Abstract
Amphiphilic block copolymers, methoxy polyethylene glycol-b-poly valerolactone (mPEGPVL) and methoxy polyethylene glycol-b-polycaprolactone (mPEGPCL), were synthesized via ring-opening polymerization of delta-valerolactone and epsilon-caprolactone in the presence of methoxy polyethylene glycol (mPEG). The mPEGPCL was end-modified with phosphorylcholine (PC) for methoxy polyethylene glycol-b-polycaprolactone (mPEGPCLPC). The mPEGPVL, mPEGPCL, and mPEGPCLPC copolymers were successfully synthesized by detection using Nuclear Magnetic Resonance, Fourier Transform Infrared spectroscopy and Gel permeation Chromatography. These copolymers could form micelle-like nanoparticles and the micellar formation was confirmed by 1H-NMR spectra in CDCl3 and D2O. The sizes of all micelles were smaller than 200 nm as measuring by dynamic light scattering detection. The morphology of micelles was spherical shap by using the transmission electron microscope and atomic force microscope analyses. The longer hydrophobic chains (PVL or PCL segments) could reduce the critical micelle concentration (CMC) of micelles, increase the particle size of micelles, and form the more stable micelle structures. Stability analysis showed that the E5000V5000 micellar solutions maintained their sizes at 37 oC for forty-two days without aggregation or dissociation, this micelle had the stable micellar structure. Using two methods to incorporate camptothecin (CPT) into the micelles, the experiments exhibited that the lyophilization method was better than the evaporation method. The CPT encapsulation efficiency was higher than 80 %, and CPT concentration was more than 0.8 mg/ml. The sizes of CPT loaded polymeric micelle formulations (CPT-PM) were smaller than 200 nm using dynamic light scattering detection. The CPT-PM could continuously release CPT for twenty-six days by using dialysis bag method. Using direct dilution method, the CPT-PM could keep 30% biologically active CPT lactone form for sixteen days. These results suggested that the CPT-PM could slow release and reduce the CPT hydrolysis rate in the water solution and maintain more CPT lactone. Using L929 mouse muscular cell line for cytotoxicity, the mPEGPVL, mPEGPCL and mPEGPCLPC copolymers showed higher than 90% relative cell viability, suggesting these copolymers have low cytotoxicity. Incubation of polymeric micelle or CPT-PM solutions with rabbit RBC showed no hemolysis phenomenon, indicating these copolymers have high hemocompatibility.The CPT-PM had similar cytotoxicity as well as CPT free drug using MTT assay with ES-2 ovarian cancer cell line, showing that the polymeric micelles did not reduce the toxicity of CPT. In the pharmacokinetic study, CPT-PMs raised the value of area under curve (AUC), form 16 hr*ng/ml increased to 62-77 hr*ng/ml, that is more than 4.8 times. CPT-PMs also elongated the blood circulation half time (t1/2), form 0.25 hr increased to 0.25-2.39 hr, that is more than 9.6 times. The CPT-PM-E5000C2000PC was the best formulation in the pharmacokinetic study. This formulation was used to do the animal efficacy study. The result of the animal efficacy study exhibited that CPT-PM-E5000C2000PC at CPT 9 mg/kg dose, Topotecan drug at 10 mg/kg dose, and CPT-PM-E5000C2000PC at CPT 18 mg/kg dose, had evident anti-tumor activity. The tumor inhibition rates (TIR %) were 88%, 77% and 97% after twenty-seven days of the cell embedding in mice. The anti-tumor activity of CPT-PM-E5000C2000PC at CPT 18 mg/kg dose was significantly better than Topotecan drug. Taken together, the mPEGPVL, mPEGPCL and mPEGPCLPC copolymers could be used as the drug delivery systems and for the therapeutic fields.