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，促进药物和非病毒性药物载体通过胎盘屏障的可重复转移。本可行性研究的中心假设是，使用噬菌体展示载体鉴定的独特肽序列通过胞吞作用提高了亲水大分子穿过人体滋养层屏障的运输效率。这些多肽与亲水性药物载体如脂质体和微球的偶联，预计将允许对未出生的婴儿进行有效的药物治疗。因此，提出了三个具体目标：(1)鉴定通过转细胞作用促进携带肽的噬菌体通过体外人滋养层屏障模型转移的肽序列；(2)确定与高效转细胞噬菌体克隆的包被肽初级序列相对应的合成肽的转细胞作用特性和大量运输限制。(3)定量不同分子大小的荧光转胞载体在双灌注人胎盘子叶中的转运。噬菌体pC89的构建和随机肽的非肽文库将在体外人滋养层屏障模型中筛选介导噬菌体有效转移的肽，包括由人滋养层细胞和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胞吞作用的标准。对具有高转移效率的噬菌体克隆的肽编码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，这些肽与四甲基罗丹明标记的右旋糖酐（3 - 500 kDa）的偶联物将被制备。这些化合物的胞吞特性将通过体外人滋养层屏障模型和体外双灌注人胎盘子叶来确定。本研究的意义在于，它将检验噬菌体展示技术鉴定人类胎盘中胞吞系统独特肽序列的可行性。这种对现有方法的创新使用可能会加速通过胞吞作用在子宫内进行微创胎儿药物治疗的新策略的发展，同时由于有利的生物分布到胎儿循环中，降低了母亲的风险。

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.