There has been tremendous recent growth in the discovery of biological therapeutic molecules, but they are often difficult to be utilized as efficacious therapies due to poor delivery and low pharmacological stability. Therefore, the delivery of macromolecules such as peptides, proteins and oligonucleotides has become a key issue in biological drug development. The additional biological barriers that evolved to protect brain cells have further restricted the application of biological therapeutics on the treatment of central nervous system disorders. In the last twenty years, a number of non-vial delivery technologies have been developed that exhibit low toxicity and immunogenicity, including cell penetrating peptides (CPPs). Although some studies have shown that the TAT peptide, one of the natural CPPs, can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivoenvironment. Here, we investigated TAT-mediated delivery of proteins and oligonucleotide-based cargos to a neuronal-like cell line and primary brain cells and offer a possible explanation for the conflicting results in the literature surrounding TAT-mediated transduction. In addition, we used a powerful tool in protein engineering, directed evolution, to identify a novel cell penetrating peptide that can be used as an efficacious penetration enhancer for lipid-based systemic drug delivery.
Using green fluorescent protein (GFP) as a model protein cargo, we demonstrated that transduction efficiency of GFP-TAT fusion protein is correlated to cellular glycosaminoglycan (GAG) expression in PC12 cells which is dictated by culture conditions. This new observation may help to resolve some of the reported controversy surrounding TAT-mediated delivery efficiency. A DNA binding domain p50 was introduced into the fluorescent fusion protein to create a biofunctional chimeric protein p50-GFP-TAT (PGT), which we used for studying TAT-mediated delivery of oligonucleotide-based cargos. The PGT construct was able to deliver 30bp and 293bp oligonucleotides to PC12 cells with an optimal ratio of 1.89 protein molecules per base pair of DNA length. This correlation was validated through the delivery of a red fluorescent protein (RFP) transgene encoded in plasmid DNA to PC12 cells, but the overall delivery efficiency of the plasmid was very low. For small interfering RNA (siRNA) delivery, PGT-mediated siGFP delivery was unable to induce detectable RNA interfere (RNAi) in a cell line stably-transfected with a GFP gene. This was probably, due to endosomal entrapment of the complex. We conclude that TAT is an efficient delivery vehicle for proteins or peptides in a fusion protein construct, but it is not an ideal vehicle for the delivery of oligonucleotides due to problems with intracellular trafficking.
We constructed a p50 based plasmid display system with a 14-mer randomized peptide library for the directed evolution of new CPPs. After four rounds of selection, we identified a novel peptide SG3, which exhibited significant penetrating abilities in both PC12 cells and primary astrocytes. In addition, the additive delivery effect of SG3 and Lipofectamine 2000 is much more significant than that of TAT and Lipofectamine, which indicates the potential use of SG3 as an efficacious penetration enhancer for systemic drug delivery platforms which have been recognized as the most promising drug delivery strategies for biological therapeutics.
Our work is one of the growing numbers of examples that demonstrate the advantages of a protein engineering methodology, directed evolution, for developing peptides or proteins with novel functions without requiring an in-depth understanding of either the structures or the detailed mechanism of their functions.
|School Location:||United States -- New York|
|Source:||DAI-B 70/12, Dissertation Abstracts International|
|Subjects:||Pharmacy sciences, Biomedical engineering, Chemical engineering|
|Keywords:||Blood-brain barrier, Cell penetrating peptides, Neuronal-like cells, Nucleotide delivery, Plasmid displays, Protein delivery|
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