Novel pH-Sensitive Cyclic Peptides

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)4WC], and asymmetric, c[E4W5C], 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[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], 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

Targeted Imaging of Urothelium Carcinoma in Human Bladders by an ICG pHLIP Peptide Ex vivo

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® peptide) conjugated with a near infrared fluorescent dye (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 non-buffered 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. ICG pHLIP® imaging agent marked high grade urothelial carcinomas, both muscle invasive and non-muscle invasive. Carcinoma in situ (CIS) was accurately diagnosed in 11 cases, whereas only 4 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

Comparative Study of Tumor Targeting and Biodistribution of pH (Low) Insertion Peptides (pHLIP® Peptides) Conjugated with Different Fluorescent Dyes

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

pH (Low) Insertion Peptide targets 4T1 mammary tumors

Malignant tumors exhibit elevated uptake and use of glucose, as well as metabolite buildup and hypoxia due to inadequate blood supply, leading to tumor acidosis. The extracellular acidity is linked to tumor progression. 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. pHLIPs® (pH (Low) Insertion Peptides) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors in BALB/c mice with three Alexa546 labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3-and Var7-pHLIPs

PHLIP-FIRE, a cell insertion-triggered fluorescent probe for imaging tumors demonstrates targeted cargo delivery in vivo

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. 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 dequenching 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. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications

Targeting Breast Tumors with pH (Low) Insertion Peptides

Extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. Thus, targeting of tumor acidity can open new avenues in diagnosis and treatment of aggressive tumors and targeting metastatic cancers cells within a tumor. pH (low) insertion peptides (pHLIPs) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)­634Mul transgenic mice with three fluorescently labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs. The Var3- and Var7-pHLIPs constructs have faster blood clearance than the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of the above breast tumor models with tumor accumulation increasing over 4 h postinjection. Staining of nonmalignant stromal tissues in transgenic mice was minimal. The pHLIPs distribution in tumors showed colocalization with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest degree of colocalization of fluorescent pHLIPs was shown to be with lactate dehydrogenase A, which is related to lactate production and acidification of tumors. In sum, the pHLIP-based targeting of breast cancer presents an opportunity to monitor metabolic changes, and to selectively deliver imaging and therapeutic agents to tumors

pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery <i>In Vivo</i>

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-<u>F</u>luorescence <u>I</u>nsertion <u>RE</u>porter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. 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 dequenching 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. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation <i>in vivo</i>. Imaging and targeted cargo delivery should each have clinical applications