While diseases of the central nervous system (CNS) are one of the leading causes of disability, the further development of neuropharmaceuticals requires the identification of better methods for delivering therapeutics to the brain. Almost all new large molecule drugs are ineffective in treating CNS disorders because they are unable to cross the brain capillary wall that forms the blood-brain barrier (BBB) and enter the brain parenchyma. Even if delivery across the BBB could be overcome, however, the difficult task of penetrating the cell membrane remains. Cell-penetrating peptides (CPPs) are one potential solution. CPPs are short, mainly basic peptides that have the ability to cross the plasma membrane and enter cells. One of the most widely studied CPPs is the TAT peptide, which is derived from a protein involved in the replication, neurotoxicity and immune activation of HIV-1. The aims of this thesis are:​ (1) to assess the ability of TAT to deliver cargoes across the plasma membrane and into brain cells, (2) to analyze the potential utility of TAT in delivering cargoes across the BBB, and (3) to develop techniques for identifying novel CPPs with improved abilities to deliver functional cargoes to brain cells.

The mechanisms underlying TAT-mediated transduction across plasma membranes have previously been investigated in cell lines, but not in primary brain cells. Here, the transduction of a green fluorescent protein (GFP)-TAT fusion protein was found to be dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes grown in the presence of serum, as experimental modulation of GAG content correlated with alterations in TAT transduction. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum-free medium and in coculture with neurons. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency.

In response to disease or injury, astrocytes undergo a phenotypic change called reactive gliosis. Activated astrocytes generate harmful radicals that exacerbate brain damage and can hinder regeneration of damaged neural circuits by secreting neuro-developmental inhibitors and GAGs. The increased expression of GAG after activation resulted in a significant increase in GFP-TAT transduction, and a peptide c-Jun N-terminal kinase (JNK) inhibitor fused to TAT significantly reduced astrocyte activation. These results suggest a potentially new, targeted therapeutic utilization of TAT as a specific delivery vehicle for activated astrocytes.

While CPPs such as TAT can enter cells, it is unclear whether CPPs can pass through cell barriers. Unlike the full length TAT protein from HIV-1, the CPP TAT did not disrupt tight junctions nor did it increase endothelial permeability. Furthermore, although TAT was capable of delivering a mock therapeutic into the endothelial cells, it could not carry the cargo across the barrier and deliver it into astrocytes on the other side. Ischemic injury significantly decreased the integrity of the endothelial monolayer, caused an increase in the transport of TAT across the monolayer by 100%, and significantly increased the delivery of GFP-TAT into astrocytes on the other side. We conclude that although TAT may not be an efficient vehicle for delivery across an intact BBB, it may have utility in delivering therapeutic cargos to endothelial cells, or in clinical situations when the BBB is disrupted.

To identify novel CPPs, a phage display library was created and screened for the ability to deliver a phagemid containing an enhanced yellow fluorescent protein (EYFP) transgene to PC12 cells. EYFP expression was never detected, likely due to the inability of the phagemid to be released from within the phage coat. A second screening system was employed which involved recovering the internalized phage through infection and amplification in E. coli. Three peptides were enriched over three rounds of screening:​ MS1, MS2, and MS3. These peptides do not contain a large number of positively charged amino acids, making them unique from most known CPPs. This phage screening technique can be used to identify custom CPPs capable of delivering cargoes under predesigned delivery conditions.

The identification of peptides with the ability to cross the BBB, without disrupting its integrity, and penetrate brain cells will have a significant and immediate impact on the treatment of neurological diseases. Furthermore, these peptides will not only increase the fundamental engineering knowledge of protein motifs necessary to penetrate cells, but also aid in future rational design of peptides with the ability to home to target organs and cell types after systemic administration.