Cell engineering biointerface focusing on cytocompatibility using phospholipid polymer with an isomeric oligo(lactic acid) segment

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.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

Generic method for modular surface modification of cellulosic materials in aqueous medium by sequential "click" reaction and adsorption

A generic approach for heterogeneous surface modification of cellulosic materials in aqueous medium, applicable for a wide range of functionalizations, is presented. In the first step, carboxymethyl cellulose (CMC) modified with azide or alkyne functionality, was adsorbed on a cellulosic substrate, thus, providing reactive sites for azide-alkyne cycloaddition click reactions. In the second step, functional units with complementary click units were reacted on the cellulose surface, coated by the click-modified CMC. Selected model functionalizations on diverse cellulosic substrates are shown to demonstrate the generality of the approach. The concept by sequentially combining the robust physical adsorption ("physical click") and robust chemical reaction ("chemical click") allows versatile, simple, and environmentally friendly modification of a cellulosic substrate with virtually any azide- or alkyne-modified molecule and even functionalization with several types of units.     

Biosynthesis of hetero-polysaccharides by Acetobacter xylinum - Synthesis and characterization of metal-ion adsorptive properties of partially carboxymethylated cellulose

Biological reconstruction of water-soluble carboxymethylated cellulose (CMC; D.S. =0.47) has been achieved by culturing Acetobacter xylinum in medium containing CMC and -glucose to give a novel hetero-polysaccharide having a carboxymethyl function. The novel extracellular polysaccharide, carboxymethylated-bacterial cellulose (CM-BC), had an ion exchange ability with enhanced specific adsorption for lead and uranyl ions compared to the original CMC and bacterial cellulose. The contribution of the hydroxy group at C-2 was confirmed by applying carboxymethylated chitin, which possesses acetamido group at C-2 of the glucose residue, as the carbon source of the incubation.     

Development of magnetically separable immobilized lipase by using cellulose derivatives and their application in enantioselective esterification of ibuprofen

Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.     

Generic Method for Attaching Biomolecules via Avidin-Biotin Complexes Immobilized on Films of Regenerated and Nanofibrillar Cellulose

We investigated the adsorption and chemical conjugation of avidin and its deglycosylated form, neutravidin, on films of regenerated and nanofibrillar cellulose. The dynamics and extent of biomolecular attachment were monitored in situ by quartz crystal microbalance microgravimetry and ex situ via surface analyses with atomic force microscopy and X-ray photoelectron spectroscopy. The installation of carboxyl groups on cellulose after modification with carboxymethylated cellulose (CMC) or TEMPO-oxidation significantly increases physisorption of avidins, which can be then covalently conjugated by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDS/NHS) coupling chemistries. The developed cellulose-avidin biointerfaces are able to scavenge biotinylated molecules from solution as demonstrated by successful surface complexation of biotinylated bovine serum albumin (Biotin-BSA) and antihuman immunoglobulin G (Biotin-anti-hIgG). Finally, we show that cellulose substrates carrying immobilized anti-hIgG are effective in detecting human immunoglobulin G (hIgG) from fluid matrices.     

Adhesion of colloidal polyelectrolyte complexes to wet cellulose

The adhesion of wet regenerated cellulose to colloidal complexes formed between carboxymethyl cellulose (CMC) and polyvinylamine (PVAm) was evaluated by measuring the force to delaminate pairs of regenerated cellulose membranes bound together with polyelectrolyte complex. The most important parameter was the surface composition of the colloidal complex particles. High delamination forces corresponded to using complexes coated with excess PVAm whereas low adhesion was observed for both CMC coated complexes and complexes in which the PVAm was replaced with polymer bearing quaternary amine groups. Adhesion with complexes was highest at pH 4 and rather insensitive to pH from 6 to 9. Finally, mild TEMPO/NaBr/NaClO oxidation of the cellulose gave much stronger adhesion when PVAm was in excess but not with the CMC rich complexes.     

SYNTHESIS OF CELLULOSE BY ACETOBACTER XYLINUM : VIII. On the Formation and Orientation of Bacterial Cellulose Fibrils in the Presence of Acidic Polysaccharides

The transfer of the glucosyl moiety from uridine diphosphate glucose in the presence of Acetobacter xylinum cell-free extracts led to the formation of a mixture of alkali-soluble and -insoluble cellodextrins. Typical cellulose fibrils could not be detected by electron microscopy in this product. Immediately after release into the medium, cellulose formed by whole cells is in a "prefibrous" form which passes through Millipore filters of 0.45 and 0.8 µ pore diameter. Non-filtrable fibrils arise from this material probably by a process of crystallization involving no extracellular enzymes. Fibrils formed in shaken cell suspensions intertwine and form aggregates visible to the naked eye. In quiet suspensions pellicles are formed which float on the surface. Soluble Na-carboxymethylcellulose (CMC) is incorporated into cellulose fibrils formed in its presence, probably by a process of co-crystallization. Aggregation of fibrils containing CMC is delayed because of electrostatic repulsion between carboxylic groups. The aggregation time depends on the amount of CMC incorporated, its degree of substitution, the pH of the medium, and the ionic strength. The amount of CMC incorporated depends on the relative concentration CMC/cellulose and on the similarity of the CMC and the cellulose molecules i.e. in molecular weight and the number of carboxyl substitutions. Cellulose pellicles formed in the presence of CMC by unshaken cell suspensions consist of crossed, superimposed layers of parallel oriented cellulose fibrils. The same phenomenon is observed when phosphomannan, but not levan, is substituted for CMC. The biogenesis of oriented cellulose fibrils is envisaged as a process comprising the following steps: polymerization of the monomeric precursor, diffusion of the molecule to crystallization sites, crystallization, and orientation. It is proposed that charged polysaccharides play a role similar to that of CMC in affecting the orientation of cellulose fibrils in the plant cell wall.     

Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.     

Biochemical Studies on Cycasin

Purification of β-glucosidase from the seeds of Japanese cycad, and properties of the purified preparation are described. The enzyme activity was determined by colorimetry using ONPG as substrate. Crude preparation was obtained easily by adsorption on fibrous CMC pulp. It was further purified by chromatography on CMC powder, and a preparation which showed an activity of 135-folds of the original extract was obtained. Influences of pH, temperature, and substrate concentration upon the enzyme activity were examined. Michaelis constant of the enzyme for ONPG was 3.3×10^–3M.     

Brief communication. on the problem of immunological detection of antigens in skeletal remains.

A detailed investigation with affinity-chromatography-purified fractions of antihemoglobin serum from rabbit shows that the hemoglobin content of human bones dating back 15 to 3,000 years may be very small. Some of the previous results (Ascenzi et al., 1985) indicating a high hemoglobin titer were +vitiated because of an unexpected cross-reactivity of bone extracts with the hemoglobin-unreactive fraction of the antiserum.     

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells.     

Purification and characterization of endoglucanase and exoglucanase components from Trichoderma viride

Major cellulase components—four endoglucanases (Endo I, II, III and IV) and one exoglucanase (Exo II)—were isolated from a commercial cellulase preparation derived from Trichoderma viride by a series of chromatographic procedures. The average molecular weights were determined by SDS-polyacrylamide gel electrophoresis. Endos I, III and IV, with M_rs of 52,000, 42,000 and 38,000, respectively, exhibited a more random hydrolytic mode on carboxymethylcellulose (CMC) than Endo II, which has an M_r of 60,000. Endo II showed low activity towards CMC, but out of the four purified endoglucanases this enzyme had the highest specific activity against Avicel. In the hydrolysis of H₃PO₄-swollen cellulose by Endos I, III and IV, cellobiose was the major product, but equimolar amounts of glucose and cellobiose were formed by Endo II. Exo II, with an M_r of 62,000, released cellobiose as the main product in the hydrolysis of H₃PO₄-swollen cellulose, but glucose was negligible. The combination of Endo I, II, III or IV with Exo II resulted in a synergistic effect in the degradation of Avicel at various combination ratios of these enzymes; the specific optimum ratio of endoglucanase to exoglucanase was largely dependent upon the random hydrolytic mode of the endoglucanase. On the other hand, adsorption of cellulase components was found apparently to obey the Langmuir isotherm, and the thermodynamic parameter (ΔH) was calculated from the adsorption equilibrium constant (K). The enthalpies of adsorption of the endoglucanases were in the range of −2.6–−7.2 KJmol⁻¹, much smaller than that of Exo II (−19.4 KJmol⁻¹). This suggest that Exo II shows stronger preferential adsorption than endoglucanases, and that the enthalpy of adsorption will be effective in distinguishing endoglucanase from exoglucanase.     

Solid media containing carboxymethylcellulose to detect CX cellulose activity of micro-organisms.

Solid media containing carboxymethyl cellulose (CMC) were developed to detect CX cellulose-producing micro-organisms. Hydrolysis of CMC was seen as a clear zone around colonies after flooding plates with 1% aqueous hexadecyltrimethyl-ammonium bromide. Tests with ten bacterial and four fungal species showed that the degree of substitution (DS) of the CMC affects both growth and enzyme production. Most of the organisms produced more CX cellulase on CMC with a DS of 0-9, but CMC with a DS of 0-4 was better for one fungus. A qualitative measure of cellulase production may be obtained by calculating the ratio of zone size to colony diameter. Solid media containing CMC provided a more rapid assay of CX cellulose production than a medium containing native cellulose.     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

Isolation and purification of isoenzymes of cellobiohydrolase I and II of trichoderma reesei using LPLC methods

Five cellulases were fractionated from a commercial cellulase preparation (Celluclast^TM) Two isoenzymes of cellobiohydrolase I (CBHI)(pI = 4.1) could be proved to be real exo-glucanases due to their activity towards MU (=methylumbelliferyl)-lactoside being inhibited by cellobiose (5 mM) and due to production of cellobiose from carboxymethylcellulose (CMC) as the sole final product.Two isoenzymes of CBHII (pI=6.15, 6.0) were shown to act as endo-glucanases because they produced glucose, cellobiose and cellotetraose from CMC and because they were not inhibited by cellobiose when decomposing MU-lactoside. Results confirm recent reports in the literature classifying CBHI and CBHII as exo-type and endo-type cellulases, respectively. Both the CBHI and the CBHII isoenzymes were shown to be active towards CMC and amorphous cellulose.CBHI and CBHII reactions could be differentiated from one another by the velocities of decomposition of CMC: CBHI acts slowly and linearly whereas CBHII acts strongly and exponentially.The fifth of the purified enzymes must be classed as a conventional endoglucanase which exhibits activity towards CMC but fails to be active towards MU-lactoside and amorphous cellulose.     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.     

Optimal preparation of immobilized liposome-bound cellulase for hydrolysis of insoluble cellulose in an external loop airlift bioreactor

Immobilized liposome-bound cellulase (ILC) was optimally prepared for the ILC-catalyzed hydrolysis of insoluble cellulose in an external loop airlift bioreactor. The liposomes with mean diameters of 200, 100, and 50 nm were used to prepare three kinds of ILCs, i.e., ILC(200), ILC(100) and ILC(50), respectively. The activity and stability of ILC(100) were examined with soluble cellulose (CMC) in addition to the insoluble substrate of cellulose powder (CC31) in a shaking flask as well as the airlift bioreactors. The experiments were carried out with 45 degrees C and pH 4.8 being found to be optimal for the activity. The activity of ILC(100) was stable in either airlift or shaking flask bioreactor during the five times repeated hydrolyses of CC31 corresponding to a total reaction time of 240 h. This confirmed that the cellulase molecules were covalently bonded to the liposomes covalently bound to the chitosan gel beads. Nevertheless, the activity of ILC(100) with CMC steadily decreased throughout the repeated reactions, suggesting an adverse effect of CMC on the ILC(100) activity. Among the three ILCs, ILC(50) was found to be the most stable and productive biocatalyst during the repeated hydrolyses of insoluble CC31 in the airlift bioreactor. More than 70% of the initial activity of ILC(50) was retained even after the six times repeated reactions for 288 h. Conversely, the ILC(200) was found to be the most unstable catalyst. Such a difference in stability among these ILCs was suggested to be caused by the difference in physical stability of their liposome membranes to the liquid shear stress due to the rising bubbles and circulating liquid as well as that in the amount of the cellulase molecules unstably incorporated in the membranes. ILC(50) was thus shown to have the most potential for an efficient hydrolysis of insoluble cellulose in a practical airlift bioreactor.     

Hybrid rigid/soft and biologic/synthetic materials: polymers grafted onto cellulose microcrystals

Rigid nanoscale polymer rods were prepared by grafting preformed amine-terminated poly(styrene) and poly(tert-butyl acrylate) onto oxidized cellulose microcrystals. Low polydispersity polymers, grown using atom transfer radical polymerization, were characterized and purified prior to cellulose attachment. Oxidation of the cellulose microcrystal led to the formation of carboxylic acids on the surface of the microcrystals. Covalent attachment of the polymers onto the cellulose microcrystals was achieved via a carbodiimide-mediated amidation reaction. The length and diameter of the polymer-cellulose composites increased upon surface modification. Typically, polymer-cellulose composites are synthesized by a grafting-from method because it can be difficult to obtain sufficient graft density using a grafting-to preparation. However, the composites reported here comprised 60-64% grafted polymer by mass. This degree of grafting-to allowed the composite to form stable suspensions in organic solvents.     

The enzymatic hydrolysis rate of cellulose decreases with irreversible adsorption of cellobiohydrolase I

Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions. In this work the adsorption behavior of intact β-1, 4-glucan-cellobiohydrolase (CBH I) from Trichoderma reesei onto microcrystalline cellulose was monitored by surface plasmon resonance and UV-spectral method. It was found that there existed reversible binding and irreversible binding of CBH I during its interaction with cellulose substrate. To evaluate the influence of adsorption on cellulose enzymatic hydrolysis, the reaction dynamics on pure cellulose were determined. A plot of the hydrolysis rate against the surface density of irreversibly adsorbed CBH I, revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density. Taken together, results presented here should be useful for modifying the binding characteristics of CBH I and making them more effective in cellulose hydrolysis.