PSS@DC NPs could be internalized efficiently into 4T1 cells.MTT assay results demonstrated that CXB significantly enhanced the sensitivity of 4T1 cells to DOX and NPs had a higher cytotoxicity than the DOX/CXB mixture.Flow cytometry data exhibited that PSS@DC NPs notably induced cell apoptosis and arrested cell cycle at G2/M phase.Experimental results of migration,invasion and adhesion assays all indicated that NPs greatly inhibited the in vitro movement of 4T1 cells.PSS@DC NPs had an excellent tumor-targeting ability and promoted the tumor accumulation of DOX in4T1 tumor-bearing mice.Experimental results of in vivo treatment showed that NPs remarkably suppressed the growth and lung metastasis of 4T1 tumors.H&E and immunohistochemistry staining showed that PSS@DC NPs exerted synergistic killing effect on tumor cells,inhibitory effect on tumor angiogenesis platelet activation,whereas had no obvious damage to normal organs.Part Ⅱ: Spectral data demonstrated that ssPBAE was synthesized successfully.TPP NPs had a regular spherical shape,an average size of 112.9±8.4 nm,and a Zeta potential of 17.2 m V.MDM@TPP NPs were also spherically shaped and displayed obvious outer membrane layer.Their size increased to 142.8±6.3 nm and Zeta potential changed to –20.4 m V.Encapsulation efficiency and loading content of m THPC in NPs were 71.69% and 5.97%,respectively.The NPs retained complete protein components on 4T1 cell membrane and greatly reduced the hemolytic toxicity of Mel.Under the 625 nm-laser irradiation,they had strong PDT performance.MDM@TPP NPs showed specific cellular internalization in 4T1 cells and strong penentration ability through tumor sphere of 4T1 cells,indicating that homologous cell membrane mediated tumor-targeted delivery via the “homing” effect.After laser irradiation,NPs triggered the generation of large amount of ROS in 4T1 cells,and also exhibited synergistic cytotoxicity and apoptosis-inducing activity.Through PDT and Mel combination treatment mediated by MDM@TPP NPs,the mitochondrial apoptosis pathway and anti-tumor immunity were activated effectively,which can promote the immunogenic death of tumor cells and stimulate the maturation and activation of immune cells by tumor antigen.MDM@TPP showed good tumor targeting in 4T1-Luc tumor-bearing mice and significantly increased the ROS production level in tumor tissues under laser irradiation.Experimental results of in vivo treatment demonstrated that MDM@TPP NPs could efficiently combine PDT and Mel to exert synergistic antitumor effects,mainly in killing primary tumors to inhibit lung metastasis,inducing the growth of distant tumors to induce immunogenic death of tumor cells and activating anti-tumor immunity.Histopathological examination results showed that the combination therapy did not cause major organ damage,indicating its high biosecurity in vivo.Conslusion:In this study,two multiple functional nanosystems were prepared to efficiently co-load and targtedly deliver antitumor drugs with different mechanisms,and achieved synergistic therapeutic effects on TNBC both in vitro and in vivo.PSS@DC NPs based on a marine sulfated polysaccharide can significantly suppress the growth and metastasis of TNBC,their antitumor mechanisms are involved in chemosensitization,antiangiogenesis and platelet activation.MDM@TPP NPs based on homologous cancer cell membrane can effectively shield the hemolytic toxicity of Mel;targetedly deliver m THPC and Mel to promote their tumor-accumulations.Upon laser irradiation,NPs have strong photodynamic efficiency and can exert synergistic effects on killing tumor cells,suppressing tumor metastasis and activating antitumor immunity in TNBC.In conclusion,this study provides the novel carrier platforms for multimode treatment and the combination strategy for TNBC treatment.