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.     

Poly(ethylene glycol)-lipase complex that is catalytically active for alcoholysis reactions in ionic liquids

Lipase-catalyzed alcoholysis was investigated in three different ionic liquids. Lyophilized native lipase had a low activity in all the ionic liquids but a poly(ethylene glycol) (PEG)-lipase complex (with a molar ratio of the polymer/enzyme of 10:1) had an increased activity of over 14-fold. Of several lipases tested, PEG-lipase PS (from Pseudomonas cepacia) exhibited the highest activity (1.07 mmol/(h g^–1 protein)) in 1-octyl-3-methylimidazolium hexafluorophosphate.     

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.     

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.     

Facile preparation of biodegradable chitosan derivative having poly(butylene glycol adipate) side chains

Various modes are being explored for the construction of functional materials from nanoparticles. Despite these efforts, the assembly of nanoparticles remains challenging with respect to the requirement of multiple component organization on varying dimensions and length scales. The graft copolymers of chitosan with poly(butylene glycol adipate) (PBGA) were prepared due to the esterification reaction between PBGA and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA) in the presence of toluene as a swelling agent. The graft copolymers are nanoparticles with the size of few hundred nanometers as observed from TEM. It is a potential method to combine chitosan with the hydrophobic synthetic polymers. The grafting reactions were conducted with various PBGA/PHCSSA feed ratios to obtain chitosan-g-PBGA copolymers with various PBGA contents. FT-IR, NMR, XRD, spectrofluorophotometer, and TEM were detected to characterize the copolymers.     

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.     

Polyethylene glycol behaves like weak organic solvent

Effect of polyethylene glycol (PEG) on protein solubility has been primarily ascribed to its large hydrodynamic size and thereby molecular crowding effect. However, PEG also shows characteristics of organic solvents. Here, we have examined the solubility of glycine and aliphatic and aromatic amino acids in PEG solutions. PEG400, PEG4000, and PEG20000 decreased the solubility of glycine, though to a much smaller magnitude than the level achieved by typical organic solvents, including ethanol and dimethyl sulfoxide. PEG4000 showed varying degree of interactions with amino acid side chains. The free energy of aliphatic side chains marginally increased by the addition of PEG4000, indicating their weak unfavorable interactions. However, it significantly decreased the free energy of the aromatic side chains and hence stabilized them. Thus, it was concluded that PEG behaves like weak organic solvents; namely PEG destabilized (interacted unfavorably with) polar and charged groups and stabilized (interacted favorably with) aromatic groups. Interestingly, the interaction of PEG20000, but neither PEG400 nor PEG4000, with glycine resulted in phase separation under the saturated concentration of glycine.     

Preparation and spectroscopic characterization of methoxy poly(ethylene glycol)-grafted water-soluble chitosan

The object of this study was to test the solubility of a methoxy poly(ethylene glycol) (MPEG)-grafted chitosan copolymer in organic solvents and aqueous solution. Water-soluble chitosan with low molecular weight (LMWSC) was used in a PEG-graft copolymerization. The MPEG was conjugated to chitosan using 4-dicyclohexylcarbodimide (DCC), and N-hydroxysuccimide (NHS). Introduction of PEG was confirmed by (1)H and (13)C NMR spectroscopy and FT-IR spectroscopy. The degree of substitution (DS) of MPEG into chitosan was calculated from (1)H NMR data and also by estimating the molecular weight (MW) using gel permeation chromatography (GPC). The DS values obtained from (1)H NMR spectroscopy and GPC were similar, indicating that MPEG-grafted LMWSC was synthesized and properly characterized. Furthermore, the introduction of PEG into chitosan increases the solubility in aqueous solutions over a range of pH values (4.0-11.0) and organic solvents such as DMF, DMSO, ethanol, and acetone.     

Separation of macromolecules by ultrafiltration: Removal of poly(ethylene glycol) from human albumin

Mixtures of albumin and poly(ethylene glycol) (PEG) were used to elucidate some of the factors which influence the separation of macromolecules by thin-channel ultrafiltration. Several membranes which readily passed PEG-4000 in the absence of protein were found to exhibit increased rejection of the synthetic polymer when albumin was added to the system. Based on a comparison of filtration flux and net sieving properties, the PM-30 membrane of Amicon was chosen for further characterization. The increased rejection of PEG-4000 in the presence of albumin was independent of albumin concentration between 1 and 100 mg/ml and persisted even after albumin was removed and the system flushed with water. Overnight incubation of the membrane with trypsin restored the original sieving properties, indicating that the ‘permanent’ effects were due to irreversible adsorption to the membrane. By measuring flux over a 10⁶-fold range of albumin concentration it was possible to resolve the effects of protein adsorption, a saturable process which occurs at low protein concentration (<0.01 mg/ml), from the effects of concentration polarization which occur at high protein concentration (>0.1 mg/ml). Only the former process has an effect on the net sieving properties in this system. In spite of the adverse effects of protein adsorption, it was still possible to obtain efficient removal of PEG-4000 from albumin. Exchange of approximately 5 vols. of solvent at room temperature resulted in a 10-fold reduction in the concentration of PEG in the sample, with no loss of albumin, and no formation of albumin dimers.     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

Preparation and characterization of poly(ethylene glycol)-g-chitosan with water- and organosolubility

A new scheme was proposed for synthesizing poly(ethylene glycol)-g-chitosan (PEG-g-CS), where methoxy poly(ethylene glycol) iodide (MPEG-I) (M_n 2000) was used for N-substitution of triphenylmethyl chitosan (TPM-CS) in organic medium. The graft copolymers were obtained by subsequent removal of protecting groups with dichloroacetic acid. By varying PEG-I/TPM-CS feed ratio, the grafting levels (GL) of PEG can be adjusted. The chitosan derivatives were characterized by FTIR, ¹H NMR, ¹³C NMR and DSC. All the copolymers were soluble in water over wide pH range. Furthermore, organosolubility of the hybrids in DMF and DMSO was also achieved when the DS value more than 24%. The lysozyme degradation rate of the copolymers in aqueous neutral medium decreased with the increase of GL value.     

Enzymatic surface modification of poly(ethylene terephthalate)

This study unambiguously confirms hydrolysis using cutinase of the persistent synthetic polymer poly(ethylene terephthalate), the most important synthetic fiber in the textile industry by direct measurement and identification of the different hydrolysis products. In this aqueous heterogeneous system, dissolved cutinase from Fusarium solani pisi acts on different solid poly(ethylene terephthalate) substrates. The extent of hydrolysis was detected by measuring the amount of (soluble) degradation products in solution using reversed-phase HPLC. Crystallinity greatly affects the capability of the enzyme to hydrolyze the ester bonds, displaying relatively high activity towards an amorphous polyester film and little activity on a highly crystalline substrate. The enzyme is sufficiently stable, hydrolysis rate on the amorphous substrate maintained at sufficient high level over a long period of time of at least five days. From an industrial point of view it is highly recommended to increase the hydrolysis rates.     

Aqueous solubilization of fullerenes using poly(amidoamine) dendrimers bearing cyclodextrin and poly(ethylene glycol)

Fullerene has a unique structure and notable chemical and physical properties, which have been studied in diverse fields including biological applications. The extremely poor solubility of fullerenes in water limits their usage for biomedical applications. In this study, we synthesized polyamidoamine dendrimers having both beta-cyclodextrin (CD) and poly(ethylene glycol) (PEG) and characterized the resulting dendrimers by (1)H NMR, IR, and gel permeation chromatography. We prepared 2.8 microM of aqueous fullerene solutions using these dendrimers. The clustering effect of CD and PEG at the surface of the dendrimer might be crucial for the solubilization of fullerene.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Synthesis and characterization of star poly(epsilon-caprolactone)-b-poly(ethylene glycol) and poly(L-lactide)-b-poly(ethylene glycol) copolymers: evaluation as drug delivery carriers

Two types of 32 arm star polymers incorporating amphiphilic block copolymer arms have been synthesized and characterized. The first type, stPCL-PEG 32, is composed of a polyamidoamine (PAMAM) dendrimer as the core with radiating arms having poly(epsilon-caprolactone) (PCL) as an inner lipophilic block in the arm and poly(ethylene glycol) (PEG) as an outer hydrophilic block. The second type, stPLA-PEG 32, is similar but with poly(L-lactide) (PLA) as the inner lipophilic block. Characterization with SEC, (1)H NMR, FTIR, and DSC confirmed the structure of the polymers. Micelle formation by both star copolymers was studied by fluorescence spectroscopy. The stPCL-PEG 32 polymer exhibited unimolecular micelle behavior. It was capable of solubilizing hydrophobic molecules, such as pyrene, in aqueous solution, while not displaying a critical micelle concentration. In contrast, the association behavior of stPLA-PEG 32 in aqueous solution was characterized by an apparent critical micelle concentration of ca. 0.01 mg/mL. The hydrophobic anticancer drug etoposide can be encapsulated in the micelles formed from both polymers. Overall, the stPCL-PEG 32 polymer exhibited a higher etoposide loading capacity (up to 7.8 w/w % versus 4.3 w/w % for stPLA-PEG 32) as well as facile release kinetics and is more suitable as a potential drug delivery carrier.     

Chitosan cross-linked with poly(ethylene glycol)dialdehyde via reductive amination as effective controlled release carriers for oral protein drug delivery

The covalently cross-linked chitosan-poly(ethylene glycol)₁₅₄₀ derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG₁₅₄₀-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2 h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12 h. The results proved that the release-and-hold behavior of the cross-linked CS–PEG₁₅₄₀H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.     

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.