Phenylboronate-salicylate ester cross-linked self-healing hydrogel composed of modified hyaluronan at physiological pH.

Several hydrogels with boronate/diol ester cross-linking have been reported. However, multiple synthetic steps or expensive reagents are required to modify some diol moieties into polymers. Therefore, diol-modified polymers, which are easily and inexpensively prepared via a single-step process, are required for the formation of boronate esters. This study reports a novel hydrogel composed of phenylboronic acid-modified hyaluronic acid and salicylic acid-modified hyaluronic acid. This hydrogel is injectable, can self-heal at physiological pH, and can be easily and inexpensively prepared. The polymer system behaved as a sol at pH 12.0 and a weak gel at pH 9.4 and 11.2, whereas it behaved as a gel over a wide pH range of 4.0-8.2. The viscoelasticity of the system decreased in response to sugar at pH 7.3. Thus, salicylic acid can be considered a promising diol moiety for hydrogel formation via boronate ester cross-linking.

Hydrogen Peroxide-Triggered Conversion of Boronic Acid-Appended Insulin into Insulin and Its Application as a Glucose-Responsive Insulin Formulation.

p-Boronophenylmethoxycarbonyl (BPmoc) is a protecting group for amines that is removable by treatment with hydrogen peroxide (H2O2). We prepared BPmoc-modified insulin (BPmoc-Ins) and subcutaneously injected the formulation into diabetic rats. The results demonstrated that BPmoc effectively sealed the blood glucose (Glc)-lowering effects of Ins. Conversely, coinjection of BPmoc-Ins and Glc oxidase (GOx) resulted in reduced blood Glc levels, indicating that Ins was generated from BPmoc-Ins through the following reactions: oxidation of endogenous Glc by GOx; production of H2O2 accompanied by Glc oxidation; removal of BPmoc residues by H2O2. These results show the potential of BPmoc-Ins for a Glc-responsive Ins release system.

Multinuclear NMR Study on the Formation and Polyol-Induced Deformation Mechanisms of Wormlike Micelles Composed of Cetyltrimethylammonium Bromide and 3-Fluorophenylboronic Acid.

We had previously confirmed a glucose-responsive decrease in the viscosity of cetyltrimethylammonium bromide (CTAB) and phenylboronic acid (PBA) wormlike micelle (WLM) systems. However, the mechanisms of the formation of WLMs and the decrease in viscosity with glucose addition have not been determined. In this study, we elucidated the mechanisms using 3-fluorophenylboronic acid (3FPBA) based on 11B NMR and 19F NMR analyses. The system in 60 mM CTAB/60 mM 3FPBA at pH 7.4 demonstrated high viscoelasticity, and the formation of WLMs in the system was confirmed by rheological characteristics. The 11B NMR spectrum at pH 7.4 revealed that 3FPBA existed in a neutral form with sp2-hybridized boron; however, the 11B signal disappeared in the presence of CTAB. In contrast, 19F NMR studies indicated that the quaternary ammonium ion of CTAB interacts with the phenyl group of 3FPBA in the sp2 form via cation-π interactions. PBA derivatives react with various polyols; thus, we investigated the change in the viscous system after the addition of sugar and sugar alcohols. The viscosity of the WLMs decreased with increased polyol concentration, especially those of fructose and mannitol, in which the decrease was apparent at 40-160 mM polyols. The 19F NMR spectra revealed that polyol addition induced decrease in the sp2 form of 3FPBA and increase in the sp3 form of 3FPBA. Based on the results, we propose the following mechanism of the polyol response: (1) The WLMs are stabilized by CTAB and 3FPBA in the sp2 form using cation-π interactions as the driving force. (2) When polyol is added to the system, the sp2 form of 3FPBA decreases and its sp3 form increases. (3) This change means that the structural component of WLMs decreases, which induces the disruption of WLMs, and the viscosity decreases. The formation and deformation mechanisms of the WLMs determined in this study are notable because 3FPBA interacts as a neutral compound, whereas CTAB often interacts with anionic aromatic compounds to form WLMs. Without 19F NMR measurements, these mechanisms would not have been discovered.

Glucose Responsive Rheological Change and Drug Release from a Novel Worm-like Micelle Gel Formed in Cetyltrimethylammonium Bromide/Phenylboronic Acid/Water System.

A novel glucose (Glc)-responsive gel formed by worm-like micelles (WLMs) has the potential to provide a self-regulating insulin delivery system. We have prepared a WLM gel system using 75 mM cetyltrimethylammonium bromide, 75 mM phenylboronic acid, and water. At pH 9.4, this gel-like system was highly viscous and supported its own weight, and dynamic viscoelasticity measurement indicated that it contained long and entangled WLMs. The visual observation of gels prepared to include >6 mM Glc revealed that these adopted a sol-like appearance, whereas those prepared to include a control compound (2-10 mM diethylene glycol) retained their gel-like appearance. The storage modulus ( G') of this system decreased as the Glc concentration increased (2-10 mM), indicating a gradual shortening of the WLMs. In vitro release was evaluated using a test compound (fluorescein isothiocyanate dextran) in a microsized flow system. By 120 min, the release of this compound from the WLM gel was around 27-fold greater in the presence of 100 mM Glc than without Glc or with 100 mM diethylene glycol. This demonstrated the successful preparation of a WLM gel that showed an altered drug release rate, depending on Glc concentration.

Sugar-Responsive Layer-by-Layer Film Composed of Phenylboronic Acid-Appended Insulin and Poly(vinyl alcohol).

Previous studies have shown that reversible chemical bond formation between phenylboronic acid (PBA) and 1,3-diol can be utilized as the driving force for the preparation of layer-by-layer (LbL) films. The LbL films composed of a PBA-appended polymer and poly(vinyl alcohol) (PVA) disintegrated in the presence of sugar. This type of LbL films has been recognized as a promising approach for sugar-responsive drug release systems, but an issue preventing the practical application of LbL films is combining them with insulin. In this report, we have proposed a solution for this issue by using PBA-appended insulin as a component of the LbL film. We prepared two kinds of PBA-appended insulin derivatives and confirmed that they retained their hypoglycemic activity. The LbL films composed of PBA-appended insulin and PVA were successfully prepared through reversible chemical bond formation between the boronic acid moiety and the 1,3-diol of PVA. The LbL film disintegrated upon treatment with sugars. Based on the results presented herein, we discuss the suitability of the PBA moiety with respect to hypoglycemic activity, binding ability, and selectivity for D-glucose.

A Pseudopolyrotaxane for Glucose-Responsive Insulin Release: The Effect of Binding Ability and Spatial Arrangement of Phenylboronic Acid Group.

A pseudopolyrotaxane (PPRX) comprising 3-carboxy-5-nitrophenylboronic acid modified γ-cyclodextrin (NPBA-γ-CyD) and naphthalene modified polyethylene glycol (Naph-PEG) as a sugar-responsive supramolecular structure is prepared. The binding of sugar by the NPBA group induced disintegration of the Naph-PEG/NPBA-γ-CyD PPRX, allowing the components to be dissolved. The Naph-PEG/NPBA-γ-CyD PPRX exhibited better sensitivity compared to that of a PPRX based on 4-carboxyphenylboronic acid modified γ-cyclodextrin (PBA-γ-CyD). We have previously reported the unique structure of Naph-PEG/PBA-γ-CyD PPRX, which formed an inclusion complex with a single-stranded PEG chain being threaded through the γ-CyD rings, with the remaining internal space being occupied by the sugar-sensing PBA moiety from a neighboring ring, thus shielding it from sugar molecules and reducing the sugar sensitivity of the PPRX. In contrast, structural analyses in this study revealed that the sugar-sensing NPBA moiety in the Naph-PEG/NPBA-γ-CyD PPRX is not included in the neighboring NPBA-γ-CyD. This spatial arrangement and the high affinity of NPBA for sugar contributed to the improved sugar responsivity. The enhanced NPBA-γ-CyD was then applied to a PPRX containing Naph-PEG-appended insulin (Naph-PEG-Ins) that showed an improved response for glucose-induced insulin release.

Effect of Physiological Changes in the Skin on Systemic Absorption of Tacrolimus Following Topical Application in Rats.

Tacrolimus (TL) ointment is a topical treatment for atopic dermatitis, a disease that exhibits various skin conditions. The effect of skin pathologies on the systemic absorption of TL and related side effects remains unknown. This study aimed to investigate factors affecting the cutaneous absorption of TL. We prepared various skin models in hairless rats by tape stripping, injection of prophlogistic material solution (PMS), and continuous subcutaneous adrenaline (Adr) infusion. In vivo absorption studies were conducted, with measurements of transepidermal water loss (TEWL) and skin blood flow as physiological parameters. Very little TL absorption was observed through intact skin. Greater TL absorption was noted in skins with high TEWL values and fully stripped skin with PMS injections. In contrast, Adr infusion, which reduced skin blood flow, resulted in decreased TL absorption through fully stripped skin. Combined use of TL and Adr on skin with PMS injections resulted in suppression of TL absorption. Our results revealed that TL absorption following topical application is affected by alterations in the skin barrier, blood flow, and vascular permeability. We propose an administration plan for TL in a flowchart as a means of preventing systemic side effects.

Preparation of polypseudorotaxanes composed of cyclodextrin and polymers in microspheres.

We prepared polypseudorotaxanes (PPRXs) composed of cyclodextrin (CyD) and polyethylene glycol (PEG) inside microspheres (MSs) by an emulsifying process using polypropylene glycol (PPG) that shows temperature-dependent hydrophilicity changes; PPG is hydrophobic at high temperatures but hydrophilic at low temperatures. An aqueous solution of CyD and PEG was dispersed as droplets in PPG at 60°C then cooled to 0°C to allow water of droplets to transfer into PPG. On removal of water in the droplets, CyD and PEG were left behind as a CyD/PEG PPRX inside the solid-state MSs. Examination of α-, ß-, and γ-CyD revealed that α-CyD was suitable for the formation of PPRX containing PEG in this MS preparation procedure. Interestingly, a new PPRX composed of α-CyD and PPG was formed in the α-CyD MSs when they were prepared in the absence of PEG from the aqueous solution of α-CyD. This MS fabrication procedure can control the size and shape of PPRX particles, and will contribute to the production of new types of CyD inclusion complexes.

Diol or Hydrogen Peroxide-responsive Micellar Systems and Their Rheological Properties.

External stimuli-responsive worm-like micelles (WLMs) have the potential for a wide range of applications. In particular, sugar (a polyol compound)-responsive WLMs have the potential for use in smartdrug release systems. Phenylboronic acid (PBA) functions as a cis-diol sensor in a similar manner it does as a glucose sensor. Thus, WLMs, primarily composed of surfactants and PBA, are expected to function as cis-diol-responsive viscoelastic systems. PBA also reacts irreversibly with hydrogen peroxide (H2O2 ) and is converted into phenol and boric acid. H2O2 is one of reactive oxygen species crucial for several physiological processes. Therefore, H2O2 -responsive WLMs have the potential for various applications. In this review, we describe cis-diol- and H2O2 -responsive micellar systems composed of cetyltrimethylammonium bromide and PBA moieties that shift their viscosities in response to stimuli.

Sugar-sensitive supramolecular structures based on phenylboronic Acid-modified cyclodextrins.

Supramolecular structures were developed from phenylboronic acid-modified cyclodextrins (PBA-CyDs). The intermolecular interaction between the PBA moiety and the CyD cavity was proved using two dimensional (2D)-NMR and powder X-ray diffraction techniques. PBA-α-CyD formed a head-to-tail supramolecular polymer, whereas PBA-ß-CyD formed a head-to-head dimer. The supramolecular structures were disintegrated in the presence of sugars owing to the resulting boronate sugar interactions.

[Structural Transitions of Micellar Systems in Response to Diol Compounds Accompanied by Changes in Viscoelastic Properties to Yield Novel Functional Materials].

External-stimuli-responsive smart viscoelastic systems have the potential for diverse applications. Worm-like micelles (WLMs) are distinct viscoelastic systems. Several stimuli-responsive WLMs have been reported thus far, in which modifications are triggered by pH variations, redox reactions, temperature shifts, and light. However, sugar-responsive WLMs have not been reported. Phenylboronic acid (PBA) reversibly forms cyclic ester with cis-diol compounds; therefore, it serves as a cis-diol sensor for compounds such as glucose (Glc) and fructose (Fru). Adding PBA to cetyltrimethylammonium bromide (CTAB) in a basic medium induces the transition of spherical micelles to WLMs. This is accompanied by a substantial increase in the viscosity of the CTAB/PBA system. Notably, the addition of Glc to the CTAB/PBA system induces the transformation of the WLMs into spherical micelles or short rod-like micelles. In this review, we describe diol-responsive micellar systems based on PBA and their rheological properties.

Anomalous glucose-responsive rheological changes in a boronic acid-modified hyaluronan.

Herein, we report anomalous glucose (Glc)-responsive gelation/solation in 3-aminophenylboronic acid-modified hyaluronic acid. With 5-20 mM Glc, gelation occurred, resulting in the formation of crosslinks via Glc, which could reversibly bind to the two boronic acid sites. Solation was induced at Glc concentrations of >80 mM.

Diol responsive viscosity increase in a cetyltrimethylammonium bromide/sodium salicylate/3-fluorophenylboronic acid micelle system.

We report a novel smart micellar system utilising a phenylboronic acid (PBA) derivative whose viscosity increases on adding diol compounds such as sugar or sugar alcohol. We prepared a typical worm-like micelle (WLM) system in 100 mM cetyltrimethylammonium bromide (CTAB)/70 mM sodium salicylate (NaSal), which showed high zero-shear viscosity (η 0). Upon the addition of 20 mM 3-fluorophenylboronic acid (3FPBA) to the WLM system, η 0 decreased by 1/300 that of the system without 3FPBA. Furthermore, upon the addition of 1.12 M fructose (Fru) and 1.12 M sorbitol (Sor) to the CTAB/NaSal/3FPBA system, η 0 increased by 50-fold and 30-fold, respectively. 19F NMR spectral results of the systems using 4-fluorosalicylic acid (FSal) instead of NaSal demonstrated that the FSal/3FPBA-complex interacts with CTAB. Moreover, the addition of sugar or sugar alcohol to the micellar system leads to a decrease in the amount of FSal/3FPBA-complex interacting with CTA+ and an increase in the amount of 3FPBA/Fru or Sor-complex, which does not interact with CTA+. These changes in molecular interactions induce the elongation of the WLMs and increase the viscosity of the system. This system utilises the competitive cyclic ester bond between the NaSal/3FPBA and 3FPBA/sugar or sugar alcohol to induce viscosity changes.

Cell Adhesive Character of Phenylboronic Acid-Modified Insulin and Its Potential as Long-Acting Insulin.

Phenylboronic acid (PBA) derivatives have attracted substantial attention owing to their unique character of forming dynamic covalent bonds with polyol compounds. Recent studies have shown interactions between PBA and sugar chains on the cell surface; they have interesting applications for sensors and drug delivery systems. In this study, we prepared phenylboronic acid-modified insulin (PBA-Ins) to evaluate its glucose-lowering activity and cell adhesiveness. In the case of intravenous injection, PBA-Ins showed longer glucose-lowering activity than native insulin. We hypothesized that this prolonged effect was the result of the interaction between the PBA moiety and sugar chains on the cell surface. Red blood cells (RBCs) were used as a cell model, and we confirmed PBA-Ins's affinity for RBCs, which induced RBC agglutination. Interestingly, using an alternative PBA-Ins administration route markedly changed its glucose-lowering activity. Unlike the intravenous injection of PBA-Ins, the subcutaneous injection showed a small effect on glucose level, which indicated that a small amount of PBA-Ins was absorbed into the bloodstream. This suggested the importance of investigating the interaction between the PBA moiety and many types of cells, such as adipocytes, in subcutaneous tissues.

Investigation of factors that cause insulin precipitation and/or amyloid formation in insulin formulations.

BACKGROUND: Multiple daily subcutaneous injections (MDSIs) are mainly used for formulating an insulin therapy for diabetic patients; however, they also cause insulin-derived amyloidosis (IDA) and lead to poor glycemic control. In addition, for the continuous subcutaneous insulin infusion system (CSII), precipitation frequently causes catheter occlusion and, if the precipitate in the formulations is amyloid, the injection of the insoluble amyloid into the subcutaneous tissue leads to IDA. The aim of this study was to conduct in vitro experiments and present a situation where insulin formulations cause precipitation and amyloid formation. METHODS: Humulin®R and NovoRapid® were used as model formulations for MDSIs and CSII, respectively. The generation of the precipitation was evaluated by measuring turbidity, and amyloid formation was evaluated by using Thioflavin T. Humulin®R was mixed with saline buffer solutions and glucose solutions to evaluate the effect of dilution. In addition, we created an experimental system to consider the effect of the time course of condition changes, and investigated the effects of insulin concentration, m-cresol existence, and pH change on the generation of the precipitate and amyloid in the formulation. RESULTS: In both the original and diluted formulations, physical stimulation resulted in the formation of a precipitate, which in most cases was an amyloid. The amyloid was likely to be formed at a near neutral pH. On the contrary, although a precipitate formed when the pH was decreased to near the isoelectric point, this precipitate was not an amyloid. Further decreases in pH resulted in the formation of amyloids, suggesting that both the positive and negative charged states of insulin tended to form amyloids. The formulation additive m-cresol suppressed amyloid formation. When additives were removed from the formulation, the amyloid-containing gel was formed in the field of substance exchange. CONCLUSIONS: To consider changes in conditions that may occur for insulin formulations, the relationship between the formation of precipitates and amyloids was demonstrated in vitro by using insulin formulations. From the in vitro study, m-cresol was shown to have an inhibitory effect on amyloid formation.

Sugar-Responsive Pseudopolyrotaxane Composed of Phenylboronic Acid-Modified Polyethylene Glycol and γ-Cyclodextrin.

We have designed a sugar-responsive pseudopolyrotaxane (PPRX) by combining phenylboronic acid-modified polyethylene glycol (PBA-PEG) and γ-cyclodextrin. Phenylboronic acid (PBA) was used as a sugar-recognition motif in the PPRX because PBA reacts with a diol portion of the sugar molecule and forms a cyclic ester. When D-fructose or D-glucose was added to a suspension of PPRX, PPRX disintegrated, depending on the concentration of the sugars. Interestingly, catechol does not show a response although catechol has a high affinity for PBA. We analyzed the response mechanism of PPRX by considering equilibria.

Development of a membrane impregnated with a poly(dimethylsiloxane)/poly(ethylene glycol) copolymer for a high-throughput screening of the permeability of drugs, cosmetics, and other chemicals across the human skin.

We aimed to develop a high-throughput screening (HTS) system for preliminary predictions of human skin permeability by using an artificial membrane that can mimic the permeation behaviour of lipophilic and hydrophilic compounds across the human skin. In this study, we synthesized a copolymer containing poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) (PEG) 6000 and impregnated it onto a supportive membrane filter to prepare a PDMS/PEG 6000 copolymer-impregnated membrane. In addition, we synthesized another polymer without PEG units and used it to prepare an impregnated membrane for determining the role of PEG 6000 units in the PDMS/PEG 6000 copolymer-impregnated membrane. The permeation characteristics of the impregnated membranes were evaluated on the basis of the permeability coefficients of 12 model compounds with different lipophilicities, by using a 2-chamber diffusion cell, and these permeability coefficients were compared with those across the human skin. We obtained a good correlation between the permeability coefficients across the PDMS/PEG 6000 copolymer-impregnated membrane and human skin. Further, we evaluated the permeation characteristics of a 96-well plate model of the PDMS/PEG 6000 copolymer by using 6 model compounds. We obtained an ideal correlation between the permeability coefficients across the PDMS/PEG 6000 copolymer using a 96-well plate and those across the human skin. Thus, the PDMS/PEG 6000 copolymer would be a good candidate for preliminary evaluation of the permeability of lipophilic and hydrophilic compounds across the human skin.

Colorimetric Sugar Sensing Using Boronic Acid-Substituted Azobenzenes.

In association with increasing diabetes prevalence, it is desirable to develop new glucose sensing systems with low cost, ease of use, high stability and good portability. Boronic acid is one of the potential candidates for a future alternative to enzyme-based glucose sensors. Boronic acid derivatives have been widely used for the sugar recognition motif, because boronic acids bind adjacent diols to form cyclic boronate esters. In order to develop colorimetric sugar sensors, boronic acid-conjugated azobenzenes have been synthesized. There are several types of boronic acid azobenzenes, and their characteristics tend to rely on the substitute position of the boronic acid moiety. For example, o-substitution of boronic acid to the azo group gives the advantage of a significant color change upon sugar addition. Nitrogen-15 Nuclear Magnetic Resonance (NMR) studies clearly show a signaling mechanism based on the formation and cleavage of the B-N dative bond between boronic acid and azo moieties in the dye. Some boronic acid-substituted azobenzenes were attached to a polymer or utilized for supramolecular chemistry to produce glucose-selective binding, in which two boronic acid moieties cooperatively bind one glucose molecule. In addition, boronic acid-substituted azobenzenes have been applied not only for glucose monitoring, but also for the sensing of glycated hemoglobin and dopamine.