Tomohiro Konno

Polymer Nanoparticles Covered with Phosphorylcholine Groups and Immobilized with Antibody for High-Affinity Separation of Proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.

Preparation of nanoparticles composed with bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer.

The poly(L-lactic acid) nanoparticles immobilized with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which has excellent blood compatibility, were prepared by a solvent evaporation technique using the water-soluble amphiphilic MPC polymer as an emulsifier and a surface modifier. The diameter and zeta-potential of the obtained nanoparticles strongly depended on the concentration of the MPC polymer. When the nanoparticles were prepared in 1.0 mg/ml of an MPC polymer aqueous solution, the diameter was 221 nm which was determined by atomic force microscopy and dynamic light scattering measurements. The X-ray photoelectron spectroscopic analysis indicated that the phosphorylcholine groups of the MPC unit were located at the surface of the nanoparticles, that is, the MPC polymer was immobilized on the PLA particles and the surface zeta-potential was -2.5 mV. Various hydrophobic fluorescence probes could permeate through the MPC polymer layer and adsorb on the PLA surface. The amount of bovine serum albumin adsorbed on the nanoparticles was significantly smaller compared with that on the conventional polystyrene nanoparticles. It is suggested that the nanoparticles immobilized with the MPC polymer have the potential for use as both a novel drug carrier and diagnostic reagent which can come in contact with blood components.

Surface modification by 2-methacryloyloxyethyl phosphorylcholine coupled to a photolabile linker for cell micropatterning

This report describes a new surface-treatment technique for cell micropatterning. Cell attachment was selectively controlled on the glass surface using a photochemical reaction. This strategy is based on combining 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to reduce non-specific adsorption, and a photolabile linker (PL) for selective cell patterning. The MPC polymer was coated directly on the glass surface using a straightforward surface modification method, and was removed by ultraviolet (UV) light illumination. All the surface modification steps were evaluated using static water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), measurements of non-specific protein adsorption, and the cell attachment test. After selective cleavage of the MPC polymer through the photomask, cells attached only to the UV-illuminated region where the MPC polymer was removed, which made the hydrophilic surface relatively hydrophobic. Furthermore, the size of the MC-3T3 E1 cell patterns could be controlled by single cell level. Stability of the cell micropatterns was demonstrated by culturing MC-3T3 E1 cell patterns for 5 weeks on glass slide. The micropatterns were stable during culturing; cell viability also was verified. This method can be a powerful tool for cell patterning research.

Microfluidic flow control on charged phospholipid polymer interface

A type of charged phospholipid polymer biointerface was constructed on a quartz microfluidic chip to control the electroosmotic flow (EOF) and to suppress non-specific protein adsorption through one-step modification. A negatively charged phospholipid copolymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), potassium 3-methacryloyloxypropyl sulfonate (PMPS) and 3-methacryloxypropyl trimethoxysilane (MPTMSi) moieties (referred to as PMBSSi) was synthesized to introduce such phosphorylcholine segments as well as surface charges onto the silica-based microchannels via chemical bonding. At neutral pH, the homogenous microchannel surface modified with 0.3 wt% PMBSSi in alcoholic solution, retained a significant cathodic EOF ((1.0 +/- 0.1) x 10(-4) cm(2) V(-1) s(-1)) with approximately one-half of the EOF of the unmodified microchannel ((1.9 +/- 0.1) x 10(-4) cm(2) V(-1) s(-1)). Along with another non-charged copolymer (poly(MPC-co-MPTMSi), PMSi), the regulation of the surface charge density can be realized by adjusting the concentration of PMBSSi or PMSi initial solutions for modification. Coincidently, the zeta-potential and the EOF mobility at neutral pH showed a monotonically descending trend with the decrease in the charge densities on the surfaces. This provides a simple but feasible approach to controlling the EOF, especially with regard to satisfying the requisites of miniaturized systems for biological applications requiring neutral buffer conditions. In addition, the EOF in microchannels modified with PMBSSi and PMSi could maintain stability for a long time at neutral pH. In contrast to the EOF in the unmodified microchannel, the EOF in the modified microchannel was only slightly affected by the change in pH (from 1 to 10). Most importantly, although PMBSSi possesses negative charges, the non-specific adsorptions of both anionic and cationic proteins (considering albumin and cytochrome c, respectively, as examples) were effectively suppressed to a level of 0.15 microg cm(-2) and lesser in the case of the 0.3 wt% PMBSSi modification. Consequently, the variation in the EOF mobility resulting from the protein adsorption was also suppressed simultaneously. To facilitate easy EOF control with compatibility to biomolecules delivered in the microfluidic devices, the charged interface described could provide a promising option.

Rapid development of hydrophilicity and protein adsorption resistance by polymer surfaces bearing phosphorylcholine and naphthalene groups.

In order to provide a protein adsorption resistant surface even when the surface was in contact with a protein solution under completely dry conditions, a new phospholipid copolymer, poly (2-methacryloyloxyethyl phosphorylcholine (MPC)- co-2-vinylnaphthalene (vN)) (PMvN), was synthesized. Poly(ethylene terephthalate) (PET) could be readily coated with PMvN by a solvent evaporation method. Dynamic contact angle measurements with water revealed that the surface was wetted very rapidly and had strong hydrophilic characteristics; moreover, molecular mobility at the surface was extremely low. When the surface came in contact with a plasma protein solution containing bovine serum albumin (BSA), the amounts of the plasma protein adsorbed on the dry surface coated with PMvN and that adsorbed on a dry surface coated with poly(MPC-co-n-butyl methacrylate) (PMB) were compared. Substantially lower protein adsorption was observed with PMvN coating. This is due to the rapid hydration behavior of PMvN. We concluded that PMvN can be used as a functional coating material for medical devices without any wetting pretreatment.

Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes.

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.

Protein adsorption resistance and oxygen permeability of chemically crosslinked phospholipid polymer hydrogel for ophthalmologic biomaterials

The biomimetic structure of a polymer hydrogel bearing phosphorylcholine groups was obtained from 2-methacryloyloxyethylphosphorylcholline (MPC) and a novel crosslinker, 2-(methacryloyloxy)ethyl-N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), to prepare biocompatible ocular materials. MMPC is a dimethacrylate with phosphorylcholine-analogous linkage. Previous reports clarified that the affinity of MMPC to MPC enables the water contents and mechanical properties of the poly(MPC) hydrogels to be varied without disturbing the bulk phases. In this study, we examined the protein adsorption resistance, water wettability, oxygen permeability, and electrolyte permeability of the mechanically enhanced poly(MPC) hydrogel crosslinked with MMPC. The amount of protein adsorbed on this hydrogel was 0.9 microg/cm(2), which accounted for 30% of Omafilcon A and 3% of Etafilcon A. Water contact angle experiments revealed the high wettability of the poly(MPC) hydrogels. The oxygen permeability and NaCl diffusion constant of the poly(MPC) hydrogels were 64 barrer and 48 x 10(-6) cm(2)/s, respectively. This high permeability resulted from the high water content, similar to the case of the human cornea. These results suggested that poly(MPC) hydrogels have good potential for use in ophthalmologic biomaterials.

Photografting of 2-methacryloyloxyethyl phosphorylcholine from polydimethylsiloxane: Tunable protein repellency and lubrication property

The phosphorylcholine group functional methacrylate monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC), was graft polymerized from the polydimethylsiloxane (PDMS) substrate using ultraviolet irradiation and using benzophenone as a photoinitiator. The varying monomer concentrations and irradiation times were investigated in order to verify the relationships between graft density and protein resistance under specific biological conditions. The ellipsometry analysis revealed that the layer thickness of the grafted polymer depended on the monomer concentrations after the irradiation for 1 min, however, it stabilized thereafter in all the specified conditions. The curve fitting of the C1s spectrum obtained by X-ray photoelectron spectroscopy analysis showed that the amount of grafted polymer increased with an increase in both monomer concentration and irradiation time. Atomic force microscopic images revealed that the terminations among the graft chains became dominant due to magnified chain mobility followed by growth of their length. In vitro albumin and fibrinogen adsorption results indicated that the resistance to protein adsorption was easily tuned by the specified conditions due to the controlled graft density. Lubrication was dramatically enhanced by the grafting and it was further promoted by an increase in the graft density in good solvents, indicating that the interactions between the graft chains and the solvents resulted in the lubrication system. These basic findings regarding the grafted PDMS surface are important for versatile applications, including its use as a biomaterial and microfluidic device.

Surface immobilization of biocompatible phospholipid polymer multilayered hydrogel on titanium alloy.

The aim of this study is to improve the biocompatibility of titanium alloy (Ti) implants by immobilization of multilayered phospholipid polymer hydrogel able to reduce protein adsorption and cell adhesion. We fabricated and characterized a multilayered hydrogel on Ti substrate via a layer-by-layer self-assembly deposition method using a phospholipid polymer bearing a phenylboronic acid moiety and poly(vinyl alcohol) (PVA). The water-soluble phospholipid polymer (PMBV) was synthesized from 2-methacrylocyloxyethyl phosphorylcholine, n-butyl methacrylate, and 4-vinylphenylboronic acid (VPBA). The PMBV reacted with PVA and formed a hydrogel due to covalent linkage between the VPBA units and hydroxyl groups of PVA. The hydrogel layer growth on the Ti surface was initialized by the deposition of one layer of photoreactive PVA bonded by UV irradiation to the Ti surface, which was modified with an alkylsilane compound. The multilayered hydrogel was built up by alternating the deposition of the PMBV and PVA; this was monitored by several methods: static contact angle measurement, X-ray photoelectron spectroscopy, and attenuated Fourier-transform infrared spectroscopy. The results revealed clearly the progressive construction of the multilayered hydrogel on the Ti substrate. The PMBV/PVA multilayer prepared on the Ti substrate reduced the adhesion of L929 cells compared with that on an untreated Ti substrate. Thus, we concluded that the formation of the multilayered hydrogel is effective to improve the biocompatibility on Ti-based medical devices.

Controlled drug release from multilayered phospholipid polymer hydrogel on titanium alloy surface

Here we describe the functionalization of a multilayered hydrogel layer on a Ti alloy with an antineoplastic agent, paclitaxel (PTX). The multilayered hydrogel was synthesized via layer-by-layer self-assembly (LbL) using selective intermolecular reactions between two water-soluble polymers, phospholipid polymer (PMBV) containing a phenylboronic acid unit and poly(vinyl alcohol) (PVA). Reversible covalent bonding between phenylboronic acid and the polyol provided the driving force for self-assembly. Poorly water-soluble PTX dissolves in PMBV aqueous solutions because PMBV is amphiphilic. Therefore, our multilayered hydrogel could be loaded with PTX at different locations to control the release profile and act as a drug reservoir. The amount of PTX incorporated in the hydrogel samples increased with the number of layers but was not directly proportional to the number of layers. However, as the step for making layers was repeated, the concentration of PTX in the PMBV layers increased. The different solubilities of PTX in PMBV and PVA aqueous solutions allow for the production of multilayered hydrogels loaded with PTX at different locations. In vitro experiments demonstrated that the location of PTX in the multilayered hydrogel influences the start and profile of PTX release. We expect that this rapid and facile LbL synthesis of multilayered hydrogels and technique for in situ loading with PTX, where the location of loading controls the release pattern, will find applications in biomedicine and pharmaceutics as a promising new technique.