Free PEG Research Articles

ТОПІЧНЕ ЛІКУВАННЯ КОНТАКТНОГО НІКЕЛЕВОГО ДЕРМАТИТУ: ПОТЕНЦІАЛ НАНОІНКАПСУЛЬОВАНОЇ СОД

Робота виконана в рамках НДР 0112U000542 «Біохімічні механізми токсичності наночастинок різної природи та інших антропогенних і біогенних токсикантів і біологічних систем» та 0116U003353 «Біохімічні механізми порушень метаболізму за умов надходження до організму токсикантів різного ґенезу». Контактний дерматит (КД) – одне з найпоширеніших захворювань на сьогодні, що розвивається у відповідь на контакт із різноманітними чинниками, одним з яких є нікель та його солі. Розвиток оксидативного та нітрооксидативного стресу є універсальним механізмом розвитку, в тому числі й захворювань шкіри та КД.Мета дослідження – вивчити ефективність наноінкапсульованої та вільної форми препарату пегільованої супероксиддисмутази (СОД) на моделі контактного нікелевого алергійного дерматиту.Матеріали і методи. Дослідження проводили на 40 щурах-самцях, яких рандомізовано на чотири групи (n=10): перша – інтактні тварини; друга – тварини з експериментальним КД; третя – щури з КД, яким для корекції застосовували ПЕГ-СОД у вільній формі (КД + СОД); четверта – особини з КД, яким наносили наноінкапсульовану форму СОД (КД + нано-СОД). Полімерні хітозанові наночастинки готували за методикою Z. Hussain et al. (2013). Інтенсивність розвитку КД оцінювали за візуальною шкалою та товщиною шкірної складки. Також досліджували показники оксидативного та нітрооксидативного стресу.Результати досліджень та їх обговорення. Аплікація 5 % сульфату нікелю викликає розвиток контактного дерматиту та оксидативного і нітрооксидативного стресу зі зростанням рівня продуктів ПОЛ та ослабленням ланок ендогенного антиоксидантного захисту. Застосування вільної форми пегільованої супероксиддисмутази супроводжується зниженням інтенсивності ураження як за візуальними ознаками, так і достовірним зниженням інтенсивності ПОЛ та активацією показників антиоксидантного захисту. Наноінкапсульвана форма пегільованої супероксиддисмутази вірогідно більш інтенсивно покращує стан уражених шкірних покривів досліджуваних тварин, у тому числі й порівняно з групою щурів, котрі отримували її вільну форму: достовірно кращими були показники як і ПОЛ, так і антиоксидантного захисту в сироватці крові та тканинах печінки.Висновки. Отримані результати дослідження свідчать по перспективність подальшого вивчення та впровадження у клінічну практику наноінкапсульованих форм СОД для лікування контактних дерматитів.

Influence of adsorbed and nonadsorbed polymer additives on the viscosity of magnesium oxide suspensions

ABSTRACTAdsorbed polymer additives have been employed to reduce water content and improve cement workability through lowering viscosity, but the influence of over‐dosage and the presence of nonadsorbed chains have yet to be fully understood. Model magnesium oxide (MgO) suspensions were used to investigate the potential processing effect of “free” chain concentration on cementitious mixtures. The rheological impact of the free chains was measured through incorporation of nonadsorbing poly(ethylene glycol) (PEG) to suspensions stabilized with an adsorbed comb‐polymer superplasticizer. Analyses of the rheological data, that showed viscosity‐increases and viscosity‐reduction due to free PEG concentrations revealed a transition from depletion flocculation to depletion stabilization that contributed to the flow properties of the suspensions. The viscosity‐reduction observed for high concentrations of free chains may be useful for improved mixing of cements with free polymer in addition to the adsorbed polycarboxylate ether‐based superplasticizer. Additionally, the influence of free PEG on the macroscale flow behavior was also examined through local velocity measurements under shear. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 45696.

Enhancement Effect of a Variable Topology of a Membrane-Tethered Anti-Poly(ethylene glycol) Antibody on the Sensitivity for Quantifying PEG and PEGylated Molecules.

Sensitive quantification of the pharmacokinetics of poly(ethylene glycol) (PEG) and PEGylated molecules is critical for PEGylated drug development. Here, we developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for PEG by tethering an anti-PEG antibody (AGP3) via tethers with different dimensions on the surface of 293T cells (293T/S-αPEG, short-type cells; 293T/L-αPEG, long-type cells; 293T/SL-αPEG, hybrid-type cells) to improve the binding capacity and detection limit for free PEG and PEGylated molecules. The binding capacity of hybrid-type cells for PEG-like molecules (CH3-PEG5K-FITC (FITC = fluorescein isothiocyanate) and eight-arm PEG20K-FITC) was at least 10-80-fold greater than that of 293T cells expressing anti-PEG antibodies with uniform tether lengths. The detection limit of free PEG (OH-PEG3K-NH2 and CH3-PEG5K-NH2) and PEG-like molecule (CH3-PEG5K-FITC, CH3-PEG5K-SHPP, and CH3-PEG5K-NIR797) was14-137 ng mL-1 in the hybrid-type cell-based sandwich ELISA. 293T/SL-αPEG cells also had significantly higher sensitivity for quantification of a PEGylated protein (PegIntron) and multiarm PEG macromolecules (eight-arm PEG20K-NH2 and eight-arm PEG40K-NH2) at 3.2, 16, and 16 ng mL-1, respectively. Additionally, the overall binding capacity of 293T/SL-αPEG cells for PEGylated macromolecules was higher than that of 293T/S-αPEG or 293T/L-αPEG cells. Anchoring anti-PEG antibodies on cells via variable-length tethers for cell-based sandwich ELISA, therefore, provides a sensitive, high-capacity method for quantifying free PEG and PEGylated molecules.

Optimization of an Anti-poly(ethylene glycol) (anti-PEG) Cell-Based Capture System To Quantify PEG and PEGylated Molecules.

Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG2K-NH2 and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL-1 levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL-1. All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum dot quantification down to 7 pM. These results suggest that the combination of 293T/15-2b cells and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules, and PEGylated proteins, whereas the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles. The optimized anti-PEG cell-based sandwich ELISA can provide a sensitive, precise, and convenient tool for the quantification of a range of PEGylated molecules.

Self dispersing mixed micelles forming systems for enhanced dissolution and intestinal permeability of hydrochlorothiazide

Mixed micelles provide promising strategy for enhancing dissolution and permeability of drugs. However, their fluid nature limited the stability of the loaded drug and hindered the development of stable oral dosage form. Accordingly, the objective was to develop solid self dispersing mixed micelle forming systems (MMFS) for enhanced dissolution and intestinal permeability of hydrochlorothiazide. Pseudoternary phase diagrams were constructed using sodium cholate, lecithin with either poloxamer 407 or PEG 4000 to determine the composition of MMFS. Both polymer free and poloxamer or PEG containing MMFS were prepared as homogenous matrices or as solid self dispersing powder. The later was developed by adsorption of MMFS on avicel-aerosil mixture. Differential scanning calorimetry provided an evidence for existence of hydrochlorothiazide as molecular dispersion in the MMFS. Dispersing polymer free, PEG 4000 or poloxamer based MMFS in aqueous medium produced micelles having size values of 119, 52.6 and 28nm, respectively. The zeta potential values were −61.8, −59.5 and −19.5mV for the same systems, respectively. Preparation of solid self dispersing MMFS enhanced the dissolution rate of hydrochlorothiazide. The intestinal absorption of hydrochlorothiazide from its aqueous solution and polymer incorporating mixed micellar systems was monitored using in situ rabbit intestinal perfusion technique. The permeability results showed a clear trend for enhanced membrane transport of the drug after being incorporated into poloxamer containing mixed micellar system. The study thus introduced a versatile easily formulated solid self dispersing system with high potential for solving the dissolution and permeability problems of class IV drugs.

Probing the Surface Hydration of Nonfouling Zwitterionic and PEG Materials in Contact with Proteins.

Zwitterionic polymers and poly(ethylene glycol) (PEG) have been reported as promising nonfouling materials, and strong surface hydration has been proposed as a significant contributor to the nonfouling mechanism. Better understanding of the similarity and difference between these two types of materials in terms of hydration and protein interaction will benefit the design of new and effective nonfouling materials. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied for in situ and real-time assessment of the surface hydration of the sulfobetaine methacrylate (SBMA) and oligo(ethylene glycol) methacrylate (OEGMA) polymer brushes, denoted as pSBMA and pOEGMA, in contact with proteins. Whereas a majority of strongly hydrogen-bonded water was observed at both pSBMA and pOEGMA surfaces, upon contact with proteins, the surface hydration of pSBMA remained unaffected, but the water ordering at the pOEGMA surface was disturbed. The effects of free sulfobetaine, free PEG chains with two different molecular weights, and PEG coated gold nanoparticles on the surface hydration of proteins were investigated. The results indicated that free sulfobetaine could strengthen the protein hydration layer, but free PEG chains greatly disrupt the protein hydration layer and likely directly interact with the protein molecules. In contrast to free PEG, the PEG chains anchored on the nanoparticles behave similarly to the pOEGMA surface and could induce strong hydrogen bonding of the water molecules at the protein surfaces.

A class of linker free amphiphilic PEG grafted polymer support for linear and cyclic peptides

PEG grafted (PEG400 and PEG1500), highly hydrophobic/hydrophilic balanced polymeric Schiff base dendron resins were synthesized by the reaction between diethylenetriamine and benzaldehyde molecules and characterized. The usefulness of the resins was demonstrated by synthesizing biologically significant linear as well as disulfide classes of endothelin peptides in high yields and purities.

PEGylation site-dependent structural heterogeneity study of monoPEGylated human parathyroid hormone fragment hPTH(1-34).

The structures of C- and N-terminally monoPEGylated human parathyroid hormone fragment hPTH(1-34) as well as their unmodified counterparts, poly(ethylene glycol) (PEG) and hPTH(1-34), have been studied by small-angle neutron scattering (SANS). The scattering results show that free hPTH(1-34) in 100 mM phosphate buffer (pH 7.4) aggregates into clusters. After conjugation with PEG, the PEG-peptide conjugates self-assemble into a supramolecular core-shell structure with a cylindrical shape. The PEG chains form a shell around the hPTH(1-34) core to shield hPTH(1-34) from the solvent. The detailed structural information on the self-assembled structures is extracted from SANS using a model of the cylindrical core with a shell of Gaussian chains attached to the core surface. On the basis of the data, because of the charge-dipole interactions between the conjugated PEG chain and the peptide, the conjugated PEG chain forms a more collapsed conformation compared to free PEG. Moreover, the size of the self-assembled structures formed by the C-terminally monoPEGylated hPTH(1-34) is about 3 times larger than that of the N-terminally monoPEGylated hPTH(1-34). The different aggregation numbers of the self-assembled structures, triggered by different PEGylation sites, are reported. These size discrepancies because of different PEGylation sites could potentially affect the pharmacokinetics of the hPTH(1-34) drug.

PEG Containing Thiol–Ene Network Membranes for CO2 Separation: Effect of Cross-Linking on Thermal, Mechanical, and Gas Transport Properties

A new family of poly(ethylene glycol) (PEG) based membranes for CO2 separation was developed using thiol–ene photopolymerization. Compared to photopolymerized PEG-containing acrylate membranes, these new thiol–ene based membranes offer improved mechanical properties and processing advantages. The starting material, a combination of a trithiol cross-linker and a PEG diene, was gradually modified with a PEG dithiol while maintaining 1:1 thiol:ene stoichiometry. This approach made it possible to decrease the network cross-link density, resulting in simultaneous increases in free volume and PEG content. Materials with high concentrations of dithiol were very stretchable, with largely, up to 500%, improved elongation at break, yet they exhibited commendable CO2/N2, O2, H2, and CH4 permeability-selectivity performance. The average molecular weight of polymer chains between cross-links, Mc, was determined experimentally by fitting the classic network affine model to stress–strain data obtained via tensile testin...

Medium Chain Triglyceride (MCT) Rich, Paclitaxel Loaded Self Nanoemulsifying Preconcentrate (PSNP): A Safe and Efficacious Alternative to Taxol<SUP>®</SUP>

The current work was aimed to develop Medium Chain Triglyceride (MCT) rich self nanoemulsifying preconcentrate of paclitaxel (PTX) for parenteral delivery. Very high concentrations of Cremophor EL and ethanol in Taxol have rendered patients to severe side effects. Years of extensive research on development of cost effective and safer vehicle for PTX, have failed to provide a promising replacement for Taxol. MCT was selected as oil owing to its parenteral acceptability, high solubilization capacity and multiple therapeutic benefits in cancer cachexia. PTX precipitation kinetics and reported toxicity profile of Kolliphor HS15 has favored its selection for PTX Self Nanoemulsifying Preconcentrate (PSNP). Presence of 30% free PEG in Kolliphor HS15 (PEG-15-hydroxystearate) restricts its miscibility with MCT, imposing significant challenge in development of MCT rich self nanoemulsifying preconcentrate. Removal of PEG layer from oil-surfactant mixture facilitated the formulation of PSNP with 51% w/w MCT. PSNP exhibited better precipitation kinetic profile, higher PTX loading with negligible hemolysis and histamine release compared to Taxol. PSNP was bioequivalent to Taxol, though V(d) and MRT was significantly higher than Taxol. PSNP showed distinctly better profile in inhibiting tumor growth and maintaining body weight with significantly higher % survival. Thus, PSNP can be a safer vehicle with potential clinical benefits.

Targeting Hepatic Cancer Cells with PEGylated Dendrimers Displaying N‐Acetylgalactosamine and SP94 Peptide Ligands

Poly(amidoamine) (PAMAM) dendrimers are branched water-soluble polymers defined by consecutive generation numbers (Gn) indicating a parallel increase in size, molecular weight, and number of surface groups available for conjugation of bioactive agents. In this article, we compare the biodistribution of N-acetylgalactosamine (NAcGal)-targeted [(14) C]1 -G5-(NH2 )5 -(Ac)108 -(NAcGal)14 particles to non-targeted [(14) C]1 -G5-(NH2 )127 and PEGylated [(14) C]1 -G5-(NH2 )44 -(Ac)73 -(PEG)10 particles in a mouse hepatic cancer model. Results show that both NAcGal-targeted and non-targeted particles are rapidly cleared from the systemic circulation with high distribution to the liver. However, NAcGal-targeted particles exhibited 2.5-fold higher accumulation in tumor tissue compared to non-targeted ones. In comparison, PEGylated particles showed a 16-fold increase in plasma residence time and a 5-fold reduction in liver accumulation. These results motivated us to engineer new PEGylated G5 particles with PEG chains anchored to the G5 surface via acid-labile cis-aconityl linkages where the free PEG tips are functionalized with NAcGal or SP94 peptide to investigate their potential as targeting ligands for hepatic cancer cells as a function of sugar conformation (α versus β), ligand concentration (100-4000 nM), and incubation time (2 and 24 hours) compared to fluorescently (Fl)-labeled and non-targeted G5-(Fl)6 -(NH2 )122 and G5-(Fl)6 -(Ac)107 -(cPEG)15 particles. Results show G5-(Fl)6 -(Ac)107 -(cPEG[NAcGalβ ])14 particles achieve faster uptake and higher intracellular concentrations in HepG2 cancer cells compared to other G5 particles while escaping the non-specific adsorption of serum protein and phagocytosis by Kupffer cells, which make these particles the ideal carrier for selective drug delivery into hepatic cancer cells.

A Small-Angle X-ray Scattering Study of α-helical Bundle-Forming Peptide–Polymer Conjugates in Solution: Chain Conformations

As a new family of soft materials, peptide/protein–polymer conjugates can lead to a wide range of potential biological and nonbiological applications. The performance of these materials depends on the protein structure and phase behavior arising from a balance between the enthalpic interactions of the components and surrounding media as well as the entropic contribution associated with polymer chain deformation. There is a great need to perform structural studies in solution that systematically investigate the polymer chain conformation upon linkage to a peptide or protein so as to evaluate how polymers affect the protein structure of the biomolecule and, consequently, its functionality. Combinations of a range of factors including low contrast, weak scattering signals in dilute solutions as well as difficulties in separating the component scattering contributions, pose significant challenges to structural characterization. Here we present a synchrotron small-angle X-ray scattering (SAXS) study of two model helix bundle forming peptide–polymer conjugates and show that with analytical modeling of the scattering intensity detailed structural information on both peptide structure and polymer conformation can be extracted. The peptide–poly(ethylene glycol) (PEG) conjugates are based on peptides that self-associate to form well-defined 3- or 4-helix bundles and the PEG chain is covalently linked either to the end or the side of the peptide (i.e. end- or side-conjugation). Using a simplified analytical geometrical body form factor model, where the peptide–polymer bundles are modeled as parallel cylinders with attached Gaussian chains, a quantitative description of the scattering behavior can be reached. On the basis of the simplified structural model, the protein tertiary structures, i.e., the α-helix bundle, remains largely intact and maintains its oligomeric state but exhibits slight swelling in solution with respect to the crystal structure. The PEG chain conformation appears to slightly depend on the conjugate architecture. In terms of the chain dimension represented by Rg, the end-conjugated PEG exhibit similar value as compared to free PEG for the molecular weight studied (2 kDa). For the side-conjugates our simple scattering model seems to indicate a systematically slightly lower values for Rg, i.e., a slight compression, in particular for the highest molecular weight (5 kDa). However, considering the limitations of the model and experimental uncertainties, further investigations, such as neutron scattering, is needed to illustrate detailed chain conformation. The present studies can be extended to other peptide–polymer or protein–polymer hybrid systems to extract information on both protein structure and polymer chain conformation. This work will thus provide valuable guidance to understand their structure and phase behavior using X-ray and neutron scattering.

Thermodynamic studies of fluphenazine decanoate solubility in PEG 200 + water mixtures

The solubility of fluphenazine decanoate (FD) in binary mixtures of polyethylene glycol 200 (PEG 200)+water at the temperature range from 298.0K to 318.0K is reported. The previously trained version of the Jouyban–Acree model for PEG 200+water, a recently proposed general cosolvency model employing partial solubility parameters, and a combination of the model with van’t Hoff equation were used to predict the solubility of FD in PEG 200+water at different temperatures. The results show that the Jouyban–Acree model can be used for solubility prediction of FD in PEG 200+water mixtures at different temperatures. The solubility data as a function of temperature were used to determine the thermodynamic properties of the dissolution process including Gibbs energy, enthalpy and entropy of the solution. An adapted version of the model is used to represent the thermodynamic properties of the solutions in the solvent mixtures and the obtained results were satisfactory. Densities of solute free PEG 200+water and FD-saturated solutions are also reported.

Characterization of PEG‐conjugated gold nanoparticles using a statistical analysis on time‐of‐flight SIMS images

Gold nanoparticles (AuNPs) and poly(ethylene glycol) (PEG) are known to be safe, biocompatible materials. In this study, AuNPs were conjugated with PEG ligands, and the suspensions of AuNPs with and without free PEG ligands were characterized using UV‐visible spectroscopy, hydrodynamic size measurement, and time‐of‐flight SIMS (ToF‐SIMS) imaging analysis. The two AuNPs solutions were each dropped onto a hydrophilic surface (bare Si wafer) and a hydrophobic surface (a plasma‐polymerized cyclohexane‐coated Si wafer), then dried to produce self‐aggregated micropatterns, from which ToF‐SIMS images were obtained. From these ToF‐SIMS images, we calculated the Pearson product–moment correlation coefficient (PMCC) of the AuNPs and PEG intensities to quantitatively measure the coexistence of both materials in the solution. Good coexistence between AuNPs and PEG ligands was obtained only from the sample film prepared by dropping the suspension of PEG‐conjugated AuNPs without free ligands onto a hydrophobic surface. The degrees of coexistence (i.e. PMCC value) seem to correlate with the amount of free PEG ligands in suspension to affect the stability of organic conjugated nanoparticles in solution. In addition, it was found that minimal interaction between the conjugated ligands and the substrate used for the sample film preparation led to an optimum PMCC statistical analysis. Our results lead us to believe that this new approach could simplify the method by which to quantitatively study the degrees of coexistence between nanoparticles and ligands, the amount of free ligands, and the stability of ligand‐conjugated nanoparticles in suspension. Copyright © 2012 John Wiley & Sons, Ltd.

Purification of a PEGylated single chain Fv

In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by 125I based cell tests. Proteins modified with 5kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification.

Poly(Ethylene Glycol) Grafted Starch Introducing a Novel Interphase in Poly(Lactic Acid)/Poly(Ethylene Glycol)/Starch Ternary Composites

The main purpose of this study is challenging to dramatically improve the toughness of poly(lactic acid) (PLA)/starch by adding poly (ethylene glycol) (PEG) into the composites and grafting PEG molecules onto the surface of starch particles. It was found that the surface grafting of PEG onto starch induced the presence of PEG-rich regions located around the starch particles, caused by migration and aggregation of free PEG molecules. A novel interphase transition layer between PLA and starch was formed, which showed great ability for cavitation and vested large-scaled plastic deformation to PLA matrix. Further mechanical properties tests indicated the formation of interphase layer significantly increase the elongation at break from 10.2 to 254.5%, and notched impact strength from 1.56 to 2.37 kJ/m2 for PLA/PEG/starch ternary composites. The influence of PEG component, ethanol extraction and annealing was also investigated.

Dual surface modification with PEG and corn trypsin inhibitor: Effect of PEG:CTI ratio on protein resistance and anticoagulant properties

The objective of this study was to investigate the bioactivity and protein-resistant properties of dual functioning surfaces modified with PEG for protein resistance and corn trypsin inhibitor (CTI) for anticoagulant effect. Surfaces on gold substrate were prepared with varying ratios of free PEG to CTI-conjugated PEG. Two methods designated, respectively, "sequential" and "direct" were used. For sequential surfaces, PEG was first immobilized on gold and the surfaces were incubated with CTI at varying concentration. For direct surfaces, a PEG-CTI conjugate was synthesized and gold surfaces were modified using solutions of the conjugate of varying concentration. The CTI density on these surfaces was measured using radiolabeled CTI. Water contact angles were measured and the thickness of PEG-CTI layers was determined by ellipsometry. Fibrinogen adsorption from buffer and human plasma, and adsorption from binary solutions of fibrinogen and α-lactalbumin were investigated using radiolabeling methods. Bioactivity of the surfaces was evaluated via their effects on FXIIa inhibition and plasma clotting time. It was found that as the ratio of CTI-conjugated PEG to free PEG increased, bioactivity increased but protein resistance was relatively constant. It is concluded that on these surfaces conjugation of PEG to CTI does not greatly compromise the protein resistance of the PEG but results in improved interactions between the CTI and the "target" protein FXIIa. At the same CTI density, sequential surfaces were more effective in terms of inhibiting FXIIa and prolonging clotting time.

Polymer-conjugated albumin and fibrinogen composite hydrogels as cell scaffolds designed for affinity-based drug delivery

Serum albumin was conjugated to poly-(ethylene glycol) (PEG) and cross-linked to form mono-PEGylated albumin hydrogels. These hydrogels were used as a basis for drug carrying tissue engineering scaffold materials, based on the natural affinity of various drugs and compounds for the tethered albumin in the polymer network. The results of the drug release validation experiments showed that the release kinetics of the drugs from the mono-PEGylated albumin hydrogels were controlled by the molecular weight (MW) of PEG conjugated to the albumin protein, the drug MW and its inherent affinity for albumin. Composite hydrogels containing both mono-PEGylated albumin and PEGylated fibrinogen were used specifically for three-dimensional (3D) cell culture scaffolds, with inherent bioactivity, proteolytic biodegradability and controlled drug release properties. The specific characteristics of these complex hydrogels were governed by the ratio between the concentrations of each protein, the addition of free PEG diacrylate (PEG DA) molecules to the hydrogel matrix and the MW of the PEG conjugated to each protein. Comprehensive characterization of the drug release and degradation properties, as well as 3D cell culture experiments using these composite materials, demonstrated the effectiveness of this combined approach in creating a tissue engineering scaffold material with controlled drug release features.

Sensitive Quantification of PEGylated Compounds by Second-Generation Anti-Poly(ethylene glycol) Monoclonal Antibodies

Poly(ethylene glycol) (PEG) is often attached to compounds to increase serum half-life, reduce immunogenicity, and enhance bioavailability. Accurate and sensitive quantification of PEG conjugates is critical for product development, pharmacokinetic measurements, and efficacy studies. However, PEGylated compounds can be difficult to quantify due to epitope masking by PEG. We previously generated two monoclonal antibodies to PEG (AGP3, IgM and E11, IgG) for quantitative detection of PEGylated proteins. We now report the identification of two second-generation mAbs to PEG (AGP4, IgM and 3.3, IgG) that bind to the repeating subunits of the PEG backbone and facilitate more sensitive quantification of a wider range of PEGylated compounds. A sandwich ELISA in which AGP4/3.3-biotin was employed as the capture/detection antibodies allowed quantification of PEG-Qdot 525 with 14-50-fold greater sensitivity than the original AGP3/E11 combination. Pegasys (PEG-interferon alpha-2a), PEG-Intron (PEG-interferon alpha-2b), Neulasta (PEG-G-CSF), and Lipo-Dox (PEGylated liposomal doxorubicin) could also be quantified with low ng/mL detection limits. The assay tolerated the presence of 50% human serum or 20% free PEG molecules. These new anti-PEG antibodies appear useful for qualitative and quantitative analysis of a wide range of PEGylated compounds.

The PEG Moiety of Certolizumab Pegol is Rapidly Cleared from the Blood of Humans by the Kidneys Once it is Cleaved from the Fabʼ

Purpose: Certolizumab pegol (CZP) is the only PEGylated anti-TNF-α and consists of a Fab' site specifically attached to a 40kDa PEG molecule. PEG is difficult to measure in bodily fluids and hence there is a paucity of information about its metabolism and clearance, despite its conjugation to proteins in many approved products. However, clearance in rats of the PEG part of CZP was previously shown using an innovative proton nuclear magnetic resonance (1H NMR) method to measure it in urine.[1] The half-life of free PEG was shown to be 23 days and the majority was cleared by the kidneys (>90%). The aim was to measure the level of CZP and total PEG in plasma of humans, and to ascertain the molecular weight (MW) of the PEG molecules excreted in urine. Methods: Blood was collected at various time points from 10 healthy volunteers after one subcutaneous dose of CZP 400 mg. CZP and total PEG levels were measured by ELISA and 1H NMR, respectively. In a separate group of 6 subjects, urine was collected at various time points up to 84 days after the same dose of CZP. The concentration of PEG in the urine was also determined by 1H NMR, although for technical reasons the absolute amount could not be calculated. The MW of the PEG in urine was assessed by SDS PAGE. Results: In the samples from the 10 subjects, at all time points measurable, the level of CZP in plasma determined by ELISA was similar to the level of PEG measured by 1H NMR. Comparison of results of the PEG assay with the plasma ELISA data for intact CZP showed that CZP accounted for 87-92% of the total PEG, as estimated by Cmax or AUC. The median half-life of CZP in this particular study was 12.3 days (range 10.4-16.2 days) versus 14.0 days (range 10.9-19.0 days) for PEG. PEG was readily detected in the urine of the 6 subjects, and it was estimated that the majority of PEG was removed via the kidneys. The MW of the PEG found in the urine was 40 kDa. Conclusion: In humans, the free PEG molecule is rapidly excreted by the kidneys. In addition, PEG is not further metabolised to lower molecular weight forms. This innovative 1H NMR method has allowed the accurate quantitation of PEG in the plasma of blood in humans and has shown that free 40 kDa PEG is rapidly removed from the blood via the kidneys. 1. Parton, et al. Gut 2007 (Suppl III)A156. Disclosure: Dr Parton is an employee of UCB; Dr King is an employee of UCB; Dr Parker is an employee of UCB; Dr Nesbitt is an employee of UCB. This research was supported by an industry grant from UCB.