Construction Of PH And H₂O₂ Double-Responsive Nanoparticles For Visualized Intracellular Release Of Protein Delivery

Compared with chemical drugs,therapeutic protein have been increasingly applied in cancer therapy due to their unique superiority including high activity,low side effects,and strong specificity.However,the application of protein drugs still faces several challenges,such as poor stability,low membrane permeability,inactivation by enzyme digestion and low bioavailability.In addition,since most protein drugs have no fluorescence,and the existing methods of protein labeling have certain limitations,it is difficult to obtain bio-distribution and metabolic information when proteins enter cells.Therefore,in this thesis,a pH and H₂O₂double-responsive nanoparticle MNPs@PDA@PEG/SHA-QCy7/Try was constructed to achieve safe intracellular protein delivery and visual monitoring of controlled release.The fabrication of MNPs@PDA@PEG/SHA-QCy7/Try includes:1)Preparation of polydopamine（PDA）-coated nanoparticle scaffolds（MNPs@PDA）via dopamine oxidation self-polymerization on the surface of Fe₃O₄ nanoparticles（MNPs）as core;2)Sequentially modification of amino-modified salicylic hydroxamic acid（SHA）and sulfhydryl-modified polyethylene glycol（PEG-SH）to the surface of PDA to prepare MNPs@PDA@PEG/SHA nanoparticles;3)MNPs@PDA@PEG/SHA-QCy7/Try Respective loading QCy7 and Try onto the nanoparticles by reacting salicylic hydroxamic acid with 4-carboxyphenylboronic acid（PBA）modified QCy7-PBA and Try-PBA to prepare the final nanoparticles of MNPs@PDA@PEG/SHA-QCy7/Try.At neutral pH,PEG forms a protective layer of the protein against enzymic degradation.At lower pH,the formed covalent bond of boric acid/salicyl hydroxamic acid breaks,which on one hand led to the release of Try-PBA for recovery of protein activity;On the other hand,led to the released QCy7-PBA,which is oxidized by H₂O₂ to generate QCy7 for the recovery of near-infrared fluorescence and visual detection of controlled protein release.The main research contents and results are as follows:1.Synthesis and Characterization of SHA and QCy7-PBA:In this thesis,the key compounds SHA and QCy7-PBA were first synthesized,and their structures were identified by nuclear magnetic resonance and mass spectrometry.The results of H₂O₂ response of QCy7-PBA using UV-Vis spectrophotometer and fluorescence spectrophotometer showed that QCy7-PBA can selectively respond to H₂O₂ and achieve near-infrared fluorescence recovery.2.Modification and activity study of trypsin:In this thesis,trypsin（Try）was used as a model protein and was modified by PBA to obtain（Try-BAB）.Polyacrylamide gel electrophoresis and ARS fluorescence titration experiment analysis results showed that the average binding of 23.5 equivalents of PBA per equivalent of Try.The results of circular dichroism and TAME colorimetric absorbance analysis showed that Try-PBA basically retained the secondary structure of theα-helix of Try and the protein modification method had no significant effect on the biological activity of Try.3.Preparation and characterization of nanoparticle MNPs@PDA@PEG/SHA-QCy7/Try:MNPS@PDA was used as the backbone.SHA and PEG-SH were covalently linked to the surface of PDA sequentially to prepare MNPs@PDA@PEG/SHA.Then,MNPs@PDA@PEG/SHA-QCy7/Try was obtained by reacting with QCy7-PBA and Try-PBA,respectively.The target nanoparticles were characterized by DLS,UV-Vis,TEM,FTIR and XPS analysis techniques.Dynamic light scattering analysis showed that the hydrated particle size was 462.0 nm and the zeta potential was-24.5 m V;The analysis of UV-Vis and FTIR showed that the characteristic peaks of the nanoparticles were consistent with the characteristic peaks of the modified compounds;XPS analysis showed that the content of Fe,O,C and N changed with the different compounds modified by the nanoparticles;transmission electron microscopy showed that the outermost of the nanoparticles MNPs@PDA@PEG/SHA-QCy7/Try has a clear gray cladding.Based on the above analysis results,this paper successfully prepared the target nanoparticles.4.Intracellular delivery of Try:The study of intracellular protein delivery of MNPs@PDA@PEG/SHA-QCy7/Try used human lung cancer cell A549.By confocal microscopy（CLSM）and flow cytometry（FACS）,the uptake of nanoparticles by A549 cells and the delivery and release of trypsin in A549 cells were studied.The results indicated that A549 cells can effective intake MNPs@PDA@PEG/SHA-QCy7/FITC-Try over time.Lyso Tracker Red was used for colocalization analysis after lysosome staining,indicating that FITC-Try-PBA can successfully escape from endosomes and lysosomes to achieve safe and efficient protein delivery.The protein visualization monitoring ability of QCy7-PBA was studied by confocal microscopy,demonstrating that near-infrared fluorescence emitted from QCy7 becomes stronger and stronger with time,enabling visual monitoring of protein delivery and controlled release by QCy7-PBA.The antitumor effect of nanoparticles MNPs@PDA@PEG/SHA-QCy7/Try was investigated by MTT assay and Annex V-FITC/PI apoptosis kit.When the concentration of MNPs@PDA@PEG/SHA-QCy7/Try was 0.25 mg m L^-1,the survival rate of A549 cells was 15.2%after incubating 3 h,showing an ideal anti-cancer effect.