基于氨基酸两性离子水凝胶库的制备与眼科应用研究

发布时间：2018-09-08 06:20

【摘要】：聚甲基丙烯酸羟乙酯(PHEMA)水凝胶和硅水凝胶在生物医学领域具有重要的应用价值,常用于制备角膜接触镜。然而,这类水凝胶表面显示出较强的疏水性,特别是硅水凝胶材料的静态水接触角达到100。左右,导致泪液中的蛋白质和脂质易于沉积在表面,临床上可能引发细菌感染等并发症。因此,提高水凝胶材料表面的亲水性和抗粘附性能,对于拓展其在眼科的临床应用非常重要。本文通过氨基引发的点击化学反应将一系列氨基酸或短肽接枝到水凝胶表面,以提高水凝胶表面的亲水性和抗粘附性能,并探索了水凝胶在眼科领域的应用。具体分为以下四个部分：第一部分,通过甲基丙烯酸羟乙酯单体(HEMA)和甲基丙烯酸缩水甘油酯单体(GMA)的自由基聚合反应,制备了一系列带有环氧基团的共聚物膜p(HEMA-co-GMA);随后,通过氨基酸的氨基与共聚物膜中环氧基团的开环反应,将一系列氨基酸接枝到PHEMA水凝胶表面,得到氨基酸两性离子PHEMA水凝胶库。衰减全反射傅里叶变换红外光谱(ATR/FTIR)和X射线光电子能谱(XPS)证明氨基酸成功地接枝到了水凝胶表面。改性后的PHEMA水凝胶的亲水性能明显提高,而蛋白沉积量则与接枝氨基酸的种类有关：中性氨基酸两性离子PHEMA水凝胶显示出较强的抵抗牛血清白蛋白(BSA)和溶菌酶(Lyz)粘附的能力；带有电荷的氨基酸两性离子PHEMA水凝胶对带有相同电荷的蛋白具有一定的抗沉积作用而对带有相反电荷的蛋白沉积量明显增加。体外细胞毒性实验表明氨基酸两性离子PHEMA水凝胶是无细胞毒性的。最后,制备了三种基于氨基酸两性离子的PHEMA水凝胶接触镜镜片,通过兔子的体内配戴实验表明该水凝胶镜片具有良好的亲水性和抗蛋白沉积的能力,同时对兔子眼组织无明显刺激性。第二部分,通过紫外光引发的硅单体和GMA的自由基聚合反应,制备了一系列带有环氧基团的共聚物膜p(Si-co-GMA);随后,通过氨基酸的氨基与共聚物膜中环氧基团的开环反应,将一系列氨基酸接枝到硅水凝胶表面,得到氨基酸两性离子硅水凝胶库。ATR/FTIR和XPS证明氨基酸成功地接枝到了硅水凝胶表面。通过接触角和含水率实验对氨基酸接枝的硅水凝胶进行表征,实验结果表明氨基酸两性离子硅水凝胶的亲水性能明显提高。随后,以BSA和Lyz作为目标蛋白来评价硅水凝胶的抗蛋白粘附性能。蛋白沉积实验结果表明中性氨基酸两性离子硅水凝胶显示出较强的抵抗BSA和Lyz粘附的能力,这要归功于由质子化的仲胺阳离子与去质子化的羧基阴离子组成的两性离子表面的形成；而带有电荷的氨基酸两性离子硅水凝胶对带有相同电荷的蛋白具有一定的抗沉积作用而对带有相反电荷的蛋白沉积量明显增加。体外细胞毒性实验表明氨基酸两性离子硅水凝胶是无细胞毒性的。最后,运用聚丙烯模具制备了三种基于氨基酸两性离子的硅水凝胶接触镜镜片。通过兔子的体内配戴实验和兔子眼球组织切片的病理学分析,表明该水凝胶镜片不仅具有良好的抗蛋白粘附性能而且对兔子眼组织无明显刺激和伤害。第三部分,首先设计并合成了一种带有亲水性基团的氟化硅氧烷大分子单体并且在不需要助溶剂的条件下能够与亲水性单体互溶,得到透明的氟化硅氧烷稳定聚合体系。通过紫外光引发的自由基聚合反应,制备了一系列带有环氧基团的共聚物膜p(FSi-co-GMA)；随后,通过氨基酸的氨基与共聚物膜中环氧基团的开环反应,将一系列氨基酸接枝到氟硅水凝胶表面,得到氨基酸两性离子氟硅水凝胶库。接触角和含水率的实验结果表明改性后的氟硅水凝胶的亲水性能明显提高。蛋白沉积实验结果表明氨基酸两性离子氟硅水凝胶的蛋白沉积量与接枝氨基酸的种类有关：中性氨基酸两性离子氟硅水凝胶显示出较强的抵抗BSA和Lyz粘附的能力；带有电荷的氨基酸两性离子氟硅水凝胶对带有相同电荷的蛋白具有一定的抗沉积作用而对带有相反电荷的蛋白沉积量明显增加。体外细胞毒性实验表明氨基酸两性离子氟硅水凝胶是无细胞毒性的,在眼科领域具有潜在的应用。第四部分,通过紫外光引发的自由基聚合反应,制备了一系列带有环氧基团的共聚物膜p(Si-co-GMA)；随后,通过短肽的氨基与共聚物膜中环氧基团的开环反应,将二甘肽(GG)和二甘氨酰甘氨酸(GGG)接枝到硅水凝胶表面,得到短肽两性离子硅水凝胶。通过ATR/FTIR,XPS和重量法对短肽两性离子硅水凝胶进行表征,实验结果表明短肽成功地接枝到了硅水凝胶的表面和内部。与此同时,短肽接枝后的硅水凝胶显示出良好的亲水性和平衡含水率。随后,以BSA和Lyz作为目标蛋白,用二喹啉甲酸法(BCA)进一步评价硅水凝胶的抗蛋白粘附性能。蛋白沉积实验结果显示所有的短肽两性离子硅水凝胶都具有较强的抵抗BSA和Lyz粘附的能力；硅水凝胶所接枝短肽的链越长,抗蛋白沉积能力越强,因此GGG接枝的硅水凝胶具有更好的抗蛋白粘附性能。体外细胞毒性实验表明短肽两性离子硅水凝胶是无细胞毒性的,表明它们具有优异的生物相容性。最后,制备了两种基于短肽两性离子的硅水凝胶接触镜镜片,通过兔子的体内配戴实验表明该水凝胶镜片具有良好的亲水性和抗蛋白沉积的能力,同时对兔子眼组织无明显刺激性。
[Abstract]:Poly (hydroxyethyl methacrylate) (PHEMA) hydrogel and silicone hydrogel have important application value in biomedical field and are often used to prepare corneal contact lenses. Therefore, it is very important to improve the hydrophilicity and anti-adhesion of the surface of hydrogel materials for expanding its clinical application in ophthalmology. In this paper, a series of amino acids or peptides were grafted onto the surface of hydrogel by click chemical reaction initiated by amino acids to improve the hydrogel coagulation. The hydrophilic and anti-adhesion properties of the adhesives were investigated, and the applications of hydrogels in ophthalmology were explored. The following four parts were included: Part I: A series of copolymer membranes P (HEM) with epoxy groups were prepared by free radical polymerization of hydroxyethyl methacrylate monomer (HEMA) and glycidyl methacrylate monomer (GMA). Amino acid amphoteric PHEMA hydrogel library was obtained by grafting a series of amino acids onto the surface of PHEMA hydrogel through the ring opening reaction of amino acids with epoxy groups in the copolymer membrane. Attenuated total reflection Fourier transform infrared spectroscopy (ATR/FTIR) and X-ray photoelectron spectroscopy (XPS) showed that amino acids were successfully grafted onto the surface of PHEMA hydrogel. The hydrophilicity of modified PHEMA hydrogel was obviously improved, while the protein deposition was related to the types of grafted amino acids: neutral amino acid amphoteric PHEMA hydrogel showed strong resistance to BSA and Lyz adhesion; charged amino acid amphoteric PHEMA water In vitro cytotoxicity test showed that amino acid amphoteric PHEMA hydrogel was non-cytotoxic. Finally, three kinds of PHEMA hydrogel contact lenses based on amino acid amphoteric ions were prepared and passed through rabbits. In the second part, a series of copolymer membranes P (Si-co-GMA) with epoxy groups were prepared by free radical polymerization of silicon monomer and GMA initiated by ultraviolet light. Amino acids were grafted onto the surface of silica hydrogels by ring-opening reaction of amino acids with epoxy groups in the copolymer membranes. Amino acids were successfully grafted onto the surface of silica hydrogels by ATR/FTIR and XPS. The results showed that the hydrophilicity of amino acid amphoteric silica hydrogel was improved obviously. Then, BSA and Lyz were used as target proteins to evaluate the anti-protein adhesion of the silica hydrogel. Protein deposition experiments showed that the neutral amino acid amphoteric silica hydrogel showed strong resistance to BSA and Lyz adhesion, which was attributed to the strong anti-protein adhesion ability of the neutral amino acid amphoteric silica hydrogel. In vitro cytotoxicity test table Amino acid amphoteric silicone hydrogel is non-cytotoxic. Finally, three kinds of silicone hydrogel contact lenses based on amino acid amphoteric ions were prepared by polypropylene mold. The wearing experiment in rabbits and pathological analysis of rabbit eyeball tissue sections showed that the hydrogel lenses not only had good anti-protein adhesion. In the third part, a kind of fluorosiloxane macromonomer with hydrophilic group was designed and synthesized, which can be soluble with hydrophilic monomer without cosolvent, and a transparent stable polymerization system of siloxane fluoride was obtained. A series of copolymer membranes P (FSi-co-GMA) with epoxy groups were prepared by radical polymerization, and then a series of amino acids were grafted onto the surface of fluorosilicone hydrogel by ring-opening reaction of amino acids with epoxy groups in the copolymer membranes. The experimental results of contact angle and water content were obtained. The results showed that the hydrophilicity of modified fluorosilicone hydrogel was improved obviously. The protein deposition of amino acid amphoteric fluorosilicone hydrogel was related to the types of grafted amino acids. Amino acid amphoteric fluorosilicone hydrogel has a certain anti-deposition effect on proteins with the same charge but a significant increase in the deposition of proteins with the opposite charge. Cytotoxicity tests in vitro show that amino acid amphoteric fluorosilicone hydrogel is non-cytotoxic and has potential applications in ophthalmology. Part IV, UV light-induced A series of copolymer membranes P (Si-co-GMA) with epoxy groups were prepared by hair radical polymerization, and then the short peptide amphoteric silica hydrogels were prepared by grafting diglyceride (GG) and diglyceryl glycine (GGG) onto the surface of the silica hydrogels via ring opening reaction of epoxy groups in the copolymer membranes with amino groups of short peptides. The short peptide amphoteric silica hydrogel was characterized by gravimetric method. The results showed that the short peptide grafted onto the surface and interior of the silica hydrogel successfully. At the same time, the short peptide grafted silica hydrogel showed good hydrophilicity and equilibrium water content. The results of protein deposition test showed that all short peptide amphoteric silica hydrogels had strong resistance to BSA and Lyz adhesion. The longer the chain of short peptide grafted on silica hydrogel, the stronger the resistance to protein deposition, so the GGG grafted silica hydrogel had better resistance to protein adhesion in vitro. Cytotoxicity test showed that short peptide amphoteric silica hydrogels were non-cytotoxic, indicating that they had excellent biocompatibility. Finally, two kinds of short peptide amphoteric silica contact lenses were prepared. The wearing experiments in rabbits showed that the hydrogels had good hydrophilicity and anti-protein deposition. It has no obvious irritation to rabbit's eye tissue.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ427.26;R318.08

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