In Situ Electrical Monitoring of Methylated DNA Based on Its Conformational Change to G-Quadruplex Using a Solution-Gated Field-Effect Transistor.

Methylated DNA is not only a diagnostic but also a prognostic biomarker for early-stage cancer. However, sodium bisulfite sequencing as a "gold standard" method for detection of methylation markers has some drawbacks such as its time-consuming and labor-intensive procedures. Therefore, simple and reliable methods are required to analyze DNA sequences with or without methylated residues. Herein, we propose a simple and direct method for detecting DNA methylation through its conformation transition to G-quadruplex using a solution-gated field-effect transistor (SG-FET) without using labeled materials. The BCL-2 gene, which is involved in the development of various human tumors, contains G-rich segments and undergoes a conformational change to G-quadruplex depending on the K+ concentration. Stacked G-quadruplex strands move close to the SG-FET sensor surface, resulting in large electrical signals based on intrinsic molecular charges. In addition, a dense hydrophilic polymer brush is grafted using surface-initiated atom transfer radical polymerization onto the SG-FET sensor surface to reduce electrical noise based on nonspecific adsorption of interfering species. In particular, control of the polymer brush thickness induces electrical signals based on DNA molecular charges in the diffusion layer, according to the Debye length limit. A platform based on the SG-FET sensor with a well-defined polymer brush is suitable for in situ monitoring of methylated DNA and realizes a point-of-care device with a high signal-to-noise ratio and without the requirement for additional processes such as bisulfite conversion and polymerase chain reaction.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands.

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

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.

Inhibition of denture plaque deposition on complete dentures by 2-methacryloyloxyethyl phosphorylcholine polymer coating: A clinical study.

STATEMENT OF PROBLEM: Denture plaque-associated infections are regarded as a source of serious dental and medical complications in the elderly population. Methods of managing this problem are needed. PURPOSE: The purpose of this clinical study was to evaluate the effects of treatment with a 2-methacryloyloxyethyl phosphorylcholine polymer, PMBPAz, on plaque deposition in complete dentures. MATERIAL AND METHODS: The study protocol was approved by the Ethics Committee of Showa University (#2013-013). Eleven individuals with maxillary complete dentures participated in this study. Their dentures were treated with PMBPAz, and the amount of denture plaque accumulation was evaluated by staining the denture surfaces with methylene blue after 2 weeks of denture usage. The same procedures were repeated to evaluate the original denture surfaces as a control. The image of the stained denture surface was captured using a digital camera, and the percentage of stained area, quantified as a pixel-based density, of the whole denture area (percentage of plaque index) was calculated for the mucosal and polished surfaces. To quantify the biofilm on the dentures, denture plaque biofilm was detached by ultrasonic vibration, resuspended in diluent, and measured with a microplate reader at an optical density of 620 nm. The effects of PMBPAz treatment on these variables were statistically analyzed with ANOVA (α=.05). RESULTS: The mean ±SD percentage of plaque index was 40.7% ±19.9% on the mucosal surfaces and 28.0% ±16.8% on the polished surfaces of the control denture. The mean percentage of plaque index of PMBPAz-treated dentures significantly decreased to 17.4%% ±12.0% on the mucosal surfaces (P<.001) and 15.0% ±9.9% on the polished surfaces (P<.05). The quantification of plaque deposition agreed with the results of these image analyses. CONCLUSIONS: These results demonstrated the effectiveness of the treatment with the PMBPAz to inhibit the bacterial plaque deposition on complete dentures.

Movement of a Quantum Dot Covered with Cytocompatible and pH-Responsible Phospholipid Polymer Chains under a Cellular Environment.

Quantum dots (QDs) were functionalized with well-defined polymer chains having both cytocompatibility and pH-responsiveness to monitor the movement of nanoparticles in a cellular environment with changing local pH. We used a triblock-type water-soluble polymer composed of three segments: (1) a pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate; DEAEMA] segment, (2) a poly[ω-(p-nitrophenyloxycarbonyl oligo(ethylene glycol)) methacrylate; MEONP] segment bearing an active ester group to react with an amino compound, and (3) a cytocompatible poly[(2-methacryloyloxyethyl phosphorylcholine; MPC) segment. Moreover, hydrophobic and carboxyl groups were attached as terminals of the polymer chain. The triblock-type polymer was attached to the QD surface through a hydrophobic layer, which was covered with the QD by hydrophobic interaction. This produced hybrid QD particles (QD/MPC polymer nanoparticles). The QD/MPC polymer nanoparticles had good water-dispersion ability after the modification. A fluorescence resonance energy transfer (FRET) phenomenon between QD and fluorescence dye (Alexa) was clearly observed at pH 7.4 and 9.0 when a fluorescence dye was reacted with the poly(MEONP) segment of the polymer. However, the efficiency decreased at pH 5.0. This was due to a change in the distance between the QD and the fluorescence dye in response to the protonation degree of the poly(DEAEMA) segment. The permeability of QD/MPC polymer nanoparticles through the cell membrane was enhanced by reacting the cell-penetrating peptide, octaarginine (R8), to the carboxyl group at the end of the polymer. The R8-QD/MPC polymer/Alexa nanoparticles attached onto the HeLa cell membrane surface within 15 min after they were added to the cell culture. This attachment initiated nanoparticle penetration of the cell membrane by endocytosis. The nanoparticles could be followed continuously as they moved in the cell culture. The change in the FRET index was determined during this process. Use of the R8-QD/MPC polymer/Alexa nanoparticle enabled us to determine nanoparticle location, based on the surrounding local pH. We concluded that QDs, modified with a cytocompatible and pH-responsible MPC polymer, provide a new imaging and transport tool in cell-based science and engineering.

Cytocompatible and multifunctional polymeric nanoparticles for transportation of bioactive molecules into and within cells.

Multifunctional polymeric nanoparticles are materials with great potential for a wide range of biomedical applications. For progression in this area of research, unfavorable interactions of these nanoparticles with proteins and cells must be avoided in biological environments, for example, through treatment of the nanoparticle surfaces. Construction of an artificial cell membrane structure based on polymers bearing the zwitterionic phosphorylcholine group can prevent biological reactions at the surface effectively. In addition, certain bioactive molecules can be immobilized on the surface of the polymer to generate enough affinity to capture target biomolecules. Furthermore, entrapment of inorganic nanoparticles inside polymeric matrices enhances the nanoparticle functionality significantly. This review summarizes the preparation and characterization of cytocompatible and multifunctional polymeric nanoparticles; it analyzes the efficiency of their fluorescence function, the nature of the artificial cell membrane structure, and their performance as in-cell devices; and finally, it evaluates both their chemical reactivity and effects in cells.

Molecular interaction forces generated during protein adsorption to well-defined polymer brush surfaces.

The molecular interaction forces generated during the adsorption of proteins to surfaces were examined by the force-versus-distance (f-d) curve measurements of atomic force microscopy using probes modified with appropriate molecules. Various substrates with polymer brush layers bearing zwitterionic, cationic, anionic, and hydrophobic groups were systematically prepared by surface-initiated atom transfer radical polymerization. Surface interaction forces on these substrates were analyzed by the f-d curve measurements using probes with the same polymer brush layer as the substrate. Repulsive forces, which decreased depending on the ionic strength, were generated between cationic or anionic polyelectrolyte brush layers; these were considered to be electrostatic interaction forces. A strong adhesive force was detected between hydrophobic polymer brush layers during retraction; this corresponded to the hydrophobic interaction between two hydrophobic polymer layers. In contrast, no significant interaction forces were detected between zwitterionic polymer brush layers. Direct interaction forces between proteins and polymer brush layers were then quantitatively evaluated by the f-d curve measurements using protein-immobilized probes consisting of negatively charged albumin and positively charged lysozyme under physiological conditions. In addition, the amount of protein adsorbed on the polymer brush layer was quantified by surface plasmon resonance measurements. Relatively large amounts of protein adsorbed to the polyelectrolyte brush layers with opposite charges. It was considered that the detachment of the protein after contact with the polymer brush layer hardly occurred due to salt formation at the interface. Both proteins adsorbed significantly on the hydrophobic polymer brush layer, which was due to hydrophobic interactions at the interface. In contrast, the zwitterionic polymer brush layer exhibited no significant interaction force with proteins and suppressed protein adsorption. Taken together, our results suggest that to obtain the protein-repellent surfaces, the surface should not induce direct interaction forces with proteins after contact with them.

Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be a suitable parameter for understanding protein adsorption.

Evaluation of the durability and antiadhesive action of 2-methacryloyloxyethyl phosphorylcholine grafting on an acrylic resin denture base material.

STATEMENT OF PROBLEM: The polymer 2-methacryloyloxyethyl phosphorylcholine is currently used on medical devices to prevent infection. Denture plaque-associated infection is regarded as a source of serious dental and medical complications in the elderly population, and denture hygiene, therefore, is an issue of considerable importance for denture wearers. Furthermore, because denture bases are exposed to mechanical stresses, for example, denture brushing, the durability of the coating is important for retaining the antiadhesive function of 2-methacryloyloxyethyl phosphorylcholine. PURPOSE: The purpose of this study is to investigate the durability and antiadhesive activity of two 2-methacryloyloxyethyl phosphorylcholine polymer coating techniques: poly-2-methacryloyloxyethyl phosphorylcholine grafting and poly-2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate coating. It was revealed that 2-methacryloyloxyethyl phosphorylcholine polymer coating of the denture base resin polymethyl methacrylate decreases bacterial biofilm formation. MATERIAL AND METHODS: Durability was examined by rhodamine staining and elemental surface analysis and by determining the wetting properties of the 2-methacryloyloxyethyl phosphorylcholine polymer-modified polymethyl methacrylate after a friction test that comprised 500 brushing cycles. Antiadhesive activity was examined by using a Streptococcus mutans biofilm formation assay. RESULTS: Poly-2-methacryloyloxyethyl phosphorylcholine-grafted polymethyl methacrylate retained 2-methacryloyloxyethyl phosphorylcholine units and antiadhesive activity even after repetitive mechanical stress, whereas co-n-butyl methacrylate-coated polymethyl methacrylate did not. CONCLUSION: These results demonstrated that graft polymerization of 2-methacryloyloxyethyl phosphorylcholine on denture surfaces may contribute to the durability of the coating and prevent microbial retention.

Inducing rapid cellular response on RGD-binding threaded macromolecular surfaces.

The rapid response of integrin ß1 molecules to an RGD peptide on a dynamic polyrotaxane surface was successfully induced. As a result, RGD peptides introduced on a highly dynamic cyclodextrin molecule enhanced the frequency of contact with specific integrin molecules on the cell membrane at the early stage of material-cell interactions.

Elastic repulsion from polymer brush layers exhibiting high protein repellency.

Hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(2-hydroxyethyl methacrylate) (PHEMA) brush layers with different thicknesses and graft densities were prepared to construct a model surface to elucidate protein-surface interactions. In particular, we focused on the steric repulsion of hydrophilic polymer layers as one of the surface properties that strongly influence protein adsorption and employed force-versus-distance (f-d) curve measurements obtained via atomic force microscopy to quantitatively evaluate the steric repulsion force, which is also referred to as the "elastic repulsion energy." We also analyzed direct interactions between the surface and proteins via the f-d curve, because these interactions trigger the protein-adsorption phenomenon. Protein-surface interactions were extremely suppressed at surfaces with high elastic repulsion energies and highly dense polymer brush structures, which is in contrast to those at surfaces with low elastic repulsion energies and low density of the grafted polymer layers. These results indicate that the elastic repulsion from the grafted polymer layer at the surface is an important parameter for controlling protein-surface interactions and protein adsorption phenomenon.

A novel electrochemical biosensing method with double-layered polymer brush modified electrode.

We developed a novel electrochemical biosensor electrode that has a potential to reduce background noise for which we constructed an original conductive substrate modified with a double-layered polymer brush structure that is water impermeable and can control biomolecules adsorption/desorption. In this study, a hydrophobic poly(tert-butyl methacrylate) brush layer was prepared on a gold electrode, and then, the tert-butyl group near the outermost surface was dissociated by the acid treatment to obtain a hydrophilic carboxy group, thereby fabricating a conductive substrate with the double-layered polymer brush structure. Formation of the double-layered polymer brush structure was indicated by surface wettability and optical analyses. The potential difference and hydrogen ion concentration, which is a typical parameter of the surrounding environment, were linearly correlated with the gold electrode having a double-layered polymer brush structure with carboxyl groups. However, there was no correlation on gold electrodes with self-assembled monolayers presenting carboxy groups. It is considered that the pH responsiveness of the carboxy groups on the outermost surface could be exhibited remarkably because the charge state in the vicinity of the surface became constant due to the hydrophobic polymer brush layer having a certain thickness. The target DNA could be captured more efficiently at the probe DNA-immobilized electrode with the double-layered polymer brush structure than when using COOH-SAM. This is the first report of the application of the double-layered polymer brush structure for the electrochemical biosensing, and it will be an excellent surface modification method to reduce background noise.

Novel polymer biomaterials and interfaces inspired from cell membrane functions.

BACKGROUND: Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials. SCOOP OF REVIEW: One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure. MAJOR CONCLUSIONS: Reduction of nonspecific protein adsorption is essential for suppression of unfavorable bioresponse and achievement of versatile biomedical applications. Simultaneously, bioconjugation of biomolecules on the phospholipid polymer platform is crucial for a high-performance interface. GENERAL SIGNIFICANCE: The biointerfaces with both biocompatibility and biofunctionality based on biomolecules must be installed on advanced devices, which are applied in the fields of nanobioscience and nanomedicine. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Effect of liposome surface modification with water-soluble phospholipid polymer chain-conjugated lipids on interaction with human plasma proteins.

Alternative liposome surface coatings for PEGylation to evade the immune system, particularly the complement system, have garnered significant interest. We previously reported poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)-based lipids (PMPC-lipids) and investigated the surface modification of liposomes. In this study, we synthesize PMPC-lipids with polymerization degrees of 10 (MPC10-lipid), 20 (MPC20-lipid), 50 (MPC50-lipid), and 100 (MPC100-lipid), and coated liposomes with 1, 5, or 10 mol% PMPC-lipids (PMPC-liposomes). Non-modified and PEGylated liposomes are used as controls. We investigate the liposome size, surface charge, polydispersity index, and adsorption of plasma proteins to the liposomes post incubation in human plasma containing N,N,N',N'-ethylenediamine tetraacetic acid (EDTA) or lepirudin by some methods such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and automated capillary western blot, with emphasis on the binding of complement protein C3. It is shown that the coating of liposome PMPC-lipids can suppress protein adsorption more effectively with an increase in the molecular weight and molar ratio (1-10 mol%). Apolipoprotein A-I is detected on PMPC-liposomes with a higher molecular weight and higher molar ratio of PMPC-lipids, whereas α2-macroglobulin is detected on non-modified, PEGylated, and PMPC-liposomes with a shorter polymer chain. In addition, a correlation is shown among the PMPC molecular weight, molar ratio, and C3 binding. The MPC10-lipid cannot inhibit C3 binding efficiently, whereas surface modifications with 10 mol% MPC20-lipid and 5 mol% and 10 mol% MPC50-lipid suppress both total protein and C3 binding. Hence, liposome modification with PMPC-lipids can be a possible strategy for avoiding complement activation.

Verification of the effect of acquisition time for SwiftScan on quantitative bone single-photon emission computed tomography using an anthropomorphic phantom.

BACKGROUND: SwiftScan single-photon emission computed tomography (SPECT) is a recently released scanning technique with data acquired when the detector is stationary and when it moves from one view to the next. The influence of scan time for using SwiftScan on quantitative bone SPECT remains unclear. This study aimed to clarify the effect of the scan time for SwiftScan SPECT on the image quality and quantification of bone SPECT compared to step and shoot mode (SSM) using 99mTc-filled anthropomorphic phantom (SIM2 bone phantom). MATERIALS AND METHODS: Phantom SPECT/computed tomography (CT) images were acquired using Discovery NM/CT 860 (GE Healthcare) with a low-energy high-resolution sensitivity collimator. We used the fixed parameters (subsets 10 and iterations 5) for reconstruction. The coefficient of variation (CV), contrast-to-noise ratio (CNR), full width at half maximum (FWHM), and quantitative value of SwiftScan SPECT and SSM were compared at various acquisition times (5, 7, 17, and 32 min). RESULTS: In the short-time scan (< 7 min), the CV and CNR of SwiftScan SPECT were better than those of SSM, whereas in the longtime scan (> 17 min), the CV and CNR of SwiftScan SPECT were similar to those of SSM. The FWHMs for SwiftScan SPECT (13.6-14.8 mm) and SSM (13.5-14.4 mm) were similar. The mean absolute errors of quantitative values at 5, 7, 17, and 32 min were 38.8, 38.4, 48.8, and 48.1, respectively, for SwiftScan SPECT and 41.8, 40.8%, 47.2, and 49.8, respectively, for SSM. CONCLUSIONS: SwiftScan on quantitative bone SPECT provides improved image quality in the short-time scan with quantification similar to or better than SSM. Therefore, in clinical settings, using SwiftScan SPECT instead of the SSM scan protocol in the short-time scan might provide higher-quality diagnostic images than SSM. Our results could provide vital information on the use of SwiftScan SPECT.

Effects of molecular architecture of photoreactive phospholipid polymer on adsorption and reaction on substrate surface under aqueous condition.

Water-soluble photoreactive polymers with both phosphorylcholine and benzophenone groups were synthesized for the reaction between the polymers and the substrate in aqueous medium. To control the polymer architecture, the living radical polymerization method was applied to the copolymerization of 2-methacryloyloxyethyl phosphorylcholine and benzophenone methacrylates. These polymers possess various architectures, such as linear polymers, polymers with hydrophobic terminals, and 4-armed star-like polymers, that could promote their adsorption on the substrate surfaces. Additionally, two types of benzophenone groups were examined. Due to the bulky phosphorylcholine group, tetra(ethylene oxide) group as a spacer between polymer main chain and benzophenone group was considered. These polymers could adsorb on the surface in an aqueous medium, followed by reaction on the surface via photoirradiation depending on the chemical structure of the benzophenone group. The thickness of the polymer layer depended on the polymer architecture, i.e. a polymer with a hydrophobic terminal could form a thick layer. After modification, the contact angle by air in the aqueous medium decreased, compared to that on the base substrate. This was due to the hydrophilic nature based on the phosphorylcholine groups at the surface. The amount of proteins adsorbed on the surface also decreased because of the surface modification. These findings indicated that these water-soluble photoreactive polymers could be applied for the safer and effective surface modification of substrates via conventional photoirradiation without using an organic solvent.

Direct photoreactive immobilization of water-soluble phospholipid polymers on substrates in an aqueous environment.

The purpose of this study is to achieve a simpler and safer surface modification of substrates using a photoreactive polymer in an aqueous environment. We synthesized water-soluble photoreactive polymers with both phenylazide groups and phosphorylcholine groups, poly(2-methacryloyloxyethyl phosphorylcholine-co-4-methacryl tetra(ethylene glycol)oxycarbonyl-4-phenylazide) (PMEPAz), via reversible addition fragmentation chain transfer polymerization. PMEPAz with different polymerization degrees were synthesized with a well-defined structure. To immobilize PMEPAz on the substrate surface by photoreaction, it is necessary to adsorb the polymer on the substrate surface in an aqueous solution because the phenylazide groups chemically bind to the substrate via a hydrogen abstract reaction. The relationship between the polymer solubilization state in the aqueous solution and the adsorption behavior at the surface was investigated. PMEPAz began to form unstable molecular aggregates at a concentration of 10-2 mg/mL and formed stable aggregates at 100 mg/mL. At a concentration of 10-1 mg/mL, unstable molecular aggregates of PMEPAz were formed in the aqueous solution, resulting in the maximization of the amount of adsorbed polymer and effective photoreaction with the substrate. The thickness of the reacted polymer layer on the substrate increased with an increase in the polymerization degree, a uniform polymer layer with a thickness of 3.4 nm was formed when the polymerization degree was 400. After surface modification, the hydrophobic surfaces of the original substrates became hydrophilic. Additionally, fibrinogen adsorption and platelet adhesion were effectively suppressed based on the characteristics of the phosphorylcholine unit.

Synthesis of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated lipids and their characterization and surface properties of modified liposomes for protein interactions.

Poly(ethylene glycol) (PEG) is frequently used for liposomal surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused on a water-soluble polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units, and synthesized poly(MPC) (PMPC)-conjugated lipid (PMPC-lipid) with degrees of MPC polymerization ranging from 10 to 100 (calculated molecular weight: 3 to 30 kDa). In addition, lipids with three different alkyl chains, myristoyl, palmitoyl, and stearoyl, were applied for liposomal surface coating. We studied the interactions of PMPC-lipids with plasma albumin, human complement protein C3 and fibrinogen using a quartz crystal microbalance with energy dissipation, and found that adsorption of albumin, C3 and fibrinogen could be suppressed by coating with PMPC-lipids. In particular, the effect was more pronounced for PMPC chains with higher molecular weight. We evaluated the size, polydispersity index, surface charge, and membrane fluidity of the PMPC-lipid-modified liposomes. We found that the effect of the coating on the dispersion stability was maintained over a long period (98 days). Furthermore, we also demonstrated that the anti-PEG antibody did not interact with PMPC-lipids. Thus, our findings suggest that PMPC-lipids can be used for liposomal coating.

Interface of Phospholipid Polymer Grafting Layers to Analyze Functions of Immobilized Oligopeptides Involved in Cell Adhesion.

The aim of this study was to design a material surface for use in the analysis of the behavior of biomolecules at the interface of direct cell contact. A superhydrophilic surface was prepared with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), which was grafted onto a substrate with controlled polymer chain density. An arginine-glycine-aspartic acid (RGD) peptide was immobilized at the surface of the polymer graft surface (PMPC-RGD surface). Initial adhesion of the cells to this substrate was observed. The PMPC-RGD surface could enable cell adhesion only through RGD peptide-integrin interactions. The density and movability of the RGD peptide at the terminal of the graft PMPC chain and the orientation of the RGD peptide affected the density of adherent cells. Thus, the PMPC graft surface may be a good candidate for a new platform with the ability to immobilize biomolecules to a defined position and enable accurate analysis of their effects on cells.

Effects of molecular interactions at various polymer brush surfaces on fibronectin adsorption induced cell adhesion.

The effects of protein adsorption on the polymer brush surfaces with well-defined chemical structures and physical properties were examined with respect to initial protein adsorption, structural changes to the adsorbed proteins, and subsequent cell adhesion. Four polymer brush surfaces with different hydrophilicities and charge states were prepared. The molecular interaction forces during adsorption-desorption processes of protein on the polymer brush surfaces depending on the chemical structure of the polymer were determined. Crucially, these molecular interactions affected the adsorption behavior and structural changes of fibronectin (FN), a cell-adhesive protein, used in this study. Adsorption of FN onto the zwitterionic polymer and anionic polymer surfaces was difficult, however significant protein adsorption to the hydrophobic and cationic surfaces was observed. Further, the structural changes to the adhered FN on these surfaces were significant. Subsequent cell adhesion experiments revealed that the adhered cell density was correlated with the amount of adsorbed FN and the degree of FN structural change. In addition, the cationic surface inhibited cell proliferation behavior. These results indicate that cellular responses can be indirectly regulated by controlling the molecular interactions which induced the structural change of adsorbed proteins via the material surface properties.