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Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH low insertion peptide (pHLIP) conjugated with a near-infrared fluorescent dye [indocyanine green (ICG)] targets low extracellular pH, allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with nonbuffered saline and instilled with a solution of the ICG pHLIP construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80% if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. The ICG pHLIP imaging agent marked high-grade urothelial carcinomas, both muscle invasive and nonmuscle invasive. Carcinoma in situ was accurately diagnosed in 11 cases, whereas only four cases were seen using white light, so imaging with the ICG pHLIP peptide offers improved early diagnosis of bladder cancers and may also enable new treatment alternatives.

A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE) 4 WC] and asymmetric, c[E 4 W 5 C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E 4 W 5 C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R 4 W 5 C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue.

Purpose Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP ® peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Procedures Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP ® variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP ® variants in multidimensional space of spectral parameters. Results The fluorescently labeled pHLIP ® variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP ® peptide. Conclusions Different cargo molecules conjugated with pHLIP ® peptides can alter biodistribution and tumor targeting. The obtained knowledge is essential for the design of novel pHLIP ® -based diagnostic and therapeutic agents targeting primary tumors and metastatic lesions.

Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP(®) peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP(®) variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP(®) variants in multidimensional space of spectral parameters. The fluorescently labeled pHLIP(®) variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP(®) peptide. Different cargo molecules conjugated with pHLIP(®) peptides can alter biodistribution and tumor targeting. The obtained knowledge is essential for the design of novel pHLIP(®)-based diagnostic and therapeutic agents targeting primary tumors and metastatic lesions.

The design and development of pH-sensitive peptides for cancer diagnostics provides an opportunity to study and address fundamental questions of in vivo bio-distribution of pH-sensitive peptides, as well as developing new targeting imaging and therapeutic agents for acidic diseased tissue such as cancer, infections, ischemia, stroke and others. The main goal of the work presented here is to investigate the following problems: - Targeting of highly metastatic mammary tumors and spontaneous breast tumors in transgenic mice with fluorescently labeled pHLIPs® (pH (Low) Insertion Peptides) pHLIP variants; - Direct imaging of pHLIP insertion and cargo translocation in vivo; - Targeting of mammary tumors using cyclic pH-sensitive peptides; - Biodistribution of different pHLIP variants conjugated with various fluorescent dyes with the main purpose to identify the best pHLIP-based constructs for clinical applications. It has been shown that extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. The most effective pH-sensitive tumor targeting agents should sense pH at the surface of cancer cells, where it is expected to be the lowest. Thus, targeting tumor acidity might represent a novel approach for the prediction of tumor aggressiveness and delivery of therapeutic agents to tumor cells with the greatest metastatic potential. pHLIP belongs to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Also, cyclic pH-sensitive peptides containing a number of Trp and Glu residues can be developed for imaging of acidity in tumors. pHLIP insertion is associated with the protonation of Asp/Glu residues in the transmembrane sequence and its inserting end. Carboxyl group protonation leads to an increase in the hydrophobicity that further triggers transmembrane formation and insertion of the peptide across a lipid bilayer. pHLIP insertion is predominantly uni-directional. In contrast to cell-penetrating peptides, pHLIP remains in the cellular membrane after insertion, translocating one end across the bilayer and comes out in the cytoplasm, while leaving the other end in the extracellular space. Therefore, pHLIP possesses dual delivery capabilities: it can translocate cell-impermeable cargo molecules into the cell cytoplasm or it can tether cargo molecules to the cell surface. Also, the process of peptide folding into a membrane ensures a much higher co-operativity of the transition compared to the pH-diffusion across a membrane. A family of novel pHLIP variants were introduced and demonstrated that tumor targeting, blood clearance and biodistribution of this peptides can be modulated by tuning their sequence and, as a result, their physical and chemical properties and their interaction with the cell membrane. For demonstrating direct imaging of pHLIP insertion and cargo translocation in vivo, the cell-inserting end of the pHLIP-FIRE peptide has a fluorophore-fluorophore or fluorophore-quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in de-quenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues, but is suppressed elsewhere due to quenching. The cyclic pH-sensitive peptides used in this work, contain: i) single cysteine (Cys, C) residue for conjugation purposes, ii) at least one tryptophan (Trp, W) for ability to record fluorescence signal, iii) 3-5 protonatable glutamic acid (Glu) residues to trigger pH-dependent interaction with membrane. As in the case of pHLIP peptides, the presence of low pH leads to the protonation of carboxyl groups of Glu residues, which increases peptides hydrophobicity and promotes partition of peptides in bilayer. Thus, there is an opportunity to develop new imaging and/or therapeutic agents, which are based on the use of pH-sensitive peptides. We have successfully established the bio-distribution of both pHLIP and cyclic pH-sensitive peptides attached to different fluorescent dyes and identified the best candidate for imaging and therapeutic applications.