Phosphorylcholine Groups Research Articles

Intermolecular Interaction of Polymer Brushes Containing Phosphorylcholine and Inverse-Phosphorylcholine.

Choline phosphate (CP) is a phosphobetaine-type zwitterionic functional group, referred to as inverse phosphorylcholine (PC) due to the reverse orientation of a positively charged quaternary amine and anionic phosphate in contrast to PC lipids in nature. The A unique dipole paring between CP and PC groups has attracted much attention in the biointerface research field. Herein, to evaluate the molecular interaction between the CP and PC groups in water, force-distance curve measurements using scanning probe microscopy (SPM) with a PC-group-functionalized cantilever was carried out on the surface of polymer brushes bearing the CP groups. Three types of methacrylate monomers bearing CP with ethyl (Et), methoxyethyl (MOE), and isopropyl (iPr) phosphates were synthesized in 42-71% yields, and polymerized by surface-initiated atom transfer radical polymerization to form polymer brushes on silicon wafers. The surface free energy of CP-polymer brushes with Et, MOE, and iPr was estimated to be 64.0, 61.4, and 57.4 mN m-1, respectively, based on contact angle measurements. Force-distance curve measurements of polymer brushes having a CP group was conducted in water at 25 °C by SPM using a spherical probe produced by attaching a silica particle (SiP; d = 25 μm) covered with PC or CP groups to a tipless cantilever. Adhesion force larger than 14 nN was observed between the CP-polymer brushes and PC-SiP, whereas PC-polymer brushes revealed extremely low adhesion force of less than 0.6 nN with PC-SiP and propylsilane-modified SiP. The specific attractive molecular interaction between CP and PC groups was quantitatively evaluated.

Blood-Compatible Surfaces with Phosphorylcholine-Based Polymers for Cardiovascular Medical Devices.

For the acquisition of blood-compatible materials, various hydrophilic polymers for surface modification have been examined. Among them, polymers with a representative phospholipid polar group, the phosphorylcholine (PC) group, are a successful example. These polymers were designed from inspiration of the cell membrane surface and provide protein adsorption resistance even following contact with plasma. This important property is based on the unique hydration state of water molecules surrounding hydrated polymer; in other words, water molecules weakly interact with the polymers and maintain their favorable cluster structure through hydrogen bonding. These polymers are not only hydrophilic, but also electrically neutral, important characteristics which make hydrogen bonding with water molecules less likely to occur and avoid hydrophobic interactions. Phosphorylcholine groups and other zwitterionic structures are significant as hydrophilic functional groups meeting these important requirements. In this review, blood compatibility of a polymer having a PC group is introduced in relation to its hydration structure, followed by a description of the applications of this polymer to cardiovascular medical devices.

Photoinduced Surface Zwitterionization for Antifouling of Porous Polymer Substrates

Surface functionalization of polymeric porous substrates is one of the most important requirements to enhance their applications in the biomedical field. In this study, we achieved photoinduced surface modification using a highly efficient reaction of hydrophilic polymers bearing phosphorylcholine groups. Polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units and 2-( N-ethylanilino)ethyl methacrylate units were synthesized with attention to the polymer architectures. The surface modification of the porous polyethylene (PE) substrates was carried out by the coating of the MPC polymers with a photochemical radical generator, followed by photoirradiation for a few minutes. Surface analysis by attenuated total reflectance Fourier transform IR spectroscopy and X-ray photoelectron spectroscopy indicated that the MPC polymer layer was generated on the PE surface. Cross-sectional confocal microscopy images showed that the MPC polymers were coated on the polymer surface, even inside the porous structure of the PE substrate. After modification, the porous PE substrates showed a significant increase in hydrophilicity and the water-penetration rate through the pores. Furthermore, the amount of protein adsorbed on the PE substrate was reduced significantly by the surface modification. These functionalities were dependent on the MPC polymer architectures. Thus, we concluded that the photoreactive polymer system developed furnished the porous substrates with antifouling properties.

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.

Smart functional polymer coatings for paper with anti-fouling properties.

Cellulose, as the main component of paper, is becoming more and more important for several high tech applications because of its beneficial properties, such as abundance, low cost, renewability, mechanical robustness and biocompatibility. To make cellulose accessable for new applications it is necessary to introduce new properties, which can be done by surface modification e.g. grafting of polymers onto surfaces. In this work, two comb copolymers, poly[(2-methyl-2-oxazoline methacrylate)-co-glycidyl methacrylate] and poly[(2-methacryloyloxyethyl phosphorylcholine)-co-glycidyl methacrylate], were synthesized by free radical polymerization of glycidyl methacrylate and oligo(2-methyl-2-oxazoline) as well as 2-methacryloyloxyethyl phosphorylcholine. After extensive characterization the polymers were covalently attached to thin cellulose model layers and filter paper using a one-step grafting-to approach. For the comprehensive analysis of these layers, thin cellulose films were fabricated on silicon wafers by spin coating of trimethylsilyl cellulose followed by acid hydrolysis which resulted in homogeneous layers as substrates for the grafting process of the functional polymers. The layers were characterized by X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM) and energy-dispersive X-ray spectroscopy (EDX). To demonstrate the high potential of such polymer-modified cellulose materials, protein repellance of the cellulose films, containing peptidomimetic 2-methyl-2-oxazoline and zwitterionic phosphorylcholine groups after successful functionalization, is shown. Cell adhesion experiments using Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae indicate the considerable anti-fouling capacity against both Gram-positive and Gram-negatve bacteria as well as the yeast fungus.

A phospholipid polymer graft layer affords high resistance for wear and oxidation under load bearing conditions.

Manipulating the surface and substrate of cross-linked polyethylene (CLPE) is an essential approach for obtaining life-long orthopedic bearings. We therefore proposed a bearing material comprised of an antioxidative substrate generated by vitamin E blending (HD-CLPE[VE]) with a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted surface, and investigated its wear resistance and oxidative stability under accelerated aging and load bearing conditions. Neither the hydration nor friction kinetics of the molecular network structure of the PMPC-grafted surface or the HD-CLPE(VE) substrate were influenced by accelerated aging but rather exhibited high stability even under high oxidation conditions. The characteristics of the PMPC-grafted surface improved the wear and impact fatigue resistance of the HD-CLPE(VE) liner regardless of accelerated aging. Notably, the PMPC-grafted surface was found to affect the potential oxidative stability at the rim part of the acetabular liner. PMPC chains serve several important functions on the surface regardless of load bearing, such as high lubricity or low lipophilicity attributed to phosphorylcholine groups and/or surrounding water-fluid film, and suppression of lipid diffusion attributed to methacrylate main chains on the surface. Together, these results provide preliminary evidence that the PMPC graft layer and vitamin E-blended substrate might positively affect the extent of orthopedic implant durability.

Critical Study of the Recognition between C-Reactive Protein and Surface-Immobilized Phosphorylcholine by Quartz Crystal Microbalance with Dissipation.

C-reactive protein (CRP), a biomarker for cardiovascular disease, has been reported to have a strong affinity to zwitterionic phosphorylcholine (PC) groups in the presence of calcium ions. In addition, PC-immobilized surfaces have been used as a nonfouling coating to prevent nonspecific protein binding. By appropriately using the features of PC-immobilized surfaces, including specific recognition to CRP and nonfouling surface, it is reasonable to create an antibody-free biosensor for the specific capture of CRP. In this study, PC-functionalized 3,4-ethylenedioxythiophene (EDOT) monomers were used to prepare PC-immobilized surfaces. The density of PC groups on the surface can be fine-tuned by changing the composition of the monomer solutions for the electropolymerization. The density of PC group was confirmed by X-ray photoelectron spectroscopy (XPS). The specific interaction of CRP with PC groups was monitored by using a quartz crystal microbalance with dissipation (QCM-D). The amount of protein binding could be estimated by the reduction in frequency readout. Through the QCM-D measurement, we revealed the nonfouling property and the specific CRP capture from our PC-immobilized surfaces. Notably, the dissipation energy also dropped during the binding process between CRP and PC, indicating the release of water molecules from the PC groups during CRP adsorption. We anticipate that surface-bound water molecules are mainly released from areas near the immobilized PC groups. Based on Hofmeister series, we further examined the influence of ions by introducing four different anions including both kosmotrope (order maker) and chaotrope (disorder maker) into the buffer for the CRP binding test. The results showed that the concentration and the type of anions play an important role in CRP binding. The present fundamental study reveals deep insights into the recognition between CRP and surface-immobilized PC groups, which can facilitate the development of CRP sensing platforms.

Fabrication of an Anti-Biofouling Plasma-Filtration Membrane by an Electrospinning Process Using Photo-Cross-linkable Zwitterionic Phospholipid Polymers.

The goal of this study is to fabricate a stable plasma filtration membrane with antibiofouling properties via an electrospinning process. To this end, a random-type copolymer consisting of zwitterionic phosphorylcholine (PC) groups and ultraviolet (UV)-cross-linkable phenyl azide groups was synthesized. The zwitterionic PC group provides antibiofouling properties, and the phenyl azide group enables the stable maintenance of the fibrous nanostructure of hydrophilic zwitterion polymers in aqueous medium via a simple UV curing process. To demonstrate the antibiofouling nature of the PC group, a polymer without antibiofouling PC groups was also prepared for comparison. The successful synthesis of the random-type copolymers containing phenyl azide groups was proven by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy, and the fibrous structure of the prepared membranes was observed by field emission scanning electron microscopy. The antibiofouling properties were analyzed by fluorescein isothiocyanate-labeled bovine serum albumin adsorption and platelet adhesion tests. The experimental results show that membranes containing zwitterionic PC groups exhibited obvious decreases in platelet adhesion and protein adsorption. Platelet-rich plasma solution was filtered using the prepared membranes to test their filtration properties. The sequential filtration process removed 80% and almost 98% of the platelets. This finding confirmed that the membrane retained its blood-inert biomaterial surface in a complex medium that included blood plasma and platelets.

Complexes Covered with Phosphorylcholine Groups Prepared by Mixing Anionic Diblock Copolymers and Cationic Surfactants.

Anionic diblock copolymers (PmAn) composed of biocompatible polybetaine, poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC), and anionic poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PAMPS) were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Two types of diblock copolymers (P24A217 and P100A99) were prepared with different compositions. The PmAn/CTAB complexes were formed by a stoichiometrically charge-neutralized mixture of anionic PmAn and cationic cetyltrimethylammonium bromide (CTAB) micelles in water. The complexes prepared using P24A217 and P100A99 were vesicles and micelles, respectively, and were covered with hydrophilic PMPC shells. The complexes dissociated upon addition of NaCl because the complex was maintained through electrostatic interactions. The P24A217/CTAB vesicles could encapsulate uncharged hydrophilic guest molecules into the interior of the aqueous phase.

Preparation and antifouling properties of 2-(meth-acryloyloxy)ethyl cholinephosphate based polymers modified surface with different molecular architectures by ATRP

Choline phosphate (CP) containing polymers modified surfaces have been shown good resist to the adhesion of proteins while prompt the attaching of mammalian cells due to the dipole pairing between the CP groups of the polymer and the phosphorylcholine (PC) groups on the cell membrane. However, the antifouling activities of CP modified surface against microbes have not been investigated at present. In addition, CP containing polymers modified surface with different molecular architectures has not been prepared and studied. To this end, glass slides surface modified with two different 2-(meth-acryloyloxy)ethyl cholinephosphate (MCP) containing polymer (PMCP) structures, i.e. brush-like (Glass-PMCP) and bottle brush-like (Glass-PHEMA-g-PMCP) architectures, were prepared in this work by surface-initiated atom transfer radical polymerization (SI-ATRP). The surface physichemical and antifouling properties of the prepared surfaces were characterized and studied. The Glass-PMCP shows improved antifouling properties against proteins and bacteria as compared to pristine glass slides (Glass-OH) and glass slides grafted with poly(2-hydroxyethyl methacrylate) (Glass-PHEMA). Notably, a synergetic fouling resistant properties of PHEMA and PMCP is presented for Glass-PHEMA-g-PMCP, which shows superior antifouling activities over Glass-PHEMA and Glass-PMCP. Furthermore, glass slides containing PMCP, i.e. Glass-PMCP and Glas-PHEMA-g-PMCP, decrease platelet adhesion and prevent their activation significantly. Therefore, the combination of antifouling PHEMA and PMCP into one system holds potential for prevention of bacterial fouling and biomaterial-centered infections.

Structurally Stable Attractive Nanoscale Emulsions with Dipole-Dipole Interaction-Driven Interdrop Percolation.

This study introduces an extremely stable attractive nanoscale emulsion fluid, in which the amphiphilic block copolymer, poly(ethylene oxide)-block-poly(ϵ-caprolactone) (PEO-b-PCL), is tightly packed with lecithin, thereby forming a mechanically robust thin-film at the oil-water interface. The molecular association of PEO-b-PCL with lecithin is critical for formation of a tighter and denser molecular assembly at the interface, which is systematically confirmed by T2 relaxation and DSC analyses. Moreover, suspension rheology studies also reflect the interdroplet attractions over a wide volume fraction range of the dispersed oil phase; this results in a percolated network of stable drops that exhibit no signs of coalescence or phase separation. This unique rheological behavior is attributed to the dipolar interaction between the phosphorylcholine groups of lecithin and the methoxy end groups of PEO-b-PCL. Finally, the nanoemulsion system significantly enhances transdermal delivery efficiency due to its favorable attraction to the skin, as well as high diffusivity of the nanoscale emulsion drops.

ホスホリルコリン基含有細胞非接着膜に及ぼす電界の影響の検討

ホスホリルコリン基含有細胞非接着膜に及ぼす電界の影響の検討

ホスホリルコリン基含有セグメント化ポリウレタンの合成と誘電緩和挙動

ホスホリルコリン基含有セグメント化ポリウレタンの合成と誘電緩和挙動

Zwitterionic phosphorylcholine grafted chitosan nanofiber: Preparation, characterization and in-vitro cell adhesion behavior

In this study, zwitterionic phosphorylcholine grafted electrospun chitosan fiber was accomplished in three steps: (1) Azide groups on the chitosan were regioselectively replaced with hydroxyl side group and then the product was electrospun. (2) Chitosan based macroinitiator was prepared using an azide-alkyne click reaction from azide-functionalized electrospun chitosan fiber. (3) Poly(2-methacryloyloxyethyl phosphorylcholine) (MPC) was grafted onto the electrospun chitosan fiber by atom transfer radical polymerization (ATRP) in order to enhance cellular viability and proliferation of 3T3, ECV and Saos. The structure of surface modified chitosan was characterized by Fourier transform infrared spectrometer (FT-IR) and 1H nuclear magnetic resonance (1H NMR). The surface morphology of the nanofibers was investigated by scanning electron microscope (SEM). In-vitro cellular attachment and spreading experiments of 3T3, ECV304 and Saos were performed on electrospun chitosan fibers in the presence and the absence of MPC grafting. Poly(MPC) grafted electrospun fiber showed an excellent performance due to phosphorylcholine groups mimicking the natural phospholipid.

Hydrophilic interaction chromatography with Fourier transform mass spectrometry of monosaccharide anhydrides.

A fast, selective, and sensitive method for the determination of three monosaccharide anhydrides (galactosan, mannosan, levoglucosan), based on hydrophilic interaction chromatography and Fourier transform mass spectrometry, was successfully developed. The simple experimental stationary phase and mass spectrometry performance screening allowed the selection of the best available chromatographic and mass spectrometry conditions. Thus, the chromatographic separation was performed on a highly selective stationary phase containing a zwitterionic phosphorylcholine group and the monosaccharide anhydrides were detected as [M+HCOO]- adduct in the negative mode. The method showed accuracy in the range of 84-111 and 89-102% with interbatch precision expressed as relative standard deviations of 5.6-15.4 and 5.0-9.0% for the aerosol extract and snow samples, respectively. The limit of quantification in absolute values ranged from 10 to 30 pg, the limit of quantification, expressed as concentration, ranged was 0.3-0.9 ng/m3 for aerosol and 10-20 ng/mL for snow samples. The method was successfully applied for the determination of monosaccharide anhydrides in aerosol and snow samples.

Calcium-independent binding of human C-reactive protein to lysophosphatidylcholine in supported planar phospholipid monolayers.

C-reactive protein (CRP), a major acute-phase pentraxin, binds to plasma membranes through the multivalent contacts with zwitterionic phosphorylcholine groups for activating classical complement systems. However, the interaction of CRP with phosphorylcholine-based biomaterials is unknown due to the lack of our understanding on the activation mechanism of CRP in altered local microenvironments. This paper reports the novel calcium-independent interaction of CRP to bioactive phospholipid lysophosphatidylcholine (LPC) in supported phospholipids monolayers as determined using SPR. Binding inhibition experiments indicate exposure of glycerophosphate moiety of LPC is responsible for the calcium-free interaction. Our study may explode the established concept that CRP requires calcium for binding to LPC on damaged cell membranes or biomaterials.

Fabrication of hydrophilic graphene film by molecular functionalization

We demonstrated the fabrication of a hydrophilic graphene film by functionalization using a phosphorylcholine group. Since the phosphorylcholine group retains water molecules, it is expected that a hydrophilic graphene film can be realized from the initial hydrophobic characteristic. To evaluate the molecular functionalization, the contact angle and four‐terminal resistivity were measured after every 1 min of phosphorylcholine group functionalization. The contact angle showed a sudden decrease of approximately 20° after 3 min. On the other hand, the resistivity gradually increased in accordance with the Langmuir adsorption isotherm. These results indicate that the phosphorylcholine group successfully modified the surface of graphene, and a hydrophilic graphene film was obtained without significant degradation of the electrical characteristics.Time dependence of the net contact angle change and resistivity change. The insets show the photographs of the contact angle measurement for the initial hydrophobic film and for the hydrophilic film obtained by functionalization.

Chitosan derivatives for gene transfer: effect of phosphorylcholine and diethylaminoethyl grafts on the in vitro transfection efficiency

The purpose of this work was to improve the functional properties of chitosan for gene transfer by inserting phosphorylcholine (PC) and diethylaminoethyl (DEAE) groups into the main chain. A series of derivatives containing increasing contents of DEAE and a fixed content of PC groups were synthesized and characterized, aiming to evaluate the effect of these groups on the nanoparticles’ properties and the in vitro transfection efficiency. The derivatives were soluble at physiological pH levels and all derivatives were less cytotoxic than the control, the lipid lipofectamine. The obtained derivatives complexed pDNA into nanoparticles with smaller sizes and higher zeta potentials than plain chitosan. The in vitro transfection was performed with nanoparticles prepared at pH 6.3 and 7.4 and the results showed that nanoparticles prepared with derivatives containing 20% of PC groups (PC18-CH) and high degrees of substitution by DEAE (PC20-CH-DEAE100, CH-DEAE80, CH-DEAE100) displayed the better transfection efficiencies in HeLa cells, reaching relative values comparable to lipofectamine. The most effective derivative, PC18CH, was selected for complexation with siRNA and its complexes demonstrated an in vitro knockdown efficiency highly dependent on the N/P ratio. Our combined results indicated that, by means of controlled modifications, the limitations of chitosan can be overcome to obtain more effective carriers based on chitosan, and the derivatives here studied hold potential for in vivo studies.

Effect of Salt on Phosphorylcholine-based Zwitterionic Polymer Brushes.

A quantitative investigation of the responses of surface-grown biocompatible brushes of poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) to different types of salt has been carried out using ellipsometry, quartz crystal microbalance (QCM) measurements, and friction force microscopy. Both cations and anions of varying valency over a wide range of concentrations were examined. Ellipsometry shows that the height of the brushes is largely independent of the ionic strength, confirming that the degree of swelling of the polymer is independent of the ionic character of the medium. In contrast, QCM measurements reveal significant changes in mass and dissipation to the PMPC brush layer, suggesting that ions bind to phosphorylcholine (PC) groups in PMPC molecules, which results in changes in the stiffness of the brush layer, and the binding affinity varies with salt type. Nanotribological measurements made using friction force microscopy show that the coefficient of friction decreases with increasing ionic strength for a variety of salts, supporting the conclusion drawn from QCM measurements. It is proposed that the binding of ions to the PMPC molecules does not change their hydration state, and hence the height of the surface-grown polymeric brushes. However, the balance of the intra- and intermolecular interactions is strongly dependent upon the ionic character of the medium between the hydrated chains, modulating the interactions between the zwitterionic PC pendant groups and, consequently, the stiffness of the PMPC molecules in the brush layer.

Novel self-assembled pH-responsive biomimetic nanocarriers for drug delivery

Novel pH-responsive biodegradable biomimetic nanocarriers were prepared by the self-assembly of N-acetyl-l-histidine-phosphorylcholine-chitosan conjugate (NAcHis-PCCs), which was synthesized via Atherton-Todd reaction to couple biomembrane-like phosphorylcholine (PC) groups, and N,N′-carbonyldiimidazole (CDI) coupling reaction to link pH-responsive N-acetyl-l-histidine (NAcHis) moieties to chitosan. In vitro biological assay revealed that NAcHis-PCCs nanoparticles had excellent biocompatibility to avoid adverse biological response mainly owing to their biomimetic PC shell, and DLS results confirmed their pH-responsive behavior in acidic aqueous solution (pH≤6.0). Quercetin (QUE), an anti-inflammatory, antioxidant and potential anti-tumor hydrophobic drug, was effectively loaded in NAcHis-PCCs nanocarriers and showed a pH-triggered release behavior with the enhanced QUE release at acidic pH5.5 compared to neutral pH7.4. The results indicated that pH-responsive biomimetic NAcHis-PCCs nanocarriers might have great potential for site-specific delivery to pathological acidic microenvironment avoiding unfavorable biological response.