电场响应纳米水凝胶的制备及抗癫痫作用研究

发布时间：2018-03-12 14:39

  本文选题：纳米水凝胶　切入点：电场响应　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：癫痫作为神经系统常见的疾病之一,其药物治疗存在靶向性差,易引起毒副作用和耐药等问题。癫痫的发作是脑内神经元阵发性异常超同步化电活动的结果,将癫痫灶点异常放电限制在局部并及时抑制,是阻止癫痫发作的关键。本文结合癫痫发作的电生理学机制和主动靶向技术,构建脑靶向电场响应纳米水凝胶。通过脑靶向肽介导纳米载体靶向累积于脑部,并在癫痫发作之际,在消弱癫痫样放电的同时快速释放药物,实现癫痫的高效治疗。本文的主要研究内容如下：1、电场响应纳米水凝胶的合成与评价。以甲基丙烯酸二甲胺基乙酯、苯乙烯和对苯乙烯磺酸钠(Sodium 4-vinylbenzene sulfonate, NaSS)为单体,以N,N'-亚甲基双丙烯酰胺为交联剂,过硫酸钾-焦亚硫酸钠为氧化还原体系引发剂,通过无皂乳液共聚法合成具有电场响应功能的纳米水凝胶。所合成的纳米水凝胶荷正电、粒径在50-146 nm之间；纳米水凝胶在水性介质中可快速(1min)响应电场变化,纳米粒溶胀,粒径变大,其变化程度与NaSS的含量有关；以苯妥英钠(PHT)为模型药物,干凝胶溶胀于药物水溶液的方法制备负载PHT纳米水凝胶；载药纳米水凝胶的体外释放速率与其粒径有关,粒径大于100 nm的载药纳米水凝药物释放速率相对较慢；同时药物从纳米水凝胶中的释放具有电场响应特性,100μA电场作用1 min后,其在0.5 h时的药物释放量最大可增加0.71倍,其增加倍数与纳米水凝胶中的NaSS含量有关。通过调整纳米水凝胶的组成筛选得到理想的电场响应纳米水凝胶用于后续研究。2、脑靶向电场响应纳米水凝胶的合成与评价。为了实现纳米水凝胶的脑部传递,利用脑靶向多肽Angiopep-2对纳米水凝胶进行修饰。在纳米水凝胶处方中添加两端具有反应活性的琥珀酰亚胺聚乙二醇丙烯酸酯,合成聚乙二醇修饰的纳米水凝胶,再利用纳米水凝胶表面聚乙二醇链段末端的琥珀酰亚胺与多肽Angiopep-2中氨基的化学反应,制备Angiopep-2修饰纳米水凝胶(ANG-ERHNPs). ANG-ERHNPs保持了电场快速响应功能；ANG-ERHNPs在bEnd.3细胞上表现为低毒性,IC50为449.0 μg/mL; ANG-ERHNPs有较好的跨体外BBB模型转运的能力,4h的累积透过百分率增加1倍,其机理可能与ANG-ERHNPs上的Angiopep-2与bEnd.3细胞表面蛋白的特异性结合,从而被bEnd.3细胞快速摄取有关；ANG-ERHNPs经腹腔注射后,通过活体成像系统可观察到其在大鼠脑组织和脑区累积；药动学研究表明：大鼠腹腔注射给药后,苯妥英钠脑靶向电场响应纳米水凝胶(ANG-PHT-ERHNPs)的相对生物利用度为PHT溶液组的1.91倍,且明显延长了PHT在体内的循环时间；组织分布结果表明：ANG-PHT-ERHNPs具有脑部转运能力,其脑靶向指数为2.56；ANG-PHT-ERHNPs腹腔注射给药后,在癫痫相关脑区(皮层、海马、杏仁核、小脑和脑干)均能测到药物,Cmax提高近1倍,提示ANG-PHT-ERHNPs可以降低给药剂量。3、脑靶向电场响应纳米水凝胶的抗癫痫药效研究。分别采用最大电休克、杏仁核电点燃和戊四氮(Pentylenetetrazol, PTZ)三种大鼠癫痫模型,评价腹腔注射给予不同剂量的PHT溶液、ANG-PHT-ERHNPs、苯妥英钠脑靶向非电场响应纳米水凝胶(ANG-PHT-HNPs)的抗癫痫作用。相比ANG-PHT-HNPs和PHT溶液,ANG-PHT-ERHNPs在三种癫痫模型中均具有更好的抗癫痫作用,不仅能降低有效治疗剂量而且可以延长药物的抗癫痫治疗时间窗；PTZ诱导的癫痫模型上测得的游离PHT结果进一步证实了PHT从ANG-PHT-ERHNPs中快速释放与发作脑电的严重程度呈正相关。
[Abstract]:One of the common diseases of nervous system as epilepsy, drug therapy has poor targeting, easy to cause the problem of side effects and drug resistance. The seizure is the brain neurons abnormal synchronization of paroxysmal electrical activity results, the abnormal discharge of epileptic foci in the local limit and prevent seizures, key this combination of seizures. The electrophysiological mechanism and active targeting technology, construct the target brain response to the electric field. The nano hydrogel brain targeting peptide mediated tumor targeting accumulation in the brain, and seizures on the occasion, rapid release of drugs in epilepsy weakened epileptiform discharge at the same time, realize the efficient treatment of epilepsy the main contents of this paper are as follows: 1. The electric field response synthesis and evaluation of nano hydrogels. The methacrylic acid two methylamino ethyl ester, styrene and sodium styrene sulfonate (Sodium 4-vinylbenzene sulfonate, NaSS) as monomer, N, N'- methylene bis acrylamide as crosslinking agent, potassium persulfate and sodium metabisulfite as redox initiator, the soap free emulsion copolymerization method to synthesis nano hydrogel electric field response function. The positively charged nano hydrogel synthesis, particle size between 50-146 nm nano hydrogels; fast in water medium (1min) in response to the electric field change, nanoparticles swelling, particle size, content of the degree of change and NaSS; with phenytoin (PHT) as a model drug, preparation method of dry gel swelling in drug solution preparation load nanostructured PHT hydrogel; drug loaded hydrogel in vitro release rate with the particle size, drug loaded hydrogel drug particle size greater than 100 nm release rate is relatively slow; at the same time from the drug with nano hydrogel release in electric field response characteristics of 100 A electric field after 1 min in 0.5 h The maximum amount of drug release can be increased by 0.71 times, the increase of NaSS content in multiple and nano hydrogel. Through screening adjustment nanohydrogels ideal electric field response nano hydrogel for further study of.2 targeting to the brain electric field response synthesis and evaluation of nano hydrogels. In order to achieve nanohydrogels brain transfer to polypeptide Angiopep-2 the nano hydrogel modified by brain target. At both ends of adding polyethylene glycol acrylate succinimide nano hydrogel prescription with reaction activity, synthesis of polyethylene glycol modified nano hydrogel, and then the chemical reaction of amino succinimide and peptide Angiopep-2 nano hydrogel surface of polyethylene glycol chain end in the preparation of Angiopep-2 modified nano hydrogel (ANG-ERHNPs). ANG-ERHNPs the electric field rapid response function ANG-ERHNPs in bEnd.3 cells on the table; 鐜颁负浣庢瘨鎬，

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