【作者】 王嫦君；

【导师】 叶娟；

【作者基本信息】 浙江大学 ， 眼科学， 2011， 博士

【摘要】 第一部分静电纺丝法制备纳米壳-核结构微胶囊的可行性研究目的：通过纳米技术-静电纺丝法探索壳-核结构微胶囊制备的可行性,进而研究各种不同的静电纺丝条件对壳-核结构微胶囊形成的影响,得出静电纺丝法制备纳米壳-核结构微胶囊的基本规律,以期为进一步载药微胶囊的研究提供实验基础,快速推动生物降解型缓释载药泪道栓的成功制备,并进一步拓宽静电纺丝纳米技术在生物医学领域的应用范围。方法：采用生物相容性良好的天然高分子材料壳聚糖、透明质酸和海藻酸钠为主要的研究原料,以生物相容性良好的合成高分子聚合物聚氧化乙烯(PEO)为辅助原料,通过调整电纺原料的浓度配比,优化静电纺丝各项参数,制备得到静电纺微粒。然后,再分别利用各种高分子聚合物的荷电性质,应用自行改良设计的静电纺丝法-溶液接收装置进行纳米壳-核结构微胶囊的制备,再将所得的接收溶液利用真空抽滤法在微孔滤膜上富集制备得到的壳-核结构微胶囊,扫描电镜检测微胶囊的形状、分散性、均一性,透射电镜检测微胶囊的壳-核结构的相互关系,并对所得的微胶囊的粒径进行统计分析,得出其粒径分布范围和粒径平均值。并在此基础上,通过改变接收溶液以及接收溶液的条件,探索静电纺丝-改良溶液接收法制备壳-核结构微胶囊的基本规律特征。结果：通过调节壳聚糖和PEO的不同浓度配比,即在3%Chi：6.5%PEO=8：2,电纺参数调整为15Kv,流速为0.1ml/h,电纺距离15cm的条件时,可用静电纺丝法制备得到纳米微粒。在此基础上,应用静电纺丝法-改良溶液接收法,使用透明质酸(HA)或者海藻酸钠(ALG)溶液作为接收溶液,利用高分子聚合物之间的荷电性质,可进一步得到分散性良好、粒径均一、表面光滑的壳-核结构的Chi/EO@HA与Chi/PEO@ALG微胶囊。对微胶囊的粒径进行统计分析结果显示：微胶囊粒径分布范围为700-1500nm,平均粒径为1123nm。通过改变接收溶液和接收溶液的浓度并进行对比研究,结果显示分别利用透明质酸或者海藻酸钠溶液作为接收溶液均可成功制备得到微胶囊产物,0.3% HA溶液和1.2% ALG溶液得到的微胶囊产物分散性最好,且接收溶液的浓度越高,微胶囊的直径也越大。结论：本研究结果首先证实了纳米技术--静电纺丝法制备Chi/PEO纳米微粒的可行性；并在此基础上,通过改良的溶液接收法对静电纺Chi/PEO微粒进行接收,利用静电相互作用的原理,制备得到了壳-核结构Chi/PEO@HA与Chi/PEO@ALG微胶囊,进一步证实了静电纺丝法-改良溶液接收法制备壳-核结构微胶囊的可行性。为载药微胶囊的研究以及为快速推动生物降解型缓释载药泪道栓的成功研制提供了可靠的实验基础。第二部分载药电纺微胶囊的制备及相关性能初步研究目的：通过将各种不同的模型药物混入Chi/PEO混合电纺液中进行静电纺丝-改良溶液接收法接收,探索纳米载药微胶囊制备的可行性及模型药物的载药量的可调整性,并进一步研究载药微胶囊的缓释性能及生物相容性,为生物降解型缓释泪道栓的研究奠定实验基础,同时也为临床上多种缓释材料、支架的制备提供一种新的方法和研究思路。方法：分别将雌激素、环孢霉素A、罗丹明、左氧氟沙星加入Chi/PEO混合电纺液内,使其浓度达到5mg/ml,接收溶液为0.3%HA溶液,调整好各项电纺参数后,进行电纺产物的接收,将接收到的电纺产物用微孔滤膜过滤制成电镜样品行扫描电镜观察。同时分别加入不同浓度的模型药物进行载药量可调整性的研究。采用《中国药典》小杯法,不同时间点取样后用紫外-分光光度法检测药物浓度,计算累计释放率,对不同条件下制备的载药微胶囊凝胶的缓释性能进行研究。采用浸提液角膜上皮细胞培养法检测载药微胶囊凝胶的生物相容性,通过MTT比色法计算细胞相对增殖率,按照国家标准GB/T 1688615—1997“医疗器械生物学评价第五部分：细胞毒性试验体外法”进行凝胶生物相容性评价。结果：将各种不同的模型药物(雌激素、环孢霉素A、罗丹明、左氧氟沙星)混入Chi/PEO混合电纺液,均可成功制备得到载药微胶囊,且载药量可根据需要进行调整。选用左氧氟沙星作为模型药物进行缓释性能研究,通过使用电纺组和非电纺组(即机械搅拌组)、1*和3*的载药浓度以及1*和3*的接收液浓度分别进行对比研究,结果显示电纺组较机械搅拌组显示出明显的缓释性能,1*载药浓度组较3*载药浓度组释放缓慢,1*接收溶液浓度组较3*接收溶液浓度组释放快速。通过载药微胶囊凝胶浸提液角膜上皮细胞培养法,采用MTT比色法计算细胞相对增殖率得到材料生物相容性属1级,即显示生物相容性良好。结论：本研究结果首先证实了不同药物载入Chi/PEO混合电纺液进行静电纺丝-改良溶液接收法制备载药微胶囊的可行性；并在此基础上,通过改变载药量及接收溶液的浓度等因素,分析研究了静电纺丝-改良溶液接收法制备的载药微胶囊凝胶的缓释性能及其影响规律。通过生物材料生物相容性的评价标准,证实了本研究制得的微胶囊凝胶具有良好的生物相容性,为生物降解型缓释泪道栓的进一步研究奠定了实验基础。第三部分微胶囊凝胶交联改性及泪道栓制作的初步实验研究目的：通过将微胶囊凝胶进行交联改性改变凝胶的溶解性、降解性,在保留其生物相容性的同时,增加其抗降解性,并最终达到延长其在体内的存留时间,实现长效缓释给药治疗的目的。初步探索利用凝胶交联改性后材料制备所得的泪道栓的理化性能、生物相容性及降解性能,以期快速促进生物降解型缓释泪道栓的产业化和临床应用,最终为干眼症患者提供一种疗效显著、使用方便、“标本兼治”价格低廉的干眼症治疗方法,改善患者的生活质量。方法：分别采用两种不同的方法[己二酸二酰肼/乙基(N,N-二甲基氨基丙基)碳二亚胺(ADH/EDCI)法和1,4-丁二醇二缩水甘油醚(BDDE)法]对静电纺丝-溶液接收法制备得到载药微胶囊凝胶进行交联改性。取微胶囊HA溶液1g,按一定比例分别加入所对应的交联剂,快速搅拌,最终得到交联完成的HA凝胶经过纯水透析、提纯后备用。将交联后的凝胶样品置于PBS中浸泡,恒温水浴箱中振荡,定期观察凝胶样品的溶胀、降解情况。将交联得到的改性凝胶样品初步制成泪道栓形状,观察其生物相容性及降解性能。结果：采用两种不同的方法(ADH/EDCI法和BDDE法)对静电纺丝-溶液接收法制备得到微胶囊凝胶进行交联改性,结果显示两种方法均可成功地对HA进行交联改性,且经ADH/EDCI交联后的凝胶块,呈透明澄清,凝胶块弹性好,在水中降解非常缓慢；经BDDE交联后的凝胶块,呈淡黄色凝胶体,略呈流动性,但粘弹性非常好,在水中降解时间延长。将交联得到凝胶体制成的泪道栓具有一定的粘弹性、生物相容性良好、降解时间延长。结论：本研究结果首先证实了不同交联剂对微胶囊HA凝胶进行交联改性的可行性；通过改性,载药微胶囊HA凝胶的降解性能明显下降,可延长其在体内存留时间,达到了长效缓释治疗的目的。同时,利用微胶囊HA凝胶交联改性后的材料制备所得的泪道栓,具有一定的粘弹性、并且生物相容性良好、降解时间延长。初步实现了生物降解型缓释泪道栓的制备,但具体研究细节仍需进一步的深入研究。更多还原

【Abstract】 PartⅠFeasibility study on the electrospinning process in fabricating core-shell structured microcapsulesObjective:The purpose of this study was to investigate the feasibility of an advanced nano-technology the electrospinning process in fabricating core-shell structured microcapsules, to reveal the basic regularity rule of electrospinning process in fabricating core-shell structured microcapsules via changing different electrospinning condition parameters, which will provide the experimental foundation for further study on drug delivered microcapsules and advance the process of successfully designed biodegradable controlled releasing drug loaded punctal plug. The results of this study will broaden the application scope of the nano-technology electrospinning process in the field of biomedicine. Methods:This was achieved by using excellent biocompatible natural polymers such as chitosan,, hyaluronic acid and alginate sodium as the main materias, and using biocompatible synthetic polymers as adjuvant material, through adjusting different concentration proportion ratio to produce electrospun microparticles. Then take advantage of the difference in charge characteristic among the polymers, using modified solution collecting method to receive the electrospun microparticles to fabricate core-shell structured microcapsules, using vacuum filtration machine to collecting the microcapsules on the surface of the micropore filter membranes, which were then examined by SEM to evaluate the shape, dispersion and uniform of the microcapsule and examined by TEM to reveal the interrelationship of the core and shell structure of the microcapsules. With statistically analyzing the particle diameter of the microcapsules, the distributing range and mean size of the microcapsules were calculated. By change the collecting solution or by changing the concentration of the collecting solution, we elucidate the basic characteristics of electrospinning-modified solution collecting method in producing core-shell structured microcapsules.Results.Through varing the concentration ratio of chitosan and PEO blends, we finally found that at the concentration ratio with 3% Chi:6.5% PEO and electrospinning parameters to be at voltage 15Kv, fluid speed 0.1ml/h and DBPC 15cm micropaticles can be produced by electrospinning. Then via electrospinning-modified solution collecting mothed, using HA or alginate as the colleting solution, take advantage of the difference between the polymers, we could further get well-dispersed, smooth-surfaced core-shell structured Chi/PEO@HAand Chi/PEO@ALG microcapsules. Statistical analysis results showed that microcapsules diameters were ranged from 700-1500nm and the mean diameter was 1123nm. Through changing the collecting solution or concentration of the collecting solution we found that 0.3% HA and 1.2% alginate solution was better in producing well-dispersed microcapsules and the diameter of the microcapsule was increasing as the concentration of the collecting solution increased.Conclusion:This study firstly testified the feasibility of nano-technology electrospinning process in produing Chi/PEO microparticles. Using modified solution collecting method to receive the electrospun Chi/PEO microparticles, utilizing the principle of electrostatic interaction, core-shell structured Chi/PEO@HA and Chi/PEO@ALG microcapsules were successfully fabricated, which further confirmed the feasibility of electrospinning modified solution collecting mothod in fabrication of core-shell structured microcapsules. The results will provide substantial experimental foundation for the further study on the drug-loaded microcapsules and for the finally successful fabrication of biodegradable controlled-releasing punctal plug. Part IIFabrication of drug-loaded microcapsule and the study on its propertiesObjective:The purpose of this study was to investigate the feasibility of electrospun microcapsule to load drug and its ability in adjusting drug loading amount. Further investigation was performed to study the releasing properties and compatibility of the drug loaded microcapsules, which will establish a substantial foundation for the manufacture of biodegradable controlled releasing punctal plug and will provide an new research approach in designing controlled releasing materials, scaffolds for clinical application.Methods:Different model drugs, such as estrodiol, cyclosporine A, rhodamine, levofloxacin, were added into the Chi/PEO blends respectively to a final concentration at 5mg/ml. The collecting solution was 0.3% HA solution. After adjusting all the electrospinning parameters, eletrospun productions were collected and then filterd through the micropore filtration membranes for SEM examination. Different concentrations of model drug were also added to the Chi/PEO blends for evaluating the ability in drug loading amount adjustment. According to the method of "Chinese Pharmacopoeias" in accessing the controlled releasing propertities, samples were fetched at different time point and measured by UV-Spectrophotometer. Drug loaded microcapsules electrospun from different conditions were also observed and accumulated releasing rates were calculated to evaluating the properties of the microcapsules. Leaching liquor of the microcapsules was used to culture the corneal epithelium cells to evaluate the biocompatility of the microcapsule hydrosols. MTT method was used to calculate the relative growth rate (RGR) and the results were evaluated according to the National Standard GB/T 1688615—1997 for the biocompatibility evaluation.Results:Different kinds of model drugs (such as estradiol, cyclosporin A, rhodamine and levofloxacin) mixed in the Chi/PEO blends were all successfully electrospun for drug loaded microcapsules and the drug loading amount could be adjusted according to different purpose. Lavofloxacin was used as a model drug for controlled releasing characteristic study. While comparing the performance of the electrospinnig group and the non-eletrospinning group (mechanically mixed group), different concentration of drug loading groups and different concentration of collecting solution groups repectively, encouraging results were showed that electrospinning group manifest greater controlled releasing ability than the contrast group. The results also showed that higher drug loading concentration displaying a higher releasing speed whereas higher collecting solution concentration displaying a lower releasing speed. Leaching liquor of the microcapsule collecting solution was used to culture corneal epithelium cells and were measured by MTT method which showed the biocompatibility belong to grade 1 according to the national standard, implying a fine biocompatibility.Conclusion:This study first demonstrated the feasibility of electrospinning-modified solution colleting method to fabricate drug loaded microcapsules via mixing different drugs into the Chi/PEO blends. The releasing profile of the microcapsules were also studied and showed a controlled releasing capability. The evaluating test of biomaterials biocompatibility also testified the fine biocompatilbility of the microcapsules hydrosel, which allow a further study on the biodegradable punctal plug for dry eye disease.PartⅢCrosslink modification of microcapsules hydrosel and the primary study on punctal plug manufactureObjective:The purpose of this study was to modify the dissolubility of the microcapsules by crosslinking in order to increase its anti-degradability, prolong its resident time for long-time controlled release while maintaining its fine biocompatibility. Process a primary study on the punctal plug using the crosslinked hydrosel materials. The physical, chemical, biocompatibility as well as the degradability were studied, which will promote the success in manufacturing biodegradable controlled-releasing punctal plug for dry eye disease and improve the life quality of the patients. Methods:Using two different methods (ADH/EDCI method and BDDE method) to crosslink the microcapsule hydrosel with electrospinning-solution collecting method, 1g microcapsule hydrosel were mixed with a certain proportion of the crosslinking agent and stirred quickly to get the crosslinked HA gelatin which was then dialysed and purified with pure water. The crosslinked gelatin sample was soaked in the PBS with constant temperature and agitating water bath for interval observation of the degrading performance. The punctal plug was manufactured using the crosslinked gelatin materials and its biocompatibility and biodegradability were studied.Results:Both of the two methods (ADH/EDCI method and BDDE method) could successfully crosslinked the microcapsule hydrosel with electrospinning-modified solution collecting method. While using ADH/EDCI method, the gelatin was transparent, with good elasticity and low biodegradability. Using BDDE method, the gelatin was lightyellow with little fluidity and good viscoelasticity, as well as low biogegradability.Conclusion:This study first testified the feasibility in using different crosslinking agent to modify microcapsule hydrosel to increase its anti-degradability and prolong resident time for long-time controlled release. The punctal plug using the crosslinked materials was with certain elasticity, fine biocompatibility and low biodegradability. The results primarily accomplished the design of biodegradable controlled releasing punctal plug. However, the details of the process of crosslink and punctal plug modification still need further study.更多还原