| Objective: The cross-linked cyclodextrin metal organic framework(CDF)loading quercetin(QUE)was coated with human hypertrophic scar fibroblast(HSF)membrane to enhance the adhesion with homologous cells.Moreover,the QUE-loaded nanocarriers were introduced by bletilla striatapoly saccharide-fabricated microneedles into dermis for improving the efficacy of QUE in treating hypertrophic scar(HS).Method: The diphenyl carbonate(DPC)was used as a crosslinking agent to prepare CDF.The infrared spectrum and contact angle were analyzed to identify the crosslinking results.The drug loading methods were optimized by single factor experiments.The HSF membrane was extracted by differential centrifugation and co-extruded by track-etched membranes to prepare cytomembrane-coated CDF(QUE@HSF/CDF).The bletilla striata polysaccharide was isolated from the traditional Chinese medicine Bletilla striata by water extracting-alcohol precipitating method,then was used to prepare the hydrosoluble microneedles(MNs-QUE@HSF/CDF).The microneedles were characterized including their solubility,stability,skin penetration,biological safety and cytomembrane proteins.Dialysis method and Franz diffusion cell were used to investigate the in vitro release and transdermal behavior of the nanoformulations and the fabricated microneedles.Then,the targeting properties of HSF/CDF were assessed by measuring HCF cellular uptake with fluorescence activated cell sorting method and laser scaning confocal microscopy.Besides,the cytotoxicity of QUE-loaded formulations were assayed by CCK-8 method.Furthermore,the apoptosis HSF cells induced by QUE was determined by flow cytometry,and the expression of apoptosis-associated proteins was determined by western blot assay.Models of HS on the back of SD rats were established by complete cortical injury.The scar elevation index(SEI)measurement,Hematoxylin&Eosin(H&E)staining isolated skin slices,ELISA,western blot assays and immunohistochemistry were carried out to evaluate the improvement of therapeutic effects on the HS-beared rats in each test preparations-treated group.Results: CDF was successfully synthesized with the identification of the contact angle and infrared spectrum.The drug loading methods were optimized as follows: 1,4-dioxane was selected as a drug solvent,the drug concentration was 150 mg/m L, drug loading temperature and time were respectively set as 45 °C and 6 h.The mean sizes,polydispersity indexes,zeta potential values of CDF and HSF/CDF were 234.8±3.2 nm and 242.5±1.6 nm,0.190±0.0728 and 0.174±0.128,and-25.1±1.1 m V and-31.0±0.6 m V,respectively.The cell membranes were clearly visually and the nanoparticles were uniformly distributed without adhesion in the field of transmission electron microscope.Bletilla striata polysaccharide had a variety of polysaccharide characteristic infrared peaks,and the polysaccharide content was 60.4±1.1%.The bletilla striata polysaccharide–fabricated microneedles in hydrogel state had pseudoplastic fluid properties,and the microneedles resembled the shapes of bullets,which were completely demoulded.Additionally,the mechanical strength of the bletilla striata polysaccharide–fabricated microneedles was larger than that of the hyaluronic acid-formed microneedles,while the hygroscopicity and solubility of the former were both lower than that of the latter.The mean depth of skin puncture of MNs-QUE@HSF/CDF was about 300 μm,with excellent biosafety was and showed no skin irritation.Notably,the HSF cytomembrane proteins were well maintained in the microneedles.Comparing with CDF,HSF/CDF had the sustained release of QUE,and the cumulative release of drug for 24 h was significantly increased by the nanocarriers in contrast with that of the free quercetin(p<0.0001).The microneedles-mediated transdermal delivery of QUE was significantly improved(p<0.0001),and the skin retention was accompanying increased(p<0.001),as compared with the groups without microneedles assisted.Due to the homologous adhesion of cell membranes,the drug retention in dermis of MNs-QUE@HSF/CDF group was significantly larger than that in epidermis(p<0.001).Compared with the CDF,the uptake of HSF/CDF by HSF cells increased(p<0.0001),followed with the increase of the IC50 value and apoptosis rate.The trend of apoptotic protein such as bax,bcl-2,caspase-3 was verified by western blot.In addition,we found that the bletilla striata polysaccharide induced apoptosis in HSF cells as compared with the untreated cells(p<0.0001).After drug intervention therapy,the HS tissue thickness and pathological features of each treatment group were improved.Among the QUE formulations treated groups,the improvement effect in MNs-QUE@HSF/CDF group was the best.Compared with the model group,the expression of inflammatory factors such as IL-6,IL-1β,b FGF in rat serum in each treatment group were significantly decreased,and the down-regulation effects in MNs-QUE@HSF/CDF group were stronger than those in the MNs-QUE@CDF group(p<0.01).Further,the western blot assay results suggested that the QUE had significant down-regulation effects on TGF-1,STAT3,and Wnt16 proteins,immunohistochemistry suggested that the QUE had significant down-regulation effects on collagen I,III proteins and the experimental results were consistent with SEI and ELISA studies.Conclusion: the MNs-QUE@HSF/CDF increased the accumulation of drugs in the lesions of the dermis,therefor improved the therapeutic effect on hypertrophic scar. |
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