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， 红细胞膜不可渗透抗镰状化的细胞内递送 药剂进入镰状红细胞（RBC）。 我们要运送的特工 包括芳香族氨基酸苯丙氨酸（Phe）和 色氨酸（Try）以及含有这些氨基酸的小肽（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）。 这些试剂是无毒的， 已知是血红蛋白S凝胶化的有效非共价抑制剂， 然而，需要相对高的细胞内浓度， 抑制胶凝（约10 mM）， 通过完整的红细胞膜的渗透性目前使其治疗 使用不切实际。 我们的研究计划包括：（1.）发展 从脂质体组成、大小等方面进行优化， 脂质体-RBC孵育条件以实现最大包封 以及随后将Phe、Try和Pep（PTP）递送到镰状RBC中，（2.） PTP抗完整红细胞镰状化作用的系统评价 和溶血产物，包括治疗诱导的以下变化： 缺氧诱导的形态学形状变化、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、细胞密度和变形能力;（3.）检查细胞内 保留外源性递送的PTP，以及（4.）的影响 PTP负载对体外产生不可逆镰状细胞的影响和（5.） 红细胞计数 我们也计划（6）。寻找可能的毒性作用 通过检查特定RBC代谢，升高细胞内PTP水平 参数，以及（7.）使用负载PTP的细胞进行体内存活研究 红细胞，在动物（兔）和人类。 此外，我们建议 （8.）利用脂质体介导的转运递送至镰状红细胞 二甲基-己二酰亚胺（DMA），已被证明具有有效的 但不幸的是，同时赋予 处理细胞产生新的抗原决定簇，导致RBC减少 体内存活率。 脂质体包封DMA，允许特异性 该试剂细胞内递送可以缓解目前的问题 与其使用有关。 脂质体转运系统的研制 可以应用于任何数量的治疗应用 临床上有用的试剂具有与它们的膜相关的问题 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.