PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine‐PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine‐PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN–PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2‐kDa FN–PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN–PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.     

Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents

Ligand conjugation is an attractive approach to rationally modify the poor pharmacokinetic behavior and cellular uptake properties of antisense oligonucleotides. Polyethylene glycol (PEG) attachment is a method to increase solubility of oligonucleotides and prevent the rapid elimination, thus increasing tissue distribution. On the other hand, the attachment of long PEG chains negatively influences the pharmacodynamic effect by reducing the hybridization efficiency. We examined the use of short PEG ligands on the in vitro effect of antisense agents. Circular dichroism showed that the tethering of PEG₁₂-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand. In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate.     

Introduction of antioxidant-loaded liposomes into endothelial cell surfaces through DNA hybridization

Ischemia–reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)–phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)₂₀) and 20-mer of complementary deoxythymidylic acid (oligo(dT)₂₀), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)₂₀ and oligo(dT)₂₀. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.     

PEG/NaPA aqueous two-phase systems for the purification of proteases expressed by Penicillium restrictum from Brazilian Savanna

The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol⁻¹ and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.     

Kinematic viscosity of therapeutic pulmonary surfactants with added polymers

The addition of various polymers to pulmonary surfactants improves surface activity in experiments both in vitro and in vivo. Although the viscosity of surfactants has been investigated, the viscosity of surfactant polymer mixtures has not. In this study, we have measured the viscosities of Survanta and Infasurf with and without the addition of polyethylene glycol, dextran or hyaluronan. The measurements were carried out over a range of surfactant concentrations using two concentrations of polymers at two temperatures. Our results indicate that at lower surfactant concentrations, the addition of any polymers increased the viscosity. However, the addition of polyethylene glycol and dextran to surfactants at clinically used concentrations can substantially lower viscosity. Addition of hyaluronan at clinical surfactant concentrations slightly increased Infasurf viscosity and produced little change in Survanta viscosity. Effects of polymers on viscosity correlate with changes in size and distribution of surfactant aggregates and the apparent free volume of liquid as estimated by light microscopy. Aggregation of surfactant vesicles caused by polymers may therefore not only improve surface activity as previously shown, but may also affect viscosity in ways that could improve surfactant distribution in vivo.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

聚乙二醇对平邑甜茶叶片气孔器超微结构的影响

 采用透射电镜技术对聚乙二醇（PEG）模拟干旱处理的平邑甜茶(Malus hupehensis Rehd (pingyitiancha))叶片气孔器超微结构进行了观察,并对叶片气孔器超微结构制片方法进行了改进。所观察结果如下：1）PEG胁迫后保卫细胞中叶绿体数量增加,而叶绿体中淀粉粒数量呈下降趋势;2）PEG胁迫降低保卫细胞中线粒体的数量及破坏线粒体的超微结构;3）PEG胁迫使平邑甜茶保卫细胞中的液泡变小以致不明显。     

Viscoelastic properties of plasticized methylcellulose and chemically crosslinked methylcellulose

Films of methylcellulose (MC), poly(ethylene glycol)400 (PEG400) plasticized MC, and MC gels (MC crosslinked with glutaraldehyde (GA)) were prepared by casting from aqueous solutions. The swelling test has shown that the MC gels were insoluble in water and that their crosslinking density increased with increasing GA and HCl concentrations. The effect of the addition of PEG400 or GA to MC was investigated through dynamic mechanical analysis (DMA). The DMA analysis of PEG400/MC blends has shown that PEG400 was compatible with MC and was an effective plasticizer since the curves of tan δ against temperature exhibited single peaks (corresponding to a single glass transition temperature), which were displaced to lower values with increasing PEG400 content. The thermogravimetric analysis (TGA) indicated that the thermal stability of MC was not affected by the chemical crosslinking. The tensile strength was slightly increased through crosslinking while the elongation was slightly decreased. The presence of moisture in MC hydrogels decreased the tensile strength and enhanced the elongation while the addition of PEG400 decreased the tensile strength but sharply increased the elongation.     

Enhancing transfection efficiency using polyethylene glycol grafted polyethylenimine and fusogenic peptide

This study presents a new formulation method for improving DNA transfection efficiency using a fusogenic peptide and polyethylene glycol grafted polyethylenimine. Succinimidyl succinate polyethylene glycol (PEG-SSA) was conjugated with polyethylenimine (PEI). PEI is well known for a good endosomal escaping and DNA condensing agent. The positively charged synthetic fusogenic peptide, KALA, was coated on the negatively charged PEG-g-PEI/DNA and PEI/DNA complexes. The KALA/PEI/DNA complexes exhibited aggregation behavior at higher KALA coating amounts with an effective diameter of around 1,000 nm. However, the KALA/PEG-g-PEI/DNA complexes were 100–300 nm in size with a surface zeta-potential (ζ) value of about +20 mV. The conjugated PEG molecules suppressed any KALA-mediated inter-particle aggregation, and thereby improved the transfection efficiency. Consequently, the transfection efficiency of the KALA/PEG-g-PEI/DNA complexes was obtained by utilizing both the fusogenic activity of KALA and the steric repulsion effect of PEC.