Construction Of Multifunctional Polymeric Nanocarriers For Gene/Drug Delivery Via Multicomponent Reactions

Nanomedicine is an important innovation field for nanotechnology in medicine such as disease prevention and treatment.Its rapid development and application play an increasingly important role in promoting the health and well-being of human beings.Gene and drug carriers based on polymeric nanomaterials have brought new dawn to patients with certain thorny diseases such as cancer and intervertebral disc degeneration(IDD).However,in the face of complex obstacles in and out of the drug or gene delivery process,the development of safe and efficient delivery vehicles still faces many challenges.From the preparation of nanomaterials to the entry into target cells,it is necessary to overcome multiple obstacles,such as colloidal stability of the nanoparticles,blood circulation,tissue accumulation and penetration,cellular uptake,endosomal escape and effective cargo release.These obstacles asked for higher design requirements for the development of new and effective nanocarriers,promoting the continuous exploration of scientists in this field.In this thesis,multi-component functional groups are introduced into polymer carriers through multicomponent reactions to effectively overcome multiple obstacles in drug or gene delivery.Firstly,a high-efficiency endosomal escape molecule containing both a hydrophobic group and a pH-responsive imidazole group was prepared by Passerini reaction.On the one hand,the molecule was combined with the polycation gene carrier molecule to improve the ability of the endosomal escape,thereby enhancing the final gene transfection efficiency;on the other hand,incorporating the endosomal escape molecule into the amphiphilic polymeric carrier of the hydrophobic small molecule prodrug,which could increase the efficiency of endosomal escape,and simultaneously activate the level of oxidation in the tumor cell oxidative responsive small molecule prodrugs,synergistically increasing antitumor effects.Furthermore,the multicomponent reaction based on thiolactone chemistry and Michael addition reaction was used to construct a series of multifunctional cationic polymers to overcome the numerous obstacles in gene delivery,which could impressively improve the delivery efficiency of plasmid DNA.Finally,in response to chronic disc degenerative diseases,a long-acting,sustained-release and highly efficient miRNA delivery system for the microenvironmental response of diseased tissues has been developed,and gene therapy for degenerative disc disease has been achieved.In detail,the research content and main conclusions of this dissertation can be further categorized into four parts as follows:1.The polymers with the capability of endosomal escape were designed and synthesized from monomers by integrating alkyl and imidazolyl via Passerini reaction and reversible addition-fragmentation chain transfer(RAFT)polymerization.Poly(2-methyIamino)ethyl methacrylate(PDMAEMA)was used as a macromolecular chain transfer agent.The polymers that integrated hydrophobic alkyl and imidazole groups in the polymer possessed the function of merging with the endosomal membrane and realizing the proton sponge effect,thereby synergistically promoting the escape process from the endosomes.After introducing the endosomal-escape polymers with proper degrees of polymerization(DPs)into poly(2-dimethylaminoethyl methacrylate)(PDMAEMA)as the gene delivery vectors,the block copolymers exhibited significantly enhanced hemolytic activity at endosomal pH,and the plasmid DNA(pDNA)-loaded polyplexes showed efficient endosomal escape compared with the homopolymer PDMAEMA,ultimately achieving dramatically increased gene transfection efficacy.These results suggest that the polymers that integrate alkyl and imidazolyl moieties for efficient endosomal escape have wide potential applications for intracellular gene delivery.2.To inhibit tumor growth effectively,a new kind of multi-functional nanoparticle was designed and prepared to achieve the synergistic effect of chemotherapy and oxidation treatment of tumors subcutaneously implanted in the mice.First,a cinnamaldehyde-containing methacrylate monomer(MMSD)linked by an acetal bond was successfully synthesized.Secondly,PEG was used as the macromolecular chain transfer agent,and the pH response amphiphilic block copolymer was obtained by the RAFT method using MMSD monomer and the endosomal escape monomer(ImOAMA)synthesized in the previous chapter.Finally,the amphiphilic block polymer was self-assembled with a camptothecin prodrug(ProCPT)that can be activated by reactive oxygen species(ROS)to obtain multifunctional nanoparticles.In vitro Experiments have shown that the nanoparticles could release cinnamaldehyde molecules in the tumor tissue or intracellular environment,which would increase the intracellular ROS level.Moreover,the significantly increased ROS could activate the prodrug molecule ProCPT to a greater extent to form the biologically toxic prodrug molecule,camptothecin(CPT),exerting its potency to promote apoptosis.Animal experiments show that this kind of nanoparticles could not only achieve better enrichment and retention in the tumor site,but also significantly inhibit the growth of tumor volume.Pathological analysis showed that the system did not cause obvious damage to normal organs of animals and had good biosafety.In summary,this dual mechanism of increasing intracellular ROS content and specifically activating prodrugs could exert synergistic therapeutic effects on tumor oxidative therapy and chemotherapy.3.Based on the multicomponent reaction of thiolactone chemistry and Michael addition,a variety of cationic polymers formed by combining different hydrophobic functional groups with amine structural compounds were obtained,and the best one which can be used as a potential gene delivery carrier was screened by experimental evaluation.Briefly,a ring-opening reaction is carried out using an amine compound containing a different number of ethyleneimine units and a thiolactone modified with a different hydrophobic group,and the thiol group produced by the reaction is followed by the poly[2-(acryloyloxy)ethyl methacrylate](PAOEMA)further undergoes a Michael addition reaction,which ultimately combines to obtain a series of functional cationic polymers containing different structural units.Subsequently,experiments such as cell uptake and intracellular distribution demonstrated that the cationic polymer modified by tetraethylenepentamine and perfluoropropyl(P3D)can effectively improve the cellular uptake ability of the complex and can significantly enhance its endosomal escape ability.Thus,it shows superior gene transfection efficiency compared to other polymers and universal transfection reagent bPEI(branched polyethyleneimine,Mw = 25 kDa).Based on the above results,we successfully used a simple and efficient synthesis method of thiolactone ring opening and Michael addition reaction to integrate different functional groups to obtain a series of potential polymer gene delivery vectors with multifunctional biological effects.The best gene delivery vector with high efficiency and low toxicity was screened by cell level experiment evaluation.4.Since intervertebral disc degeneration(IDD)is a chronic pathological process,targeted interventions based on miRNA gene therapy require long-lasting effects to produce the desired therapeutic effect.Considering that the high expression of matrix metalloproteinases(MMPs)in fibrotic intervertebral disc tissue greatly promotes the occurrence and development of intervertebral disc degeneration,a hydrogel system with dual response of injectable MMPs was designed and constructed.The hydrogel system achieves stable,long-lasting and intelligent delivery of the therapeutic gene miR-29a for inhibition of disc degeneration studies.In detail,cationic block copolymer PEG-GPLGVRG-PAsp(DET)-Chole(PGPC)that responded to the MMP enzyme was designed to complex with miR-29a.Specially,the PAsp(DET)component has been demonstrated to display high gene transfection efficiency and negligible cytotoxicity due to remarkable endosomal escape ability and degradable properties.The peptide linkage(GPLGVRG)can be cleaved specifically by a series of MMP enzymes.The terminal moiety,cholesterol,was introduced to stabilize the complexes between the cationic block copolymer and small miRNA via introduction of hydrophobic interactions.Additionally,for sustained release,the polyplexes loading miR-29a were encapsulated into MMP-responsive PEG/CGPLGVRGC hydrogels that can be formed rapidly via Michael addition reaction in an injectable manner.In general,the mixture of miR-29a/PGPC polyplex micelles and CGPLGVRGC was mixed with eight-arm maleimide-terminated star PEG,followed by immediate injection into IVD for in situ formation of the complexes-encapsulated hydrogels.Upon exposure to high MMP level during IDD,the two-stage miRNA delivery can be achieved in a responsive and sustained manner,including first MMP-responsive degradation of hydrogels for release of miR-29a polyplex micelles and MMP-responsive dePEGylation of the micelles for enhanced cellular internalization into nucleus pulposus cells and efficient endosomal escape.The mechanism of fibrosis inhibition by miR-29a was demonstrated to silence MMP-2 expression and block P-catenin translocation pathway from the cytoplasm to the nucleus.The devised polyplex micelle-encapsulated hydrogels finally realized the sustained and bioresponsive miRNA delivery for treatment of chronic IDD,which represents a novel and promising strategy to perform local gene therapy of chronic diseases.