Synthesis And Biomedical Properties Of Ph-responsive Poly (α,β-L-aspartic Acid) Derivatives

Poly(amino acid)s have been extensively studied in biomedical applications,due to their unique regular secondary structures, rigid backbone, biocompatibility,biodegradability and various side groups for further functionalization. Most ofpolypeptides reported so far were prepared by ring-opening polymerization of aminoacid N-carboxyanhydrides (NCAs). However, the NCA approach has a costdisadvantage and a production problem because the pendant reactive groups carriedby the amino acids need to be protected prior to polymerization and deprotectedunder harsh conditions after polymerization to give the functional side groups. Poly(α, β-L-aspartic acid)(α, β-L-PAsp) can be synthesized by thermal polycondensationof L-aspartic acid (L-Asp), which is easily controlled and not costly. It would be agood candidate for preparing polypeptide derivatives.In this thesis, Poly (amino acid)-based amphiphilic copolymer was utilized tofabricate a better micellar drug delivery system (DDS) with improved compatibilityand sustain release of Doxorubicin (DOX). First, Poly (ethylene glycol) monomethylether (MPEG) and DOX were conjugated onto polyasparihyazide, prepared via thereaction of poly (succinimide)(PSI) with hydrazine, to afford an amphiphilicpolymer [PEG-hyd-P (AHy-hyd-DOX)] with acid-liable hydrazone bonds. The DOX,chemically conjugated to the water-soluble polymer of the PAHy throughpH-sensitive hydrazone bond, was designed to supply hydrophobic segments andimprove the compatibility between the core and physically entrapped DOXmolecules. PEG segments grafted to the polymer via hydrazone bond was designedto prolong its lifetime in blood circulation. Free DOX molecules could be entrappedinto the nanoparticles of such an amphiphilic polymer via-interaction betweenthe conjugated and free DOX molecules to obtain a drug delivery system with highDOX load. The drug loading capacity, drug release behavior, and morphology of themicelles were investigated. The micelles’ biological activity was evaluated in vitro.Results showed that drug loading capacity was intensively augmented by adjusting the feed ratio, and the maximum loading capacity was as high as38%. Besides, theDOX-loaded system exhibited a pH-dependent drug release profile in vitro. Thecumulative release of DOX was much faster at pH5.0than that of the release at pH7.4. The DOX-loaded system kept highly antitumor activity for a long time,compared with free DOX. This easy-prepared DDS, with features ofbiocompatibility, biodegradability, high loading capacity and responsiveness to pH,was a promising candidate for the DOX delivery and controlled release.In order to accelerate the rate of DOX release, a facile micelle-formingcopolymer-drug conjugate mPEG/IM-g-(PAHy-DOX) were reported. The DOX,chemically conjugated to the pH-responsive poly (amino acid)-based amphiphilicgraft polymer mPEG/IM-g-PAHy by hydrozone bonds, was designed to have a fasterrelease rate due to the presence of pH-sensitive group of imidazole. The drug loading,drug release, and morphology of the micelles were investigated. Compared with theamphiphilic polymer [PEG-hyd-P (AHy-hyd-DOX)], DOX-loaded system reportedhere has obviously enhanced pH-sensitivity and superior drug release behaviors, atpH5.0, the cumulative release of DOX from mPEG/IM-g-(PAHy-DOX)was muchfaster than that of the release from [PEG-hyd-P (AHy-hyd-DOX)].An injectable poly (α,β-L-aspartic acid)-based hydrogel via hydrazonecrosslinking strategy was also studied in this thesis. The gelation occurs underphysiological conditions immediately upon mixing of the two aqueous poly(α,β-L-aspartic acid) derivatives specifically derivatized through hydrazone linkageswith aldehyde (PAPDAL) and hydrazide (PAHy) functional groups, respectively,without addition of crosslinker or catalyst. The fast gelation provides this systemwith injectable property. PAsp hydrogels were synthesized in PBS solution andcharacterized by different methods including gel content and swelling, Fouriertransformed infrared spectra, in vitro degradation and biocompatibility experiments.A scanning electron microscope viewed the interior morphology of gel whoseporous three-dimensional structure enabled it to efficiently encapsulate the drugs.Sustained and stable DOX release from the PAsp hydrogel was observed during invitro delivery experiments and exhibited its potentially high application prospect inthe field of protein drug delivery. In addition, a novel zwitterionic polypeptide derivative, denoted as His-PAsp/PAsp,has been successfully synthesized by amidation of Poly (α, β-L-aspartic acid) withL-histidine methyl ester. Turbidity, zeta potential and1H NMR measurements wereused to study the aggregation behaviors of His-PAsp/PAsp under different pH values.The modified polypeptide derivative composed of certain negatively and positivelycharged residues randomly, exhibiting an isoelectric point (IEP) and bearing oppositecharges at pH values far high or below the isoelectric point. Silicon wafer, as a modelsubstrate material, was then coated with zwitterionic polypeptide. The amount ofprotein adsorbed to the coated surface could be controlled, depending on the dose ofthe polypeptide. Since its good biodegradability and superior anti-protein-foulingproperty, this pH-responsive zwitterionic polypeptide is a promising candidate forsurface modification in many biomedical applications, including medical implants,drug delivery carriers, and biosensors.A series of zwitterionic dimethyl aminopropyl amine-grafted poly(α, β-L-asparticacid)s (DMAP-PASP) with pH-dependent charge profiles, were synthesized bysubsequent aminolysis polysuccinimide with3-dimethyl aminopropyl amine andalkali. The zwitterionic polypeptides had an IEP, which could be easily tuned frompH3.8to9.5and showed opposite charges below and above the IEP. Because of theflexibility of controlling pH dependence, it is expected that zwitterionicpolyaspartamide would have a number of biomedical applications, where the abilityto respond to delicate pH changes is required. The zwitterionic polypeptide was ableto inhibit the protein adsorption to the anionic surface at physiological pH. Theseresults warrant further exploration of zwitterionic polypeptides as a stealth coatingfor tumor-targeted nanocarriers to overcome the current limitation of PEG.