Construction of Unimolecular Micelle Aggregates in Nanoscale and Its Application in Drug Delivery and Cell Imaging

Author:Sun Pei

Supervisor:zhu xin yuan lu yun feng


Degree Year:2018





Recent years have witnessed tremendous progress in the area of nano-delivery system.Up to now,as a promising delivery vehicle,synthetic polymeric nanocarriers have been widely applied to deliver small molecule drugs,aiming to achieve better water solubility,prolong blood retention time,and improve the accumulation of agent in the desired location.However,the efficacy of the polymer micelles based on linear amphiphilic block polymers is often limited by their instability in vivo due to the change of external environment,especially the dilution of blood,which will lead to the burst release of the escaped agents and the loss of their nano-characters.In addition,considering the stability of nano-delivery system in vivo,small molecular drugs are usually covalently conjugated to polymer carriers via a“post-synthesis”strategy,which requires multiple reaction steps with limited yield,and has limited control of the site and degree of functional reagents loading.A preferable approach is,therefore,the“bottom-up”strategy,which proceeds by reversing the conventional“post-synthesis”approach and instead directly incorporates the drugs during the synthesis of the polymer by the polymerization of polymerizable prodrug monomers.With this strategy,the resulting polyprodrug can load drugs into the polymer matrix homogeneously and control the drug loading readily by adjusting the feed ratio of monomer/chain transfer agent,which is very suitable for the preparation of biomaterials with higher requirements for repeatability.In this thesis,we have constructed a series of amphiphilic hyperbranched polymers-based unimolecular micelles through the"bottom-up"strategy,and realized their applications in drug delivery and cell imaging.This thesis is divided into four parts,and the specific research contents and conclusions are summarized as follows:1.“Bottom-up”construction of drug-loaded unimolecular micelle aggregates and its anticancerr activity evaluationDespite the great advantages of polymer-drug conjugates(PDC)in cancer therapy,control of the drug loading site and degree via a facileapproach remains a great challenge.Herein,by combining the controllability of the“bottom-up”strategy and the stability of multiarm hyperbranched amphiphiles,we have developed novel multi-polyprodrug-arm hyperbranched amphiphiles(H40-star-(PHCPTMA-b-PMPC),hPCM)via reversible addition-fragmentation chain transfer(RAFT)polymerization for cancer therapy.The hPCM was constructed via two-step polymerization of an acid-labile prodrug monomer and a zwitterionic monomer,respectively.By using an H40 macroRAFT agent,10-hydroxycamptothecine(HCPT)prodrug monomers were directly polymerized via the“bottom-up”strategy as a polyprodrug-arm inner-shell of hPCM with homogeneous drug distribution.The drug loading content can be facilely tuned through variation of the feed ratio of HCPTMA/H40 macroRAFT agent.Finally,the poly-zwitterionic hydrophilic outer-shell of hPCM was formed by RAFT polymerization of zwitterionic monomer to ensure preferable biocompatibility.By dissolving in dilute solution,unimolecular micelles of hPCM can be obtained,which endow desirable stability for the micelles.The effective cellular internalization,extended blood retention time,considerable accumulation in tumor tissue,and excellent anticancer activity of the hPCM micelles have been evaluated both in vitro and in vivo.2.“Bottom-up”construction of hyperbranched poly(prodrug-co-photosensitizer) amphiphiles unimolecular micelles and its application in chemo-photodynamic dual therapyDespite the great advantages of chemo-photodynamic combination therapy,tedious synthesis steps and laborious purification procedures make the fabrication of chemo-photodynamic combined therapeutic platforms rather difficult.In this study,we develop a facile “bottom-up” strategy to fabricate hyperbranched poly(prodrug-co-photosensitizer)amphiphiles,hPCBE,for chemo-photodynamic dual therapy.The easily prepared hPCBE possess a bottom-up-constructed hydrophobic core hPCB direct copolymerized from reduction-responsive CPT prodrug monomerand boron dipyrromethene-based photosensitizer monomer,as well as abiocompatible shell polymerized from hydrophilic monomers.Because of the covalently interconnected core-shell structure,hPCBE exists as unimolecular micelles in aqueous solution and exhibits excellent structural stability under dilution condition.The hPCBE micelles can be effectively internalized by MCF-7 cells and release CPT triggered by the reductive milieu.In addition,photosensitizer moieties embedded in the hPCB core could generate singlet oxygen(1O2)effectively under irradiation,endowing hPCBE with the boosting of chemotherapeutic efficacy.As compared to the single chemotherapy of hyperbranched polyprodrug amphiphiles hPCE and photodynamic therapy of hyperbranched polyphotosensitizer amphiphiles hPBE,hPCBE shows higher in vitro cytotoxicity.We expect that our approach will further boost research on the design of multifunctional drug delivery systems via the facile“bottom-up”strategy.3.Construction of BODIPY-functionalized fluorescent hyperbranched glycopolymers for hepatoma-targeted imagingGlycopolymers-based targeted fluorescent nanoparticles(FNPs)based have good prospects in the field of cancer diagnosis and treatment because of their designability,processability,as well as high biocompatibility and especially enhanced protein binding affinity termed as the“glycoside cluster effect”.However,the synthesis of such there are many shortcomings in the current synthetic methods,such as FNPs often suffer from complicated synthetic steps with limited yield,and has limited control of the site and degree of functional reagents loading,which will lead to the dense packing of the fluorescent molecules and thus fluorescence quenching.Herein,a novel type of multivalent and highly specific fluorescent hyperbranched glycopolymers hPGVB has been designed and prepared successfully via a facile“bottom-up”strategy.The acetylated hPGVB was prepared by one-pot RAFT copolymerization of acrylate-type galactose monomers AcGalEA and methacrylate-type fluorescent monomers BYMA in presence of a inimer-type RAFT chain transfer agent.After deacetylation,the resulting amphiphilic hPGVB could self-assemble into stable nanoparticles in aqueous media,showing strong green fluorescence with relative high quantum yields and good photostability.The cell viability study indicates the excellent biocompatibility of the hPGVB FNPs against HepG2 and NIH3T3 cells.More importantly,comparing with the galactose-free fluorescent hyperbranched polymers hPEVB,hPGVB FNPs can be selectively internalized by asialoglycoprotein(ASGP)receptor-rich HepG2cells,indicating their potential application in the bioimaging fields.4.Stable AIE-active unimolecular micelles of hyperbranched conjugated copolymer for long-term cell imagingThis unique feature of the AIE fluorophores makes them ideal candidates in the construction of FPNs for bioapplication.However,most of these AIE-based FNPs are formed from the amphipathic linear polymers above their critical aggregate concentration(CAC).When these polymeric micelles are subjected to physiological diluted solution below their CAC and alterations in other factors such as temperature,pH,and ionic strength,they may disassemble into free polymeric chains,and worse yet,the aggregation state of the AIE fluorophore would be disturbed,which will heavily hinder their fluorescence emission under physiological conditions.Herein,we take the intrinsic advantage of unimolecular conjugated polymeric micelles,develop a stable AIE-active fluorescent unimolecular micelles HCP-star-PEG.The easily prepared HCP-star-PEG possess a tetraphenylethene-based hyperbranched conjugated polymer core HCP,as well as a biocompatible PEG shell.The as-prepared HCP-star-PEG micelles feature strong green fluorescence with robust photo,pH,temperature,storage,biological medium,and especially dilution stability even below the CAC owing to their covalently interconnected core-shell structure.We further demonstrate that the resultant stable HCP-star-PEG micelles with favorable biocompatibility are promising biological probes for long-term cell imaging.