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Samarium-sodium double sulfate crystalline hydrate NaSm(SO4)2·H2O was obtained by the crystallization from an aqueous solution containing equimolar amounts of ions. The anhydrous salt of NaSm(SO4)2 was formed by a thermally induced release of the equivalent of water from NaSm(SO4)2·H2O. The kinetic parameters of thermal decomposition were determined (Ea = 102 kJ/mol, A = 9·106). The crystal structures of both compounds were refined from the X-ray powder diffraction data. Sulfate hydrate NaSm(SO4)2·H2O crystallizes in the trigonal symmetry, space group P3121 (a = 6.91820(3) and c = 12.8100(1) A, V = 530.963(7) A3). The anhydrous salt crystallizes in the triclinic symmetry, space group P-1 (a = 6.8816(2), b = 6.2968(2) and c = 7.0607(2) A, α = 96.035(1), β = 99.191(1) and γ = 90.986(1)°, V = 300.17(1) A3). The vibrational properties of compounds are mainly determined by the sulfate group deformations. The luminescence spectra of both sulfates are similar and are governed by the transitions of samarium ions 4G5/2 → 6HJ (J = 5/2, 7/2, 9/2 and 11/2). The anhydrous sulfate is stable up to 1100 K and undergoes an almost isotropic expansion when heated. After 1100 K, the compound decomposes into Sm2(SO4)3 and Na2SO4.

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation., T-cell activation primarily occurs in the lymph nodes, highly organized and specialized secondary lymphoid organs. Here the authors show that the acidic extracellular pH in lymph node paracortical zones limits cytokine production by effector T-cells, but does not alter their activation by antigen-presenting cells.

The process of high-temperature oxidation of EuS in the air was explored in the temperature range of 500–1000 °C. The oxidation reaction enthalpy was determined (ΔH0exp = −1718.5 kJ/mol). The study of oxidation products allowed to establish the mechanism of EuS oxidation with oxygen. At 500–600 °C, EuS is oxidized to a mixture of Eu3+-containing compounds (Eu3S4, Eu2O2S). In the range of 700–1000 °C, only europium oxysulfate Eu2O2SO4 is formed. The structure refinement for Eu2O2SO4 was performed by the Rietveld method. The luminescence intensity of europium oxysulfate Eu2O2SO4 with characteristic 4f-4f transitions from the 5D0 state was investigated as a function of oxidation temperature.

The crystal structures of the complex sulfides SrLnCuS3 (Ln = Sm, Gd, Er and Lu) have been determined and refined using powder X-ray diffraction. The crystals are found to be orthorhombic, with the structure type changing consecutively in the order BaLaCuS3 → Eu2CuS3 → KZrCuS3 as the Ln3+ ionic radius decreases in the order La/Pr → Sm/Gd → Er/Lu. Variations of the structure parameters along the series of compounds studied are analyzed, and an effect caused by crystallochemical contraction on the stabilization of the respective structure types is demonstrated.

PURPOSE: To develop a tool to measure the pH at the surfaces of individual cells. PROCEDURES: The SNARF pH-sensitive dye was conjugated to a pHLIP® peptide (pH-Low Insertion Peptide) that binds cellular membranes in tumor spheroids. A beam splitter allows simultaneous recording of two images (580 and 640 nm) by a CCD camera. The ratio of the two images is converted into a pH map resolving single spheroid cells. An average pH for each cell is calculated and a pH histogram is derived. RESULTS: Surface pH depends on cellular glycolytic activity, which was varied by adding glucose or deoxy-glucose. Glucose was found to decrease the surface pH relative to the pH of the bulk solution. The surface pH of metastatic cancer cells was lower than that of non-metastatic cells indicating a higher glycolytic activity. CONCLUSIONS: Our method allows cell surface pH measurement and its correlation with cellular glycolytic activity.

We consider theoretically the process of two-electron one-photon (TEOP) transitions to the ground state with the emission of a single ${K}_{\ensuremath{\alpha}\ensuremath{\alpha}}$ photon in a He-like sequence of atomic ions: from boron to uranium. The corresponding transition probabilities and transition energies are calculated within the QED theory. The calculations of the transition probabilities are performed in various gauges. The intensity ratios between the TEOP transitions and the other major transitions are presented.

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner., Graphical Abstract Graphical AbstractCBX-12 is a pH-sensitive peptide-drug conjugate that selectively delivers the TOP1 inhibitor exatecan into tumor cells in an antigen agnostic manner. The peptide forms an alpha helix only in the low pH tumor microenvironment, allowing directional insertion of the peptide across the tumor membrane, cleavage of the linker within the cytosol, and release of free exatecan within the tumor cell. This pH-based delivery approach results in a high therapeutic index universally across solid tumors.

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.

The europium oxysulfate crystal structure was solved by X-ray powder diffraction. The compound crystallizes in monoclinic crystal system. The unit cell parameters are a = 13.6952(1) A, b = 4.1929(4) A, c = 8.1393(2) A, β = 107.455(4)°, space group C2/c. The three major structural elements are recognized to be Eu atoms, SO4 tetrahedra, and μ-O atoms, and their coordination environments were analyzed. The specific structural elements are infinite screw chains composed of trigonal prisms [EuO9]; sulfate tetrahedra [(SO4)Eu8] enclosed into cubes built of europium atoms; and infinite chains composed of tetrahedra [OEu4]. Rationale was given to the splitting of the IR absorption bands corresponding to the vibrations of sulfate groups.

Acidity at surface of cancer cells is a hallmark of tumor microenvironments, which does not depend on tumor perfusion, thus it may serve as a general biomarker for targeting tumor cells. We used the pH (low) insertion peptide (pHLIP) for decoration of liposomes and niosomes. pHLIP senses pH at the surface of cancer cells and inserts into the membrane of targeted cells, and brings nanomaterial to close proximity of cellular membrane. DMPC liposomes and Tween 20 or Span 20 niosomes with and without pHLIP in their coating were fully characterized in order to obtain fundamental understanding on nanocarrier features and facilitate the rational design of acidity sensitive nanovectors. The samples stability over time and in presence of serum was demonstrated. The size, ζ-potential, and morphology of nanovectors, as well as their ability to entrap a hydrophilic probe and modulate its release were investigated. pHLIP decorated vesicles could be useful to obtain a prolonged (modified) release of biological active substances for targeting tumors and other acidic diseased tissues. Electronic supplementary material The online version of this article (10.1186/s11671-018-2807-8) contains supplementary material, which is available to authorized users.

Silver–europium double sulfate AgEu(SO4)2 was obtained by solid-phase reaction between Ag2SO4 and Eu2(SO4)3. The crystal structure of AgEu(SO4)2 was determined by Monte Carlo method with simulated ...

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane’s hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.

Reactions of europium sulfates Eu 2 (SO 4 ) 3 ·8H 2 O and EuSO 4 complete decomposition were studied by Simultaneous Thermal Analysis. It was revealed that one-step dehydratation of Eu 2 (SO 4 ) 3 ·8H 2 O crystallohydrate is accompanied by the formation of amorphous anhydrous europium sulfate Eu 2 (SO 4 ) 3 . Crystallization of amorphous europium (III) sulfate occurs at 381.1 °С (in argon) and 391.3 °C (in air). The average enthalpy values for dehydratation reaction of Eu 2 (SO 4 ) 3 ·8H 2 O (ΔH° = 141.1 kJ/mol), decomposition reactions of Eu 2 (SO 4 ) 3 (ΔH = 463.1 kJ/mol), Eu 2 O 2 SO 4 (ΔH = 378.4 kJ/mol) and EuSO 4 (ΔH = 124.1 kJ/mol) were determined. The step process mechanisms of thermal decomposition of europium (III) sulfate in air and europium (II) sulfate in inert atmosphere were established and justified. The kinetic parameters of complete thermal decomposition of europium (III) sulfate octahydrate were calculated by Kissinger model. The standard enthalpies of compound formation were calculated using thermal effects and formation enthalpy data for binary compounds.

Samples of Ln2S3 (Ln = Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) polymorphs were prepared from Ln2O3 in a CS2 + H2S flow at 1050–1150 °C. DSC melting traces were recorded for Ln2S3 compounds of yttrium lanthanides for the first time. Samples for DSC were prepared by melting and then recrystallizing polycrystalline samples in crucibles of the DSC setup. The melting peak shapes and melting ranges signified the congruent melting of Ln2S3 compounds. The temperatures and enthalpies of melting were determined for the prepared Ln2S3 polymorphs: γ-Gd2S3 (t m = 1794 °C; ΔH = 56.3 kJ mol−1), γ-Tb2S3 (t m = 1753 °C; ΔH = 55.8 kJ mol−1), γ-Dy2S3 (t m = 1698 °C; ΔH = 57 kJ mol−1), δ-Ho2S3 (t m = 1697 °C; ΔH = 47.7 kJ mol−1), δ-Er2S3 (t m = 1662 °C; ΔH = 42.2 kJ mol−1), δ-Tm2S3 (t m = 1665 °C; ΔH = 46.9 kJ mol−1), and e-Lu2S3 (t m = 1755 °C; ΔH = 53.6 kJ mol−1). The Ln2S3 samples experienced thermal dissociation near their melting temperatures; the relevant mass losses were 0.1–0.24 mass%. After DSC, the samples remained single phases; their concentration changed to Gd2S2.98, Tb2S2.99, Dy2S2.98, Ho2S2.97, Er2S2.97, and Lu2S2.98. A δ-Tm2S3 sample experienced a greater mass loss (0.77 mass%) at 1500–1670 °C to acquire the concentration Tm2S2.91. e-Yb2S3 decomposed to YbS at temperatures above 1500 °C. The melting temperatures show an inner periodicity, and the enthalpies of melting of Ln2S3 show the tetrad effect as a function of rLn+3.

Novel approaches in synthesis of spherical and multispiked gold nanoparticles coated with polyethylene glycol (PEG) and pH Low Insertion Peptide (pHLIP®) were introduced. The presence of a tumor-targeting pHLIP® peptide in the nanoparticle coating enhances the stability of particles in solution and promotes a pH-dependent cellular uptake. The spherical particles were prepared with sodium citrate as a gold reducing agent to form particles of 7.0±2.5 nm in mean metallic core diameter and ∼43 nm in mean hydrodynamic diameter. The particles that were injected into tumors in mice (21 µg of gold) were homogeneously distributed within a tumor mass with no staining of the muscle tissue adjacent to the tumor. Up to 30% of the injected gold dose remained within the tumor one hour post-injection. The multispiked gold nanoparticles with a mean metallic core diameter of 146.0±50.4 nm and a mean hydrodynamic size of ~161 nm were prepared using ascorbic acid as a reducing agent and disk-like bicelles as a template. Only the presence of a soft template, like bicelles, ensured the appearance of spiked nanoparticles with resonance in the near infrared region. The irradiation of spiked gold nanoparticles by an 805 nm laser led to the time- and concentration-dependent increase of temperature. Both pHLIP® and PEG coated gold spherical and multispiked nanoparticles might find application in radiation and thermal therapies of tumors.

The advantages of targeted therapy have motivated many efforts to find distinguishing features between the molecular cell surface landscapes of diseased and normal cells. Typically, the features have been proteins, lipids or carbohydrates, but other approaches are emerging. In this discussion, we examine the use of cell surface acidity as a feature that can be exploited by using pH-sensitive peptide folding to target agents to diseased cell surfaces or cytoplasms.

Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remain elusive, and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as pH [low] insertion cycles, or pHLIC) can permit accurate in vivo visualization of acidosis. Methods: Acid-sensitive cyclic peptide c[E(4)W(5)C] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with (64)Cu (half-life, 12.7 h). C57BL/6J mice were administered lipopolysaccharide (15 mg/kg) or saline (vehicle) and serially imaged with [(64)Cu]Cu-c[E(4)W(5)C] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non–pH-sensitive cell-penetrating control peptide (c[R(4)W(5)C]) was used to confirm specificity of [(64)Cu]Cu-c[E(4)W(5)C]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [(64)Cu]Cu-c[E(4)W(5)C] PET signal. Results: [(64)Cu]Cu-c[E(4)W(5)C] radiochemical yield and purity were more than 95% and more than 99%, respectively, with molar activity of more than 0.925 MBq/nmol. Significantly increased [(64)Cu]Cu-c[E(4)W(5)C] uptake was observed in lipopolysaccharide-treated mice (vs. vehicle) within peripheral tissues, including blood, lungs, liver, and small intestines (P < 0.001–0.05). Additionally, there was significantly increased [(64)Cu]Cu-c[E(4)W(5)C] uptake in the brains of lipopolysaccharide-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of lipopolysaccharide-treated mice (vs. vehicle). Immunohistochemical analysis revealed microglial or macrophage infiltration, suggesting activation in brain regions containing increased tracer uptake. [(64)Cu]Cu-c[R(4)W(5)C] demonstrated significantly reduced uptake in the brain and periphery of lipopolysaccharide mice compared with the acid-mediated [(64)Cu]Cu-c[E(4)W(5)C] tracer. Conclusion: Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven proinflammatory responses. This study suggests that [(64)Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.

Crystalline hydrate of double cesium europium sulfate [CsEu(H2O)3(SO4)2]·H2O was synthesized by the crystallization from an aqueous solution containing equimolar amounts of 1Cs+:1Eu3+:2SO42− ions. Anhydrous salt CsEu(SO4)2 was formed as a result of the thermal dehydration of the crystallohydrate. The unusual effects observed during the thermal dehydration were attributed to the specific coordination of water molecules in the [CsEu(H2O)3(SO4)2]·H2O structure. The crystal structure of [CsEu(H2O)3(SO4)2]·H2O was determined by a single crystal X-ray diffraction analysis, and the crystal structure of CsEu(SO4)2 was obtained by the Rietveld method. [CsEu(H2O)3(SO4)2]·H2O crystallizes in the monoclinic system, space group P21/c (a = 6.5574(1) Å, b = 19.0733(3) Å, c = 8.8364(2) Å, β = 93.931(1)°, V = 1102.58(3) Å3). The anhydrous sulfate CsEu(SO4)2 formed as a result of the thermal destruction crystallizes in the monoclinic system, space group C2/c (a = 14.327(1) Å, b = 5.3838(4) Å, c = 9.5104(6) Å, β = 101.979(3) °, V = 717.58(9) Å3). The vibration properties of the compounds are fully consistent with the structural models and are mainly determined by the deformation of non-rigid structural elements, such as H2O and SO42−. As shown by the diffused reflection spectra measurements and DFT calculations, the structural transformation from [CsEu(H2O)3(SO4)2]·H2O to CsEu(SO4)2 induced a significant band gap reduction. A noticeable difference of the luminescence spectra between cesium europium sulfate and cesium europium sulfate hydrate is detected and explained by the variation of the extent of local symmetry violation at the crystallographic sites occupied by Eu3+ ions, namely, by the increase in inversion asymmetry in [CsEu(H2O)3(SO4)2]·H2O and the increase in mirror asymmetry in CsEu(SO4)2. The chemical shift of the 5D0 energy level in cesium europium sulfate hydrate, with respect to cesium europium sulfate, is associated with the presence of H2O molecules in the vicinity of Eu3+ ion.

Complex sulfides BaDyCuS3, BaHoCuS3 and BaYbCuS3 were synthesized in a flow of sulfiding gases (CS2, H2S) at 900оC from standard solutions of lanthanide and copper nitrates, as well as from the same standard Ba(OH)2 solution. The crystal structures of BaDyCuS3, BaHoCuS3 and BaYbCuS3 were obtained by the Rietveld refinement method. All three compounds crystallize in the Cmcm space group (KZrCuS3 structural type) as predicted by the tolerance factor analysis. Their micromorphological, thermal and spectroscopic properties are evaluated. BaDyCuS3 and BaHoCuS3 melt congruently at 1376.5 °C and 1363.8 °C. BaYbCuS3 melts incongruently at 1353.3 °C. The optical band gap is 2.45 eV for BaDyCuS3, 2.37 eV for BaHoCuS3 and 1.82 eV for BaYbCuS3. The low bandgap of BaYbCuS3 is explained by the charge transfer band of Yb at the bottom of conduction band. The vibrational parameters of BaDyCuS3, BaHoCuS3 and BaYbCuS3 crystals were determined with the use of Raman and Infrared spectroscopies.

SrLnCuS3 (Ln = La–Lu) compounds melt incongruently. Their thermochemical parameters are determined. The melting temperatures and the enthalpies of melting are: for SrLaCuS3, T = 1513 K and ΔH = 6.9 kJ mol−1; for SrCeCuS3, T = 1468 K and ΔH = 5.2 kJ mol−1; for SrPrCuS3, T = 1459 K and ΔH = 13.2 kJ mol−1; for SrNdCuS3, T = 1429 K and ΔH = 16.8 kJ mol−1; and for SrSmCuS3, T = 1605 K and ΔH = 2.8 kJ mol−1. Three high-temperature polymorphic transitions are found to occur in SrLnCuS3 (Ln = Sm, Gd–Lu) compounds. The parameters of these transitions are determined: for SrSmCuS3, Tα ↔ β = 1452 K, ΔHα ↔ β = 3.0 kJ mol−1, Tβ ↔ γ = 1464 K, ΔHβ ↔ γ = 0.2 kJ mol−1, Tγ ↔ δ = 1476 K, and ΔHγ ↔ δ = 1.1 kJ mol−1; for SrDyCuS3, Tα ↔ β = 1530 К, Tβ ↔ γ = 1568 К, and Tγ ↔ δ = 1585 K; for SrTmCuS3, Tα ↔ β = 1580 K, Tβ ↔ γ = 1618 K, and Tγ ↔ δ = 1631 K; and for SrYbCuS3, Tα ↔ β = 1567 K, Tβ ↔ γ = 1608 K, and Tγ ↔ δ = 1621 K. The transitions are observed both upon heating and upon cooling. The high-temperature phases are not quenchable. Phase-transition temperature versus r(Ln3+) curves for SrLnCuS3 (Ln = La–Lu) feature the tetrad effect. The SrLnCuS3 (Ln = La–Nd) compounds are classified as thiocuprates; their melting temperatures decrease systematically from La to Nd. The SrCuLnS3 (Ln = Sm, Gd–Lu) compounds are classified as thiolanthanates; their melting temperatures increase in the order from Sm to Tm and from Tm to Lu.

The investigation of pH-dependent membrane-associated folding has both fundamental interest and practical applications for targeting of acidic tumors and specific delivery of therapeutic molecules across membrane of cancer cells. We and others investigated molecular mechanism and medical uses of class of water soluble membrane peptides, pH (Low) Insertion Peptides (pHLIP® peptides). Here we employed optical spectroscopy methods to study interactions of the truncated pHLIP® peptide (Short pHLIP®) with lipid bilayer of membrane. Tryptophan fluorescence, CD and OCD data indicate on pH-triggered formation of transmembrane helical structure. Dual quenching and FRET assays demonstrated that Short pHLIP® peptide spans lipid bilayer of membrane similar to Long pHLIP® peptides. Truncated pHLIP® peptides with multiple charged and protonatable residues in their sequences potentially can make these peptides to be less hydrophobic compared to Long pHLIP® peptides, and might have utility in tumor imaging, and potentially, in pH-regulated cytoplasmic delivery of moderately hydrophobic drugs., Highlights • Protonation of single Asp triggers pH-dependent insertion into membrane. • Truncated pHLIP® peptide adopts TM helical orientation at low pH. • Truncated pHLIP® peptide spans lipid bilayer similar to full-length long peptide. • Potential applications in cytoplasmic delivery of small hydrophobic molecules.

pH (Low) Insertion Peptides (pHLIP peptides) find application in studies of membrane-associated folding because spontaneous insertion of these peptides is conveniently triggered by varying pH. Here, we employed small-angle X-ray scattering (SAXS) to investigate a wild-type (WT) pHLIP peptide oligomeric state in solution at high concentrations and monitor changes in the liposome structure upon peptide insertion into the bilayer. We established that even at high concentrations (up to 300 µM) the WT pHLIP peptide at pH 8.0 does not form oligomers larger than tetramers (which exhibit concentration-dependent transfer to the monomeric state, as was shown previously). This finding has significance for medical applications when high concentration of the peptide is injected into blood and diluted in blood circulation. The interaction of WT pHLIP peptide with liposomes does not alter the unilamellar vesicle structure upon peptide adsorption by the lipid bilayer at high pH or upon insertion across the bilayer at low pH. At the same time, SAXS data clearly demonstrate the insertion of the peptide into the membrane at low pH, which opens the possibility of investigating the kinetic process of polypeptide insertion and exit from the membrane in real time by time-resolved SAXS.

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.

In order to synthesize single crystals of europium-silver double sulfate monohydrate, a hydrothermal reaction route was used. It was found that the crystallization cannot be performed under standard conditions. The compound AgEu(SO4)2·H2O crystallizes in the trigonal crystal system, space group P3221 (a ​= ​6.917(1), c ​= ​12.996(2) A, V ​= ​538.53(17) A3). The structure consists of triple-capped trigonal prisms [EuO9], in which one oxygen atom belongs to crystalline water, silver octahedra [AgO6], and sulfate tetrahedra [SO4]. The hydrogen bonds in the system additionally stabilize the structure. The electronic band structure wasstudied by density functional theory calculations which show that AgEu(SO4)2·H2O is an indirect band gap dielectric. Temperature dependent photoluminescence spectroscopy shows emission bands of transitions from the 5D0 state to the spin-orbit components of the 7FJmultiplet (J ​= ​0–6).The ultranarrow transition 5D0 - 7F0 shows a red shift with respect to other europium-containing water-free sulfates that is ascribed to the presence of OH group in the crystal structure in the close vicinity of the Eu3+ ion. An effect of abnormal sensitivity of the Ω4 intensity factor to minor distortions of the local environment is detected for the observed low local symmetry of C2.

Solid solutions of oxysulfides (RE0.95Ln0.05)2O2S (RE = La, Y; Ln = Ho, Tm) were obtained by hydrogen reduction of the co-precipitated sulfates followed by sulfidation of the reaction products. The crystal chemical characteristics of the obtained compounds were refined by the Rietveld method. Morphological certification of particles in the dynamics of synthesis was performed. Most of the particles produced by chemical reactions have a cut that indicates the formation of a compound with a hexagonal syngony with angles of 60 and 120°. This indicates that the thermal effect of gaseous reagents H2, H2S on sulfates leads to heterogeneous reactions of thermal dissociation and the formation of new phases. Steady state luminescence properties displayed characteristic sharp bands corresponding to 4f-4f transitions. Luminescence decay curves of all studied samples showed monoexponential decay with microsecond and hundreds microsecond lifetimes depending on doping ions. Calculated color coordinates of Ho3+ and Tm3+-doped powders make them promising candidates to be used as phosphors.

The oxidation process of samariumoxysulfide was studied in the temperature range of 500&ndash, 1000 &deg, C. Our DTA investigation allowed for establishing the main thermodynamic (∆H&ordm, exp = &minus, 654.6 kJ/mol) and kinetic characteristics of the process (Ea = 244 kJ/mol, A = 2 &times, 1010). The enthalpy value of samarium oxysulfate (&Delta, H&ordm, f (Sm2O2SO4(monocl)) = &minus, 2294.0 kJ/mol) formation was calculated. The calculated process enthalpy value coincides with the value determined in the experiment. It was established that samarium oxysulfate crystallizes in the monoclinic symmetry class and its crystal structure belongs to space group C2/c with unit cell parameters a = 13.7442 (2), b = 4.20178 (4) and c = 8.16711 (8)&Aring, &beta, = 107.224 (1)&deg, V = 450.498 (9)&Aring, 3, Z = 4. The main elements of the crystalline structure are obtained and the cation coordination environment is analyzed in detail. Vibrational spectroscopy methods confirmed the structural model adequacy. The Sm2O2SO4luminescence spectra exhibit three main bands easily assignable to the transitions from 4G5/2 state to 6H5/2, 6H7/2, and 6H9/2 multiplets.

OBJECTIVE: To improve visualization of upper tract urothelial carcinomas. Previous studies using the novel pH low insertion peptide (pHLIP) variant 3 (Var3) conjugated to indocyanine green (ICG) have demonstrated high sensitivity and specificity for imaging of bladder urothelial carcinoma. Here, we describe a novel approach for the imaging of upper tract urothelial carcinomas using ICG-Var3 pHLIP. METHODS: Twelve ex-vivo upper urinary tract specimens were irrigated with ICG-Var 3 pHLIP for 15 minutes and then examined using a white light laparoscopic camera followed by near infrared fluorescent (NIRF) imaging using a Stryker 1588 AIM imaging system. Standard histopathologic evaluation was performed and findings were correlated with white light and ICG-Var3 NIRF imaging. One patient who underwent radical nephrectomy for renal cell carcinoma was used as a negative control. RESULTS: Nineteen lesions were identified on histopathologic evaluation in 10 patients, including 82% high-grade urothelial carcinoma and 18% low-grade urothelial carcinoma. Nineteen (100%) malignant lesions were identified using NIRF imaging, while 15 (78.9%) lesions were identified using conventional white light examination. The sensitivity of ICG-Var3 pHLIP NIRF imaging was 100% compared to 78.9% white light examination. Both modalities are 100% specific. Benign collecting systems and ureters did not show uptake of the pHLIP construct. CONCLUSION: In this feasibility study, the ICG-Var3 pHLIP imaging agent demonstrated superior diagnostic performance compared to conventional white light examination. While additional studies are required for validation and in-vivo translation, pHLIP-based imaging represents a promising tool to improve the evaluation and management of upper tract urothelial carcinoma. UROLOGY 139: 134–140, 2020.

The pH (low) insertion peptide (pHLIP) is a family of peptides that are able to insert into a lipid bilayer at acidic pH. The molecular mechanism of pHLIPs insertion, folding and stability in the membrane at low pH is based on multiple protonation events, which are challenging to study at molecular level. More specifically, the relation between the experimental pK of insertion (pKexp) of pHLIPs and the pKa of the key residues is yet to be clarified. We carried out a computational study, complemented with new experimental data, and established the influence of (de)protonation of key titrable residues on the stability of the peptide membrane-inserted state. Constant-pH molecular dynamics simulations were employed to calculate the pKa values of these residues along the membrane normal. In the wt-pHLIP, we identified Asp14 as the key residue for the stability of the membrane-inserted state, and its pKa value is strongly correlated with the experimental pKexp measured in thermodynamics studies. Also, in order to narrow down the pH range at which pHLIP is stable in the membrane, we designed a new pHLIP variant, L16H, where Leu in the 16th position was replaced by a titrable His residue. Our results showed that the L16H variant undergoes two transitions. The calculated pK and experimentally observed pKexp values are in good agreement. Two distinct pKexp values delimit a pH range where the L16H peptide is stably inserted in the membrane while, outside this range, the membrane-inserted state is destabilized and the peptide exits from the bilayer. pHLIP peptides have been successfully used to target cancer cells for the delivery of diagnostics and therapeutic agents to acidic tumors. The fine tuning of the stability of pHLIP inserted state and its restriction to a narrow well-defined pH range might allow the design of new pHLIPs, able to discriminate between tissues with different extracellular pH values.

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 (i) 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; (ii) 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 (iii) 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.

Two methods of Lu-177 production are reviewed: irradiation of isotopically enriched Lu- 176 (direct way) and irradiation of ytterbium enriched with Yb-176 (indirect way). Based on neutronphysical calculations Lu-177 yield and specific activity were estimated for both methods. Lu-177 specific activity strongly depends on neutron flux density in the direct way, that is 75,000 Ci/g for 10- days irradiation in a neutron flux of 2.10(15) cm(-2) s(-1), and only 13,000 Ci/g after 30 days irradiation at neutron flux 1.10(14) cm(-2) s(-1). Irradiation of Yb-176 provides Lu-177 specific activity close to theoretical value (110,000 Ci/g). Neutron flux density effect Lu-177 yield, that is 530 Ci/g for 2.1015 cm(-2) s(-1) neutron flux density after 30 days irradiation. A procedure of isolation and purification of Lu-177 from irradiated targets is described based on combination of galvanostatic extraction of ytterbium followed by cation-exchange chromatography from alfa-hydroxyisobutirate solutions on BioRad AG(®)50W-X8 resin.

The pH (low) insertion peptide (pHLIP) belongs to a family of peptides originated from a segment of the transmembranar C helix of bacteriorhodopsin. The peptide has three major states: state I - soluble and unstructured; state II - adsorbed at the membrane surface and unstructured; state III - inserted in the bilayer as an α-helix at low pH values. One of the major applications of pHLIP befalls on its ability to insert into membrane cells with an acidic vicinity, such as tumoral cells, thus working as an efficient tumor-specific biomarker1. However, wt-pHLIP has a significant limitation, since it accumulates in the kidneys in considerable amounts due to their naturally acidic extracellular pH. This limitation led to a need for increased pHLIP specificity by delimiting the pH range of insertion, further strengthening its application as a biomarker and possible drug-delivery system for inflammatory tissues. The stochastic titration constant-pH molecular dynamics (CpHMD) method has been successfully used to sample protonation behaviour of titrable amino acids inserted in a lipid bilayer, presenting, however, an insufficient amount of data to extensively describe pKa profiles2. The newly developed pH-replica exchange (pHRE) method, allows the exchange of pH values between replicas within a certain probability. This approach enhances the transitions between energy minima, improving the sampling of non-favorable protonation states, which leads to a better description of the pKa profiles. This new method was applied to simulations of wt-pHLIP and L16H variants. The pHRE simulations led to more detailed, accurate and consistent pKa profiles and allowed the identification of Asp14 as the key residue whose protonation state triggers the insertion process. The calculated insertion pKa value of this residue is in good agreement with the experimental insertion pK value for the wt sequence. Moreover, the simulations of L16H showed that this variant exhibits a second insertion pKa, at lower pH, indicating that, below this value, the peptide would exit the membrane. These results were corroborated by new experimental data performed by our collaborators, Prof. Oleg Andreev in Rhode Island, USA. We acknowledge financial support from FCT through projects UID/MULTI/00612/2013, PTDC/QEQ-COM/5904/2014 and grant SFRH/BPD/110491/2015.

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.

pH-Low Insertion Peptides (pHLIP® peptides) are a platform technology for targeting of acidity at the surface of individual cells within tumors. Most proteins that are located across membranes have one or more helical segments that cross the lipid bilayer, and studies of these membrane proteins revealed pHLIPs, which could fold from disorganized (unstructured) states outside of a membrane to form a helix across the membrane in response to local acidity. Because tumor cell surfaces are acidic as a result of their active metabolism, versions of pHLIP® peptides were extensively investigated and tested as possible tumor targeting agents. Our studies showed that an extensive variety of cargoes can be targeted to cell surfaces and that a surprising range of cargoes can be delivered across membranes into cells if their surfaces are acidic, creating opportunities to detect and treat tumors. pHLIP® peptides can be used to target and tether cargo molecules to the surfaces of cells in low pH environments of acidic diseased tissues. The cargo can be an optical marker, a PET, a SPECT, or a MR imaging agent, or an antigen or a protein delivered to induce certain cellular or immune responses. We are translating pHLIP®-based imaging agents to the clinic for diagnostic PET imaging, fluorescence-guided surgical procedures and photo-acoustic pre-operative imaging. pHLIP® peptides can also be used for the intracellular delivery of payloads, facilitating the translocation of therapeutic cargoes across the membranes of cells with low extracellular pH, such as those cells found in acidic diseased tissue. These payloads are conjugated to the membrane-inserting end of pHLIP® peptide, typically via a link that is unstable inside the cell. Thus, pHLIP® peptide targets a payload to acidic diseased cells and flips the cargo across the plasma membrane, releasing it in the cytoplasm. Therapeutic cargoes may include toxins, metabolic activators/inhibitors, chemotherapeutic agents, or agents to alter gene expression. *YKR, OAA, and DME are founders of pHLIP, Inc. and have financial interests in the company. Citation Format: Yana K Reshetnyak, Oleg A Andreev, Donald Engelman. pHLIP technology for targeting acidity at surface of tumor cells and extracellular and intracellular delivery of imaging and therapeutic agents [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A102. doi:10.1158/1535-7163.TARG-19-A102

Introduction: The detection of urothelial carcinomas of the bladder and upper tract is not satisfactory. Incomplete findings by contemporary imaging modalities provide a challenge in patient care, so there is a definitive need to improve diagnosis and treatment methods for urothelial carcinomas. Experimental procedures: ICG (indocyanine green) near infrared fluorescent (NIRF) dyes were conjugated with a pH-Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells. In a few cases IR800 pHLIP® was used. Urinary neoplasm (kidney, ureter and bladder) specimens obtained through radical surgery were irrigated and incubated ex-vivo with ICG pHLIP® followed by fluorescence imaging. Pathology findings were correlated with the ICG pHLIP® NIRF imaging. Results: In the upper urothelial tract, nine of ten patients with nephroureterectomy had urothelial carcinomas (UTUC). Eighteen (100%) malignant lesions were found in NIRF imaging. Only thirteen (72%) of malignant lesions were found under white light macroscopic examination. ICG pHLIP® NIRF imaging gave a 28% increase in diagnosis of UTUC in ex-vivo specimens. There was no non-specific uptake in the non-tumoral tissue. The ICG pHLIP® imaging agent identified malignant upper tract urothelial lesions with 100% specificity and sensitivity. In the lower urinary tract, thirty-eight radical cystectomy specimens were incubated with pHLIP® conjugated with one of the NIRF dyes (ICG or IR800). In the thirty-eight bladders, eighty-one lesions were identified using fluorescent pHLIP®(specificity (97%) and sensitivity (100%)). White light cystoscopy did not detect 20.5% of lesions identified under NIRF cystoscopy (p Conclusions: The ICG pHLIP® NIRF revealed additional lesions not seen with white light ex-vivo cystoscopy or preoperative ureteroscopy. pHLIP® based agents promise to improve diagnostic accuracy for urothelial carcinomas and may enable targeted treatment. Citation Format: Borivoj Golijanin, Anna Moshnikova, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak, Ali Amin, Dragan Golijanin. Ex-vivo targeting of urothelial carcinomas by fluorescent pHLIP imaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1399.

Introduction: Fluorescence imaging has applications in medicine for many image-guided procedures such as mapping and visualization of lymph nodes, targeting and marking cancerous lesions and assessment of tumor margins. Several fluorescent molecules are approved for clinical use including the ICG (indocyanine green) near infrared (NIR) fluorescent dye. We conjugated ICG to a pH Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells, and evaluated ICG pHLIP® as a targeted imaging agent for tumor visualization. Our construct is progressing toward clinical trials. Experimental Procedures: ICG pHLIP® was manufactured for proof-of-concept tumor-targeting imaging studies in mice, and toxicity evaluation in mice, dogs and rats. Tumor targeting was assessed in various human and murine tumor models. Biodistribution was evaluated at different time points from 5 min to 48 hours after a single intravenous administration of 0.5 mg/kg of ICG pHLIP®. The distribution of the agent within tumors was evaluated in tumor sections. Results: The fluorescence of ICG is enhanced about 15 times when pHLIP® tethers it to a cell membrane, significantly improving the contrast index. No toxicity is seen at 25-30x of the proposed human dose, evaluated in mice and dogs. ICG pHLIP® demonstrates excellent tumor targeting, including targeting of 1-3 mm sized tumors in breast. About 10-15% ID/g was observed within tumors at 4 hours p.i., and the signal persists for up to 24 hours with a slight decline at 48 hours. The background signal in major organs decayed after agent administration. The highest uptake, of about 50% ID/g, was observed in liver at 4 hrs p.i. and decayed after that. An excellent correlation was established between ICG pHLIP® NIRF imaging and H&E histopathology at the indicated tumor location. Conclusions: ICG pHLIP® is a promising new imaging agent for fluorescence-guided surgical applications. It is on the path to clinical translation for accurate resection of breast tumors. Since pHLIP® targeting of tumors is based on the general principle of cancer cell surface acidity, ICG pHLIP® is an agent that may be used in a range of fluorescence-guided surgical interventions. Citation Format: Troy Crawford, Anna Moshnikova, Sean Roles, Lukas M. Carter, Jason S. Lewis, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak. ICG pHLIP: A novel agent for fluorescence-guided surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1956.

We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, “fusogenic” pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells.

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide–lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.

Acidity is a biomarker of cancer that is not subject to the blunting clonal selection effects that reduce the efficacy of other biomarker technologies, such as antibody targeting. The pH (low) insertion peptides (pHLIP®s) provide new opportunities for targeting acidic tissues. Through the physical mechanism of membrane-associated folding, pHLIPs are triggered by the acidic microenvironment to insert and span the membranes of tumor cells. The pHLIP platform can be applied to imaging acidic tissues, delivering cell-permeable and impermeable molecules to the cytoplasm, and promoting the cellular uptake of nanoparticles. Since acidosis is a hallmark of tumor development, progression, and aggressiveness, the pHLIP technology may prove useful in targeting cancer cells and metastases for tumor diagnosis, imaging, and therapy.

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.

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.

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.

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.

Previously we provided evidence that myosin subfragment 1 (S1) can bind either one (state 1) or two actin monomers (state 2) in solution and in muscle fiber. Here we present results of the kinetics study of binding of S1 to F-actin labeled with fluorescent dye pyrene. A transition from state 1 to state 2 depends on probability that the second actin is free, which is high when molar ratio of S1/actin ( R ) is less than 0.5, and it decreases dramatically when R > 2.0 due to the parking problem. The kinetics data obtained at different molar ratios were well fitted by two binding states model. The sequential binding of myosin head initially with one actin monomer and then with the second actin monomer in F-actin can play a key role in force generation by actin–myosin and their directed movement.

We have used the pHLIP® peptide to study the roles of carboxyl groups in transmembrane peptide insertion. The pH (low) insertion peptide (pHLIP) binds to the surface of a lipid bilayer as a disordered peptide at neutral pH, and when the pH is lowered it inserts across the membrane to form a transmembrane helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event facilitating the membrane insertion is the protonation of aspartic (Asp) and/or glutamic (Glu) acid residues, since at neutral pH their negatively charged side chains hinder membrane insertion. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ~pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP peptides.

We find that pH-(low)-insertion-peptide (pHLIP)-facilitated translocation of phalloidin, a cell-impermeable polar toxin, inhibits the proliferation of cancer cells in a pH-dependent fashion. The monomeric pHLIP inserts its C terminus across a membrane under slightly acidic conditions (pH 6–6.5), forming a transmembrane helix. The delivery construct carries phalloidin linked to its inserting C terminus via a disulfide bond that is cleaved inside cells, releasing the toxin. To facilitate delivery of the polar agent, a lipophilic rhodamine moiety is also attached to the inserting end of pHLIP. After a 3 h incubation at pH 6.1–6.2 with 2–4 μ M concentrations of the construct, proliferation in cultures of HeLa, JC, and M4A4 cancer cells is severely disrupted (> 90% inhibition of cell growth). Treated cells also show signs of cytoskeletal immobilization and multinucleation, consistent with the expected binding of phalloidin to F actin, stabilizing the filaments against depolymerization. The antiproliferative effect was not observed without the hydrophobic facilitator (rhodamine). The biologically active delivery construct inserts into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayers with an apparent pK a of ∼6.15, similar to that of the parent pHLIP peptide. Sedimentation velocity experiments show that the delivery construct is predominantly monomeric (> 90%) in solution under the conditions employed to treat cells (pH 6.2, 4 μM). These results provide a lead for antitumor agents that would selectively destroy cells in acidic tumors. Such a targeted approach may reduce both the doses needed for cancer chemotherapy and the side effects in tissues with a normal pH.