pH-sensitive Function Research Articles

Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins.

Transient intracellular pH dynamics 1 regulate mammalian proliferation 2,3 , migration 4 , and differentiation 5 . However, for many pH-dependent cell processes, the molecular mediators are unknown 6 . Prior work identified histidine residues as molecular switches in pH-sensitive proteins, but how other ionizable residues contribute to pH-dependent protein allostery is understudied. Here, we develop an in silico computational pipeline to identify putative pH-sensitive proteins and their molecular mechanisms. We first apply this pipeline to SHP2, a known pH-sensitive signaling protein with an uncharacterized molecular mechanism. We show wild-type SHP2 phosphatase activity is pH-sensitive in vitro and in cells, and mutation of identified H116 and E252 to non-titratable alanine residues abolishes pH-sensitive function. We also show that c-Src is a previously unrecognized pH-dependent kinase, and mutation of the identified ionizable network again abolishes pH-sensitive activity. Constant pH molecular dynamics simulations support a conserved allosteric mechanism of pH-dependent binding of inhibitory SH2 domains to the functional catalytic domains of SHP2 and c-Src. We apply our computational pipeline across SH2 domain-containing signaling proteins and identify evolutionarily conserved putative pH-sensing networks. Our results reveal that pH is an allosteric regulator of SH2 domain-containing signaling proteins providing insight into normal pH-dependent cell biology and diseases where pHi is dysregulated, such as cancer.

Charactering ionizable residue networks at SH2 interfaces in pH-sensitive proteins.

Intracellular pH (pHi) dynamics (7.0 to 7.8) regulate myriad cell processes but the molecular mechanisms driving these behaviors are largely unknown. While some pH-sensitive proteins use titratable histidine residues to sense physiological changes in pHi, others have unknown or histidine-independent mechanisms. Charged amino acids (Asp, Glu, and Lys) can have pKas that are up- or down-shifted into the physiological range depending on protein environment, but the role for these residues in regulating pH-dependent protein function is understudied. Here, we use computational and biochemical approaches to investigate pH sensors that use networks of cooperative ionizable residues with a cumulative physiological pKa. We performed computational analyses of two pH sensitive signaling proteins with unknown mechanisms: (phosphatidylinositol-3-kinase (PI3K) and SHP-2 tyrosine-protein phosphatase). In both proteins, we identified networks of ionizable residues with upshifted pKas at the inhibitory SH2 domain interface. We next used biochemical mutational analyses to probe the accuracy of our computational approaches by measuring pH sensitive activity of wild-type and mutant proteins in vitro. Importantly, because SH2 domains are structurally conserved, this pH-sensing mechanism could be conserved in a subset of kinases and phosphatases with inhibitory SH2 domains. We tested this hypothesis by identifying a network of ionizable residues in other signaling proteins that contain inhibitory SH2 domains. We analyzed JAK2 kinase, which has not been shown to have pH sensitive function, and identified an ionizable network at the SH2 domain interface. Future work will validate this JACK2 result biochemically and explore whether our computational approaches can be applied to identify pH sensitive proteins across the human proteome. Improved understanding of how ionizable networks respond to pH could reveal paths to better therapeutic targeting of these proteins in diseases with dysregulated pHi.

Fabrication of Polysaccharide-Based Halochromic Nanofibers via Needle-Less Electrospinning and Their Characterization: A Study of the Leaching Effect.

Responsive materials, i.e., smart materials, have the ability to change their physical or chemical properties upon certain external signals. The development of nanofibrous halochromic materials, specifically combining the pH-sensitive functionality and unique nanofiber properties, could yield interesting new applications, especially when the common problem of dye leaching is successfully tackled. Therefore, in this article, we studied the fabrication process of polysaccharide-based halochromic nanofibrous materials by using a combination of various halochromic dyes (bromothymol blue, bromocresol green, and thymol blue) and cellulose acetate in a spinning solution using a one-pot strategy. The inhibition of leaching was addressed by using a complexing agent: poly-diallyl-dimethylammonium chloride (PDADMAC). The preparation of hybrid spinning solutions, their characterization, and ability to form continuous nanofibers were studied using a high production needle-less electrospinning system. The produced hybrid solutions and nanofibers were characterized, in terms of their rheological properties, chemical structure, morphology, and functionality. Fabricated nanofibrous halochromic structures show a clear color change upon exposure to different pH values, as well as the reduced leaching of dyes, upon the addition of a complexing agent. The leaching decreased by 61% in the case of bromocresol green, while, in the case of bromothymol blue and thymol blue, the leaching was reduced by 95 and 99%, respectively.

Modulation of Tumor Hypoxia by pH-Responsive Liposomes to Inhibit Mitochondrial Respiration for Enhancing Sonodynamic Therapy.

Background and PurposeSonodynamic therapy (SDT) has been widely used for the noninvasive treatment of solid tumors, but the hypoxic tumor microenvironment limits its therapeutic effect. The current methods of reoxygenation to enhance SDT have limitations, prompting reconsideration of the design of therapeutic approaches. Here, we developed a tumor microenvironment-responsive nanoplatform by reducing oxygen consumption to overcome hypoxia-induced resistance to cancer therapy.MethodsA pH-responsive drug-loaded liposome (MI-PEOz-lip) was prepared and used to reduce oxygen consumption, attenuating hypoxia-induced resistance to SDT and thereby improving therapeutic efficiency. Photoacoustic imaging (PAI) and fluorescence imaging (FI) of MI-PEOz-lip were evaluated in vitro and in breast xenograft tumor models. The pH-sensitive functionality of MI-PEOz-lip was applied for pH-triggered cargo release, and its capacity was evaluated. The MI-PEOz-lip-mediated SDT effect was compared with other treatments in vivo.ResultsMI-PEOz-lip was demonstrated to specifically accumulate in tumors. Metformin molecules in liposomes selectively accumulate in tumors by pH-responsive drug release to inhibit the mitochondrial respiratory chain while releasing IR780 to the tumor area. These pH-responsive liposomes demonstrated PAI and FI imaging capabilities in vitro and in vivo, providing potential for treatment guidance and monitoring. In particular, the prepared MI-PEOz-lip combined with ultrasound irradiation effectively inhibited breast tumors by producing toxic reactive singlet oxygen species (ROS), while the introduction of metformin inhibited mitochondrial respiration and reduced tumor oxygen consumption, resulting in excellent sonodynamic therapy performance compared with other treatments.ConclusionIn this study, we present a novel strategy to achieve high therapeutic efficacy of SDT by the rational design of multifunctional nanoplatforms. This work provides a new strategy that can solve the current problems of inefficient oxygen delivery strategies and weaken resistance to various oxygen-dependent therapies.

A Bioinspired Hierarchical Underwater Superoleophobic Surface with Reversible pH Response

AbstractThe development of oil‐repellent surfaces in liquid environments has received considerable attention because of the urgent demand for antifouling coatings in marine industry. Inspired by the unique nanostructure surface of filefish scale, hierarchical films that consist of poly(pentafluorophenyl acrylate) free standing micropillars grafted with pH responsive poly(methacrylic acid) nanobrushes are fabricated by anodic aluminum oxide templating method combined with a subsequent post‐polymerization modification strategy. The obtained films exhibit constantly underwater superoleophobicity, furthermore, a pH sensitive functionality, which enables reversible switching between low and high oil adhesion as a result of the adjustable oil sliding angle. This particular study provides a very mild method for the facile fabrication of bioinspired nanostructures with excellent oil‐repellent performance and switchable oil‐adhesion properties, thus paving the way toward novel functional materials with smart structures for promising applications, such as smart microfluidics, controllable bioadhesion, and intelligent materials for oil removal treatment and marine antifouling.

Synthesis and application of near-infrared absorbing morpholino-containing aza-BODIPYs

Morpholino-containing aza-BODIPYs at 3,5-positions were synthesized. The maxima absorption and emission of these dyes locate at the near-infrared region. Aza-BODIPY 1 with the morpholino group as a pH-sensitive functionality could be used to be a pH probe, and the dramatic increase in fluorescence intensity at 675 nm by about 1500 folds. Moreover, the singlet oxygen generation of PS 2 with the dibromo groups at 2,6-positions was more effective than that of the parent dye 1.

A pH-Sensitive Polymer for Cancer Targeting Prepared by One-Step Modulation of Functional Side Groups

Nanocarriers with pH-sensitive functionality are of great interest in the development of pH-dependent drug release compounds in acidic tumor microenvironments. A new polyelectrolyte block copolymer, poly[(benzyl-L-aspartate)-co-(N-(3-aminopropyl) imidazole-L-aspartamide)]-poly(ethylene glycol) (PABI-PEG), was prepared by one-step modulation to produce pH-sensitive nanocarriers. PABI-PEG formed a stable nanocarrier at pH values above 7.4 and was destabilized in acidic conditions (pH 6.5) through the protonation of the imidazole groups. Docetaxel loaded micelle (DLM) exhibited pH-dependent drug release through structural conversion due to the protonation of the imidazole groups on the PABI block. The critically low micelle concentration of PABI-PEG at physiological pH and the pH-dependent drug release would result to high stability and restrict drug loss during systemic circulation which may lower the toxicity of normal tissue to physiological pH. Additionally, the extracellular tumor pH (<7.0) and early endosomal pH (<6.5) environments triggered the disintegration of micelles, producing higher drug release compared to other normal tissues and blood (pH 7.4). Therefore, PABI-PEG may be a pHsensitive drug delivery method for cancer chemotherapy.

A pH-activatable fluorescent aptamer probe for imaging of target cancer cells

Early cancer diagnosis in molecular level shows great promise in relevant prevention and clinical treatment. Herein, we developed a novel method to detect and evaluate biomarkers on cancer cell surface. Based on the excellent selectivity of AS1411 aptamer to targeted nucleolin-overexpressed cells, we used a fluorescent probe with a pH-sensitive function to label the AS1411 aptamer modified with azide by click-reaction. The spectral characteristics of fluorophore naphthalimide has a good pH dependence. By this way, nucleolin-overexpressed cell could be discriminated from normal cell. Further, this strategy could also discriminate the breast cancer tissue from the adjacent benign tissue in formalin-fixed and parrffin- embedded (FFPE) tissue specimens. It is considered that our method has the potential to be applied in medical detection.

Digital pH Test Strips for In-Field pH Monitoring Using Iridium Oxide-Reduced Graphene Oxide Hybrid Thin Films

While pH test strips are not decimally accurate and only semiquantitative, the more accurate and automated laboratory-based pH meters lack disposability and portability. Benefiting from advantages of both sides and filling the gap of unmet needs in between, we integrated a miniaturized paper-fluidic electrochemical pH platform coupled with a portable pH device powered by consumer batteries to make digital pH test strips for truly in-field measurements. To overcome the inability to form the smooth and uniform pH-sensitive functionality on screen-printed carbon, i.e., anodically electrodeposited iridium oxide thin films (AEIROFs), reduced graphene oxide was used as substrate for synthesis of homogeneous metal oxide–nanocarbon hybrid thin films, IrO2-reduced graphene oxide (IrO2-rGO), as the sensing moiety with strong substrate adhesion. A hydrophobic barrier-patterned paper micropad (μPAD) with programmed flow rates can locally accelerate linear wicking speed to boost delivery and response time. IrO2-rGO sh...

Targeted Dual pH-Sensitive Lipid ECO/siRNA Self-Assembly Nanoparticles Facilitate In Vivo Cytosolic sieIF4E Delivery and Overcome Paclitaxel Resistance in Breast Cancer Therapy.

RNAi-mediated knockdown of oncogenes associated with drug resistance can potentially enhance the efficacy of chemotherapy. Here, we have designed and developed targeted dual pH-sensitive lipid-siRNA self-assembly nanoparticles, RGD-PEG(HZ)-ECO/siRNA, which can efficiently silence the oncogene, eukaryotic translation initiation factor 4E (eIF4E), and consequently resensitize triple-negative breast tumors to paclitaxel. The dual pH-sensitive function of these nanoparticles facilitates effective cytosolic siRNA delivery in cancer cells, both in vitro and in vivo. Intravenous injection of RGD-PEG(HZ)-ECO/siRNA nanoparticles (1.0 mg-siRNA/kg) results in effective gene silencing for at least one week in MDA-MB-231 tumors. In addition, treatment of athymic nude mice with RGD-PEG(HZ)-ECO/sieIF4E every 6 days for 6 weeks down-regulates the overexpression of eIF4E and resensitizes paclitaxel-resistant MDA-MB-231 tumors to paclitaxel, resulting in significant tumor regression at a low dose, with negligible side effects. Moreover, repeated injections of the RGD-PEG(HZ)-ECO/siRNA nanoparticles in immunocompetent mice result in minimal immunogenicity, demonstrating their safety and low toxicity. These multifunctional lipid/siRNA nanoparticles constitute a versatile platform of delivery of therapeutic siRNA for treating cancer and other human diseases.

Morpholino-terminated dendrimer shows enhanced tumor pH-triggered cellular uptake, prolonged circulation time, and low cytotoxicity

Short circulation is a disadvantage of dendrimers as drug delivery carriers due to their small size. In this work, pH-sensitive morpholino-terminated generation 5 poly(amido amine) (PAMAM) is prepared. At neutral pH, the dendrimer has a hydrophilic neutral surface and thereby stealth property. Under tumor acidic environments, the dendrimer develops positive charges due to protonation of the morpholino groups, and cellular uptake of the dendrimer is subsequently enhanced. The dendrimer is predicted to penetrate deeper within the tumor due to its small size (∼9 nm), and thereby the pH-sensitive function can be efficiently achieved because the microenvironment in the deeper sites is more acidic. Moreover, the morpholino-terminated PAMAM exhibited longer in vivo circulation time compared to PAMAM and hydroxyl-terminated PAMAM, partly compensating for the disadvantage of short circulation of dendrimers. Furthermore, the morpholino-terminated PAMAM also showed lower cytotoxicity than PAMAM and hydroxyl-terminated PAMAM.

The development of polyamide 6.6 nanofibres with a pH-sensitive function by electrospinning

The development of a pH-sensitive nanofibrous sensor could contribute to interesting applications thanks to the combination of the pH-sensitive functionality and the unique characteristics of nanofibres. The effect of the addition of pH-sensitive dyes to the polymer solution is however unknown. Moreover also the halochromic behaviour of the resulting nonwovens can be influenced by this incorporation. Therefore, we studied the production of halochromic nano nonwovens by adding various pH-indicator dyes to a polyamide 6.6 polymer solution prior to electrospinning. Next, the halochromism of two selected dyes (Bromocresol Purple and Brilliant Yellow) was investigated as case studies. Our results show that the pH-indicator addition has no influence on the average fibre diameter. Poorly dissolved dyes cause however instabilities in the process as seen by the droplet formation. The investigated nanofibrous structures showed a clear colour change with a change in pH. This halochromic behaviour was however different from the behaviour of the dye in solution due to dye–fibre interactions. Generally it can be concluded that a nanofibrous pH-sensor can be developed by electrospinning with incorporated pH-indicator dyes.

Acidic Extracellular Environments Strongly Impair ABCA1-Mediated Cholesterol Efflux From Human Macrophage Foam Cells

In the deep microenvironments of advanced human atherosclerotic lesions, the intimal fluid becomes acidic. We examined the effect of an acidic extracellular pH on cholesterol removal (efflux) from primary human macrophages. When cholesterol efflux from acetyl-low-density lipoprotein-loaded macrophages to various cholesterol acceptors was evaluated at pH 7.5, 6.5, or 5.5, the lower the pH the more was cholesterol efflux reduced. The reduction of efflux to lipid-free apolipoprotein A-I was stronger than to high-density lipoprotein(2) or to plasma. Cholesterol efflux to every acceptor was severely compromised also at neutral pH when the macrophages had been loaded with cholesterol at acidic pH, or when both loading and efflux were carried out at acidic pH. Compatible with these observations, the typical upregulation of ABCA1 and ABCG1 mRNA levels in macrophages loaded with cholesterol at neutral pH was rapidly attenuated in acidic medium. The secondary structure of apolipoprotein A-I did not changed over the pH range studied, supporting the notion that the inhibitory effect of acidic pH on cholesterol efflux rather impaired the ability of the foam cells to facilitate ABCA1-mediated cholesterol release. Secretion of apolipoprotein E from the foam cells was fully inhibited when the pH was 5.5, which further reduced cholesterol efflux. An acidic pH reduces cholesterol efflux via different pathways and particularly impairs the function of the ABCA1 transporter. The pH-sensitive function of human macrophage foam cells in releasing cholesterol may accelerate lipid accumulation in deep areas of advanced atherosclerotic plaques where the intimal fluid is acidic.