The In Vitro Injectable Chitosan-based Thermosensitive Hydrogel Scaffold Loaded With Chitosan Nanoparticles Was Used To Evaluate The Sustained Release Performance And Stability Of PDNA-BMP2 In Vitro

Purpose: The aim of the study was to prepare the hydrogel complex which was designed by embedding chitosan nanoparticles(CSn) loaded with bone morphogenetic protein2 plasmid DNA(p DNA-BMP2) into a chitosan(CS)-based hydrogel with ?,?-glycerophosphate(?,?-GP), termed CS/CSn(p DNA-BMP2)-GP. Characterization, in vitro release profile for p DNA-BMP2, structural stability of p DNA-BMP2, were then conducted.Methods: 1. CSn were prepared via ionotropic gelation and morphology were observed by scanning electron microscopy(SEM); The binding of p DNA with CS was visualized using a 0.8% agarose gel electrophoresis method; The encapsulation efficiency(EE) and loading capacity(LC) of p DNA-BMP2 inside the nanoparticles were determined by high-speed centrifugation. 2. The thermosensitive hydrogel CS/?,?-GP was prepared by physical crosslinking method, the complex system CS/CSn(p DNA-BMP2)-GP was synthesize by adding moderate amount CSn(p DNA-BMP2) to CS/?,?-GP under magnetic stirring. Gelation time was determined by the test tube inverting method at 37°C; morphological studies of CS/CSn(p DNA-BMP2)-GP were observed using a scanning electron microscope(SEM). Weighting method was used to evaluate the swelling and degradation of CS/?,?-GP. 3. Phosphate-buffered saline(PBS) and artificial saliva(AS) were used as the release media, DNA content released from CSn(p DNA-BMP2) and CS/CSn(p DNA-BMP2)-GP were determined at specified time points in order to investigate the release kinetics. 4. Samples A, B, C, D were selected from AS and their concentrations were adjusted to 0.4?g/m L, the structural stability of recombinant plasmid p DNA-BMP2 was determined through the evaluation of transformation efficiency of competent E.coli and DNA sequence.Results: 1. Typically prepared CSn and CSn(pDNA-BMP2) showed a spherical and polydispersed nature. The particle size of CSn(p DNA-BMP2) was 500 nm bigger than CSn with 200 nm. The result of agarose gel electrophoresis showed that CSn could bind effectively p DNA-BMP2. Encapsulation efficiency and loading capacity are more than 80% and 30%, respectively. 2. The sol-gel transition time was 5 min in the CS-?,?-GP system, but only 3min when CSn(p DNA-BMP2) was added to the CS-?,?-GP system. The substructures CS/CSn(p DNA-BMP2)-GP displayed a crosslinked network with regular pores and a porous structure(CSn(p DNA-BMP2) was uniformly dispersed in a 3D matrix in the CS hydrogel. Weighting method showed the swelling ratio and degradation was faster in medium of p H4.0 than p H6.8. 3. An in vitro p DNA-BMP2 release test showed that the cumulative release rate of p DNA-BMP2 was much slower from CS/CSn-GP than from CSn in identical release media. In release media with different p H, p DNA-BMP2 release was much slower at p H6.8 than at p H4.0?4. Transformation efficiency of samples were higher than controlled group; DNA sequence analysis showed that there were no insertion, deletions, rearrangements, modifications.Conclusion: 1. CSn has good shape, appropriate particle size and can bind effectively p DNA-BMP2 with high EE and LC. 2. The thermosensitive complex system CS/CSn(p DNA-BMP2)-GP displayed a crosslinked network and good swelling and degradation. The complex had a much slower release rate of DNA compared with CSn(p DNA-BMP2). 3. The thermosensitive complex system CS/CSn(p DNA-BMP2)-GP could protect the structural stability of recombinant plasmid p DNA-BMP2 in order to pave the later functions of p DNA-BMP2.