Synthesis And Studies On Injectable Thermosensitive PLGA-PEG-PLGA Triblock Copolymers

Temperature-responsive polymers are a kind of novel smart polymer carriers as a result of the development of the biomaterials science. They are soluble in water below the gelation temperature and form free flowing liquid, but respond to small change in temperature stimuli and form physically crosslinked hydrogels by sol-gel phase transition. The polymer aqueous solutions undergo sol-gel transition by physical process avoiding of the usage of organic solvent or chemical reaction which may be harmful to the sensitive drugs. Thus, this kind of polymers hold high potential as injectable sustained release carriers of drug delivery systems. We have synthesized the temperature-responsive PLGA-PEG-PLGA triblock copolymers possessing the suitable gelation temperature and investigated the safety, biocompatibility, degradability in vitro and in vivo and the ability to solubilize the water-insoluble drugs. We also investigated the drug release behavior from the hydrogel and the parameters influencing the drug release. We selected interleukin-2 (rhIL-2) as model drug to study the feasibility of the copolymers used as the sustained carriers of sustained delivery of protein and peptide drugs.The PLGA-PEG-PLGA copolymers with DL-lactide/glycolide molar ratio ranging from 6/1 to 15/1 were synthesized by the copolymerization of DL-lactide, glycolide and polyethylene glycol (PEG) for the first time and characterized by IR, ¹³C-NMR, and ¹H-NMR. The ¹H-NMR spectrum data indicated that the DL-lactide/glycolide ratio in the copolymers were closed to the monomers added. The copolymer aqueous solution have suitable gelation temperatures between 30 and 37℃. The increase of the copolymer concentration in solutions and DL-lactide/glycolide molar ratio will lead to a decrease of gelation temperature. Some water-soluble additives such as PEG will influence the gelation temperature of the copolymer solutions.Dynamic rheological experiments were used to characterize the state of the copolymer solution at different temperatures. The copolymer solution was low viscosity free flowing liquid far below the gelation temperature. The sol-gel transition process was rapid and characterized by the rapid increase of elastic modulus and viscous modulus. The copolymer solution completed the phase transition at 37℃, and the formed hydrogel showed higher elastic modulus with the increase of DL-lactide/glycolide molar ratio, indicating that the increase of DL-lactide/glucolide molar ratio can enhance the hydrogel strength. The normalization method showed that the purity of copolymer was 99.7ï¼…. The results of copolymer stability tests indicated that the copolymer were stable in the conditions of high temperature and intense light but were sensitive to high humidity.The copolymers have low toxicity and the maximal tolerance dose (MTD) of subcutaneous and intramuscular injection of copolymers in mice was 1000 and 300 times as human clinical dose, respectively. Intramuscular injection of the copolymer solutions had a mild inflammatory reaction and the inflammation was relieved in the fourth week, indicating that the copolymer had a good compatibility.The in vitro erosion period of the copolymer-based hydrogel was about 30 days and the erosion rate decreased with the increasing of the DL-lactide/glycolide molar ratio. The molecular weight decrease of the copolymer during the in vivo degradation test was slower than the in vitro test.The CMC of the copolymers at 25℃and 37℃were determined by the surface tension method. The CMC of the copolymer decreased with the increase of the DL-lactide/glycolide molar ratio and temperature. The copolymer showed good ability to solubilize the water-insoluble drugs such as indomethacin etc. The solubility of the water-insoluble drugs in the copolymer solution enhanced more than several hundreds times compared with that in water. But the copolymer can not solubilize the stradiol and norehtisterone due to the worse compatibility between the loaded drug and core-forming PLGA block. The different drug loaded method showed significant effect on the solubilization efficacy. The solubilization efficacy increase in the order of solvent evaporation method, emulsion-solvent evaporation method and physical mixing method.The 5-fluorouracil (FU), indomethacin (IM) and bee venom peptides (BVP) were selected as model drug to investigate the drug release behavior and the parameters influencing the drug release. The FU (a model hydrophilic drug) was released from the hydrogel over 5 days and IM (a model hydrophobic drug) was released over 30 days with an S shape release profile. The former is diffusion dominant while the latter is diffusion dominant initially followed by the hydrogel erosion dominant release at later stage. The DL-lactide/glycolide molar ratio has less effect on drug release dominated by diffusion but significantly affected drug release dominated by hydrogel erosion and the release rate of IM at later stages decreased with the increase of the DL-lactide/glycolide molar ratio. The results of FTIR analysis and X-ray analysis indicated that the N-H group of BVP was hydrogen bonded with the C=O group of the copolymer. The BVP was released from the hydrogel over 40 days dominated by diffusion. The drug-copolymer interaction was found to significantly retard the BVP release and the degradation of BVP loaded hydrogel and the hydrogel erosion has little effect on the drug release.Interleukin-2 (rhIL-2) was released from the hydrogel over 20 days in vitro without burst effect. The released rhIL-2 in the first week remained high biological activity but decreased with time after 1 week. SDS-polyacrylamide gel electrophoresis showed that IL-2 was released as monomer from the copolymer-based hydrogels and remained the structure integrity. The C_max, T_Max and t_1/2 of the subcutaneous injection of the rhIL-2 sustained formulation (5×10⁶U/kg) was 206.1pg/ml, 36h and 188.4h, respectively. The fraction absorbed in vivo generated by the Wagner-Nelson method was plotted versus the fraction released in vitro at the same time. The in vitro/in vivo correlation equation is Y=0.019X- 0.20, r=0.9870. Correlation coefficient calculated from the calibration curve was more than the critical correlation coefficients, indicating that the in vivo/in vitro correlation is good.The antitumor activity of the rhIL-2 sustained release formulation on hepatic cancer (H₂₂) was investigated by local application in mice and compared with the rhIL-2 solution applications on five consecutive days. Compared with the negative control, the low, medium and high dose of sustained formulation significantly reduced the tumor weight (P＜0.01). The medium and high dose sustained formulation showed no significant difference compared with rhIL-2 solution (P＞0.05).