Neutral Physiological Conditions Research Articles

Ag-La(OH)3@Dy2O3 hybrid composite modified laser-induced graphene surface for simultaneous electrochemical detection of bisphenol A and tartrazine

Laser-induced graphene (LIG), created through CO2 laser irradiation of polyimide film (PI) substrates, has emerged as a versatile and promising material in numerous fields. However, despite its potential, a comprehensive exploration of LIG’s surface structure and reactivity remains absent in the literature, which is crucial for advancing its applications in electrochemical and electroanalytical fields. In this study, we developed and examined the interfacial structure and electronic behavior of an Ag-La(OH)3@Dy2O3 hybrid composite-modified LIG system as a model. We employed scanning electrochemical microscopy (SECM) to investigate these properties, extending our analysis to the simultaneous detection of organic pollutants, such as bisphenol A (BPA) and tartrazine (TRZ), in real samples. Compared to previous LIG production techniques, the LIG-modified electrode demonstrated a high electron transfer rate at the interface. The tailored LIG substrate showed excellent electrochemical activity towards electrochemical oxidation and simultaneous electroanalytical applications for BPA and TRZ with low detection limits (BPA: 9.2 nM, TRZ: 0.96 nM) in neutral physiological conditions. As a proof of concept, an extension to the real-time application of simultaneous monitoring of BPA and TRZ in various food samples was successfully demonstrated.

Poly(l-Ornithine)-Based Polymeric Micelles as pH-Responsive Macromolecular Anticancer Agents.

Anticancer peptides and polymers represent an emerging field of tumor treatment and can physically interact with tumor cells to address the problem of multidrug resistance. In the present study, poly(l-ornithine)-b-poly(l-phenylalanine) (PLO-b-PLF) block copolypeptides were prepared and evaluated as macromolecular anticancer agents. Amphiphilic PLO-b-PLF self-assembles into nanosized polymeric micelles in aqueous solution. Cationic PLO-b-PLF micelles interact steadily with the negatively charged surfaces of cancer cells via electrostatic interactions and kill the cancer cells via membrane lysis. To alleviate the cytotoxicity of PLO-b-PLF, 1,2-dicarboxylic-cyclohexene anhydride (DCA) was anchored to the side chains of PLO via an acid-labile β-amide bond to fabricate PLO(DCA)-b-PLF. Anionic PLO(DCA)-b-PLF showed negligible hemolysis and cytotoxicity under neutral physiological conditions but recovered cytotoxicity (anticancer activity) upon charge reversal in the weakly acidic microenvironment of the tumor. PLO-based polypeptides might have potential applications in the emerging field of drug-free tumor treatment.

Lactate and glucose induced self‐assembly of hydrophobic boronic acid‐substituted polymers

AbstractAmphiphilic boronic acid (BA)‐substituted block copolymers have been utilized in biomedical applications through binding to saccharides, including to glucose whereas binding to the anaerobically produced metabolite lactate is unreported. Glucose requires basic conditions, while lactate binding occurs at neutral physiological conditions. Herein, BA binding to glucose and lactate is used for the first time to convert solvated hydrophobic homopolymer and hydrophobic block copolymers to amphiphilic core‐shell nanoparticle spheres and worms.

An Enzymatic Antibiotic Adjuvant Modulates the Infectious Microenvironment to Overcome Antimicrobial Resistance of Pathogens.

The emergence and evolution of antimicrobial resistance (AMR) pose a significant challenge to the current arsenal to fight infection. Antibiotic adjuvants represent an appealing tactic for tackling the AMR of pathogens, however, their practical applications are greatly constrained by the harsh infectious microenvironment. Herein, it is found that silver nanoclusters (Ag NCs) can possess tunable enzymatic activities to modulate infectious microenvironments. Based on this finding, an enzymatic nanoadjuvant (EnzNA) self-assembled from Ag NCs, which is inert under neutral physiological conditions but can readily disassemble into isolated Ag NCs exhibiting biofilm destructive oxidase-mimetic activity in the acidic biofilm microenvironment, is developed. Once internalized into the neutral cytoplasm of bacteria, Ag NCs switch to reveal the thiol oxidase-mimetic activity to suppress ribosomal biogenesis for AMR reversal and evolution inhibition of pathogens. Consequently, EnzNAs revitalize various existing antibiotics against methicillin-resistant Staphylococcus aureus, and potentiate the antibiotic efficacy against biofilm-mediated skin infection and lethal lung infection in mice. These findings highlight the capability of enzyme-mimetic nanomaterials to modulate the infectious microenvironment and potentiate antibiotics, providing a paradigm shift for anti-infection therapy.

BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: An example of snake venom phospholipases A2 versatility

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.

Facile preparation of pH/redox dual-responsive biodegradable polyphosphazene prodrugs for effective cancer chemotherapy

In order to maximize the therapeutic effect and and minimize the systemtic side effect of the small molecule anticancer drugs, biodegradable drug delivery systems (DDSs) that respond to tumor microenvironment (TME) have attracted significant attention. Herein, a novel redox/pH dual-responsive and biodegradable polyphosphazene (PPZ) nano-prodrugs have been prepared via one-pot crosslinking of vanillin modified DOX (VMD, acid-sensitive) and 4,4′-dihydroxydiphenyl disulfide (HPS, GSH-responsive) with hexachlorocyclotriphosphazene (HCCP). The phenol groups of the as-synthesized VMD and HPS have high nucleophilic substitution activity towards HCCP under base catalyst and afforded PPZ nano-prodrugs, denoted as HCCP-VMD-HPS, with a high drug loading ratio of up to 56.4 %. As expected, the skeleton of the PPZ consisting of imine bonds in VMD and the disulfide bonds in HPS and cyclotriphosphazenes inclined to be decomposed in low pH conditions and high level of GSH environments. The antitumor drug DOX was found to be controlled released in TME conditions (extracellular, pH∼6.8 and endosomes, lysosomes pH∼5.0 with ∼10 mM GSH), rather than neutral physiological conditions (pH 7.4 with ∼20 μM GSH). Moreover, the resulting HCCP-VMD-HPS nano-prodrug have obvious cytotoxicity to cancer cells while a negligible side effect to normal cells. We therefore believe that the prepared redox/pH dual-responsive and biodegradable PPZ DDSs have great potential in various field.

Antineoplastic Drug‐Free Anticancer Strategy Enabled by Host‐Defense‐Peptides‐Mimicking Synthetic Polypeptides

An antineoplastic drug-free anticancer strategy enabled by host defense peptides (HDPs)-mimicking synthetic polypeptides is reported. The polypeptide exhibits a broad spectrum of anticancer activity in 12 cancer cell lines, including drug-resistant and highly metastatic tumor cells. Detailed mechanistic studies reveal that the cationic anticancer polypeptide (ACPP) can directly induce rapid necrosis of cancer cells within minutes through a membrane-lytic mechanism. Moreover, a pH-sensitive zwitterionic derivative of ACPP (DA-ACPP) is prepared for in vivo application. DA-ACPP shows negligible hemolysis under neutral physiological conditions, and can be converted back to ACPP in slightly acidic tumor environments, resulting in selective killing of cancer cells. Consequently, DA-ACPP shows an effective inhibition of tumor growth in both 4T1 orthotopic breast tumor models and B16-F10 melanoma pulmonary metastatic models. Overall, these findings demonstrate that synthetic HDPs-mimicking polypeptides represent safe and effective antineoplastic agents, which sheds new light on the development of drug-free synthetic polymers for cancer therapy.

Improved delivery and antimetastatic effects of Stattic by self-assembled amphiphilic pendant-dendron copolymer micelles in breast cancer cell lines

Stattic is a signaling pathway inhibitor that could potentially impede the progression of tumor metastasis. However, the pharmaceutical applications of Stattic are hampered by its poor water solubility. In this study, we explored the integration of Stattic within amphiphilic pendant-dendron copolymeric (P71D3) micelles to improve its water solubility and delivery towards tumor cells. The P71D3/Stattic micelles with a 1:1.2 (P71D3:Stattic) ratio optimized for subsequent studies were 164 nm in diameter with a drug loading percentage of 52% w/w. These P71D3/Stattic micelles demonstrated a higher drug release (32%) in acidic tumor conditions compared to neutral physiological conditions (14%). However, rapid and high Stattic release was observed when dialyzed against a simulated plasma solution containing 4% albumin. Interestingly, when the same Stattic-loaded micelles and 4% albumin solution were in direct contact, the albumin molecules bound to the surface of the micelles, forming a protein corona. P71D3/Stattic micelles were found to be 30- to 90-fold more potent and to decrease the effective antimotility concentration by 3- to 6-fold compared to free Stattic in metastatic human and murine mammary cancer cells. The P71D3/Stattic micelles effectively enhanced the potency of the drug as determined from the in vitro studies, suggesting the use of P71D3/Stattic micelles as a promising antimetastatic agent worthy of further study.

Injectable and Self-Healing Nanocomposite Hydrogels with Ultrasensitive pH-Responsiveness and Tunable Mechanical Properties: Implications for Controlled Drug Delivery.

Injectable, self-healing, and pH-responsive hydrogels are great intelligent drug delivery systems for controlled and localized therapeutic release. Hydrogels that show pH-sensitive behaviors in the mildly acidic range are ideal to be used for the treatment of regions showing local acidosis like tumors, wounds and infections. In this work, we present a facile preparation of an injectable, self-healing, and supersensitive pH-responsive nanocomposite hydrogel based on Schiff base reactions between aldehyde-functionalized polymers and amine-modified silica nanoparticles. The hydrogel shows fast gelation within 10 s, injectability, and rapid self-healing capability. Moreover, the hydrogel demonstrates excellent stability under neutral physiological conditions, while a sharp gel-sol transition is observed, induced by a faintly acidic environment, which is desirable for controlled drug delivery. The pH-responsiveness of the hydrogel is ultrasensitive, where the mechanical properties, hydrolytic degradation, and drug release behaviors can alter significantly when subjected to a slight pH change of 0.2. Additionally, the hydrogel's mechanical and pH-responsive properties can be readily tuned by its composition. Its excellent biocompatibility is confirmed by cytotoxicity tests toward human dermal fibroblast cells (HDFa). The novel injectable, self-healing, and sensitive pH-responsive hydrogel serves as a promising candidate as a localized drug carrier with controlled delivery capability, triggered by acidosis, holding great promise for cancer therapy, wound healing, and infection treatment.

Rational Design of β-sheet Peptides with Self-Assembly into Nanofibres on Remineralisation of Initial Caries Lesions.

To explore the self-assembly and gelation properties of synthetic peptides, and their efficacy on hydroxyapatite (HAP) nucleation and in situ remineralisation of initial caries lesions. Mass spectrometry and reversed-phase high performance liquid chromatography (RPHPLC) were used to confirm the successful synthesis of peptides. Their self-assembly properties and conformation stability were evaluated using circular dichroism (CD) spectroscopy and Fourier-transform infrared spectroscopy (FTIR). Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate their cytotoxicity. The efficacy of the peptides on HAP nucleation and in situ remineralisation of initial caries lesions was explored using FTIR, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction and transverse microradiography analysis. Two kinds of self-assembly β-sheet peptides named ID4 and ID8, respectively, were successfully synthesised with purities greater than 95%. Both were stable under neutral physiological conditions and had low cytotoxicity. ID4 and ID8 showed calcium responsive self-assembly properties and could self-assemble into nanofibres. Compared with ID4, ID8 resulted in the rapid formation of hydrogel with a lower concentration of calcium, and self-assembled ID8 hydrogel induced the formation of flower-like HAP and significantly promoted the remineralisation of initial enamel caries. ID8 could serve as the template to induce HAP nucleation and promote biomimetic remineralisation of initial caries lesions. These results underpin future research on peptide design, and ID8 may be a promising bioactive component for anti-caries applications.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Responsive Assembly of Silver Nanoclusters with a Biofilm Locally Amplified Bactericidal Effect to Enhance Treatments against Multi-Drug-Resistant Bacterial Infections.

Bacterial biofilms pose a major threat to public health because they are resistant to most current therapeutics. Conventional antibiotics exhibit limited penetration and weakened activity in the acidic microenvironment of a biofilm. Here, the development of biofilm-responsive nanoantibiotics (rAgNAs) composed of self-assembled silver nanoclusters and pH-sensitive charge reversal ligands, whose bactericidal activity can be selectively boosted in the biofilm microenvironment, is reported. Under neutral physiological conditions, the bactericidal activity of rAgNAs is self-quenched because the toxic silver ions’ release is largely inhibited; however, upon entry into the acidic biofilm microenvironment, the rAgNAs not only exhibit charge reversal to facilitate local accumulation and retention but also disassemble into small silver nanoclusters, thus enabling deep penetration and accelerated silver ions release for dramatically amplified bactericidal activity. The superior antibiofilm activity of rAgNAs is demonstrated both in vitro and in vivo, and the mortality rate of mice with multi-drug-resistant biofilm-induced severe pyomyositis can be significantly reduced by rAgNAs treatment, indicating the immense potential of rAgNAs as highly efficient nanoscale antibacterial agents to combat resistant bacterial biofilm-associated infections.

Liposomes of dimeric artesunate phospholipid: A combination of dimerization and self-assembly to combat malaria

Artemisinin and its derivatives are highly effective drugs in the treatment of P. falciparum malaria. However, their clinical applications face challenges because of short half-life, poor bioavailability and growing drug resistance. In this article, novel dimeric artesunate phospholipid (Di-ART-GPC) based liposomes were developed by combination of dimerization and self-assembly to address these shortcomings. Firstly, Di-ART-GPC conjugate was synthesized by a facile esterification of artesunate (ART) and glycerophosphorylcholine (GPC) and confirmed by MS, 1H NMR and 13C NMR. The conjugate was then assembled to form liposomes without excipient by thin film hydration method. The assembled Di-ART-GPC liposomes have typical multilamellar vesicle structure with bilayer morphology as determined by transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM). Moreover, the liposomes displayed an average hydrodynamic diameter of 190 nm and negative zeta potential at −20.35 mV as determined by Zetasizer. The loading capacity of ART was calculated approximately 77.6% by weight with this liposomal formulation after a simple calculation. In vitro drug release and degradation results showed that the Di-ART-GPC liposomes were stable in neutral physiological conditions but effectively degraded to release parent ART in simulated weakly acidic microenvironment. In vivo pharmacokinetics study revealed that Di-ART-GPC liposomes and conjugate have longer retention half-life in bloodstream. Importantly, Di-ART-GPC liposomes (IC50 0.39 nM) and the conjugate (IC50 1.90 nM) demonstrated excellent in vitro antiplasmodial activities without causing hemolysis of erythrocytes, which were superior to free ART (IC50 5.17 nM) and conventional ART-loaded liposomes (IC50 3.13 nM). Furthermore, the assembled liposomes resulted in enhanced parasites killing in P. berghei-infected mice in vivo with delayed recrudescence and improved survivability, compared to free ART administration. Based on these encouraging results, Di-ART-GPC liposomal formulation could be a replacement to parent ART in clinical malarial therapy after thorough investigation.

Star Polymer-Drug Conjugates with pH-Controlled Drug Release and Carrier Degradation

In this study, we describe the design, synthesis, and physicochemical and preliminary biological characteristics of new biodegradable, high-molecular-weight (HMW) drug delivery systems with star-like architectures bearing the cytotoxic drug doxorubicin (DOX) attached by a hydrazone bond-containing spacer. The star polymers were synthesized by grafting semitelechelic N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers on a 2,2-bis(hydroxymethyl)propionic acid- (bis-MPA-) based polyester dendritic core. The molecular weight of the star polymers ranged from 280 to 450 000 g/mol and could be adjusted by proper selection of the bis-MPA dendrimer generation and by considering the polymer to dendrimer molar ratio. The biodegradation of the polymer conjugates is based on the spontaneous slow hydrolysis of the dendritic core in neutral physiological conditions. Hydrazone spacers in the conjugates were fairly stable at neutral pH (7.4) mimicking blood stream conditions, and DOX was released from the conjugates under mild acidic conditions simulating the tumor cell microenvironment in endosomes and lysosomes (pH 5). Finally, we have shown the significant in vitro cytotoxicity of the star polymer-DOX conjugate on selected cancer cell lines with IC50 values almost comparable with that of the free drug and higher than that observed for a linear polymer-DOX conjugate with much lower molecular weight.

An NBD-based Fluorescent Probe with High Selectivity to Cysteine over Homocysteine under Neutral Physiological Conditions

Abstract 7-nitro-2,1,3-benzoxadiazole (NBD)-based fluorescent probe for the detection of cysteine (Cys) over homocysteine (Hcy) and amino acids under neutral physiological conditions based on thiol-induced aromatic nucleophilic substitution reaction was synthesized by the reaction of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) with p-thiocresol. The proposed sensing mechanism was proved by mass spectrometry of the reaction mixtures. This probe was successfully applied to the fluorescence imaging of Cys in HeLa cells.

Benzoboroxole‐Functionalized Magnetic Core/Shell Microspheres for Highly Specific Enrichment of Glycoproteins under Physiological Conditions

Efficient enrichment of specific glycoproteins from complex biological samples is of great importance towards the discovery of disease biomarkers in biological systems. Recently, phenylboronic acid-based functional materials have been widely used for enrichment of glycoproteins. However, such enrichment was mainly carried out under alkaline conditions, which is different to the status of glycoproteins in neutral physiological conditions and may cause some unpredictable degradation. In this study, on-demand neutral enrichment of glycoproteins from crude biological samples is accomplished by utilizing the reversible interaction between the cis-diols of glycoproteins and benzoboroxole-functionalized magnetic composite microspheres (Fe3O4/PAA-AOPB). The Fe3O4/PAA-AOPB composite microspheres are deliberately designed and constructed with a high-magnetic-response magnetic supraparticle (MSP) core and a crosslinked poly(acrylic acid) (PAA) shell anchoring abundant benzoboroxole functional groups on the surface. These nanocomposites possessed many merits, such as large enrichment capacity (93.9 mg/g, protein/beads), low non-specific adsorption, quick enrichment process (10 min) and magnetic separation speed (20 s), and high recovery efficiency. Furthermore, the as-prepared Fe3O4/PAA-AOPB microspheres display high selectivity to glycoproteins even in the E. coli lysate or fetal bovine serum, showing great potential in the identify of low-abundance glycoproteins as biomarkers in real complex biological systems for clinical diagnoses.

Reversible immobilization of drugs on a hydrogel matrix, 3. Hydrolysis of chloramphenicol precursors

AbstractKinetics of hydrolytic release of chloramphenicol (1) chemically immobilized on HEMA‐based ophthalmic hydrogels were examined. The rate constants of hydrolysis of the phenolic ester and acetal bonds of the spacer immobilizing the drug were determined at 37°C, and essentially at neutral pH, for the monomeric drug, the soluble copolymers and the cross‐linked hydrogels, respectively. A drastic decrease in reactivity of the ester bond was observed from the small monomeric molecule to the soluble chains and then to the swollen network (relative reactivities ≈ 1300/25/1), whereas the acetal bond is less reactive, with a rate constant apparently independent of the size of the carrier. It is suggested that in all cases the ester bond is cleaved at first, liberating a drug bearing acetal intermediate which is then slowly hydrolysed to free chloramphenicol. All reactions were observed to be first order with respect to the hydrolysable precursor. When the rate constants are low enough, which is the case when considering the hydrogels, a pseudo‐zero order is obtained and the release rate of chloramphenicol is constant over a long period of time and depends only on the initial concentration of immobilized, drug. Nevertheless, the too slow acetal hydrolysis in neutral physiological conditions limits the daily delivery to a few micrograms for a standard corneal lens with a 5 mol‐% drug loading.

2-[[(4-Amino-2-pyridyl)methyl]sulfinyl]benzimidazole H+/K+-ATPase inhibitors. The relationship between pyridine basicity, stability, and activity

The benzimidazole sulfoxide class of antisecretory H+/K+-ATPase inhibitors need to possess high stability under neutral physiological conditions yet rearrange rapidly at low pH to the active sulfenamide 2. Since the initial reaction involves internal nucleophilic attack by the pyridine nitrogen, control of the pyridine pKa is critical. In this paper we show that by utilizing the powerful electron-donating effect of a 4-amino substituent on the pyridine, moderated by the electron-withdrawing effect of a 3- or 5-halogen substituent, a combination of high potency (as inhibitors of histamine-stimulated gastric acid secretion) and good stability under physiological conditions can be obtained. Furthermore, the role of the steric interaction between the 3/5-substituents and the 4-substituent in modifying the electron-donating ability of the 4-amino group is exemplified, and additional factors affecting stability are identified. One compound, in particular, 2-[[(3-chloro-4-morpholino-2- pyridyl)methyl]sulfinyl]-5-methoxy-(1H)-benzimidazole (3a, SK&F 95601), was chosen for further development and evaluation in man.

Immobilization of microbial cells in crosslinked, prepolymerized, linear polyacrylamide gels: Antibiotic production by immobilized Streptomyces clavuligerus cells

AbstractA mild new method for the immobilization whole microbial cells has been developed. Cells were suspended in a solution of preformed, linear, water‐soluble Polyacrylamide chains, partially substituted with acylhydrazide groups. The Prepolymerized backbone polymer was crosslinked, in the presence of viable cells, by stoichiometric amounts of dialdehydes such as glyoxal, glutardialdehyde, and period ate‐oxidized polyvinyl alcohol. The crosslinking reaction, carried out in cold, neutral physiological conditions resulted in cells entrapped in gels with physical properties similar to those of the common Polyacrylamide gels. However, cell damage generally caused by the acrylamide monomer was avoided. Resting Streptomyces clavuligerus cells, possessing a high capacity for antibiotic production, were entrapped according to this procedure. These immobilized cells produced cephalosporins continuously for 96 h with yields similar to those of free resting cells. The same cells, when immobilized by direct polymerization acrylamide monomers, yielded significantly lower amount of antibiotics.