Construction And Evaluation Of Biomimetic Nano-Drug Delivery System For Cancer Cell Membrane

Conventional nano-carriers are usually lack of specificity and are easy to be recognized by the immune system and removed as foreign bodies,resulting in low efficiency in cancer treatment.Clearance of the immune system,poor antigen presentation of tumor tissues and lack of specific targets are the biggest obstacles that limit the nano-drug delivery system.The new nano-drug delivery system should have the ability of long blood circulation,immune escape and specific tumor targeting.Cancer cells have the potential of unlimited replication and the ability of immune escape and homologous adhesion dependent on membrane proteins,based on the ability of immune escape and homologous adhesion of cancer cells,cancer cell membrane coating can well overcome the dilemma of immune clearance and non-specific binding of nano-drug delivery system.Therefore,in this study,paclitaxel(PTX)was used as a model drug to prepare biomimetic poly(lactic-co-glycolic acid)(PLGA)nanoparticles of H460-NCI human cell lung cancer cell membrane,in order to achieve immune escape and efficient tumor targeting.PLGA drug-loadednanoparticles(PLGANPs)were prepared by solvent volatilization.Cancer cell membrane(CCM)was extracted and purified by hypotonic lysis,mechanical fragmentation and differential centrifugation.Cancer cell membrane biomimetic nanoparticles(CCMNPs)were prepared by nano-membrane extrusion method.The particle size distribution,polydispersity index(PDI)and Zeta potential of PLGANPs and CCMNPs were measured by dynamic light scattering laser particle size analyzer(DLS),and their morphologies were observed by transmission electron microscope(TEM).Cancer cell lysate(CCL),CCM and CCMNPs were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The membrane proteins on CCMNPs were qualitatively analyzed by Western blotting(WB)to verify the retention of related functional membrane proteins on CCMNPs.Through the single factor investigation,taking the film coating rate as the evaluation index,the CCM extrusion times(A),CCM/PLGANPs ratio(B)and co-extrusion times(C)which have great influence on the CCMNPs coating rate were selected as the investigation factors,three levels were determined,and the orthogonal experimental design was carried out,and the optimum process conditions were determined as follows:CCM extrusion times 50 times,CCM and PLGANPs feed ratios 6:2,total extrusion times 30 times.The results showed that the average particle size of PLGANPs was(81.56 ± 1.84)nm,PDI was 0.076 ±0.0008,Zeta potential was(29.5± 1.53)mV,the average particle size of CCMNPs was(94.13±1.67)nm,PDI was 0.071 ±0.0005,Zeta potential was(29.5 ± 1.53)mV.The difference of particle size between the two groups was 12.57nm,which was consistent with the thickness of two-layer cancer cell membrane(10～16nm).TEM observed that both PLGANPs and CCMNPs are regular spherical structures with uniform size.CCMNPs has a "core-shell" structure,and there is a translucent film on the outside,which is about the thickness of 7nm,which coincides with the thickness of monolayer CCM,indicating that CCM has been successfully coated on PLGANPs.The results of SDS-PAGE electrophoresis and WB showed that there was no HistoneH3,as a nuclear marker protein on CCMNPs,but it still retained the related functional protein(CD47,N-Cadherin,Galectin-3,CD44,CD326)which played immune escape,homologous adhesion and homologous targeting.The existing forms of drugs in CCMNPs were studied by differential scanning calorimeter(DSC),X-ray powder diffractometer(XRD)and Fourier transform infrared spectroscopy(FTIR).Through methodological investigation,a feasible method for the determination of paclitaxel by HPLC was determined.The entrapment efficiency,drug loading,stability and in vitro release of CCMNPs were investigated.The results showed that PTX was successfully loaded in CCMNPs and encapsulated in CCMNPs in an amorphous state,the entrapment efficiency of CCMNPs was(84.42 ± 1.82)%,the drug loading was(14.16 ±0.31)%,the stability was good within seven days,the particle size and Zeta potential had no obvious change,and the cumulative release rate in 168 h was(78.85±1.29)%.In the in vitro cell experiment,coumarin-6 was encapsulated in PLGANPs and CCMPNs as a fluorescent probe.The CCMNPs of PLGANPs and LLC mouse lung cancer cells were nurtured with RAW264.7 mouse macrophages respectively,and the CCMNPs of PLGANPs and NCI-H460 human cell lung cancer cells were nurtured with NCI-H460 human cell lung cancer cells respectively.The cell uptake was observed by confocal microscope imaging and flow cytometry to verify the immune escape and homologous targeting of CCMNPs in vitro.The cytotoxicity was detected by CCK-8 method.The results showed that the fluorescence intensity of mononuclear macrophages of RAW264.7 mice in PLGANPs group was 7.3 times higher than that in CCMNPs group,indicating that the cancer cell membrane coating could effectively reduce the uptake of nanoparticles by macrophages by disguising CD47 proteins on nanoparticles as autologous cells.The fluorescence intensity of NCI-H460 human cell lung cancer cells in CCMNPs group was 9.6 times higher than that in PLGANPs group.The results showed that the cancer cell membrane coating could effectively increase the uptake of nanoparticles by cancer cells through the homologous targeting and adhesion of cancer cell membrane proteins,and the results of CCK-8 cytotoxicity showed that the IC50 values of paclitaxel injection,PLGANPs and CCMNPs on NCI-H460 human cell lung cancer cells were 38.33±0.60,17.37±0.64,3.02±0.47ng/mL,respectively.In the experiment of homologous targeting and anticancer efficacy in vivo,DiR was encapsulated in PLGANPs and CCMNPs as a fluorescent probe.After the drug was injected into the tail vein of tumor-bearing nude mice,the in vivo distribution of the drug was determined by in vivo imaging technique.The tumor-bearing nude mice were randomly divided into(1)normal saline group,(2)commercial paclitaxel injection group,(3)PLGANPs group,(4)CCMNPs group.After 21 days of tail vein injection,the tumor volume and tumor inhibition rate of each group were calculated.The results showed that the fluorescence intensity of liver,spleen,lung and kidney in PLGANPs group was higher than that in CCMNPs group,and the difference among liver,lung and kidney was significant.The fluorescence intensity of tumor tissue in CCMNPs group was 2.9 times higher than that in PLGANPs group,indicating that CCMNPs has stronger tumor targeting in vivo.After 21 days of administration,the tumor volume of the CCMNPs group changed significantly compared with other groups.The tumor inhibition rate of the CCMNPs group was 22.72%higher than that of the PLGANPs group and 42.44%higher than that of the commercial paclitaxel injection group.To sum up,CCMNPs was successfully prepared by nano-film extrusion.Through related experiments in vivo and in vitro,it was confirmed that CCMNPs could evade the recognition and clearance of immune system by disguising the CD47 protein on the membrane as autologous cells,and realize the active targeting of tumor through the homologous adhesion characteristics of N-Cadherin,Galectin-3,CD44,CD326 protein retained on the membrane.So that the aim of immune escape and tumor targeting are achieved,a good tumor treatment effect is realized,and acorresponding theoretical basis is provided for the future clinical application of the tumor.