The Application And Mechanism Of Radioactive Skin Injury Therapy With Vascular Growth Factor Loaded Chitosan Nanoparticles

Objective:To prepare chitosan nanoparticles loaded with vascular growth factor and explore its application and mechanism in radiation skin injury therapy.Methods:(1) The auther established the adioactive skin injury model that the buttock skin of rats was irradiated by 45 Gy X-ray irradiation in order to observe the gross changes and microcirculation changes of radiation region.(2) Only one vascular growth factor was screened from vascular endothelial growth factor(VEGF), basic fibroblast growth factor(bFGF) and hepatocyte growth factor(HGF) including compared with model of gross view, microvessel count and one-step TUNEL staining results, after local subcutaneous injections of above three growth factors into radiation regions.(3)Ionic crosslinking method was adopted to prepare nanoparticles combined with Sodium tripolyphosphate(TPP) and chitosan(CS) and screening of vascular endothelial growth factor. Then we detected the physicochemical properties and investigate its in vitro release rules.(4)Vascular growth factor loaded chitosan nanoparticles after dried and resuspended were subcutaneously injected into rat radiation regions in which skin changes were evalued compare with the methods of continuous injection and one-time injection of the equal amount of vascular growth factor.(5) The mechanism of adioactive skin injury therapy with vascular growth factor loaded chitosan nanoparticles was further investigated through the comparison of monitor of microcirculation, the content of vascular endothelial cells damage maker(vascular von Willebrand factor, vWF), the expression of apoptosis protein(Caspase3) in different groups. The qualitative and quantitative analyses of vascular growth factor were detected with immune tissues of science and Technology(IHC) and immunoblotting(Western blotting).Results:(1) After 45 Gy X-ray irradiation, 2 weeks of hair removal, 4 weeks of skin ulcers, two months of ulcer wound healing, microcirculation disturbance gradually aggravated with time.(2) Two weeks after irradiation, irradiation area in skin wounds ofVEGF group did not show significant changes in two weeks after irradiation, while alopecia universalis was observed in both bFGF group and HGF group. Further study showed that wounds of the VEGF group showed more microvessel, fewer apoptotic cells.The difference was statistically significant(P < 0.01).(3) Particle size of the VEGF165-loaded chitosan nanoparticles prepared by ionic crosslinking is about 400 nm.The Zeta potential is 25.3V. The encapsulation rate is about 85%. The release rate is reach to 90% with in-vitro release steadily for 30 days.(4) Compared to the continuous injection of VEGF165 group, there was no significant difference in time of alopecia and ulceration in subcutaneous injection of VEGF165-loaded chitosan nanoparticles group(P > 0.05).Compared to the control group and the same dosage of one-time injection VEGF165 group,time of alopecia and ulceration in subcutaneous injection of VEGF165-loaded chitosan nanoparticles group was much longer and the time of wound healing was much shorter(P< 0.05).(5) After two weeks’ injection, the microvessel count and vWF content of VEGF165 loaded chitosan nanoparticles group were more than those of the control group and simple one-time injection of VEGF165 group(P < 0.01), while expression level of capase 3 is weaker than that of the other two groups. It is verified that the content of VEGF165 in tissues injected with VEGF165-loaded nanoparticles and in tissues with continuous VEGF165 injection were higher than that in tissues of control group and in tissues with simple one-time VEGF165 injection group.Conclusions:(1) After the 45 Gy X-ray radiated, obvious skin wounds of rats can be formed. Microvascular injury and microthrombus formation gradually aggravated with time with natures of long-term, potentiality, persistence and progression.(2) VEGF165 can effectively intervent the occurrence and development of the adioactive skin injury rat model.(3) VEGF165-loaded chitosan nanoparticles prepared by nic crosslinking method has high entrapment efficiency, stable physicochemical property, steady release in vitro,and a certain sustained-release of VEGF165 in vivo rat model.(4) VEGF165-loaded chitosan nanoparticles may play an important part in protecting vascular endothelial cells,inhibiting the apoptosis of endothelial cells, delaying the occurrence and development of radiation-induced skin injury, and promoteing the healing of radiation induced skin damage.