Polyethylene Glycol Moiety Research Articles

Biochemical and Pharmacokinetic Properties of PEGylated Cystathionine γ-Lyase from Aspergillus carneus KF723837

Cystathionine γ-lyase (CGL) was purified to its electrophoretic homogeneity from Aspergillus carneus by various chromatographic approaches. The purified enzyme has four identical subunits of 52 kDa based on SDS and native PAGE analyses. To improve its structural stability, purified CGL was modified by covalent binding to polyethylene glycol moieties. The specific activity of free-CGL and PEG-CGL was 59.71 and 48.71 U/mg, respectively, with a PEGylation yield of 81.5 and 70.7% modification of surface ε-amino groups. Free- and modified CGL have the same pattern of pH stability (8.0-9.0). At 50°C, the thermal stability [half-life time (T_1/2)] of PEG-CGL was increased by 40% in comparison to free-CGL. The activity of CGL was completely inhibited by hydroxylamine and Hg⁺², with no effect by EDTA. Free-CGL (0.04 m<smlcap>M</smlcap>^-1s^-1) and PEG-CGL (0.03 m<smlcap>M</smlcap>^-1s^-1) have a similar catalytic efficiency to <smlcap>L</smlcap>-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µ<smlcap>M</smlcap> for the free- and PEG-CGL, respectively. By in vitro proteolysis, PEG-CGL retains >50% of its initial activity compared to <10% of the free-CGL for acid protease for 30 min. From in vivo pharmacokinetics in New Zealand white rabbits, the T_1/2 was 19.1 and 28.9 h for the Holo free-CGL and PEG-CGL, respectively, ensuring the role of PEGylation on shielding the CGL surface from proteolytic attack, reducing its antigenicity, and stabilizing its internal Schiff base. By external infusion of pyridoxal 5′-phosphate (10 µ<smlcap>M</smlcap>), the T_1/2 of free- and PEG-CGL was prolonged to 24 and 33 h, respectively, so dissociation of pyridoxal 5′-phosphate was one of the main causes of loss of enzyme activity. The biochemical and hematological responses of rabbits to free- and PEG-CGL were assessed, with relative similarity to the negative control, confirming the nil toxicity of enzymes. The titer of IgG was duplicated in response to free- versus PEG-CGL after 45 days. To the best of our knowledge, this is the first report concerned with purification and PEGylation of CGL from fungi, with higher affinity for <smlcap>L</smlcap>-cystathionine. With further molecular studies, CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H₂S).

Poly(ethylene glycol)-or silicone-modified hyaluronan for contact lens wetting agent applications.

Hyaluronan (HA) is a hydrophilic biopolymer that has been explored as a wetting agent in contact lens applications. In this study, HA was modified with siloxy or polyethylene glycol moieties using click chemistry to make it more soluble in monomer solutions used to synthesize model contact lens materials; unmodified HA was not soluble in the same monomer solutions. The water contents of the silicone hydrogels were not increased by the presence of modified HA, nor was there a decrease in the surface contact angle. However, modified HA did lead to a reduction in lysozyme adsorption in some cases. The leaching rate of HA modified with polyethylene glycol from a 78:22 DMA:TRIS(OH) hydrogel was significantly slower than for unmodified HA.

Synthesis and evaluation of anticancer activity in cells of novel stoichiometric pegylated fullerene-doxorubicin conjugates.

To synthesize pegylated stoichiometrically and structurally well-defined conjugates of fullerene (C60) with doxorubicin (DOX) and investigate their antiproliferative effect against cancer cell lines. Stoichiometric (1:1 and 1:2) pegylated conjugates of C60 with DOX were synthesized using the Prato reaction to create fulleropyrrolidines equipped with a carboxyl function for anchoring a polyethylene glycol (PEG) moiety and either a hydroxyl group for attaching one molecule of DOX or a terminal alkyne group for attaching two molecules of DOX through a click reaction. In both conjugates, the DOX moieties are held through a urethane-type bond. Drug release was studied in phosphate buffer (PBS, pH7.4) and MCF-7 cancer cells lysate. The uptake of the conjugates by MCF-7 cancer cells and their intracellular localization were studied with fluorescence microscopy. The antiproliferative activity of the conjugates was investigated using the WST-1 test. One or two DOX molecules were anchored on pegylated C60 particles to form DOX-C60-PEG conjugates. Drug liberation from the conjugates was significantly accelerated in the presence of tumor cell lysate compared to PBS. The conjugates could be internalized by MCF-7 cells. DOX from the conjugates exhibited much delayed, compared to free DOX, localization in the nucleus and antiproliferative activity. Pegylated DOX-C60 conjugates (1:1) and (2:1) with well-defined structure were successfully synthesized and found to exhibit comparable, but with a delayed onset, antiproliferative activity with free DOX against MCF-7 cancer cells. The results obtained justify further investigation of the potential of these conjugates as anticancer nanomedicines.

Separation of PEGylated variants of ribonuclease A and apo-α-lactalbumin via reversed phase chromatography

The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, “PEGylation”, often results in a population of conjugate species that includes differing numbers and locations of attached PEG chains. As some portion of this population may be biologically inactive, a challenging separation problem arises. An interesting alternative to the size-based resolution of these conjugates involves the use of reversed phase chromatography (RPC), treating the PEG moieties as hydrophobic purification tags. The use of RPC raises concerns about protein denaturation in the mobile and on the stationary phase. Here, the potential dual role of conjugated PEG chains as both group-specific separation tags and as steric or structural stabilizers in RPC was explored. In this work, RPC with C18-based media was used to resolve PEGylation number variants of ribonuclease A (RNase A) and apo-α-lactalbumin (apo-αLac) in a neutral pH mobile phase. While the attachment of 20kDa PEG molecules did not modify the structures of RNase A and apo-αLac, as confirmed by structural analysis using circular dichroism, exposure to the mobile phase modifier, acetonitrile, and to the C18 media during separation resulted in perturbations to both the secondary and tertiary structures of all species studied. RNase A experienced small perturbations that were mediated to some extent by PEGylation; these results were consistent with activity assays which showed that PEGylated RNase A species retained native-like activity after RPC separation. Apo-αLac, a more hydrophobic and less stable protein than RNase A, experienced extensive structural perturbations regardless of PEGylation state. The temperature of the mobile phase was found to strongly influence chromatographic separation of PEG-conjugates with conjugate species becoming more strongly retained with increasing temperature. This work shows that it is feasible to employ RPC with neutral pH mobile phases to resolve PEG conjugate number heterogeneity.

Quantitative analysis of polyethylene glycol (PEG) and PEGylated proteins in animal tissues by LC-MS/MS coupled with in-source CID.

The covalent conjugation of polyethylene glycol (PEG, typical MW > 10k) to therapeutic peptides and proteins is a well-established approach to improve their pharmacokinetic properties and diminish the potential for immunogenicity. Even though PEG is generally considered biologically inert and safe in animals and humans, the slow clearance of large PEGs raises concerns about potential adverse effects resulting from PEG accumulation in tissues following chronic administration, particularly in the central nervous system. The key information relevant to the issue is the disposition and fate of the PEG moiety after repeated dosing with PEGylated proteins. Here, we report a novel quantitative method utilizing LC-MS/MS coupled with in-source CID that is highly selective and sensitive to PEG-related materials. Both (40K)PEG and a tool PEGylated protein (ATI-1072) underwent dissociation in the ionization source of mass spectrometer to generate a series of PEG-specific ions, which were subjected to further dissociation through conventional CID. To demonstrate the potential application of the method to assess PEG biodistribution following PEGylated protein administration, a single dose study of ATI-1072 was conducted in rats. Plasma and various tissues were collected, and the concentrations of both (40K)PEG and ATI-1072 were determined using the LC-MS/MS method. The presence of (40k)PEG in plasma and tissue homogenates suggests the degradation of PEGylated proteins after dose administration to rats, given that free PEG was absent in the dosing solution. The method enables further studies for a thorough characterization of disposition and fate of PEGylated proteins.

Pegylated Interferons

Most multiple sclerosis (MS) therapies are injectable drugs, and the frequency of injections has been shown to be inversely proportional to overall compliance. One method of improving therapeutic compliance and thus clinical outcomes is to develop medications that require less frequent dosing. One of the most promising modification techniques to extend the bioavailability of a drug is poly(ethylene glycol) conjugation (pegylation), which increases the size of a molecule by attaching polyethylene glycol moieties to the parent compound, resulting in slower clearance and metabolism. This approach has been used to improve the efficacy of a number of therapeutic molecules, including interferons. Peginterferon beta-1a, a pegylated form of interferon beta-1a, is currently in phase III clinical trials for relapsing MS and has the potential to improve patient compliance by reducing the number of injections while maintaining clinical efficacy. The role of nurses in educating patients about the effective use of this new MS therapy is discussed.

Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG2000 conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH2CH2) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics.

Development and evaluation of PEGylated Enoxaparin: A novel approach for enhanced anti-Xa activity

Enoxaparin (ENX) is one of the most widely prescribed low molecular weight heparin inprophylaxis and treatment of venous thromboembolism. In this study, Enoxaparin–PEG conjugate (P-ENX) was synthesized from Enoxaparin and polyethylene glycol (PEG) and evaluated for its potential for extended duration of action. The esterification of the carboxyl groups of the drug moiety with the hydroxyl groups of mPEG-2000 was done by employing carbodiimide coupling chemistry. P-ENX conjugate was purified by dialysis and characterized by Fourier transform infrared spectroscopy (FTIR), Proton-Nuclear magnetic resonance (1H NMR) and matrix-assisted laser desorption/ionization (MALDI) mass analysis techniques. FTIR analysis revealed frequency of the carbonyl group in accord with ester linkage formation between the drug and the PEG moiety. 1H NMR of the conjugate showed significant change in the chemical shift further indicative of ENX and PEG chemical interaction. In MALDI spectra, small peaks at 12,907 and 16,137 m/z confirmed the probability of conjugation of ENX and PEG. P-ENX exhibited considerable enhancement in anti-Xa activity (by three-folds) in comparison to free ENX. Further, an increase in AUC (over four-folds) was observed in P-ENX. Thus, PEGylation of ENX is a novel approach for extended and enhanced activity of ENX with a potential for decreased dosing frequency.

Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94‐9027) for haemophilia A

Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05IUmL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.

LY2605541—A Preferential Hepato-Specific Insulin Analogue

The first generation of basal insulin analogs, insulin glargine and detemir, are characterized by a more predictable day-to-day insulin absorption rate, a flatter time/action profile, and a longer duration than the older, intermediate-acting NPH insulin (1). The duration of insulin glargine is longer than insulin detemir (1). The second-generation basal insulin analog insulin degludec has an even longer half-life of about 25 h, a very flat profile of action with a duration exceeding 42 h, and a reduced risk of nocturnal hypoglycemia in both type 1 and type 2 diabetic patients compared with insulin glargine (2). Yet, there is still room for further refinements of the basal insulin analogues. Insulin treatment commonly results in weight gain; thus, an insulin analog associated with no weight gain or weight loss will be a great therapeutic advance. The peripheral administration of insulin does not replicate the two- to threefold higher portal versus systemic circulating insulin levels, causing an imbalance between hepatic and peripheral metabolic actions. Therefore, an insulin analog with hepatic specificity could be of interest. In this issue, Moore et al. (3) report on the effect of the novel basal insulin LY2605541 on hepatic and nonhepatic glucose uptake in their elegant dog model. During peripheral intravenous infusion of LY2605541, a switch from hepatic glucose output to uptake was reported, and nonhepatic glucose uptake increased less than in control experiments with human insulin, indicating that LY2605541 possesses preferential hepatic effects, thereby mimicking endogenously secreted insulin (3). The active component of LY2605541 is insulin lispro, a short-acting insulin analog, which is covalently coupled to a single 20-kilodalton polyethylene glycol (PEG) moiety via an urethane bound to lysine B28 (Fig. 1) (4,5). This results in …

New biodegradable bisphosphonate vinylic monomers and near infrared fluorescent nanoparticles for biomedical applications

Bisphosphonates (BPs) are non‐hydrolyzable pyrophosphate analogs with high affinity to hydroxyapatite due to their ability to create bidentate or tridentate chelates with calcium ions. Consequently, BP lead to strong interactions with dentin, enamel, and bones. BPs (especially those bearing OH) accelerate osteoblasts action, while strongly inhibiting osteoclasts, thus contributing to enhanced bone formation. Recently, several novel BP non‐biodegradable and biodegradable vinylic monomers were synthesized in our laboratory. This article provides a detailed description regarding the synthesis of a unique biodegradable BP monomer and near infrared (NIR) fluorescent nano/microparticles for diagnosis and therapy of bone malignancy. These particles were prepared by dispersion copolymerization of three monomers: methacrylate polyethylene glycol (PEG) BP, N‐(3‐aminopropyl) methacrylamide, and the crosslinker monomer tetra(ethyleneglycol) diacrylate. The PEG‐BP nanoparticles size was controlled by changing various polymerization parameters. These BP particles possess dual functionality: covalent attachment of a dye (e.g., NIR fluorescent dye) or drug to the nanoparticles through the primary amine groups belonging to the aminopropyl methacrylamide monomeric units and chelation to the bone mineral hydroxyapatite through the BP groups belonging to the methacrylate PEG‐BP monomeric units. Body distribution of the optimal crosslinked BP nanoparticles was tested on a chicken embryo model via intravenous administration. This study indicated that the fluorescence intensity of all the organs except the bones decreased significantly within 48 h (p &lt; 0.05) while that of the bones hardly changed over that time (p &gt; 0.05). In addition, a relatively long half‐life time of these nanoparticles in blood has been demonstrated, probably due to the PEG moiety. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Combination of Nanoparticles with Physical Stimuli toward Cancer Therapy

Drug delivery systems represent an important strategy for cancer treatment. The targeted delivery of drugs is required for effective and safe cancer therapy. In cancer therapy, the target cells include cancer cells and immunocompetent cells such as antigen presenting cells. Anticancer drugs utilized include small molecular drugs, proteins and nucleic acid medicines. In order to deliver these drugs into the target cells, various nanoparticles have been developed. However, the efficacy of the nanoparticulate system itself is generally insufficient for the safe and effective treatment of cancer. For example, polyethylene glycol (PEG)-modified (PEGylated) nanoparticles accumulate in cancerous tissues; however, the PEG moiety on the surface of the nanoparticles disturbs cellular uptake, which is known as the 'PEG dilemma.' Thus, additional strategies such as receptor-mediated targeting are necessary to improve the delivery and cellular uptake of nanoparticles. Among additional strategies, in this review we have focused on the combination of nanoparticles with various physical stimuli, such as electric pulse and ultrasound, to improve the targeted delivery of the nanoparticles.

A multifunctional amphiphilic polymer as a platform for surface-functionalizing metallic and other inorganic nanostructures.

We designed a new set of polymer ligands that combine multiple metal-coordinating groups and short polyethylene glycol (PEG) moieties in the same structure. The ligand design relies on the controlled grafting of a large number of amine-terminated histamines and PEG short chains onto a poly(isobutylene-alt-maleic anhydride) backbone, via a one-step nucleophilic addition reaction. This addition reaction is highly efficient, can be carried out in organic media and does not require additional reagents. We show that when imidazole groups are used the resulting polymer ligand can strongly ligate onto metal nanostructures such as nanoparticles (NPs) and nanorods (NRs) made of gold cores. The resulting polymer-coated NPs and NRs exhibit good colloidal stability to pH changes and added electrolytes. This constitutes a departure from the use of thiol-based ligands to coordinate on Au surfaces. The present chemical approach also opens up additional opportunities for designing hydrophilic and reactive platforms where the polymer coating can be adjusted to various metal and metal oxide surfaces by simply modifying or combining the addition reaction with other metal coordinating groups. These could include iron oxide NPs and semiconductor QDs. These polymer-capped NPs and NRs can be used to develop biologically-active platforms with potential use for drug delivery and sensing.

A quantitative and site-specific chemoenzymatic glycosylation approach for PEGylated MUC1 peptides

Full control over complex post-translational modifications (PTMs), such as O-glycosylation, is a prerequisite for testing and understanding the biological role of these modifications in protein function. Despite considerable progress over the last years, high throughput and easy-to-use methods for the synthesis of complex glycosylated peptides are still missing. We present here an efficient methodology to produce homogeneous site-specifically O-glycosylated peptides. Sequential chemoenzymatic glycosylation and separation from the reaction components are achieved via the temporary attachment of a monodisperse polyethylene glycol (PEG) polymer to the N-terminus of these peptides. Subsequent proteolytic removal of the PEG moiety allows quantitative recovery of homogeneous O-glycopeptides, suitable as building blocks for glycoprotein synthesis. Here, we demonstrate the preparation of glucuronylated variants of MUC1, a well-known member of the human mucin family. Homogeneously O-glycosylated variants were synthesized and will be used to study the role of O-linked glucuronic acid epitopes within the functional environment of the human MUC1 tandem repeat.

Preparation and Characterization of Decyl-Terminated Silicon Nanoparticles Encapsulated in Lipid Nanocapsules

In this Article, we report on the encapsulation of decyl-modified silicon nanoparticles (decyl-SiNPs) into ∼80 nm lipid nanocapsules (LNCs). The decyl-SiNPs were produced by thermal hydrosilylation of hydride-terminated SiNPs (H-SiNPs) liberated from porous silicon. Various techniques, including Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), UV-vis absorption, dynamic light scattering (DLS), and photoluminescence (PL), were used to characterize their size, shape, colloidal, and optical properties. The results indicate that these nanocapsules feature controllable size, good dispersity, high loading rate of SiNPs, colloidal stability in various media, and bright PL. The PL of decyl-SiNPs loaded LNCs was stable upon heating to 80 °C, but was sensitive to basic solutions due to proton-gated emission of the SiNPs arranged at the LNCs interface between the oil phase and the hydrophilic polyethylene glycol moieties of the surfactant. These luminescent nanocapsules are therefore promising candidates as cellular probes for fluorescence imaging. In addition, it was found that TEM imaging of small-sized decyl-SiNPs could be greatly improved by preliminary negative staining of TEM grids with phosphotungstic acid.

Polynitroxylated-Pegylated Hemoglobin Attenuates Fluid Requirements and Brain Edema in Combined Traumatic Brain Injury Plus Hemorrhagic Shock in Mice

Polynitroxylated-pegylated hemoglobin (PNPH), a bovine hemoglobin decorated with nitroxide and polyethylene glycol moieties, showed neuroprotection vs. lactated Ringer's (LR) in experimental traumatic brain injury plus hemorrhagic shock (TBI+HS). Resuscitation with PNPH will reduce intracranial pressure (ICP) and brain edema and improve cerebral perfusion pressure (CPP) vs. LR in experimental TBI+HS. C57/BL6 mice (n=20) underwent controlled cortical impact followed by severe HS to mean arterial pressure (MAP) of 25 to 27 mm Hg for 35 minutes. Mice (n=10/group) were then resuscitated with a 20 mL/kg bolus of 4% PNPH or LR followed by 10 mL/kg boluses targeting MAP>70 mm Hg for 90 minutes. Shed blood was then reinfused. Intracranial pressure was monitored. Mice were killed and %brain water (%BW) was measured (wet/dry weight). Mice resuscitated with PNPH vs. LR required less fluid (26.0±0.0 vs. 167.0±10.7 mL/kg, P<0.001) and had a higher MAP (79.4±0.40 vs. 59.7±0.83 mm Hg, P<0.001). The PNPH-treated mice required only 20 mL/kg while LR-resuscitated mice required multiple boluses. The PNPH-treated mice had a lower peak ICP (14.5±0.97 vs. 19.7±1.12 mm Hg, P=0.002), higher CPP during resuscitation (69.2±0.46 vs. 45.5±0.68 mm Hg, P<0.001), and lower %BW vs. LR (80.3±0.12 vs. 80.9±0.12%, P=0.003). After TBI+HS, resuscitation with PNPH lowers fluid requirements, improves ICP and CPP, and reduces brain edema vs. LR, supporting its development.

AB0285 First real life experience with certolizumab pegol in rheumathoid arthritis (ra) in latinamerica

BackgroundCertolizumab pegol (CZP) is a novel TNF inhibitor, consisting of a humanized Fab’ fragment fused to a 40-kd polyethylene glycol moiety with proved efficacy and safety in multiple RA clinical...

The Immunogenicity of Polyethylene Glycol: Facts and Fiction

An increasing number of pegylated therapeutic proteins and drug targeting compounds are being introduced in the clinic. Pegylation is intended to increase circulation time and to reduce an immunogenic response. Recently however a number of publications have appeared claiming that the polyethylene glycol (PEG) moiety of these products in itself may be immunogenic and that the induced anti-PEG antibodies are linked to enhanced blood clearance and reduced efficacy of the products. A critical review of the literature shows that most, if not all assays for anti-PEG antibodies are flawed and lack specificity. Also the biological effects induced by anti-PEG antibodies lack the characteristics of a bona fide antibody reaction. Standardization of the anti-PEG assays and the development of reference sera are urgently needed.

Chemical Modification of Polymer Brushes via Nitroxide Photoclick Trapping

The preparation of polymer brushes (PBs) bearing α-hydroxyalkylphenylketone (2-hydroxy-2-methyl-1-phenylpropan-1-one) moieties as photoreactive polymer backbone substituents is presented. Photoreactive polymer brushes with defined thicknesses (up to 60 nm) and high grafting densities are readily prepared by surface initiated nitroxide mediated radical polymerization (SINMP). The photoactive moieties can be transformed via Norrish-type I photoreaction to surface-bound acyl radicals. Photolysis in the presence of a persistent nitroxide leads to chemically modified PBs bearing acylalkoxyamine moieties as side chains resulting from trapping of the photogenerated acyl radicals with nitroxides. Application of functionalized nitroxides to the photochemical PB postmodification provides functionalized PBs bearing cyano, polyethylene glycol (PEG), perfluoroalkyl, and biotin moieties. As shown for one case, photochemical postfunctionalization of the PB through a mask using a biotin-conjugated nitroxide as the trapping reagent leads to the corresponding site-selective chemically modified PB, which is successfully used for site-specific streptavidin immobilization. Surface analysis of PBs was performed by contact angle (CA) measurements, X-ray photoelectron spectroscopy (XPS), attenuated total reflection (ATR), fourier transform infrared (FTIR) spectroscopy, and fluorescence microscopy.

Conjugating poly(ethylene) glycol (PEGylation) to a high‐affinity anti‐methamphetamine antibody fragment

Methamphetamine (METH) is considered one of the top drug problems in the United States today, yet there are no FDA approved pharmacological treatments available for METH abuse. To this end, we have designed and produced an anti‐METH single chain antibody fragment (scFv7F9Cys) as a potential anti‐METH pharmacological treatment. However, scFv7F9Cys has a short half‐life in its native state, limiting its clinical use. Thus, the aim of this study was to examine the in vitro effects of conjugating scFv7F9Cys to poly(ethylene) glycol (PEG) as a potential method of increasing half‐life. Three different PEG moieties were tested: linear 5 kDa and 20kDa PEGs and a branched 40 kDa PEG. ScFv7F9Cys was conjugated to each PEG forming three different scFv7F9Cys‐PEG conjugates (scFv7F9Cys‐PEG5K, ‐PEG20K, or ‐PEG40K). ScFv7F9Cys‐PEG conjugates were then purified from unreacted scFv7F9Cys and PEG using affinity chromatography (IMAC). PEGylation and purification were analyzed by SDS‐PAGE and size exclusion chromatography. METH binding affinity and capacity were tested for each scFv7F9Cys‐PEG conjugate and compared to native scFv7F9Cys using saturation binding assays. Both PEGylation of scFv7F9Cys and purification of scFv7F9Cys‐PEG were successful and binding analysis suggests PEGylation did not alter METH affinity or binding capacity. NIDA R01 DA026423, NIH CSTA 1UL1RR029884, NIDA T32 DA022981