| Polyurethanes are one of the most important synthetic biomaterials that are widely used in various medical devices including heart valves, catheters, vascular grafts, and prostheses. Taking advantage of their excellent biocompatibility and physical properties, polyurethanes in particular degradable polyurethanes have been explored for different biomedical applications such as controlled drug release and tissue engineering. In order to attain degradability, polyurethanes are typically prepared by polyaddition reaction of degradable polyester or polycarbonate diols with diisocyanate. It should be noted, however, that these degradable polyurethanes generally exhibit a slow degradation rate, which does not match controlled drug release applications. In this thesis, triblock copolymer micelles based on novel acid degradable poly(acetal urethane)(PAU) were designed and developed for p H-triggered intracellular delivery of doxorubicin(DOX). Through c RGD conjugation, c RGD-decorated PEG-PAU micelles exhibited enhanced internalization and anticancer drug efficacy. Besides p H-sensitivity, dual responsive PEG/PU nanocarriers showed p H and reduction-sensitivities in the cytosol, leading to improved drug release behavior and anticancer activity. The contents in this thesis are as follows:In chapter 1, we give a brief introduction about nanocarriers, polyurethanes and its application in biomedical field, stimuli-sensitive nanoparticles, as well as the advantage of targeted micelles and its application.In chapter 2, we developed p H-responsive biodegradable micelles with novel acid degradable PAU as the core and PEG as the shell for p H-triggered intracellular delivery of DOX. PAU with Mn ranging from 4.3 to 12.3 kg/mol was conveniently prepared from polyaddition reaction of lysine diisocyanate(LDI) and a novel diacetal-containing diol, terephthalilidene-bis(trimethylolethane)(TPABTME) using dibutyltin dilaurate(DBTDL) as a catalyst in N,N-dimethylformamide(DMF). The conjugation of PAU with PEG(Mn = 5 kg/mol) at both ends afforded PEG-PAU-PEG triblock copolymers that readily formed micelles with average sizes of about 90-120 nm in water. The dynamic light scattering(DLS) measurements revealed fast swelling and disruption of micelles under acidic p H. UV/vis spectroscopy corroborated that acetal degradation was accelerated at p H 4.0 and 5.0. The in vitro release studies showed that doxorubicin(DOX) was released in a controlled and p H-dependent manner, in which ca. 96 %, 73 %, and 30 % of drug was released within 48 h at p H 4.0, 5.0, and 7.4, respectively. Notably, MTT assays displayed that DOX-loaded PEG-PAU-PEG micelles had a high in vitro antitumor activity in both RAW 264.7 and drug-resistant MCF-7/ADR cells. The confocal microscopy and flow cytometry experiments demonstrated that PEG-PAU-PEG micelles mediated efficient cytoplasmic delivery of DOX. Importantly, blank PEG-PAU-PEG micelles were shown to be non-toxic to RAW 264.7 and MCF-7/ADR cells even at a high concentration of 1.5 mg/m L. Hence, micelles based on p H-sensitive degradable poly(acetal urethane) can be regarded as a biocompatible and efficient nanocarrier for intracellular drug delivery.In chapter 3, we designed and synthesized the c RGD-conjugated p H-responsive biodegradable c RGD-PEG-PAU-PEG-c RGD micelles for high efficient internalization and intracellular DOX release. The conjugation of PAU with dual-funtionalized PEG(HOOC-PEG-SH, Mn = 6.5 kg/mol) at both ends afforded HOOC-PEG-PAU-PEG-COOH triblock copolymers. Finally, the polymer c RGD-PEG-PAU-PEG-c RGD was obtained via amidation reaction of HOOC-PEG-PEG-PAU-PEG-COOH and c RGD-NH2. The c RGD-conjugated micelles were constructed from PEG-PAU-PEG and c RGD-PEG-PAU-PEG-c RGD block copolymers, in which c RGD-PEG-PAU-PEG-c RGD was designed with a longer PEG than that in PEG-PAU-PEG(6.5 vs 5.0 k Da) to fully expose c RGD ligands onto the surface of micelles for effective targeting to human glioblastoma U87 MG cells. PEG-PAU-PEG combining with 0, 10, 20 or 30 % of c RGD-PEG-PAU-PEG-c RGD formed uniform micelles with average sizes of 117-128 nm(denoted as PAU, PAU/10 T, PAU/20 T, PAU/30 T micelles). MTT assays in U87 MG and MCF-7 cells showed that DOX-loaded PAU/30 T micelles exhibited apparent targetability and significantly enhanced antitumor efficacy toward αvβ3 intergin overexpressing U87 MG cells with a particularly low half maximal inhibitory concentration(IC50) of 4.21 μg DOX equiv/m L which was 2-fold higher than free DOX·HCl and approximately 4-fold lower than that for nontargeting counterparts under otherwise the same conditions. Interestingly, confocal microscopy observations using DOX-loaded PAU/30 T micelles showed that DOX was efficiently delivered and released into the perinuclei of U87 MG cells in 4 h. Flow cytometry revealed that cellular DOX level in U87 MG cells treated with DOX-loaded PAU/30 T micelles was much greater than that with nontargeting micelles, signifying the importance of active targeting. Ligand-directed, p H-responsive biodegradable micelles have emerged as a versatile and potent platform for targeted cancer chemotherapy.In chapter 4, we designed c RGD-conjugated p H and reduction dual responsive PEG/PU micelles for high efficient internalization and quick intracellular drug release. Dual responsive polyurethane(SSPU(TMBPE)) developed from polyaddition reaction of novel disulfide-containing diisocyanate(CDI) and 2,4,6-trimethoxybenzylidene-pentaerythritol(TMBPE) using dibutyltin dilaurate(DBTDL) as catalyst in N,N-dimethylformamide(DMF). After conjugation with PEG at both side, PEG-SSPU(TMBPE)-PEG was successfully synthesized. By conjugation with dual-funtionalized PEG(NHS-PEG6.5k-NCO)(Mn = 6.5 kg/mol) and further amidation reaction with c RGD-NH2, c RGD-PEG-SSPU(TMBPE)-PEG-c RGD was obtained. The c RGD-conjugated dual responsive micelles were constructed from PEG-SSPU(TMBPE)-PEG and c RGD-PEG-SSPU(TMBPE)-PEG-c RGD block copolymers for effective targeting to human glioblastoma U87 MG cells. PEG-SSPU(TMBPE)-PEG combining with 0, 10, 20 or 30 wt % of c RGD-PEG-PAU-PEG-c RGD formed uniform micelles with average sizes about 120 nm(denoted as SSPU(TMBPE), SSPU(TMBPE)/10 T, SSPU(TMBPE)/20 T, SSPU(TMBPE)/30 T micelles). The in vitro release studies showed that about 100 % of DOX was released in 12 h from dual sensitive micelles at p H 5.0 in the presence of 10 m M(GSH), while 50 % of DOX release was observed from single sensitive micelles under the same conditions. In contrast, minimal DOX(<15 %) was observed for and dual sensitive micelles under the physical condition(p H 7.4). MTT assays in U87 MG showed that DOX-loaded SSPU(TMBPE)/30 T micelles exhibited apparent targetability and significantly enhanced antitumor efficacy toward U87 MG cells with a particularly low IC50 of 2.16 μg DOX equiv/m L, which was comparable to free DOX·HCl, 4-fold lower than nontargeting dual sensitive micelles and 8-fold lower than single sensitive micelles under otherwise the same conditions. Interestingly, confocal microscopy observations using DOX-loaded SSPU(TMBPE)/30 T micelles showed that DOX was more efficiently delivered and released into U87 MG cells than nontargeting single sensive and dual sensitive micelles in 4 h. Flow cytometry revealed that cellular DOX level in U87 MG cells treated with DOX-loaded SSPU(TMBPE)/30 T micelles was much greater than that with nontargeting dual sensitive micelles and single sensitive micelles as controls, signifying the importance of combining dual responsiveness and active targeting. Ligand-directed, p H and reduction dual responsive biodegradable micelles have emerged as a versatile and potent platform for targeted cancer chemotherapy.In chaper 5, a comprehensive summary and a future perspective of this thesis work were provided. |
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