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Induction of oxidative stress is an effective approach to causing apoptotic death of cancer cells. Since oxidative stress is generally caused by an intracellular redox imbalance, altering the intracellular redox is a promising strategy toward the growth suppression of cancer cells. Here, we attempted to induce apoptosis in MDA-MB-231 human breast cancer cells by adding a cell-membrane permeable redox phospholipid polymer that can alter the intracellular redox. We found that apoptosis and the deactivation of oxidative phosphorylation were induced in the MDA-MB-231 cells in the presence of the oxidized form of the redox polymer. Remarkably, such phenomena were not observed in the presence of the reduced form of the redox polymer that cannot intercept metabolic electrons. These results indicate that the redox polymer that mediates extracellular electron transfer (EET) generates oxidative stress, leading to the apoptosis of the cancer cells.

This work presents a salt-responsive interpenetrating network (IPN) hydrogel with effective antimicrobial properties and surface regeneration. The hydrogels were engineered using the double network strategy to form loosely cross-linked zwitterionic poly(sulfobetaine vinylimidazole) (pSBVI) networks into the highly cross-linked cationic poly((trimethylamino)ethyl methacrylate chloride) (pTMAEMA) framework via photopolymerization. The pTMAEMA/pSBVI hydrogel has strong mechanical properties, with a fracture stress 120× higher than single network pTMAEMA hydrogel. In addition, there is inverse correlation between elastic modulus and elastic strain of pTMAEMA/pSBVI hydrogels as a function of ionic strength. The cationic pTMAEMA and zwitterionic pSBVI show opposite swelling behaviors in salt solutions due to the polyelectrolyte effect and antipolyelectrolyte effect. Therefore, the pTMAEMA/pSBVI hydrogel elicits a significant interfacial transition in solutions with different ionic strengths. The IPN hydrogels have switchable lubrication and optical transmittance between deionized water and 1.0 M NaCl solution. The protein adsorption tests further confirmed the switchable interface of salt-responsive IPN hydrogels. In addition, bacterial attachment test on pTMAEMA/pSBVI hydrogels with Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli) show bacterial killing rates of the IPN hydrogel over 80% for S. epidermidis and 90% for E. coli after incubating the hydrogels in the bacterial solutions for 24 h. The bacterial release rate from the IPN hydrogel reached 96% after washing with 1.0 M NaCl solution. Furthermore, the excellent reusability of the pTMAEMA/pSBVI hydrogels was demonstrated by the high bacterial killing and bacterial release rates after five kill/release cycles. The work presents a new stimuli-responsive IPN hydrogel with structural modulation, tunable antimicrobial properties, and surface regeneration by ionic strength. Integrating two salt-responsive polymers with mutually independent actions into a single material provides a new direction for smart materials with potential medical and industrial applications.

Reversible addition-fragmentation chain transfer (RAFT) radical polymerization, mediated by 4-cyanopentanoic acid dithiobenzoate and 4,4'-azobis(4-cyanovaleric acid) (V-501) in water at 70 degrees C, of biocompatible 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) yields a macro-chain transfer agent (CTA) that was employed in the synthesis of a range of stimulus-responsive AB diblock copolymers in protic media. Well-defined block copolymers of varying molar composition, with narrow molecular weight distributions (M(w)/M(n) = 1.10-1.24) were prepared with N,N-diethylacrylamide (DEAm), 4-vinylbenzoic acid (VBZ), N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine (DMAPS), and the newly synthesized N,N-di-n-propylbenzylvinylamine (DnPBVA) in either methanol, 2,2,2-trifluoroethanol, or aqueous media. When a combination of (1)H NMR spectroscopy and dynamic light scattering is used, it is shown that all block copolymers are capable of existing as molecularly dissolved chains in aqueous media with average hydrodynamic diameters of approximately 6-7 nm provided the aqueous environment is appropriately tuned. Similarly, these unimers can be induced to undergo self-assembly in the same aqueous environment provided the correct external stimulus (change in temperature, pH, or electrolyte concentration) is applied. In such instances, aggregates with average sizes in the range of approximately 22-180 nm are formed and are most likely due to the formation of polymeric micelles and vesicles. Such self-assembly is also completely reversible. Removal, or reversal, of the applied stimulus results in the reorganization to the unimeric state.

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.

This study aimed to develop a sensitive and reliable immunoassay by applying a highly functional phospholipid polymer biointerface. We synthesized a phospholipid polymer--poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). MEONP contains active ester groups on the side chains for immobilization of antibodies via oxyethylene. PMBN with different compositions and oxyethylene chain lengths were synthesized; their effects on nonspecific and specific values in the immunoassay were evaluated. MPC units reduce the background by preventing nonspecific protein adsorption. MEONP units could conjugate antibodies and enhance the specific signal. The specific signal was independent of the oxyethylene chain length, but long oxyethylene chains increased the background. Specific signals corresponding to the antigen were observed with the PMBN coating, and a liner standard curve was obtained. The PMBN-coated surface maintained residual activity after long-term storage. This surface affords a low background without requiring blocking treatment and is suitable for immobilized antibodies.

Polymer nanoparticles for sequential enzymatic reactions were prepared by combining a phospholipid polymer shell with a polystyrene core. The active ester groups for the bioconjugation and phospholipid polar groups were incorporated into the phospholipid polymer backbone using a novel active ester monomer and 2-methacryloyloxyethyl phosphorylcholine. For the sequential enzymatic reactions, acetylcholinesterase, choline oxidase, and horseradish peroxidase-labeled IgG were immobilized onto the nanoparticles. As substrates, acetylcholine chloride, choline chloride, and tetramethylbenzidine were added to the nanoparticle suspension, the acetylcholine chloride was converted to choline chloride, the choline chloride was oxidized by choline oxidase, and hydrogen peroxide was then formed as an enzymatic degradation product. The hydrogen peroxide was used for the next enzymatic reaction (oxidized by peroxidase) with tetramethylbenzidine. The sequential enzymatic reactions on the nanoparticles via degradation products (hydrogen peroxide) were significantly higher than that of the enzyme mixture. This result indicated that the diffusion pathway of the enzymatic products and the localization of the immobilized enzyme were important for these reactions. These nanoparticles were capable of facilitating sequential enzymatic reactions.

A biodegradable organo hydrogel hybrid material is presented, which is formed through the water uptake of a phosphoryl choline zwitterionomer (PC ionomer). The water uptake and subsequent swelling is induced by the phosphoryl choline (PC) end group functionality. The nonfunctional poly(trimethylene carbonate) is hydrophobic and as such does not absorb any water. Disks of the PC ionomer showed significant water uptake, typically above 90 wt % when fully swollen. This high water uptake triggered us to utilize the material for drug and protein loading and subsequent release. Fluorescein and fluorescein-labeled proteins were used as simple models for the loading and release characteristics of the material which was studied by fluorescence spectroscopy. The rate of release of the loaded molecules was compared, and it was shown that the release rate was similar for FITC and insulin but slightly slower for albumin. These results suggest that the PC ionomer may be used as a biodegradable and low elastic modulus material with an additional drug and/or protein release capacity. Such materials are of particular interest for use in a variety of applications in vivo, for example as drug eluting stents.

To enable the visualization of the distribution and dynamics of intracellular biomolecules and thereby understand the mechanisms of intracellular bioreactions, we developed a specific functional nanoprobe through the combination of a well-designed, cytocompatible phospholipid polymer and molecular beacons (MBs). A water-soluble, amphiphilic phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl tetra(ethylene glycol) methacrylate] (PMBS), was synthesized and conjugated with MBs to form nanoprobes via a chemical reaction between the ester group of N-hydroxysuccinimide and the amine group of the MBs. Surface tension measurements indicated that the polymeric nanoprobes had different conformations in aqueous solution, specifically at a concentration of 1.0 mg/mL. The PMBS, containing the large, hydrophobic BMA, formed polymer aggregates. The carcinoma cells used to test the probes remained 100% viable after incubation with PMBS-MB probes. The polymeric nanoprobes demonstrated not only a high target specificity but also resistance to nonspecific adsorption of proteins compared with unconjugated MBs and were able to penetrate the cytoplasm of the cells, allowing the live imaging of mRNA. In summary, MPC polymer-MB nanoprobes have great potential for practical application for the noninvasive monitoring of intracellular biomolecules and bioreactions in real time.

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.

Initial contact between a biological environment and a biomaterial ultimately decides the in vivo performance. Therefore, the fabrication of a delicate biointerface is important because it can be utilized as a platform for novel biomaterials. For the preparation of advanced biomedical devices such as biochips, nanoparticles, and cell engineering devices, the surface properties may be modified by the design of polymeric biomaterials. Anomalous phospholipid polymers with an isomeric oligo(lactic acid) segment were designed and evaluated as a biointerface. The phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine was easily copolymerized with isomeric oligo(lactic acid) macromonomers, and the obtained polymer could easily form thin coating membranes as biointerfaces. The oligo(lactic acid) involves three kinds of isomers: dl-, d-, and l-forms. The favorable characteristic on the surface provides regulation of cell-material interactions on the biointerface. The oligo(lactic acid) segment could form hydrophobic domains, which were considered to be located on the interface, to enhance protein adsorption and cell adhesion. The most favorable characteristics on the biointerface were dual functions of cytocompatibility by the phospholipid polymer and cell adhesion property by the oligo(lactic acid) segment. In this study, we focused on the biological responses such as protein adsorption and cell adhesion by change in the oligo(lactic acid) component. The cell viability on the confluent stage was evaluated in terms of metabolic activity.

To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. The "living" polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPC(m)-BMA(n)) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPC(m)-BMA(n), solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPC(m)-BMA(n) as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPC(m)-BMA(n) may find application as a promising reagent to make a good formulation with a hydrophobic drug.

To obtain a novel biodegradable cross-linker, polymerizable polyphosphate (PIOP) was synthesized by ring-opening polymerization of 2-i-propyl-2-oxo-1,3,2-dioxaphospholane with 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl methacrylate) (OPEMA). The number averaged molecular weight of the PIOP was 1.2 x 10(4), and the number of OPEMA units in one PIOP molecule was 2.2. Nonenzymatic degradation of the PIOP was evaluated in various pH aqueous media. The degree of hydrolysis was dependent on the pH; that is, it increased with an increase in the pH of the medium. At pH 11.0, the PIOP completely degraded in only 6 days. The poly[2-methacryloyloxyethyl phosphorylcholine (MPC)] cross-linked with the PIOP was prepared by radical polymerization. This polymer could form hydrogel, and the free water fraction in the hydrogel was high. The enzymatic activity of trypsin in contact with the hydrogel was similar to that in buffer solution. There is no adverse effect caused by the hydrogel to reduce the function of the trypsin. The cytotoxicity of poly(MPC) and degraded PIOP was evaluated using v79 cells, and it was not observed in either case. In conclusion, PIOP is a hydrolyzable polymer, which can be used as a cross-linker, and novel hydrogels having biodegradability and biocompatibility were prepared from poly(MPC) cross-linked with the PIOP.

To recognize and control ligand-receptor interactions at the interface between cells and polymer materials, we investigated a model system with an enzyme and a substrate conjugated with a biocompatible phospholipid polymer in an aqueous medium. We explored the regulation of enzyme-substrate (ES) complexation using horseradish peroxidase (HRP) as the enzyme and 4-aminoantipyrine (AAP) and 3-(p-hydroxyphenyl) propionic acid (HPPA) as substrates. The phospholipid polymer (PMBN), composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl poly(oxyethylene)methacrylate, was prepared and conjugated with AAP (PMBN-AAP conjugate). The formation and dissociation of the ES complex were investigated using capillary electrophoresis and fluorescence spectroscopy. In the chart of the capillary electrophoresis, a much longer retention time of HRP was observed in the PMBN-AAP conjugate-coated capillary compared with that in a nontreated capillary. The retention time was significantly longer in comparison with the case of a mixed solution of HRP and AAP. This result clearly shows that HRP forms an ES complex with the immobilized PMBN-AAP conjugate and that the addition of AAP to the medium inhibits the interactions between HRP and the PMBN-AAP conjugate. Though HRP forms an ES complex with both AAP and the PMBN-AAP conjugate, the ES complex with the PMBN-AAP conjugate was easily dissociated by addition of HPPA as an alternative substrate because HRP started to react with the HPPA immediately. However, the HRP that formed an ES complex with AAP fell behind in reacting with the HPPA. The activity of HRP was maintained at the initial level in the presence of the PMBN-AAP conjugate at 25 degrees C for 1 week. Additionally, even under H(2)O(2) conditions, HRP stored with the PMBN-AAP conjugate maintained 40% of the initial activity whereas HRP was deactivated within 6 h. This result indicates that the PMBN-AAP conjugate could block the active sites by formation of an ES complex. This is due to the formation of the ES complex, which retained the structure of HRP by blocking the active sites. On the basis of these results, we considered that the reversible attachment and detachment by PMBN conjugated with specific ligands from cellular receptors will be realized.

We investigated the bioconjugation of enzymes on polymer nanoparticles covered with bioinert phosphorylcholine groups. A water-soluble amphiphilic phospholipid polymer (PMBN) was specially designed for preparation of nanoparticles and conjugation with enzymes on them. The PMBN was prepared by random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenylester bearing methacrylate. The PMBN was used as an emulsifier and a surface modifier to prepare the poly(l-lactic acid) nanoparticles by a solvent evaporation technique in aqueous medium. The nanoparticles covered with phosphorylcholine groups were stably dispersed in an aqueous solution and a phosphate buffered saline. The diameter and surface zeta-potential of the nanoparticles were ca. 200 nm and -6 mV, respectively. The p-nitrophenyl ester groups, which are active ester units for the amino groups of the protein, were located at the surface of the nanoparticles. Both acetylcholine esterase and choline oxidase were co-immobilized (dual-mode conjugation) by the reaction between the p-nitrophenyl ester group and the amino group of these enzymes. The enzymatic reactions on the nanoparticles were followed using a microdialysis biosensor system with a microtype hydrogen peroxide electrode in the probe. The nanoparticles conjugated with these enzymes could detect the acetylcholine chloride as hydrogen peroxide, which is a product of the enzymatic reactions on the surface of the nanoparticles in the probe. Namely, continuous enzyme reactions could be occurring on the surface of the nanoparticles. It is concluded that the nanoparticles are a promising tool for a highly sensitive and microdiagnostic system.

Poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA) macromonomers were synthesized for preparation of a novel cytocompatible polymer. The cytocompatible polymer was composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and the enantiomeric PLLA (or PDLA) macromonomer. The degree of polymerization of the lactic acid in the PLLA and PDLA segments was designed to be ca. 20. The copolymer-coated surface was analyzed with static contact angle by water. From the result, the PLLA (or PDLA) segment and MPC unit were located on the coated surface, and the monomer unit in the copolymer was reconstructed by contacting water. Fibroblast cell culture was performed to evaluate cell adhesion on the coated surface, and the cell morphology was observed. The number of cell adhesion is correlated with the PL(D)LA content, and the cell morphology is correlated with the MPC unit content. The porous scaffold was prepared by the formation of a stereocomplex between the PLLA and PDLA, and the cell adhesion and following cell intrusion was then evaluated. The fibroblast cells adhered on the surface and intruded into the scaffold through the connecting pores after 24 h. The cell morphology became round shape from spreading with the decreasing PLLA (or PDLA) content in the copolymer. It is considered that the change in the cell morphology would be induced by the MPC unit as cytocompatible unit. These findings suggest that the porous scaffold makes it possible to have cytocompatibility and to produce three-dimensional tissue regeneration.

A porous scaffold as a cell-compatible material was designed and prepared using a phospholipid copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and enantiomeric macromonomers, the poly(L-lactic acid) (PLLA) macromonomer, and poly(D-lactic acid) (PDLA) macromonomer. On the basis of the wide-angle X-ray diffraction and differential scanning calorimetry measurements, the formation of a stereocomplex between the PLLA and PDLA segments of the copolymer was observed on the porous scaffold. The porous structure was prepared by a sodium chloride leaching technique, and the pore was linked to the scaffold. The pore size was confirmed by scanning electron microscopy and found to be ca. 200 microm. These observations suggest that the porous scaffold makes it possible to produce cell-compatible materials, which may involve the following advantages for tissue engineering: (i) cell compatibility using phospholipid copolymer, (ii) adequate cell adhesion by poly(lactic acid), and (iii) complete disappearance of scaffold by dissociation of stereocomplex. The cell experiment using the porous scaffold will be the next subject and reported in a forthcoming paper.