Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol)

Hybrid gels constructed from proteins and polymers have attracted a wide range of attention in the field of biomedicine and bioengineering. We report herein the enzymatic preparation of polymer–protein hybrid hydrogels composed of terminally bis-functionalized linear poly(ethylene glycol) (PEG) and streptavidin (SA). PEG was conjugated with tyramine to introduce terminal phenolic hydroxyl (Ph-OH) groups. A peptide tag containing a tyrosine residue (G₄Y-tag) was genetically introduced at the C-terminus of SA. The Ph-OH-modified PEG and G₄Y-tagged SA (SA-G₄Y) were treated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) to yield (PEG-Ph-OH)–(SA-G₄Y) hybrid gels. Biotinylated enhanced green fluorescent protein (biotin-EGFP) was selectively captured in the obtained hybrid gels, indicating that SA-G₄Y retained its biological function. The amount of biotin-EGFP immobilized in the hybrid gels depended on the concentration of SA-G₄Y. In addition, biotinylated bacterial alkaline phosphatase (biotin-BAP) was immobilized in the hybrid gel. The immobilized biotin-BAP exhibited more than 95% of the initial activity after 5 rounds of recycling. The results suggest the facile functionalization of the hybrid gel with a variety of biotinylated functional molecules.     

Analytical partitioning of poly(ethylene glycol)-modified proteins

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.     

Heterogeneity of synaptosomal membrane preparations from different regions of calf brain studied by partitioning and counter-current distribution.

1. Membranes obtained by lysis and Yeda-press treatment of synaptosomes (nerve endings) from cortex, caudateus nucleus, and hippocampal region of calf brain have been studied by partitioning within a liquid-liquid aqueous two-phase system consisting of water, dextran, Ficoll, and poly(ethylene glycol). 2. The partitioning of membranes was sensitive to the presence of a dextran-bound dye, Procion yellow HE-3G, in the lower phase. 3. The two-phase system was used for counter-current distribution to study the heterogeneity of the synaptic membranes from the three regions of the brain and to separate the membranes into fractions. 4. The obtained counter-current distribution profiles strongly depended on the region of the brain from which the membranes were isolated. 5. The membrane fractions obtained showed marked differences in their SDS electrophoresis pattern.     

Subfractionation of cell populations by partitioning in dextran-poly (ethylene glycol) aqueous phases

Partitioning of cells in dextran-poly(ethylene glycol) aqueous two-phase systems depends on the interaction between the surface properties of the cells and the physical properties of the phases. The latter can be manipulated to a considerable extent by selection of polymer concentrations and ionic composition and concentration. If salts (e.g., phopshate) are used that have an unequal affinity for the two phases, an electrostatic potential difference between the phases results and, at appropriately high polymer concentrations, the partition coefficient of cells is determined predominantly by membrane charge-associated properties. By “balancing” the magnitude of the electrostatic potential difference against that of the interfacial tension (primarily a function of polymer, but also phosphate, concentrations) one can obtain phase systems that give usable partition coefficients for most cell populations (1). In work under way in our laboratory on the effects of different chemical and enzymatic modifications on the relative surface properties of rat red blood cells of different ages, we have now found that certain phase compositions did not resolve such treated cell subpopulations while other phase compositions did. Thus not all charged phase systems in which cell populations as a whole have usable partition coefficients are equally capable of detecting or subfractionating cell subpopulations. It is therefore essential, before drawing conclusions on the nonseqarability of cell subpopulations, to test cell separability in charged phase systems of different compositions if the system initially chosen does not afford a subfractionation.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles

The critical micelle concentrations of 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 microM, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG-DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.     

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.     

Lipase-catalyzed transformations using poly(ethylene glycol) as solvent

Abstract

Candida antarctica lipase catalyzes a number of elementary reactions like alcoholysis, ammoniolysis and aminolysis in poly(ethylene glycol) (PEG) media. Reaction rates were comparable to or better than those observed in conventional organic reaction media and ionic liquids. It is envisaged that PEGs could have added benefits for performing biotransformations with highly polar substrates, which are sparingly soluble in common organic solvents.     

Novel poly(ethylene glycol) derivatives for preparation of ribosome-inactivating protein conjugates

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.     

Poly(ethylene glycol) quantitation by laser nephelometry

When poly(ethylene glycol) 3350 is estimated by the method of Skoog [(1979) Vox Sang. 37, 345-349], fine particles form. The particles are not attributable to residual protein but to a poly(ethylene glycol)/barium/iodine complex that can be quantitated by means of a laser nephelometer. The method is sensitive to at least 10 mg% poly(ethylene glycol) 3350 (4 micrograms in the cuvette) in 2500 mg% protein, and nephelometer response is approximately linear between 30 and 200 mg% of the polymer. The coefficient of variance is about 8%. Triton X-100, Pluronic F-68, Varonic 1000MS, and poly(ethylene glycol) of higher and lower molecular weight react well. Alkylated celluloses, dextrans, glycerol, glycine, and sodium dodecyl sulfate do not react significantly. Barium can be replaced with Mg, Ca, Ni, Fe, and other divalent cations in the reaction, but other than for Hg, light-scattering is most intense with Ba. The reaction goes to completion in about 5 min and is most intense when the barium is added before the iodine.     

Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M(r) = 5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M(r) = 2000, 5000 or 20000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M(r) = 5000, or 20000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; [M(r) = 20000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (相似文献   

Synthesis of a library of benzoindolizines using poly(ethylene glycol) as soluble support

A library of benzoindolizines (pyrrolo [1,5-a] quinolines 10 and pyrrolo [1,5-a] quinolines 9) has been synthesized using poly(ethylene glycol) (PEG) as soluble polymer support. The PEG-supported isoquinolinium salt 4 reacted, respectively, with active alkenes 11 using tetrakispyridinecobalt(II) dichromate (TPCD) as oxidant or alkynes 12 to give 10, of which yields were from moderate to high. By analogy, the reaction of PEG-supported quinolinium salt 3 with 12 was to produce 9. However, in the presence of TPCD the reaction of 3 with 11 afforded indolizines 8, which was discovered firstly.     

Depth profile of free volume in a mixture and copolymers of poly(N-vinyl-pyrrolidone) and poly(ethylene glycol) studied by positron annihilation spectroscopy

Effect of hydrogen bonding on the depth profile of the free-volume in a mixture (weight ratio of 65:35) of poly(N-vinyl-pyrrolidone) (PVP) and poly(ethylene glycol) (PEG) and the copolymers of vinyl pyrrolidone with poly(ethylene glycol) diacrylate (PVP-PEGDA) and monomethacrylate (PVP-PEGMMA) was studied using positron annihilation spectroscopy. Doppler broadening energy spectra of annihilation radiation and positron annihilation lifetime were measured as a function of positron incident energy (0-30 keV). Significant variations of the free-volume depth profile in terms of the S parameter, ortho-positronium lifetime, intensity, and lifetime distribution are observed as a result of the hydrogen-bonding replacement of covalent bonds. The polymer mixture with hydrogen bonding through two sides of PEG short chains has a larger free volume and a wider distribution than the comb-structured PVP-PEGMMA and the network structured PVP-PEGDA. A longer ortho-positronium lifetime is observed near the surface than in the bulk. This is interpreted in terms of surface effect, free volume, and hydrogen bonding for drug delivery applications of polymeric materials.     

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes

A decrease in the number of binding sites for¹²⁵I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.     

Novel prodrugs of SN38 using multiarm poly(ethylene glycol) linkers

CPT-11, also known as irinotecan, is a prodrug that is approved for the treatment of advanced colorectal cancer. The active metabolite of CPT-11, SN38 (7-ethyl-10-hydroxy-camptothecin), has 100- to 1000-fold more potent cytotoxic activity in tissue cell culture compared with CPT-11. However, parental administration of SN38 is not possible because of its inherently poor water solubility. It is reported here that a multiarm poly(ethylene glycol) (PEG) backbone linked to four SN38 molecules (PEG-SN38) has been successfully prepared with high drug loading and significantly improved water solubility (400- to 1000-fold increase). Three different protecting strategies have been developed in order to selectively acylate the 20-OH of SN38 to preserve its E-ring in the lactone form (the active form of SN38 with cytotoxic activities) while PEG is still attached. One chemical process has been optimized to make a large quantity of the PEG-SN38 conjugate with a high yield that can be readily adapted for scale-up production. The PEG-SN38 conjugates have shown excellent in vitro anticancer activity, with potency similar to that of native SN38, in a panel of cancer cell lines. The PEG-SN38 conjugates also have demonstrated superior anticancer activity in the MX-1 xenograft mice model compared with CPT-11. Among the four conjugates, PEG-Gly-(20)-SN38 (23) has been selected as the lead candidate for further preclinical development.     

Dynamics of water in supercooled aqueous solutions of glucose and poly(ethylene glycol)s as studied by dielectric spectroscopy

The dielectric behaviour of aqueous solutions of glucose, poly(ethylene glycol)s (PEGs) 200 and 600, and poly(vinyl pyrrolidone) (PVP) has been examined at different concentrations in the frequency range of 10(6)-10(-3) Hz by dielectric spectroscopy and by using differential scanning calorimetry down to 77 K from room temperature. The shape of the relaxation spectra and the temperature dependence of the relaxation rates have been critically examined along with temperature dependence of dielectric strength. In addition to the so-called primary (alpha-) relaxation process, which is responsible for the glass-transition event at T(g), another relaxation process of comparable magnitude has been found to bifurcate from the main relaxation process on the water-rich side, which continues to the sub-T(g) region, exhibiting relaxation at low frequencies. The sub-T(g) process dominates the dielectric measurements in aqueous solutions of higher PEGs, and the main relaxation process is seen as a weak process. The sub-T(g) process was not observed when water was replaced by methanol in the binary mixtures. These observations suggest that the sub-T(g) process in the aqueous mixtures is due to the reorientational motion of the 'confined' water molecules. The corresponding dielectric strength shows a noticeable change at T(g), indicating a hindered rotation of water molecules in the glassy phase. The nature of this confined water appears to be anomalous compared to most other supercooled confined liquids.     

Preparation of cibacron blue F3G-A (polyethylene glycol) in large scale for use in affinity partitioning

The reaction parameters for preparation of Cibacron Blue F3G-A polyethylene glycol have been studied, including temperature, concentration of reactants, and addition of neutral salt (Na(2)SO(4)) as well as ethanol. The yield of dypolymer is strongly dependent on temperature and time of reaction. Preparation in large scale has been done under optimal conditions binding more than 30% of the dye to polyethylene glycol in a low-cost procedure. The effectiveness of this dye-polymer for use in liquid-liquid extraction of enzymes is demonstrated by partition of glucose 6-phosphate dehydrogenase and phosphofructokinase when an extract of baker's yeast is included in a dextran-polyethylene glycol-water two-phase system.     

Ultrastructural study of echinocytes induced by poly (ethylene glycol)-cholesterol

Poly (ethylene glycol)-cholesterol (PEG-Chol) consists of a hydrophilic PEG and hydrophobic cholesterol moiety. When PEG-Chol was applied to erythrocytes, the reagent quantitatively induced protrusions by exclusively distributing in the outer monolayer of the membrane. This kind of response has been regarded as a general response that reduces the stress of expansion of the outer monolayer. However, the relationship between the membrane architecture and the distribution of such molecules is unknown. In this study, we examined the distribution of tagged PEG-Chol along the shape change pathway. The echinocytic shape was initiated by the initial formation of bumps on the rim of the discoid, which subsequently elongated as protrusions. These protrusions contained aggregates of granular structures, which appeared to accommodate the increase in the outer monolayer area. At higher concentrations, PEG-Chol further induced sphero-echinocytosis that resulted in numerous branched protrusion processes. We found that PEG-Chol was exclusively distributed in these protrusions and, in particular, accumulated at the tips. These results suggested that externally intercalated PEG-Chol was sequestrated from erythrocytes as membrane protrusions through an as-yet-unknown mechanism.     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.