RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells.
BACKGROUND
To facilitate selective drug delivery to hepsin (Hpn)-expressing cancer cells, the RIPL peptide (IPLVVPLRRRRRRRRC; 16mer; 2.1 kDa) was synthesized as a novel cell penetrating/homing peptide (CPHP) and conjugated to a liposomal carrier.


METHODS
RIPL peptide-conjugated liposomes (RIPL-Lipo) were prepared by conjugating RIPL peptides to maleimide-derivatized liposomal vesicles via the thiol-maleimide reaction. Vesicle size and zeta potential were examined using a Zetasizer. Intracellular uptake specificity of the RIPL peptide, or RIPL-Lipo, was assessed by measuring mean fluorescence intensity (MFI) after treatment with a fluorescent marker in various cell lines: SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaT for Hpn(-). FITC-dextran was used as a model compound. Selective translocational behavior of RIPL-Lipo to LNCaP cells was visualized by fluorescence microscopy and confocal laser scanning microscopy. Cytotoxicities of the RIPL peptide and RIPL-Lipo were evaluated by WST-1 assay.


RESULTS
RIPL peptides exhibited significant Hpn-selectivity. RIPL-Lipo systems were of positively charged nanodispersion (165 nm in average; 6-24 mV depending on RIPL conjugation ratio). RIPL-Lipo with the conjugation of 2300 peptide molecules revealed the greatest MFI in all cell lines tested. Cellular uptake of RIPL-Lipo increased by 20- to 70-fold in Hpn(+) cells, and 5- to 7-fold in Hpn(-) cells, compared to the uptake of FITC-dextran. Cytosolic internalization of RIPL-Lipo was time-dependent: bound instantly; internalized within 30 min; distributed throughout the cytoplasm after 1 h. Cytotoxicities of RIPL peptide (up to 50 μM) and RIPL-Lipo (up to 10%) were minor (cell viability >90%) in LNCaP and HaCaT cells.


CONCLUSIONS
By employing a novel CPHP, the RIPL-Lipo system was successfully developed for Hpn-specific drug delivery.
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Min Hyung
MH
Kang
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Min Jung
MJ
Park
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Hyun Joon
HJ
Yoo
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Kwon Yie
KY
hyuk
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Sang Gon
SG
Lee
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Sung Rae
SR
Kim
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
Dong Woo
DW
Yeom
College of Pharmacy, Dankook University, Cheonan-Si, Republic of Korea.
Myung Joo
MJ
Kang
College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea. Electronic address: ywchoi@cau.ac.kr.
Young Wook
YW
Choi
D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000008:administration & dosage; D016503:Drug Delivery Systems; D006801:Humans; D008081:Liposomes / Q000008:administration & dosage*; D061986:MCF-7 Cells; D008301:Maleimides / Q000008:administration & dosage; D008856:Microscopy, Fluorescence / Q000379:methods; D010455:Peptides / Q000008:administration & dosage*; D012697:Serine Endopeptidases / Q000378:metabolism*
D004337:Drug Carriers; D008081:Liposomes; D008301:Maleimides; D010455:Peptides; C043592:maleimide; D012697:Serine Endopeptidases; C055645:hepsin
Cell penetrating/homing peptide; Hepsin; IPL; Intracellular delivery; Liposome; Polyarginine; Targeting