Synthesis Of Biodegradable Thermosensitive PCLA-PEG-PCLA Hydrogels, Encapsulation Of Or Modified By Peptides, And Potential Medical Applications

Injectable hydrogels are very promising in regenerative medicine due to their high water content, similar modulus to the soft tissues, and their "minimal invasiveness". Among those hydrogels, biodegradable and thermo-sensitive block copolymers composed of poly(ethylene glycol) (PEG) and polyesters are especially attractive, because their building blocks have been approved by FDA as components in implanting materials, and also because they exhibit tunable biodegradability and any chemical reaction has been avoided in gel formation. This kind of physical hydrogels have been tried as vehicles for controlled release of drugs, yet little report concerns their applications as tissue regenerative materials, where more complicated factors such as cell adhesion and wound healing process etc should be taken into consideration.This thesis was initially aimed to extend the physical hydrogel to tissue engineering and make corresponding fundamental investigations of material preparation and modification. In the course of our studies, we also tried its application in post-operational anti-adhesion. We focused on triblock copolymer composed of hydrophilic PEG and hydrophobic poly(?-caprolactone-co-lactide) (PCLA). The dissertation attempted to combine the polymer science, synthetic chemistry, material chemistry and physics, and medicine application together to investigate novel polymeric biomaterials.The main work includes synthesis of PCLA-PEG-PCLA, sol-gel transition of its aqueous solution, evaluation of in vitro and in vivo biocompatibility and biodegradability, synthesis and immobilization of bioactive peptides to the hydrogel as potential tissue engineering material, and the application as barrier materials for the prevention of post-operative adhesion. The main innovative achievements are listed as follows:1. Synthesis of triblock copolymer PCLA-PEG-PCLA, and enabling the amorphous state during storage of the corresponding physical hydrogel and the mild acidity of its degradation product. A series of block copolymers with centered hydrophilic PEG and two hydrophobic polyester blocks were synthesized via ring-opening polymerization. The sol-gel transition temperature of the aqueous polymer solution was controlled between room and body temperatures by adjusting the length of polyester blocks etc. The resulting "room-temperature sol, body-temperature gel" is thus injectable, which brings much convenience in potential medical applications. The polyester block was obtained via random copolymerization of caprolactone (CL) and lactide (LA), to minimize the side-effect of acidic degradation products of using PLA alone, or the non-controllable crystallization-induced gelation by using PCL alone.2. Confirmation of satisfactory biocompatibility of synthetic polymers, good gel persistence and adjustable degradation rate of the corresponding physical hydrogel. To evaluate the basic biocompatibility of the materials, we first carried out in vitro cytotoxicity experiments according to the GB standard protocols, and little cytotoxicity was abserved in the presence of the synthesized polymer. No obvious hemolysis and pyrogen reaction was observed in animal experiments as well. The tissue in the implant site was examined by HE staining analysis, and neither obvious pathological changes nor severe inflammatory reaction was found, which also indicated good biocompatibility of the hydrogel. We detected the degradation process of the polymers under biomimetic physiological condition, and found that the hydrogel could persist for 12 weeks in vitro with a roughly linear decrease of molecular weight of the remaining polymers, different from the exponential decrease of polyesters in a solid and porous form. The hydrogel was also implanted subcutaneously in rabbits, and the in vivo degradation was faster than in vitro, which might be due to the enzyme-assisted degradation besides hydrolysis. Nevertheless the physical hydrogel still kept its integrity for about 6 weeks in vivo, one order of magnitude longer than the conventional Pluronic (PEG-PPG-PEG) hydrogel.3. First trying the thermoreversible PCLA-PEG-PCLA hydrogel in prevention of post-operative adhesions with a success in animal experiments. Post-operative adhesions are very common in surgery, and barrier materials are recognized as the most effective anti-adhesion solution. The biodegradable polymer films have been widely used as barriers; however their solid state limits their applications in laparoscopic surgery, and the anti-adhesion efficacy still remains controversial. Our injectable hydrogel is, in contrast, suitable even for complicated shapes. In vivo applications of this hydrogel in a rabbit model indicates that the hydrogel is very convenient in operation and highly effective in reducing the formation of post-operative intestinal adhesion, which is even better than commercialized PLA films. The excellent anti-adhesion may be from the cell- and protein-resisting PEG segments, which are rich on the hydrogel surface after the amphiphilic polymer self-assembled into micelle and the micelles are percolated into the physical gel.4. Finding that free peptides of adhesive RGD sequence enhanced the efficacy of post-operational anti-adhesion instead of enhancing adhesion, after the peptides were encapsulated in thermoreversible PCLA-PEG-PCLA hydrogel and then released out of the hydrogel. We encapsulated the bioactive peptide cycle(-RGDfK-) in the PCLA-PEG-PCLA hydrogel. The peptides were released in a sustained manner for one week in vitro. A better efficacy of prevention of post-operative adhesion was found in the group of peptide-loaded hydrogels than the group of hydrogels in the rabbit experiments. Although the immobilized RGD peptide enhances cell adhesion on the material surface as reported in the literature, cell adhesion could also be inhibited by RGD if the peptides were free; the released RGD peptide could be further conjugated with the integrins of the inflammatory cells, resulting in a weaker tissue adhesion.5. First parallel immobilization of peptide to the hydrophilic or hydrophobic blocks of amphiphilic polymers, and revealing the effect of immobilizing site in peptide modification of block copolymers on the cell adhesion efficacy. As a potential tissue engineering materials, the hydrogel should be modified to enhance cell viability. To enhance cell adhesion on the gel, a bioactive peptide cyclo(-RGDfK-) was synthesized through Fmoc strategy in the formulism of solid-phase peptide synthesis. To immobilize the peptide onto the polymer chain, we further synthesized an asymmetric photo-reactive linker, with one end to insert into the PEG chain and another end of NHS ester to conjugate with the amino groups from the peptides. Then, we immobilized the peptide to either hydrophilic or hydrophobic blocks of the polymer chain, and compared the cell adhesions on the resulting hydrogel surfaces. Our study has revealed that for the modification of the amphiphilic materials, the bioactive molecules immobilized to the hydrophilic parts is the better choice in molecule design.In spite of injectable hydrogels, some of my studies also concerned manufacture of the pre-shaped porous scaffolds. Double-layered PLGA scaffolds were designed and successfully fabricated, which have provided a reliable technology platform for the further application of such kind of scaffolds in tissue engineering.