Secretory function of adrenal chromaffin cells cultured on polypyrrole films.

Polypyrrole (PPy) is a conducting polymer and is obtained electrochemically on an electrode such as indium-tin oxide (ITO). In this study, in order to develop a novel cell-culture system which makes it possible to communicate with cultured mammalian cells, bovine adrenal chromaffin cells were cultured on PPy-coated ITO plates for 7 days and the influence of PPy-coating on the cell functions was investigated. Since the chromaffin cells synthesize and secrete catecholamines such as adrenaline and noradrenaline, the amount of synthesized and released catecholamines from the chromaffin cells cultured on PPy-coating and ITO itself were measured. The cells on the PPy-coated ITO plate could be kept in culture, without any significant changes in morphology and in the secretory responsiveness to acetylcholine as compared with those of the cells cultured on collagen. On the contrary, the cells on the ITO plate lost the responsiveness, while the amount of catecholamines synthesized was affected little by both PPy and ITO surfaces. It is suggested that PPy supports the secretory function of the chromaffin cells when they are cultured on it. This paper describes that PPy films are applicable as a polymer-modified electrode which support the cell function without collagen.

Change in cytoplasmic free Ca2+ concentration in rabbit platelets contacting with albumin coated and uncoated polystyrene surfaces.

Direct measurement of cytoplasmic free Ca2+ concentration in rabbit platelets in contact with polystyrene latexes was examined by monitoring an intracellular trapped fluorescent indicator dye, Fura 2, as a quantitative evaluation of platelet activation on material surfaces. It was found that an increase in cytoplasmic free calcium levels in platelets in contacting latexes was strongly reduced by the adsorption of albumin on the latex surfaces. Further, such an elevation of cytoplasmic free calcium levels was found to require a calcium influx from an extracellular medium across the platelet-plasma membrane.

In vitro and in vivo studies on antithrombogenicity of poly(propylene oxide) segmented nylon 610 in relation to its crystalline-amorphous microstructure.

In vitro and in vivo antithrombogenicity of poly(propylene oxide) segmented nylon 610 was investigated. For the in vitro simulation of thrombosis on copolymer surfaces, a modified microsphere column method evaluated the thrombosis time of the copolymer column. For the in vivo evaluation of the antithrombogenicity of copolymer surfaces, small diameter tubing, precoated with copolymer, was implanted in rabbits as an arteriovenous shunt to determine the time of patency for the copolymer tubing. In both the in vitro and in vivo tests, the copolymer having a particular size and distribution of crystalline and amorphous microphases on its surface exhibited excellent antithrombogenicity; the copolymer with a long period of approximately 12 nm and a crystallite thickness of 6.5 nm suppressed the activation process of adhering platelets to show thromboresistant property. This paper emphasizes the importance of controlling the crystalline-amorphous microstructure for the antithrombogenicity of polymer surfaces with a semicrystalline microstructure.

Reversibility of granulocyte adhesion using polyamine-grafted nylon-6 as a new column substrate for granulocyte separation.

Reversibility of leucocytes adhered onto the surface of polyamine-grafted nylon-6 was investigated to estimate its feasibility as a new column substrate for granulocyte separation from whole blood. Polyamine-grafted nylon-6 was synthesized by a radical polymerization of 2-diethylaminoethyl methacrylate monomer onto nylon-6. The surfaces of these graft copolymers were found to show a microphase-separated structure composed of island-like phases of cationic polyamine and continuous phases of nonionic nylon-6. Interaction between leucocytes and these copolymer surfaces was studied by passing rabbit heparinized blood through a column packed with glass beads precoated with these copolymers. Columns of these copolymers showed a selective adhesion of granulocytes among leucocyte populations. Also, the adhering granulocytes were able to be recovered from the column by a gentle elution procedure. From these results, it was concluded that polyamine-grafted nylon-6 having a microphase-separated structure is suitable for use as a column substrate in granulocyte separation from whole blood.

Temperature-modulated platelet and lymphocyte interactions with poly(N-isopropylacrylamide)-grafted surfaces.

Temperature-responsive semitelechelic poly(N-isopropylacrylamide) (PIPAAm) bearing a carboxyl end group has been chemically immobilized on aminated polystyrene particle surfaces via condensation reaction. PIPAAm-grafted particles were uniformly suspended in aqueous media at lower temperatures. With increasing temperature, PIPAAm-grafted particles aggregated and precipitated. Such reversible changes in particle colloidal behaviour was correlated to temperature-modulated hydrophilic/hydrophobic changes of particle surfaces modified by PIPAAm hydration/dehydration with temperature changes. Interactions between platelets and PIPAAm-grafted surfaces were studied by monitoring cytoplasmic free Ca2+ concentration ([Ca2+]i) changes in platelets using intracellularly-trapped Ca2+ indicator dye, Fura 2, at various temperatures. Although changes in [Ca2+]i in platelets in contact with PIPAAm-grafted particles were not observed below the critical temperature of PIPAAm, significant changes in [Ca2+]i in platelets were induced by contact with particles above this critical temperature. Furthermore, temperature-modulated cell adsorption/desorption control by PIPAAm-grafted particles was investigated using a particle aggregation assay in the presence of lymphocytes. Below the critical temperature of PIPAAm, mixed suspensions were completely homogeneous due to minimal interaction between lymphocytes and hydrated particles. In contrast, aggregated precipitates were observed by increasing the suspension temperature above the critical temperature of PIPAAm resulting from strong hydrophobic interactions between particles with lymphocytes. These precipitates are reversibly resuspended in cold buffer. The feasibility of cell activation/inactivation or cell attachment/detachment control by temperature-modulated surface changes is attractive for suspension cell culture and drug delivery at targeted sites in vivo.

Temperature modulated solubility-activity alterations for poly(N-isopropylacrylamide)-lipase conjugates.

Chemical modification of proteins by use of functional polymers is expected to endow them with new properties without destroying their native functions, thus providing useful materials for application in different fields. We have synthesized poly(N-isopropylacrylamide) [poly(IPAAm)] co-oligomer with N,N-dimethylacrylamide (DMAAm) and reactive end groups by telomerization of IPAAm. This co-oligomer exhibits a lower critical solution temperature (LCST) at 37 degrees C. Using this temperature-responsive semitelechelic co-oligomer, we prepared polymer-enzyme conjugates of lipase by covalent coupling via carboxyl end-groups. This bioconjugate exhibits a LCST at 37 degrees C, having rapid, reversible hydration-dehydration changes due to highly mobile free polymer end groups. The conjugate retained its native enzymatic activity below this critical temperature, above which it precipitated and its catalytic function was shut off. This conjugate can be readily separated from reaction mixtures as a precipitate by simple temperature changes after reaction and reused in cycles without denaturation. Such a modulated system is attractive for application as a novel bioreactor system.

Effect of microstructure of poly(propylene-oxide)-segmented polyamides on platelet adhesion.

The relationship between microstructure and platelet adhesivity of six types of poly(propylene oxide) (PPO)-segmented polyamides based on the polyamide segments nylon 210, 310, 410, 510, 610, and 710 were investigated. These multiblock PPO-segmented copolymers were prepared by interfacial polycondensation. Physical characterization of these copolymers was by means of thermal analysis, transmission electron microscope, wide-angle X-ray diffraction (WAXD), and small-angle X-ray scattering (SAXS). The WAXD and SAXS measurements showed that the copolymers had microstructures containing crystalline and amorphous phases and that these microstructures, represented by means of crystallite thickness and long period, varied with incorporation of PPO segments. Blood compatibility of these copolymers was evaluated by estimating the amount of adhering platelets on the copolymer surfaces. The amount of adhering platelets was minimum for the surfaces of the copolymers having a crystallite thickness of 6.0-6.5 nm and a long period of 12-13 nm. This result suggests that the particular size and distribution of the crystalline and amorphous phases in the copolymer could be determining factors for suppressing platelet adhesion on the copolymer surface, and that the control of these factors could lead to ideal antithrombogenic polymers.

Temperature-responsive bioconjugates. 2. Molecular design for temperature-modulated bioseparations.

We have synthesized carboxyl semitelechelic oligo(N-isopropylacrylamide) (OIPAAm) using radical telomerization with 3-mercaptopropionic acid. This telomerization is also effective for the synthesis of carboxyl semitelechelic co-oligomers of IPAAm with butyl methacrylate (BMA) as hydrophobic or N,N-dimethylacrylamide (DMAAm) as hydrophilic comonomers. All co-oligomers are highly water-soluble at lower temperatures and exhibit phase separation with increasing temperature. Pure OIPAAm exhibits a lower critical solution temperature (LCST) at 32 degrees C, and the LCST for co-oligomers can be controlled to increase over 32 degrees C with increasing DMAAm composition and to decrease below 32 degrees C with increasing BMA composition. OIPAAm was grafted to bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) by activated ester-amine coupling. These OIPAAm-biomolecule conjugates maintain their temperature responses, are soluble in cold water, and precipitate over a range of temperatures related to oligomer content. Conjugates could be selectively precipitated and independently separated from conjugate solution mixtures with increasing temperature. In this case, the number of OIPAAm molecules attached to a conjugate affects the aggregate sizes of precipitated conjugates in mixtures. Both conjugate mixture ratios and solution concentrations influence the contamination of oligo(IPAAm-co-DMAAm)-BSA conjugates in precipitated oligo(IPAAm-co-BMA)-BPF conjugates. Furthermore, precipitated conjugates separated using centrifugation and filtration redissolve in water and maintain their biofunctionality, indicating the potential of strategy in reversible bioreactors and protein separations.

Effect of molecular architecture of poly(N-isopropylacrylamide)-trypsin conjugates on their solution and enzymatic properties.

Polymer-enzyme hybrid conjugates modified by a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), have been synthesized. We have investigated the molecular architecture of PIPAAm-enzyme conjugates by preparing two types of PIPAAm-trypsin conjugates, wherein PIPAAm chains are attached by either single-end or multipoint chemistry. A semitelechelic co-oligomer (IDc) was attached to trypsin by single-point conjugation (IDc-trypsin). A copolymer (PIDAAc) consisting of acrylic acid and IPAAm randomly linked in polymer chains was attached to trypsin using multipoint conjugation (PIDAAc-trypsin). Both conjugates exhibited reversible temperature-responsive phase separation. The IDc-trypsin conjugate exhibited phase separation at the same temperature as pure IDc, due to the highly mobile free polymer end group which remains sensitive to small temperature changes. The PIDAAc-trypsin conjugate precipitated at higher temperatures than pure PIDAAc, whose movement was restricted by multiple binding points. Enzyme stability in solution was improved after introduction of PIPAAm chains, which prevented autolysis attributed to conjugate steric hindrance. Stability under repeated temperature cycling was also dependent on the architecture of conjugates; the IDc-trypsin conjugate was more stable than the PIDAAc-trypsin. As a consequence, single-end conjugation of polymer to enzyme provides novel bioconjugate with novel functionality attributed to attached polymer while retaining native biological function with high stability.

Temperature-responsive bioconjugates. 1. Synthesis of temperature-responsive oligomers with reactive end groups and their coupling to biomolecules.

Using 3-mercaptopropionic acid (MPA) as a chain-transfer agent, the radical oligomerization of N-isopropylacrylamie (IPAAm) was carried out with varying molar ratios of MPA to IPAAm and DMF. The molecular weight of oligo-IPAAm (OIPAAm) could be controlled by the ratio of MPA to IPAAm. The OIPAAm was confirmed to average one carboxyl end group per chain. All OIPAAms samples were highly water-soluble at lower temperatures and exhibited phase separation near 32 degrees C. The optical transmittance of the OIPAAms aqueous solutions changed drastically at 32 degrees C which was independent of OIPAAms molecular weight. In aqueous solutions of OIPAAm having concentrations higher than 1 wt% and molecular weight of 6100, the oligomers were precipitated and recovered in 85 wt% yield of their original content. Further, OIPAAm was grafted to atelo collagen by activated ester-amine coupling. The OIPAAm-collagen conjugates were able to dissolved in cold water and precipitated at 34 degrees C. Temperature-responsive OIPAAm-collagen conjugates are expected to maintain native collagen functionality in the solution state, react at lower temperatures, and be easily removed from the system with small temperature increases.

Endothelial cell differentiation into capillary structures by copolymer surfaces with phenylboronic acid groups.

A ternary copolymer composed of m-acrylamidophenylboronic acid, N,N-dimethylaminopropylmethacrylamide and N-isopropylacrylamide was synthesized. Long-term culture of bovine aortic endothelial cells on this copolymer substrate demonstrated adhesion and proliferation of the cells. After 26 days in culture, endothelial cells spontaneously developed into capillary networks. The interactions between phenylboronic acid groups in copolymer and glycoconjugates on endothelial cell plasma membranes are proposed to regulate the induction of tissue formation, since phenylboronic acid groups are known to specifically form reversible complexes with cis-diol compounds such as glucose. This copolymer is a novel material capable of mediating specific signals analogous to extracellular matrix to promote proliferation of endothelial cells, inducing capillary structures and prompt angiogenesis.

Effect of phenylboronic acid groups in copolymers on endothelial cell differentiation into capillary structures.

Copolymers (IABb) composed of N-isopropylacrylamide (I), N-(3-dimethylaminopropyl)-acrylamide (A), 3-acrylamidophenylboronic acid (B), and a hydrophobic comonomer, n-butyl methacrylate (b), were synthesized as cell culture substrata since we previously learned that bovine aortic endothelial cells (BAECs) cultured on the phenylboronic acid-containing copolymer (IAB) differentiated into capillary structures after 26 days culture. The synthesized IABb copo ymers contained higher boron and amine moieties than the IAB copolymer. The results of the dynamic contact angle measurement revealed that IABb copolymer-coated surfaces showed a relatively hydrophobic nature, changing to hydrophilic in response to the aqueous environment. BAECs cultured on the copolymer substrata developed into capillary networks after 7 days. This is probably due to the enrichment of boron and amine segments in the vicinity of the hydrophilic copolymer surface, enhancing more pronounced interaction of boronates with cell membrane glycocalyx. The introduction of n-butyl methacrylate into the polymers might enhance the diffusion of the hydrophobic segments to the bulk polymers and the concentration of relatively hydrophilic segments at the outermost polymer surfaces by contact with water. A copolymer (IAP) without boronic acid groups was also prepared using N-phenylacrylamide (P) as a comonomer instead of 3-acrylamidophenylboronic acid in the IAB copolymer to investigate the effect of boronic acid moieties on the capillary formation of the cultured cells. Although the endothelial cells seeded on the copolymer without boronic acid groups adhered during the early culture period, these cells showed neither proliferation nor differentiation and detached from the surface after 13 days. These results strongly support the opinion that the phenylboronic acid groups in the copolymers are responsible for the specific induction of tissue formation of BAECs through the interaction with glycoconjugates on the cell membranes.

Postadsorptive behavior of plasma proteins on poly(propylene oxide)-segmented nylon-610 surfaces and its implication in preventing contact-induced activation of platelets on these surfaces.

The influence of adsorbed plasma proteins on preventing contact-induced activation of platelets on poly(propylene oxide) (PPO)-segmented nylon-610 surfaces was investigated by monitoring changes in cytoplasmic free Ca2+ concentrations in platelets and adsorption/desorption of albumin and fibrinogen on these copolymer surfaces. Direct measurement of cytoplasmic free Ca2+ concentration in platelets in contact with copolymer surfaces was achieved by monitoring spectral changes of a fluorescent indicator dye, Fura 2. These copolymers were characterized by a surface microstructure composed of coexisting crystalline and amorphous phases. An increase in cytoplasmic free Ca2+ concentration in platelets interacting with polymer surfaces was observed, and this increase was found to be strongly reduced both by the adsorption of plasma proteins into the polymer surface and by modifying the surface microstructure of the polymer itself. Transient changes in cytoplasmic free Ca2+ concentration were observed in platelets in contact with the surface of copolymer 61P3-25, which exhibited excellent nonthrombogenicity in our previous studies, depending on the residence time of plasma and plasma concentration. Additionally, adsorption/desorption of albumin and fibrinogen on copolymer surfaces was estimated using 125I-labeled proteins. Exchange of the adsorbed albumin with fibrinogen and minimum fibrinogen adsorption were observed particularly on the 61P3-25 surface. Exchange of adsorbed fibrinogen with plasma proteins and/or increased fibrinogen adsorption were also observed on all other polymer surfaces examined. Finally, we conclude that controlled formation of a defined protein adsorption layer on the 61P3-25 surface via the transient exchange of adsorbed albumin with fibrinogen from plasma, can be a dominant factor in preventing platelet adhesion and activation on this surface.

Temperature-responsive bioconjugates. 3. Antibody-poly (N-isopropylacrylamide) conjugates for temperature-modulated precipitations and affinity bioseparations.

Immunoglobulin G (IgG) has been modified by poly(N-isopropylacrylamide) (PIPAAm) to create a novel bioconjugate which exhibits reversible phase transition behavior at 32 degrees C in aqueous media. A terminal carboxyl group introduced into PIPAAm molecule by polymerization of IPAAm with 3-mercaptopropionic acid was used for conjugation to IgG via coupling reaction of activated ester with protein amino group. These conjugates exhibited rapid response to changes in solution temperature and significant phase separation above a critical solution temperature corresponding to that for the original PIPAAm. These conjugates bound to antigen quantitatively in aqueous system, and antigen-bound complex also demonstrated phase separation and precipitation above a critical temperature. Precipitate was reversibly redissolved in cold buffer. Though particular conjugate which includes 12 molecules of PIPAAm with 6,100 molecular weight suppressed more than 95% of Fc-dependent binding with protein A, it retained approximately 60% of original specific antigen binding activity. It was manifested that polymer content of conjugate was 20-30 wt% for the case of 6,100 molecular weight of PIPAAm to demonstrate specific antigen binding activity most effectively and to reduce Fc-dependent binding with protein A. IgG-PIPAAm conjugates were soluble in water and formed antigen-bound complex in homogeneous solution system below a critical temperature. These conjugates were separated from solution and other solutes corresponding to PIPAAm nature and scarcely bound to antigen above a critical temperature. It is revealed that temperature-responsive PIPAAm conjugated to biomolecule operated as a switching molecule. These phenomena are attractive for not only reversible bioreactors and protein separations but also carrier substrate to localize biomolecules such as drugs, peptides and hormones in a living body.