Encapsulation of multiple enzymes within a microgel via water-in-water emulsions for enzymatic cascade reactions.

Enzyme-loaded spherical microgels with diameters of several micrometers have been explored for use in therapeutic microreactors and biosensors. Conventional preparation strategies for enzyme-loaded microgels utilized water-in-oil emulsions or flow chemistry techniques. The former damage enzyme activity using organic solvents and the latter are expensive and difficult to expand because of the complex system. In this study, we present a simple strategy for creating multiple enzyme-loaded gelatin-based microgels with tunable diameters in a single flask. This strategy was based on our finding that enzymes spontaneously partitioned in a dispersed methacryloyl gelatin aqueous solution in a poly(vinylpyrrolidone) (WGelMA/WPVP) aqueous solution. The method achieved an encapsulation efficiency of over 70% even with four types of enzymes and retained their activity owing to the full aqueous system. Additionally, the encapsulated ß-galactosidase activity was maintained for 24 hours at pH 6, although naked ß-galactosidase lost approximately 60% of its activity, which was superior to that of previous enzyme-loaded gelatin gels. Moreover, this simple method enabled the production of 10 g-scale or more microgels in one batch. We also demonstrated that multiple enzyme-loaded gelatin microgels functioned as cascade microreactors for lactose and glucose sensing. This versatile strategy enables the production of enzyme-loaded microgels while maintaining the enzyme activity using very low technologies. This result contributes to the easy preparation of enzyme-loaded microgels and their applications in the biomedical and green catalytic fields.

Photoassisted Surface Modification with Zwitterionic Phosphorylcholine Polymers for the Fabrication of Ideal Biointerfaces.

Surface modification using zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers is commonly performed to fabricate interfaces that reduce nonspecific fouling by biomolecules and cells. Accordingly, several clinically used devices, such as guide wires, stents, oxygenators, left ventricular assist devices, and microcatheters have been modified using MPC polymers. The specific types of surface modifications vary across substrates and applications. Recently, photoreactions have garnered attention for surface modification due to their stability and tunability. This review highlights various studies that employed photoreactions to modify surfaces using MPC polymers, especially photoinduced graft polymerization of MPC. In addition to antifouling materials, several micromanipulated, long-lasting hydrophilic, and super antiwear surfaces are summarized. Furthermore, several photoreactive MPC polymers that can be used to control interactions between biomolecules and materials are presented along with their potential to form selective recognition surfaces that target biomolecules for biosensors and diagnostic devices.

Gas-Phase Biosensors (Bio-Sniffers) for Measurement of 2-Nonenal, the Causative Volatile Molecule of Human Aging-Related Body Odor.

The molecule 2-nonenal is renowned as the origin of unpleasant human aging-related body odor that can potentially indicate age-related metabolic changes. Most 2-nonenal measurements rely on chromatographic analytical systems, which pose challenges in terms of daily usage and the ability to track changes in concentration over time. In this study, we have developed liquid- and gas-phase biosensors (bio-sniffers) with the aim of enabling facile and continuous measurement of trans-2-nonenal vapor. Initially, we compared two types of nicotinamide adenine dinucleotide (phosphate) [NAD(P)]-dependent enzymes that have the catalytic ability of trans-2-nonenal: aldehyde dehydrogenase (ALDH) and enone reductase 1 (ER1). The developed sensor quantified the trans-2-nonanal concentration by measuring fluorescence (excitation: 340 nm, emission: 490 nm) emitted from NAD(P)H that was generated or consumed by ALDH or ER1. The ALDH biosensor reacted to a variety of aldehydes including trans-2-nonenal, whereas the ER1 biosensor showed high selectivity. In contrast, the ALDH bio-sniffer showed quantitative characteristics for trans-2-nonenal vapor at a concentration range of 0.4-7.5 ppm (with a theoretical limit of detection (LOD) and limit of quantification (LOQ) of 0.23 and 0.26 ppm, respectively), including a reported concentration (0.85-4.35 ppm), whereas the ER1 bio-sniffer detected only 0.4 and 0.8 ppm. Based on these findings, headspace gas of skin-wiped alcohol-absorbed cotton collected from study participants in their 20s and 50s was measured by the ALDH bio-sniffer. Consequently, age-related differences in signals were observed, suggesting the potential for measuring trans-2-nonenal vapor.

pH-Responsive Association Behavior of Biocompatible Random Copolymers Containing Pendent Phosphorylcholine and Fatty Acid.

Well-defined pH-responsive biocompatible random copolymers composed of 2-(methacryloyloxy)ethyl phosphorylcholine and varying quantities of sodium 11-(acrylamido)undecanoate (AaU) (fAaU = 0-58 mol %) were synthesized via reversible addition-fragmentation chain transfer radical polymerization. The pH-responsive association and dissociation behavior of the random copolymers was studied via turbidity, 1H nuclear magnetic resonance relaxation time, dynamic light scattering, static light scattering (SLS), and fluorescence measurements. At basic pH levels, the random copolymers dissolved in water in a unimer state because the AaU units behaved in a hydrophilic manner as a result of the ionization of the pendent fatty acids. The random copolymers with fAaU < 52 mol % associated intramolecularly within a single polymer chain to form unimer micelles at pH 3 because of the protonation of the pendent fatty acids. On the other hand, the random copolymer with fAaU ≥ 52 mol % formed intermolecular aggregates composed of four polymer chains at pH 3, as established by the SLS measurements. The random copolymers displayed the ability to solubilize hydrophobic guest molecules, such as N-phenyl-1-naphthylamine, into the hydrophobic microdomain formed by the pendent dehydrated fatty acids at acidic pHs. At pH 4, 1-pyrememethanol is captured by the random copolymer with fAaU = 52 mol %, and it is released from the random copolymer above pH 9. Furthermore, the mucoadhesive properties of the random copolymer with fAaU = 9 mol % were studied using a surface plasmon resonance technique. The copolymer was adsorbed onto mucin at pH 3; however, the adsorption decreased at pH 7.4.

Nanofluidic Model Membrane for the Single-Molecule Observation of Membrane Proteins.

Membrane proteins play essential roles in the cell, and they constitute one of the most important targets of drugs. Studying membrane proteins in a controlled model membrane environment can provide unambiguous, quantitative information on their molecular properties and functions. However, reconstituting membrane proteins in a model system poses formidable technological challenges. Here, we developed a novel model membrane platform for highly sensitive observation of membrane proteins by combining a micropatterned lipid membrane and a nanofluidic channel. A micropatterned model membrane was generated by lithographically integrating a polymerized lipid bilayer and a natural (fluid) lipid bilayer. A nanofluidic channel having a defined thickness was formed between the fluid bilayer and a polydimethylsiloxane (PDMS) slab by attaching the polymeric bilayer and PDMS slab using an adhesion layer composed of silica nanoparticles that are coated with a biocompatible polymer brush. As we reconstituted rhodopsin (Rh), a G-protein-coupled receptor (GPCR), from a detergent-solubilized state into the fluid bilayer, only successfully reconstituted Rh molecules diffused laterally in the lipid bilayer and migrated into the nanogap junction, where they could be observed with a vastly improved signal-to-background ratio. The nanogap junction effectively separates the sites of reconstitution and observation and provides a novel platform for studying the molecular properties and functions of membrane proteins at the single-molecular level.

Control of Structural Coloration by Natural Sunlight Irradiation on a Melanin Precursor Polymer Inspired by Skin Tanning.

Natural melanin affects the reflection and absorption of light, and it is known as an important element in producing bright structural colors in nature. In this study, we prepared core-shell particles using a melanin precursor polymer, that is, polytyrosine (PTy), as a shell layer by the oxidative polymerization of tyrosine ethyl ester (Ty) in the presence of cerium oxide (CeO2) core particles. Inspired by skin tanning, irradiating the CeO2@PTy core-shell particles with UV or natural sunlight caused melanization by extending the π-conjugated length of PTy, producing colloidal particles with the ability to absorb light. The pellet samples consisting of CeO2@PTy particles appeared whitish because of multiple scattered light. In contrast, the light absorption capacity of CeO2@PTy UV or CeO2@PTy Sun particles after light irradiation suppressed scattered light, dramatically improving the visibility of the structural color of the pellet samples made from these particles. Thus, a new method has been developed to control the visualization of structural colors to the human eye by irradiating the melanin precursor polymer with light.

Controlled biointerfaces with biomimetic phosphorus-containing polymers.

Phosphorus is a ubiquitous and one of the most common elements found in living organisms. Almost all molecules containing phosphorus in our body exist as analogs of phosphate salts or phosphoesters. Their functions are versatile and important, being responsible for forming the genetic code, cell membrane, and mineral components of hard tissue. Several materials inspired from these phosphorus-containing biomolecules have been recently developed. These materials have shown unique properties at the biointerface, such as nonfouling ability, blood compatibility, lubricity, mineralization induction capability, and bone affinity. Several unfavorable events occur at the interface of materials and living organisms because most of these materials have not been designed while taking host responses into account. These unfavorable events are directly linked to reducing functions and shorten the usable periods of medical devices. Biomimetic phosphorus-containing polymers can improve the reliability of materials in biological systems. In addition, phosphorus-containing biomimetic polymers are useful not only for improving the biocompatibility of material surfaces but also for adding new functions due to the flexibility in molecular design. In this review, we describe the recent advances in the control of biointerfacial phenomena with phosphorus-containing polymers. We especially focus on zwitterioninc phosphorylcholine polymers and polyphosphoesters.

Biochemical Methanol Gas Sensor (MeOH Bio-Sniffer) for Non-Invasive Assessment of Intestinal Flora from Breath Methanol.

Methanol (MeOH) in exhaled breath has potential for non-invasive assessment of intestinal flora. In this study, we have developed a biochemical gas sensor (bio-sniffer) for MeOH in the gas phase using fluorometry and a cascade reaction with two enzymes, alcohol oxidase (AOD) and formaldehyde dehydrogenase (FALDH). In the cascade reaction, oxidation of MeOH was initially catalyzed by AOD to produce formaldehyde, and then this formaldehyde was successively oxidized via FALDH catalysis together with reduction of oxidized form of ß-nicotinamide adenine dinucleotide (NAD+). As a result of the cascade reaction, reduced form of NAD (NADH) was produced, and MeOH vapor was measured by detecting autofluorescence of NADH. In the development of the MeOH bio-sniffer, three conditions were optimized: selecting a suitable FALDH for better discrimination of MeOH from ethanol in the cascade reaction; buffer pH that maximizes the cascade reaction; and materials and methods to prevent leaking of NAD+ solution from an AOD-FALDH membrane. The dynamic range of the constructed MeOH bio-sniffer was 0.32-20 ppm, which encompassed the MeOH concentration in exhaled breath of healthy people. The measurement of exhaled breath of a healthy subject showed a similar sensorgram to the standard MeOH vapor. These results suggest that the MeOH bio-sniffer exploiting the cascade reaction will become a powerful tool for the non-invasive intestinal flora testing.

Ultra-Sensitive Isopropanol Biochemical Gas Sensor (Bio-Sniffer) for Monitoring of Human Volatiles.

Our groups have previously developed a biochemical gas sensor to measure isopropanol (IPA) in exhaled air and have applied it for breath IPA investigation in healthy subjects and diabetes patients. In this study, the original bio-sniffer was modified with a series of components that improved the limit of detection (LOD). First, the modified IPA bio-sniffer used a C8855-type photomultiplier tube (PMT) that performed well in the photon sensitivity at the peak wavelength of nicotinamide adenine dinucleotide (NADH) fluorescence. Second, the multi-core bifurcated optical fiber, which incorporated 36 fibers to replace the previous dual-core type, enhanced the fluorescence collection. Third, the optical fiber probe was reinforced for greater width, and the flow-cell was redesigned to increase the area of the enzyme-immobilized membrane in contact with the air sample. These modifications lowered the detection limit to 0.5 ppb, a significant increase over the previous 1.0 ppb. Moreover, the modified bio-sniffer successfully analyzed the IPA concentration in exhaled air from a volunteer, which confirmed its capability for real-world sample detection. The modified bio-sniffer is more applicable to breath measurement and the detection of other extremely-low-concentration samples.

Bone Mineral Affinity of Polyphosphodiesters.

Biomimetic molecular design is a promising approach for generating functional biomaterials such as cell membrane mimetic blood-compatible surfaces, mussel-inspired bioadhesives, and calcium phosphate cements for bone regeneration. Polyphosphoesters (PPEs) are candidate biomimetic polymer biomaterials that are of interest due to their biocompatibility, biodegradability, and structural similarity to nucleic acids. While studies on the synthesis of PPEs began in the 1970s, the scope of their use as biomaterials has increased in the last 20 years. One advantageous property of PPEs is their molecular diversity due to the presence of multivalent phosphorus in their backbones, which allows their physicochemical and biointerfacial properties to be easily controlled to produce the desired molecular platforms for functional biomaterials. Polyphosphodiesters (PPDEs) are analogs of PPEs that have recently attracted interest due to their strong affinity for biominerals. This review describes the fundamental properties of PPDEs and recent research in the field of macromolecular bone therapeutics.

Label-Free Specific Detection and Collection of C-Reactive Protein Using Zwitterionic Phosphorylcholine-Polymer-Protected Magnetic Nanoparticles.

In this study, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)]-protected Fe3O4 nanoparticles were prepared and used for the label-free specific detection and collection of an acute inflammation marker, C-reactive protein (CRP), in a simulated body fluid. The Fe3O4 nanoparticle surface was modified using poly(MPC) by surface-initiated atom-transfer radical polymerization. The density of poly(MPC) was 0.16 chains/nm2, and the colloidal stability of the nanoparticles in aqueous media and human plasma was effectively improved by surface modification. The size of the as-prepared poly(MPC)-protected Fe3O4 nanoparticles was ∼200 nm. After coming into contact with CRP, the nanoparticles aggregated as CRP comprises five subunits, and each subunit can bind to a phosphorylcholine group with two free Ca2+ ions. The change in the nanoparticle size exhibited a good correlation with the CRP concentration in the range of 0-600 nM. A low limit of detection of 10 nM for CRP was observed. The particles effectively reduced the adsorption of nonspecific proteins, and the change in the nanoparticle size with CRP was not affected by the coexistence of bovine serum albumin at a concentration 1000 times greater than that of CRP. Nanoparticle aggregates formed using CRP were dissociated using ethylenediamine- N, N, N', N'-tetraacetic acid, disodium salt, thereby regenerating poly(MPC)-protected Fe3O4 nanoparticles. In addition, CRP was collected from aqueous media using an acidic buffer solution and human plasma. CRP-containing aqueous solutions were treated with poly(MPC)-protected Fe3O4. After poly(MPC)-protected Fe3O4 nanoparticles were separated using a neodymium magnet and centrifugation, the concentration of CRP in the media dramatically decreased. In stark contrast, the concentration of albumin present in the test solution did not change even after treatment with the nanoparticles. Therefore, nanoparticles specifically recognize CRP from complex biological fluids. Although inhibition tests in the presence of 1,2-dioleoyl- sn-glycero-3-phosphocholine liposomes or free poly(MPC) were also carried out, the binding of poly(MPC)-protected Fe3O4 to CRP was not affected by these inhibitors. In conclusion, poly(MPC)-brush-bearing magnetic nanoparticles can serve not only as reliable materials for detecting and controlling the levels of CRP in simulated body fluids but also as diagnostic and therapeutic materials.

Surface Grafting Polyphosphoesters on Cellulose Nanocrystals To Improve the Emulsification Efficacy.

Particle-stabilized emulsion systems have been developed to address the problematic properties of conventional surfactants. However, the nature and properties of the fine particles used in such systems remain a critical issue for stability enhancement. Herein, we describe a thermoswitchable oil-in-water (O/W) particle-stabilized emulsion that exhibits improved stability due to the addition of cellulose nanocrystals (CNCs) modified with poly[2-isopropoxy-2-oxo-1,3,2-dioxaphospholane] (PIPP), which exhibits relatively good biocompatibility and biodegradability. Various parameters, such as surface activity, concentration of particles, polarity of solvents, and temperatures, on the formation of emulsions with CNCs grafted with PIPP (CNC-g-PIPP) were investigated. Results showed that the surface activity of CNC-g-PIPP was significantly improved compared with the unmodified material. Heptane-in-water particle-stabilized emulsions with CNC-g-PIPP were stably formed, and the effect of temperature on the stability of the emulsions was characterized. CNC-g-PIPP exhibited function as an effective particulate emulsifier at 4 °C because of the strong adsorption at the oil-water interface. However, the emulsions rapidly disintegrated at 45 °C, which is above the low critical solution temperature of PIPP on CNC, as the hydrophobized CNC-g-PIPP desorbed from the oil-water interface. Based on these findings, a thermally induced reversible emulsification/demulsification was presented. The resulting switchable particle-stabilized emulsion based on CNC-g-PIPP shows promise for the ability to control the stability of an emulsion in response to temperature, which is attractive for use in biological applications.

Surface Modification of Macrophages with Nucleic Acid Aptamers for Enhancing the Immune Response against Tumor Cells.

Antigen-presenting cells play a dominant role in cancer immunotherapy. Tumor cells, however, can still resort to several mechanisms of immune evasion that ultimately lead to the development of tumor tissues. In the current study, we performed surface modification of live macrophages with nucleic acid aptamers with the aim to enhance their affinity for tumor cells. Intercellular adhesion of tumor cells to surface-modified macrophages and the functions of the macrophages when in contact with tumor cells were investigated. To immobilize thiol-terminated nucleic acid aptamers that showed high affinity for the membrane protein of the tumor cells, methacryloyl groups were delivered into the sialic acids of the macrophages via metabolic glycoengineering (MGE). The proposed surface modification was cytocompatible and did not induce any undesirable activation of macrophages. According to the cell proliferation assay, the density of aptamers immobilized on a macrophage was found to decrease over time. However, the presence of aptamers on the cell surface was observed for more than 24 h after the immobilization. The number of adherent tumor cells on aptamer-immobilized macrophages was significantly larger than that of non-immobilized macrophages. Although the number of adherent tumor cells on aptamer-immobilized macrophages was not influenced by the pretreatment of doxorubicin to induce apoptosis in tumor cells, the apoptosis-induced tumor cells were highly phagocytosed by the aptamer-immobilized macrophages. The secretion amount of proinflammatory cytokines (TNF-α and IL-12) from the macrophages was coincident with the phagocytic index, which increased with the phagocytic uptake of tumor cells by the macrophages. In addition, the expression level of the major histocompatibility complex (MHC) class I and II molecules, required for antigen presentation, increased in nucleic acid aptamer-immobilized macrophages. Overall, the surface modification of macrophages with nucleic acid aptamers improved the tumor cell recognition of macrophages, indicating that the combination of cell surface engineering and anticancer drug treatment could constitute a promising strategy for tumor cell elimination.

Biochemical Gas Sensors (Biosniffers) Using Forward and Reverse Reactions of Secondary Alcohol Dehydrogenase for Breath Isopropanol and Acetone as Potential Volatile Biomarkers of Diabetes Mellitus.

This study describes two biosniffers to determine breath acetone and isopropanol (IPA) levels and applies them for breath measurement in healthy subjects and diabetic patients. Secondary alcohol dehydrogenase (S-ADH) can reduce acetone and oxidize nicotinamide adenine dinucleotide (NADH to NAD+) in a weak acid environment. NADH can be excited by 340 nm excitation lights and subsequently emit 490 nm fluorescence. Therefore, acetone can be measured by the decrease in NADH fluorescence intensity. S-ADH can also oxidize IPA and reduce NAD+ to NADH when it is in an alkaline environment. Thus, IPA can be detected by the increase of fluorescence. The developed biosniffers show rapid response, high sensitivity and high selectivity. The breath acetone and IPA analysis in healthy subjects shows that the mean values were 750.0 ± 434.4 ppb and 15.4 ± 11.3 ppb. Both acetone and IPA did not show a statistical difference among different genders and ages. The breath acetone analysis for diabetic patients shows a mean value of 1207.7 ± 689.5 ppb, which was higher than that of healthy subjects (p < 1 × 10-6). In particularly, type-1 diabetic (T1D) patients exhaled a much higher concentration of acetone than type-2 diabetic (T2D) patients (p < 0.01). The breath IPA also had a higher concentration in diabetic patients (23.1 ± 20.1 ppb, p < 0.01), but only T2D patients presented a statistical difference (23.9 ± 21.3 ppb, p < 0.01). These findings are worthwhile in the study of breath biomarkers for diabetes mellitus diagnosis. Additionally, the developed biosniffers provide a new technique for volatolomics research.

Hybrid Model Membrane Combining Micropatterned Lipid Bilayer and Hydrophilic Polymer Brush.

Substrate-supported planar lipid bilayers (SPBs) are being utilized as a versatile model system of the biological membrane. However, the proximity between the solid support and membrane limits utility of SPBs for the functional analyses of membrane proteins. Here, we present a model membrane that can enlarge the distance between the substrate surface and the membrane by combining a stable scaffold of polymerized lipid bilayer with a hydrophilic polymer brush. A micropatterned SPB was generated by the lithographic polymerization of diacetylene lipids and subsequent incorporation of natural (fluid) lipid bilayers. Hydrophilic polymer brush of poly-2-methacryloyloxyethyl phosphorylcholine (poly(MPC)) was formed on the surface of polymeric bilayer by the in situ atom transfer radical polymerization (ATRP) in aqueous solution, in the presence of embedded fluid lipid bilayers. A model membrane protein (Haloquadratum walsbyi bacteriorhodopsin: HwBR) could be reconstituted into the polymer brush-supported bilayers with significantly reduced immobile molecules. Furthermore, the polymer brush terminals could be functionalized by successively polymerizing MPC and 2-aminoethyl methacrylate (AMA). The reactive amine moiety of poly(AMA) enables to conjugate a wide range of biological molecules and surfaces to the membrane. The combination of micropatterned bilayer and polymer brush mimics the two- and three-dimensional structures of the biological membrane, providing a platform to assay membrane proteins in a truly biomimetic environment.

Antimicrobial Silver Nanoclusters Bearing Biocompatible Phosphorylcholine-Based Zwitterionic Protection.

Infection is one of the most serious issues in medical treatments leading to the development of several antimicrobial agents. In particular, silver ions released from silver substrates is well-known as a reliable antimicrobial agent that either kills the microorganisms or inhibits their growth. Unfortunately, many reports have shown that silver-based antimicrobial agents are toxic for human cells as well. To improve the biocompatibility of silver antimicrobial agents, we have synthesized thiol-terminated phosphorylcholine (PC-SH)-protected silver nanoclusters (PC-AgNCs) via strong thiol-metal coordination with controlled ultrasmall size of the clusters. A change in plasmon-like optical absorption was studied to affirm the successful synthesis of small thiolated AgNCs through the absorption spectra that become molecular-like for the AgNCs. We observed that PC-AgNCs were spherical with an average diameter of <2 nm. The ultrasmall size clusters were exceedingly immobilized by the PC-SH on the surface, resulting in excellent biocompatibility and antibacterial activity simultaneously. The biocompatible, antimicrobial PC-AgNCs exhibit interesting advantages compared with other silver antimicrobial agents for medical applications.

Clickable and Antifouling Platform of Poly[(propargyl methacrylate)-ran-(2-methacryloyloxyethyl phosphorylcholine)] for Biosensing Applications.

A functional copolymer platform, namely, poly[(propargyl methacrylate)-ran-(2-methacryloyloxyethyl phosphorylcholine)] (PPgMAMPC), was synthesized by reversible addition-fragmentation chain-transfer polymerization. In principle, the alkyne moiety of propargyl methacrylate (PgMA) should serve as an active site for binding azide-containing molecules via a click reaction, i.e., Cu-catalyzed azide/alkyne cycloaddition (CuAAC), and 2-methacryloyloxyethyl phosphorylcholine (MPC), the hydrophilic monomeric unit, should enable the copolymer to suppress nonspecific adsorption. The copolymers were characterized using Fourier transform infrared (FTIR) and (1)H NMR spectroscopies. Thiol-terminated, PPgMAMPC-SH, obtained by aminolysis of PPgMAMPC, was immobilized on a gold-coated substrate using a "grafting to" approach via self-assembly. Azide-containing species, namely, biotin and peptide nucleic acid (PNA), were then immobilized on the alkyne-containing copolymeric platform via CuAAC. The potential use of surface-attached PPgMAMPC in biosensing applications was shown by detection of specific target molecules, i.e., streptavidin (SA) and DNA, by the developed sensing platform using a surface plasmon resonance technique. The copolymer composition strongly influenced the performance of the developed sensing platform in terms of signal-to-noise ratio in the case of the biotin-SA system and hybridization efficiency and mismatch discrimination for the PNA-DNA system.

Low-Temperature Processable Block Copolymers That Preserve the Function of Blended Proteins.

Low-temperature processable polymers have attracted increasing interest as ecological materials because of their reduced energy consumption during processing and suitability for making composites with heat-sensitive biomolecules at ambient temperature. In the current study, low-temperature processable biodegradable block copolymers were synthesized by ring-opening polymerization of l-lactide (LLA) using polyphosphoester as a macroinitiator. The polymer films could be processed under a hydraulic pressure of 35 MPa. The block copolymer films swelled in water because the polyphosphoester block was partially hydrated. Interestingly, the swelling ratio of the films changed with temperature. The pressure-induced order-to-disorder transition of the block copolymers was characterized by small-angle X-ray scattering; a crystallinity reduction in the block copolymers was observed after application of pressure. The crystallinity of the block copolymers was recovered after removing the applied pressure. The Young's modulus of the block copolymer films increased as the LLA unit content increased. Moreover, the modulus did not change after multiple processing cycles and the recyclability of the block copolymers was also confirmed. Finally, polymer films with embedded proteinase K as a model protein were prepared. The activity of catalase loaded into the polymer films was evaluated after processing at different temperatures. The activity of catalase was preserved when the polymer films were processed at room temperature but was significantly reduced after high-temperature processing. The suitability of low-temperature processable biodegradable polymers for making biofunctional composites without reducing protein activity was clarified. These materials will be useful for biomedical and therapeutic applications.

Inhibition of matrix metalloproteinases and toxicity of gold and platinum nanoparticles in L929 fibroblast cells.

This study evaluated the inhibition of matrix metalloproteases (MMPs) and cellular responses elicited by gold (Au) and platinum (Pt) nanoparticles (NPs). The interaction of MMP-1 and NPs was evaluated using an MMP assay kit. The cultured L929 cells were exposed to various concentrations of NPs. The cellular responses to NPs were examined using a cytotoxicity assay (that evaluated cell viability and lactic dehydrogenase production), real-time polymerase chain reaction (RT-qPCR), and transmission electron microscopy. Both types of NPs, when used at concentrations above 10 µg ml(-1), inhibited MMP-1 activity. No cytotoxic effects were found when the cells were exposed to AuNPs. In contrast, PtNPs, at both 100 and 400 µg ml(-1), induced cytotoxicity. No inflammatory responses (production of interleukin-6 and tumor necrosis factor-alpha) to NPs were identified by RT-qPCR. The negative surface charge of NPs (COOH(-)) binds to the Zn(2+) of the MMP active center by chelation, leading to MMP inhibition. Gold nanoparticles are plausible candidates for MMP inhibitors in resin-bonding materials because they effectively inhibit MMP-1 activity without cytotoxic or inflammatory effects.

Crosslinked duplex DNA nanogels that target specified proteins.

Specific detection of protein biomarkers plays an important role in diagnostics and therapeutics. We have fabricated polymeric nanogels, which can specifically interact with the cancer biomarker thrombin to serve as a model. Two types of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers bearing a thrombin-binding oligonucleotide aptamer and its complementary chain were independently synthesized by redox-initiated radical polymerization. These MPC polymers associate in a complimentary fashion due to double strand formation of the oligonucleotides in aqueous media, leading to the spontaneous formation of spherical nanogels. Nanogel formation was confirmed by dynamic light scattering (DLS) and transmittance microscopy. The average size of nanogel particles was 124 ± 2 nm and the nanogels were mono-dispersed (polydispersity index 0.21). Functional intercalators could be stably incorporated into nanogels through the physical interaction between the intercalators and the oligonucleotides. The ethidium bromide (EtBr)-incorporating nanogels were used as detectors for thrombin. The fluorescence intensity of solutions containing the EtBr-incorporating nanogels was decreased with an increase in the concentration of thrombin. The transformation of quadruplex-thrombin structure from complementary double-stranded structures resulted in the decrease in fluorescence intensity. In contrast, the intensity did not change when the nanogels were incubated with albumin. Thrombin is only one such model used to demonstrate this technique; oligonucleotide aptamers can be freely designed to interact with versatile bio-substances. Therefore, aptamer-crosslinked nanogels can be appropriate nanomaterials for disease diagnosis and therapy.