Although there are many very effective reagents available to introduce transcriptionally active DNA into viable cells, approaches to deliver functional peptides and proteins into living cells are limited. For this reason, we developed a lipid-mediated protein delivery reagent, called BioPORTER, which can deliver fluorescently labeled antibodies, functional enzymes (caspases and b-galactosidase) and other macromolecules into the cytoplasm of a variety of different adherent and suspension cells. Although BioPORTER is very effective for delivering many different macromolecules into cells it has some limitations. The lipid-mediated delivery system is not effective for delivering hydrophilic proteins and small peptides. For example, using BioPORTER we tried to deliver several peptides including, SV4O NLS and a p53 peptide, without success. Since synthetic peptides are readily available and have the potential to exert intracellular effects on viable cells, it is unfortunate that BioPORTER is not effective for this purpose. Therefore, the objective of this proposal is to understand why BioPORTER is less effective for peptide delivery, and to use this knowledge to engineer a modified reagent that is effective. A series of fluorescently labeled synthetic peptides will be synthesized with different physical properties and their intracellular uptake will be monitored by confocal microscopy and FACS analysis. The understanding gained from this analysis will be applied to the design of peptides and reagent compositions to demonstrate functional intracellular peptide delivery.
Thesaurus Terms: intracellular transport, peptide, protein transport, reagent /indicator, technology /technique development apoptosis, cytoplasm, liposome, oligonucleotide, peptide structure, synthetic peptide confocal scanning microscopy, flow cytometry, fluorescence microscopy, high throughput technology, tissue /cell culture
