Targeting acidic pre-metastatic niche in lungs by pH low insertion peptide and its utility for anti-metastatic therapy.

Dysregulated extracellular pH, the universal feature of tumor, works as an evolutional force to drive dissemination of tumor cells. It is well-established that tumor acidity is associated with tumor growth and metastasis. However, the pH of pre-metastatic niche remains unclear. We hypothesized that primary tumor cells remotely prime acidity in secondary organ to achieve metastatic colonization. Herein, we demonstrated that the pH responsive probe pH Low Insertion Peptide (pHLIP) was notably accumulated in pre-metastatic lungs of 4T1.2 breast tumor-bearing mice. The pHLIP-targeted lungs showed high amounts of lactate and overexpressed glycolysis-related proteins. Pharmacological inhibition of glycolysis suppressed the lung acidification induced by 4T1.2 cancer cell culture supernatant and delayed subsequent metastatic burden of disseminated tumor cells. In the acidic lungs, pHLIP was primarily localized in alveolar type 2 cells which strongly expressed glycolysis-related proteins. 4T1.2-derived extracellular vesicles expressed some of the glycolysis-related proteins, and their administration increased pHLIP accumulation and glycolytic enhancement in lungs. pHLIP-conjugated dexamethasone effectively attenuated lung metastatic burden by disrupting pro-inflammatory response in the acidic lungs. From these results, targeting the metastasis-supporting microenvironment by pHLIP technology creates possibility to identify pre-metastatic organ and prevent metastatic recurrence.

MicroRNA 29a therapy for CEACAM6-expressing lung adenocarcinoma.

BACKGROUND: Non-coding microRNAs (miRNAs) play critical roles in tumor progression and hold great promise as therapeutic agents for multiple cancers. MicroRNA 29a (miR-29a) is a tumor suppressor miRNA that inhibits cancer cell growth and tumor progression in non-small cell lung cancer. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which plays an important role in lung cancer progression, has been identified as a target of miR-29a. Here, we evaluated the therapeutic efficacy of a peptide vector capable of delivering miR-29a intracellularly using the acidic tumor microenvironment in a lung adenocarcinoma xenograft mouse model. METHODS: A miRNA delivery vector was constructed by tethering the peptide nucleic acid form of miR-29a to a peptide with a low pH-induced transmembrane structure (pHLIP) to enable transport of the miRNAs across the plasma membrane. Tumor suppressive effects of pHLIP-miR29a on lung adenocarcinoma development in vivo were assessed using a BALB/c xenograft model injected with A549 cells. RESULTS: Incubation of A549 cells with pHLIP-miR-29a at an acidic pH downregulated endogenous CEACAM6 expression and reduced cell viability. Intravenous injection of the mice with pHLIP-miR-29a inhibited tumor growth by up to 18.1%. Intraperitoneal injection of cisplatin reduced tumor volume by 29.9%. Combined pHLIP-miR-29a + cisplatin treatment had an additive effect, reducing tumor volume up to 39.7%. CONCLUSIONS: Delivery of miR-29a to lung adenocarcinoma cells using a pHLIP-mediated method has therapeutic potential as a unique cancer treatment approach.

Recent Advances in Imaging Agents Anchored with pH (Low) Insertion Peptides for Cancer Theranostics.

The acidic extracellular microenvironment has become an effective target for diagnosing and treating tumors. A pH (low) insertion peptide (pHLIP) is a kind of peptide that can spontaneously fold into a transmembrane helix in an acidic microenvironment, and then insert into and cross the cell membrane for material transfer. The characteristics of the acidic tumor microenvironment provide a new method for pH-targeted molecular imaging and tumor-targeted therapy. As research has increased, the role of pHLIP as an imaging agent carrier in the field of tumor theranostics has become increasingly prominent. In this paper, we describe the current applications of pHLIP-anchored imaging agents for tumor diagnosis and treatment in terms of different molecular imaging methods, including magnetic resonance T1 imaging, magnetic resonance T2 imaging, SPECT/PET, fluorescence imaging, and photoacoustic imaging. Additionally, we discuss relevant challenges and future development prospects.

Targeting cancer cells in acidosis with conjugates between the carnitine palmitoyltransferase 1 inhibitor etomoxir and pH (low) insertion peptides.

Targeting enzymes involved in tumor metabolism is a promising way to tackle cancer progression. The inhibition of carnitine palmitoyltransferase 1 (CPT1) by etomoxir (Eto) efficiently slows down the growth of various cancers. Unfortunately, the clinical use of this drug was abandoned because of hepatotoxic effects. We report the development of pH-sensitive peptide (pHLIP)-drug conjugate to deliver Eto selectively to cancer cells exposed to acidic microenvironmental conditions. A newly designed sequence for the pHLIP peptide, named pHLIPd, was compared with a previously published reference pHLIP peptide, named pHLIPr. We showed that the conjugate between pHLIPd and Eto has a better pH-dependent insertion and structuration than the pHLIPr-based conjugate inside POPC vesicles. We observed antiproliferative effects when applied on acid-adapted cancer cells, reaching a larger inhibitory activity than Eto alone. In conclusion, this study brings the first evidence that pHLIP-based conjugates with a CPT1 inhibitor has the potential to specifically target the tumor acidic compartment and exert anticancer effects while sparing healthy tissues.

A Dual pH-Responsive DOX-Encapsulated Liposome Combined with Glucose Administration Enhanced Therapeutic Efficacy of Chemotherapy for Cancer.

BACKGROUND: The acidic microenvironment of cancer can promote tumor metastasis and drug resistance. Acidic tumor microenvironment-targeted therapy is currently an important means for treating tumors, inhibiting metastasis, and overcoming drug resistance. In this study, a dual pH-responsive DOX-encapsulated liposome (DOPE-DVar7-lip@DOX) was designed and fabricated for targeting the acidic tumor microenvironment. On the one hand, the response of acid-sensitive peptide (DVar7) to the acidic tumor microenvironment increased the uptake of liposomes in tumors and prolonged the retention time; on the other hand, the response of acid-sensitive phospholipid (DOPE) to the acidic tumor microenvironment improved the controlled release of DOX in tumors. METHODS: The acid-sensitive peptide DVar7 modified liposomes can be obtained by simple incubation of DSPE-DVar7 with DOX-loaded DOPE liposomes (DOPE-lip@DOX). The tumor targeting of the dual pH-responsive liposome was investigated in vitro and in vivo by near-infrared fluorescence imaging. The tumor therapeutic efficacy of DOPE-DVar7-lip@DOX was evaluated in breast cancer mouse model using the traditional liposome as a control. Moreover, we regulated the tumor microenvironment acidity by injecting glucose to further enhance the therapeutic efficacy of cancer. RESULTS: DVar7 can allosterically insert into the tumor cell membrane in the acidic tumor microenvironment to enhance the tumor uptake of liposomes and prolong the retention time of liposomes in tumor. In addition, the therapeutic efficacy of pH-responsive liposomes can be further enhanced by glucose injection regulating the acidity of tumor microenvironment. DISCUSSION: DVar7 modified acid-sensitive nanocarriers combined with acidity regulation have great potential to improve drug resistance in clinical practice, thus improving the response rate and therapeutic effect of chemotherapy.

Long non-coding RNAs: From disease code to drug role.

Enormous studies have corroborated that long non-coding RNAs (lncRNAs) extensively participate in crucial physiological processes such as metabolism and immunity, and are closely related to the occurrence and development of tumors, cardiovascular diseases, nervous system disorders, nephropathy, and other diseases. The application of lncRNAs as biomarkers or intervention targets can provide new insights into the diagnosis and treatment of diseases. This paper has focused on the emerging research into lncRNAs as pharmacological targets and has reviewed the transition of lncRNAs from the role of disease coding to acting as drug candidates, including the current status and progress in preclinical research. Cutting-edge strategies for lncRNA modulation have been summarized, including the sources of lncRNA-related drugs, such as genetic technology and small-molecule compounds, and related delivery methods. The current progress of clinical trials of lncRNA-targeting drugs is also discussed. This information will form a latest updated reference for research and development of lncRNA-based drugs.

Pharmacokinetic modeling reveals parameters that govern tumor targeting and delivery by a pH-Low Insertion Peptide (pHLIP).

A pH-Low Insertion Peptide (pHLIP) is a pH-sensitive peptide that undergoes membrane insertion, resulting in transmembrane helix formation, on exposure to acidity at a tumor cell surface. As a result, pHLIPs preferentially accumulate within tumors and can be used for tumor-targeted imaging and drug delivery. Here we explore the determinants of pHLIP insertion, targeting, and delivery through a computational modeling approach. We generate a simple mathematical model to describe the transmembrane insertion process and then integrate it into a pharmacokinetic model, which predicts the tumor vs. normal tissue biodistribution of the most studied pHLIP, "wild-type pHLIP," over time after a single intravenous injection. From these models, we gain insight into the various mechanisms behind pHLIP tumor targeting and delivery, as well as the various biological parameters that influence it. Furthermore, we analyze how changing the properties of pHLIP can influence the efficacy of tumor targeting and delivery, and we predict the properties for optimal pHLIP phenotypes that have superior tumor targeting and delivery capabilities compared with wild-type pHLIP.

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

The applications of the pH low insertion peptide (pHLIP) in cancer diagnosis and cross-membrane cargo delivery have drawn increasing attention in the past decade. With its origin as the transmembrane (TM) helix C of bacteriorhodopsin, pHLIP is also an important model for understanding how pH can affect the folding and topogenesis of a TM α-helix. Protonations of multiple D/E residues transform pHLIP from an unstructured coil at membrane surface (known as state II, at pH ≥ 7) to a TM α-helix (state III, pH ≤ 5.3). While these initial and end states of pHLIP insertion have been firmly established, what happens at the intervening pH values is less clear. However, the intervening pH range is most relevant to pHLIP-cell interactions in the acidic extracellular tumor environment (and in the endosomes within cells). Here, using advanced solid-state NMR spectroscopy with palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine unilamellar vesicles as the model membrane, we systematically examined the state of pHLIP-membrane interactions (in terms of the membrane locations of D/E residues, as well as lipid dynamics) at the intervening pH values of 6.4, 6.1, and 5.8, along with the known states at pH 7.4 and 5.3. Thermodynamic intermediate states distinct from the initial and end states were discovered to exist at each of the intervening pH examined. They support a multistage model of pHLIP insertion in which the D/E titrations occur in a defined sequence at distinct intermediate pH values. This multistage model has important ramifications in pHLIP applications.

pH low insertion peptide mediated cell division cycle-associated protein 1 -siRNA transportation for prostatic cancer therapy targeted to the tumor microenvironment.

Prostate cancer (PCa) is a common malignancy in male urinary system. Cell division cycle-associated protein 1 (CDCA1) is expressed highly in many cancer cells. Yet, whether CDCA1 play an important role in PCa progression is uncertain. pH low insertion peptide (pHLIP), a PH-induced transmembrane structure, can pass through the cell membrane into intracellular in an acidic environment. In this study, we try to confirm the expression status of CDCA1 in the PCa patients' tissues and PCa cell line. In addition, to make the CDCA1-siRNA efficiently targeting the PCa cells, pHLIP and CDCA1-siRNA were combined with disulfide bond to become effector molecules. By the characteristics of the pHLIP allosteric occurring in cancer tissue acidic microenvironment, CDCA1-siRNA may be transported specificity into prostatic cancer cells and released in the cytoplasm. The interference effect of the effector molecules on the CDCA1 was detected in vitro and in vivo. The results showed that CDCA1 was highly expressed in PCa cell line and human PCa clinical samples. Knock down CDCA1 significantly inhibit the growth and promote the apoptosis of prostatic cancer cells. In the intracellular translocation experiment, CDCA1-siRNA could be delivered into cytoplasma at pH 6.2, but not at pH 7.4. In the in vivo test, the tumor size was reduced obviously in the NOD/SCID mice treated with pHLIP-CDCA1-siRNA compared to the CDCA1-siRNA and the bioluminescent signal of Cy5-pHLIP-CDCA1-siRNA was focused detected in the tumor site. Our findings indicated that CDCA1 might be a very key molecule regulating survival and proliferation of PCa. pHLIP-CDCA1-siRNA might be a promising targeting therapy for PCa.

pH-responsive pHLIP (pH low insertion peptide) nanoclusters of superparamagnetic iron oxide nanoparticles as a tumor-selective MRI contrast agent.

Superparamagnetic iron oxide nanoparticles (SPION) are contrast agents used for noninvasive tumor magnetic resonance imaging (MRI). SPION with active targeting by tumor-specific ligands can effectively enhance the MRI sensitivity and specificity of tumors. However, the challenge remains when the tumor specific markers are yet to be determined, especially in the case of early tumor detection. In this study, the effectiveness of pH-responsive SPION via a pH low insertion peptide (pHLIP) to target tumor acidic microenvironments was investigated. Polylysine polymers were first successfully modified with pHLIP to have the pH-responsive capability. SPION pHLIP nanoclusters of 64, 82, 103, and 121nm size were then assembled by the pH-responsive polymers in a size-controlled manner. The pH-responsive SPION nanoclusters of the 64nm size exhibited the most effective pH-responsive retention in cells and tumor selective imaging in MRI. More importantly, the unique contrast enhancement of tumor inner core by the pH-responsive SPION in three different tumor models demonstrated the clinical potential to target tumor acidic microenvironment through pHLIP for tumor early detection and diagnosis by MRI. STATEMENT OF SIGNIFICANCE: Detection and diagnosis of tumors at early stage are critical for the improvement of the survival rate of cancer patients. However, the challenge remains when the tumor specific markers are yet to be determined, especially in early tumor detection. pH low insertion peptide (pHLIP) has been used as a specific ligand to target the tumor acidic microenvironment for tumors at early and metastatic stages. Superparamagnetic iron nanoparticles (SPION) are contrast enhancing agents used in the noninvasive magnetic resonance imaging for tumors. This research has demonstrated that pH-responsive pHLIP nanoclusters of SPION were able to target different tumors and facilitate the noninvasive diagnosis of tumors by MRI.

Probe for the measurement of cell surface pH in vivo and ex vivo.

We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH-measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7-6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1-6.4, whereas in nonmetastatic tumors, it was 6.7-6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible.

A smart tumor targeting peptide-drug conjugate, pHLIP-SS-DOX: synthesis and cellular uptake on MCF-7 and MCF-7/Adr cells.

Doxorubicin (DOX) is a potent anticancer drug for the treatment of tumors, but the poor specificity and multi-drug resistance (MDR) on tumor cells have restricted its application. Here, a pH and reduction-responsive peptide-drug conjugate (PDC), pHLIP-SS-DOX, was synthesized to overcome these drawbacks. pH low insertion peptide (pHLIP) is a cell penetrating peptide (CPP) with pH-dependent transmembrane ability. And because of the unique cell membrane insertion pattern, it might reverse the MDR. The cellular uptake study showed that on both drug-sensitive MCF-7 and drug-resistant MCF-7/Adr cells, pHLIP-SS-DOX obviously facilitated the uptake of DOX at pH 6.0 and the uptake level on MCF-7/Adr cells was similar with that on MCF-7 cells, indicating that pHLIP-SS-DOX had the ability to target acidic tumor cells and reverse MDR. In vitro cytotoxicity study mediated by GSH-OEt demonstrated that the cytotoxic effect of pHLIP-SS-DOX was reduction responsive, with obvious cytotoxicity at pH 6.0; while it had poor cytotoxicity at pH 7.4, no matter with or without GSH-OEt pretreatment. This illustrated that pHLIP could deliver DOX into tumor cells with acidic microenvironment specifically and could not deliver drugs into normal cells with neutral microenvironment. In summary, pHLIP-SS-DOX is a promising strategy to target drugs to tumors and provides a possibility to overcome MDR.

pHLIP-mediated targeting of truncated tissue factor to tumor vessels causes vascular occlusion and impairs tumor growth.

Occluding tumor blood supply by delivering the extracellular domain of coagulation-inducing protein tissue factor (truncated tissue factor, tTF) to tumor vasculature has enormous potential to eliminate solid tumors. Yet few of the delivery technologies are moved into clinical practice due to their non-specific tissue biodistribution and rapid clearance by the reticuloendothelial system. Here we introduced a novel tTF delivery method by generating a fusion protein (tTF-pHLIP) consisting of tTF fused with a peptide with a low pH-induced transmembrane structure (pHLIP). This protein targets the acidic tumor vascular endothelium and effectively induces local blood coagulation. tTF-pHLIP, wherein pHLIP is cleverly designed to mimic the natural tissue factor transmembrane domain, triggered thrombogenic activity of the tTF by locating it to the endothelial cell surface, as demonstrated by coagulation assays and confocal microscopy. Systemic administration of tTF-pHLIP into tumor-bearing mice selectively induced thrombotic occlusion of tumor vessels, reducing tumor perfusion and impairing tumor growth without overt side effects. Our work introduces a promising strategy for using tTF as an anti-cancer drug, which has great potential value for clinical applications.

Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity.

The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH50 of 6.1, while most solid tumors have extracellular pH (pH(e)) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.