Construction Of PCL-b-PEG-b-PCL Polymer Drug Delivery System And Antitumor Study

Cancer is the second worldwide cause of death, exceeded only by cardiovascular diseases. Chemotherapy is one of the most widely used forms in the treatment of metastatic cancers. However, conventional cancer chemotherapy is not effective enough and lacks selectivity between cancer cells and normal cells, thus producing significant side effects to normal tissues and organs. Rapid growth in nanotechnology towards the development of nanomedicine products holds great promise to improve therapeutic strategies against cancer. They can improve the pharmacokinetic and pharmacodynamic profiles of conventional therapeutics and may thus optimize the efficacy of existing anti-cancer compounds. Some representative drug delivery systems in the treatment of cancer include liposome, nanoparticle, nanoemulsion, polymeric micelles, polymersomes, etc.Amphiphilic block copolymers can self assemble into various nanosized vehicles, such as various types of micelles and polymersomes. In this study, two kinds of nano-sized drug delivery system (DDS) including polymeric micelles and polymersomes were successfully developed from poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-b-PEG-b-PCL) copolymers with different hydrophilic/hydrophobic block. Meanwhile, lipid-polymer hybrid nanopaticles were successfully developed based on PCL-b-PEG-b-PCL and was formed as the third nano-sized DDS. Paclitaxel and doxorubicin were used as model hydrophobic and hydrophilic anticancer drugs, both of which are effective and widely used chemotherapeutic drugs. Three kinds of anticancer DDS including PTX-loaded polymeric micelles, folate-modified lipid-polymer hybrid nanoparticles for paclitaxel delivery, and polymersomes loaded with both PTX and DOX were developed and evaluated for their efficiency in cancer chemotherapy.The main research contents are as follows:1. Study on different type of nanosized vehicles formed from PCL-b-PEG-b-PCL amphiphilic block copolymersIn aqueous solutions, amphiphilic block copolymers can self assemble into various nanosized vehicles, such as micelles (spherical, prolate, or oblate) and polymersomes. The type of structure formed is dependent on the relative lengths of the hydrophobic and hydrophilic blocks of the copolymer, molecular weight, as well as on the method of preparation. As for PCL-b-PEG-b-PCL amphiphilic block copolymers, no literature reported its rules of forming polymeric micelles or polymersomes with different hydrophilic/hydrophobic block ratio and molecular weight. In the present study, a series of biodegradable PCL-b-PEG-b-PCL triblock copolymers was synthesized by ring-opening polymerization of ε-CL initiated by PEG. PCL-b-PEG-b-PCL triblock copolymers with ∫ PEG (w) of 50% was found to form polymeric micelles, which was characterized with apparent core-shell morphology. Whereas PCL-b-PEG-b-PCL triblock copolymers with ∫ PEG (w) of 33% was found to form polymersomes, which was characterized with a hydrophobic bilayer membrane and aqueous core.2. Study on paclitaxel-loaded polymeric micelles as intravenous drug delivery system for cancer chemotherapyThe purpose of this study was to develop polymeric nanoscale drug-delivery system (nano-DDS) for paclitaxel (PTX) from poly(εcaprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-b-PEG-b-PCL, PCEP484) copolymers, intended to be intravenously administered, able to improve the therapeutic efficacy of the drug and devoid of the adverse effects of Cremophor EL. Paclitaxel-loaded polymeric micelles (PTX-PM) were prepared by thin-film hydration and ultrasonic method. SEM and TEM indicated that the obtained PTX-PM exhibiting homogeneous spherical shapes with apparent core-shell morphology, which was characterized by a hydrophobic core surrounded by a hydrophilic corona. Hydrophobic PTX was effectively incorporated into the hydrophobic PCL core through hydrophobic interaction. The drug-loading content (DLC) and theencapsulation efficiency (EE) was 28.98% and 94.36%, respectively. The prepared PTX-loaded polymeric micelles exhibited a narrow size distribution with an average diameter of 93 nm and polydispersity index of 0.19, which was favorable for intravenous injection. Differential scanning calorimetry (DSC) study indicated that PTX was in solid amorphous state after being encapsulated in the polymeric micelles. In vitro release study indicated that PTX was released from 1M sodium salicylate solutions in a controlled and sustained fashion, with zero-order release kinetics in the first 5 days. In vitro cytotoxicity demonstrated that the cytotoxic effect of PTX-loaded polymeric micelles was lower than that of Taxol(?) at the same PTX content. The cytotoxicity of Taxol(?) and PTX-loaded polymeric micelles all showed time-and dose-dependent cell proliferation inhibition, confirming that higher drug concentration and longer incubation time are essential for the drug to effectively kill tumor cells. Pharmacokinetic results indicated that the PTX-loaded polymeric micelles had longer systemic circulation time, slower plasma elimination rate and higher bioavailability than those of Taxol(?). PTX-loaded polymeric micelles showed greater tumor growth-inhibition effect in vivo on EMT-6 breast tumor in comparison with that of Taxol(?), with tumor growth inhibition of 85.79% and 63.37%, respectively (P<0.05). In conculsion, the prepared PTX-loaded polymeric micelles showed high anti-tumoral efficacy and low toxicity, as well as long circulation time and higher accumulation in tumors due to EPR effects, and might have the potential to be developed as an effective anticancer drug-delivery system for cancer chemotherapy.3. Study on folate-modified lipid-polymer hybrid nanoparticles for paclitaxel delivery in cancer chemotherapyLipid-polymer hybrid nanoparticles (LPNPs) are polymeric nanoparticles enveloped by lipid layers that combine the highly biocompatible nature of lipids with the structural integrity afforded by polymeric nanoparticles. The purpose of this study was to develop a novel lipid-polymer hybrid drug carrier comprised of folate (FA) modified lipid-shell and polymer-core nanoparticles (FLPNPs) for sustained, controlled, and targeted delivery of paclitaxel (PTX). The NPs consist of 1) a poly(ε-caprolactone) hydrophobic core based on self-assembly of poly(s-caprolactone)-poly(ethylene glycol)-poly(s-caprolactone) (PCL-PEG-PCL) amphiphilic copolymers,2) a lipid monolayer formed with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000),3) a targeting ligand (FA) on the surface, and were prepared using a thin-film hydration and ultrasonic dispersion method. TEM image indicated that the PTX-FLPNPs exhibited homogeneous spherical shapes with an apparent lipid monolayer on the surface. The "core-shell-shell" structure might be that the core is formed with PCL and PTX, while the inner and outer layers are formed with DSPE and extended PEG molecules, respectively. The confocal laser scanning fluorescence image of rhodamine-PE labeled FLPNPs further confirmed the "core-shell-shell" structure created by this procedure. There was an approximate 8 nm increase in the particle size of FLPNPs as compared to the diameter of LPNPs (279.9 nm vs 271.5 nm). The polydispersity of PTX-LPNPs, and FLPNPs demonstrated narrow size distribution, indicating that homogenous hybrid NPs could be developed using this thin film method. The higher negative zeta potential value of the FLPNPs rather than that of the LPNPs (-17.5 mV vs-14.2 mV) results from the deprotonation of the carboxylic groups of folate. Using the thin-film hydration and ultrasonic dispersion method, high DL of more than 26%(w/w) in both of the hybrid NPs could be achieved with high EE of more than 90% when the PTX feed ratio was 30%(w/w). The in vitro drug release profiles showed that PTX was released from both of the formulations in a controlled and sustained fashion with no apparent initial drug burst. The qualitative and quantitative results of cellular uptake of the NPs provide strong evidence for the internalization efficiency and targeting ability of the folate conjugated on the lipid monolayer for the EMT-6 cancer cells which overexpress folate receptor. In vitro cytotoxicity assay against H1299 lung cancer and EMT-6 breast cancer demonstrated that the cytotoxic effect of PTX-loaded FLPNPs was lower than that of Taxol(?), but higher than that of PTX-loaded LPNPs (without folate conjugation). In EMT-6 breast tumor model, intratumoral administration of PTX-loaded FLPNPs showed simiilar antitumor efficacy but low toxicity compared to Taxol(?). More importantly, PTX-loaded FLPNPs showed greater tumor growth inhibition (65.78%) than the nontargeted PTX-loaded LPNPs (48.38%) (P<0.05). These findings indicated that the PTX loaded-FLPNPs with mixed lipid monolayer shell and biodegradable polymer core would be a promising nanosized drug formulation for tumor-targeted therapy.4. Preliminary study on polymersomes loaded with both hydrophobic and hydrophilic drugs for cancer chemotherapyThe polymersome architecture, with its large hydrophilic reservoir and its thick hydrophobic lamellar membrane, provides significant storage capacity for both water soluble and insoluble substances in the synergistic treatment of cancer. In this study, polymersomes with obvious hydrophobic lamellar membrane were successfully developed from PCL-b-PEG-b-PCL (PCEP888) using thin-film hydration and ultrasonic method. PTX was loaded into the core of the polymersome membrane through hydrophobic interactions, while DOX was loaded into the hydrophilic reservoir of the polymersomes by ammonium sulfate gradient method established for liposomes. TEM image of the PS-PTX-DOX showed that the polymersomes had a bilayered lamellar structure and core forming from aggregates of DOX. The confocal laser scanning fluorescence image of further confirmed DOX was encapsulated in the hydrophilic reservoir. The prepared PS-PTX-DOX exhibited a narrow size distribution with an average diameter of 169.7 nm and polydispersity index of 0.211, which was favorable for intravenous injection in the treatment of cancer. In vitro cellular uptake study revealed that the fluorescence of PS-PTX-DOX in the nucleus was much weak than that of DOX after incubating for the same time and dosage of DOX. The red fluorescence intensity in the nucleus increased with the incubation time increased, probably due to the more DOX released from the PS-PTX-DOX and entered in to the nucleus. Ex vivo DOX fluorescence imaging revealed that PS-PTX-DOX had highly efficient targeting and accumulation at the implanted site of EMT-6 xenograft tumor in vivo. The improved delivery of DOX to tumor for PS-PTX-DOX might be attributed to reduced uptake by the RES, the excellent construct stability during the blood circulation and the EPR effect, which would contribute to the enhanced antitumor efficiency.