LIPOSOME-MEDIATED DELIVERY OF ANTI-SICKLING AGENTS

我们建议开发一种脂质体介导的转运系统，用于 红细胞膜不透性抗病药物的细胞内递送 毒剂进入镰状红细胞(RBC)。我们计划交付的代理商 这项技术包括芳香族氨基酸苯丙氨酸(Phe)和 色氨酸(Try)以及含有这些氨基的小肽(PEP) 酸(L-苏氨酸-L-苯丙氨酸和 L-赖氨酸-L-苯丙氨酸-L-苯丙氨酸)。这些药剂无毒，而且 都是已知的有效的非共价血红蛋白S凝胶抑制剂， 然而，相对较高的细胞内浓度需要 抑制凝胶化(约10 mm)，再加上其有限的 目前，通过完整的红细胞膜的渗透性使它们具有治疗作用 使用不切实际。我们的研究计划包括：(1)新技术的发展 理想的脂质体组成和大小及优化 脂质体-红细胞孵育条件对最大包封率的影响 随后将Phe、Try和Pep(PTP)导入镰刀状红细胞，(2) PTP对完整红细胞抗病原性的系统评价 和溶血物，包括治疗引起的抗药性变化 低氧引起的形态改变、血红蛋白氧亲和力和 相对溶解度、聚合物形成百分比、红细胞指数、阳离子 渗漏、细胞密度和变形性；细胞内的检查 保留外源传递的PTP，以及(4.)的效果 PTP负载对体外生成不可逆镰状细胞的影响及(5) 红细胞粘附性。我们还计划(6)。寻找可能的毒副作用 通过检测特定的红细胞代谢来提高细胞内PTP水平 参数，以及(7.)使用负载的PTP进行体内存活研究 红细胞，在动物(兔子)和人类中都是如此。此外，我们建议 (8)利用脂质体介导导入镰刀形红细胞的研究 己二酸二甲酯(DMA)，已被证明具有 但不幸的是，同时赋予了 导致红细胞减少的处理细胞新的抗原决定簇 活体内存活。脂质体包封法制备允许特异性的DMA 这种药物的细胞内释放可能会缓解目前的问题 与它的使用有关的。脂质体转运系统的研究进展 可应用于任何数量的潜在治疗应用 临床上有用的药物存在与其膜相关的问题 渗透性和/或非特异性反应性。

We propose to develop a liposome-mediated transport system for the intracellular delivery of red cell membrane impermeable anti-sickling agents into the sickle erythrocyte (RBC). The agents we propose to deliver by this technique include the aromatic amino acids phenylalanine (Phe) and tryptophan (Try) as well as small peptides (Pep) containing these amino acids (L-threonine-L-phenylalanine and L-lysine-L-phenylalanine-L-phenylalanine). These agents are non-toxic and are known to be effective non-covalent inhibitors of hemoglobin S gelation, however, the relatively high intracellular concentrations required to inhibit gelation (approximately 10 mM) coupled with their limited permeability across intact RBC membranes currently makes their therapeutic use impractical. Our research plan involves: (1.) Development of the ideal liposome, in terms of liposome composition and size, and optimizaton of liposome-RBC incubation conditions to effect the maximal encapsulation and subsequent delivery of Phe, Try and Pep (PTP) into sickle RBC, (2.) Systematic evaluation of the anti-sickling properties of PTP in intact RBC and hemolysates, including treatment-induced alterations in: resistance to hypoxia-induced morphological shape changes, hemoglobin oxygen affinity and relative solubility, percent polymer formation, red cell indices, cation leak, cell density and deformability; (3.) Examination of the intracellular retention of exogeneously delivered PTP, as well as the (4.) Effect of PTP-loading on in vitro generation of irreversibly sickled cells and (5.) RBC adhesiveness. We also plan to (6.) look for possible toxic effects of elevated intracellular PTP levels by examining specific RBC metabolic parameters, and (7.) Perform in vivo survival studies using the PTP-loaded RBC, both in animals (rabbits) and in humans. In addition, we propose to (8.) Utilize liposomal-mediated transport to deliver into sickle RBC dimethyl-adipimidate (DMA), which has been shown to possess potent anti-sickling properties but unfortunately simultaneously confers to the treated-cells new antigenic determinates which lead to decreased RBC survival in vivo. Liposomal encapsulation of DMA allowing specific intracellular delivery of this agent may alleviate problems currently associated with its use. The development of a liposome transport system can be applied to any number of therapeutic applications where potentially clinically useful agents have problems associated with their membrane permeability and/or non-specific reactivity.