Grafted Poly Ethylene Glycol Chains Research Articles

Synthesis of polyethylene glycol-grafted magnetic nanoparticles for the fast and efficient extraction of anabolic substances from human urine

Anabolic steroids and β-agonists are commonly prohibited substances found in doping control studies; therefore, the determination of anabolic substances in biological samples is crucial. To analyze the anabolic compounds in urine, an adsorbent, polyethylene glycol (PEG)-grafted magnetic nanoparticle material (Fe3O4@SiO2-PEG), with low toxicity and strong biocompatibility was prepared in this investigation. Compared to those of Fe3O4 and Fe3O4@SiO2, the grafted PEG chains (approximately 5.4 wt.%) on the magnetic nanoparticles improved the extraction efficiencies by factors of 3.9–17.0 and 2.5–2.9, respectively, likely due to the electrostatic attraction and hydrogen bonding. To achieve maximum extraction efficiency, several extraction parameters were optimized, including the kind and volume of desorption solvent, pH, and the extraction and desorption time. The standard curves were linear within the range of 0.5–20 μg/L for methyltestosterone and trenbolone, and 0.02–5 μg/L for clenbuterol. The limits of detection for the three drugs were 0.01–0.12 μg/L. The limits of quantification were 0.02–0.40 μg/L. The levels of precision of the optimized method were assessed based on the respective intra- and inter-day and batch-to-batch relative standard deviations in the ranges of 3.2–5.2 % (n = 5), 5.9–11.3 % (n = 4), and 6.7–9.2 % (n = 3). The Fe3O4@SiO2-PEG nanoparticles could exclude urine matrix interferences (matrix effect of 91.8–98.1 %) and achieve satisfactory recoveries (75.5–116.1 %), affording sensitive and accurate determination of trace anabolic substances in urine.

Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.

Polymer nanocomposites from natural clay: understanding clay-PEG interactions and their effect on spacing between clay-plates

The interactions between clay-plates and hydrophilic polymer are investigated assuming that the polyethylene glycol (PEG) chains are grafted onto face-to-face clay-plates. Besides the usual van der Waals attractive interaction, the clay-plates experience a repulsive effective interaction, due to the excluded volume force between monomers along the grafted PEG chains. The face-to-face clay-plates then play the role of polymer brushes. The free energy (per unit area) of a clay-plate pair is the sum of these interactions, and from its expression, we determine the minimal inter-sheet distance, after intercalation, which corresponds to a critical percentage of PEG. Finally, our result is in good agreement with some recent experimental work.

Adsorption of Plasma Proteins onto PEGylated Lipid Bilayers: The Effect of PEG Size and Grafting Density.

Lipid bilayers grafted with polyethylene glycol (PEG) of different sizes (Mw = 750, 2000, and 5000) and grafting densities (1.6-25 mol % of PEGylated lipid in dipalmitoylphosphatidylcholine (DPPC) lipid molecules) were simulated with human serum albumin (HSA) using coarse-grained force fields. At low enough grafting density, the PEG has a conformation similar to that of an isolated chain in water, and its Flory radius RF is smaller than the distance between the grafting points (d), which is the so-called "mushroom" regime. In contrast, densely grafted PEG chains (RF > d) extend like brushes, with brush layer thickness given by the Alexander-de Gennes theory. A nearly spherical HSA added to this simulation migrates to the bilayer surface because of the charge interactions between anion residues of HSA and cationic cholines of DPPC, but this HSA-bilayer binding can be sterically suppressed by the PEG chains to an extent that depends on the PEG size and grafting density. In particular, regardless of the extent of the coverage of the PEG on the bilayer, the binding between HSAs and bilayers is suppressed by the PEG layer in a brush but not in a mushroom, indicating that the attractive force between proteins and bilayers can overcome the steric effect of the PEG layer in the mushroom state or in the transition region from mushroom to brush. This helps explain and clarify experiments that show much less adsorption of plasma proteins onto the particle or membrane surface when PEGs are in the brush rather than in the mushroom state.

Nanotechnology in Therapeutics – Current Technologies and Applications

Nanotechnology is an area of science devoted to the design, construction and utilization of functional structures on the nanometer scale (often 100nm or smaller). Therapeutic applications of nanotechnologies include the treatment of cancer liver diseases. Avoiding the recognition by the liver is also possible by developing long circulating polymeric colloidal carriers (“stealth” systems) able to avoid the opsonisation process and the recognition by the macrophages. The design of such carriers is based on the physico-chemical concept of the “steric repulsion” by grafting polyethylene glycol chains at the surface of nanoparticles, the adsorption of steric proteins may be dramatically reduced due to steric hindrance. Such an approach allows maintaining the drug carrier for a longer time into the circulation and the resulting extravasation towards non reticuloendothelial-located cancers may become possible. Now, new applications and exciting perspectives are proposed for the delivery of drugs to previously non-accessible diseased sanctuaries, like the brain (treatment of glioma and autoimmune diseases of the brain) or the ocular tissues (treatment of the autoimmune uveitis). Ocular autoimmunity is especially intriguing because of the unique immunological characteristics of the eye. On the basis of laboratory data, the feeding of ocular antigens, has been proposed as a safe and efficacious therapy for uveitis. This review highlights how these obstacles can be overcome by polymer science and nanotechnology. New developments in polymer science coupled with cell-based delivery strategies support the notion that diseases that now have limited therapeutic options can show improved outcomes by advances in nanomedicine. Keywords: Nanomedicine, cancer therapy, dendrimers, neurodegenerative disorders, osteoporosis

Nanotechnologies for drug delivery: Application to cancer and autoimmune diseases

Abstract Polymer-based nanotechnologies are now proposed as an alternative to classical formulations for drug administration, delivery and targeting. Therapeutic applications of the first generation of nanotechnologies include the treatment of cancer liver diseases. Avoiding the recognition by the liver is also possible by developing long circulating polymeric colloidal carriers (“stealth” systems) able to avoid the opsonization process and the recognition by the macrophages. The design of such carriers of second generation is based on the physico-chemical concept of the “steric repulsion”: by grafting polyethyleneglycol chains at the surface of nanoparticles, the adsorption of seric proteins may be dramatically reduced due to steric hindrance. Such an approach allows maintaining the drug carrier for a longer time into the circulation and the resulting extravasation towards non reticuloendothelial-located cancers may become possible. Now, new applications and exciting perspectives are proposed for the delivery of drugs to previously non accessible diseased sanctuaries, like the brain (treatment of glioma and autoimmune diseases of the brain) or the ocular tissues (treatment of the autoimmune uveitis). Finally, the use of nanotechnologies for the delivery of nucleic acids (oligonucleotides) is also discussed in this review.

Adsorption of poly(ethylene glycol)‐modified ribonuclease A to a poly(lactide‐co‐glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.

A genetically modified recombinant tumor necrosis factor-α conjugated to the distal terminals of liposomal surface grafted polyethyleneglycol chains

A genetically modified recombinant tumor necrosis factor (TNF)-α (rKRKTNF) was conjugated to the terminal carboxyl groups of liposome grafted polyethyleneglycol (PEG) chains. The long-circulating liposomes were composed of egg phosphatidylcholine, cholesterol (chol) and 7% carboxyl PEG-phosphatidylethanolamine. The conjugation efficiency of the genetically modified rKRKTNF under the conditions described in the text was approximately 55%. The biological activity of liposomal rKRKTNF, as tested with an in vitro cytotoxicity assay was reduced compared to the free, unconjugated rKRKTNF. In vivo biodistribution studies showed that conjugation of as little as 0.13% of the grafted PEG chains resulted in a rapid elimination of the formulation from the blood stream. It is speculated that both non-selective conjugate chemistry and inherent recognition of the TNF by the components of the reticuloendothelial system (RES) are responsible for the short blood half life of the rKRKTNF-PEG-liposomes. The result suggest that conjugating a rapidly clearing recombinant cytokine to long-circulating liposomes provides little advantage in modifying the pharmacokinetic parameters of the cytokine.

Experimental and Monte Carlo Simulation Studies of the Thermodynamics of Polyethyleneglycol Chains Grafted to Lipid Bilayers

Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a “mushroom” regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for conjugate binding by the mobile PE-PEG polymer chains.

Preliminary characterization of bovine beta-lactoglobulin after its conjugation to polyethylene glycol

The major component of the whey fraction of bovine milk, beta-lactoglobulin (betaLG), has been transformed by grafting polyethylene glycol chains either on the thiol group (free and after reduction of the S-S bridges) of the cysteine residues, or on the amino group of the lysine residues and/or of the N-terminal amino acid. Acylation of the protein was achieved at a controlled pH of 7.0 using increasing ratios of activated PEG to betaLG. Transformation of the dimeric form into the monomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of the free amino functions still present after reaction. The incidence of pegylation on the secondary structure of betaLG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studies with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorimetric measurements, showed an identical behavior of native as well as of pegylated betaLG.The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native betaLG. The pegylated protein exhibited less than 1/100 of the native betaLG inhibition capacity, that could moreover never be complete. This is thus demonstrating the loss of accessibility for at least multiple conformational epitopes through pegylation procedure.Spectrofluorimetric measurements showed that betaLG-N-PEG(7) was still able to bind retinol while no effect on the intrinsic fluorescence could be detected by adding palmitic acid. Whether this last ligand binds or not to pegylated betaLG is discussed.