Controlled Synthesis And In Vitro Evaluation Of Hyperbranched Polymers For Anticancer Drug Delivery

The stimuli-responsive core-shell nanoparticles that can respond to the relevant biological signals of the tumor microenvironment,such as local high fever,weakly acidic pH,and reducing environments,have a wide range of applications in the field of drug delivery and controlled release.Polymers with(hyper)branched topologies have attracted extensive attention and research interest since their discovery because of their unique physical and chemical properties,such as low molecular chain entanglement,low viscosity,and easy multifunctionalization.Due to its unique advanced topological structure,hyperbranched polymers can form stable monomolecular micelles,which can effectively solve the stability of polymer drug carriers in body fluids and blood circulation.This outstanding advantage further makes hyperbranched polymers as carrier materials have great research value and application prospects in drug loading and controlled release.Studies have shown that when the molecular weight(M_w)is constant,hyperbranched polymers(HPs)with higher degree of branching(DB)and lower molecular weight distribution index(PDI)are more suitable for biomedical applications.Currently,two reported strategies are used to prepare hyperbranched polymers:(1)Self-condensing vinyl polymerization mediated by bifunctional monomers or copolymerization of vinyl monomers and crosslinking agents,(2)The stepwise polymerization of AB_x(x?2)monomer or A₂ and B_n(n?3)monomer.However,these methods all exist to varying degrees in the process of preparing hyperbranched polymers as the target polymer M_w or DB increases,the larger polymer PDI appears.These problems undoubtedly restrict the convenient and controllable synthesis of hyperbranched polymers and its biomedical applications.In order to solve the above problems,this dissertation focuses on the two preparation methods mentioned above from the construction of secondary in-situ branching and template hyperbranched polymers.Hyperbranched polymer with no significant change in PDI with increasing M_w and DB,precise control of the degree of branching(DB)and adjustable molecular weight M_w was prepared.The structures of monomers and polymers were characterized by nuclear magnetic resonance spectroscopy(¹H NMR,¹³C NMR)and size exclusion chromatography combined with multi-angle laser light scattering analyzer(SEC-MALLS).Dynamic light scattering(DLS)and Transmission electron microscopy(TEM)were used to study the size and morphology of polymer micelles.Finally,the anti-tumor effects of drug-loaded nanoparticles in vitro were evaluated through in vitro drug loading and release studies,cytotoxicity and in vitro cell uptake.The specific research contents are as follows:1)In the second chapter of the dissertation,we designed and prepared a two-headed initiator 2-((2-((2-bromo-2-methylpropionyl)oxy)ethyl)disulfanyl)ethyl methacrylate(BSSMA),this monomer uses disulfide bonds to bridge polymerizable double bonds and ATRP initiators.BSSMA and the small molecule cross-linking agent ethane-1,2-diacrylate(DDA)have undergone RAFT copolymerization to obtain a reduction-sensitive hyperbranched polymer BP-P(BSSMA-st-DDA)(BP),then we reduced the dithiocarbonate groups around the BP with a large amount of AIBN,because the branched structure contains vinyl residues of the crosslinking agent,secondary branching reactions occur simultaneously during the reduction process to generate secondary in-situ branched copolymer SBP-P(BSSMA-st-DDA)(SBP).Compared with BP,the M_w of the secondary in-situ branched copolymer increased by nearly two times,while the increase of PDI was very small.In order to provide sufficient micelle stability,SBP was used as a macroinitiator to perform ATRP polymerization of OEGMA to prepare an amphiphilic hyperbranched block statistical copolymer SBP-P(BSSMA-st-DDA)-b-POEGMA.In this study,a simple and robust strategy was developed to prepare stimuli-responsive hyperbranched polymers with no significant change in PDI with increasing M_w and DB for effectively carry out anticancer drug delivery,polymer micelles loaded with doxorubicin(DOX)showed intracellular reduction-promoted drug release,effective cell uptake,and inhibition of HeLa cell proliferation.2)To reduce the cumulative release of DOX of the drug-loaded polymer micelles prepared in the second chapter under physiological conditions of pH=7.4,in the third chapter of the paper,we introduced not only hydrophilic segments but also polymerization of hydrophobic monomers on the periphery of the hyperbranched core.Similarly,dihydroxy disulfide was used as the starting material,and a single-end esterification reaction with methacryloyl chloride was used to obtain the reduction-sensitive double-headed reagent 2-((2-hydroxyethyl)disulfanyl)ethyl methacrylate ester(HDSMA),the monomer and small molecule crosslinking agent are copolymerized at 70?to produce hyperbranched polymer HBP-P(HDSMA-st-DDA),and then using the polymer as the hyperbranched core,the ring-opening of caprolactone and the RAFT polymerization of DMAEMA were successively obtained to obtain the amphiphilic multi-sensitive hyperbranched star copolymer HBP-PCL-PDMAEMA.The variable temperature particle size of the polymer under different pH conditions was studied by DLS,the morphology of the self-assembly of the amphiphilic hyperbranched copolymer was characterized at different temperatures by TEM.Finally,to confirm the potential of HBP-PCL-PDMAEMA in the controlled release of drugs,the drug release of the drug-loaded micelles under the four conditions of PBS(pH 7.4+10 mM),pH 2.2,pH7.0 and PBS(pH 7.4)was measured by fluorescence spectrophotometer and ultraviolet-visible spectrophotometer respectively.The system is based on the preparation of multiple sensitive polymers based on hyperbranched structures,and it has research significance in applications such as self-assembly and controlled drug release.3)In order to better prepare hyperbranched polymers with precisely controlled branching degree(DB)and adjustable molecular weight M_w,in the fourth chapter of the paper,we successfully designed and synthesized trifunctional AB₂ monomer,which has both A-SS-B₂ chain transfer agent(CTA-AB₂)with multifunctional reagents and high reactivity.These include a reduction-sensitive disulfide bond for polymer degradation to promote intracellular release of drug-carrying compounds,a general trithiocarbonate group that can be used for RAFT polymerization of vinyl monomer,and a terminal active group consisting of an azide group and two alkynyl groups.In the presence of CuSO₄?5H₂O and sodium ascorbate(NaVc),AB₂ monomer generates a hyperbranched polymer template(HPT)through a step-by-step polymerization reaction.HPT has a precisely adjusted DB and multiple CTA units,which can be used for any vinyl RAFT polymerization of monomers.Next,HPT was used as a macromolecular chain transfer agent for the RAFT polymerization of OEGMA to prepare the amphiphilic hyperbranched copolymer HPT-POEGMA,and the in vitro properties of anticancer drugs were studied.This trifunctional AB₂monomer provides a powerful way to easily prepare bioreducible hyperbranched polymers with precisely controlled DB and can be used for controlled release applications.4)Due to the cumbersome preparation steps of AB₂ monomers and the unavailability of raw materials will limit the application of hyperbranched polymers,in the fifth chapter of the paper,we respectively synthesized the A₂ monomer with reduction-sensitive disulfide bond and the trithiocarbonate group that can undergo RAFT polymerization and the B₃ monomer with three alkynyl groups,and the two monomers are gradually polymerized by clicking to obtain the hyperbranched polymer HBP.Subsequently,we prepared the acid-sensitive hydrophilic monomer?-OEGMA containing acetal bonds,the monomer uses the hyperbranched polymer prepared above to carry out RAFT polymerization to prepare the amphiphilic dual-sensitive hyperbranched copolymer HBP-P(?-OEGMA).Finally,the polymer as a drug carrier in vitro drug loading and release under different conditions and the cytotoxicity to HeLa cells were studied.The experimental results show that the breaking of the disulfide bond on the main chain can achieve the degradation of drug-loaded micelles more than the breaking of the acetal bond on the side chain,thereby increasing the cumulative release of the drug.