Cell Membrane Mimetic Chitosan-based Nanocarriers For Pulmonary Protein Delivery

In this paper,from the perspective of cell membrane bionics,in view of the poor stability and low bioavailability of protein drugs during use,a protein drug cell membrane biomimetic nanocarrier was constructed by ion-crosslinking method based on cell membrane biomimetic phosphorylcholine modified chitosan PCCs.We studied the loading and release of the biomimetic nanocarriers on the extracellular domain of recombinant FGF receptor(msFGFR2c),and the interaction mechanism between biomimetic nanocarriers and airway epithelial cells Calu-3 and human embryonic lung fibroblasts MRC-5.The rat model of pulmonary fibrosis induced by bleomycin was used as an animal model to evaluate the improvement of anti-fibrosis treatment of msFGFR2 c by bionic nanocarriers by pulmonary administration.First,the extracellular protein of msFGFR2 c was used as the loaded target protein,and the drug-loaded nanoparticles PCCs/msFGFR2 c and HTCC/msFGFR2 c were prepared by ion-crosslinking,and HTCC/msFGFR2 c was constructed with water-soluble quaternary ammonium chitosan HTCC as control.The sizes of PCCs/msFGFR2 c and HTCC/msFGFR2 c were measured at about 190 nm and 210 nm respectively,the corresponding surface potentials are about 0.81 mV and 9.7mV,respectively.The encapsulation rate of PCCs/msFGFR2 c and HTCC/msFGFR2 c was47.2% and 27.19%,respectively,and their drug loading percentages were about 9.4%and 5.8%,respectively.Atomic force microscopy and transmission electron microscopy showed that the morphology of the nanoparticle was spherical.In vitro drug release test showed that,within 24 h,the cumulative release rate of PCCs/msFGFR2 c at pH=5.5 and pH=7.4 reached 84% and 76%,respectively,and the cumulative release rate of HTCC/msFGFR2 c at pH=5.5 and pH=7.4 was 76% and70%,respectively,indicating that the cumulative release efficiency of PCCs/msFGFR2 c was higher than that of HTCC/msFGFR2 c,and drug-loaded nanoparticles have a good sustained release effect.The results of in vitro cytotoxicity experiments reflect that when theconcentration of PCCs-NP reached 2.0 mg/mL,it was not toxic to MRC-5 and Calu-3cells,but when the concentration of HTCC-NP reached 0.5 mg/mL,it was already toxic to MRC-5 cells and Calu-3 cells,indicating that PCCs-NP is more biocompatible than HTCC-NP.The results of cell uptake experiments showed that the MRC-5 and Calu-3 cells uptake the PCCs and HTCC and their nanocarrier particles were time and concentration dependent.The uptake efficiency is positively correlated with uptake time and uptake concentration within a certain range.The distribution experiments of fluorescent nanocarrier particles at the subcellular level showed that the nanocarrier particles ingested by Calu-3 cell were mainly distributed in the cytoplasm within 2.0h,and the fluorescent nanocarrie particles were not founded in the cell lysosome.A transwell epithelial airway transport model was established.The results of TEER experiment showed that the TEER value could be completely recovered after24 hours,when the fluorescent labeled nanoparticles with a concentration of 0.125mg/mL were added to the AP end,indicating that the effect of PCCs-NP and HTCC-NP at a concentration of 0.125mg/mL on the structure of Calu-3 monolayer in4 h was temporary and reversible;however,when fluorescently labeled nanoparticles with a concentration of 0.25mg/mL were added to the AP end,the effect of PCCs-NP on the TEER value of the Calu-3 monolayer was recoverable,while the effect of HTCC-NP on the TEER value of the Calu-3 monolayer was unrecoverable,indicating that the effect of PCCs-NP on the structure of Calu-3 monolayer is reversible at this concentration,while the effect of HTCC-NP on the structure of Calu-3 monolayer is irreversible.The results of transport experiments showed that when the chitosan derivatives PCCs and HTCC were made into nano-particles PCCs-NP and HTCC-NP,and their transport efficiency and apparent permeability coefficient(Papp)were improved in Calu-3 monolayer.Immunofluorescence experiments showed that PCCs-NP had little effect on the expression of tight junction protein ZO-1 at a concentration of 0.25mg/mL,and both PCCs and HTCC could attenuate the expression of ZO-1 to varying degrees,however,the expression of ZO-1 can be completely inhibited by HTCC-NP,which suggests that the two nanocarriers havedifferent transepithelial cell layer mechanisms.Finally,the bleomycin-induced pulmonary fibrosis model of Wistar rats was established,and the effect of drug-loaded nanocarriers on pulmonary fibrosis rats was studied by pulmonary administration.Rat body weight,lung tissue HE and Massion staining,X-ray results showed that PCCs/msFGFR2 c nanosystems had a significant inhibitory effect on pulmonary fibrosis,and its therapeutic effect was superior to that of free msFGFR2 c group.In general,the biomimetic nanocarriers prepared in this study have good biocompatibility,and can improve the therapeutic effect of pulmonary drug delivery of protein drugs.So they are expected to be an excellent drug carrier material for pulmonary administration.