Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors

Abstract

The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including () biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; () cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and () tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

pH-responsive high stability polymeric nanoparticles for targeted delivery of anticancer therapeutics

“…The measured membrane insertion pK a of ATRAM is 6.5 (see Supplementary Note 3) [26][27][28] , making the peptide ideal for targeting cancer cells in the acidic microenvironment of solid tumors 45,53 , which results from the high glycolytic rates of tumor cells 54 . Indeed, ATRAM was shown to efficiently target tumors in mice 55 .…”

pH-dependent thermodynamic intermediates of pHLIP membrane insertion determined by solid-state NMR spectroscopy

“…This family of peptides can detect tumors (and other diseased tissues) based on their local acidosis (2)(3)(4)(5)(6)(7)(8). In addition, pHLIP can translocate polar cargo molecules across cellular membranes both in vitro and in vivo (9)(10)(11)(12)(13). These applications are feasible because, in response to mild acidity, pHLIP inserts into membrane (1,14).…”

“…Membrane insertion of pHLIP has been followed by tracking pHdependent changes in Trp fluorescence or circular dichroism (CD) signals (1,10,14,(18)(19)(20). Such plots often appear as sigmoidal twostate transitions between the initial state II at pH ≥ 7 and the final state III at pH ≤ 5 (1,20).…”

“…Thus, it was thought that 50% of pHLIP molecules are in the inserted state III at pH 6 (21,22). This apparent pH 50 value (also known as the pK) has been a key parameter for guiding pHLIP optimization studies, with mixed results (10,13,21,22). A recent study by Barrera and coworkers (20) showed that this pH 50 value varied depending on how the Trp fluorescence signal was analyzed.…”

pHLIP(Var7)-P1AP suppresses tumor cell proliferation in MDA-MB-231 triple-negative breast cancer by targeting protease activated receptor 1

“…Many studies have shown that pHLIPs can link a variety of different molecules, such as fluorescent dyes, toxins, drugs, peptides, and peptide nucleic acids (PNAs) [14][15][16][17][18][19][20][21]. Among the pHLIP variants studied, four sequences have high tumortargeting properties, namely, wild type (WT), variant 3 (Var3), variant 7 (Var7), and ATRAM [22]. One study [19] linked pHLIP (WT) to the P1AP peptide by a non-cleavable linker (chloroacetyl chloride) to obtain pHLIP(WT)-P1AP, which can inhibit the growth of human breast cancer cells (MDA-MB-231 and MCF7 cells) that highly express PAR1 receptor.…”