Construction Of High Performance Gene Delivery Systems Based On Assembly

Gene therapy is a therapeutic method by counteracting or replacing genes adversely affact the body.Successeful gene therapy needs to overcome the obstacles such as cell and tissue barriers to perform the function without disrupting essential regulatory mechanisms.In addition,the gene-accepted cells should achieve certain quantities of genes to reverse the sick condition.Since the first clinical trial in 1989,almost three decades have passed with more and more trials in inherited diseases,cancer and chronic infections.Although the results were not so positive,a number of clinical gene-therapy trials in phase I/II showed remarkable evidence of efficacy and safety in serious diseases in blood,immune and nervous systems.It should be noted that the therapy trials were all performed with dielivery technologies due to the degradability of ncucleic acids(NAs)in body.Effective gene therapies depend on the high performance delivery systems.In comparation with drawbacks of virial vectors,non-viral vectors came to people’s view in an irresistible force.The most common investigated non-virius vestors are polycations.The current synthetic polycations-based NAs delivery systems are more versatile and safe,but the efficiency is substantially less than viral vectors.The current delivery systems mainly focus on improving the cellular uptake of NA-vector complexes.In fact,the expression of gene is influenced by various additional factors,including stability of NA in the cytoplasm and release of intracellular NA.In this thesis,we mainly focused on preparation of high performed polycation gene delivery systems with high cellular uptake and accelerating release of NAs utilizing non-covalent self assembly method based on biocompatible and biodegradable biomolecules.Firstly,we developed a series of poly(aspartic acid)(PAsp)-based host-guest supramolecular assemblies for gene delivery.Due to its good properties such as low cytotoxicity,degradability,and biocompatibility,PAsp is a good candidate for the development of new drug delivery systems.From this proof of concept,a series of new PAsp-based degradable supramolecular assemblies were prepared for effective gene therapy via the host-guest interactions between the cyclodextrin(CD)-cored PAsp-based polycations and the pendant benzene group-containing PAsp backbones.Such supramolecular assemblies exhibited good degradability,enhanced pDNA condensation ability,and low cytotoxicity.Furthermore,the supramolecular assemblies expressed much higher transfection efficiencies than CD-cored PAsp-based counterparts at various N/P ratios.In addition,the effective antitumor ability of assemblies was demonstrated with a suicide gene therapy system.The present study would provide a new means to produce degradable supramolecular drug delivery systems.Secondly,as we have investigated the potential of the assembly,a non-covalent methodin the design of high efficiency delivery system,we then tried to introduce heparin into the assemblies to accelerate NA release.As representative biomolecules,polysaccharide-based copolymers have attracted much attention due to their effective performances.Heparin,as a kind of polysaccharide with highly negative charge densities,has attracted much attention in biomedical fields.However,the modification and application of heparin are limited due to its poor solubility.In this work,we report a flexible way to adjust the solubility of heparin from water to oil via the introduction of tetrabutylammonium groups for further functionalization.A range of heparin-based comb copolymers were readily synthesized in a MeOH/dimethylsulfoxide mixture via atom-transfer radical polymerization.The heparin-based polymer nanoparticles were produced due to the electrostatic interaction between the negatively charged heparin backbone and polycation grafts.Then the pDNA condensation ability,cytotoxicity,and gene transfection efficiency of the nanoparticles were characterized in comparison with the reported gene vectors.The nanoparticles were proved to be effective gene vectors with low cytotoxicity and high transfection efficiency.The introduction of negative charged heparin preserved the efficiency of the delivery system and effectively decreased the cytoxicity.Moreover,it is proved that by adjusting the solubility of heparin,polymer graft functionalization of heparin can be readily realized for wider applications,and modification methods are also enlarged.Finally,we prepared novel low-cytotoxic core-shell nanocomplexes(Hep@PGEA)based on assembly strategy.The assembled nanocomplexes with unlockable negatively charged heparin cores(HepNP),upon the breakdown of HepNP cores by reductants,unlocked heparin would interact with outer cationic PGEA shells and accelerate release of condensed NAs.Heparin,as one well-known negatively charged polysaccharide,posseses abundant sulfate groups,which could promote angiogenesis,anticoagulant and anti-inflammatory,except strongly interaction with polycations.The delivey system was proposed for effective miRNA-pDNA staged gene therapy of myocardial infarction(MI),one of the most serious cardiovascular disease(CVDs).Hep@PGEA was proved to be a robust delivery vector of both miRNA and pDNA.With the progression of MI,glutathione(GSH)amounts in heart tissues increase.Thus,staged gene therapy was proposed for systemic treatment of MI by using Hep@PGEA to sequentially deliver miR-499(for the inhibition of cardiomyocyte apoptosis to decrease heart damages in the initial stage)and pVEGF(for the promotion of angiogenesis to restore blood supply in the later stage).Such staged NA delivery produced very impressive results in restoring heart function,decreasing infarct area,promoting angiogenesis and suppressing cardiac hypertrophy.Due to the wide existence of redox agents in cells,the proposed unclockable heparin nanocmoplexes and staged therapy strategy would provide new methods to effectively treat serious diseases.Overall,the polycationic vectors with NA accelerating release constructed with biocompatible and biodegradable biomolecules-PAsp and heparin,and combinations of assembly and atom transfer radical polymerization(ATRP)can efficiently deliver and release the genes for therapeutics.These positive results are of great significance for new delivery systems with high performances,and also provide new avenues for cancer and cardiovascular diseases treatments.