Preparation Of RhBMP-2-rhVEGF Loaded Chitosan Nanospheres And Its Properties In Vitro

ObjectiveUsing chitosan as a carrier to prepare nanospheres, which involved with Recombinant Human Bone Morphogenetic Protein-2(rhBMP-2) and vascular endothelial growth factor (VEGF), at the basic of this, then the properties in vitro of chitosan nanospheres are evaluated for being the basic of researching the influence on biological function and the effect of rhBMP-2-rhVEGF in body.Methods:1.The preparation of chitosan nanospheres:which use ionic Crosslinking method and Tripolyphosphate (TPP) is used of Crosslinking agent. Each nanospheres with rhBMP-2、rhVEGF、rhBMP-2-rhVEGF are prepared after defined the shape range by preparing nothing loaded chitosan nanospheres. Firstly, these factors are tested by orthogonal comparison, such as the concentration of protein, CTS and TPP, and the mass ratio of CTS and TPP. Secondly, The morpha and distribution of nanospheres are observed by Electro Microscope, particle size is tested by particle size Analyzer。2. the entrapment is tested by ELISA kit. Lastly, the accumulation release ratio and its curve are calculated and drew at different timeResults1.The operation and technology of Preparation chitosan nanospheres by ionic Crosslinking method is easy and stability. And chitosan nanospheres are displaying rules of spherical, good dispersibility and particle sizes concentrated.2.The best crafamanship of prepare rhVEGF loaded chitosan nanospheres are defined. The concentration of rhVEGF, CTS and TPP, and the mass ratio of CTS and TPP are lOng/ml,2mg/ml,1.5mg/ml,5:1. The particle size,PDI, Zet-potentio and the entrapment of rhVEGF loaded chitosan nanospheres are346.2±2.4nm,0.219±0.017,24.0±0.9and98.43±1.10%at this condition. 3.The best crafamanship of prepare rhBMP-2loaded chitosan nanospheres are defined. The concentration of rhBMP-2, CTS and TPP, and the mass ratio of CTS and TPP are50ng/ml,3mg/ml,1.5mg/ml,5:1. The particle size,PDI, Zet-potentio and the entrapment of rhBMP-2loaded chitosan nanospheres are330±2.3nm,0.463±0.013,30.0±0.8and92.63±1.33%at this condition.4.The best crafamanship of prepare rhBMP-2-rhVEGF loaded chitosan nanospheres are defined. The concentration of rhBMP-2, rhVEGF,CTS and TPP, and the mass ratio of CTS and TPP are50mg/ml,10mg/ml1mg/ml,1.5mg/ml,5:1. The particle size,PDI, Zet-potentio and the entrapment of rhBMP-2and rhVEGF of rhBMP-2-rhVEGF loaded chitosan nanospheres are396.0±1.8nm,0.403±0.005,29.8±1.8,86.6±0.8%and93.72±1.30%at this condition.5. The vitro of sustained-release curves of three kinds of drug loaded chitosan nanospheres is very commonly. Release doses is1%～2%at24th hour and90%at30th day. And it keeps constant speed increase after24h and gentle after20days.Conclusion:1. The operation and technology of Preparation of rhBMP-2and rhVEGF loaded chitosan nanospheres by ionic Crosslinking method is easy and stability. The concentration of protein、CTS and TPP and the mass ratio of CTS and TPP have a apparent effect on the nanospheres’ size、zeta-potential and the ratio of actual drug loading, and so on.2. The optimization of rhBMP-2、rhVEGF and rhBMP-2-rhVEGF chitosan nanospheres are spherical in shape, well distributed, high entrapment efficiency and well properties in vitro.