| Intracerebral hemorrhage(ICH) is a fatal subtype of stroke with high morbidity and mortality, which threats human health seriously. After ICH, the spilled blood will flow into brain tissue, format hematoma, and damage brain tissue. For the patients with large blood loss(>30m L), the preferred treatment is surgery, which can remove hematoma, reduce intracranial pressure and protect nerve from compression from hematoma. However, surgery still exists some shortcomings, such as incomplete hematoma removal and poor prognosis recovery, which even lead to a series of secondary injury and cause severe neurologic deficits. Among the secondary injury factors, red blood and their degradation products’ toxic effects have caused scientists wide attention. Red blood cells in hematoma can release a large amount of hemoglobin, which can produce harmful substances such as carbon monoxide, iron and bilirubin. Among these harmful degradation products, iron, as the main degradation product of red blood cells, will gather in brain tissue, produce free radicals, attack cell proteins and nucleic acids and then cause cell death. Therefore, intervention of secondary damage caused by overload iron has gradually become the treatment options to ICH.Based on the secondary damage to nerve cells caused by overload iron produced by incompletely remove hematoma in the operation process, a kind of novel hydrogel with iron chelating properties has been constructed in this paper. The hydrogel could adsorb overload iron around it and then chelate them, reduce iron concentration in brain hemorrhage and reduced secondary injury to nerve cells, which provided a new treatment strategy to adjuvant therapy of ICH. The main research work and corresponding results were done in this thesis:(1) Extracted keratin proteins from human hair, and get the extraction yield 3.52 ±0.56%. We analyzed the keratins by SDS-PAGE, which showed that they contained both high molecular weight(~ 40–60 k Da) alpha keratins and low molecular weight(~ 10–15 k Da) gamma keratins, and most of them were high sulfur amino acids with good hydrophilic. Then we analyzed the keratins amino acid composition, the result showed that 17 kinds of amino were detected, and the total content of amino was 64.50±4.08% of the total keratin quality. In addition to tryptophan, glutamine and asparagine, all amino acids were detected. Among these amino acids, the most were glutamic acid and arginine, they accounted for 11.08±1.15% and 7.01±0.57%, respectively. The least were methionine, histidine, phenylalanine and lysine, and they accounted for 0.61±0.15%,1.03±0.06%,1.68±0.14% and 1.93±0.41%.(2) We prepared blank keratin hydrogel by physical deposition, and examined the effect of keratin hydrogel concentration on their iron adsorption. We knew that the hydrogel with the concentration of 30% showed the best iron adsorption: the adsorption capacities increased rapidly during the first 120 minutes and iron adsorption rate was 27.47±1.71%; between 120 and 360 min, iron concentration decreased gently, and the adsorption quantity of hydrogel was 4.64±0.49μg/mg; after 360 min, both iron concentration and hydrogel adsorption quantity were getting balance. Then we simulated the changes of adsorbed amount with time with a pseudo-first-order model and a pseudo-second order model, and knew that they fitted the later better. Furthermore, we evaluated the diffusion mechanism using intraparticle diffusion kinetics model, which showed that external mass transfer and intraparticle diffusion both controlled the adsorptive removal of iron. Finally, we characterized hydrogels with SEM, FT-IR, and XRD, and found that the hydrogels were highly porous with 20-100μm pore sizes which contributed to drug loading and iron adsorption. Both XRD and FT-IR results matched our observation of the process in which iron adsorption was successful.(3) We prepared minocycline loaded keratin hydrogel by physical deposition, and examined the effect of drug-loading rate on their iron chelation. We knew that the gel with 30% drug-loading rate showed the best iron chelation: the chelation capacities increased rapidly during the first 120 minutes and iron chelation rate was up to 44.16±2.35%, and hydrogel chelation quantity was 5.18±0.24μg/mg; between 120 and 360 min, iron concentration decreased gently; at the time of 360 min, iron chelation rate was 59.70±3.37%, the drugs were nearly completely used; by the end of the 720 minutes, the iron chelation rate was 62.38±4.14%, and the hydrogel chelation quantity was 7.20±0.50μg/mg. Finally, we characterized hydrogels with SEM, FT-IR, and XRD. All the similar pro structures in SEM images indicated that the keratin had not precipitated during formation, and minocycline was successfully loaded into the keratin hydrogels. Both XRD and FT-IR results showed that drug embedding process was physical process and hydrogels had good iron chelation capability.(4) To further investigate the keratin hydrogels toxicity and protective effect to nerve cells, we carried out cell experiment. When the hydrogel concentration increased to 200 μg/m L, cell survival rate significantly decreased, and when the concentration was rising to 800 μg/m L, cell survival rate decreased to 51.55±10.09%. The results showed that keratin hydrogel had slight cytotoxicity to nerve cells. We established nerve cell damage model by adding 25 μM hemoglobin. After incubation by drug loaded hydrogels for 24 h, survival rate of damaged cells gradually increased. When the hydrogel concentration reached 400 μg/m L, compared with the control group(50.88±15.62%), the cell survival rate increased to(94.22 ± 8.57%), which showed that keratin gel had good protective effect on neurons. Finally, Map2 immunofluorescence cytochemistry staining and DAPI staining showed that the keratins hydrogels had antagonism effect on hemoglobin toxicity. |
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