| Cancer has become a major killer which threatens the human’s life seriously, and chemotherapy is the primary means of cancer therapy. However, chemotherapeutics usually has the characteristics of poor water solubility, high toxicity and low bioavailability. To solve these problems, research on drug delivery system (DDS) based on nanocarriers emerges as the times require. Among them, as the most common form of DDS, polymeric micelles can well solubilize insoluble chemotherapeutics, and realize targeted drug delivery and controlled release. This dissertation aims to construct the rumor-targeted and stimuli-responsive polymeric micelles. The properties, tumor targeting and therapy in vitro and in vivo of these micelles have been evaluated.In the chapter 2, the thermo-responsive Pluronic F127-poly(D,L-lactic acid) (F127-PLA, abbreviated as FP) copolymers were successfully synthesized through ring-opening polymerization. The micelles were prepared by a solvent evaporation method. The drug loading content (LC), encapsulation efficiency (EE), particle size, lower critical solution temperature (LCST), opaque temperature and precipitate temperature of micelles increased with increasing of PLA block. The critical micelle concentration (CMC) of micelles decreased with increasing of PLA block. The drug loaded in micelles can be triggered release by both acid pH and hyperthermia. However, only the FP100 micelles with a LCST between 37℃ and 40℃ possess an ideal drug release property which could remain stable under normothermia but rapidly release drug upon hyperthermia. Therefore, FP100 micelles were chosen to decorate with folate (FA) for the cell test. FA-FP100 micelles have almost the same particle size, morphologies, thermo-responsive property and drug release profile compared with FP100 micelles. The cellular uptake study indicated that FA-FP100 micelles could be specifically internalized by folate receptor (FR) overexpressed HeLa cells instead of FR-negative A549 cells. Live/dead staining and Alamar blue assay demonstrated that the FP100 and FA-FP100 blank micelles possess excellent cytocompatibility. The viability of 3T3 cells and HeLa cells treated with micelles were above 90% up to the micellar concentration of 100μg/mL. The hyperthermia of 40℃ significantly improved the cytotoxicity of DOX-loaded micelles against HeLa cells. Finally, the Annexin V-FITC/FI Apoptosis Detection kit was used to stain the HeLa cells after incubation with DOX-loaded micelles, and the apoptotic cells were enumerated by FACS. The result indicated that the DOX-loaded micelles at 40℃ induced more cells apoptosis than that at 37℃.In the chapter 3, the FA-targeted and pH-responsive polymer-drug conjugate FA-hyd was successfully synthesized by decorating folate on the hydrophilic segment of poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL), and subsequently conjugating DOX to the hydrophobic segment of polymers by an acid-instable hydrazone linker. The micelles were prepared by a solvent evaporation method, and they are uniform spheres with a particle size around 70 nm. At pH 5.0, the hydrazone bonds in FA-hyd could be broken, the micellar size increased and drug release could be accelerated; the polymer-drug conjugate remained stable at pH 7.4. The hemolysis assay, coagulation measurements and Alamar blue assay indicated that the FA-PECL blank micelles possess good biocompatibility. The cellular uptake and cytotoxicity studies showed that FA-hyd micelles could specifically recognize and kill FR-positive KB cells. The drug biodistribution indicated that FA-hyd micelles significantly increased the accumulation of drug in tumor but decreased in normal tissues. The pharmacokinetic parameters showed that the area under the curve (AUC) of FA-hyd micelles was greater than other groups, while the volume of distribution (Vd) and clearance (CL) were slower and smaller, and the elimination half-life (τ1/2β) was only faster than that of FA-cbm micelles. These results indicated that FA-hyd micelles obviously prolonged the blood circulation of drug. The FA-hyd group showed the slowest growing of tumors, little changes in body weight and the longest survival time after continuous treatment of the FA-targeted and pH-responsive polymer-drug conjugates to the Balb/c mice bearing 4T1 tumor. The results indicated that the FA-hyd micelles possess the best antitumor activity and highest safety. The tumors were excised at the end of the treatment period for H&E staining. The FA-hyd group showed the most obvious characteristic of apoptosis, which further confirmed its excellent therapeutic effect at tissue level.In the chapter 4, the FA-targeted and pH/reduction-dual responsive mPEG-PLA-ss-PEI/FA-DMMA(PELE/FA-DA) terpolymers were successfully synthesized. Material Studio software was used to stimulate the reaction process between PEI and FA, the result proved that FA was more likely to react with the end primary amine of PEI, which was conductive to be exposed on the surface of micelles for FR recognition. The MS stimulation correlates well with experimental results. The PELE/FA-DA micelles could resistant BSA absorption, resulting in a good stability in blood circulation. At pH 6.8, the amide bonds in PELE/FA-DA could be broken, the micellar size increased, and the surface negative charge reversed to positive charge; the terpolymer remained stable at pH 7.4. The disulfide bond in PELE/FA-DA could be cleaved triggered by 10 mM GSH, PEI was subsequently desheilded from the micelles, resulting in the increase of particle size and disassembly of micelles. From the TEM images, we can see the micellar structure was destroyed and the micelles rapidly aggregated when the PELE/FA-DA micelles were incubated with 10 mM GSH at pH 5.0. The DOX released from the micelles could be obviously accelerated by acid pH and reductive agent. PELE/FA-DA micelles possessed higher cellular internalization rate and cytotoxicity at pH 6.8 than that at pH 7.4. The drug biodistribution indicated that PELE/FA-DA micelles significantly increased the accumulation of drug in tumor but decreased in normal tissues. The pharmacokinetic parameters showed that the AUC of PELE/FA-DA group was greatest, while the Vd and CL were slowest and smallest, and the T1/2β was longest among all groups. These results indicated that PELE/FA-DA micelles obviously prolonged the blood circulation of drug. The in vivo antitumor study showed that the mice bearing 4T1 tumor treated with the DOX-loaded FA-targeted and pH/reduction-dual responsive micelles possessed the slowest tumor volume, slightly increased body weight and the longest survival time compared with other groups, indicating the best antitumor activity and highest safety of these micelles. The tumors were excised at the end of the treatment period for H&E and TUNEL staining. The PELE/FA-DA group showed the fewest tumor cells and most obvious characteristic of apoptosis as well as the highest apoptotic rate, which further confirmed its excellent therapeutic effect at tissue level.In the chapter 5, the molar ratio of polymers was adjusted based on chapter 4 to design the micelles with nuclear targeting ability. mPHG-PI.A-ss-PEI-DMMA (PHLEss-DA) was also chosen as the carriers, the difference from chapter 4 was that the molecule weight and molar ratio of mPEG and PLA were changed. We found that the PELA micelles formed by mPEG5000-PLA4000 possessed a particle size smaller than nuclearpores. Thus, this copolymer was further reacted with PEI and DMMA to synthesize PELESS-DA. The micelles prepared by solvent evaporation method were uniform spheres with a particle size around 40 nm, and the LC and EE were above 10% and 70%, respectively. When the pH decreased from 7.4 to 4.5, the PELEss-DA micelles showed a change in the surface charge from negative to positive and increased in particle size. The micellar size decreased after incubation with 10 mM GSH, and became the same as that of PELA micelles after 2 h. The disulfide bond in PELEss-DA could be cleaved triggered by 10 mM GSH, PEI was subsequently desheilded from the micelles. The DOX released from the micelles could be accelerated by acid pH. PELEss-DA micelles could be internalized more by MCF-7 cells and MCF-7/ADR cells at pH 6.8 than that at pH 7.4, and DOX could be delivered to the nucleus of the cells by the size changeable micelles. Besides, the cytotoxicity of the drug-loaded micelles was bigger at acidic condition. The Annexin V-FITC/PI staining proved that the DOX-loaded PELEss-DA micelles at pH 6.8 induced more MCF-7/ADR cells apoptosis than other groups. The CLSM images of tumor frozen tissue sections showed that the DOX-loaded PELEss-DA micelles accumulated in the tumor tissue at the highest levels among all groups, and were mostly located in the nucleus. The in vivo antitumor study showed that the nude mice bearing MCF-7/ADR tumor treated with the DOX-loaded size changeable micelles possessed the slowest tumor volume, slightly increased body weight and the longest survival time compared with other groups, indicating the best antitumor activity and highest safety of these micelles. The tumors were excised at the end of the treatment period for histological analysis as well as Western blot and RT-PCR test. H&E and TUNEL staining showed that the PELEss-DA group presented significant nuclear condensation and fragmentation, and the highest level of cell apoptosis, which confirmed its excellent therapeutic effect at tissue level. Western blot and RT-PCR proved that the DOX-loaded PELEss-DA micelles led to the most significant up-regulation of Caspase-3 and down-regulation of Bcl-2 at protein and gene levels, respectively. |
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