Study Of Novel Amphiphilic Block Copolymers Instructed By “Molecular Glue” And Its Micellization

Amphiphilic block copolymers, can self-assemble into nanomicelles in aqueous, has been widely used in anticancer drugs delivery and controlled release system. Synthesis of block copolymers, with molecular weight controlled, narrow distribution and molecular structure, composition can be designed, is one of the difficulties in copolymers study. Therefore, how to study and explore a novel and efficient synthesis method for block copolymers is one of the important research work in this field. In this dissertation, based on the review and summarization of previous outstanding research works of block copolymers synthesis, mainly about covalent and supramolecular interactions for block copolymers connection, proposes a synthetic strategy: bolck copolymers instructed by double H-bonding association duplex of “molecular glue”. In this study, four kinds of intelligent block copolymers instructed by “molecular glue” system: redox-responsive copolymers PEG-PLA, targeted and redox-responsive copolymers CSO-PLA/FA-CSO-PLA, dual reductive and oxidative stress responsive copolymers PEG-DMTK-SS-PLA, tribolck copolymers PEG-PLA-PEG/PLA-PEG-PLA, and studied their self-assembled micelles for drug delivery. The main results are shown as follows.1) Dynamic covalent bolck copolymers instructed by double H-bonding association duplex of “molecular glue”Instructed by double H-bonding association duplex of “molecular glue” that allow reversible and unsymmetrical disulfide bond formation, hydrophilic(PEG) and hydrophobic(PLA) polymer chains are efficiently coupled into amphiphilic diblock copolymers. The presence of complementary H-bonding duplex in the linking units serves to desymmetrize the otherwise symmetrical disulfide-bond forming process, which ensures the controlled coupling of the PEG and PLA blocks under thermodynamic conditions, while minimizing undesired self-coupling of the same blocks. With its thermodynamic(reversible) nature and mild conditions involved, this strategy should not be limited to the PEG-PLA diblock copolymers described here. Instead, the same coupling method should be general to the controlled coupling of many other polymer blocks that otherwise are not compatible. By taking advantage of the sequence-programmability of the H-bonded and disulfide-linked duplexes we developed, association units having different specificities should allow the efficient coupling of oligomeric or polymeric blocks of desired number and properties into redox-responsive block copolymers under reversible conditions.2) Redox-responsive micelles self-assembled from dynamic covalent block copolymers PEG-PLA for intracellular drug delivery.Redox-responsive micelles self-assembled from dynamic covalent block copolymers PEG-PLA with double disulfide linkage in the backbone have been developed successfully. The amphiphilic block copolymers PEG-PLA associated with complementary double H-bonding duplex of “molecular glue” can self-assemble into spherical micelles in aqueous media with size from 34 nm to 107 nm. Moreover, in vitro drug release analyses indicate that reductive environment(DTT) can result in triggered drug release profiles. The glutathione(GSH) mediated intracellular drug delivery was investigated against HeLa human cervical carcinoma cell line. Flow cytometry and fluorescence microscopy measurements demonstrated that the micelles exhibited faster drug release to nucleus with intracellular glutathione(GSH). Cytotoxicity assay(MTT) of DOX-loaded micelles indicated the higher cellular proliferation inhibition against HeLa cells with intracellular GSH-mediated. These reduction-responsive, biodegradable and biocompatibility micelles could provide a favorable platform to construct excellent drug delivery systems for cancer therapy.3) Chitosan oligosaccharide copolymer micelles CSO-PLA/FA-CSO-PLA instructed by “molecular glue” for targeted intracellular drug delivery.Folic acid(FA) conjugated chitosan oligosaccharide(CSO) polylactic acid(PLA) copolymer CSO-PLA/FA-CSO-PLA with double disulphide linkage in the backbone directed by double H-bonding association duplex of “molecular glue” was synthesized, its self-assembled micelles was evaluated as smart targeted drug delivery carrier. Both of the intermediates and the terminal copolymers were characterized by 1H-NMR and gel permeation chromatography(GPC). The critical micelle concentration(CMC) value is 0.071 and 0.045 mg/m L which suggests the micelle is highly stable in dilute solution. TEM and DLS measurement further confirmed the successful formation of micelle with average size of 61 and 100 nm for blank and DOX loaded micelles, respectively. The micelle was destructed under reductive environment(DTT) led to encapsulated drug released. Moreover, fluorescence microscope measurements demonstrated that micelle exhibited both of passive and active targeting ability in He La cells due to EPR effect and folate-mediated endocytosis. These results suggested the micelle would provide a favourable platform for constructing excellent drug delivery systems for cancer therapy.4) Dual reductive and oxidative stress responsive block copolymer micelles PEG-DMTK-SS-PLA instructed by “molecular glue” for drug delivery.Novel amphiphilic block copolymer PEG-DMTK-SS-PLA with dimethyl thioketal(DMTK) and disulfide linkage incorporated into the backbone were synthesized, which can self-assemble into dual stimuli-responsive micelles. The CMC values of these copolymer micelles with different molecular weights from 0.051 to 0.087 mg.m L-1 suggest that they are highly stable in dilute solution, the conclusion was further confirmed by stability evaluation with DLS measurements. TEM measurements showed well defined micellar structures with average diameters of 74 nm and 55 nm for DOX-loaded and blank micelles respectively. Moreover, in vitro drug release analyses demonstrated that both oxidative(H2O2) and reductive(GSH) environment can trigger drug release from these micelles. The low cytotoxicity of these micelles was confirmed by the MTT assay using NIH 3T3 cells even for concentrations up to 500 μg/m L. Fluorescence microscopy, flow cytometry and MTT activity analysis further showed that the PEG-DMTK-SS-PLA micelles were degraded faster in the presence of reactive oxygen species(ROS) and reductants glutathione(GSH) in HeLa cells than that of mPEG-PLA(without any sensitive linkage) or PEG-PLA(with only double disulfide linkage in the backbone) micelles. These results indicate that the dual-responsive micelles are promising for efficient anticancer drug delivery.5) Triblock copolymer PLA-PEG-PLA/PEG-PLA-PEG instructed by “molecular glue” and its micellization.Triblock copolymers PLA-PEG-PLA/PEG-PLA-PEG with complementary double H-bonding duplex of “molecular glue” and disulfide linkage in the backbone have been developed successfully. The amphiphilic triblock copolymer PEG2000-PLA5000-PEG2000 can self-assemble into spherical micelles in aqueous media with the critical micelle concentration(CMC) value is 0.022 mg/m L which suggests the micelle is highly stable in dilute solution. TEM and DLS measurement further confirmed the successful formation of micelle with average size of 47 and 84 nm, for blank and DOX loaded micelles, respectively. The micelle was destructed by DTT indicate that reductive environment can result in triggered drug release profiles. So, these reduction-responsive micelles could provide a favorable platform to construct drug delivery systems for cancer therapy.