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.
| Basha, Saleem; Vaidhyanathan, Shruthi; Pauletti, Giovanni M (2011) Selection of peptide ligands for human placental transcytosis systems using in vitro phage display. Methods Mol Biol 716:141-56 |
