New Strategies On Self-assembly Of Glycopolymers And Their Bio-applications

Glycopolymer has been widely studied recent years, including polymerization, self-assembly and bio-application. However, some aspects do not receive enough attention, for example, lots of glycopolymers do not have clear configuration which makes it difficult to establish structure-function relationships; glycopolymer just act as a hydrophilic stabilizer in self-assemble systems in which carbohydrate chemistry has not been fully utilized; application of the self-assembled entities was limited to drug delivery other than immune system in which carbohydrate plays extremely important role. As we have keen interest in macromolecular self-assembly for years and developed "block-copolymer-free" strategy recent years, new strategies in self-assembly of glycopolymers were developed as well as their bio-application. Based on this, the research in this thesis was carried out, with contents as follows:1:Glyco-Inside Micelles and Vesicles directed by Protection-Deprotection Chemistry.Protection-deprotection of carbohydrate is often required in preparation of glycopolymers, which causes an obvious polarity change of the polymers, but it has been neglected in the studies of self-assembly. A new strategy for self-assembly of sugar-containing block copolymers is suggested based on the protection-deprotection chemistry. To be specific, a series of well-defined block copolymers of PS-b-PManAc (PS, polystyrene block; PManAc, "sugar block" with acetylated a-mannopyranoside side groups) were obtained via two-steps RAFT (Reversible Addition Fragmentation chain Transfer) polymerization. The acetyl groups were removed as soon as the catalyzer TABOH (tetrabutylammonium hydroxide) was added which resulted in glyco-inside structures of the deprotected copolymer PS-b-PMan, i.e. vesicles with a sugar wall and micelles with a sugar core since hydroxyl group has little solubility in THF. Besides, vesicle-to-micelle transition of the assemblies with decreasing the relative length of the sugar block was observed. These unique glyco-inside assemblies show interesting functions such as generating homogeneous Au nanoparticles (1-3 nm) within the membrane from AuCl4- without any additional reducing reagents or energy input. If the solution was switched from THF to water what we observed is the concomitance of glyco-inside to glyco-outside inversion and the transformation between vesicles and micelles.2:Polymeric vesicles mimicking glycocalyx (PV-Gx) for studying carbohydrate-protein interactions in solution.Dynamic covalent bond could be formed between carbohydrate and phenyl boronic acid in basic solution which could be used as the driving force to construct the non-covalently connected micelles (NCCM) or vesicles (NCCV). Based on that the following system was built, trying to establish structure-function relationships of glycopolymers. To be specific, for one thing, two novel P-D-pyranosyl acrylamide monomers β-MGal and β-MGlc were synthesized and polymerized into two well-defined homo-polymers via RAFT; for another, the temperature sensitive polymer poly(N-isopropyl acrylamide) with phenyl boronic acid end BA-PNIPAM was obtained. Polymeric vesicles mimicking glycocalyx PVG (a carbohydrate coat on cell surface which is extremely important in a variety of biological processes) could be achieved once the temperature was above the LCST of PNIPAM. PVG is composed of hydrophilic glycopolymer and hydrophobic PNIPAM with the outer and inner surface layers of sugars, where the two homo-polymers are connected by the dynamic covalent bonds between phenylboronic acid and sugars. The distinctive anomeric linkage of carbohydrates renders the PVGs the specificities in their interactions with different proteins. Three plant lectins PNA (Peanut agglutinin lectin), ECA (Erythrina cristagalli) and Con A (Concanavalin A) were chosen and then dynamic light scattering (DLS) was employed for the first time to monitor the binding process between the sugars on PVGs and the lectins in situ, showing remarkable advantages of DLS as a versatile, label-free and solution-based method.3:In vitro and in vivo targeting of peritoneal macrophages via glyco-nanoparicles.Immunocyte has lots of carbohydrate targets in the membrane including Mannose Receptor (MR) and Macrophage Galactose Lectin (MGL) et al. As the receptors expressed in the immunocyte membrane were so sensitive to the biological environment that this aspect was not fully understood yet. In this thesis, four glyco-nanoparticles M-Man, M-Fuc, M-Gal and M-Lac were prepared as well as the PEG modified one M-PEG as control, trying to screen the carbohydrate recognition domain (CRD) expressed in murine peritoneal macrophages. For one thing, the thick carbohydrate shell of the glyco-nanoparticles avoided the hard-detection caused by low local concentration of carbohydrate in lots of cases. For the other, the multi glyco-nanoparticles’screen get rid of the illusion caused by the non-specific interaction. It was found that all of the glyco-nanoparticles could be recognized by the macrophages and endocytosed mediated by the recepors both in vitro and in vivo which confirmed that there are both MR and MGL in macrophages. What’s more, the endocytosis was mediated by both caveolin and clathrin.4:Tailoring the immune response by targeting C-type lectin receptors on peritoneal macrophages using glyco-nanoparticles.The expression of CD206 down-regulated as the co-culture of glyco-nanoparticles with peritoneal macrophages which mean the phenotype changes from M2 to M1.14 cytokines and chemokines were chosen for the real-time PCR which demonstrated that the glyco-nanoparticles could increase the expression of pro-inflammatory cytokines and varied from sugar to sugar.