Fendos and Engelman: pHLIP and acidity as universal biomarker for cancer 33 out changing tumor labeling, in agreement tion that results in retention of cargo mole- with the interpretation. cules in cells even after the peptide has been washed away (Figure 3A-B) [14]. This plat- form has been used to translocate phalloidin pHLIP’s In Se r t Io n Me CHAn ISM toxin into cells and stimulate cell death in The mechanism of pHLIP’s pH-depen- culture (Figure 3C-D) [19], a very direct dent insertion intimately involves the side demonstration of the high potential pHLIP chain topology of the peptide [11,17]. pHLIP has for use in translocating and targeting is a hydrophobic peptide with a long stretch drug molecules to tumor tissues. of hydrophobic residues from positions 9 to 30 (Figure 1B). From a number of experi- SIg n IFICAn Ce An D o Ut Lo o k ments, we have determined that pHLIP’s pH- dependence is largely due to the titration of In addition to solid tumors, areas of in- the two aspartic residues at positions 14 and flammation such as arthritis also have been 25. When acid is added, these two residues shown to exhibit a locally restricted acidic become protonated, converting from charged milieu [16]. To see if pHLIP could be used forms to polar, uncharged forms. This proto- as a probe for these other areas of localized nation effectively converts each residue into acidity, pHLIP conjugates were tested in a a more hydrophobic form, facilitating and rat arthritis model [15], where inflammation stabilizing peptide insertion thermodynami- was induced using a solution of Freud’s ad- cally (Figure 1C), with the two aspartates juvant and BSA injected into the rat’s knee. likely participating directly in spanning the Much to our satisfaction, we found pHLIP- membrane. In addition to the two aspartic fluorophore conjugates preferentially local- residues in the spanning domain, it is likely ized to the areas of induced inflammation the three charged residues at the C-terminal, with similar kinetics as for tumors [15], inserting end of the peptide (Figure 1B) and opening the door for pHLIP’s use in a wider the free carboxyl at the C-terminus also need range of diagnostic and drug-delivery appli- to be protonated (perhaps only transiently) to cations. facilitate insertion [18]. We have reviewed here some of the key characteristics and properties of pHLIP, which uses acidity as a marker for disease Mo Le CULAr CAr g o tissue. Not only does pHLIP allow for spe- t r An SLo CAt Io n USIn g pHLIP cific localization of fluorophores to tumors One of the implications of the insertion and sites of inflammation, but it also offers process is that the C-terminal (inserting end) immense potential as a translocation and of the peptide makes contact with the other drug delivery platform. The obvious nonin- side of the bilayer after insertion (Figure vasive nature of the technology is a key at- 1C). We were interested to see if this translo- traction, along with the fact that pHLIP cation of the C-terminal could be used to appears to be benign even when injected into translocate cargo molecules from one side mice in moderate quantities. Unlike most of the membrane bilayer to the other. By other diagnostic methods dependent on spe- adding a cysteine residue to the C-terminus, cific protein antigens such as CA-2 (prostate we have, in fact, been able to attach mole- cancer) or CA-125 (ovarian cancer), pHLIP cules such as dansyl dye and PNAs by disul- offers the potential for use as a broader-spec- fide bonding or bonds and translocate them trum diagnostic tool that does not simply across synthetic liposome bilayers and cell identify proteins specific to a limited subset plasma membranes [14]. Since disulfide of cancer types but actually localizes in vivo bonds are cleaved when they make contact to the site of the tumor, giving pHLIP power with the highly reducing environment of the in not only diagnosing the presence of solid cytosol, our pHLIP-conjugate platform of- tumors but also in labeling their location and fers a cleavable, releasable cargo transloca- size. We expect our ongoing efforts in con-