pH-sensitive Units Research Articles

A biomimetic nanocarrier facilitates glucose consumption and reactive oxide species accumulation in enzyme therapy for colorectal cancer

Glucose oxidase (GOx)-based enzyme therapeutics are potential alternatives for colorectal cancer (CRC) treatment via glucose consumption and accumulation of hydrogen peroxide (H2O2). Given that H2O2 can be eliminated by cytoprotective autophagy, autophagy inhibitors that can interrupt autolysosome-induced H2O2 elimination are promising combination drugs of GOx. Here, we developed a multifunctional biomimetic nanocarrier for effective co-delivery of an autophagy inhibitor-chloroquine phosphate (CQP) and GOx to exert their synergistic effect by irreversibly upregulating intracellular reactive oxygen species (ROS) levels. Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were used to encapsulate both GOx and CQP using a W/O/W multi-emulsion method. Calcium phosphate (CaP) was used to “fix” CQP to GOx in the internal water phase, where it served as a pH-sensitive unit to facilitate intracellular drug release. Folic acid-modified red blood cell membranes (FR) were used to camouflage the GOx/CQP/CaP encapsulated PLGA NPs (referred to as PLGA/GCC@FR). In an AOM/DSS-induced CRC mouse model, PLGA/GCC@FR exhibited improved antitumor effects, in which the number of tumor nodes were only a quarter of that in the free drug combination group. The enhanced therapeutic effects of PLGA/GCC@FR were attributed to the prolonged tumor retention which was verified by both dynamic in vivo imaging and drug biodistribution. This multifunctional biomimetic nanocarrier facilitated combined enzyme therapeutics by depleting glucose and augmenting intracellular ROS levels in tumor cells, which exerted a synergistic inhibitory effect on tumor growth. Therefore, this study proposed a novel strategy for the enhancement of combined enzyme therapeutics, which provided a promising method for effective CRC treatment.

Synthesis and spectroscopic characterization of multifunctional D-π-A benzimidazole derivatives as potential pH sensors

We report multifunctional D-π-A molecular systems having the N,N-diethylamino group as a pH sensitive donor unit connected to the electron accepting substituents such as the cyano moiety and the pH responsive benzimidazole fragment. For that purpose we have synthesized three types of derivatives, namely acrylonitrile, Schiff base and iminocoumarin derived benzimidazoles, whose photophysical characterization has been carried out in several polar and non-polar organic solvents as well as aqueous solution buffers with different pH. Computationally supported determination of species involved in prototrophic equilibria, including their respective pKa values, has been performed to aid in understanding structural and substituent effects on their UV–Vis and fluorescence spectral properties and pH sensing potentials. Both sets of results jointly indicate that all molecules are predominantly unionized under neutral conditions, while their transition towards monocationic forms, responsible for spectroscopic response changes, occurs at pH ≈ 1–4 for acrylonitriles 4–5, pH ≈ 4–5 for Schiff bases 11–12, and pH ≈ 4.5–6.5 for iminocoumarins 7–8, thus increasing their practical attractiveness for a broad range of applications in the same order.

Single-wavelength-excited fluorogenic nanoprobe for accurate realtime ratiometric analysis of broad pH fluctuations in mitophagy

Mitophagy has a critical role in maintaining cellular homeostasis through acidic lysosomes engulfing excess or impaired mitochondria, thereby pH fluctuation is one of the most significant indicators for tracking mitophagy. Then such precise pH tracking demands the fluorogenic probe that has tailored contemporaneous features, including mitochondrial-specificity, excellent biocompatibility, wide pH-sensitive range of 8.0–4.0, and especially quantitative ability. However, available molecular probes cannot simultaneously meet all the requirements since it is extremely difficult to integrate multiple functionalities into a single molecule. To fully address this issue, we herein integrate two fluorogenic pH sensitive units, a mitochondria-specific block, cell-penetrating facilitator, and biocompatible segments into an elegant silica nano scaffold, which greatly ensures the applicability for real-time tracking of pH fluctuations in mitophagy. Most significantly, at a single wavelength excitation, the integrated pH-sensitive units have spectra-distinguishable fluorescence towards alkaline and acidic pH in a broad range that covers mitochondrial and lysosomal pH, thus enabling a ratiometric analysis of pH variations during the whole mitophagy. This work also provides constructive insights into the fabrication of advanced fluorescent nanoprobes for diverse biomedical applications.

NIPAAm-containing amphiphilic block copolymers with tailored LCST: Aggregation behavior, cytotoxicity and evaluation as carriers of indomethacin, tetracycline and doxorubicin

Nanocarriers based on amphiphilic block copolymers, with tailored temperature and pH responsiveness, were prepared. The hydrophilic blocks consist of temperature-sensitive [N-isopropylacrylamide (NIPAAm)] or NIPAAm plus pH-sensitive units [5-methacryloyloxy pentatonic acid (5MPA) or 4-methacryloyloxy benzoic acid (4MBA)], while the hydrophobic block is composed of n-hexyl acrylate (HA) or styrene (ST). Particle sizes were within the suitable range for the desired application (30–182 nm). Drug loading was achieved via an organic solvent-free method and a THF-buffer method leading to drug loadings of indomethacin, tetracycline and doxorubicin of up to 11, 11 and 60 wt%, respectively. In vitro release kinetics were performed under simulated physiological conditions (pH 7.4 and 37 °C; pH 6.0 and 40 °C) and show differences depending on the copolymer composition. The average kinetic data were well fitted to the mathematical model of Peppas. NIPAAm-containing copolymers were slight or non-cytotoxic for rat primary hepatocytes at concentrations less than 200 µg mL−1. Some of the polymeric aggregates prepared may find application as pharmaceutical carriers.

Multi-faced investigation for pH-sensitivity and solvent polarity in highly charged thermo-responsive hydro- and cryogels with strongly dissociated groups: A comparative evaluation of physico-chemical properties

Different series of strong polyelectrolyte poly(N-isopropylacrylamide-co-sodium acrylate) P(NIPA-co-NaA) hydrogels and cryogels were prepared in aqueous solution. With changing the ionic comonomer NaA ratio in reaction mixture, the resulting physico-chemical and swelling properties varied significantly. The findings elucidated that ionic P(NIPA-co-NaA) gels exhibited multi-stimuli performances with pH-, temperature-, solvent-, surfactant- and salt-responsivity, showing more sensitivity than non-ionic PNIPA. Their swollen/collapsed behaviors were strongly dependent on various synthesis/external parameters, including the content of ionic comonomer, temperature and solvent polarity. The effects produced by non-solvents were qualitatively compared considering the aspects related to co-nonsolvency phenomenon and re-entrant swelling transition on the basis of weakening of PNIPA-water interactions. Measurements of the elastic properties and swelling degree of samples having different NaA content were treated by combining Flory-Rehner theory and rubber elasticity to interpret the experimental observations. The polymer-solvent interaction parameter χ of hydrogels was calculated from the swelling studies in water at different temperatures and was related to the composition and temperature. The ionic strength-reversibility and on-off switching kinetics of hydrogels and cryogels were studied in 10−5 M and 1.0 M of NaBr solutions. Diffusion kinetics showed that the swelling of ionic hydrogels is of non-Fickian-type transportation, however, non-ionic PNIPA hydrogels and P(NIPA-co-NaA) cryogels showed pseudo-Fickian diffusion. The resulting hydrogels and cryogels containing both temperature and pH-sensitive units are able to interact with oppositely charged groups via hydrophobic and electrostatic interactions.

Tunable Molecular Logic Gates Designed for Imaging Released Neurotransmitters.

Tunable dual-analyte fluorescent molecular logic gates (ExoSensors) were designed for the purpose of imaging select vesicular primary-amine neurotransmitters that are released from secretory vesicles upon exocytosis. ExoSensors are based on the coumarin-3-aldehyde scaffold and rely on both neurotransmitter binding and the change in environmental pH associated with exocytosis to afford a unique turn-on fluorescence output. A pH-functionality was directly integrated into the fluorophore π-system of the scaffold, thereby allowing for an enhanced fluorescence output upon the release of labeled neurotransmitters. By altering the pH-sensitive unit with various electron-donating and -withdrawing sulfonamide substituents, we identified a correlation between the pKa of the pH-sensitive group and the fluorescence output from the activated fluorophore. In doing so, we achieved a twelvefold fluorescence enhancement upon evaluating the ExoSensors under conditions that mimic exocytosis. ExoSensors are aptly suited to serve as molecular imaging tools that allow for the direct visualization of only the neurotransmitters that are released from secretory vesicles upon exocytosis.

Poly(N-isopropylacrylamide-co-methacrylic acid) pH/thermo-responsive porous hydrogels as self-regulated drug delivery system

A dual drug delivery system based on a “biosensor” (pH-sensitive unit) and a delivery component (thermosensitive hydrogel) was developed. The pH/thermosensitive hydrogel is able to restore the thermosensitive characteristics after electrostatic interaction of the pH-sensitive units with selected biologically active compounds that act as triggering agents.The poly(N-isopropylacrylamide-co-methacrylic acid) (poly(NIPAAm-co-MA)) was synthesized as an interesting pH/thermo-responsive copolymer by free radical polymerization method. Due to the presence of carboxylic groups in MA units, the copolymer loses its thermosensitivity at physiological pH and temperature. However, when the negatively-charged carboxylic groups of the pH-sensitive units interact electrostatically with the positively-charged drugs with hydrophobic character propranolol, lidocaine or metoclopramide, taken as model biologically active compounds, the copolymer restores the thermosensitive properties around the physiological pH and temperature. The poly(NIPAAm-co-MA) linear copolymer was converted into pH/thermo-responsive porous microgels using oligomers of NIPAAm above their LCST, as porogens. Accordingly, the swelling/collapsing processes of the microgels occur only after the interaction with the positively-charged hydrophobic drugs. The hydrophobic drug acts as a triggering agent and the pH/temperature sensitive hydrogel turns as a biosensor (pH-sensitive units) and a delivery component (thermosensitive hydrogel).

The thermosensitivity of pH/thermoresponsive microspheres activated by the electrostatic interaction of pH‐sensitive units with a bioactive compound

pH/thermosensitive hydrogels whose thermosensitivity is activated by the electrostatic interaction of the pH-sensitive units with a hydrophobic bioactive compound are proposed here as sensor-based self-regulated drug delivery systems. Poly(N-isopropylacrylamide-co-acrylamide-co-aminoethylacrylamide) (poly(NIPAAm-co-AAm-co-AEAAm)) was prepared as a new pH/thermoresponsive polymer by hydrolysis of poly(N-isopropylacrylamide-co-acrylamide (poly(NIPAAm-co-AAm)) at high temperature (120°C) in the presence of ethylenediamine. Owing to the hydrophilicity of the inserted amine, the copolymers lose the thermosensitivity at physiological temperature and have a slow-phase transition at temperatures much higher than that of the human body. However, when the positively charged amine groups of the pH-sensitive units bind electrostatically with the negatively charged drug diclofenac, the copolymers retrieve the thermosensitivity. Poly(NIPAAm-co-AAm-co-AEAAm) was transformed into pH/thermoresponsive stable microspheres by an original approach based on crosslinking of the amine groups of aminoethylacrylamide (AEAAm) with glutaraldehyde at a temperature slightly below the lower critical solution temperature. The swelling/deswelling processes of microspheres occur only after the interaction of poly(NIPAAm-co-AAm-co-AEAAm) with the negatively charged drug diclofenac. The pH/temperature-sensitive microgels and dicofenac act as sensors and as the triggering agent, respectively. Conductometric titration reveals that the thermosensitivity of poly(NIPAAm-co-AAm-co-AEAAm) is retrieved when just half of its amino groups are complexed with diclofenac.

Prediction of the appropriate size of drug molecules that could be released by a pulsatile mechanism from pH/thermoresponsive microspheres obtained from preformed polymers

Preparation of cross-linked pH/thermoresponsive microspheres from preformed polymers is still lacking in literature since copolymers possessing both temperature- and pH-sensitive units together with a cross-linkable moiety in appropriate ratios are required. Moreover, choosing of the appropriate drugs able to be loaded and then released in a pulsatile manner is randomly performed. Here, we report the synthesis of pH/thermoresponsive cross-linked microspheres based on N-isopropylacrylamide and N-alloc-ethylenediamine. A chromatographic method was developed to predict the appropriate size of drug molecules that could be loaded and then released in a pulsatile manner. Accordingly, it was established that common drugs (salicylic acid, benzoic acid, nicotinic acid, lidocaine and diclofenac), with molecular weights ranging between 100 and 1000gmol−1, could be loaded and released in a pulsatile manner. Biologic molecules with higher molecular weights (heparin, lysozyme and bovine serum albumin) are completely excluded from the pores of cross-linked pH/thermoresponsive microspheres both below and above the volume phase transition temperature.

PH‐Sensitive Pentablock Copolymer Nanocapsules as Nontoxic and Efficient Gene Carriers

A biodegradable amphiphilic pentablock copolymer PAE-PCL-PEG-PCL-PAE with a pH-sensitive unit was synthesized for use as a nontoxic, biodegradable carrier for gene delivery by forming nanocapsules entrapping nucleic acid drugs. The PAE block can interact with plasmid DNA to form polyelectrolyte complexes in an acidic environment. At physiological pH, the PAE blocks are deprotonated and form an insoluble skin, resulting in the formation of nanocapsules that encapsulate plasmid DNA. The surface charges of the nanocapsules became almost neutral at pH = 7.4, and their size ranged from 210 to 280 nm. The nanocapsule maintained most of its transfection efficiency even in the presence of serum. These nanocapsules are therefore potential carriers for systemic gene therapy.

Smart controlled preparation of multilayered hydrogel for releasing bioactive molecules

We focused on layer-by-layer (LBL) method which has emerged as a versatile and efficient technique. The preparation of multilayered hydrogel can resolve the problems. We fabricated and characterized pH-controlled preparation of polymer multilayer via LbL method on titanium alloy surface for releasing the growth factor. To achieve this purpose, we synthesized water-soluble phospholipid polymer (PMDV) having both N, N-dimethylaminoethyl methacrylate (DMAEMA) as pH-sensitive unit and 4-vinylphenylboronic acid (VPBA) moiety as sugar binding units. To construct the multilayered hydrogel, alginate (ALG), which possesses a polysaccharide comprised of two repeating carboxylated monosaccharide units was used. We could construct multilayered hydrogel easily by using two interaction types. The first is the electrostatic interaction between positively charged DMAEMA and negatively charged carboxyl group of ALG, and the second is the covalent bonding between VPBA and diol of ALG. Multilayered PMDV and ALG hydrogel was characterized by contact angle measurement, surface plasmon resonance. The results demonstrate that the surface composition of the multilayered hydrogel can be simply constructed via LbL process and VEGF release can be released during 1 week. We expect this system will be applied in biomedical and drug delivery system.

Tunable phase transition behaviors of pH-sensitive polyaspartamides having various cationic pendant groups

New pH-sensitive graft copolymers based on poly(2-hydroxyethyl aspartamide) (PHEA) were prepared by attaching various cationic monomers, such as 4-(aminomethyl)pyridine (PY), 1-(3-aminopropyl)imidazole (IM), and N-(3-aminopropyl)dibuthylamine (BU), as pH-sensitive units and octadecylamine (C18) as a hydrophobic segment on poly(succinimide). Phase transition of each copolymer solution occurred at a vicinity of the pK a value of the cationic groups, and their insoluble pH ranges were broadened as the feed amount of pH-sensitive moieties was increased. Depending on the cationic grafts having different pK a values, the pH ranges where the copolymer became insoluble could be tuned. Copolymers PHEA-g-C18-PY, PHEA-g-C18-IM, and PHEA-g-C18-BU exhibited phase separations in solutions at pH ranges of 4∼6, 6∼8, and 9∼12, respectively. These polymers have the unique feature of their pH sensitivity profiles being identified to three regimes. Under low pH conditions (below pK a ), the polymer solution is transparent. At medium pH (around pK a ), polymer precipitation occurred in solution. At pH > pK a , the polymer solution is gradually dissolved again.

Preparation and characterization of pH- and temperature-sensitive pullulan microspheres for controlled release of drugs

Most part of pH- and temperature-sensitive microspheres used for the controlled delivery of drugs are not biodegradable. Therefore, the aim of this work is to prepare pH- and temperature-sensitive microspheres from biodegradable and biocompatible natural polymers. Pullulan microspheres were prepared by suspension cross-linking with epichlorohydrin of an aqueous solution of the polymer. In order to confer them temperature sensitivity, poly( N-isopropylacrylamide- co-acrylamide) was grafted onto pullulan microspheres. Then, the pH-sensitive units (–COOH) were introduced by reaction between the remaining –OH groups of the pullulan with succinic anhydride. The grafted pullulan microspheres are more hydrophilic than pullulan microspheres, their swelling degree as well as water regain increase significantly. The thermo-sensitivity of the carboxylated microspheres depends to the number and the ionization form (–COOH/–COO −) of carboxylic groups. At a low exchange capacity (0.35 meq/g), microspheres are thermo-sensitive both in the protonated and deprotonated form of –COOH groups. At a higher exchange capacity (2.25 meq/g), microspheres are almost unswellable in the protonated form and swell extensively in the ionized form (up to 28 times than their dried form) loosing in a great extent the thermo-sensitive properties. In isotonic phosphate buffer pH = 7.4, both thermo-sensitive and pH/thermo-sensitive microspheres possess a phase transition temperature close to that of the human body temperature. Loading and release profiles of lysozyme, taken as a molecular model system, were investigated.

Synthesis and properties of pH and temperature sensitive P(NIPAAm- co-DMAEMA) hydrogels

In order to investigate the influence of the continuous alkylamide sequence having pH sensitive unit on the temperature sensitivity of poly( N-isopropylacrylamide) (PNIPAAm)-based hydrogel, a monomer, N-(2-(dimethylamino) ethyl)-methacrylamide (DMAEMA), having an ethylamide group as well as an aliphatic tertiary amino group, was designed and synthesized. Hydrogels based on NIPAAm and DMAEMA were prepared via free radical polymerization. The resulted P(NIPAAm- co-DMAEMA) hydrogels were characterized in terms of maximum swelling ratio, swelling kinetics, temperature response kinetics, and effect of pH. The data obtained show that the novel hydrogels have the strong desire to respond to external temperature and pH stimuli. Importantly, because the P(NIPAAm- co-DMAEMA) hydrogels have the continuous alkylamide sequence containing isopropylamide pendant groups from PNIPAAm and ethylamide pendant groups from PDMAEMA, the incorporation of DMAEMA moiety not only provides the pH sensitivity, but also maintains the thermal properties of P(NIPAAm- co-DMAEMA) hydrogels, even as the molar percentage of DMAEMA moiety reaches 14 mol%.

Thermo- and pH-sensitive gel membranes based on poly-(acryloyl- L-proline methyl ester)- graft-poly(acrylic acid) for selective permeation of metal ions

Thermo- and pH-responsive gel membranes were synthesized by γ-ray grafting of pH-responsive poly(acrylic acid) (AAc) onto thermo-responsive polymer gel of acryloyl- L-proline methyl ester (A-ProOMe). The gel membranes of poly(A-ProOMe) with 15 mol% graft chains of AAc exhibited both thermo- and pH-responses. Under the condition (pH 6.0, 30°C) in which the thermo-sensitive unit shrinks and the pH-sensitive unit swells, the selective permeation of Li ion over Co and Ni ions can be achieved.

Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH.

There are many potential applications of 'intelligent' aqueous polymer systems in medicine, biotechnology, industry and in environmental problems. Many of these polymer systems undergo reversible phase transitions--for example, abrupt changes in volume--in response to external stimuli such as temperature, pH or the nature of the solvent. Most of the polymers studied previously are responsive to only one kind of stimulus. But for some applications, independent responsiveness to several factors, such as temperature and pH, may be required. Here we describe a polymer that undergoes marked solubility changes in water in response to temperature and/or pH changes. The polymer is prepared by grafting temperature-sensitive side chains onto a pH-sensitive backbone. We also find that block copolymers, in which the temperature- and pH-sensitive units alternate along the chain, show similar behaviour.

Neurophysiological studies on superficial medullary chemosensitive area for respiration

In cats anesthetized with chloralose-urethane (40 mg/kg chloralose; 200 mg/kg urethane) pH sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata was examined while monitoring phrenic nerve activity simultaneously. pH was varied by superfusion of the ventral medullary surface with mock cerebrospinal fluid (CSF) of different pH (pH 7.4 control, 7.0 and 7.8). A total of 130 units from 21 cats changed their firing rate in response to CSF-pH changes. These were subdivided into 3 groups. In Group I, 31 respiratory pH sensitivie units increased their firing rate in response to decreased mock CSF-pH, noxious pinch, joint movement in contralateral forelimb, and increased inspired CO 2. These responses may have originated from respiratory center neurons. Group II consisted of 59 non-respiratory pH sensitive units whose firing rate changed in an inverse manner with CSF-pH changes. Of these, 30 responded to contralateral distal forelimb movement, 15 to hair manipulation, 9 to heavy pressure and 5 to noxious pinch. Increased inspired CO 2 (rebreathing) did not modify activity. The response to pH is believed to be from non-specific neurons. Group III consisted of 40 non-respiratory pH sensitive units responding to CSF-pH changes and to increased inspired CO 2. The firing rate was irregular, the interval distribution approaching an exponential function. It may be postulated that the impulse frequency of chemosensitive impulses may be irregular at the site of impulse generation, the irregularity decreasing by convergence during transmission to the respiratory centers. The time course of Group III chemosensitive units was similar to phrenic nerve responses.