Leukocyte/platelet Hybrid Membrane Based Biomimetic Nanocarrier For Targeted Treatment Of Triple-negative Breast Cancer By Combining PTT/PDT And Chemotherapy

ObjectiveTriple negative breast cancer（TNBC）is a subtype of breast cancer,which is more prone to recurrence,metastasis and poor prognosis as compared to other breast cancer subtypes,is one of the difficult problems in cancer clinical treatment.In this study,we constructed a multi-functional biomimetic nanotherapeutic system named as WPDDI,in which dendritic large pore mesoporous silica nanospheres（DLMSNs）were used for the co-loading of near-infrared photosensitizer IR780 and chemotherapeutic drug doxorubicin（DOX）,and the mixed membranes of white blood cells（WBC）and platelets（PLT）were used for surface-modification.We hope to realize the efficient combination of photothermal（PTT）/photodynamic therapy（PDT）and chemotherapy through tumor-targeted delivery of IR780 and DOX mediated by WPDDI,thus exert synergistic effects against TNBC.ContentsThis study was divided into the following three parts.In the first part,WPDDI were prepared and characterized.The main contents included the preparation of WPDDI and investigations of their surface protein expressions,optical properties and loading capabilities for IR780 and DOX.In the second part,we systematically evaluated the in vitro properties of WPDDI,which included its PTT/PDT efficiencies,drug release properties,immune evasion ability,cellular internalization,cytotoxicity and apoptosis induction in breast cancer cells.In the the third part,a mouse model of breast cancer was constructed by subcutaneous injection of 4T1 cells into the mice and then used for investigating the tumor-targeting ability,PTT/PDT efficiencies and synergistic efficacy of WPDDI,thus to evaluate the poteintal of this novel biomimetic nanotherapeutic system for TNBC treatment.Methods1.Preparation and characterization of WPDDI.DLMSNs were synthesized by a dual template method.Next,DLMSNs were separately mixed with DOX and IR780under stirring at room temperature,thus obtained to DOX and IR780 co-loaded DLMSNs,named as DDI.Cell membranes isolated separately from mouse platelets and macrophage Raw264.7 cells were physically mixed and then coated on the surfaces of DDI by the sonication method,and therefore aquired WPDDI.DLMSNs,DDI and WPDDI were characterized morphologically using a transmission electron microscope（TEM）,and their particle size,size distribution and surface charge property were detected by a Malvern particle size analyzer monitor.The in vitro stability of WPDDI was also evaluated through monitoring their size change during a storage period.The total proteins and functional protein components on WPDDI were analyzed respectively by the Coomassie brilliant blue staining and flow cytometry,and the platelet and Raw264.7 cell membranes were used as the comparisons.The optical properties of DDI and WPDDI were characterized by an Ultraviolet spectrophotometer,and furthermore the loading contents of DOX and IR780 in WPDDI were also measured.2.In vitro evaluations of WPDDI.The temperature changes of DDI and WPDDI dispersions under a 808 nm laser irradiation at a power density of 2 W/cm² were monitored by an infrared thermal imaging camera,thus to evaluate their photothermal efficiencies.And furthermore,Singlet Oxygen Sensor Green（SOSG）was used as a fluorescence probe to monitor the generations of singlet oxygen（¹O₂）in DDI and WPDDI dispersions upon laser irradiation,thus to evaluate their photodynamic efficiencies.The dynamic dialysis method was used to investigate in vitro drug release properties of DDI and WPDDI at different pH values with or without laser irradiation.The cellular uptakes of WPDDI in mouse breast cancer 4T1 cells and mouse macrophage Raw264.7 cells were assessed by the flow cytometry and confocal microscopy,thus to evaluate their tumor-targeting property and immune escape ability.To evaluate the synergistic effects of PTT/PDT and chemotherapy mediated by WPDDI on breast cancer in vitro,the MTT assay and dead/living cell staining were applied to detect the cytotoxicity of WPDDI in 4T1 cells,and also the flow cytometry was used analyzed the apoptosis of 4T1 cells after combination treatment.3.In vivo evaluations of WPDDI.A mouse model of breast cancer was constructed by subcutaneous injection of 4T1 cells into of the right groin of mice.The tumor-targeting property of WPDDI in 4T1 tumor-bearing mice after intravenous injection was assessed by an in vivo fluorescence imaging system.The temperature change at the tumor site under local laser irradiation was monitored using the infrared thermography,thus to evaluate the in vivo PTT efficiency of WPDDI.Meanwhile,the productions of ¹O₂ in tumor tissues were also detected by the SOSG fluorescence probe method,thus to evaluate the in vivo PDT effiency of WPDDI.After intravenous injection of WPDDI and followed by local laser irradiation,the tumor volumes and body weights of 4T1 tumor-bearing mice were monitored for at least 20d,thus to investigate the in vivo antitumor activity and biosafety of WPDDI.At the end of treatment,the major organs and tumors were isolated form the mice and then examined histopathologically by haematoxylin and eosin（H&E）staining.Cell apoptosis and angiogenesis in tumor tissues were further detected by Tunel analysis and immunofluorescence staining for CD31.Results1.DLMSNs were successfully prepared and used for co-loading IR780 and DOX efficiently,thus obtained DDI.The mixed cell membranes isolated from mouse platelets and macrophage Raw264.7 cells were effectively coated on the surfaces of DDI,thus aquired WPDDI.The particle sizes of DLMSNs,DDI and WPDDI were141.1,143.6 and 153.9 nm,and their Zeta potentials were–20.5,–5.03 and–26.9 mV,respectively.The TEM images confirmed the surface coating of the mixed cell membranes on DDI.Besides,WPDDI exhibited characteristics of total proteins and functional protein components of the membranes from platelets and Raw264.7 cells.During storage period,the size of WPDDI did not changed significantly,demonstrating their excellent stability in vitro.The loading contents of IR780 and DOX in WPDDI were 13.3%and 10%,respectively.2.Upon laser irradation,the temperature of WPDDI dispersion rapidly increased to 55.5°C,indicating their high photothermal effiency.Meanwhile,the laser irradiation notably improved the generation of ¹O₂ in WPDDI dispersion,demonstrating their high photodynamic efficiency.WPDDI exhibited obvious pH and photothermal dual-responsive drug release behavior.At 120 h,the release amounts of DOX from WPDDI were 57.2%and 33.1%at pH 6.5 and pH 7.4 respectively,and further enhanced to be 70.5%and 49.4%after laser irradiation.WPDDI were specifically uptaken by mouse breast cancer 4T1 cells,suggesting their good tumor-targeting ability.As compared to DDI,WPDDI showed evidently reduced cellular uptake in Raw264.7 cells,meaning that WPDDI could evade immune recognition and clearance to some extent.In 4T1 cells,WPDDI with laser irradiation possessed very high PTT/DT efficiencies and significantly inhibited the cell growth and induced the cell apoptosis as compared to both free DOX and free IR780 with laser irradiation.It indicated that WPDDI exerted synergistic effects against breast cancer by combining PTT/PDT and chemotherapy.3.A mouse model of breast cancer was successfully constructed by subcutaneous injection of 4T1 cells.After intravenous injection,WPDDI showed evidently prolonged in vivo circulation time and excellent tumor-targeting property,and furthermore exhibited very high PTT/PDT effiencies at tumor site after laser irradiation.Single treatment of WPDDI with laser irradiation notably inhibited tumor growth and angiogenesis,and induced tumor cell apoptopsis,suggesting their synergisitc effects against breast cancer.No obvious weight loss and pathological injuries in major organs were observed during the whole experiment,indicating that WPDDI could not give rise to a significant in vivo cytotoxicity.ConclusionsIn this study,we prepared a biomimetic nanotherapeutic system WPDDI using DLMSNs as a nanocarrier for efficient co-loading of IR780 and DOX and followed by surface-modification with the mixed membranes of platelets and macrophagocytes for tumor-targeted delivery.Both in vitro and in vivo,WPDDI exhibited significant tumor-targeting ability and synergistic effects against breast cancer by combining PDT/PTT and chemotherapy upon laser irradiation.Altogether,WPDDI showed a great potential for combination treatment of TNBC in clinic.