Construction Of Multi-Functional Polymeric Nanoparticles And Studies On Their Properties

With the development of nanotechnology, polymeric nanoparticles with their unique properties had become the research hot spot in the fields of biomedicine, energy storage materials, electronic devices and nanobiological components. The advances of living radical polymerization and coupling chemistry provided a versatile platform for the preparation of polymers with complex structures and advanced functions. In this thesis, RAFT polymerization was combined with click chemistry and dynamic covalent chemistry to construct glycopolymer-peptide bioconjugates, multi-functional core-cross-linked polymeric nanoparticles, multi-responsive protein nanocarriers and polymer-chymotrypsin nanoparticles.1. Comb-shaped glycopolymer-peptide bioconjugates vis thiol-ene click chemistry and studies on their solution behaviors1) RAFT polymerization had been employed to synthesize the protected glycopolymer PMAIpGlc-b-PHEMA, The acrylate groups were introduced to the PMAIpGlc-b-PHEMA diblock copolymer by post-polymerization modification. The block glycopolymer with pendant glucose moieties was achieved by the transformation of PMAIpGlc to the corresponding hydrophilic polymer PMAGlc under acidic conditon.2) As a model peptide, reduced glutathione (GSH) was conjugated to the PMAGlc-b-P(HEMA-acrylate) via thiol-ene "click" chemistry to generate glycopolymer-peptide bioconjugate PMAGlc-GSH. The solution behavior of the PMAGlc-GSH was investigated by dynamic light scattering (DLS), zeta potentiometric titration, and transmission electron microscopy (TEM). It was found that PMAGlc-GSH self-assembled to monodispersed spherical micelle in the aqueous solution because of the inter/intra-molecular hydrogen bonding between the pendant GSH moieties of PMAGlc-GSH. The micelles could specially recognize protein Con A due to the presence of glucose moieties on the shells. Under endosomal pH value (pH=5.5), PMAGlc-GSH micelles dissociated into unimers because of the cleavage of the β-thiopropionate linkages.2. Construction of multi-functionalizable core-cross-linked polymeric nanoparticles via dynamic covalent bond.1) A series of P(PEGMA-co-MAO) copolymers with different compositions were prepared via RAFT polymerization. By treatment with excess hydrazine hydrate, the copolymers were converted to corresponding hydrazide-containing copolymer (P(PEGMA-co-MAH)).2) Well-defined glycoconjugated copolymer and biotinylated copolymer were synthesized because of the versatile reactivity of hydrazides. The glycoconjugated copolymer could specially recognize Con A. The amount of available biotin to avidin was calculated to be ca.1.1μmol biotin/mg polymer by an HABA/avidin competitive binding assay.3) The core-cross-linked nanoparticles were prepared through the acylhydrazone formation of hydrazide containing copolymers and terephthaldicarboxaldehyde (TDA). DLS and’H NMR results demonstrated the pH-reverisible and dynamic characters of the cross-linked nanoparticles. In vitro cytotoxicity of the cross-linked nanoparticles and the corresponding hydrazide copolymers was investigated in HeLa cells by CCK-8assays. The results revealed that the copolymer with higher hydrazide contents showed toxic to HeLa cells. However the cross-linked nanoparticles exhibited high cell viability and no concentration-dependent toxicity at the tested concentrations up to300p.g/mL4) Excess hydrazide functions not involved in the cross-linking could be utilized as handles for the post-functionalization of nanoparticles, which were further adorned with biotin and FITC.5) With hydrophobic dye nile red as a model drug, these nanoparticles encapsulated a cargo of dye molecules, which demonstrate a pH-triggered release behavior. The dye-loaded nanoparticles prepared by the copolymer with higher hydrazide contents exhibited better pH-responsive behavior.3. Muti-responsive protein nanocarrier from dynamic covalent polymer1) RAFT polymerization had been employed to synthesize the diblock copolymer P(PEGMA)-b-PMAO. By reacted with hydrazine hydrate, the diblock copolymer was converted to corresponding hydrazide-containing copolymer P(PEGMA)-b-PMAH.2) The dynamic covalent copolymer was generated by the bioconjugation of pyridoxal5’-phosphate (PLP) to the pendant hydrazide groups through reversible acylhydrazone linkages. The results revealed that temperature had a pronounced effect on the cleavage of acylhydrazone bonds of the PLP-conjugated dynamer. In vitro cytotoxicity study demonstrated that the PLP conjugated dynamer and the polymer precursor were nontoxic to HeLa cells at the tested concentrations up to200μg/mL.3) The PLP-conjugated dynamer was negatively charged at physiological pH. Polyion complex (PIC) micelles were formed through the electrostatic interaction between positively charged protein lysozyme and negatively charged PLP-conjugated dynamer.4) These PIC micelles demonstrated pH-, salt-, and enzyme-responsive features. The enzymatic activity of PIC micelles toward the hydrolysis of the bacterial substrate Micrococcus luteus cells was evaluated. A reduced activity was observed after lysozyme was entrapped into the core of the PIC micelles because of the shielding effect of P(PEGMA) corona, the dissociation of the micelles, triggered by the increase in ionic strength of the milieu, resulted in the recovery of lysozyme activity.4. Constructon of polymer-protein nanoparticles based on dynamic covalent bonds and studies on their properties1) Aldehyde-modified chymotrypsin (M-CT) was prepared by the esterification reaction between lysine residues of chymotrypsin and succinimidyl4-formylbenzoate (S-4FB). Polymer-chymotrypsin nanoparticles were prepared through the acylhydrazone formation of hydrazide containing block copolymers P(PEGMA)-b-PMAH and M-CT.2) SDS-PAGE, DLS and TEM results demonstrated the formation the polymer-chymotrypsin nanoparticles. The secondary structure and conformation of CT and polymer-chymotrypsin nanoparticles were studied by CD spectroscopy and fluorescence emission spectroscopy. 3) The polymer-chymotrypsin nanoparticle demonstrated pH-responsive and dynamic characters. The enzymatic activity of polymer-chymotrypsin nanoparticles toward the hydrolysis of the substrate N-succinyl-L-phenylalanine p-nitroanilide (SPNA) was evaluated. A reduced activity was observed after chymotrypsin was entrapped into the nanoparticle because of the shielding effect of the polymer chains. The M-CT and polymer-chymotrypsin nanoparticles exhibited excellent thermal and stored stability.