Fabrication Of Stimuli-responsive Conjugated Block Copolymer For Drug Delivery And Cell Imaging

Cancer as a malignant disease seriously endangers people's health.Although with the development of medicine and technology,more and more new treatment methods have been developed and applied to the treatment of cancer,chemotherapy is still the main treatment for the time being.Among multitudinous chemotherapeutics,doxorubicin?DOX?has been used as a broad-spectrum antitumor drug and widely used in the clinical treatment of various cancer due to a wide range of biochemical effects on the body which can embed DNA and inhibit nucleic acid synthesis.Unfortunately,the toxicity caused by poor specificity and solubility limits its applications in cancer therapy.By virtue of the continuous development of nanomedicine technology,a new kind of nanoparticle drug delivery system has been generated which effectively overcomes poor solubility and instability of small-molecule drugs by encapsulating anticancer drugs into nanoparticles.Nanocarriers can not only effectively reduce the probability of phagocytosis by reticuloendothelial system but also prolong the circulation time in vivo.Besides,nanoparticles with a diameter smaller than 200 nm can enrich at the tumor site by passively targeting via high permeability and high retention effect?EPR effect?,and the modifiability of nanocarriers can introduce various active targeting ligands to enhance targeting and therapeutic effect.The intricacy of tumor tissue microenvironment brings dilemma to drug delivery and also provides certain opportunities that the introduction of various structural units which can respond to the tumor microenvironment and corresponding changes into polymer carriers can improve the efficacy of chemotherapy by the intracellular destabilization of the carriers and the rapid release of the loaded therapeutic drug.Besides,the introduction of small molecule fluorescent groups or fluorescent polymer segments into the nanoparticles is a desirable approach to fluorescence imaging-based real-time monitoring of intracellular drug release to achieve integration of diagnosis and treatment.Compared with the photobleaching and poor photostability of organic small molecule fluorescent dyes,high toxicity of inorganic quantum dots as well as serious background and photobleaching of fluorescent proteins and aptamers,conjugated polymers?CPs?by virtue of large extinction coefficients,outstanding photostability,tunable optical characteristics,simple synthesis and modification and low toxicity have been extensively adopted for in vitro and in vivo bioimaging applications.Besides,the introduction of hydrophilic groups or polymers to fabricate nanomicelles can improve stability that micelles can be passively targeted by EPR effect while avoiding being swallowed by the RES system,also the conjugated polymers of micelles can contain drugs as a hydrophobic core so as to achieve integration of diagnosis and treatment to improve treatment efficiency.However,the existing research based on conjugated polymers is mainly focused on the synthesis of binary block copolymers containing conjugated segments.Due to the low end-initiation or chain transfer group activity of the polymer segments prepared by continuous controlled radical polymerization,it is difficult to synthesize a ternary block copolymer with a precisely controlled structure and composition containing a conjugated segment.At the same time,due to the instability of dynamic chemical bonds,it is also challenging to synthesize tumor microenvironment-responsive copolymer carriers by introducing dynamic chemical bonds into the structure of conjugated polymers.In order to solve the above two problems,in chapter 2 of this dissertation,we firstly coupled PF-N₃ and Alkyne-PNIPAAm₁₂₀-Br through efficient molecular clicks to obtain PF₁₁-b-PNIPAAm₁₂₀-Br,which was coupled with Alkyne-POEGMA₁₇-Br again to successfully obtain the triblock copolymer PF₁₁-b-PNIPAAm₁₂₀-b-POEGMA₁₇ after azidation.An effective method for the synthesis of ternary block copolymers with conjugated chain segments was developed.In Chapter 3,by directly polymerizing oligo?ethylene glycol?monomethyl ether methacrylic monomer?-OEGMA monomers with acetal bonds,a pH-responsive conjugated block copolymer was successfully prepared.The dynamic bond is introduced into the structure of conjugated copolymer by monomer design.The specific research contents and results are as follows:?1?The first chapter introduces the research background of multifunctional polymer micelle drug delivery system in detail,including the current status of cancer,treatment methods,tumor microenvironment and the advances of various nanoparticle drug carriers,as well as the advances active targeting-based,environmental stimulus-response-based and theranostic-based multifunctional polymeric micellar drug delivery systems.?2?In chapter 2,on the basis of the local high temperature of tumor microenvironment,an amphiphilic triblock copolymer PF₁₁-b-PNIPAAm₁₂₀-b-poly?oligo?ethylene glycol?monomethyl ether methacrylate?₁₇(PF₁₁-b-PNIPAAm₁₂₀-b-POEGMA₁₇)with fluorescence and temperature response was fabricated.We reported the design and controlled synthesis of a PF-and PNIPAAm-based amphiphilic triblock copolymer(PF₁₁-b-PNIPAAm₁₂₀-b-POEGMA₁₇),with a well-defined structure by a strategy of sequential click couplings between suzuki-coupling-generated PF₁₁-N₃ and atom-transfer radical polymerization?ATRP?-produced Alkyne-PNIPAAm₁₂₀-Br and Alkyne-POEGMA₁₇-Br,both of which were initiated by propargyl2-bromoisobutyrate and polymerization-controllable.The successful synthesis of polymer was verified by ¹H NMR,size exclusion chromatography and multiangle laser light scattering?SEC-MALLS?analyses,as the morphology and size of the self-assembled micelles were verified by DLS and TEM that the micelles were 30 nm sphere with narrow distributions.And the lower critical solution temperature?LCST?of the micelles was about 33?investigated by the temperature-dependent changes in hydrodynamic size and optical transmittance.The quantum yield of 12.74%was investigated by ultraviolet absorption spectrum and fluorescence emission spectrum.The drug loading capacity?DLC?of DOX-loaded micelles fabricated at 25 and 37? were respectively 3.5%and 1.9%investigated by UV absorption.The hydrophobicity of PNIPAAm above the LCST contributes to the drug loading capacity of the CSC micelles constructed at 37?.On the contrary,the lower stability of this formulation actually accounts for the significantly decreased drug loading capacity.The optimal micelle formulation should therefore balance the trade-off between the drug loading capacity and the stability issue of micelles.Then the imaging performance and endocytosis were investigated by a fluorescence confocal microscope.Finally,the cytotoxicity and drug release study of drug-loaded micelles at different temperatures were investigated that the drug-loaded micelles incubated in the simulated tumor environment demonstrated faster and more drug release attributed to the hydrophilic to hydrophobic transition of the PNIPAAm middle layer and subsequently somewhat to the aggregation of the CSC micelles.The excellent drug release performance and the low cytotoxicity as well as the good optical properties of the polymer confirmed excellent prospects of PF₁₁-b-PNIPAAm₁₂₀-b-POEGMA₁₇7 in the diagnosis and treatment of cancer.?3?In chapter 3,on the basis of the subacidity of tumor microenvironment,an amphiphilic block copolymer PF₁₁-b-poly??-oligo?ethylene glycol?monomethyl ether methacrylate?₁₅(PF₁₁-b-P??-OEGMA?₁₅)with fluorescence and pH response was fabricated.We reported the design and controlled synthesis of a PF-and P??-OEGMA?-based amphiphilic block copolymer PF₁₁-b-P??-OEGMA?₁₅ with a well-defined structure by a strategy of click couplings between Suzuki-coupling-generated PF₁₁-N₃ and atom-transfer radical polymerization?ATRP?-produced Alkyne-P??-OEGMA?₁₅-Br,as the monomer of?-OEGMA possesses acetal bond previously prepared then initiated by propargyl2-bromoisobutyrate and polymerization-controllable.The successful synthesis of polymer was verified by ¹H NMR,size exclusion chromatography and multiangle laser light scattering?SEC-MALLS?analyses,as the size of about 33 nm,narrow size distribution and sensitive pH response of the self-assembled micelles were verified by DLS.In addition,the excellent optical properties and the drug loading capacity?DLC?of about 4.1%were investigated by ultraviolet absorption and fluorescence emission spectroscopy.Finally,the drug release study of drug-loaded micelles at different pH were investigated that the drug-loaded micelles incubated in PBS buffer at pH 5.0demonstrated faster and more drug release attributed to breakage of the acetal bond in?-OEGMA and subsequently somewhat collapse of the micelles,which confirmed excellent prospects of PF₁₁-b-P??-OEGMA?₁₅ in the diagnosis and treatment of cancer.