Funtional Polyaspartamide Derivatives By Click Chemistry For Drug/Gene Delivery

In recent years, the development of new active pharmaceutical ingredients and treatment techniques (eg:gene therapy) is rapid, but how to deliver these drugs and therapeutic genes with good effect into cells is still a problem. The interest in poly(amino acid)s has increased significantly due to their biocompatibility, controlled biodegradability, and nontoxicity of its degradation products. In addition, poly(amino acid)s contain chemical functionality, such as hydroxyl, amine, and carboxyl groups, which can be used for further modification. Thus, a series of novel drug/gene polymeric carriers based on amino acid polymers received widespread attention. The physical or chemical properties of smart polymeric carriers responding to the external environment such as temperature, pH and reductant, which can be utilized for the loading and controlled release of drug. Because of the high selectivity and high fidelity,"click" chemistry has rapidly become a very popular method for polymer synthesis and modification. In this study, a series of thermo/pH/reductive-responsive biodegradable polyaspartamide derivatives were designed and synthesized via click chemistry for drug/gene delivery.In Charpt1, synthesis of poly(amino acid)s, applications of click chemistry in drug/gene delivery, and the progress and applications of stimuli-responsive polymer in drug/gene delivery systems were reviewed.In Charpt2, thermosensitive poly(aspartic acid) derivatives containing pendant azido groups (denoted as P(Asp-Az)x-HPA) were prepared from poly(L-succinimide) via the ring-opening reaction with2-azidoethylamine and5-diethylamino-2-pentanol. Then, aromatic ring was introduced to the side chains of P(Asp-Az)39-HPA by click reaction to obtained P(Asp-Az)39-HPA-PEA. The structure of resulting polymers was characterized using FTIR and1H NMR. The cloud point of P(Asp-Az)39-HPA and P(Asp-Az)56-HPA is58?and29?, respectively. Thermoresponsive behaviour of the poly(aspartic acid) derivatives containing pendant azido groups was confirmed by dynamic light scattering (DLS) and transmittance measurements, and the cloud point can be tuned by introducing varying amounts of aromatic ring. Based on the thermosensitive of azido-containing P(Asp-Az)x-HPA and P(Asp-Az)39-HPA-PEA, polymers with hydrophilic long PEG chain or targeted moieties can be designed and synthesized further via click chemistry for drug delivery. In Charpt3, based on the thermosensitive azido-containing P(Asp-Az)39-HPA-PEA described in Chaprt2, a series of thermosensitive polyaspartamide derivatives containing pendant aromatic structures and hydrophilic PEGs [PEG-P(Asp-Az)39-HPA-PEA] were synthesized by introducing long PEG chains via click reaction. The micelles and drug loaded micelles based on PEG-P(Asp-Az)39-HPA-PEA were prepared by quick heating method. This simple drug loading method showed high encapsulation efficiency (>90%), avoiding the use of toxic organic solvents. The drug-loaded micelles showing a mean size around80nm with narrow size distribution (PDI<0.2) in PBS by DLS were stable upon dilution under physiological conditions. The drug loading content of PEG-P(Asp-Az)39-HPA-PEA increased with increasing the amout of aromatic structures in polymer. The PTX-loaded micelles showed obvious anti-cancer activity similar to Taxol (the commercial paclitaxel formulation) against HeLa cells, while blank micelles were non-toxic. The present results suggest that the polyaspartamide derivatives containing aromatic structures PEG-P(Asp-Az)x-HPA-PEA are a promising delivery system for the hydrophobic anticancer drugs.In Charpt4, a series of double-stimuli (pH/thermo) sensitive polyaspartamide derivatives containing pendant imidazole rings PEG-P(Asp-Az)39-HPA-IMZ were synthesized via click reaction for drug delivery. The micelles based on PEG-P(Asp-Az)39-HPA-IMZ polymer and DOX-loaded micelles were prepared by a quick pH-changing method. This easy drug loading method showed high encapsulation efficiency (>90%) avoiding the use of toxic organic solvents. The DOX-loaded micelles showing a mean size around65nm with narrow size distribution (PDI<0.1) in PBS by DLS were stable in PBS within24h. The DOX-loaded micelles displayed much faster release at pH5.0than that at pH7.4. In vitro experiments revealed that the DOX-loaded micelles showed obvious anti-cancer activity against HeLa cells, while blank micelles were non-toxic even at polymer concentrations up to1000mg/L. Thus, these non-toxic, dual pH-and thermo-sensitive polyaspartamide derivatives containing imidazole rings may be a promising anti-cancer drug delivery system.In Charpt5, polyaspartamide-based disulfide-containing brushed polyethylenimine derivatives P(Asp-Az)x-SS-PEIs were synthesized via click chemistry and evaluated as nonviral gene delivery carrier. First, azide-functional poly(aspartic acid) derivative with various azide-group densities and mono-alkyne-terminated PEI with disulfide linkage were synthesized. Then click reaction between the azide-functional poly(aspartic acid) derivative as main chain and the mono-alkyne-terminated PEI as branched chain resulted in high-molecular-weight disulfide-containing brushed PEI derivative. The structure of obtained polymers was confirmed by1H NMR and FTIR. It was shown that the disulfide-containing P(Asp-Az)x-SS-PEIs were able to bind plasmid DNA and condense DNA into small positive nanoparticles. The reduction-sensitivity of the P(Asp-Az)x-SS-PEI/DNA polyplexes was confirmed by gel retardation assay and dynamic light scattering (DLS) in the presence of DTT. In vitro experiments revealed that the reducible P(Asp-Az)x-SS-PEI not only had much lower cytotoxicity, but also posed high transfection activity (both in the presence and absence of serum) as compared to the control non-degradable25-kDa PEI. This study indicates that a reducibly degradable brushed polymer P(Asp-Az)x-SS-PEI composed of low-molecular-weight (LMW) PEI via a disulfide-containing linkage can be a promising gene delivery carrier.