pH-温度双响应rtPA复合物的合成及在亚低温溶栓治疗中的研究

Clinical studies have shown that lowering the body temperature down to 30-35℃ during the treatment of ischemic stroke can effectively reduce the damage to the brain caused by the thrombolytic process, namely, Hypothermia Treatment, and near the thrombus is weak acid pH. Modifying recombinant tissue plasminogen activator (rtPA), the only effective drug for treating stroke currently, to achieve the pH-temperature dual response is undoubtedly the key to the treatment. However, most of the protein modification methods reported in the literature cannot be met the protein thrombolytic activity and safety requirements in therapy. Applicant combines polymer chemistry and clinical practices to propose a “safe” protein modification strategy using pH and temperature two types of responsive polymers. This enables an effective dual “on-off” control of the rtPA biological activity from the hypothermia temperature range to normal body temperature, the weak acid pH to physiological pH, by modifying surface amino group of protein Lys residue, regulating the precise ratio of pH and temperature two type of responsive polymers, the molecular weight of polymer and the grafting rate. In this study, the protein surface grafting, precise control of the number of grafted polymers and the relationship between structure and performance of polymer/protein complex will be investigated. Therapy models will be established to ascertain the thrombolytic process and mechanisms of the pH-temperature dual responsive rtPA composite, which is expected to a reliable theoretical basis and material basis for the application of such dually responsive polymer-protein conjugates in the thrombolytic therapy.

临床研究证明患缺血性脑卒中(脑血栓)后降低人体温度至30-35℃,可有效缓解治疗中的脑部损害(亚低温治疗),且血栓附近呈弱酸性。作为脑血栓的唯一特效药,针对重组组织型纤溶酶原激活剂（rtPA）改性使其具有pH和温度双响应无疑是治疗的关键。但目前文献报道的蛋白修饰方法大多无法满足rtPA活性和安全性要求。申请人结合高分子化学与临床医学的工作实践,提出利用pH和温度两种响应高分子对蛋白进行修饰的方法。通过对rtPA表面氨基的修饰、精确调控pH和温度两种响应高分子配比、分子量、接枝率等手段,实现对rtPA生物活性从血栓周围弱酸性pH至人体正常生理pH、从亚低温治疗温度范围至人体正常温度的双重“开-关”式控制。本项目将重点对蛋白表面接枝反应、接枝数量的精确控制和高分子/蛋白复合体系结构-性能关系进行系统考察；同时建立治疗模型,明确此双响应复合物的溶栓过程和机制,为响应性溶栓制剂的应用提供材料基础。

鉴于目前溶栓蛋白药物易被各种水解酶降解、半衰期短、无靶向性，需加大剂量才达到血药浓度，而增加剂量溶栓的同时也增加了机体出血风险等普遍问题，本研究结合血栓组织特殊的生理微环境（即血栓局部pH值低于组织正常代谢的细胞外环境、血小板糖蛋白受体在血栓中血小板表面高度密集表达），同时考虑到亚低温对脑血栓治疗的意义，设计并制备了三维结构可控的pH/温度双敏感高分子/溶栓蛋白药物复合结构新体系，能够在血栓局部位点释放药物和产生溶栓作用，并减少由常规溶栓药剂引起危及生命的出血并发症，以实现溶栓治疗的安全性和有效性。主要包括：.1.pH敏感溶栓纳米制剂的制备及性能研究.将尿激酶和氧化葡聚糖通过亚胺键结合，并用RGD多肽进行修饰，制备了pH敏感的血栓靶向性蛋白-高分子键合物（uPA-Oxd），并对其结构、形貌和性能进行了表征。RGD修饰的键合物能与血栓中血小板上活化的GPIIb/IIIa受体结合，使其具有较好的血栓靶向性和更好的溶栓性能。该uPA-Oxd键合物这种pH触发靶向溶栓性能无需外源触发剂，对于降低溶栓药物尿激酶由于无靶向性需大量使用所引起的并发症具有积极意义。.2.用于亚低温治疗的pH/温度双敏感溶栓纳米制剂.制备了pH/温度双敏感溶栓药物体系（uPA/PEG-PPG-PEG/PEG），并对其结构和性能进行了系统表征。结果表明，uPA/PEG-PPG-PEG/PEG具有良好的pH/温度敏感性，且由于溶栓药物蛋白表面聚合物的空间阻碍作用，uPA/PEG-PPG-PEG/PEG具有较好的抗酶水解能力。另一方面，由于温敏聚合物PEG-PPG-PEG和pH敏感亚胺键的引入，uPA/PEG-PPG-PEG/PEG的酶活性受温度和pH值双开关控制，即在正常体温及生理pH下（37℃，pH 7.4），uPA/PEG-PPG-PEG/PEG的活性被部分隐藏，而在亚低温治疗条件下（低于37℃）或弱酸性环境中，uPA/PEG-PPG-PEG/PEG的显示正常酶活性。.项目按照预期计划有序开展，总体执行情况顺利，发表高质量的SCI论文8篇，授权5项发明专利。.技术指标 .（1）获得可用于亚低温治疗的pH/温度双敏感溶栓纳米制剂，并对其结构进行全面的表征；.（2）揭示所制备的pH/温度双敏感溶栓药物体系生物活性、抗酶水解性能与pH和温度之间的关系。