Construction And Evaluation Of MCF-7 Cell Membrane Camouflaged PLGA Nanoparticle Loaded With Curcumin And Chlorin E6 For Tumor Targeting Drug Delivery

Objective: The objective of this project is to develop a biomimetic nanoparticle with low toxicity and high anti-tumor efficiency which is based on the combination of photodynamic therapy(PDT)and chemotherapy.Curcumin and the chlorin e6 were encapsulated in PLGA nanoparticle to inherit the advantages of PDT and the safety of natural medicine for improving anti-tumor efficacy.By coating the homologous tumor cell membranes on the outer surface of the nanoparticles,the nanoparticles can achieve immune evasion by means of the EPR effect.The specific adhesion of homologous tumor cells endows biomimetic nanoparticles with active tumor-targeting ability.Method: PLGA nanoparticles loaded with curcumin and chlorin were prepared by nanoprecipitation method.The MCF-7 tumor cell membranes was coated onto the nanoparticles by co-extrusion method to obtain biomimetic nanoparticles MNP.Physical characterization,western blotting,in vitro cellular experiments,pharmacokinetic study were investigated to confirm the tumor targeting mechanism,the anti-tumor effect and long-blood-circulation characteristics of MNP.In vitro release behavior,safety,stability and biocompatibility were evaluated.Result: The optimal formulation was obtained by orthogonal test: drug to PLGA ratio of 0.06,water to acetone ratio of 5,and RH40 to PLGA ratio of 2.The encapsulation efficiency of curcumin and chlorin e6 in Cur/Ce6-NP prepared according to the optimal prescription was 76.54 % and 73.97 %,respectively,and the drug loading was 5.10 %.The particle size and zeta potential of Cur/Ce6-NP and Cur/Ce6-MNP was 193 nm、-34 m V,and 202 nm、-25 m V,respectively.Core-shell structure of biomimetic nanoparticle can be clearly observed by the transmission electron microscopy.Western blot result confirmed the presence of the specific cell adhesion molecules on MNP,demonstrating that the protein markers Ep CAM,N-cadherin and Galectin-3 were successfully transformed from MCF-7 tumor cells to MNP surface by membrane coating,whih is a support for homologous cell-specific targeting property.Flow cytometry and laser confocal microscopy were used to demonstrate the specific bingding ability of MNP,MNP tend to be uptaked by homologous MCF-7 cells.The reactive oxygen species(ROS)generation of Ce6-NP and Ce6-MNP was verified by laser confocal microscopy to provid support for subsequent photodynamic therapy,and MCF-7 cell treated with MNP produced more ROS.Cytotoxicity results demonstrated that the cytotoxicity of Cur/Ce6-NP is more strongly than Cur-NP and Ce6-NP,Cur/Ce6-NP showed strongest cytotoxicity towards MCF-7 cells while the ratio of Cur to Ce6 was ten to one(w/w).Besides,Cur/Ce6-MNP shows higher inhibition rate towards MCF-7 cells than Cur/Ce6-NP.Cur/Ce6-NP and Cur/Ce6-MNP mainly depend on Ce6 to induce apoptosis,and the apoptosis rate of Cur/Ce6-MNP group is higher.The results of the scratch test indicated that both Cur/Ce6-NP and Cur/Ce6-MNP have the ability to inhibit the migration of MCF-7 tumor cells,and Cur/Ce6-MNP showed higher inhibition ability.Through the stability evaluation,it was verified that NP and MNP did not coagulate in the plasma.There was no significant change in the size of NP and MNP in water,PBS and plasma within six days.There was no apparent hemolysis when the PLGA concentration of Cur/Ce6-NP and Cur/Ce6-MNP below 400 μg/m L,suggesting the safety of NP and MNP is good.There was no obvious cytotoxicity while the NP and MNP concentrations ranged from 10 μg/m L to 125 μg/m L,representing the favorable biocompatibility.The in vitro release rate of Cur/Ce6-MNP was lower than that of Cur/Ce6-NP,indicating the tumor cell membrane coating can prevent the rapid leakage of the loaded drug during blood circulation,and its sustained release performance provides a prerequisite for targeting drug delivery.In the PK study,Cur/Ce6-MNP showed decreased elimination rate,prolonged blood circulation time and improved bioavailability,compared with Cur/Ce6-NP and free drugs.Conclusion: In this study,MCF-7 breast cancer cell membrane camouflaged biomimetic PLGA nanoparticle loaded with curcumin and chlorin e6 was successfully prepared.This biomimetic nano drug delivery system is a promising tumor treatment modality which showed expected anti-tumor effect on MCF-7 breast cancer cells.