Synthesis, Characterization And Application In Drug Delivery System Of PH-responsive MPEG-PCL-PGA

Since the rapid development achieved in synthetic chemistry, synthetic biodegradable polymer has been widely applied in biomedical field. Poly-L-lactic acid (PLA)、poly(ε-caprolactone)(PCL)、poly(lactide-co-glycolide)(PLGA) and other conventional synthetic polymeric materials are easily to be obtained and non-toxic, these blocks are very promising in constructing biomedical materials. Recently, a rising interest has been paid to a new polymer-polypeptide, which is not only because of their metabolizable degradation units, but also their ability of changing secondary conformation in response to the outer stimulates, such as pH and temperature. The ability of changing secondary conformation of polypeptide will lead to the reversal of solubility, which usually cause the dissolution/precipitation transition of aggregates and this make the polymer cself-assemble into more controllable aggregates.Amphiphilic copolymers usually comprise two or more homopolymer blocks which contain at least one hydrophilic and one hydrophobic block. This amphipathy make them be able to self-assemble into different aggregates such as micelle, polymersome and nanoparticle as long as they have the appropriate hydrophilic molar percentage. Different from micelle and nanoparticle, polymersome is a hollow sphere that contains an aqueous cell in the core surrounded by a bi-layer polymeric membrane. The aqueous core can be utilized for encapsulation of the therapeutic molecules such as proteins and peptides to protect then from being deactivated by outer stimuli. Polymersomes have high membrane stability and low membrane permeability compared to liposomes. These are mainly due to the thicker membrane of polymersomes. It is reported that the membrane thickness of liposomes is typically4～5nm, while the hydrophobic membrane thickness of polymersomes can be engineered to exceed10nm by simply varying the hydrophobic block molecular weight. Besides, the membrane properties of polymersomes can be controlled and modulated by changing the species and lengths of copolymer blocks.Doxorubicin hydrochloride is widely used in clinic for cancer patients. It is a highly effective anti-neoplastic agent and usually used to treat several of adult and pediatric cancers, such as leukemia, lymphomas and breast cancer. The successful use of doxorubicin has been hampered by its toxicities such as hematopoietic suppression, nausea, vomiting, extravasation, alopecia, and the main side-effect is cardiotoxicity. In order to reduce the toxicities of doxorubicin the drug molecules are usually encapsulated into the drug vehicles, which can not only decrease the amount of drug distributed in normal tissues, but also can take advantage of the lower pH value of cancer tissue to deliver more drug molecules to the target spots. Therefore, using a pH-sensitive material to carry doxorubicin is very promising. In addition, it has been reported that encapsulateing doxorubicin in polymersome can decrease the doxorubicin-induced cardiotoxicity.In the present thesis, we synthesize the pH-responsive copolymer, mPEG-PCL-PGA, which can spontaneous self-assemble into polymersomes in aqueous solution. Monomethyl poly (ethylene glycol) mPEG is selected as the hydrophilic block because it can resist plasma protein adsorption, and the carriers with a PEG brush on the surface are general to obtain a prolonged blood circulation. Hydrophobic poly (ε-caprolactone)(PCL) generally presents a soft coil structure, and the ester linkage made it easy to be metabolized by the various enzymes in the body. Poly (L-glutamic acid)(PGA) can change its secondary conformation with the change of pH. At acidic pH, the PGA block is neutralized and its secondary conformation changes from a charged coil to a neutral and more compact a-helical structure. The structure variation is accompanied with a decreased solubility and this change makes the polymersome release more drug molecules in cancer tissues. The main methods and results are as follows.1. Synthesis, charaterization and aggregation behaviour of mPEG-PCL-PGAmPEG-PCL-PGA is synthesized in three steps. Firstly, block copolymer PEG-PCL-OH is prepared by ring-opening polymerization (ROP) of ε-caprolactone using methoxy-poly (ethylene glycol) as an initiator and stannous octanoate as a catalyst. It is reported synthesizing a polypeptide with low polydispersity requires a much faster initiation step than the growing step, which can be met only for sterically unhindered primary aliphatic amines. As the synthesis of homo-polypeptides is generally initiated by primary amines, we need to convert the-OH end-group of mPEG-PCL-OH into-NH2by linking mPEG-PCL-OH with Phe-NBOC, after removing the tert-butoxycarbonyl (BOC) group, mPEG-PCL-NH2is obtained. α-amino acid N-carboxyanhydrides (NCAs) are highly reactive amino acid derivatives which have been widely used for synthesizing polypeptides. Ring-opening polymerization (ROP) of NCAs is the most favorite route to synthesize polypeptides. NCAs have the advantages of simultaneously possessing the activated CO group and the protected amino group. Thus we synthesize mPEG-PCL-PGA by using mPEG-PCL-NH2as macroinitiator and carry out the ROP of y-benzyl-L-glutamate-N-carboxyanhydrides (BLG-NCA). After removing the benzyl protecting group, mPEG-PCL-PGA is obtained. The structures of the polymers are characterized by’H-nuclear magnetic resonance (1H-NMR), fourier transform infrared spectrophotometry (FTIR). Surface tension measurement is performed to determine the surface activity and critical aggregation concentration (C AC) of the copolymer. The results indicate that the surface tension of water can be reduced to about46mN/m and the CAC of the amphiphilic copolymer is about3.6mg/mL. The morphology of aggregate is detected by transmission electron microscopy (TEM).2. Preparation, properties and drug release of doxorubicin-Ioaded polymersome In the present thesis, we prepare doxorubicin-loaded polymersome by nanopreparation method, and observe the morphology of the aggregates by negative dying using2.0wt%phosphotungstic acid solutions under the transmission electron microscopy (TEM). The effect of feed weight ratio of polymer and drug to the encapsulation efficiency and drug loading capacity is also investigated. The best weight ratio of copolymer and doxorubicin is about10:1.5considering the economic benefit and the stability of the polymersome. At this ratio, the doxorubicin loading capacity and encapsulation efficiency are (10.4±0.5)%and (78.7±1.4)%, respectively. During the in vitro drug release, three different phosphate buffer solution (pH7.4、6.5and5.0) are selected to simulate the nomal physiological environment and internal environment of tumor cells. It can be seen that more amount of drug are released from the polymersomes with the decrease of the pH value, indicating that the polymersome is pH-dependent. In addition, drug release from the polymersome is much slower than that from the stock solution. Therefore, doxorubicin release from the polymersome is controllable.3. In vitro anti-tumor evaluation of the doxorubicin-loaded polymersomeThe cytotoxicity of polymer solution and doxorubicin-loaded polymersome on the growth of MCF-7, A549are investigated using WST-1colorimetric method to evaluate the antineoplasmic activity in vitro. The results show that the polymer solution is far less toxic comparing with the doxorubicin stock solution and doxorubicin-loaded polymersome. The inhibition rates of polymer to both MCF-7and A549are higher than doxorubicin stock solution. The data are listed as follows: for MCF-7, the inhibition rate of highest doxorubicin concentration(500μg/mL) of polymer were (25.7±1.5)%(72h)、(17.1±3.4)%(48h)、(12.8±2.7)%(24h); for A549, the corresponding data were (36.3±1.7)%(72h)、(19.6±3.4)%(48h)、(11.3±2.7)%(24h). While that value of highest doxorubicin concentration from doxorubicin stock solution and polymersome are as below:to MCF-7, the value are (86.7±4.1)%(72h)、(79.9±7.3)%(48h)、(71.2±4.9)%(24h) for doxorubicin stock solution and (93.4±3.4)%(72h)、(89.7±2.3)%(48h)、(79.0±3.4)%(24h) for doxorubicin-loaded polymersome; for A549, the value are (84.2±2.2)%(72h)、(76.5±6.7)、(70.2±3.0)%(24h) for doxorubicin stock solution and (86.3±0.9)%(72h)、(84.4±7.2)%(48h、(75.0±2.5)%(24h) for doxorubicin-loaded polymersome. From the above data, it also can be seen that doxorubicin-loaded polymersome can kill more cancer cells than doxorubicin stock solution within the same time.4. In vivo studies of multifunctional polymersome delivery systemWistar rats with about (200±20) g body weight are used in this study. Both free Dox solution and Dox-loaded polymersome are administrated by IV route at single dose (7mg/kg body weight). At designated times, the blood samples are drawn from the sinus jugularis immediately separated by centrifugation. Acetonitrile is used to remove the endogenous proteins. Finally, the supernate is injected for HPLC analysis. The data for pharmacokinetics in rats are analyzed with DAS2.0. According to the data given by DAS2.0, it can be seen that the area under the curve (AUC) of drug-loaded polymersome (2081.621) is much higher than that of doxorubicin (1591.873). In addition, the mean residence time (MRT) of polymersome (1.897h) was twice longer than that of Dox (0.948). These results clearly show that the PolyDox exhibits a good controlled-release property in vivo.In general, we successfully synthesize the pH-responsive amphiphilic block copolymer mPEG-PCL-PGA which could self-assemble into stable polymersome for carrying doxorubicin. It could not only increase drug concentration in vivo, but also release more drugs in cancer tissues to kill the cancer cells. This study provides some research foundation for studying more effective and safer pH-responsive drug vehicle.