Synthesis And Characterization Of Acid-sensitive Copolymer Or Polymeric Prodrug For Dox Delivery

Due to the significant biodegradability and biocompatibility, stimuli-responsive polymeric materials have been extensively used in biomedical research field, such as drug delivery, gene therapy as well as tissue engineering. The acid-sensitive polymers possess a smart property which endow them a brilliant potential application in cancer treatment. On one hand, they are stable in physiological neutral environment while the nanoparticles formed by self-assembly can be used to load anti-tumor drugs. On the other hand, the drug-loaded nanoparticles can be dissociated in the acidic micro-environment in tumor tissues, resulting in efficiency and rapid treatment. This thesis is focused on acid-sensitive biomaterials, using different polymers including poly(ethylene glycol), poly(ε-caprolactone), polyphosphoesters and cellulose nanocrystals to fabricate a series of acidic-sensitive drug or gene carriers as well as water-soluble polymeric prodrugs which can be used in cancer therapy. The characterization of chemical structures, self-assembly behavior, drug-loading ability, biocompatibility and hydrolysis behavior were also studied. The main content of present work can be summarized as follows:(1) A new kind of acid-sensitive amphiphilic copolymers have been prepared by using acidic-cleavable linkage to connect the hydrophilic poly(ethylene glycol)(PEG) chain and hydrophobic poly(ε-caprolactone)(PCL) chain. This novel copolymer can be used as drug carrier to deliver the anti-tumor drug doxorubicin(DOX) and release DOX rapidly under the determined condition. First, the terminal of start material PEG was modified by azide group and acetal group through chemical reactions to obtain N3-a-PEG-a-N3. Then using propargyl alcohol as initiator, ε-CL as monomer, Sn(Oct)2 as catalyst to prepare the propargyl-terminated homopolymer(propargyl-PCL) via ring-opening polymerization(ROP). Finally, the modified PEG was used to react with propargyl-PCL via the Cu-Catalyzed Azide-Alkyne Cycloaddition(CuAAC “click” reaction) to prepare the acidic-sensitive polymeric drug carrier. In the neutral condition, these copolymers can self-assemble into micelles to encapsulate hydrophobic anti-tumor drug DOX, while these drug-loaded micelles would dissociate as the result of the cleavage of acetal group in the acidic environment to release the encapsulated DOX. The chemical structures of copolymers were investigated with 1H NMR, 13 C NMR, FT-IR and GPC measurement. TEM and DLS test were employed to observe the self-assembly behavior of copolymers. The biological properties of copolymers were studied by the MTT assay, in vitro drug release and live cell imaging system. The hydrolysis behavior of copolymers was also studied with GPC and DLS measurement. What’s more, the in vitro drug release and intracellular release were also compared with pH-insensitive copolymer.(2) In part II, N3-a-PEG-a-N3 was used to react with 4-nitrophenyl progargyl carbonate(4-NPC) via the CuAAC “click” reaction to prepare the 4-nitrophenyl end-capped and acid-sensitive PEG(NC-a-PEG-a-NC) which was further used to react with DOX through the ammonolysis reaction to get the PEGylated acidic-sensitive DOX prodrug(DOX-a-PEG-a-DOX). The acidic-labile acetal and carbamate groups were used as linkages to connect the hydrophilic PEG and hydrophbic DOX. To avoid some side effects caused by the acidic degradation product of the hydrophobic domains, DOX was directly conjugated to PEG chain without using any hydrophobic polymers. The chemical structures of prodrugs were investigated with 1H NMR and FT-IR measurement. The content of DOX was determined by fluorescence spectrum and HPLC spectrum. The self-assembly behavior of prodrugs was observed with TEM and DLS. The biological properties of prodrugs were studied by the MTT assay, in vitro drug release and live cell imaging system. The hydrolysis behavior of prodrugs under different pH values was investigated through DLS measurement.(3) In part III, the sustainable material, cellulose nanocrystals(CNC), was used to construct a pH-sensitive drug carrier. Firstly, azide group was modified onto the surface of CNC after defulfation and chloridation treatments to obtain CNC-N3. Propargyl-PEEP was then synthesized by ROP using propargyl alcohol as the initiator and EOP as the monomer, followed by CuAAC “click” reaction withCNC-N3. Regarding that this material is negatively charged, it owns a possibility to bind positive charged DOX via electrostatic interaction while this interaction will be destroyed by the attack from ions in cytoplasm and the PEEP will be degraded under the acidic environment, resulting in a pH-dependent release of encapsulated DOX. The chemical structures of materials were investigated with 1H NMR and FT-IR measurement, while the in vitro drug loading and release behavior was studied by fluorescence spectrum. TEM test and zeta-potential were employed to observe the morphologies of materials. The biological properties of materials were studied by the MTT assay and live cell imaging system.(4) To achieve a synergistic gene and drug therapy, in part IV, a combined drug and DNA carrier has been prerared. A functional copolymer, alkynyl-PDMAEMA-co-PHEMA, was first synthesized via atom transfer radical polymerization(ATRP), and then used to react with azide modified cellulose nanocrystals(CNC-N3) through CuAAC “click” reaction to obtain a type of cellulose nanocrystals grafted cationic copolymer. This kind of materials can not only encapsulate DOX, but also condense DNA via electrostatic interaction. The chemical structures of copolymers were investigated with 1H NMR and FT-IR measurement, and the in vitro drug loading and release behavior were investigated by fluorescence test. The morphologies of nanocrystals were observed by TEM while the surface potential of nanocrystals was detected through zeta-potential measurement. The biological properties of materials were preliminary studied by the MTT assay and agarose gel retardation assay.