Biol. Pharm. Bull., 30(2),218-223, February 2007

Regular Articles

Creation of Novel Cell-Penetrating Peptides for Intracellular Drug Delivery Using Systematic Phage Display Technology Originated from Tat Transduction Domain

Haruhiko KAMADA,^*,a Takayuki OKAMOTO,^b,c Maki KAWAMURA,^a,b Hiroko SHIBATA,^a,b Yasuhiro ABE,^a,b Akiko OHKAWA,^a,b Tetsuya NOMURA,^a,b Masaki SATO,^a,b Yohei MUKAI,^a,b Toshiki SUGITA,^a,b Sunao IMAI,^a,b Kazuya NAGANO,^a,b Yasuo TSUTSUMI,^a,b Shinsaku NAKAGAWA,^b Tadanori MAYUMI,^d and Shin-ichi TSUNODA^a

^a Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation; 7-6-8 Asagi, Saito, Ibaraki, Osaka 567-0085, Japan: ^b Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University; 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan: ^c Department of Molecular Pathobiology, Mie University School of Medicine; 2-174 Edobashi, Tsu, Mie 514-8507, Japan: and ^d Department of Cell Therapeutics, Graduate School of Pharmaceutical Sciences, Kobe Gakuin University; 518 Arise, Ikawadani, Nishi-ku, Kobe 651-2180, Japan. * To whom correspondence should be addressed. e-mail: kamada@nibio.go.jp

Many biologically active proteins need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm. Cell penetrating peptides (CPPs) have been developed to efficiently deliver a wide variety of cargo in a fully biological active form into a range of cell types for the treatment of multiple preclinical disease models. To further develop this methodology, we established a systematic approach to identify novel CPPs using phage display technology. Firstly, we screened a phage peptide library for peptides that bound to the cell membrane. Secondly, to assess functionality as intracellular carriers, we recombined cDNAs of binding peptides with protein synthesis inhibitory factor (PSIF) to create fusion proteins. Randomly chosen clones were cultured and expression of peptide-PSIF fusion proteins induced, followed by screening of protein synthesis activity in cells. Using this systematic approach, novel and effective CPPs were rapidly identified. We suggest that these novel cell-penetrating peptides can utilized as drug delivery tools for protein therapy or an analytical tool to study mechanisms of protein transduction into the cytoplasm.

Key words cell penetrating peptide; phage display; Tat