Peptide Ligands for Placental Transcytosis Receptors

描述(由申请人提供)：我们研究的长期目标是开发创新的给药策略，促进药物和非病毒药物载体通过胎盘屏障的可重复转移。在这项可行性研究中要检验的中心假设是，使用噬菌体展示载体鉴定的独特的肽序列通过跨细胞作用提高了亲水性大分子通过人类滋养层屏障的运输效率。这些多肽与脂质体和微球等亲水性药物载体的结合有望实现对新生儿的有效药物治疗。因此，提出了三个具体的目标：(1)鉴定通过跨细胞作用促进含肽噬菌体通过人滋养层屏障体外模型的转移的多肽序列-(2)确定与高效跨细胞噬菌体克隆的衣肽初级序列相对应的合成肽的跨细胞特性和跨体外人滋养层屏障模型的批量转运极限。(3)定量不同分子大小的荧光跨细胞载体在双灌流胎盘子叶中的跨胎盘转运。将对噬菌体pC89的构建和随机多肽的非肽库进行筛选，以寻找能够介导高效的噬菌体跨体外模型的人滋养层屏障的噬菌体转移，包括由人足月细胞滋养层和BeWo细胞单层建立的合胞体融合层。将设计实验来确定噬菌体转移是否满足细胞穿透的标准。将对具有高转移效率的噬菌体克隆的多肽编码DNA插入片段进行测序，并合成相应的线性和环状类似物。此外，还将制备这些多肽与四甲基罗丹明标记的右旋糖苷(3-500 kDa)的偶联物。这些化合物的跨细胞特性将使用人滋养层屏障和体外双重灌流的人胎盘子叶的体外模型来确定。这项研究的意义在于，它将检验噬菌体展示技术识别人胎盘跨细胞系统的独特多肽序列的可行性。这种对现有方法的创新使用可能会加速开发新的策略，通过细胞穿透在子宫内进行侵入性较小的胎儿药物治疗，同时降低母亲的风险，因为良好的生物分布进入胎儿循环。

DESCRIPTION (provided by applicant): The long-term goal of our research is to develop innovative delivery strategies that facilitate reproducible transfer of drugs and non-viral drug carriers across the placental barrier. The central hypothesis to be tested in this feasibility study is that unique peptide sequences identified using phage-display vectors increase transport efficiency of hydrophilic macromolecules across the human trophoblast barrier via transcytosis. Conjugation of these peptides with hydrophilic drug carriers such as liposomes and microspheres is anticipated to allow effective pharmacotherapy of the unborn. Thus, there are three specific aims proposed: (1) To identify peptide sequences enhancing transfer of peptide-bearing phages across in vitro models of the human trophoblast barrier via transcytosis- (2) To determine transcytosis properties and bulk transport limits of synthetic peptides corresponding to the primary sequences of coat peptides of efficiently transcytosed phage clones across in vitro models of the human trophoblast barrier. (3) To quantify transplacental transport of fluorescent transcytotic carriers of different molecular sizes across dually perfused human placental cotyledons. Constructs of the phagemid pC89 and a nonapeptide library of random peptides will be screened for peptides that mediate efficient phage transfer across in vitro models of the human trophoblast barrier, including confluent layers of syncytium established from human term cytotrophoblasts and BeWo cell monolayers. Experiments will be designed to determine whether phage transfer satisfies criteria for transcytosis. Peptide-encoding DNA inserts of phage clones exhibiting high transfer efficiency will be sequenced and corresponding linear and cyclic analogs synthesized. In addition, conjugates of these peptides with tetramethylrhodamine- labeled dextrans (3 - 500 kDa) will be prepared. Transcytosis properties of these compounds will be determined using in vitro models of the human trophoblast barrier and ex vivo dually perfused human placental cotyledons. The significance of this research is that it will test the feasibility of phage display technology to identify unique peptide sequences for transcytosis systems in the human placenta. This innovative use of an existing methodology may accelerate the development of novel strategies for less invasive fetal pharmacotherapy in utero via transcytosis while reducing the risk for the mother because of favorable biodistribution into the fetal circulation.