Endoplasmic Reticulum Membrane-coated Liposomes As Targeted Drug Delivery System In The Treatment Of Breast Cancer:A Preliminary Study

Cell membrane-coated drug delivery systems（CMCDDs）assembles nanoparticles with cell membrane materials to completely retain the original protein expression and structural functions of the cell membrane.Since the core drug delivery system coated the cell membrane materials to make CMCDDs with good biocompatibility,and targeting capability.Therefore,the cell membrane-coated drug delivery systems have attracted significant research attention.In recent years,the selection of cell membrane materials has mainly focused on the outer membrane of eukaryotic cells in the majority of studies,such as erythrocyte membrane,macrophage membrane,tumor cell membrane,etc.However,the cell membranes can be divided outer membrane system and endomembrane system,both of which are composed of the same components.The endomembrane systems have higher content of functional proteins,which can provide the drug delivery system with plentiful biological functions.The cell endomembrans has not been fully recognized and used as cell membrane materials of CMCDDSs.Hence,this study utilized that the charge asymmetry of cell membranes and core drug delivery systems,developed that the endoplasmic reticulum membrane coated DSPE-mPEG₂₀₀₀ modified liposomes（ER@PLip）and the cancer cell membrane coated DSPE-mPEG₂₀₀₀ modified liposomes（CM@PLip）.To compare the in vivo and in vitro functions of two cell membrane coated drug delivery systems and the therapeutic efficacy of breast cancer treatment.Compared to the CM@PLip,ER@PLip as a novel cell intracellular membrane coated drug delivery system exhibits the following characteristics:（1）the endoplasmic reticulum is an largest cell intracellular endomembrane system in cell membrane systems,which has high extraction yields,effectively reducing preparation expenses.（2）ER membrane materials with abundant functional protein expression on the surface,such as CD47 protein and a variety of adhesion molecule protein molecules that provide ER@PLip with extended circulation time in vivo and active tumor targeting capabilities.（3）combination with PD-1/PD-L1antagonist peptides has shown excellent breast cancer treatment efficacy.（4）expression of calreticulin（CRT）on the surface of ER membranes can promote positive regulation of the tumor immune microenvironment and further improve therapeutic efficacy.1.Preparation and characterization of ER@PLip4T1 cells were fragmented using hypotonic solution and extracted by differential centrifugation to obtain ER membranes and CM membranes,respectively.The extraction yields of ER membranes and CM membranes were investigated through measuring the phospholipid content of the main components of cell membranes.The Elisa phospholipid quantification experiment showed that the extraction yield of ER membranes is 2.48 times as higher as CM membranes.Western blotting analysis was conducted to confirm the presence of CD47 and specific homologous-binding adhesion molecules on ER membranes and CM membranes.The results demonstrated that the ER membranes and CM membranes both possessed these cellular adhesion molecules（e.g.,EpCAM,N-cadherin）and CD47,there was no significant difference.Elisa analysis was conducted to confirm the presence of CRT on ER membranes and CM membranes.The results demonstrated that CRT expression was significantly higher in ER membranes than CM membranes.Therefore,ER@PLip are expected to positive regulation of tumor immune microenvironment through CRT protein.ER@PLip and were obtained by liposome extruder preparation.The hydrodynamic sizes of the ER@PLip was 138 nm.The surface potentials of ER@PLip was similar to ER membrane materials,which preliminary evidence that the ER membrane materials were successfully coated on the surface of PLip.Membrane coating around the polymeric core was visualized using transmission electron microscopy（TEM）,and the ER@PLip was spherical in shape and exhibited a core-shell structure.The laser confocal experiment confirmed that the ER membrane has been successfully modified to the PLip surface.Protein gel electrophoresis exhibited the modulation of the protein profile,indicating that ER@PLip and ER membrane materials,CM@PLip and CM membrane materials both possessed a similar protein profile,respectively.2.Evaluation of in vitro functions of ER@PLipThrough fluorescence microscopy observation and flow cytometry assay to analyze the ability of RAW 264.7 cells to phagocytose ER@PLip,and showed that the retention of CD47 on ER membrane materials ensured effective macrophage escape of ER@PLip.The 4T1 cells uptake study showed that ER@PLip and CM@PLip are expected to obtain homologoustargeting ability,because ER membrane and CM membrane expressing surface adhesion molecules（EpCAM and N-cadherin）.ER@PLip effectively avoids lysosomal enzymatic digestion in Lysosomal fluorescence co-localization assay.VP16 was chosen,as the model drug.We successfully prepared ER@PLip/VP16,which showed good stability after being left4°C for 72 h.In vitro drug release results showed that ER@PLip/VP16 has an obvious trend of sustained drug release compared to PLip/VP16.In vitro anti-tumor experiments showed that ER@PLip/VP16 has significant concentration-dependent cytotoxicity to 4T1 cells and can effectively inhibit 4T1 cell proliferation.The in vivo safety of the formulation was evaluated by haemolysis,the ER@PLip/VP16 was found to be effective in reducing the haemolytic effect of VP16.3.Evaluation of long circulation and active targeting function of ER@PLipIn pharmacokinetic experiments,the ER@PLip/VP16 exhibited significantly prolonged in vivo circulation compared with that of PLip/VP16,because the CD47 on the ER membrane to reduced immunogenicity of PLip.In 4T1 tumor-bearing mice model,ER@PLip exhibited specific accumulation in 4T1 tumor based on live imaging,and CLSM images of tumor site fluorescence sections indicated that ER@PLip of the tumor site fluorescence intensity was significantly higher than that of PLip.They demonstrated that ER@PLip has good active tumor targeting ability.Quantitative analysis of tumor targeting by tissue distribution experiments,which showed that ER@PLip not only achieved active enrichment of tumor sites through homologous adhesion proteins,but also effectively reduced VP16 content in liver,kidney,spleen and other metabolic tissues,which can further evidence of the active targeting ability and long circulation ability in vivo of ER@PLip.4.Anticancer effect evaluation of ER@PLip with ^DPPA-1 peptideThe in vivo pharmacodynamic evaluation treatment groups were divided into single treatment groups and combination treatment groups.Based on the analysis of tumor growth curve results,it found that the tumor inhibition rate of ER@PLip/VP16group was 73.38%,and ER@PLip/VP16+^DPPA-1 group has best treatment effect in the combination treatment groups.The reason for the difference in treatment effect may be that ER@PLip/VP16 promotes the proliferation and infiltration of T cells through ER membrane material CRT protein expression.The ^DPPA-1peptide can inhibit the PD-1/PD-L1 interaction for cancer immunotherapy.This study combines chemotherapy with immunotherapy to treat breast cancer,which can maximise the effectiveness of breast cancer treatment.By immunofluorescence staining,immune cells CD45,CD8 cells were further observed in treated tumors.The H&E stained images of major organs and serum biochemistry assay showed that ER@PLip/VP16presents high biosafety.We have conducted a preliminary study on the mechanism of its modulation of the tumor immune microenvironment.By flow cytometry analysis,found that improved content of dendritic cells,CD4⁺T cells,and CD8⁺T cells were further observed in ER@PLip treated tumor.The immunogenic cell death of cancer cells induced by ER@PLip surface CRT protein could promote antigen releasing from tumors,taken up and presented by dendritic cells（DCs）,and the subsequent activation of cytotoxic T lymphocytes（CTLs）.In summary,based on the endoplasmic reticulum’s expression of abundant functional proteins and carrying powerful biological functions,we have developed an endoplasmic reticulum membrane coated liposomes as targeted drug delivery system in the treatment of breast cancer.This study found that no significant difference between ER@PLip and CM@PLip in extend circulation time in vivo and tumor active targeting capability.However,endoplasmic reticulum-coated liposomes have advantages in terms of preparation,with higher extraction yields for ER membrane materials.Meanwhile,expression of CRT on the surface of ER membranes can promote positive regulation of the tumor immune microenvironment.The combination with PD-1/PD-L1 antagonist peptides to realize the combinatorial antitumor effect.This study provides a reference for the study of novel cell membrane-coated drug delivery systems in the treatment of cancer,and offers new inspiration for the exploration of novel therapeutic approaches for breast cancer.