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递送策略，促进药物和非病毒药物载体穿过胎盘屏障的可重复转移。在该可行性研究中待检验的中心假设是，使用噬菌体展示载体鉴定的独特肽序列通过转胞吞作用增加亲水性大分子穿过人滋养层屏障的转运效率。这些肽与亲水性药物载体如脂质体和微球的缀合预期允许对胎儿的有效药物治疗。因此，提出了三个具体目标：（1）鉴定通过转胞吞作用增强携带肽的噬菌体跨体外人滋养层屏障模型转移的肽序列-（2）确定与有效转胞吞作用的噬菌体克隆的外壳肽的一级序列对应的合成肽跨体外人滋养层屏障模型的转胞吞作用性质和大量转运限制。(3)定量不同分子大小的荧光转胞吞载体通过双重灌注的人胎盘子叶的跨胎盘转运。将筛选噬菌粒pC 89的构建物和随机肽的九肽文库中介导有效噬菌体转移穿过人滋养层屏障的体外模型的肽，所述体外模型包括从人足月细胞滋养层和BeWo细胞单层建立的合胞体的汇合层。将设计实验以确定噬菌体转移是否满足转胞吞作用的标准。将对表现出高转移效率的噬菌体克隆的编码肽的DNA插入物进行测序，并合成相应的线性和环状类似物。此外，将制备这些肽与四甲基罗丹明标记的葡聚糖（3 - 500 kDa）的缀合物。将使用人滋养层屏障的体外模型和离体双重灌注的人胎盘子叶来确定这些化合物的转胞吞性质。本研究的意义在于，它将测试噬菌体展示技术的可行性，以确定独特的肽序列的转胞吞系统在人类胎盘。现有方法的这种创新性使用可以加速通过转胞吞作用在子宫内进行微创胎儿药物治疗的新策略的开发，同时降低母亲的风险，因为有利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.