PNAS：击杀肿瘤的新技术

Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo

Oleg A. Andreev*,, Allison D. Dupuy, Michael Segala*, Srikanth Sandugu*, David A. Serra, Clinton O. Chichester, Donald M. Engelman,¶, and Yana K. Reshetnyak*,,¶

*Physics Department, University of Rhode Island, 2 Lippitt Road, Kingston, RI 02881; Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, New Haven, CT 06520; Research Office, University of Rhode Island, 70 Lower College Road, Kingston, RI 02881; and Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Fogarty Hall, 41 Lower College Road, Kingston, RI 02881

Contributed by Donald M. Engelman, March 17, 2007 (received for review December 19, 2006)

Abstract

The pH-selective insertion and folding of a membrane peptide, pHLIP [pH (low) insertion peptide], can be used to target acidic tissue in vivo, including acidic foci in tumors, kidneys, and inflammatory sites. In a mouse breast adenocarcinoma model, fluorescently labeled pHLIP finds solid acidic tumors with high accuracy and accumulates in them even at a very early stage of tumor development. The fluorescence signal is stable for >4 days and is approximately five times higher in tumors than in healthy counterpart tissue. In a rat antigen-induced arthritis model, pHLIP preferentially accumulates in inflammatory foci. pHLIP also maps the renal cortical interstitium; however, kidney accumulation can be reduced significantly by providing mice with bicarbonate-containing drinking water. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an -helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The replacement of two key Asp residues located in the transmembrane part of pHLIP by Lys or Asn led to the loss of pH-sensitive insertion into membranes of liposomes, red blood cells, and cancer cells in vivo, as well as to the loss of specific accumulation in tumors. pHLIP nanotechnology introduces a new method of detecting, targeting, and possibly treating acidic diseased tissue by using the selective insertion and folding of membrane peptides.

cancer targeting | imaging | peptide insertion

Fig. 1. Imaging tumors and inflammation. (a) The mechanism of pHLIP interaction with lipid bilayers. The peptide has three states: soluble in water, bound to the surface of a membrane (at normal pH 7.4), and inserted across the membrane as an -helix (at low pH). (b) Overlay of pHLIP-Cy5.5 fluorescence and light images of mice bearing a tumor (7 mm in diameter, 12 d after 10⁶ cell implant) in the right flank obtained on the homemade imager (i.p. injection of 500 µg/kg of pHLIP-Cy5.5 1 d before imaging). (c) pHLIP-Alexa750 fluorescent image (excitation 750 nm, emission 800 nm, artificial green color) of mice bearing a tumor (2 mm in diameter 6 d after 10⁶ cell implant) in the right flank obtained on the IR scanner with focal distance set at 3 mm, which allows for the collection of light from the interior of the body (i.p. injection of 300 µg/kg of pHLIP-Alexa750 1 d before imaging). Reflectance is denoted in red (excitation 680 nm). (d) pHLIP-Cy5.5 given as a single i.p. injection (200 µg/kg) into the left side of mice initially diffused into the left flank, but 20 h later it accumulated in a tumor on the right flank. The fluorescent image of the back part of each mouse is presented. Blue color represents the background fluorescent signal, and the red color represents a high intensity of the fluorescence signal. (e–h) Overlay of pHLIP-Cy5.5 (500 µg/kg) fluorescence and light images of back part of mice bearing tumors of different sizes in right flanks: (e) undetectable by eye at time of imaging 5 d after 10⁵ cell implant, (f) 3 x 4 mm (8 d after 10⁶ cell implant), (g) 5 x 6 mm (12 d after 10⁶ cell implant), (h) 8 x 9 mm (18 d after 10⁶ cell implant). (i) Accumulation of pHLIP- Cy5.5 (2 days after i.p injection, 30 µg/kg) in inflammation sites is shown by overlay of pHLIP fluorescence and photo images of rat right (arthritis) and left (control) legs. The arthritis was induced in the right leg by injection of methylated BSA and Freund's complete adjuvant (the left knee of the rat received a sham injection of saline and was used as a control). A substantial fluorescence signal (4–5 times higher than in the left knee joint) was detected in the right knee (1), especially in the knee joint (2) (red color represents high fluorescence intensity).

英文全文链接：

http://www.pnas.org/cgi/content/full/104/19/7893?