The Study Of Tumor Microenvironment PH-sensitive Liposome Coated With Erythrocyte Membrane

The ideal drug delivery system is able to achieve optimal biocompatible,extend blood circulation time,active targeting ability and environmental responsiveness.However,nanocarriers are easily engulfed by the reticuloendothelial system after entering the blood circulation,resulting in rapid clearance of nanoparticles.Recently,RBC membrane-coated drug delivery system have been developed with the abilities of low immunogenicity,high biodegradability and effectively evade clearance of reticuloendothelial system.However,the intact RBC membrane would limit the drug release and uptake by targeted cells when the drug carrier approach to the target tumor site.In this work,the liposome containing Cholesteryl hemisuccinate(CHEMS)and Solamargine(SM)coated with RBC membrane(RBC-S-C-LIP),which is extremely stable under physiological conditions to obtain the extended blood circulation.When approach to the acidic tumor site,the membrane-coated liposome removing the coat because of the partly released Solamargine.The discharged liposome was modified with rhamnose on the surface could be further taken up by tumor cells specifically,and via predominate lipid-mediated endocytosis to bypass endosomal trafficking.In addition,the nanoplatform could achieve synergistic antitumor effects by remodulation of tumor microenvironment.First,RBC-S-C-LIP was prepared.The results showed CHEMS could tune the in vitro SM release in different p H values and the optimal molar ratio of PC:CHEMS was10:6.After the RBC membrane decoration,the hydrodynamic diameter of RBC-S-C-LIP was larger than that of S-C-LIP.Theδpotential was close to the RBC membrane.Transmission electronic microscope(TEM)measurements showed that RBC-S-C-LIP was nanometer-sized spherical particles with a core-shell structure,and a few nanogolds specifically bound to the extracellular domain of red blood cells.When the RBC-membranes were labeled with Di D and liposomes were labeled with C6,the complete overlay was showed in confocal laser scanning microscope(CLSM)images.In SDS-PAGE electrophoresis assay,RBC-S-C-LIP retained the same protein with RBC membrane.RBC-S-C-LIP showed good stability after being left 4℃for 72 h.To further understand the p H-responsive properties,various characterizations of RBC-S-C-LIP were determined.The SM release of RBC-S-C-LIP was rapidly increased in p H 6.5.TEM images suggested it had a morphological change.As a result,RBC membrane was successful wrapped on the liposome.Moreover,the nanoplatform have p H-responsive properties.Next,In vitro behavior of RBC-S-C-LIP was studied.The results examined by fluorescence microscopic and flow cytometry showed that the uptake of RBC-S-C-LIP by macrophage cells was obviously reduced.To ensure the SM modification rate on liposome,fluorescence intensity was displayed.When molar ratio of SM:PC was 1:20,the uptake of PC-3 cells was dramatically increased.Flow cytometry results showed that S-C-LIP mainly rely on lipid raft mediated endocytosis entry cells.Subcellular colocalization study further indicated that S-C-LIP efficiently escape from the endosome followed by trafficking to the Golgi apparatus or endoplasmic reticulum.In addition,when the RBC-S-C-LIP incubated with cells in p H 6.5,it has the same cellular internalization behavior with S-C-LIP.Based on the above results,in vitro tumor penetration efficiency study was further conducted by tumor spheroids,indicating that the tumor penetration efficiency of liposomes containing SM was dramatically improved.PTX was chosen as the model drug to evaluate the formulation’s delivery capacity and therapeutic effects.RBC-S-C-LIP/PTX was around 105 nm with a polydispersity index of 0.054.The entrapment efficiency of prepared liposomes was higher than 90%by HPLC.In vitro release experiments showed that it had good sustained-release properties,and after being left at 4°C for 72 h,no significant change in particle size was observed.In pharmacokinetic experiment,RBC-S-C-LIP/PTX had extend blood circulation time.In cell proliferation experiment,SM and PTX could exert a synergistic effect in inhibiting PC-3 cell proliferation.In vivo imaging,tumor slices and in vivo distribution experiments all showed that RBC-S-C-LIP/PTX displayed remarkable accumulation in tumor.Antitumor efficacy of prepared liposomes was conducted on PC-3 tumor-bearing mice.RBC-S-C-LIP/PTX demonstrated higher tumor inhibition effect than other groups.H&E stained images of major organs and serum biochemistry assay showed that RBC-S-C-LIP/PTX presents high biosafety.Tumor immune microenvironment of SM was analyzed after different liposomes treatment.Immunofluorescence and Western blot assay both showed free SM could induce the calreticulin exposure.Flow cytometry demonstrated the increased number of DCs,CD4~+T cells,CD8~+T cells,NK cells was observed in RBC-S-C-LIP/PTX group.As a major immunosuppressive cell,myeloid-derived suppressor cells(MDSCs)were reduced.The increased ratio of M1/M2 also observed.In ELISA analysis,when treated with RBC-S-C-LIP/PTX,immunostimulatory cytokines IL-2,IL-12 and IFN-γwere increased than that of other groups.Inhibitory cytokines,IL-10 and TGF-β,inhibit the effect of antitumor immune system,were greatly reduced after RBC-S-C-LIP/PTX treatment compared to the control group.In summary,we have developed a membrane-coated nanoplatform based on the natural glycoalkaloid’s characteristic.This nanoplatform could prolong the circulation time;then remove the coat with high penetration efficiency when approaching to tumor site.In addition,this nanoplatform stimulate the anti-tumor immune response,regulate the immunosuppressive microenvironment,and has a good anti-tumor effect.The combination of biological membrane nanoparticle with natural products can contribute to study of the rational design of membrane-coating systems,has broad application prospects in clinical treatments.