Abstract Development of living cell-based devices holds great promise in many biomedical and industrial applications. To increase our understanding of the process, we investigated the biological and ...electrochemical properties of a redox phospholipid polymer hydrogel containing an electron-generating bacteria ( Shewanella oneidensis MR-1). A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine- co - n -butyl methacrylate- co - p -vinylphenylboronic acid- co -vinylferrocene) (PMBVF), was our choice for incorporation into a hydrogel matrix that promotes encapsulation of bacteria and acts as an electron transfer mediator. This hydrogel formed spontaneously and encapsulated Shewanella in three-dimensional structures. Visual analysis showed that the encapsulated Shewanella maintained viability and metabolic activity even after long-term storage. Cyclic voltammetry measurement indicated that the PMBVF/poly(vinyl alcohol) (PMBVF/PVA) hydrogel had stable and high electron transfer efficiency. Amperometric measurement showed that the hydrogel could maintain the electron transfer efficiency even when Shewanella was encapsulated. Thus, the PMBVF/PVA hydrogel not only provides a mild environment for long-term bacterial survival but also maintains electron transfer efficiency from the bacteria to the electrode. We conclude that hydrogel/bacteria hybrid biomaterials, such as PMBVF/PVA/ Shewanella , may find application in the fabrication of living cell-based devices.
Abstract We fabricated multi-layered hydrogels on titanium alloy (Ti) surfaces by applying alternating layers of a water-soluble phospholipid polymer (PMBV) and polyvinyl alcohol (PVA). This was ...accomplished by a layer-by-layer (LbL) process that is based on the formation of reversible covalent bonds between the boronic acid subunits in the PMBV and the hydroxyl groups in the PVA. When placed in an aqueous medium, PMBV acquires a polymeric aggregate structure with hydrophobic domains that can effectively solubilize hydrophobic molecules such as the anticancer drug paclitaxel (PTX) used in this study. The PTX-containing PMBV layer acted as a reservoir in the multi-layered hydrogels. To obtain diverse release profiles, the PTX was loaded in either the top layer (top-type) or the bottom layer (bottom-type) of the hydrogels; additional layers of PMBV and PVA, without PTX, functioned as a diffusion-barrier. In cell culture experiments, top-type hydrogels demonstrated excessive suppression of human epidermal carcinoma A431 cell proliferation over 5 days due to the initial high concentration of released PTX. However, bottom-type hydrogels were able to maintain a constant cell number profile. The release of PTX from multi-layered hydrogels was governed by both diffusion through the diffusion-barrier and dissociation of the hydrogel through an exchange reaction of phenylboronic acid subunits with the low-molecular weight d -glucose in the cell culture medium. In the cell culture experiments, the cell cycle was arrested in S and G2/M phases, as expected following PTX-mediated growth inhibition; control hydrogels did not demonstrate any appreciable cell cycle arrest. We concluded that cell proliferation could be controlled by the concentration of PTX released from the multi-layered hydrogels prepared through the LbL process. This system when used to solubilize bioactive agents at an appropriate layer within the hydrogel has potential for localized and surface-mediated delivery of bioactive molecules from biomedical devices.
Hydration of polymer chains plays a key role for determining the extent of protein adsorption on polymeric materials. Here we investigated the hydration of poly(2-methacryloyloxyethyl ...phosphorylcholine (MPC)) chains, which resist protein adsorption and following cell adhesion effectively. The hydration was compared with that of poly(methoxy oligo(ethylene glycol)-monomethacrylate (Me(EG)nMA)) chains, which also have hydrophilic units. The poly(MPC) and poly(Me(EG)nMA) hydrogels with equilibrium water contents (EWCs) in the range from 86 to 97wt% were prepared. By differential scanning calorimetric measurements, water in both the hydrogels was classified into two states: freezable and nonfreezable water. The poly(MPC) hydrogels had larger nonfreezable water than the poly(Me(EG)nMA) hydrogels even when their EWCs were similar, which indicated the higher hydrating ability of poly(MPC) chains. We suggested that the difference in the amount of nonfreezable water around polymer chains may influence the degree of protein adsorption resistance after contact with body fluid for a long period.
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Magnetic polymer beads, composed of a polystyrene core and hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) graft layer, containing magnetic nanoparticles were prepared for analyzing ...genes in the cells. The initiator group for atom transfer radical polymerization (ATRP) was provided on the surface of the beads and polymerization of MPC created the poly(MPC) graft layer. The terminal bromine group of poly(MPC) chains converted to a reactive group. Streptavidin was immobilized using the terminal reactive group in the poly(MPC) chains for capturing the biotinated DNA primer of the polymerase chain reaction (PCR). After selective binding of three kinds of messenger RNA from the cell lysate, PCR was carried out to increase complementary DNA. The polymer beads were stable even under the PCR thermal cycling conditions, and dispersed again easily. The magnetic polymer beads can be candidate solid support for PCR of cell lysates.
Water-soluble and cytocompatible polymers were investigated to enhance a transporting efficiency of biomolecules into cells in vitro. The polymers composed of a 2-methacryloyloxyethyl ...phosphorylcholine (MPC) unit, a hydrophobic monomer unit, and a cationic monomer unit bearing an amino group were synthesized for complexation with model biomolecules, siRNA. The cationic MPC polymer was shown to interact with both siRNA and the cell membrane and was successively transported siRNA into cells. When introducing 20–50 mol% hydrophobic units into the cationic MPC polymer, transport of siRNA into cells. The MPC units (10–20 mol%) in the cationic MPC polymer were able to impart cytocompatibility, while maintaining interaction with siRNA and the cell membrane. The level of gene suppression of the siRNA/MPC polymer complex was evaluated in vitro and it was as the same level as that of a conventional siRNA transfection reagent, whereas its cytotoxicity was significantly lower. We concluded that these cytocompatible MPC polymers may be promising complexation reagent for introducing biomolecules into cells, with the potential to contribute to future fields of biotechnology, such as in vitro evaluation of gene functionality, and the production of engineered cells with biological functions.
► Polymeric electron transfer mediator/immobilized enzyme multilayer was fabricated. It was achieved by layer-by-layer processed for molecular assembly. ► The multilayer showed good electrochemical ...performance on Au electrode. ► Concentration of glucose could be measured using the electrode efficiently.
The layer-by-layer (LBL) construction of an enzyme electrode covered with a multilayer structure alternately composed of a polymeric electron transfer mediator and a polymer-modified enzyme was examined. Poly(2-methacryloyloxyethyl phosphorylcholine-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMVF) was synthesized and used as a polymeric electron transfer mediator. Glucose oxidase (GOx) was selected as a model enzyme and poly(vinyl alcohol) (PVA) chains were bound to the GOx (GOx-PVA) under mild conditions. The PMVF and PVA formed a gel spontaneously through a selective reaction between phenylboronic acid units and hydroxyl groups in both polymers. Using the spin coating technique, a repeating PMVF/GOx-PVA multilayer was fabricated on the surface of an Au electrode. The thickness of each PMVF/GOx-PVA layer was around 5.8nm, corresponding to the dimensions of GOx. The electrochemical performance of the electrode was evaluated in glucose concentration measurement. The oxidation current of glucose by GOx was measured at 0.38V (vs. Ag/AgCl), verifying that ferrocene units in the PMVF of the hydrogel electrically wired the immobilized GOx. Moreover, the current increased with the number of PMVF/GOx-PVA layers. That is, both intermolecular electron transfer between each individual layer and the presence of a freely diffusing substrate in the hydrogel were achieved. We conclude that a LBL structure constructed from PMVF and a PVA-modified enzyme is effective for use in developing bioelectronic devices that employ enzyme molecules.
A redox‐active phospholipid polymer with a phospholipid‐mimicking structure (2‐methacryloyloxyethyl phosphorylcholine; MPC) was synthesized to construct a biocompatible electron mediator between ...bacteria and an electrode. In this study, a copolymer of MPC and vinylferrocene VF; poly(MPC‐co‐VF) (PMF) is synthesized. When PMF is added to cultures of the bacterial species Escherichia coli (Gram negative) and Lactobacillus plantarum (Gram positive), which have different cell wall structures, a catalytic current mediated by PMF is observed. In addition, growth curves and live/dead assays indicate that PMF does not decrease metabolic activity or cell viability. These results indicate that PMF mediates extracellular electron transfer across bacterial cell membranes without associated cytotoxicity.
Biocompatible electron shuttle: A redox‐active phospholipid polymer with a phospholipid‐mimicking structure is synthesized to construct a biocompatible electron mediator between bacteria and an electrode.
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► Easily applicable and generic approach to prepare multilayer hydrogels with controlled thickness was developed. ► We demonstrated the 3D precise spatial multicellular hydrogel ...matrix with cells could maintain their activity for at least 24h. ► This method can be used to quantitatively study cell-to-cell interactions mediated by the diffusion of soluble factors.
A precise spatial multicellular polymer hydrogel matrix was prepared by successive assembly of cell-laden hydrogel layers alternated by hydrogel layers without cells based on the spontaneous hydrogel formation between 2 aqueous polymer solutions. The polymers used were a water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA). Each cell-laden layer was deposited as a cell-laden PMBV solution on a PMBV/PVA precursor film. PMBV/PVA multilayer hydrogel was stacked on the top of a cell-laden layer by sequential coating with spinning of the PMBV and PVA solutions. This process allowed the formation of the PMBV/PVA multilayer hydrogel with finely controlled thickness. Finally, we succeeded in cell patterning by using a multilayer hydrogel matrix, forming a sandwich of 2 cell-laden layers separated by a PMBV/PVA multilayer hydrogel. The cells remained alive during the spinning process and maintained their metabolism for at least 24h. This precise spatial multicellular PMBV/PVA hydrogel can be used to examine interactions between many different cells and construct customized microenvironments for multicellular co-cultures.
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•Spontaneously forming cytocompatible polymer hydrogels are reviewed.•The special properties of PMBV/PVA hydrogels are briefly explained.•Spinning-assisted LbL method was used to ...fabricate multilayered PMBV/PVA hydrogels.•This multilayered hydrogel was applied for regulating cell functions in 3D matrix.
Given the importance of regulating living cell functions in three-dimensional (3D) environments, we have designed a cytocompatible and spontaneously forming polymer hydrogel matrix. Water-soluble phospholipid polymers bearing a phenylboronic acid unit, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV), and poly(vinyl alcohol) (PVA), are candidate systems for preparing the hydrogel matrices. Aqueous solutions of PMBV and PVA can be used to form hydrogels based on reversible complexation between the phenylboronic acid groups in PMBV and the diol groups in PVA, even under cell culture conditions. Uniform cell encapsulation is easily achieved with hydrogel formation, and cells survived well in the hydrogel. By applying a spinning-assisted layer-by-layer (LbL) process, multilayered PMBV/PVA hydrogels containing living cells can be assembled. This multilayered hydrogel, which mimics the stratified structure of in vivo tissues, allows the layer-specific encapsulation of cells and temporary storage of bioactive molecules. Distance-dependent cell–cell interactions are investigated using the multilayered hydrogel where two cell-laden layers are separated by a finely controlled multilayered hydrogel. In addition, dual-crosslinked multilayered hydrogels are also assembled by alternative depositions of PMBV and photoreactive-PVA solutions, followed by photoirradiation. The dual-crosslinked hydrogel has a lower diffusivity of bioactive molecules than that of single-crosslinked hydrogel and therefore acts as a diffusion-controlling barrier. We demonstrate the utility of this dual-crosslinked hydrogel by examining ways to regulate the diffusion of bioactive molecules in the hydrogel and investigating the diffusion-dependent effects on cell behavior. In conclusion, these hydrogel matrices can provide insights into the regulation of cell behavior in 3D matrices. In turn, our results may contribute to the future design of 3D cell culture systems and tissue regenerated medicine based on cell engineering.
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