Simultaneous enhancement of cell proliferation and thermally induced harvest efficiency based on temperature-responsive cationic copolymer-grafted microcarriers

The development of large-scale suspension cell cultures using microcarriers has long been a focus of attention in the fields of pharmacy and biotechnology. Previously, we developed cell-detachable microcarriers based on temperature-responsive poly(N-isopropylacrylamide) (PIPAAm)-grafted beads, on which adhering cells can be noninvasively harvested by only reducing the temperature without the need for proteolytic enzyme treatment. In this study, to improve the cell harvest efficiency from bead surfaces while maintaining cell adhesion and proliferation properties, we prepared temperature-responsive cationic copolymer-grafted beads bearing a copolymer brush consisting of IPAAm, positively charged quaternary amine monomer (3-acrylamidopropyl trimethylammonium chloride; APTAC), and hydrophobic monomer (N-tert-butylacrylamide; tBAAm). The incorporation of positively charged APTAC into the grafted copolymer brush facilitated bead dispersibility in a cell culture system containing Chinese hamster ovary (CHO-K1) cells and consequently allowed for enhanced cell proliferation in the system compared to that of unmodified CMPS and conventional PIPAAm homopolymer-grafted beads. Additionally, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted beads exhibited the most rapid and efficient cell detachment behavior after the temperature was reduced to 20 °C, presumably because the highly hydrated APTAC promoted the overall hydration of the P(IPAAm-co-APTAC-co-tBAAm) chains. Therefore, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted microcarriers are effective in facilitating both cell proliferation and thermally induced cell detachment in a suspension culture system.     

Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands

In this study, specific interactions between immobilized RGDS (Arg-Gly-Asp-Ser) cell adhesion peptides and cell integrin receptors located on cell membranes are controlled in vitro using stimuli-responsive polymer surface chemistry. Temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(IPAAm-co-CIPAAm)) copolymer grafted onto tissue culture grade polystyrene (TCPS) dishes permits RGDS immobilization. These surfaces facilitate the spreading of human umbilical vein endothelial cells (HUVECs) without serum depending on RGDS surface content at 37 degrees C (above the lower critical solution temperature, LCST, of the copolymer). Moreover, cells spread on RGDS-immobilized surfaces at 37 degrees C detach spontaneously by lowering culture temperature below the LCST as hydrated grafted copolymer chains dissociate immobilized RGDS from cell integrins. These cell lifting behaviors upon hydration are similar to results using soluble RGDS in culture as a competitive substitution for immobilized ligands. Binding of cell integrins to immobilized RGDS on cell culture substrates can be reversed spontaneously using mild environmental stimulation, such as temperature, without enzymatic or chemical treatment. These findings are important for control of specific interactions between proteins and cells, and subsequent "on-off" regulation of their function. Furthermore, the method allows serum-free cell culture and trypsin-free cell harvest, essentially removing mammalian-sourced components from the culture process.     

Terminally functionalized thermoresponsive polymer brushes for simultaneously promoting cell adhesion and cell sheet harvest

For preparing cell sheets effectively for cell sheet-based regenerative medicine, cell-adhesion strength to thermoresponsive cell culture surfaces need to be controlled precisely. To design new thermoresponsive surfaces via a terminal modification method, thermoresponsive polymer brush surfaces were fabricated through the surface-initiated reversible addition-fragmentation chain transfer (RAFT) radical polymerization of N-isopropylacrylamide (IPAAm) on glass substrates. The RAFT-mediated grafting method gave dithiobenzoate (DTB) groups to grafted PIPAAm termini, which can be converted to various functional groups. In this study, the terminal carboxylation of PIPAAm chains provided high cell adhesive property to thermoresponsive surfaces. Although cell adhesion is generally promoted by a decrease in the grafted PIPAAm amount, the decrease also decelerated thermally-induced cell detachment, whereas the influence of terminal modification was negligible on the cell detachment. Consequently, the terminally modified PIPAAm brush surfaces allowed smooth muscle cells (SMCs) to simultaneously adhere strongly and detach themselves rapidly. In this study, SMCs were unable to reach a confluent monolayer on as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm(2)) without terminal carboxylation due to their insufficient cell-adhesion strength. On the other hand, though a decrease in the PIPAAm amount allowed SMCs to form a confluent cell monolayer on the PIPAAm brush surface, the SMCs were unable to be harvested as a monolithic cell sheet by low-temperature culture at 20 °C. Because of their unique property, only terminal-carboxylated PIPAAm brush surfaces achieved rapid harvesting of complete cell sheets by low-temperature culturing.     

Immobilization of galactose ligands on acrylic acid graft-copolymerized poly(ethylene terephthalate) film and its application to hepatocyte culture

Surface modification of argon-plasma-pretreated poly(ethylene terephthalate) (PET) films via UV-induced graft copolymerization with acrylic acid (AAc) was carried out. Galactosylated surfaces were then obtained by coupling a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the AAc graft chains with the aid of a water-soluble carbodiimide (WSC) and N-hydroxysulfosuccinimide (sulfo-NHS). The modified PET films were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact-angle measurements. The galactosylated PET films were used as substrates for hepatocyte culture. The effects of surface carboxyl group concentration on the extent of galactose ligand immobilization, the extent of hepatocyte attachment, and the surface morphology were investigated. The amount of the galactose ligands immobilized on the PET surface increased with the AAc polymer graft concentration. AFM images revealed that the surface roughness of the PET film increased after graft copolymerization with AAc, but did not change appreciably with the subsequent immobilization of the galactose ligands. At the surface carboxyl group concentration of about 0.56 micromol/cm(2) or galactose ligand concentration of about 0.51 micromol/cm(2), the hepatocyte culture on the galactosylated surface exhibited the optimum concentration and physiological functions and formed aggregates or spheroids after just 1 day of culture. The albumin and urea synthesis functions of these hepatocytes were comparable to or higher than those of the hepatocytes cultured on the collagen-modified PET substrates.     

Synthesis and characterization of injectable poly(N-isopropylacrylamide-co-acrylic acid) hydrogels with proteolytically degradable cross-links

Hydrogels composed of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) were prepared by redox polymerization with peptide cross-linkers to create an artificial extracellular matrix (ECM) amenable for testing hypotheses regarding cell proliferation and migration in three dimensions. Peptide degradable cross-linkers were synthesized by the acrylation of the amine groups of glutamine and lysine residues within peptide sequences potentially cleavable by matrix metalloproteinases synthesized by mammalian cells (e.g., osteoblasts). With the peptide cross-linker, loosely cross-linked poly(N-isopropylacrylamide-co-acrylic acid) [P(NIPAAm-co-AAc)] hydrogels were prepared, and their phase transition behavior, lower critical solution temperature (LCST), water content, and enzymatic degradation properties were investigated. The peptide-cross-linked P(NIPAAm-co-AAc) hydrogels were pliable and fluidlike at room temperature and could be injected through a small-diameter aperture. The LCST of peptide-cross-linked hydrogel was influenced by the monomer ratio of NIPAAm/AAc but not by cross-linking density within the polymer network. A peptide-cross-linked hydrogel with a 97/3 molar ratio of NIPAAm/AAc exhibited a LCST of approximately 34.5 degrees C. Swelling was influenced by NIPAAm/AAc monomer ratio, cross-linking density, and swelling media; however, all hydrogels maintained more than 90% water even at 37 degrees C. In enzymatic degradation studies, breakdown of the peptide-cross-linked P(NIPAAm-co-AAc) hydrogels was dependent on both the concentration of collagenase and the cross-linking density. These results suggest that peptide-cross-linked P(NIPAAm-co-AAc) hydrogels can be tailored to create environmentally-responsive artificial extracellular matrixes that are degraded by proteases.     

Thermoresponsive protein adsorption of poly(N-isopropylacrylamide)-modified streptavidin on polydimethylsiloxane microchannel surfaces

The control of protein adsorption on microchannel surfaces is important for biosensors. In this study, we demonstrated protein adsorption method that is controlled through temperature change, i.e., thermoresponsive protein adsorption, on polydimethylsiloxane (PDMS) microchannel surfaces using a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). To provide general protein adsorption control method, we adopted biotin-streptavidin chemistry and synthesized streptavidin covalently modified with PNIPAAm (PNIPAAm-StAv). Modification of streptavidin, a hydrophilic protein, with PNIPAAm induced successful thermoresponsive adsorption on a PDMS microchannel surfaces: PNIPAAm-StAv adsorbed at 37 degrees C and desorbed at 10 degrees C on the surfaces. We also demonstrated the thermoresponsive adsorption of biotinylated immunoglobulin G (IgG-b) using PNIPAAm-StAv. Conjugation of IgG-b with PNIPAAm-StAv induced successful thermoresponsive IgG-b adsorption on PDMS. Modification of PDMS surfaces with PNIPAAm reduced physical adsorption of the partially hydrophobic IgG-b on the surface and contributed to the high-contrast thermoresponsive adsorption of IgG-b: less than 1% of the IgG-b adsorbed at 37 degrees C was detected after the PNIPAAm-PDMS surface was washed at 10 degrees C. The controllable adsorption of this system is expected to be applied to the regeneration of biosensor chips and to on-chip protein manipulation.     

Poly(N-isopropylacrylamide)-based semi-interpenetrating polymer networks for tissue engineering applications. 1. Effects of linear poly(acrylic acid) chains on phase behavior

Poly(N-isopropylacrylamide)-based [P(NIPAAm)-based] semi-interpenetrating polymer networks (semi-IPNs), consisting of P(NIPAAm)-based hydrogels and linear poly(acrylic acid) [P(AAc)] chains, were synthesized, and the effects of the P(AAc) chains on semi-IPN injectability and phase behavior were analyzed. In P(NIPAAm)- and P(NIPAAm-co-AAc)-based semi-IPN studies, numerous reaction conditions were varied, and the effects of these factors on semi-IPN injectability, transparency, phase transition, lower critical solution temperature (LCST), and volume change were examined. The P(AAc) chains did not significantly affect the LCST or volume change of the semi-IPNs, compared to control hydrogels. However, the P(AAc) chains affected the injectability, transparency, and phase transition of the matrices, and these effects were dependent on chain amount and molecular weight (MW) and on interactions between the P(AAc) chains and the solvent and/or copolymer chains in P(NIPAAm-co-AAc) hydrogels. These results can be used to design "tailored" P(NIPAAm)-based semi-IPNs that have the potential to serve as functional scaffolds in tissue engineering applications.     

Surface-grafted, environmentally sensitive polymers for biofilm release

Controlling bacterial biofouling is desirable for almost every human enterprise in which solid surfaces are introduced into nonsterile aqueous environments. One approach that is used to decrease contamination of manufactured devices by microorganisms is using materials that easily slough off accumulated material (i.e., fouling release surfaces). The compounds currently used for this purpose rely on low surface energy to inhibit strong attachment of organisms. In this study, we examined the possible use of environmentally responsive (or "smart") polymers as a new class of fouling release agents; a surface-grafted thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAAM), was used as a model compound. PNIPAAM is known to have a lower critical solubility temperature of approximately 32 degrees C (i.e., it is insoluble in water at temperatures above 32 degrees C and is soluble at temperatures below 32 degrees C). Under experimental conditions, >90% of cultured microorganisms (Staphylococcus epidermidis, Halomonas marina) and naturally occurring marine microorganisms that attached to grafted PNIPAAM surfaces during 2-, 18-, 36-, and 72-h incubations were removed when the hydration state of the polymer was changed from a wettability that was favorable for attachment to a wettability that was less favorable. Of particular significance is the observation that an organism known to attach in the greatest numbers to hydrophobic substrata (i.e., H. marina) was removed when transition of PNIPAAM to a more hydrated state occurred, whereas an organism that attaches in the greatest numbers to hydrophilic substrata (i.e., S. epidermidis) was removed when the opposite transition occurred. Neither solvated nor desolvated PNIPAAM exhibited intrinsic fouling release properties, indicating that the phase transition was the important factor in removal of organisms. Based on our observations of the behavior of this model system, we suggest that environmentally responsive polymers represent a new approach for controlling biofouling release.     

Protein-reactive, thermoresponsive copolymers with high flexibility and biodegradability

A family of injectable, biodegradable, and thermosensitive copolymers based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide, and a macromer polylactide-hydroxyethyl methacrylate were synthesized by free radical polymerization. Copolymers were injectable at or below room temperature and formed robust hydrogels at 37 degrees C. The effects of monomer ratio, polylactide length, and AAc content on the chemical and physical properties of the hydrogel were investigated. Copolymers exhibited lower critical solution temperatures (LCSTs) from 18 to 26 degrees C. After complete hydrolysis, hydrogels were soluble in phosphate buffered saline at 37 degrees C with LCSTs above 40.8 degrees C. Incorporation of type I collagen at varying mass fractions by covalent reaction with the copolymer backbone slightly increased LCSTs. Water content was 32-80% without collagen and increased to 230% with collagen at 37 degrees C. Hydrogels were highly flexible and relatively strong at 37 degrees C, with tensile strengths from 0.3 to 1.1 MPa and elongations at break from 344 to 1841% depending on NIPAAm/HEMAPLA ratio, AAc content, and polylactide length. Increasing the collagen content decreased both elongation at break and tensile strength. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Degradation products were shown to be noncytotoxic. Cell adhesion on the hydrogels was 30% of that for tissue culture polystyrene but increased to statistically approximate this control surface after collagen incorporation. These newly described thermoresponsive copolymers demonstrated attractive properties to serve as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications.     

Regulation of cell adhesion using a signal-responsive membrane substrate

We have developed a novel cell culture material that regulates cell adhesion by changes in potassium ion concentration. The material is a polyethylene substrate grafted to a copolymer of the thermoresponsive polymer N-isopropylacrylamide (NIPAM) and benzo-18-crown- 6-acrylamide (BCAm), with a pendant crown ether as sensor. The crown ether recognizes potassium ion concentrations and NIPAM conformational changes lead to changes in the hydrophobicity/hydrophilicity balance of the entire polymer at constant cell culture temperatures. Although cells were successfully cultured on the ion recognition material in normal culture medium at 37 degrees C, the cells could be detached from the material surface by adding potassium ions alone, without proteolytic enzymes, because the surface to which the cells were attached altered its surface characteristics to a more hydrophilic state. Therefore, cell layers with intact cell-to-cell junctions and high activities were successfully recovered. Furthermore, by changing the target sensors, this material will be able to control cell adhesion through various cellular signals.     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Surface functionalization of polypyrrole film with glucose oxidase and viologen

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

Role of a membranous sialyltransferase complex in the synthesis of surface polymers containing polysialic acid in Escherichia coli. Temperature-induced alteration in the assembly process.

Membrane-associated sialyltransferase complexes of Escherichia coli K-235 catalyze the synthesis of sialyl polymers which remain associated with the cell envelope. Sialyl monophosphorylundecaprenol is an intermediate in the formation of these unique surface structures, and fluidity of the lipid phase is required for the proper function of the enzyme complex (Troy, F.A., Vijay, I.K., and Tesche, N. (1975) J. Biol. Chem. 250, 156-163, 164-170). In membranes containing an increased unsaturated fatty acid content of the phospholipids, obtained by growing cells at 15 degrees C, synthesis of polysialic acid was uncoupled from synthesis of the sialyl lipid-linked intermediate. Using reconstruction experiments, the importance of the role of an endogenous acceptor in polymer formation was suggested by the unexpected finding that polysialic acid synthesis could be reactivated in inactive membranes by the addition of an exogenous acceptor which contained sialic acid. Concomitant with polymer synthesis was a rapid loss of labeled sialic acid from the lipid phase. The activated sialic acid was shown to be transferred directly to the exogenous acceptor. These results establish: 1) that the temperature-induced alteration in polymer synthesis resulted from the inability of cells grown at 15 degrees C to either synthesize or assemble a functional endogenous acceptor and not from a defect in the synthesis of the sialyltransferase; 2) the intermediate precursor role of lipid-soluble sialic acid in sialyl polymer synthesis; and 3) that the exogenous acceptor served directly as an "acceptor" and not as a catalytic "effector" which stimulated an inactive membrane-enzyme complex. These results are in accord with the possibility that the low temperature-induced derangement in polymer formation is a consequence of the altered lipid structure resulting from the greater unsaturated fatty acid content in the membrane phospholipids. U-14C-labeled exogenous acceptor was isolated from the culture filtrate of cells grown at 37 degrees C and purified to homogeneity by preparative polyacrylamide gel electrophoresis. The pure acceptor was characterized structurally as a homopolymer of sialic acid with a degree of polymerization of approximately 12. Potassium borohydride reduction of the acceptor prior to complete hydrolysis with neuraminidase established that the polymer possessed a free reducing terminus of sialic acid. Subsequent structural studies showed that these oligomers of sialic acid appeared in the culture filtrate as a result of acid-catalyzed hydrolysis from membrane-associated polysialic acids of about 150 to 200 sialyl residues. Marked diminution of several membrane proteins was observed for cells grown at 15 degrees C. The possible relationship of these alterations to the upward shift in unsaturated lipids and to the loss of a functional endogenous acceptor is currently under study.     

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> ATCC 43961

The marine bacterium Saccharophagus degradans was investigated for the synthesis of polyhydroxyalkanoates (PHAs), using glucose as the sole source of carbon in a two-step batch culture. In the first step the microorganism grew under nutrient balanced conditions; in the second step the cells were cultivated under limitation of nitrogen source. The biopolymer accumulated in S. degradans cells was detected by Nile red staining and FT-IR analysis. From GC-MS analysis, it was found that this strain produced a homopolymer of 3-hydroxybutyric acid. The cellular polymer concentration, its molecular mass, glass transition temperature, melting point and heat of fusion were 17.2+/-2.7% of dry cell weight, 54.2+/-0.6 kDa, 37.4+/-6.0 degrees C, 165.6+/-5.5 degrees C and 59.6+/-2.2 J g(-1), respectively. This work is the first report determining the capacity of S. degradans to synthesize PHAs.     

Glucose-responsive polymer gel bearing phenylborate derivative as a glucose-sensing moiety operating at the physiological pH

The work attempts to prepare a totally synthetic, glucose-responsive polymer gel bearing a phenylborate derivative as a sensor moiety to glucose, for future use as a self-regulated insulin delivery system. The molecular strategies to enable the system to be operated under physiological conditions (pH 7.4, 37 degrees C) are presented that involve the use of a novel phenylborate derivative [4-(1,6-dioxo-2,5-diaza-7-oxamyl) phenylboronic acid: DDOPBA] possessing an appreciably low pK(a) ( approximately 7.8), the adoption of poly(N-isopropylmethacrylamide) (PNIPMAAm) for the main chain, which itself undergoes a sharp thermo-induced phase transition at its LCST around 40 degrees C, as well as the introduction of a carboxyl group of methacrylic acid as the third comonomer. Glucose-responsive behaviors of the obtained gels were evaluated based on the changes in the equilibrium swelling degree determined in the presence and the absence of glucose, for various pH and temperature conditions. As a consequence of the combined molecular effects, a sufficient sensitivity of the system was accomplished at physiological pH and in the temperature range close to the physiological condition such as 30 degrees C. Furthermore, the glucose-induced continuous volume changes of the gels were demonstrated under those conditions, which occurred in a remarkably concentration-dependent manner. In these experiments, the critical glucose concentrations to induce the gels' responses in the range of normoglycemic sugar level were observed. These observations may provide us with an excellent prospect for the use of the gel as a self-regulated, insulin-delivery system discretely switching the release at the normoglycemia.     

Preparation of thermoresponsive cationic copolymer brush surfaces and application of the surface to separation of biomolecules

We have prepared poly( N-isopropylacrylamide (IPAAm)- co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) brush-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) using the CuCl/CuCl 2/Me 6TREN catalytic system in 2-propanol at 25 degrees C for 16 h. The prepared temperature-responsive surfaces were characterized by chromatographic analysis using the modified silica beads as stationary phases. Chromatographic retention times for adenosine nucleotides in aqueous mobile phases were significantly increased compared to that previously reported for other cationic hydrogel surfaces, indicating that strong electrostatic cationic copolymer brush interactions occur between the surfaces and nucleotide analytes. Retention times for adenosine nucleotides significantly decreased with increasing column temperature, explained by the decreasing basicity in the copolymer with increasing temperature. Step-temperature gradients from 10 to 50 degrees C shorten ATP retention times. These results indicate that cationic copolymer brush surfaces prepared by ATRP can rapidly alter their electrostatic properties by changing aqueous temperature.     

Carbon nanotubes promote neuron differentiation from human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine and transplantation therapy due to their self-renewal and pluripotent properties. We report that 2D thin film scaffolds composed of biocompatible polymer grafted carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells into neuron cells while maintaining excellent cell viability. According to fluorescence image analysis, neuron differentiation efficiency of poly(acrylic acid) grafted CNT thin films is significant greater than that on poly(acrylic acid) thin films. When compared with the conventional poly-l-ornithine surfaces, a standard substratum commonly used for neuron culture, this new type thin film scaffold shows enhanced neuron differentiation. No noticeable cytotoxic effect difference has been detected between these two surfaces. The surface analysis and cell adhesion study have suggested that CNT-based surfaces can enhance protein adsorption and cell attachment. This finding indicates that CNT-based materials are excellent candidates for hESCs’ neuron differentiation.     

The kinetics of formation and structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine

A combination of differential scanning calorimetry (DSC) and X-ray diffraction have been used to study the kinetics of formation and the structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine (18:0-lysoPC). For water contents greater than 40 weight %, DSC shows a sharp endothermic transition at 27 degrees C (delta H = 6.75 kcal/mol) corresponding to a low-temperature phase----micelle transition. This sharp transition is not reversible, but is regenerated in a time and temperature-dependent manner. For example, with incubation at 0 degrees C the maximum transition enthalpy (delta H = 6.75 kcal/mol) is generated in about 45 min after an initial slow nucleation process of approx. 20 min. The kinetics of formation of the low-temperature phase is accelerated at lower temperatures and may be related to the disruption of 18:0-lysoPC micelles by ice crystal formation. X-ray diffraction patterns of 18:0-lysoPC recorded at 10 degrees C over the hydration range 20-80% are characteristic of a lamellar gel phase with tilted hydrocarbon chains with the bilayer repeat distance increasing from 47.6 A at 20% hydration to a maximum of 59.4 A at 39% hydration. At this maximum hydration, approx. 19 molecules of water are bound per molecule of 18:0-lysoPC. Electron density profiles show a phosphate-phosphate distance of 30 A, indicating an interdigitated lamellar gel phase for 18:0-lysoPC at all hydration values. The angle of chain tilt is calculated to be between 20 and 30 degrees. For water contents greater than 40%, this interdigitated lamellar phase converts to the micellar phase at 27 degrees C in a kinetically fast process, while the reverse (micelle----interdigitated bilayer) transition is a kinetically slower process (see also Wu, W. and Huang, C. (1983) Biochemistry 22, 5068-5073).     

Cell wall composition and protoplast regeneration in Candida albicans

The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.