Cy5.5-labeled pH low insertion peptide (pHLIP)

The pH low insertion peptide (pHLIP) is a peptide of 37 amino acids that inserts across the cell membrane as an α-helix when the extracellular pH (pHe) is acidic (1-4). pHLIP labeled with the near-infrared (NIR) fluorescent marker Cy5.5 (pHLIP-Cy5.5) was developed by Andreev et al. for optical mapping of areas (tumor and arthritis) of elevated acidity in the small animals (1).Tumor microenvironment is characterized by low pHe (5, 6). Almost all solid tumors have a neutral to alkaline intracellular pH (pHi), but they develop an acidic pHe (known as the Warburg effect, Nobel Prize in 1931 ). The average pHe could be as low as 6.0 (7-9). A pH gradient (pHi > pHe) exists across the cell membrane in tumors. This gradient is contrary to that found in normal tissues, in which pHi is lower than pHe (7.2–7.4) (7-9). Diffusion of the H+ ions along concentration gradients from tumors into adjacent normal tissues creates a peritumoral acid gradient (10). The mechanisms responsible for the low pHe include anaerobic glycolysis because of hypoxia, aerobic glycolysis (the Warburg effect), increased metabolic CO2 production associated with uncontrolled cell growth, and increased activity of ion pumps on the cell membrane (5, 7).Low pHe affects many aspects of tumor physiology. It is one of the driving forces in the clonal selection leading to invasive and metastatic diseases (11, 12). Rofstad et al. have shown that lowering culture pH to 6.8 results in a promotion of in vivo metastasis of treated human melanoma cells compared with controls (cultured at pH 7.4) after tail vein injection of the cells in mice (13). Exposure of tumor cells to an acidic environment leads to increased expression of various factors that contribute to tumor progression (12). Tumor cells are able to maintain a high proliferation rate in the acidic environment, whereas the peritumoral acid gradient limits immune response to tumor antigens and induces normal cell apoptosis, extracellular matrix degradation, and angiogenesis (7, 11). The passage of noncarrier-mediated weak drugs through the cell membranes is also influenced by the acidic pHe (14-16). Typically, the drugs in an uncharged state (lipophilic form) pass more efficiently through the cell membranes. This leads to the hypothesis of ‘ion-trapping’ that weakly basic drugs will concentrate in more acidic compartments (14, 15). The acid pHe of tumors will therefore hinder weakly basic drugs from reaching their intracellular targets, thereby reducing cytotoxicity (16). Conversely, the acid pHe of tumors will improve uptake of weak acids into the relatively neutral intracellular space (17). The currently used chemotherapeutic drugs such as mitoxantrone, doxorubicin, daunorubicin, anthracyclines, anthraquinones and vinca alkaloids are all weak bases (pKa 5.5–6.8), while cyclophosphamide, 5-fluorouracil and chlorambucil are weak acids (pKa 7.8–8.8) (15). Both in vitro and in vivo studies have shown that the activities of those weak bases are inhibited by the low pHe (14-16). On the contrary, the actions of the weak acids are enhanced by the low pHe. The pH gradient in tumors exerts a protective effect upon the cells from weak-base drugs as well as acts to potentiate the action of weak acid drugs (17). Studies have consistently shown that selective tumor alkalinization in vivo is likely to result in an enhancement in the anti-tumor activity of weakly basic chemotherapeutic drugs (18, 19). Low pHe has also been shown to impair the effectiveness of some drugs such as paclitaxel in that their chemical structures do not predict pH-dependent ionization (7). In addition, radiation therapies are known to be significantly less effective at the hypoxic and acidic regions of tumor (20).An understanding of the mechanisms involved in tumor-specific low pHe leads to the development of targeted therapeutic approaches (6, 7). Low pHe is also considered a promising marker for tumor targeting detection (4, 8). The pHLIP interacts with the surface of membranes as an unstructured peptide at neutral pHe, but at acidic pHe (<7.0) it inserts across the membrane and forms a stable transmembrane α-helix (1, 2, 21, 22). The pHLIP affinity for membranes at low pH (5.0) is 20 times higher than that at high pH (8.0). Studies by Zoonens et al. showed that the pHLIP could translocate cell-impermeable cargo molecules across a cell membrane and release them in the cytoplasm (23). The process is mediated by the formation of a transmembrane α-helix because of increased peptide hydrophobicity associated with the protonation of Asp residues at low pH (1, 22). Replacement of the two key Asp residues located in the transmembrane part of pHLIP with Lys or Asn leads to the loss of pH-sensitive membrane insertion (3). Andreev et al. labeled the pHLIP with Cy5.5 and tested its feasibility for optical mapping of tumor and arthritis which were characterized by elevated acidity (1).Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets in small animals (24-26). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light are used. Near-infrared (NIR) fluorescence detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive alternative to radionuclide imaging in small animals (27, 28).