Preparation Of Recombinant Protein Marked Erythrocyte Membrane-cloaked Microspheres And Evaluation Of Colon Cancer Targeting In Vivo And In Vitro

BackgroundMost of the conventional chemotherapeutic drugs have systemic toxicity and many side effects because of their non-specific in vivo distribution.With the advent of drug delivery system,it overcomes the non-specific targeting rather than traditional medicines.They can preferentially accumulate at sites of action and hold great promise to improve the therapeutic index of varieties of drugs.With the combination of synthetic nanoparticles and natural biomaterials have recently gained attention from medical field.The cell membrane-camouflaged nanoparticle platform has emerged as a novel delivery strategy with the potential to improve the therapeutic efficacy for the treatment of a variety of diseases.Objective1.Establising recombinant protein anti-EGFR-iRGD marked erythrocyte membrane-cloaked poly(lactic-coglycolic acid)(PLGA)microspheres,to evaluate their physicochemical characters and the stability in vitro.2.Using 3D multicellular spheroids(MCS)in vitro to evaluate tumor penetrating ability3.Evaluate tumor targeting performance in vivo.Methods1.Establishing RBCm-derived vesicles with hypotonic medium treatment andsonicate using a bath sonicator.Afterwards this the membrane vesicles were subsequently extruded repeatedly through polycarbonate porous membranes with different pore size using a mini-extruder.Blank PLGA nanoparticles were prepared using O/W emulsion.After that,RBCm-derived vesicles was coated onto the polymeric cores using the extrusion approach.The anti-EGFR-iRGD recombinant protein was derivatized with the N-hydroxysuccinimide ester of palmitic acid.With the protein being chemically lipidated with palmitate and subsequently gain the ability to anchor onto RBCm-PLG A micro spheres via the lipid chain.The structure,size and surface zeta potential,and the stability of anti-EGFR-iRGD-RBCm-PLGA microspheres in vitro were verified using transmission electron microscopy,dynamic light scattering,respectively.2.Three different colorectal cancer cell lines(SW480、HT29、Caco-2)were cultured to test the expression level of EGFR with Western blot.3.Developing colorectal cancer cell 3D multicellular spheroids(MCS),evaluating the permeability of anti-EGFR-iRGD-RBCm-PLGA microspheres in multicellular spheroids with confocal microscopy.4.The animal model of human colorectal cancer transplanted in nude mice was established to observe the tumor targeting ability of anti-EGFR-iRGD-RBCm-PLGA micro spheres.Results1.Anti-EGFR-iRGD-RBCm-PLGA microspheres was successfully established.A spherical core-shell structure was visualized by transmission electron microscopy.The particle size is approximately 180 nm measured by dynamic light scattering(DLS).2.Compared with SW480,HT29 and Caco-2 human colorectal cancer cell lines have higher expression levels of EGFR.3.Anti-EGFR-iRGD-RBCm-PLGA microspheres was visualized to penetrate into HT29 cell 3D multicellular spheroids in vitro.4.In vivo imaging of the anti-EGFR-iRGD-RBCm-PLGA microspheres exhibited the remarkable tumor targeting ability in nude mice.ConclusionThe anti-EGFR-iRGD-RBCm-PLGA microspheres are safe,stable and it shows the significant targeting and penetrating ability in vivo and in vitro.This will be a promising drug delivery system of anti-cancer drugs.