Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

Development of novel poly(ethylene glycol)-based vehicles for gene delivery

The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.     

Optimization of DNA-directed immobilization on mixed oligo(ethylene glycol) monolayers for immunodetection

The development of protein chips has suffered from problems regarding long-term protein stability and activity. We present a protein sensor surface for immunodetection that is prepared by a DNA-directed protein immobilization method on a mixed self-assembled monolayer (SAM). By this approach, an immobilized single-stranded DNA (ssDNA) surface can be transferred/modified into a protein chip by flowing in ssDNA-conjugated protein when the protein chip measurement is needed. Therefore, the long-term stability of the protein chip will not be a problem for various applications. We tried various compositions for the SAM layer, the length of the ssDNA spacer, the end-point nucleotide composition, and the processes of ssDNA immobilization of the SAM for an optimized condition for shifting the DNA chip to a protein chip. The evaluations were made by using surface plasmon resonance. Our results indicated that a 50:1 ratio of oligo(ethylene glycol) (OEG)/COOH-terminated OEG and DNA sequences with 20mer are the best conditions found here for making a protein chip via a DNA-directed immobilization (DDI) method. The designed end-point nucleotide composition contains a few guanines or cytosines, and ssDNA immobilization of the SAM by dehybridizing immobilized double-stranded DNA (dsDNA) can improve the hybridization efficiency.     

Self-assembled, cation-selective ion channels from an oligo(ethylene glycol) derivative of benzothiazole aniline

This paper describes the spontaneous formation of well-defined pores in planar lipid bilayers from the self-assembly of a small synthetic molecule that contains a benzothiazole aniline (BTA) group attached to a tetra-ethylene glycol (EG4) moiety. Macroscopic and single-channel current recordings suggest that these pores are formed by the assembly of four BTA-EG4 monomers with an open pore diameter that appears similar to the one of gramicidin pores (~0.4 nm). The single-channel conductance of these pores is modulated by the pH of the electrolyte and has a minimum at pH~3. Self-assembled pores from BTA-EG4 are selective for monovalent cations and have long open channel lifetimes on the order of seconds. BTA-EG4 monomers in these pores appear to be arranged symmetrically across both leaflets of the bilayer, and spectroscopy studies suggest that the fluorescent BTA group is localized inside the lipid bilayers. In terms of biological activity, BTA-EG4 molecules inhibited growth of gram-positive Bacillus subtilis bacteria (IC50~50 μM) and human neuroblastoma SH-SY5Y cells (IC50~60 μM), while they were not toxic to gram-negative Escherichia coli bacteria at a concentration up to 500 μM. Based on these properties, this drug-like, synthetic, pore-forming molecule with a molecular weight below 500 g mol(-1) might be appealing as a starting material for development of antibiotics or membrane-permeating moieties for drug delivery. From a biophysical point of view, long-lived, well-defined ion-selective pores from BTA-EG4 molecules offer an example of a self-assembled synthetic supramolecule with biological function.     

Functionalization of poly(oligo(ethylene glycol) methacrylate) films on gold and Si/SiO2 for immobilization of proteins and cells: SPR and QCM studies

Thin films of a biocompatible and nonbiofouling poly(oligo(ethylene glycol) methacrylate) ( pOEGMA) with various thicknesses were formed on gold and Si/SiO 2 substrates by a combination of the formation of self-assembled monolayers (SAMs) terminating in bromoester-an initiator of atom transfer radical polymerization (ATRP)-and surface-initiated ATRP. After the formation of the pOEGMA films, terminal hydroxyl groups of side chains divergent from the methacrylate backbones were activated with N, N'-disuccinimidyl carbonate (DSC), and the DSC-activated pOEGMA films were reacted with (+)-biotinyl-3,6,9-trioxaundecanediamine (Biotin-NH 2) to form biotinylated pOEGMA films. By surface plasmon resonance experiments with the target protein (streptavidin) and model proteins (fibrinogen and lysozyme), we verified that the resulting films showed the enhanced signal-to-noise ratio ( approximately 10-fold enhancement) for the biospecific binding of streptavidin compared with the biotinylated substrate prepared from carboxylic acid-terminated SAMs. Quartz crystal microbalance measurements were also carried out to obtain the surface coverage of streptavidin and fibrinogen adsorbed onto the biotinylated pOEGMA films with various thicknesses and to investigate the effect of film thicknesses on the biospecific binding of streptavidin. Both the binding capacity of streptavidin and the signal-to-noise ratio of streptavidin/fibrinogen were found to be saturated at the 20 nm thick pOEGMA film. In addition, to demonstrate a wide applicability of the pOEGMA films, we constructed micropatterns of streptavidin and cells by microcontact-printing biotin-NH 2 and poly- l-lysine onto the DSC-activated pOEGMA films, respectively.     

Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin

A simple and rapid continuous-flow immunosensor based on surface plasmon resonance (SPR) has been developed for detection of insulin as low as 1 ng ml-1 (ppb) with a response time of less than 5 min. At first, a heterobifunctional oligo(ethyleneglycol)-dithiocarboxylic acid derivative (OEG-DCA) containing dithiol and carboxyl end groups was used to functionalize the thin Au-film of SPR chip. Insulin was covalently bound to the Au-thiolate monolayer of OEG-DCA for activating the sensor surface to immunoaffinity interactions. An on-line competitive immunosensing principle is examined for detection of insulin, in which the direct affinity binding of anti-insulin antibody to the insulin on sensor surface is examined in the presence and absence of various concentrations of insulin. Immunoreaction of anti-insulin antibody with the sensor surface was optimized with reference to antibody concentration, sample analysis time and flow-rate to provide the desired detection limit and determination range. With the immunosensor developed, the lowest detectable concentration of insulin is 1 ng ml-1 and the determination range covers a wide concentration of 1-300 ng ml-1. The developed OEG-monolayer based sensor chip exhibited high resistance to non-specific adsorption of proteins, and an uninterrupted highly sensitive detection of insulin from insulin-impregnated serum samples has been demonstrated. After an immunoreaction cycle, active sensor surface was regenerated simply by a brief flow of an acidic buffer (glycine.HCl; pH 2.0) for less than 1 min. A same sensor chip was found reusable for more than 25 cycles without an appreciable change in the original sensor activity.     

Development of an AMBER-compatible transferable force field for poly(ethylene glycol) ethers (glymes)

An all-atom force field consistent with the general AMBER force field (GAFF) format for poly(ethylene glycol) dimethyl ether (diglyme or G2) was developed by fitting to experimental liquid densities and dielectric constants. Not surprisingly, the new force field gives excellent agreement with experimental liquid phase densities and dielectric constants over a wide temperature range. Other dynamic and thermodynamic properties of liquid G2 such as its self-diffusion coefficient, shear viscosity, and vaporization enthalpy were also calculated and compared to experimental data. For all of the properties studied, the performance of the proposed new force field is better than that of the standard GAFF force field. The force field parameters were transferred to model two other poly(ethylene glycol) ethers: monoglyme (G1) and tetraglyme (G4). The predictive ability of the modified force field for G1 and G4 was significantly better than that of the original GAFF force field. The proposed force field provides an alternative option for the simulation of mixtures containing glymes using GAFF-compatible force fields, particularly for electrochemical applications. The accuracy of a previously published force field based on the OPLS-AA format and the accuracies of two modified versions of that force field were also examined for G1, G2, and G4. It was found that the original OPLS-AA force field is superior to the modified versions of it, and that it has a similar accuracy to the proposed new GAFF-compatible force field.

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Graphical abstract Transferability of an AMBER-compatible force field parameterized for G2 to other glymes

     

In vitro cytotoxicity of redox radical initiators for cross-linking of oligo(poly(ethylene glycol) fumarate) macromers

A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate-2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

Development of poly(ethylene glycol) conjugated lactoferrin for oral administration

Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.     

Effect of the oligo(ethylene glycol) group on the antioxidant activity of manganese salen complexes

The synthesis and antioxidant activity of oligo(ethylene glycol)-modified manganese salen complexes are reported. Their SOD activities were similar and 2- to 3-fold more potent than the standard compound EUK-134. Their catalase-like activity was lower than that of EUK-134 in the initial conversion rate; however, some analogs exhibited a better catalytic turnover number.     

Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment

Abstract An NADP(H)-specific glutamate dehydrogenase of Haloferax mediterranei has been purified to apparent homogeneity and characterised. The purified enzyme was stabilized by glycerol in absence of salt. Glutamate dehydrogenase from Hf. mediterranei is a hexameric enzyme with a native molecular mass of 320 kDa composed of monomers each with a molecular mass of 55 kDa. At pH 8.5 the enzyme has K _ms of 0.018, 0.34 and 4.2 mM for NADP+, 2-oxoglutarate and ammonium, respectively. Amino acid composition and sequence of the first 16 residues of the N-terminus have been determined.     

Antifouling properties of oligo(lactose)-based self-assembled monolayers

The antifouling (AF) properties of oligo(lactose)-based self-assembled monolayers (SAMs), using four different proteins, zoospores of the green alga Ulva linza and cells of the diatom Navicula incerta, were investigated. The SAM-forming alkylthiols, which contained 1, 2 or 3 lactose units, showed significant variation in AF properties, with no differences in wettability. Non-specific adsorption of albumin and pepsin was low on all surfaces. Adsorption of lysozyme and fibrinogen decreased with increasing number of lactose units in the SAM, in agreement with the generally observed phenomenon that thicker hydrated layers provide higher barriers to protein adsorption. Settlement of spores of U. linza followed an opposite trend, being greater on the bulkier, more hydrated SAMs. These SAMs are more ordered for the larger saccharide units, and it is therefore hypothesized that the degree of order, and differences in crystallinity or stiffness between the surfaces, is an important parameter regulating spore settlement on these surfaces.     

Synthesis of oligo(ethylene glycol) substituted phosphatidylcholines: Secretory PLA2-targeted precursors of NSAID prodrugs

A series of new phosphatidylcholine analogues with structurally modified sn-2-substituents have been prepared. The synthetic compounds include oligo(ethylene glycol) derivatives with chain-terminal pharmacophores that upon catalytic hydrolysis by phospholipase A₂ yielded a series of oligo(ethylene glycol)-conjugates of the respective drugs. The approach here outlined may open a new way to employ OEG derivatives of phospholipids for therapeutic applications as secretory PLA₂-targeted precursors of prodrugs.     

Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG₃) and hexa(ethylene glycol) (EG₆) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.     

Biomimetic poly(ethylene glycol)-based hydrogels as scaffolds for inducing endothelial adhesion and capillary-like network formation

The extracellular matrix (ECM) is an attractive model for designing synthetic scaffolds with a desirable environment for tissue engineering. Here, we report on the synthesis of ECM-mimetic poly(ethylene glycol) (PEG) hydrogels for inducing endothelial cell (EC) adhesion and capillary-like network formation. A collagen type I-derived peptide GPQGIAGQ (GIA)-containing PEGDA (GIA-PEGDA) was synthesized with the collagenase-sensitive GIA sequence attached in the middle of the PEGDA chain, which was then copolymerized with RGD capped-PEG monoacrylate (RGD-PEGMA) to form biomimetic hydrogels. The hydrogels degraded in vitro with the rate dependent on the concentration of collagenase and also supported the adhesion of human umbilical vein ECs (HUVECs). Biomimetic RGD/GIA-PEGDA hydrogels with incorporation of 1% RGD-PEGDA into GIA-PEGDA hydrogels induced capillary-like organization when HUVECs were seeded on the hydrogel surface, while RGD/PEGDA and GIA-PEGDA hydrogels did not. These results indicate that both cell adhesion and biodegradability of scaffolds play important roles in the formation of capillary-like networks.     

Thermally cross-linked oligo(poly(ethylene glycol) fumarate) hydrogels support osteogenic differentiation of encapsulated marrow stromal cells in vitro

A novel polymer, oligo(poly(ethylene glycol) fumarate) (OPF), cross-linked with a thermal radical initiation system has recently been developed in our laboratory as an injectable, biodegradable cell carrier for regeneration of orthopaedic tissues. The cross-linking, swelling, and degradative properties of hydrogels prepared from OPF with poly(ethylene glycol) of two different chain lengths were assessed. The two OPF types had similar gelation onset times ( approximately 3.6 min) but, when cross-linked for 8 min at 37 degrees C, exhibited significantly different swelling characteristics (fold swelling: 17.5 +/- 0.2 vs 13.4 +/- 0.4). Rat marrow stromal cells (MSCs) were then directly combined with the hydrogel precursors and encapsulated in a model OPF formulation at approximately 14 million cells/mL, cultured in vitro in the presence of osteogenic supplements (dexamethasone), and monitored over 28 days via histology. MSC differentiation in these samples (6 mm diameter x 0.5 mm thick before swelling), as determined by Von Kossa staining for calcified matrix, was apparent by day 21. At day 28, mineralized matrix could be seen throughout the samples, many microns away from the cells. These experiments strongly support the usefulness of thermally cross-linked OPF hydrogels as injectable cell carriers for bone regeneration.     

Synthesis of amphiphilic alternating polyesters with oligo(ethylene glycol) side chains and potential use for sustained release drug delivery

Novel amphiphilic alternating polyesters, poly((N-phthaloyl-l-glutamic anhydride)-co-(2-(2-(2-methoxyethoxy)ethoxy)methyl)oxirane) (P(PGA-co-ME(2)MO)), were synthesized by alternating copolymerization of PGA and ME(2)MO. The structures of the synthesized polyesters were characterized by (1)H NMR, (13)C NMR, FT-IR, and GPC analyses. Because of the presence of oligo(ethylene glycol) (OEG) side chains, the polyesters could self-assemble into thermosensitive micelles. Dynamic light scattering (DLS) showed that these micelles underwent thermoinduced size decrease without intermicellar aggregation. In vitro methyl thiazolyl tetrazolium (MTT) assay demonstrated that the polyesters were biocompatible to Henrietta Lacks (HeLa) cells, rendering their potential for drug delivery applications. Two hydrophobic drugs, rifampin and doxorubicin (DOX), were loaded into the polyester micelles and observed to be released in a zero-order sustained manner. The sustained release could be accelerated in lower pH or in the presence of proteinase K, due to the degradation of the polyester under these conditions. Remarkably, in vitro cell experiments showed that the polyester micelles accomplished fast release of DOX inside cells and higher anticancer efficacy as compared with the free DOX. With enhanced stability during circulation condition and accelerated drug release at the target sites (e.g., low pH or enzyme presence), these novel polyesters with amphiphilic structures are promising to be used in sustained release drug delivery systems.     

Development of an immunosensor for the detection of vitellogenin using impedance spectroscopy

A simple and direct immunosensor for the determination of carp (Carassius auratus) vitellogenin (Vtg), a female-specific protein, has been proposed based on an antibody-captured conducting polymer-coated electrode. The monoclonal antibody specific to carp (C. auratus) Vtg was immobilized by covalent coupling to the carboxylic acid group on the polymer. The antibody immobilization and antibody-antigen interaction have been demonstrated by means of quartz crystal microbalance and impedance spectroscopic techniques. The impedance change occurred at the sensor surface due to the specific immuno-interaction was utilized to determine Vtg. The sensor showed high selectivity and sensitive response to Vtg in a buffered medium without redox probe. Vtg was determined in the linear range from 1.0 to 8.0 microg/l with the standard deviation of +/-0.13 (n =3) and the detection limit was determined to be 0.42 microg/l. This method was applied to the determination of Vtg in real male and female carp (C. auratus) serum samples.     

Strong repulsive forces between protein and oligo (ethylene glycol) self-assembled monolayers: a molecular simulation study

Restrained molecular dynamics simulations were performed to study the interaction forces of a protein with the self-assembled monolayers (SAMs) of S(CH2)4(EG)4OH, S(CH2)11OH, and S(CH2)11CH3 in the presence of water molecules. The force-distance curves were calculated by fixing the center of mass of the protein at several separation distances from the SAM surface. Simulation results show that the relative strength of repulsive force acting on the protein is in the decreasing order of OEG-SAMs > OH-SAMs > CH3-SAMs. The force contributions from SAMs and water molecules, the structural and dynamic behavior of hydration water, and the flexibility and conformation state of SAMs were also examined to study how water structure at the interface and SAM flexibility affect the forces exerted on the protein. Results show that a tightly bound water layer adjacent to the OEG-SAMs is mainly responsible for the large repulsive hydration force.