Preparation Of Macrophage Membrane Coated Drug-loaded PLGA Nanoparticles And Experimental Study On Targeting Of CT26 Cells

Part ? Preparation and Characterization of Macrophage Membrane-coated PLGA NanoparticlesPurpose: Macrophage membrane was extracted and purified,HCPT-PLGA nanoparticles were prepared and co-extruded with macrophage membrane to prepare nanoparticles(HCPT-MCNP),and characterized.Methods: Hypoosmotic lysis and mechanical destruction with liposome extruder were used to destroy cell structure.Cell membranes with complete sequence of membrane proteins were separated by differential centrifugation,and purified cell membranes without nucleus were confirmed by fluorescence staining.HCPT-PLGA nanoparticles were prepared by emulsification method.HCPT-MCNP was prepared by co-extruding purified macrophage membrane and prepared HCPT-PLGA with liposome extruder.The morphology,particle size,distribution,potential,stability,drug encapsulation efficiency,drug loading rate and drug release rate of PLGA nanoparticles were characterized in vitro.The retention of macrophage membrane proteins on MCNP nanoparticles was detected by Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Results:(1)Cell lysis products,including cell membrane,nucleus and some other cell substructures,were obtained by mechanical destruction of cell structure with hypoosmotic buffer and liposome extruder.Blue nuclear fluorescence and orange-red cell membrane fluorescence were observed in the extracts after centrifugation at a single time by differential centrifugation,and almost only red cell membrane and no blue cell nucleus were observed after repeated centrifugation.(2)HCPT-PLGA nanoparticles prepared by emulsification were with regular morphology and uniform particle size.The average particle size was 201.5 ± 2.7 nm,and the zeta-potential was-25.78 ± 3.78 m V.The morphology was round by TEM,and the encapsulation efficiency was 69.34 ± 3.31% and drug loading rate was 0.81 ± 0.02% by UV-spectrophotometry(n=3).(3)The average particle size of HCPT-PLGA was 208.2 ± 1.4 nm,and its zeta-potential was-12.33 ± 1.08 m V.The morphology was observed and identified by TEM,showing a circular shape with a film wrapped on the outer surface with a core-shell structure.The thickness of the shell was measured under the electron microscope at about 7 nm,which was in line with the difference between the particle size of PLGA and MCNP.UV-spectrophotometry was used to determine the encapsulation efficiency and drug loading rate was 57.80 ± 8.75% and 0.59 ± 0.10%(n=3).(4)Both PLGA and MCNP were with good stability in PBS buffer in vitro,the particle size of which maintained around 200 nm.(5)Fluorescence spectrophotometry was used to detect the drug release rate.HCPT-PLGA and HCPT-MCNP had similar release curves,and the drug release rate within 24 hours reached 58.91% and 57.19%,respectively.After that,the drug release rate almost stopped releasing further and was in a drug concentration plateau.(6)SDS-PAGE showed that MCNP had similar protein profile with the extracted macrophage membrane,that is,MCNP retained the membrane protein of macrophages.Conclusion: In this study,PLGA nanoparticles loaded with HCPT were successfully prepared and coated with macrophage membranes to prepare MCNP.MCNP nanoparticles with stable particle size were obtained.The drug loaded on PLGA nanoparticles could be slowly released in PBS,thus having the potential to treat tumors.Part ? Cell Targeting and Immune Escape VerificationPurpose:The targeting effect of PLGA and MCNP nanoparticles on CT 26 tumor cells and the ability of avoiding macrophage RAW 264.7 phagocytosis were verified.Methods: The prepared fluorescent labeled PLGA and MCNP were co-incubated with CT 26 cells,respectively.The phagocytosis rate of CT 26 cells was observed by fluorescent microscopy and quantitatively verified by flow cytometry.At the same time,they were co-incubated with RAW 264.7 cells,respectively.The phagocytosis rate of RAW 264.7 cells were observed by fluorescent microscopy,and quantitatively verified by flow cytometry.Results: The fluorescence overlap area of MCNP group and CT26 cells was significantly larger than that of PLGA group,and the fluorescence overlap area of MCNP group and RAW 264.7 cells was significantly smaller than that of PLGA group,under fluorescence microscope,.The relative uptake rates of PLGA and MCNP by CT 26 cells were 74.83 ± 1.14% and 95.80 ± 0.79%,respectively,with a significant statistical difference(p=0.0019).The relative uptake rates of PLGA and MCNP in RAW264.7 cells were 23.5±0.45% and 12.4±1.88%,respectively,with significant statistical difference(p=0.0156).Conclusion: Macrophage membrane coating can promote the uptake of PLGA NPs by CT 26 cells to achieve the function of biomimetic nanoparticles targeting cancer cells,and reduce the uptake of nanoparticles by RAW 264.7 cells thus achieving immune evasion.