Bioactive Galactose-functionalized Double- Hydrophilic Copolymers With And Their Thermoresponsive Self-assembly Dynamics

Glycopolymers with sugar moieties are attractive as functional biomaterial because of excellent biocompatibility and ability to selectively recognize lectin proteins.Thermoresponsive double-hydrophilic block glycopolymers contain pendant sugar residues that can be exposed or hidden via an externally controlled coil-to-globule phase transition around the lower critical solution temperature.When the temperature is above the LCST,the glycopolymers self-assemble into micelles,and conversely,the micelles dissociate into individual molecules below the LCST.The reversible display of specific sugar moieties in the glycopolymers can easily be regulated by changing the system temperature,and the LCST value of a thermoresponsive glycopolymers can be properly modulated by adjusting the relative amounts of monomers.Although various studies have shown that the double-hydrophilic block glycopolymers can assemble into different structures,in the case of thermoresponsive double-hydrophilic block glycopolymers,their self-assembling behavior has never been systematically investigated.Thus a series of thermoresponsive double-hydrophilic block glycopolymers were synthesized by reversible addition-fragmentation chain transfer polymerization,composed of a thermoresponsive block and a galactose-functionalized block.Then,we systematically investigate their thermoresponsive self-assembly behavior,to demonstrate the existence of strong interactions between the micellization and LCST,depending on the concentration and structure of the macromolecules and system temperature and the study on thermoresponsive self-assembly behavior would provide theoretical basis for biology application.Three galactose monomers with different linkers of carbon numbers prepared by enzymatic synthesis,then thermosensitive monomer poly（diethyleneglycol methacrylate）as macro chain transfer agent,we obtain a serial of thermosensitive double-hydrophilic block glycopolymers with different linkers of carbon numbers such as PDEGMA-b-POVSGal,PDEGMA-b-POVNGal-1,2,3,PDEGMA-b-POVZGal by RAFT polymerization.FT-IR and ¹H-NMR analysis for the structure and composition of glycopolymers,GPC test to get molecular weight and molecular distribution of polymers.The results showed that well-defined thermosensitive block glycopolymers with narrow polydispersity were prepared by RAFT polymerization,PDI was about 1.3,which indicated RAFT polymerization was controllable.Thermoresponsive double hydrophilic block glycopolymers PDEGMA-b-POVNGal-1,2,3with increasing OVNGal content were selected as our research objects.We systematically investigated their thermoresponsive self-assembly behavior and micelle morphology in aqueous solution,using UV-Vis spectrophotometer,static and dynamic light scattering techniques,combing with TEM imaging.Due to presence of two possible transitions,micellization and LCST,their interactions could generate much interesting and richer self-assembling phenomena.The low critical solution temperature（LCST）of PDEGMA-b-POVNGal-1,2,3 was investigated by two methods,UV-Vis spectrophotometer and static light scattering technique,this temperature-dependent transition of thermosensitive glycopolymers is completely reversible,The LCST value decreases as OVNGal increases.At f_OVNG=8.8%,the LCST value is 36.0?C,at f_OVNG=11.0%,the LCST value reduce to 30.5?C,The LCST value of a thermoresponsive copolymer can be properly modulated by adjusting the relative amounts of monomers.The CMT value increases and the R_g value of Micelle-1 decreases as OVNGal increases,indicating that the POVNGal block is unfavorable in forming micelles.It is due to the increase of hydrogen bonds between hydroxyl groups of galactose and water which retard the process of phase transition with the increase in OVNGal content.The results of light scattering and TEM show that,for high concentrations glycopolymers,starting from individual molecules,as temperature increases,they first form micelles after reaching the critical micellization temperature（CMT）,with a R_g value around 30 nm,which are very similar to those typical amphiphilic surfactants,referred to as Micelle-1.Then,as temperature increases to reach the LCST,a new type of bigger micelles are formed,with substantially larger sizes,which are referred to as Micelle-2.The shape of Micelle-2 depends on the size of the POVNGal block,the ration of R_g/R_h can conjecture the morphology of the micelles,TEM images further confirm generate micelles which is prolate ellipsoids or hollow spheres or non-hollow spheres,but the thermoresponsive self-assembly behavior of Micelle-2 does not depend on the polymer concentration.For relatively low concentrations of polymer,the LCST-type phase transition proceeds from individual molecules,instead of Micelle-1.In this case,the LCST-induced micelles are different from those formed from Micelle-1,which are referred to as Micelle-3.The shape of Micelle-3 depends not only on the size of the POVNGal block but also on the glycopolymer concentration,which can form micelles with either crystal-like or spherical or hollow-spherical.With the tunable LCST value,together with the systematically study of thermoresponsive self-assembly,we believe that the synthesized thermoresponsive double-hydrophilic block glycopolymers would have potential applications in biological processes.Finally,we explored the possibility of thermoresponsive double-hydrophilic block glycopolymers in biological applications,the micelle solutions of PDEGMA-b-POVNGal-2 were selected as research object,we studied specific recognition to lectin,cytocompatibility and the influence of hepatoma cells after binding with lectin.The results showed the lectin-binding ability of our synthesized thermoresponsive double-hydrophilic block glycopolymers is still active after RAFT polymerization,which can selective recognize RCA₁₂₀ and PNA,and the recognition ability binding with RCA₁₂₀ is stronger.Glycopolymers we synthesized has good cytocompatibility because of including a large quantity galactose units and can promote the cells growth well.But when polymers bind with RCA₁₂₀ and PNA,L-02 cells are normal growth,they are able to promote the human hepatoma SMMC-7721 cells aggregation and death,and binding with RCA can inhibit growth SMMC-7721 cells more effectively.Confocal laser scanning microscope and flow cytometry also proved that PDEGMA-b-POVNGal-2-RCA₁₂₀/PNA could targeted with SMMC-7721 cells,and PDEGMA-b-POVNGal-2-RCA₁₂₀ can targeted with SMMC-7721 cells and induce cells death effectively.