Parenteral Delivery of Peptides and Proteins - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 583

such as nuclei, lysosomes, and mitochondria in order to exert biological action. Being
selectively permeable, the cell membrane prevents such drugs and their nanoparticu-
late drug carriers to enter the cell. Thus, targeting P/P drugs inside the cell to achieve
therapeutic action in such conditions is very difficult unless the drug is highly lipo-
philic or very small or there is an active transport mechanism involved, which hap-
pens mainly with very short peptides [436] . The existing methods for delivery of
macromolecules, such as viral vectors and membrane perturbation techniques, can
result in high toxicity, immunogenicity, and low delivery yield [437] . Nonviral vec-
tors with targeted ligands, because of their physicochemical characteristics like size,
are more susceptible to lysosomal degradation [438-443] . Thus, to deliver P/P drugs
into the cytoplasm by bypassing the endocytic pathway to protect the drug from lyso-
somal degradation is an essential and challenging task for pharmaceutical research
scientists.
A promising approach to overcome the cellular barrier for intracellular P/P drug
delivery is “cell-penetrating peptides (CPPs).” This approach involves molecular
engineering of P/P to tie certain proteins or peptides to the hydrophilic P/P drug of
interest. The construct possesses the ability to translocate across the plasma mem-
brane and deliver the cargo intracellularly; the process is termed protein transduction
[444,445] .
The property of translocation across the cell membrane was introduced in 1988
by Green [448] and Frankel [447] , who demonstrated that the 86-mer transactivating
transcriptional activator, Tat protein encoded by HIV-1, was shown to enter the HeLa
cells in a nontoxic and efficient manner in vitro when introduced in the surrounding
media. Using mutagenesis studies, the biological activity and penetrating capability
of Tat was found to reside in the region between residues 48 and 60 [446] . In 1999,
it was found that Tat peptide is able to transduce large proteins like -galactosidase,
horseradish peroxidase (HRP), and RNAase. In light of such properties, Tat became
known as the first “CPP.” Subsequently, a number of peptides, either derived from
proteins or synthesized chemically, demonstrated the property of translocation. Protein
transduction domains (PTDs) or CPPs consist of short sequences of amino acids (less
than 30), highly positively charged—the crucial factor for activity of CPP—contrib-
uted by basic residues lysine (transportan, model amphipathic peptide [MAP]) or
arginine (TAT peptide, penetratin, and pVEC) that are able to penetrate almost any
cell, carrying with them a relatively large and wide range of cargoes such as proteins,
oligonucleotides, liposomes (200 nm), and drugs [449] . In 2003, the first CPP-based
drug reached phase II clinical trials. Other examples of CPPs are antennapedia (Antp-
43-58) [450] , VP22 [451] , transportan [452] , MAP [453] , and synthetic polyarginines
such as R9 analogue of TAT (R9-TAT), HIV-1 Rev, flock house virus coat peptide,
and DNA-binding peptides like c-Fos [138-163] and yeast GCN4 [454,455] .
CPPs have the ability to enter cells independent of a membrane receptor. They
show no cell-type specificity [456] and are not affected by size or low temperature,
and are only saturable at very high protein concentrations [457-462] . Protein trans-
duction with CPP occurs efficiently into almost 100% of cells such as peripheral blood
lymphocytes, all blood cells, bone marrow stem cells, diploid human fibroblasts,
osteoclasts, osteosarcoma, fibrosarcoma cells, glioma, heptocellular carcinoma, renal