| Objective:To investigate the effect of chitosan(CS)nanoparticles loaded with tegcycline(TGC)on corneal neovascularization after alkali burn.Methods: TGC/CS nanoparticles were prepared by ion crosslinking method.After centrifugation at low temperature and high speed,high performance liquid chromatography was used The content of TGC in supernatant was detected by chromatography,and the entrapment rate and drug loading of different initial TGC concentrations were calculated to select the best initial concentration.Then the drug release of 6h,12 h,18h,24 h,30h,36 h,42h and 48 h were measured and calculated under the condition of constant temperature water bath oscillation,and repeated three times.The shape and particle size of TGC were measured by transmission electron microscope.Six New Zealand white rabbits were selected.TGC/CS nanoparticles were used in the right eye as the experimental eye and the same amount of normal saline was dripped into the left eye as the control eye.After 1h,6h and 24 h,slit lamp was used to observe and observe.According to Draize test score,cornea and conjunctiva of two eyes were taken after 24 h for HE staining to detect the ocular surface toxicity.40 New Zealand white rabbits were randomly divided into TGC/CS nanoparticles group(group A),TGC group(group B),blank CS nanoparticles group(Group C)and normal saline control group(Group D),10 eyes in each group,alkali burn model in the right eye,no treatment in the left eye;TGC/CS nanoparticles eyes in group A,TGC eyes in group B,CS nanoparticles eyes in group C,normal saline eyes in group D,2/day,28 days in total;fissure Corneal neovascularization(CNV)was observed on the 7th,14 th and 28 th day,and the length of CNV was measured macroscopically.28 days after pentobarbital anesthesia,corneal tissues were taken for HE staining to observe the pathological changes and the expression of nuclear transcription factor-κB(NF-κB),vascular endothelial growth factor(VEGF)and matrix metalloproteinase-9(MMP-9)were detected by Western blot.Results: Most of TGC/CS nanoparticles are regular spherical,with particle size of(235.84 ± 34.59)nm,encapsulation efficiency of(67.17 ± 0.29)% and drug loading of(59.67 ± 0.29)% respectively.The system is relatively stable and has sustained-release function.TGC / CS nanoparticles had no damage to the cornea and conjunctiva of rabbits.The area of CNV in group A was significantly less than that in group B,C and D at 7d,14 d and 28 d,and that in group B was significantly less than that in group C and D,the difference was statistically significant(P < 0.05).The area in group C was smaller than that in group D,but the difference was not significant(P > 0.05).He staining showed that on the 7th day after alkali burn,some epithelial cells were damaged,matrix edema,various inflammatory cells were infiltrated widely,and CNV was generated in some of them;on the 14 th day,corneal structure was partially recovered,with medium amount of inflammatory cells infiltrated,CNV area reached the peak,and on the 28 th day,all layers of structure were basically recovered,inflammatory cells were largely reduced,neovascularization was atrophic,the degree of C and D groups was the most serious and the difference was not obvious,group B was in the middle,group A was the most Light.Western blot results showed that the relative contents of mmp-9 in four groups of NF-κB were 0.36±0.01,0.55±0.03,0.77±0.02,and 0.97±0.03,respectively,and the relative contents of mmp-9 were 0.29±0.02,0.40±0.02,0.53±0.02,and 0.68±0.01.The relative content of VEGF in each group was 0.38±0.02,0.45±0.01,0.76±0.02,and 0.77±0.02,respectively.The differences between groups C and D were insignificant(P>0.05),and the pantwise differences between the remaining groups were statistically significant(P<0.05).Conclusion: TGC/CS nanoparticles have no damage to the ocular surface.Both TGC/CS nanoparticles and free TGC can effectively inhibit the growth of CNV.The mechanism of inhibition is to down regulate the expression of NF-κB,VEGF and MMP-9 in corneal tissue. |
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