Objective:To prepare the multilayer alginate chitosan microspheres loading vascular endothelial growth factor (VEGF) and vancomycin (VAN), and to study in vitro release characteristics.Methods:The microspheres were prepared by emulsion cross-linking and self-assembly techniques.The effects of sodium alginate concentration, calcium chloride concentration, oil/water ratio and span80 concentration on the entrapment efficiency(EE) and drug loading(DL) of VEGF and VAN were investigated by orthogonal experimental design to optimize the preparation process.The surface morphology and particle size of microspheres were observed by scanning electron microscope (SEM).Self-assembly was detected by Fourier transform infrared spectroscope (FTIR).The EE, DL and in vitro release of VEGF and VAN were detected by ELISA double antibody sandwich method and ultraviolet spectrophotometry,and the cumulative release curve was drawn.Results:The prepared microspheres were yellowish brown powder.The SEM results showed that the microspheres were spherical, the surface was smoothy, and the dispersity was better.The average particle size was about 50 μm.Sodium alginate concentration of 1.0 g·mL-1, CaCl2 concentration of 8 g·mL-1, oil to water ratio of 3∶1, and span80 concentration of 2% were the best formula.The EE of VEGF and VAN were 49.63% and 16.67%, respectively.In vitro, the cumulative release last 16.5 d and 12.5 d respectively and the amount reached up to 95%.Conclusion:The multilayer alginate chitosan microspheres loading VEGF and VAN present several advantages, such as smaller particle size, higher EE and better controlled release.

OBJECTIVE: To explore whether the differentiation of vascular stem cells (VSC) into smooth muscle cells (SMC) in aortic dissection (AD) is dysregulated, and to verify the role of Notch3 pathway in this process. METHODS: Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery, Guangdong Provincial People's Hospital Affiliated to Southern Medical University. VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads. The cells were divided into normal donor-derived VSC group (Ctrl-VSC group) and AD-derived VSC group (AD-VSC group). The presence of VSC in the aortic adventitia was detected by immunohistochemical staining, and VSC was identified by stem cell function identification kit. The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1 (10 μg/L) for 7 days. They were divided into normal donor VSC-SMC group (Ctrl-VSC-SMC group), AD VSC-SMC group (AD-VSC-SMC group) and AD VSC-SMC+Notch3 inhibitor DAPT group (AD-VSC-SMC+DAPT group,DAPT 20 μmol/L was added during differentiation induction). The expression of contractile marker Calponin 1 (CNN1) in SMC derived from aortic media and VSC were detected by immunofluorescence staining. The protein expressions of contractile markers α-smooth muscle actin (α-SMA), CNN1 as well as Notch3 intracellular domain (NICD3) in SMC derived from aortic media and VSC were detected by Western blotting. RESULTS: Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels, and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes. Compared with normal donor vascular tissue, the expressions of SMC markers α-SMA and CNN1 of tunica media contraction in AD were down-regulated (α-SMA/β-actin: 0.40±0.12 vs. 1.00±0.11, CNN1/β-actin: 0.78±0.07 vs. 1.00±0.14, both P < 0.05), while the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 2.22±0.57 vs. 1.00±0.15, P < 0.05). Compared with Ctrl-VSC-SMC group, the expressions of contractile SMC markers α-SMA and CNN1 were down-regulated in AD-VSC-SMC group (α-SMA/β-actin: 0.35±0.13 vs. 1.00±0.20, CNN1/β-actin: 0.78±0.06 vs. 1.00±0.07, both P < 0.05), the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 22.32±1.22 vs. 1.00±0.06, P < 0.01). Compared with AD-VSC-SMC group, the expressions of contractile SMC markers α-SMA, CNN1 were up-regulated in AD-VSC-SMC+DAPT group (α-SMA/β-actin: 1.70±0.07 vs. 1.00±0.15, CNN1/β-actin: 1.62±0.03 vs. 1.00±0.02, both P < 0.05). CONCLUSIONS Dysregulation of VSC differentiation into SMC occurs in AD, while inhibition of Notch3 pathway activation can restore the expression of contractile proteins in VSC-derived SMC in AD.
Humans ; Actins ; Platelet Aggregation Inhibitors ; Signal Transduction ; Aortic Dissection ; Cell Differentiation ; Myocytes, Smooth Muscle ; Stem Cells