| Backgrounds: Regional anesthesia has always been a crucial part of clinical anesthesia and pain medicine. And nerve block owns the characteristics that easy to operate, keep patients conscious and the protective reflex still, cheap, relatively high safety and analgesic effect, which hold extremely an important position in clinical anesthesia. However, single injection of local anesthetics can merely maintain 6-8 h, which cannot meet the demand of postoperative analgesia. To extend the local anesthetic effect from a single injection, we made hydrophobic alkaline of bupivacaine from clinically common ultilized water-soluble local anesthetics, and package it into biodegradable, temperature-sensitive hydrogels. Applying the new kind of local anesthetics encapsulation agents to extend the effect of sciatic nerve block has been researched, at the same time, the degradation of hydrogels and the release rate of encapsulated anesthetics has also been explored.Methods: 1. Preparation and characterization of bupivacaine free baseWe made hydrophobic alkaline of bupivacaine from clinically common ultilized water-soluble local anesthetics.The morphology was obtained by scanning electron microscopy(SEM) technology and the difference between BUP and BFB was also compared. BFB was added to hydrogels at 4 ° C kept stirring until completely mixed.The change of morphology and rheological mechanics about hydrogels with or without BFB were also compared.In vitro drug release was carried out at the temperature of 37 ° C, PBS was taken out regularly and the concentration of BFB released into the PBS media was measured and the release curve was thus gotten.2. Evaluation of block effect Rats underwent sciatic nerve block, sensory blockade was tested through the hot plate experiment and motor block was evaluated by observing body movements of block side at different time points after the treatment. Criteria was set to evaluate the effect of motor block.3. Biocompatibility evaluationBy observing local tissue adhesion, biological rejection such as fibrous capsules at the site of injection, the biocompatibility was evaluated. The muscle and nerve tissue biopsiesnear the injection site were obtained, local response of inflammatory,toxicity of muscles and nerves were examined, a comprehensive evaluation of the new formutions ‘safety was known. Our approach utilizing a hydrophobic local anesthetic and a bio-erodible thermoresponsive hydrogel matrix together to yield a promising multi-faceted delivery system with the ability to provide long-term analgesia.Results: 1. Preparation and characterization of BFBWe obtained a low water soluble BFB by adding Na OH to commercial BUP·HCl. Rapid precipitation of the amorphous, insoluble BUP base was observed during the alkalization. Through SEM analysis, the hydrochloride salt form of BUP appears as a thick crystal bulk with dimension of about 100- 187 μm(Fig. 1A). After alkalization, we found that the microstructure of BUP appears on a nano-scale, with a diameter < 1 μm and a length ranging from 17- 30 μm(Fig. 1B).2. Assessments of thermogels without and with BFBA phase graph(Fig. 2) was used to determine the proper concentration(Pluronic F127 20 wt.%., PAla-PEG-PAla 8 wt.%. and PLGA-PEG-PLGA 20 wt.%.) to make sure the hydrogels existed in a flowing sol state at room temperature and turned to non-flowing gel states once injected.As shown in Fig. 3, the critical temperature based on rheological measurements coincided well with what obtained by means of vial inversion. After BFB was added, the temperature associated with sol-gel transition increased as expected.Moreover, the micron-scale surface patterns and protrusions of freeze-dried hydrogels with or without BFB were detected by SEM(Fig. 4). All of the hydrogels presented a continuous and porous channel structure. Once BFB was added, a tentacle-like appearance was evident within the matrices, except for the PLGA-PEG-PLGA hydrogelMoreover, the effectiveness of the encapsulated formulation was further investigated by in vivo and in vitro release behavior. None of the groups demonstrated burst release during the initial 2 h. Pluronic F127 persisted less than 12 hours with a cumulative release amount of 92.52%. During the first 12 h, 19.01% and 14.76% of the encapsulated BFB were released(respectively) from PAla-PEG-PAla and PLGA-PEG-PLGA hydrogels. As seen in Fig. 5, proteolytic activity accelerates the degradation rate of the hydrogel matrix and release of BUP. Among the three hydrogels, PAla-PEG-PAla was most influenced by elastase.3.In vivo nerve blockade efficiencyAll animals injected with BFB hydrogels had maximal sensory blockade during the first 10 minutes, which implies the onset time was not affected or prolonged by the effect of controlled release. The order of sensory recovery(earliest to last) was as follows: BUP·HCl > PLGA-PEG-PLGA > PAla-PEG-PAla > Pluronic F127; this reflected the recovery process from complete sensory block to normal. Motor function appears to return to normal faster than sensory function.4.Local tissue responses at the injection siteNo local abnormality or gelatinous residue was observed in the group treated with BUP·HCl or Pluronic F127 +BFB. The gross appearance of the surgical sites in rats treated with PAla-PEG-PAla and PLGA-PEG-PLGA was notable: hydrogel residue was notably apparent adjacent to the sciatic nerve and tissue planes were adherent to one another.As shown in Fig. 10. Pluronic F127 Group showed a greater extent of inflammation compared to the BUP·HCl Group. No obvious histological abnormality was seen in the BUP·HCl group. An inflammatory response was apparent in the PAla-PEG-PAla and PLGA-PEG-PLGA groups, demonstrating profound fibroblastic hyperplasia, necrosis, and myositis. Hyperemia and collagenous hyperplasia could be found widely in the PAla-PEG-PAla group. Nerve damage was not present in any group.Conclusion: Choosing a biomaterial with a residue time similar to that of the drug’s intended duration may improve the therapeutic benefits while minimizing toxic side effects. |
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