Kyoko Fukazawa

Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing.

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 microm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.

Development of a method to evaluate caspase-3 activity in a single cell using a nanoneedle and a fluorescent probe.

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).

Direct observation of selective protein capturing on molecular imprinting substrates

A sensing interface for specific protein capture was fabricated using a novel molecular imprinting (MIP) process. Bovine serum albumin (BSA) and ovalbumin (OVA) were imprinted on a quartz substrate with modified alkyl groups, and target protein capture was detected using a deep-UV fluorescence image microscope (UVFLIM). The imprinted protein was immobilized to silica beads (diameter: 15 μm) using a phospholipid polymer containing both active ester groups and silane coupling groups, which were used as protein stamps to prepare the imprinting surface. Protein recognition sites were constructed by integrating sodium dodecyl sulfate (SDS) as the ligand, which was immobilized with a biocompatible photoreactive phospholipid polymer. When BSA solution was added to the BSA-based MIP substrate, strong fluorescence was observed from the tryptophan residue of BSA. In contrast, for the OVA-based MIP substrate and non-MIP substrate, no fluorescence was observed. The surface showed good selectivity of BSA against OVA. The phospholipid polymer layer prevented non-specific protein adsorption, resulting in highly selective protein recognition. Further, when the protein-imprinted substrate was constructed without ligands, neither protein was captured on the substrate. We demonstrated the importance of ligand integration for capturing target proteins at specific positions. UVFLIM can be used to detect biomolecules at the single-molecule level by using intrinsic fluorescence without molecular labeling. Our new protein-imprinted surface used with UVFLIM is a versatile tool for capturing biomolecules.

Effects of 3,4-dihydrophenyl groups in water-soluble phospholipid polymer on stable surface modification of titanium alloy

The surface of a titanium (Ti) alloy substrate was modified by a simple and quick process using a water-soluble polymer, and the effects of 3,4-dihydroxyphenyl (DHP) groups in the polymer side chain on the modification process were examined. The polymers (PMDP) composed of both 2-methacryloyloxyethyl phosphorylcholine (MPC) unit and 3,4-dihydroxyphenyl methacrylate unit were synthesized for surface anchoring. The Ti alloy substrate was coated with PMDP using an aqueous solution of the polymer. A PMDP layer with a thickness of 20 nm was formed on the Ti alloy substrate simply by dip coating for 10s without drying. Even when the Ti alloy substrate with PMDP coating was immersed in the aqueous medium for 1 week, no change in the thickness was observed, i.e., the PMDP layer was bound to the surface very stably. Oxidation of the DHP groups reduced the stability of the polymer layer significantly. Thus, the DHP groups play a significant role in achieving stable binding. Protein was adsorbed on the Ti alloy substrate; however, this was not observed for the PMDP-coated Ti alloy substrate. In conclusion, we confirmed the effects of DHP groups in PMDP on the stability of the coating on the Ti alloy substrate. Moreover, we found that surface treatment using PMDP was simple, quick, and reliable, and thus, it has great potential for improving biofouling of Ti alloy substrates used in medical devices.

Photoreactive Polymers Bearing a Zwitterionic Phosphorylcholine Group for Surface Modification of Biomaterials

Photoreactive polymers bearing zwitterionic phosphorylcholine and benzophenone groups on the side chain were synthesized and used as surface modification reagents for biomaterials. A photoreactive methacrylate containing the benzophenone group, 3-methacryloyloxy-2-hydroxypropyl-4-oxybenzophenone (MHPBP), was synthesized via a ring-opening and addition reaction between glycidyl methacrylate and 4-hydroxybenzophenone. Then, water-soluble, amphiphilic polymers poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-MHPBP) (PMH) and poly(MPC-co-n-butyl methacrylate-co-MHPBP), with different monomer unit compositions, were synthesized through radical polymerization. Ultraviolet-visible (UV/vis) absorption spectra of these polymer solutions showed that these polymers have maximum absorption peaks at 254 and 289 nm that can be attributed to the benzophenone unit. The intensity of UV adsorption at 289 nm was decreased with increased UV irradiation time, and it was saturated within a few minutes, indicating that the polymers are highly sensitive to UV irradiation. A commercial material (i.e., cyclic polyolefin) was simply modified by a UV irradiation for 1.0 min. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analysis results indicated that the stability of the polymer on the surface was dramatically enhanced because of the photochemical reaction of the benzophenone moiety. The air contact angles of PMH surfaces measured in water were up to 160°. Thus, highly hydrophilic surfaces were obtained. The critical surface tension of the PMH-modified surface was 45.7 mN/m. By evaluating the biological reactivity of the treated surface, protein adsorption and cell adhesion were completely inhibited on the surface, which was prepared using a photopatterning procedure using PMH. In conclusion, photoreactive MPC polymers with a benzophenone moiety could be used as a novel and effective surface modifier.

Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.

Synthesis of Photoreactive Phospholipid Polymers for Use in Versatile Surface Modification of Various Materials to Obtain Extreme Wettability

We synthesized photoreactive phospholipid polymers for use in the versatile surface modification of various materials. The photoreactive methacrylate derivative 2-methacryloyloxyethyl-4-azidobenzoate (MPAz) was synthesized. MPAz was copolymerized with 2-methacryloyloxyethyl phosphorylcholine (MPC) to obtain poly(MPC-co-MPAz) (PMPAz) and with both MPC and n-butyl methacrylate (BMA) to obtain poly(MPC-co-BMA-co-MPAz) (PMBPAz). PMPAz and PMBPAz were bound to the surfaces of various materials, including polymers, glass, and metals, by exposure to ultraviolet irradiation. The azide groups in the MPAz units played a role in the surface anchoring; the polymer was bound covalently to the substrate. After the photoreaction, the surfaces were converted from hydrophobic to superhydrophilic, and their cell adhesion was effectively suppressed.

Simple surface treatment using amphiphilic phospholipid polymers to obtain wetting and lubricity on polydimethylsiloxane-based substrates

Simple surface treatment of polydimethylsiloxane (PDMS) substrates was performed using an aqueous-ethanolic solution of amphiphilic phospholipid polymers to reduce the hydrophobic and high friction characteristics of PDMS. The phospholipid polymers, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-2-ethylhexyl methacrylate (EHMA)-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED) and poly(MPC-co-EHMA) (PMEH) were synthesized, and the effects of the electric charge of the polymer chain on the stability of the attachment to the PDMS surface was investigated. The polymers were dissolved in a mixed solvent of ethanol and water, and the PDMS samples were treated by a simple dipping method using the polymer solution. Pure ethanol as the solvent was ineffective for the attachment of the polymers to the PDMS surface. It was considered that the hydrophobic interactions and electrostatic attraction forces between the polymer chains and the PDMS surface were too weak for efficient interaction in this solvent. On the other hand, the surface wettability and lubricity of PDMS could be improved by treatment with an aqueous-ethanolic solution of PMED. The static contact angle was decreased from 90° to 20° by this treatment, and the dynamic friction coefficient against a Co-Cr ball was decreased by nearly 80% compared with that of the untreated PDMS. The hydrophobic interactions and electrostatic attraction forces generated by PMED were both essential for the stable adsorption of the polymer layer on PDMS. Furthermore, the solubilized state of the polymers affected the adsorption of the polymer. We concluded that the surface of PDMS could be stably modified using aqueous-ethanolic solutions of PMED without the need for pretreatments.

CHAPTER 5:2-Methacryloyloxyethyl Phosphorylcholine Polymers

The synthesis and properties of phosphorylcholine group-bearing methacrylate, 2-methacryloyloxyethyl phosphorylcholine (MPC), and its polymers are described. Molecular design of the MPC polymers with significant functions for biomedical and medical applications is summarized in detail. Some MPC polymers have been provided commercially for these applications. The MPC polymer can provide an artificial cell membrane-like structure at the surface as an excellent interface between artificial systems and biological systems. The adsorption of proteins and adhesion of the cells are suppressed on the surface of the MPC polymers. In other words, the specific biomolecules immobilized on the MPC polymer surface show their original functions. In the clinical medicine field, MPC polymers have been used for surface modification of medical devices, including long-term implantable artificial organs to improve biocompatibility. The use of MPC polymers has increased worldwide as a result of their specific properties at the interface of artificial cell membrane-like structures.

Selection of highly osteogenic and chondrogenic cells from bone marrow stromal cells in biocompatible polymer-coated plates.

To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.