mPEG Derivatives Research Articles

Poly(Ethylene Glycol)/β-Cyclodextrin Pseudorotaxane Complexes as Sustainable Dispersing and Retarding Materials in a Cement-Based Mortar.

Pseudorotaxane complexes between β-CD and mPEG derivatives bearing a carboxylic acid function (mPEG–COOH) were synthesized and investigated for their dispersing properties in a cement-based mortar. The formation of mPEG–COOH derivatives and their pseudorotaxanes was investigated by 1D nuclear magnetic resonance, diffusion ordered spectroscopy, and thermogravimetric analysis experiments. Mortar tests clearly indicate that mPEG–COOH@β-CD-interpenetrated supramolecules show excellent dispersing abilities. In addition, the supramolecular complexes show a retarding effect, analogously to other known β-CD-based superplasticizers in which the β-CD is covalently grafted on a polymeric backbone.

Macroporous chitosan/methoxypoly(ethylene glycol) based cryosponges with unique morphology for tissue engineering applications

Three-dimensional porous scaffolds are widely employed in tissue engineering and regenerative medicine for their ability to carry bioactives and cells; and for their platform properties to allow for bridging-the-gap within an injured tissue. This study describes the effect of various methoxypolyethylene glycol (mPEG) derivatives (mPEG (-OCH3 functionality), mPEG-aldehyde (mPEG-CHO) and mPEG-acetic acid (mPEG-COOH)) on the morphology and physical properties of chemically crosslinked, semi-interpenetrating polymer network (IPN), chitosan (CHT)/mPEG blend cryosponges. Physicochemical and molecular characterization revealed that the –CHO and –COOH functional groups in mPEG derivatives interacted with the –NH2 functionality of the chitosan chain. The distinguishing feature of the cryosponges was their unique morphological features such as fringe thread-, pebble-, curved quartz crystal-, crystal flower-; and canyon-like structures. The morphological data was well corroborated by the image processing data and physisorption curves corresponding to Type II isotherm with open hysteresis loops. Functionalization of mPEG had no evident influence on the macro-mechanical properties of the cryosponges but increased the matrix strength as determined by the rheomechanical analyses. The cryosponges were able to deliver bioactives (dexamethasone and curcumin) over 10 days, showed varied matrix degradation profiles, and supported neuronal cells on the matrix surface. In addition, in silico simulations confirmed the compatibility and molecular stability of the CHT/mPEG blend compositions. In conclusion, the study confirmed that significant morphological variations may be induced by minimal functionalization and crosslinking of biomaterials.

Evolution of Reactive mPEG Polymers for the Conjugation of Peptides and Proteins

The covalent attachment of methoxy-poly(ethylene glycol) (mPEG) is a well established strategy used to improve the pharmaceutical properties of several biomolecules. Since the pioneering work of Abuchovsky, PEGylation has emerged as a powerful technology of significant relevance, not only for the development of new and better drugs, but also for application in material science. Peptides and proteins are the most traditional targets for PEGylation due to their intense and diverse biotechnological applications. The terminal amino group, as well as the ε-amino group of lysine and the thiol group of cysteine, are all well known nucleophilic sites that have traditionally been used to couple peptides and proteins to mPEG derivatives. Advances in the methods for preparation of the mPEG starting materials, together with a careful selection of new mPEG functional end-groups have allowed new reactive mPEGs to emerge, which show narrow polydispersity and controlled reactivity, providing more homogeneous conjugates. In the last few years the trend has moved towards site-selective, reversible and enzymatic PEGylation using a new generation of tailor-made reagents and strategies. The main goal of this article is to present some of the most relevant achievements obtained in the PEGylation of peptides and proteins. The chemistry underlying the current methods used for the preparation of mPEG reagents, as well as the chemistry involved in the PEGylation reactions are presented in detail, in order of stimulating the synthetic and polymer chemist to turn their attention in this fascinating multi and interdisciplinary field of research. Keywords: PEGylation, Poly(ethylene glycol), PEG reagents, Conjugation, Protein PEGylation, Controlled drug release, Activated PEG.

Facile spectrophotometric assay of molar equivalents of N-hydroxysuccinimide esters of monomethoxyl poly-(ethylene glycol) derivatives

BackgroundA new method is developed to quantify molar equivalents of N-hydroxysuccinimide (NHS) esters of derivatives of monomethoxyl poly-(ethylene glycol) (mPEG) in their preparations with NHS acetate ester as the reference.ResultsNHS ester of succinic monoester or carbonate of mPEG of 5,000 Da was synthesized and reacted with excessive ethanolamine in dimethylformamide at 25°C for 15 min. Residual ethanolamine was subsequently quantified by absorbance at 420 nm after reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS) at pH 9.2 for 15 min at 55°C followed by cooling with tap water. Reaction products of ethanolamine and NHS esters of mPEG caused no interference with TNBS assay of residual ethanolamine. Reaction between ethanolamine and NHS acetate ester follows 1:1 stoichiometry. By the new method, molar equivalents of NHS esters of carbonate and succinic monoester of mPEG in their preparations were about 90% and 60% of their theoretical values, respectively. During storage at 37°C in humid air, the new method detected spontaneous hydrolyses of the two NHS esters of mPEG more sensitively than the classical spectrophotometric method based on absorbance at 260 nm of NHS released by reaction with ammonia in aqueous solution.ConclusionThe new method is favorable to quantify molar equivalents of NHS esters of mPEG derivatives and thus control quality of their preparations.

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).

Preparation and characterization of mono‐PEGylated consensus interferon by a novel polyethylene glycol derivative

AbstractTraditional PEGylation strategy of peptide and protein usually results in heterogeneous products with wide molecular weight distribution. Mono‐PEGylated consensus interferon (C‐IFN) with homogeneous molecular weight was prepared by a novel methoxypolyethylene glycol (mPEG) derivative. 2‐Fluoro‐1‐methylpyridinium toluene‐4‐sulfonate (FMP) was applied to activate mPEG and a novel functionalized mPEG derivative (FMP‐mPEG) was obtained. The reactivity of FMP‐mPEG toward amino groups of C‐IFN was evaluated, and the heterogeneity of PEGylated protein was identified by high‐performance size‐exclusion chromatography (HPSEC) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS). Finally, the PEGylated C‐IFN was separated from intact protein by cation‐exchange chromatography. The results indicated that PEGylation process was dependent on pH dramatically and the modification degree was increased with the increasing molar excess of FMP‐mPEG. Under optimized condition, mono‐PEGylated C‐IFN with molecule weight of 24.64 kDa was achieved. Further investigation of in vitro biological activities demonstrated that about 88% of antiviral activity was maintained by the mono‐PEGylated C‐IFN. Copyright © 2006 Society of Chemical Industry

Novel polyethylene glycol derivative suitable for the preparation of mono-PEGylated protein

A novel methoxypolyethylene glycol (mPEG) derivative, containing a reactive group of 1-methyl pyridinium toluene-4-sulfonate, was synthesized and characterized. The mPEG derivative was successfully conjugated with two proteins: recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN). Homogeneous mono-PEGylated proteins were obtained which were identified by high performance size-exclusion chromatography and MALDI-TOF mass spectrometry. The biological activities of the mono-PEGylated rhG-CSF and the mono-PEGylated C-IFN were maintained at 90% and 88%, respectively.

PEGylation of G-CSF using cleavable oligo-lactic acid linkage

A new class of methoxy-poly(ethylene glycol) (mPEG) derivatives having a cleavable group of oligo-lactic acid (OLA) was synthesized by ring opening polymerization of l-lactide using a terminal hydroxyl end of mPEG as an initiator. The synthesized mPEG derivatives had 0.8 and 3.6 lactic acid units based on the MALDI-TOF data. A terminal end group of mPEG-OLA was further activated with p-nitrophenyl chloroformate to produce mPEG-OLA- p-nitrophenyl carbonate (PC). mPEG-OLA-PC derivatives were conjugated to primary amine groups of recombinant human granulocyte-colony stimulating factor. PEGylated G-CSF conjugated with mPEG-OLA-PC derivatives consisted of native, mono-, di-, and tri-PEGylated species, and they did not show any apparent conformational changes even after PEGylation. When incubating in pH 7.4 buffer at 37 °C for 2 days, mPEG-OLA-G-CSF conjugates liberated mPEG by cleaving the OLA spacer, resulting in the gradual regeneration of G-CSF.

Novel poly(ethylene glycol) derivatives for preparation of ribosome-inactivating protein conjugates.

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.

Poly(ethylene glycol)–human serum albumin–paclitaxel conjugates: preparation, characterization and pharmacokinetics

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.

B-Domain deleted recombinant coagulation factor VIII modified with monomethoxy polyethylene glycol.

Recombinant coagulation factor VIII (r-VIII SQ) was chemically modified with monomethoxy poly(ethylene glycol) (mPEG). Three mPEG derivatives were used for coupling to the r-VIII SQ lysines, a mixed anhydride of monomethoxy poly(ethylene glycol) succinic acid (mPEG-SAH), monomethoxy poly(ethylene glycol) succinimidyl succinate (mPEG-SS), and monomethoxy poly(ethylene glycol) tresylate (mPEG-TRES). A consequence of the modification with all derivatives was a substantial reduction in coagulant activity, even at very low degrees of modification. A method was developed with the purpose of avoiding conjugation at certain important biological sites on the factor VIII and thereby producing conjugates with better retained activity. This was achieved by immobilizing the protein onto a solid matrix during the modification reaction. Characterization of conjugates by SDS-PAGE, western blots, interaction with von Willebrand factor (vWf), and thrombin activation/inactivation analyses was undertaken. The SDS-PAGE and western blots revealed coupling heterogeneity regarding degree of modification. The amount of factor VIII able to bind to vWf decreased with the conjugation. Thrombin activated the modified factor VIII to essentially the same extent as the reference preparation of r-VIII SQ. Inactivation of the modified factor VIII was, however, slower than inactivation of the unmodified protein. Finally, an in vitro study was performed to evaluate the influence of the mPEG modification on the protein stability in extract of porcine tissue. Despite that conjugates with low degrees of modification were included in the study, the coagulant activity was preserved to a significantly higher extent in all incubation mixtures containing conjugates compared to that with unmodified protein.

Active Site Protection of Proteolytic Enzymes by Poly(ethylene glycol) Surface Modification

A method to prevent the loss of enzymatic activity of proteolytic enzymes toward high molecular weight substrates that occurs upon derivatiza tion with monomethoxypoly(ethylene glycol) (mPEG) is described. It is based on the heterogenous phase enzyme modification after the enzyme is bound to an active site inhibitor immobilized on an insoluble resin. This procedure pro tects the active site and the surrounding area from mPEG linkage. Trypsin modified by mPEG in a heterogeneous phase, using benzamidine bound to Sepharose maintained a high degree of its ability to hydrolize large molecular weight substrates, such as bovine serum albumin or casein, compared to the mPEG derivatives obtained without any protection or with free benzamidine in solution.

Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent

The experimental conditions for the preparation of conjugates of ovalbumin (OA) and monomethoxypolyethylene glycol (mPEG) of a preselected average degree of conjugation, n, using cyanuric chloride as the coupling agent, have been investigated with emphasis on purification and characterization of the products. These conjugates served as prototypes of tolerogenic mPEG derivatives of antigenic proteins which were capable of suppressing in mammals the immunological response to the corresponding unmodified antigens. In other studies in this laboratory, the tolerogenicity of OA(mPEG) n conjugates was found to be a function of n. The reproducibility of the reaction leading to the production of OA(mPEG) n conjugates was shown to depend primarily on the reactivity of the mPEG-cyanuric chloride intermediate, which—for best results—had to be synthesized under completely anhydrous conditions. Isolation of the OA(mPEG) n conjugates was optimized by the use of ion-exchange chromatography whereby rapid removal of large amounts of uncoupled intermediate from the conjugate was achieved; the conditions of fractionation were affected by the degree of conjugation. This method of purification was superior to dialysis, ultrafiltration, and gel filtration. Furthermore, by the application of analytical hydrophobic interaction HPLC it was possible to differentiate among conjugates of different degrees of conjugation and to establish the absence of any detectable free OA in any of the preparations. The quantity of mPEG in the conjugates was determined directly by NMR.

Tolerance induction in mice by conjugates of monoclonal immunoglobulins and monomethoxypolyethylene glycol. Transfer of tolerance by T cells and by T cell extracts.

The specific tolerance induced in mice by conjugates of human monoclonal IgG (HIgG) with monomethoxypolyethylene glycol (mPEG) was transferred to normal mice by spleen cells or a surface immunoglobulin negative (sIg-) Lyt-2+ subpopulation of these cells. Although transferable tolerance was demonstrable 6 to 14 days after treatment of the cell donors with tolerogen, the state of tolerance persisted in the treated mice for at least 43 days. Moreover, an extract prepared by freezing and thawing of the sIg- spleen cells obtained from mice 6 days after treatment with HIgG(mPEG)20 was capable of reducing (greater than 85%) the immune response of normal mice to heat aggregated HIgG. On the basis of these results, it is suggested that similar tolerogenic mPEG derivatives of xenogeneic monoclonal immunoglobulins (XIg) may prove to be useful therapeutic agents in man when administered before treatment with the unmodified XIg.