| Basic fibroblast growth factor and vascular endothelial cell growth factor can promote the proliferation of myoblasts and vascular endothelial cells,with accelerating the formation of new blood vessels.However,the control of release and dosage are two crucial factors for promoting the optimal regeneration of vascularized muscle regeneration.This thesis demonstrates the utilization of supercritical fluid-based anti-solvent process(SAS)to construct bFGF and VEGF nano-embeded microparticles(NEMs)using silk fibroin and polylactic acid-polyethylene glycol-polylactic acid(PPP)as materials for promoting the effect of growth factor on the repair of muscle defect in mice in situ.It is expected that bFGF and VEGF can accelerate the repair of muscle defects by promoting the increase of blood vessel density through the temporal release behavior in the muscle defects,which provides an idea for solving the problem of vascularized muscle regeneration.Initially,the preparation and characterization of silk fibroin nanoparticles(SF NPs),PPP microparticles,and SF/PPP NEMs are demonstrated.The full-factor design results of Minitab showed that when the concentration of silk fibroin solution is 1.0%(w/v),the flow rate of solution is 0.5 mL/min,and the carbon dioxide flow rate is 40 g/min,the obtained SF NPs possessed excellent morphology and particle size distribution with an average particle size of 84 nm.The characterization of the relevant physicochemical properties of PPP particles and SF/PPP NEMs shows that the three particles are all spherical,and their particle sizes are mostly distributed around 90 nm,1.0 ?m and 1.5 ?m.The surface electronegativity of the three particles is in the negative range.Fourier transform infrared spectroscopy(FTIR)results showed that the physical and chemical structure of the three particles have no substantial changes compared to the raw material.X-ray powder diffraction spectrum results showed that compared to silk fibroin raw materials,the ?-helix of SF NPs transitions to ?-sheet.The diffraction peaks of PPP particles and SF/PPP NEM are similar,and the crystallinity is lower than that of their raw materials.Differential scanning calorimetry results showed that the glass transition temperature of SF NPs is higher than the raw material,but the decomposition temperature of SF NPs and PPP particles and SF/PPP NEM are all reduced.The experimental results of in vitro degradation for 8 weeks showed that the morphology of the three kinds of particles had changed to some extent,indicating that the slow degradation of SF/PPP NEM.Further,the preparation and characterization of drug-loaded SF/PPP NEMs aredemonstrated.Choosing bovine serum albumin(BSA)as a model,BSA@SF/PPP NEM and SF/BSA@PPP NEMs are constructed,and the drug loading and in vitro release effects of BSA under different dosages and drug loading models are investigated.The results showed that when the dosage is 10%(w/w),the drug loadings of BSA@SF/PPP NEM and SF/BSA@PPP NEM are(1.56±0.17)%and(2.05±0.30)%respectively.When the dosage is 20%(w/w),the drug loading amounts of BSA@SF/PPP NEM and SF/BSA@PPP NEM are(2.03±0.44)%and(2.88±0.05)%respectively.In vitro drug release results show that BSA loading of SF/PPP NEM has a sustained release effect,and the release rate of BSA in PPP micro-layer is faster than that in SF nano-layer.Moreover,the biocompatibility evaluation in vitro of the vector is explored.The cytotoxicity and hemolytic properties of SF NPs,PPP microparticles and SF/PPP NEM are investigated respectively.The experimental results showed that the carrier has shown no signs of cytotoxicity,no hemolytic properties and good biochemical indicators.And the acute systemic toxicity results show that the carrier has no visceral toxicity.In summary,the carrier material has excellent biocompatibility.Finally,bFGF-VEGF nano-embedded microparticles with different spatial distribution are prepared by the SAS technology,and related animal experiments were carried out.A systematic investigation is conducted on the in vitro release performance,the activity of growth factors in the release solution,their effects on the relative proliferation rates of mouse myoblast cells(C2C12)and human umbilical vein endothelial cells(HUVECs)and their role in promoting repair of muscle defects in mice.The results showed that when the dosage of bFGF is 1.5×10-8(w/w),the drug loadings of bFGF@SF/VEGF@PPP NEM and VEGF@SF/bFGF@PPP NEM are(3.41±0.26)×10-11 and(2.08±0.18)×10-11 respectively.Moreover,at the VEGF dosage of 0.5×10-8(w/w),the loading of bFGF@SF/VEGF@PPP NEM and VEGF@SF/bFGF@PPP NEM are(1.51±0.06)×10-11 and(2.37±0.04)×10-11 respectively.The release behavior in vitro results showed that the growth factor in the microlayer is released faster than that of in the nanolayer.The growth factor activity assay in the release solution showed that the bFGF and VEGF are active after two weeks of release.In addition,the effects of bFGF-VEGF for C2C12 and HUVECs proliferation rates showed that the growth factors in the two systems significantly promoted the proliferation of C2C12 and HUVECs compared with the control group,but the effect of VEGF@SF/bFGF@PPP NEM is better After SF/PPP NEM loaded bFGF-VEGF was injected in situ into muscle defect sites of mice,H&E staining results showed that inflammatory cells infiltrate in the defect muscles of mice in the later system group,indicating that the sequential release of bFGF and VEGF played a better role in promoting the repair.In summary,different spatially distributed bFGF-VEGF nano-embedded microparticles with regular morphology and uniform particle size are constructed using the SAS technology.Moreover,the in vitro release behavior of model protein BSA provides a reference for the study of growth factors,indicating that the delivery system has the characteristics of time-space and sequentially controlled release.The carrier has excellent biocompatibility.BFGF and VEGF could promote the repair of muscle defect sites in mice,providing an idea for solving the problem of clinical vascularized muscle regeneration. |
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