High Molecular Weight Polyethylene Glycol Research Articles

Partition of plasmid DNA in polymer–salt aqueous two-phase systems

The partition of plasmid DNA (pDNA) in polyethylene glycol (PEG)–phosphate aqueous two-phase systems (ATPS) is presented. A high molecular weight (HMW) and a low molecular weight (LMW) polymer, PEG-1450 and -300, were used in combination with di-potassium hydrogen phosphate. The experimental results demonstrated that the plasmid pTX0161 displays a varied partition behaviour in PEG–phosphate ATPS. In HMW PEG (PEG-1450–phosphate systems), pDNA partitioned to the bottom phase only. In LMW PEG (PEG-300–phosphate systems), pDNA partitioned to all of the phases with respect to the phase composition, system temperature and concentration of lysate used in the ATPS. In systems with volume ratios higher than one, pDNA was mainly recovered in the top phase. For volume ratios between 0.5 and 1, pDNA mainly partitioned to the interface. In systems with volume ratios below 0.5, most of the pDNA was recovered in the bottom phase. For temperatures between 4 and 25 °C, the partition to the top phase decreased whereas partition to the interface steadily increased. At 25 °C, over 80% of pDNA was recovered in the interface. The partition to the bottom phase increased steadily with increasing temperatures up to 40 °C and the partition to the interface decreased. At 20 °C, the recovery of pDNA in the interface gradually increased and reached a maximum at 60% (w/w) lysate with 80% recovery recorded. At 25 °C, over 80% of pDNA was recovered in the interface from lysate concentrations greater than 35% (w/w). At 30 °C, the top phase preference changed to an interface preference between 0 and 20% (w/w) lysate.

Effects of Macromolecular Crowding on Glycoprotein Processing Enzymes

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.

Marked Therapeutic Efficacy of a Novel Polyethyleneglycol-SN38 Conjugate, EZN-2208, in Xenograft Models of Non-Hodgkin's Lymphoma.

Background: The clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin), the active moiety of CPT-11 (Camptosar®), has been severely compromised due to its poor solubility. EZN-2208 is a novel, water-soluble, polyethyleneglycol (PEG)-SN38 conjugate generated by linking SN38 with a multi-arm high molecular weight PEG, 40k 4-arm-PEG, via a glycine linker. Here, we compare the efficacy of EZN-2208 and CPT-11 in the treatment of animals bearing non-Hodgkin's lymphomas.Methods: The in vitro cytotoxicity of EZN-2208 and CPT-11 were tested in human lymphoma (Raji, Daudi, DoHH2) cancer cell lines using the tetrazolium assay. The pharmacokinetics and maximum tolerated dose (MTD) of EZN-2208 and CPT-11 were evaluated in tumor-free severe combined immunodeficient (SCID) mice and the therapeutic efficacy was evaluated in xenograft models of non-Hodgkin's lymphoma (Raji and Daudi).Results: In vitro, the IC50 of EZN-2208 ranged from 2–20nM and the in vitro potency of EZN-2208 was 30 to 50-fold more than CPT-11. The MTDs of EZN-2208 and CPT-11 in SCID mice were 30 and 60 mg/kg, respectively, when injected as a single dose. When administered in a multiple dose regimen (q2d x 5), the MTDs of EZN-2208 and CPT-11 were 10- and 40 mg/kg respectively. The pharmacokinetic profile of EZN-2208 was biphasic showing a rapid distribution phase (t1/2a =0.6h) and a slow elimination phase (t1/2b =19.3h for conjugate and 14.2h for SN38). In the Raji xenograft model, treatment with a single MTD of EZN-2208 resulted in a 500% improvement in life span (ILS) and 50% cures of animals compared with 19% ILS observed for mice treated with a single MTD of CPT-11. Multiple dose treatment of EZN-2208 resulted in 90% cures of animals compared with 63% ILS and no cures observed for the CPT-11 group. In the Daudi xenograft model, a single injection of EZN-2208 given as early treatment resulted in cures of 100% of animals in contrast to 66% ILS and no cures observed for CPT-11 group. When treatment was delayed, a single MTD of EZN-2208 caused 90% cures of animals, whereas CPT-11 treatment was completely ineffective.Conclusions: EZN-2208 demonstrated excellent efficacy in preclinical models of non-Hodgkin's lymphoma. Ongoing Phase I studies will determine the optimal dose and schedule of EZN-2208.

Cell wall water content has a direct effect on extensibility in growing hypocotyls of sunflower (Helianthus annuus L.)

It has been proposed that spacing between cellulose microfibrils within plant cell walls may be an important determinant of their mechanical properties. A consequence of this hypothesis is that the water content of cell walls may alter their extensibility and that low water potentials may directly reduce growth rates by reducing cell wall spacing. This paper describes a number of experiments in which the water potential of frozen and thawed growing hypocotyls of sunflower (Helianthus annuus L.) were altered using solutions of high molecular weight polyethylene glycol (PEG) or Dextran while their extension under constant stress was monitored using a creep extensiometer (frozen and thawed tissue was used to avoid confounding effects of turgor or active responses to the treatments). Clear reductions in extensibility were observed using both PEG and Dextran, with effects observed in hypocotyl segments treated with PEG 35 000 solutions with osmotic pressures of > or =0.21 MPa suggesting that the relatively mild stresses required to reduce water potentials of plants in vivo by 0.21 MPa may be sufficient to reduce growth rates via a direct effect on wall extensibility. It is noted, therefore, that the water binding capacity of plant cell walls may be of ecophysiological importance. Measurements of cell walls of sunflower hypocotyls using scanning electron microscopy confirmed that treatment of hypocotyls with PEG solutions reduced wall thickness, supporting the hypothesis that the spatial constraint of movement of cellulose microfibrils affects the mechanical properties of the cell wall.

Liposomal approach towards the development of a longer‐acting factor VIII

Prevention of spontaneous bleeding in patients with severe haemophilia A usually requires therapeutic infusions every 2-3 days because of the short half-life of factor VIII (FVIII). Longer-acting FVIII products that require less frequent infusions would be beneficial and might obviate the need for central catheters in most patients. Liposomal formulation can enhance the efficacy of some therapeutic products. The incorporation of high-molecular weight polyethylene glycol (PEG) can extend the circulatory half-life of the liposome. These combined approaches led to the development of BAY 79-4,980, a PEG-containing liposomal version of Kogenate FS (rFVIII-FS). Results from preclinical models and early clinical trials have shown that BAY 79-4,980 prolongs the time to the next bleed. Further clinical evaluation of the efficacy and long-term safety of BAY 79-4,980 are planned.

Evaluation of Hydroperoxides in Common Pharmaceutical Excipients

While the physical properties of pharmaceutical excipients have been well characterized, impurities that may influence the chemical stability of formulated drug product have not been well studied. In this work, the hydroperoxide (HPO) impurity levels of common pharmaceutical excipients are measured and presented for both soluble and insoluble excipients. Povidone, polysorbate 80 (PS80), polyethylene glycol (PEG) 400, and hydroxypropyl cellulose (HPC) were found to contain substantial concentrations of HPOs with significant lot-to-lot and manufacturer-to-manufacturer variation. Much lower HPO levels were found in the common fillers, like microcrystalline cellulose and lactose, and in high molecular weight PEG, medium chain glyceride (MCG), and poloxamer. The findings are discussed within the context of HPO-mediated oxidation and formulating drug substance sensitive to oxidation. Of the four excipients with substantial HPO levels, povidone, PEG 400, and HPC contain a mixture of hydrogen peroxide and organic HPOs while PS80 contains predominantly organic HPOs. The implications of these findings are discussed with respect to the known manufacturing processes and chemistry of HPO reactivity and degradation kinetics. Defining critical HPO limits for excipients should be driven by the chemistry of a specific drug substance or product and can only be defined within this context.

Effect of Molecular Size of PEGylated Recombinant Human Epidermal Growth Factor on the Biological Activity and Stability in Rat Wound Tissue

The purpose of this study was to investigate the effect of size of polyethylene glycol (PEG) conjugated to recombinant human epidermal growth factor (rhEGF) on its stability in skin wound tissue and in vitro biological activity to find the desirable conjugate as topical therapeutic agent for wound healing. Site-specific PEGylation at N-terminus of rhEGF was performed with monomethoxy PEG-Butyraldehyde derivatives (MW 2, 5, and 20 kDa). Mono-PEG-rhEGFs retained 60–70% of biological activity of native rhEGF, and the effect of PEG size was not significant. The improvement of stability in the rat skin wound tissue was dependent on the increase of the PEG size attached. The degradation half-lives of native rhEGF, mono-PEG-2K-, −5K-, and −20K-rhEGFs were 1.1, 3.1, 5.2, and 41.5 hr, respectively. Therefore, mono-PEG-20K-rhEGF was considered to be the most desirable in terms of the increase of stability and the preservation of biological activity. This study suggests that the high molecular weight PEG at N-terminus of rhEGF would give a satisfactory stabilizing effect and thus may improve therapeutic efficacy in clinical use.

A polymer solution process for synthesis of (Y,Gd) 3Al 5O 12:Ce phosphor particles

Cerium-doped yttrium-gadolinium aluminum garnet, (Y,Gd) 3Al 5O 12:Ce, shortened here to (Y,Gd)AG:Ce, was systematically prepared by a polymer complex method, using high molecular weight polyethylene glycol (PEG). Without PEG, only large flakes several micrometers in size containing nearly pure (Y,Gd)AlO 3 were obtained. Adding PEG dramatically reduced the particle size. However, the (Y,Gd)AG phase was not obtained by merely adding PEG. Instead, mainly (Y,Gd) 2O 3 and small amount of Ce 2O 3 were obtained. Simultaneous use of PEG and a flux (simple salt) such as BF 2 resulted in the formation of small YAG particles. The presence of the (Y,Gd)AG phase results in intense yellow–green luminescence. By selecting various heating routes, the fractions of cubic and hexagonal phases were controlled. It was found that the luminescence intensity is proportional to the fraction of cubic phase, and is probably independent of the hexagonal phase.

Novel Processing for Softly Agglomerated Luminescent Y2O3: Eu3+ Nanoparticles Using Polymeric Precursors

Synthesis of europium ion doped yttrium oxide (Y2O3:Eu3+) phosphor nanoparticles using a relatively high molecular weight polyethylene glycol is reported. Y2O3: Eu3+ materials could be prepared by simply heating in air provided that, water-soluble polymer be added into solutions containing metal nitrates. The polymer was expected to form carbonaceous materials around the produced primary particles to reduce the tendency of those particles to agglomerate. The carbonaceous materials could be removed by heating at higher temperatures, resulting in softly-agglomerated particles in the size range of 20-100 nm. The effect of synthesis parameters on particle morphology, crystallinity, and photoluminescence properties was investigated.

Pharmacokinetics and Biodistribution of Novel Aptamer Compositions

Aptamers are highly selective nucleic acid-based drugs that are currently being developed for numerous therapeutic indications. Here, we determine plasma pharmacokinetics and tissue distribution in rat of several novel aptamer compositions, including fully 2'-O-methylated oligonucleotides and conjugates bearing high-molecular weight polyethylene glycol (PEG) polymers, cell-permeating peptides, and cholesterol. Levels of aptamer conjugates in biological samples were quantified radiometrically and by a hybridization-based dual probe capture assay with enzyme-linked fluorescent readout. Intact aptamer in urine was detected by capillary gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). Aptamer compositions examined exhibited a wide range of mean residence times in circulation (0.6-16 h) and significant variation in distribution levels among organs and tissues. Among the conjugates tested, in vivo properties of aptamers were altered most profoundly by conjugation with PEG groups. Complexation with a 20 kDa PEG polymer proved nearly as effective as a 40 kDa PEG polymer in preventing renal clearance of aptamers. Conjugation with 20 kDa PEG prolonged aptamer circulatory half-life, while reducing both the extent of aptamer distribution to the kidneys and the rate of urinary elimination. In contrast, the fully 2'-O-Me aptamer composition showed rapid clearance from circulation, and elimination with intact aptamer detectable in urine at 48 h post-administration. We find that conjugation and chemical composition can alter fundamental aspects of aptamer residence in circulation and distribution to tissues. Though the primary effect of PEGylation was on aptamer clearance, the prolonged systemic exposure afforded by presence of the 20 kDa moiety appeared to facilitate distribution of aptamer to tissues, particularly those of highly perfused organs.

Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing

The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (14)C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The 14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH.

Water vapor permeability, tensile properties and solubility of methylcellulose-based edible films

Water vapor permeability (WVP), tensile strength (TS), % elongation ( E), adsorption capacity and % soluble matter (SM) were investigated in methylcellulose (MC) films plasticized by polyethylene glycol (PEG). The WVP of films was determined to be 0.232 × 10 −10–1.160 × 10 −10 g/m s Pa, TS between 17 and 44 N/mm 2 and % E between 14% and 97%, depending on composition. Film formation was affected by MC concentration, ethanol percent in the solution and the presence of PEG. Incorporation of various molecular weight (MW) PEGs to the polymer matrix increased both WVP and % E, decreased TS. Increase in PEG400 concentration had similar effects. Solubility studies indicated that MC films were water-soluble and PEG containing samples had higher solubilities. Results suggest that mainly hydrogen bonding between PEG and MC or blocking effect in the case of high MW PEGs determine the film properties.

Effect of water potential on the development of an haploid strain of Sporisorium reilianum f.sp. zeae

Sporisorium reilianum f.sp. zeae, the causal agent of head smut, infects the roots of the maize plantlets. Little information is available concerning the development of the fungus in soil, although this saprophytic phase is an important part of the life cycle. This paper reports that water potential also affects hyphal induction, and this effect on the fungus may influence disease transmission. In response to a decrease in water potential from 0 to −1.52 MPa in presence of variable molecular weight polyethylene glycols, haploid hyphae develop from the haploid yeast. Hyphal extension is fastest at low water potentials (−1.2 MPa) controlled with high molecular weight polyethylene glycols, PEG-3350 and PEG-8000. Formation of parasitic dikaryotic hyphae following fusion between haploid hyphae was possible at low water potential (−1.2 MPa) and was not inhibited by water stress. These results are consistent with the hypothesis that the effects of low soil water potential on yeast–hyphal transition and hyphal growth facilitate the convergence of compatible haploid strains, and that this may increase disease severity.

Anaerobic Treatment of Polyethylene Glycol of Different Molecular Weights

Biodegradation of high molecular weight polyethylene glycol has been found difficult under aerobic conditions. The use of a chemical treatment prior to the aerobic one was necessary to remove high molecular weight of polyethylene glycol. Anaerobic treatment of polyethylene glycol may present an alternative to integrated chemical and biological treatment for the elimination of this compound. Anaerobic biodegradation of polyethylene glycol from 200 to 10000 molecular weight was studied. It showed that, unlike aerobic treatment, the size of the molecule up to 10000 does not affect the process efficiency. The critical retention time and the optimum retention time were estimated and ranged from 10 to 14 and from 16 to 19 days, respectively. Under these operational conditions percentage of removal ranged from 85 to 90%. Thus, anaerobic degradation of polyethylene glycol presents removal percentages and hydraulic retention times similar to those of biodegradable compounds tested at the same operational conditions.

Investigation of Carrying Agents on Microstructure of Electroplated Cu Films

Adding high molecular-weight polyethylene glycol (PEG) as a carrying agent benefits Cu electroplating from the viewpoint of an increase in both filling capability and films' conductivity, when plated in a lower current-density region. On electroplating in a higher current-density region, a gradually decreasing grain size from the top to bottom of Cu films occurs in the electrolyte without PEG or with PEG200. In comparison, sharp grains are formed for the mentioned multi-domain crystallization for those films deposited by the electrolyte containing PEG2000 or PEG6000 due to its lower surface tension which facilitates grain growth along the higher electric field. The used concentration of PEG was flexible due to the fact that the surface morphology, resistivity and deposition rate of deposited films remained almost unchanged over a wide PEG concentration range.

In vitro screening for increased drought tolerance in rice

In vitro screening at the cellular level was performed with mature seed-derived callus from five rice varieties, viz. IR 18351-229-3, IR 3185-6-3-3-2, SR 26-B, Nona Bokra, and C 14-8 of diverse geographical origin and with differential drought resistance at the in planta level. Callus was induced from mature seeds on Murashige and Skoog medium supplemented with 2.0 mgl−1 (9 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0, 10.0, and 15.0 gl−1 of high molecular weight polyethylene glycol (PEG, 6000) as stressing agent to create chemical drought. Simultaneous efforts were also made to assess the effects of chemical drought in altering morphogenetic response in different varieties under in vitro culture. Seed germination was almost unaffected in SR 26-B and C 14-8, unlike in other varieties where germination was seriously affected. In general, seed germination was found to be decreased in three genotypes, viz. IR 18351-229-3, IR 3185-6-3-3-2, and Nona Bokra, with increased PEG concentrations. All genotypes displayed callus induction percentage in decreasing order with increased PEG concentrations supplemented in the callus induction medium (CIM), except SR 26-B and Nona Bokra. Callus induction was found to be more on CIM fortified with 5.0 gl−1 PEG. In general, embryogenic callus induction and plantlet regeneration was found to be indirectly proportional to increased PEG concentrations used in CIM. Considering all characters, C 14-8 was found to be most appropriate in developing drought-tolerant lines under in vitro culture conditions followed by SR 26-B and Nona Bokra. A number of putative drought-tolerant plants were developed in C 14-8, SR 26-B, Nona Bokra, and IR 18351-29-3, and forwarded for field evaluation. In the majority of the progenies, a monogenic inheritance pattern for the drought tolerance character was observed.

Apparent membrane pore-formation by portuguese Man-of-war ( Physalia physalis) venom in intact cultured cells

Intracellular, ratiometric microfluorimetry with fura-2 reveals that low doses of Portuguese Man-of-war ( Physalia physalis) venom cause a linear increase in intracellular calcium accumulation by cultured L-929 cells. The influx of calcium is preceded by a lag period that is relatively independent of venom concentration, except at very low concentrations. Electron micrographs of negatively stained preparations of membranes from venom-treated L-929 and GH 4C 1 cells exhibit 10–80 nm diameter lesions. The number and diameter of these lesions correlate with venom concentration. The venom forms lesions in GH 4C 1 cells at much lower concentrations than in L-929 cells. Osmotic protectants such as sucrose and polyethylene glycol (PEG), reduce the extent of lactate dehydrogenase (LDH) release from venom-treated cells with the higher molecular weight PEG causing a greater inhibition of LDH release than sucrose. These results imply that Man-of-war venom produces pore-like structures in the membranes of target cells, which leads to colloid osmotic swelling with subsequent release of intracellular proteins and cell lysis.

Removal of aqueous phenol by Arthromyces ramosus peroxidase

The removal of aqueous phenol by Arthromyces ramosus peroxidase (ARP), in the presence of high molecular weight polyethylene glycol (PEG) as a protective additive, was investigated to study (i) the stoichiometry between phenol removal and peroxide consumption, (ii) enzyme inactivation in the presence of excess hydrogen peroxide, and (iii) the effect of reactor operating mode on phenol removal. The stoichiometry was observed to increase with increasing phenol conversion, and reached a maximum value of 0.9 mol phenol/mol peroxide when nearly complete phenol conversion was achieved. Spectral scans indicated that P-670 did not form with ARP in the presence of high concentrations of peroxide and in the absence of a reducing substrate. As long as phenol concentration was not limiting, the turnover capacity varied by approximately 3% of its average value of 76 400. This indicates that only marginal improvement in the efficiency of enzyme use would be achieved by stepped addition of enzyme. Similarly, sequential addition of hydrogen peroxide over an extended period of time yielded only a slight improvement in phenol removal from the reaction mixture. Key words: Arthromyces ramosus peroxidase, enzyme inactivation, phenol removal, reactor operation, stoichiometry.

Partitioning of pectinolytic enzymes in polyethylene glycol/potassium phosphate aqueous two-phase systems

Four different pectinolytic enzymes have been partitioned in polyethylene glycol (PEG)-potassium phosphate aqueous two-phase systems (20–15%). The influence of PEG molecular weight and the addition of sodium chloride were investigated. The best results concerning to the partition coefficients ( K p and K e) and the yield recovery ( R) were: PEG-400/phosphate with NaCl for exo-polygalacturonase ( K e=5.35 and R=89.5%), PEG-600/phosphate for pectin lyase (PL) ( K e=43.18 and R=98.5%) and pectinesterase (PE) ( K e =1.51 and R=69.6%), and PEG-10,000/phosphate for endo-polygalacturonase ( K e =1.35 and R=53.5%). The best purification factors were observed in the upper phase for the systems containing high molecular weight PEG without NaCl: PEG-6000 for exo-polygalacturonase (5.49 fold) and PEG-10,000 for endo-polygalacturonase (16.28 fold), PE (16.64 fold) and PL (14.27 fold).

Thermal Adaptability and Hand of Wool Fabric Treated with High Molecular Weight Polyethylene Glycol and a Softener

The aim of this study is to enhance the thermal adaptability of wool fabric by a treatment with high molecular weight polyethylene glycol (PEG-8000), and to improve the PEG-finished fabric's hand by treatment with a softener. Using the pad-dry-cure method, the fabric is treated in aqueous baths with 20-45% owb concentrations of PEG and 8.0-9.0% DMDHEU, resulting in 12-38% PEG add-ons proportional to the PEG concentrations. Subsequently, using the pad-cure method, the fabric is treated in aqueous solutions with 2-6% owb softener concentrations. Thermal adaptability, measured by heat of fusion and crystallization on a DSC, improves as PEG add-on increases. The PEG-treated fabric is stiffer and less smooth, soft, and full than when untreated, according to measurements with the KES-FB system. The PEG-treated fabric's thermal storage and release are 30-60% lower after softening, but the hand improves. Optimum conditions for maximizing thermal adaptability and achieving a hand equivalent to the untreated fabric are 45% PEG-8000/9.0% DMDHEU in the finishing bath and 6% softener solution concentration.