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.     

Poly(ethylene glycol) amphiphile adsorption and liposome partition

Surface localized poly(ethylene glycol) (PEG) amphiphiles of type C_16:0-EO₁₅₁ and C_18:2-EO₁₅₁ were studied via ellipsometry at macroscopic, flat methylated silica (MeSi), phosphatidic acid (PA), and phosphatidylcholine (PC) surfaces. At these surfaces the amphiphiles adsorb similarly, in a non-cooperative manner, achieving a plateau (≈0.1 PEG chains/nm²) well below amphiphile critical micelle concentration (CMC). The resultant PEG-enriched layers were 10–15 nm thick, with a polymer concentration (≈0.07 g/cm³) greater than the PEG-enriched phase of many dextran, PEG aqueous two-phase systems. PEG-amphiphile adsorption (mg/m²) at hydrophobic and phospholipid flat surfaces correlated with changes in the partition (log K) of PC liposomes in such two-phase systems. PEG-amphiphile adsorption at macroscopic surfaces appears to represent a balance between hydrophobic attraction and repulsive intra-chain interactions which promote chain elongation normal to the surface.     

A gaussian model for substrates of entangled cross-linked poly(ethylene glycol) in biomedical applications

Cells usually spread on a synthetic substrate through bonds between receptors and chemical groups on the substrate (ligands). Therefore, it is valuable to study the effects of the average number density of these chemical groups and the average distance between them to model and predict the cell behavior. Poly(ethylene glycol) [PEG] modified with peptide groups has been used widely in biomedical applications as a substrate material. In this study, a coarse-grained model is proposed for PEG to predict the average number density of ligands and the average distance between them. Molecular information such as initial molecular weight distribution, average molecular weight between cross-links, and average molecular weight between entanglements is used as input parameters. Based on simulation results, it is concluded that both entanglement and cross-link densities are required to create a network structure. The results suggest that an average initial molecular weight 2-3 times the average molecular weight between entanglements and a moderate cross-link density are sufficient to create a closed network structure with a high ligand density and a small average distance between them.     

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL)

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.     

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.     

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.     

Spectroscopic investigation of lipase from Pseudomonas cepacia solubilized in 1,4‐dioxane by non‐covalent complexation with methoxypoly(ethylene glycol)

Lipase from Pseudomonas cepacia was made soluble in 1,4‐dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme–PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far‐ and near‐UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near‐UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase–PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme–substrate interactions. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 624–629, 1999.     

Low versus high molecular weight poly(ethylene glycol)-induced states of stem bromelain at low pH: stabilization of molten globule and unfolded states

The effect of low, medium, and high molecular weight poly(ethylene glycol) (e.g., PEG-400, -6000, and -20,000) on the structure of the acid unfolded state of unmodified stem bromelain (SB) obtained at pH 2.0 has been studied by various spectroscopic methods. The conformation of stem bromelain at pH 2.0 exhibits substantial loss of secondary structure and almost complete loss of native tertiary contacts and has been termed the acid unfolded state (A(U)). Addition of PEG-400 to A(U) led to an increase in the mean residue ellipticity (MRE) value at 222 nm, indicating formation of alpha-helical structure. On the other hand, PEG-6000 and 20,000 led to a decrease in the MRE value at 222 nm, indicating unfolding of the A(U) state. Interestingly, at 70% (w/v) PEG-400 and 40% (w/v) PEG-20,000, MRE values at 222 nm almost approach the native state at pH 7.0 and the unfolded state (6 M GnHCl) of stem bromelain, respectively. The probes for tertiary structure showed formation of nonnative tertiary contacts in the presence of 70% (w/v) PEG-400, while 40% (w/v) PEG-6000 and 20,000 were found to stabilize the unfolded state of SB. An increase in binding of 1-anilino 8-naphthalene sulfonic acid and a decrease in fractional accessibility of tryptophan residues (f(a)) compared to A(U) in the presence of 70% PEG-400 indicate that the PEG-400-induced state has a significant amount of exposed hydrophobic patches and is more compact than A(U). The results imply that the PEG-400-induced state has characteristics of molten globule, and higher molecular weight PEGs led to the unfolding of the A(U) state.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.     

Biocatalytic surface modification of knitted fabrics made of poly (ethylene terephthalate) with hydrolytic enzymes from Thermobifida fusca KW3b

Knitted fabrics made from poly(ethylene terephthalate) fibres were treated with an enzyme preparation from Thermobifida fusca KW3b showing hydrolytic activity on p-nitrophenyl butyrate. The fabrics were also treated with NaOH and the results were compared. Both enzyme- and alkaline-treated fabrics showed an increase in reactive dye uptake, vertical wicking height and water absorption capacity of the fabrics indicating an increased surface hydrophilicity. However, X-ray photoelectron (XPS) and energy-dispersive spectroscopy (EDS) did not give conclusive results on the presence of newly introduced hydrophilic groups on the surface of the fibres. Analysis of the enzyme-treated poly(ethylene terephthalate) fibres by scanning electron microscopy (SEM) and by atomic force microscopy (AFM) indicated an increase in surface roughness of the fibres, which may contribute to the observed increased hydrophilicity of the PET fabrics. However, much longer treatment times (24 h) were required to obtain these effects with the enzymes compared to the chemical treatment (1 h).     

Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2_IIA3_IIB_IIA1_II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.