PEGylation Methods Research Articles

Lipegfilgrastim: A long-acting, once-per-cycle, glycopegylated recombinant human filgrastim.

e13548 Background: Lipegfilgrastim is a once-per-cycle fixed-dose glycoPEGylated granulocyte-colony stimulating factor (G-CSF) under development for the prevention of severe neutropenia in cancer patients receiving chemotherapy (CTx). PEGylation of a molecule extends its half-life in the body, requiring less frequent dosing and allowing for administration of G-CSF once per CTx cycle, making treatment potentially less expensive and enhancing patient compliance and safety. Briefly, traditional PEGylation methods use chemical conjugation through reactive groups on amino acids, which may reduce protein activity and result in non-uniform chemical and pharmaceutical properties. Here, we describe a highly site-specific GlycoPEGylation technology for site-directed PEGylation and summarize preclinical findings compared with pegfilgrastim (Neulasta). Methods: Glycosylation sequon scanning was used to identify the glycoPEGylation site with least impact on protein activity. E. coli-expressed G-CSF was selectively glycosylated at natural O-glycosylation sites and a polyethylene glycol (PEG) sialic acid derivative attached using a sialyltransferase (glycoPEGylation technology). Ligand binding affinity was assessed using the BIACORE 3000 system. Biologic activity of lipegfilgrastim was assessed in an NFS-60 cell line proliferation assay vs filgrastim and pegfilgrastim. PK and PD properties were studied in healthy and neutropenic animal models. Results: GlycoPEGylation produces long-acting G-CSF with improved PK profiles. In vitro, lipegfilgrastim had binding affinity and specific activity comparable to pegfilgrastim, and both were lower than non-PEGylated filgrastim. Comparable increases in leukocytes, neutrophilic granulocytes, and monocytes were seen with lipegfilgrastim and pegfilgrastim in rats and monkeys and were consistent with the effects expected for a long-lasting G-CSF. Conclusions: GlycoPEGylation technology platform is used to produce lipegfilgrastim – a novel, biologically active G-CSF with greater structural homogeneity and comparable pharmacologic properties to conventionally PEGylated G-CSFs.

Preparation of radioactive praseodymium oxide as a multifunctional agent in nuclear medicine

Many studies have attempted to assess the significance of the use of the β(-)particle emitter praseodymium-142 ((142)Pr) in cancer treatment. As praseodymium oxide (Pr(2)O(3)) powder is not water soluble, it was dissolved in HCl solution and the resultant solution had to be pH adjusted to be in an injectable radiopharmaceutical form. Moreover, it was shown that the nanosized neodymium oxide (Nd(2)O(3)) induced massive vacuolization and cell death in non-small-cell lung cancer. In this work, the production of (142)Pr was studied and water-dispersible nanosized Pr(2)O(3) was proposed to improve the application of (142)Pr in nuclear medicine. Data from different databases pertaining to the production of (142)Pr were compared to evaluate the accuracy of the theoretical calculations. Water-dispersible nanosized Pr(2)O(3) was prepared using a poly(ethylene glycol) (PEG) coating or PEGylation method as a successful mode of drug delivery. Radioactive (142)Pr(2)O(3) was produced via a (142)Pr(n,γ)(142)Pr reaction by thermal neutron bombardment of the prepared sample. There was good agreement between the reported experimental data and the data based on nuclear model calculations. In addition, a small part of nano-Pr(2)O(3) particles remained in suspension and most of them settled out of the water. Interestingly, the PEGylated Pr(2)O(3) nanoparticles were water dispersible. After neutron bombardment of the sample, a stable colloidal (142)Pr(2)O(3) was formed. The radioactive (142)Pr(2)O(3) decays to the stable (142)Nd(2)O(3). The suggested colloidal (142)Pr(2)O(3) as a multifunctional therapeutic agent could have dual roles in cancer treatment as a radiotherapeutic agent using nanosized (142)Pr(2)O(3) and as an autophagy-inducing agent using nanosized (142)Nd(2)O(3).

PEGylation of fluoridated hydroxyapatite (FAp):Ln3+ nanorods for cell imaging

PEGylation is a popular approach for the surface functionalization of nanoparticles to achieve improved properties and better performance. Herein, we developed a facile method for surface PEGylation of hydrophobic fluoridated hydroxyapatite (FAp):Ln3+ (Ln = Eu or Tb) nanorods via hydrophobic interactions between oleic acid and amphiphilic synthetic copolymers, which were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization using stearyl methacrylate (SMA) and poly(ethylene glycol) methacrylate (PEGMA) as monomers. Our results demonstrated that the morphology and fluorescent properties of the FAp nanorods are not significantly changed by the PEGylation procedure, and the resulting FAp nanorods were found to be stable in aqueous solution. More importantly, these PEGylated FAp nanorods are biocompatible with cells and could be utilized for cell imaging applications. Therefore, we believe that the method described in this work is a simple, efficient and general strategy for the surface PEGylation of hydrophobic nanoparticles.

Site-Specific PEGylated Exendin-4 Modified with a High Molecular Weight Trimeric PEG Reduces Steric Hindrance and Increases Type 2 Antidiabetic Therapeutic Effects

The purpose of this study was to optimize an Exendin-4 (Ex4-Cys) site-specific PEGylation method with a high-molecular-weight trimeric PEG. Here, we describe the preparation of C-terminal specific PEGylated Ex4-Cys (C40-tPEG-Ex4-Cys), which was performed using cysteine and amine residue specific coupling reactions between Ex4-Cys and activated trimeric PEG. The C40-PEG-Ex4-Cys was obtained at high yields (~83%) and characterized by MALDI-TOF mass spectrometry. The receptor binding affinity of C40-PEG(5K)-Ex4-Cys was 3.5-fold higher than that of N-terminal PEGylated Ex4-Cys (N(ter)-PEG(5K)-Ex4-Cys), and receptor binding by the trimeric PEG (tPEG; 23, 50 kDa) adduct was much higher than that of branched PEG (20 kDa). Furthermore, C40-tPEG(50K)-Ex4-Cys was found to have greater blood circulating t(1/2) and AUC(inf) values than native Ex4-Cys by 7.53- and 45.61-fold, respectively. Accordingly, its hypoglycemic duration was much greater at 59.2 h than that of native Ex4-Cys at 7.3 h, with a dose of 25 nM/kg. The results of this study show that C-terminal specific PEGylation using trimeric PEG is effective when applied to Ex4-Cys and suggest that C40-tPEG(50K)-Ex4-Cys has considerable potential as a type 2 antidiabetic agent.

Facile surface PEGylation via tyrosinase-catalyzed oxidative reaction for the preparation of non-fouling surfaces

The control of biological interactions that occur at material-cell/blood interfaces is of great importance to help maximize in vitro and in vivo performance of biomedical devices. PEGylation has been extensively used as an effective surface modification tool that can alter biological responses on device surfaces. Herein, we report a new surface PEGylation method using a tyrosinase-catalyzed oxidative reaction. Tyramine (TA), an enzymatically active phenolic compound, was chemically conjugated to methoxy poly(ethylene glycol) (mPEG). Surface immobilization of mPEG-TA onto various substrates was accomplished simply and rapidly by adding tyrosinase under mild conditions. It was shown that the water contact angles on all surfaces modified with mPEG-TA were decreased, indicating successful introduction of hydrophilic PEG. In addition, the X-ray photoelectron spectroscopy (XPS) spectra demonstrated the differences in the atomic composition on the TiO2 surface after treatment with mPEG-TA. Non-fouling surfaces prevent non-specific interactions with proteins and cells; consistently, the PEGylated TiO2 surface clearly showed a decrease in both levels of bovine serum albumin (BSA) adsorption and NIH3T3 cell attachment. Therefore, the facile surface PEGylation using a tyrosinase-catalyzed oxidative reaction should be useful for designing non-fouling surfaces of biomedical devices.

PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation

Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin–biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I2–BaCl2 for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography.

A hexon-specific PEGylated adenovirus vector utilizing blood coagulation factor X

We previously developed a hexon-specific PEGylated adenovirus (Ad) vector by utilizing avidin-biotin interaction. However, the Ad vector was aggregated due to the multiple interactions between avidin and biotin, resulting in a reduction in the transduction efficiencies in the organs following systemic administration. In this study, we developed a new method for hexon-specific PEGylation by mixing Ad vectors with PEGylated blood coagulation factor X (FX) (PEG-FX). FX specifically binds to the hexon protein, suggesting that FX serves as an adaptor molecule for hexon-specific modification. Intravenous administration of the PEG-FX-associated Ad (PEG-FX-Ad) vector into conventional mice resulted in prolonged blood retention. However, the transduction efficiencies in the liver were not reduced by PEG-FX. On the other hand, in the warfarinized mice, the PEG-FX-Ad vectors exhibited a significant reduction in the liver transduction. In addition, incubation of the PEG-FX-Ad vector with unmodified FX resulted in dissociation of PEG-FX from the Ad vector, indicating that a substitution of PEG-FX with endogenous FX occurs in the blood following administration. This study demonstrates that FX can be used as an adaptor molecule for hexon-specific modification; however, modified FX might be substituted with endogenous FX in the blood.

Synthesis of Micron Sized Gold Network Structure by Assembling Individual Gold Nanoparticles by Laser Assisted PEGylation Method

Abstract: In this work we have successfully fabricated & assembled free Gold nanoparticles (diameter 8-10nm) into mi-cron size structured network (with unbroken lengths often more than few microns) by the bottom up method through the process of PEGylation. This was done using only a solid metallic Au target, MEG (Mono Ethylene Glycol) (combined with DI water with fixed proportion) and a pulsed excimer laser (
=248nm). The Au networked structure formed in liquid medium is mechanically as well as thermally stable and can be transferred unchanged into a solid substrate which can span a large surface area. The nanochains show a broad optical absorption covering almost the complete visible spectrum. NMR analysis was carried out to understand the formation mechanism of these chains. The process reported does not use any precursor, reducing agent or surfactant and thus can be described as one pot synthe-sis route and the complete synthesis can be done well within few minutes and instantly br ready to use as there is no fur-ther processing is required. It is also biocompatible (as Gold, PEG are non toxic materials for living beings) so can be functionalised and used inside the body for various purposes. This method can be used in mass production of such net-work structures.

PEGylation 기술을 이용한 단백질 의약품 개발

PEGylation, the attachment of polyethylene glycol (PEG) to proteins, is currently main technology for improving efficacy of protein drugs. This technology can prolong the plasma half-life, augment the in vivo stability, and diminish the immunogenicity of therapeutic proteins. Therefore, PEGylated proteins have the enhanced therapeutic efficacy and the reduced undesirable effects versus their native therapeutics. Since the first PEGylated protein product appeared on the market in the early 1990s, currently ten PEGylated protein products have been launched. These marketed drug products have proved the applicability and safety of the PEGylation technology. This review presents overview of PEGylation technology and addresses characteristics of PEGylation methods applied for the development of several protein drugs.

Opsonization, Biodistribution, Cellular Uptake and Apoptosis Study of PEGylated PBCA Nanoparticle as Potential Drug Delivery Carrier

For nanocarrier-based targeted delivery systems, preventing phagocytosis for prolong circulation half life is a crucial task. PEGylated poly(n-butylcyano acrylate) (PBCA) NP has proven a promising approach for drug delivery, but an easy and reliable method of PEGylation of PBCA has faced a major bottleneck. PEGylated PBCA NPs containing docetaxel (DTX) by modified anionic polymerization reaction in aqueous acidic media containing amine functional PEG were made as an single step PEGylation method. In vitro colloidal stability studies using salt aggregation method and antiopsonization property of prepared NPs using mouse macrophage cell line RAW264 were performed. In vitro performance of anticancer activity of prepared formulations was checked on MCF7 cell line. NPs were radiolabeled with 99mTc and intravenously administered to study blood clearance and biodistribution in mice model. These formulations very effectively prevented phagocytosis and found excellent carrier for drug delivery purpose. In vivo studies display long circulation half life of PBCA-PEG20 NP in comparison to other formulations tested. The PEGylated PBCA formulation can work as a novel tool for drug delivery which can prevent RES uptake and prolong circulation half life.

Integrated Solid-Phase Synthesis and Purification of PEGylated Protein

We describe an integrated method for solid-phase protein PEGylation and the purification of mono-PEGylated protein thus synthesized. Lysozyme was used as model protein in this study. Methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) was first immobilized on a stack of microporous hydrophobic interaction membranes housed in a module. The membrane-bound m-PEG propionaldehyde was then contacted with lysozyme solution, which also contained sodium cyanoborohydride as a reducing agent. The PEGylated lysozyme thus synthesized remained attached to the membrane, whereas unreacted protein could easily be removed from the module. PEGylated protein was then eluted from the membrane in a partially purified form using salt-free buffer. Two separate steps were thus integrated into a single process: protein PEGylation, followed by purification of mono-PEGylated protein. This solid-phase method is likely to be suitable for PEGylating any protein because it is based on the immobilization of the activated PEG and not the protein being PEGylated.

Poly(acrylic acid)‐graft‐poly(ethylene glycol) preparation and adsorption on polyelectrolyte multilayers (PEMs) for custom‐made antiadhesive surfaces

AbstractIn this study, we report on a simple and versatile method for PEGylation of surfaces which is applicable practically independent of the substrate material: a poly(acrylic acid)‐graft‐poly(ethylene glycol) (PAA‐g‐PEG) copolymer was synthesized with grafting ratios g in the range of g = 20–2 [acrylic acid (AA) monomers to PEG side chains] and adsorbed either on bare stainless steel or on stainless steel precoated with different types of polyelectrolyte multilayers (PEMs) from aqueous solution. For PAA‐g‐PEG synthesis two principal routes were compared with respect to yield and control of g: the active ester route using different carbodiimide coupling agents and the route via poly(acrylic acid chloride) (PAA‐Cl). Measurements based on Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR‐ATR) showed that the adsorbed amount of PAA‐g‐PEG copolymer is in general drastically enhanced, compared to bare stainless steel as a substrate, when a suitable PEM is used as an interlayer. The amount was also found to be strongly dependent on the type of PEM and to increase with increasing thickness of the PEM interlayer. The stability of highly PEGylated PEMs and the influence of thermal crosslinking on stability were investigated by immersing the PEM‐PEG samples for 1 week into 1 M NaCl solution at pH 4.5 and 0.15 M phosphate buffered saline (PBS) at pH 7.4, respectively.

Thermal characterisation of PEGylated mucin

Objective To investigate the characteristics of PEGylated mucin and its potential usage. Methods Mucin was extracted from giant African land snails and PEGylated mucin was prepared with different ratios of PEG 2000-Mwt and mucin (1 : 1, 0 : 1, 2 : 1, 1 : 3 and 3 : 1 to form batch A-E) using solvent technique. The physicochemical properties of mucin were identified and the solubility of mucin was assessed. The thermal properties of PEGylated mucin were measured by differential scanning calorimetry (DSC). Results Carbohydrates, proteins and trace amounts of fats were present in snail mucin. The mucin powder was water-soluble at 30°C and more water-soluble at 35°C, but not soluble; in acetone, ethanol, 0.1 M NaOH, 0.1M H 2SO 4 and 0.1 NH 4OH was water-soluble. The melting point T m ranged from 58.58 °C to 61.17 °C, crystallization temperature Tc 37.08 °C to 39.83 °C, and glass transition temperature Tg 126.85 °C to 138.39 °C. The variation in T m, T c, and T g with the composition in the PEGylaton showed that an interaction between PEG and mucin occured. Conclusions This result can serve as a basis for further evaluation of the PEGylation method and be used for drug delivery.

PEGylation of interleukin‐10 for the mitigation of enhanced pain states

The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of approximately 3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.

A solid-phase adsorption method for PEGylation of human serum albumin and staphylokinase: preparation, purification and biochemical characterization

A solid-phase adsorption method was developed to circumvent the disadvantage of the conventional liquid-phase PEGylation, i.e. the heterogeneity of the PEGylated products. The model proteins, human serum albumin (HSA) and staphylokinase (SAK), were adsorbed on the ion exchange chromatography media, followed by PEGylation with succinimidyl carbonate (SC)-mPEG5K and salt elution. Since PEGylation with SC-PEG5K alters the positive charge of the proteins, Q-Sepharose Big Beads and DEAE Sepharose Fast Flow were used for adsorption of HSA and SAK, respectively. Size exclusion chromatography and SDS-PAGE studies demonstrated that solid-phase PEGylation of proteins generate monoPEGylated proteins with the yield of 35-47%. Circular dichroism and intrinsic fluorescence studies showed that solid-phase PEGylation led to little conformational change of the proteins. Solid-phase PEGylation resulted in 35% loss in the biological activity of SAK, which is lower than the liquid-phase PEGylation (70%).

The development of stable aqueous suspensions of PEGylated SPIONs for biomedicalapplications

We report here the development of stable aqueous suspensions of biocompatiblesuperparamagnetic iron oxide nanoparticles (SPIONs). These so-called ferrofluids are usefulin a large spectrum of modern biomedical applications, including novel diagnostic tools andtargeted therapeutics. In order to provide prolonged circulation times for the nanoparticlesin vivo, the initial iron oxide nanoparticles were coated with a biocompatible polymerpoly(ethylene glycol) (PEG). To permit covalent bonding of PEG to the SPION surface,the latter was functionalized with a coupling agent, 3-aminopropyltrimethoxysilane (APS).This novel method of SPION PEGylation has been reproduced in numerous independentpreparations. At each preparation step, particular attention was paid to determine thephysico-chemical characteristics of the samples using a number of analytical techniquessuch as atomic absorption, Fourier transform infrared (FT-IR) spectroscopy and Ramanspectroscopy, transmission electron microscopy (TEM), photon correlation spectroscopy(PCS, used for hydrodynamic diameter and zeta potential measurements) andmagnetization measurements. The results confirm that aqueous suspensions of PEGylatedSPIONs are stabilized by steric hindrance over a wide pH range between pH 4 and 10.Furthermore, the fact that the nanoparticle surface is nearly neutral is in agreementwith immunological stealthiness expected for the future biomedical applicationsin vivo.

Reproductible production of a PEGylated dual-acting peptide for diabetes

A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.

Site-Specific PEGylation for High-Yield Preparation of Lys21-Amine PEGylated Growth Hormone-Releasing Factor (GRF) (1−29) using a GRF(1−29) Derivative FMOC-Protected at Tyr1 and Lys12

PEGylation has been viewed as an effective means of overcoming the therapeutic restriction of growth hormone-releasing factor (1-29) (GRF(1-29)) due to its short biological lifetime caused by severe proteolysis and rapid glomerular filtration. Of three isomers according to the PEGylation sites (Tyr1, Lys12, or Lys21), PEGylated GRF(1-29) at Lys21-amine (Lys21-PEG-GRF(1-29)) was shown to have the highest bioactivity. In this report, we propose a unique two-step site-specific PEGylation method capable of producing only Lys21-PEG-GRF(1-29) with a single composition in high yield using a GRF(1-29) derivative protected at Tyr1 and Lys12 and remained available at Lys21 (FMOC1,12-GRF(1-29)). The first step of this reaction involved the PEG attachment to FMOC1,12-GRF(1-29), and the second step involved the removal of FMOC moieties. This PEGylation process was optimized at the following conditions: 0.2-0.3% (v/v) triethylamine concentration, 5.0-6.0-fold molar amount of PEG, reaction temperature of 25-45 degrees C, and reaction time of 30 min. Under these conditions, the maximum yield of Lys21-PEG-GRF(1-29) produced was ca. approximately 95%, 6.3-fold higher than that by nonspecific PEGylation at pH 8.5. Significantly, this site-specific Lys21-PEG-GRF(1-29) was found to have greatly increased resistance to rat plasma, liver, and kidney homogenates, with 7.0-, 25.4-, and 16.4-fold longer half-lives vs GRF(1-29), respectively. Furthermore, 125I-Lys21-PEG-GRF(1-29) displayed significantly reduced liver and kidney distributions and extended blood presence vs 125I-GRF(1-29) in rats. Due to these benefits, Lys21-PEG-GRF(1-29) displayed an enhanced initial growth hormone release in vivo despite having 15% remaining activity in vitro. This devised PEGylation method using an FMOC-protection/deprotection strategy would provide great usefulness for PEGylating bioactive peptides in terms of improved biological potency, elevated production yield, and a uniform composition.

High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation

The purpose of this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of mono-PEGylated (Lys18) salmon calcitonin (Lys18-PEG-sCT), which was previously demonstrated to have superior pharmaceutical properties to other conjugates. For the site-specific PEGylation, this study used the sCT derivative (FMOC1,11-sCT), which was FMOC protected at Cys1- and Lys11-amines among three PEGylation sites including Lys18-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC1,11-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC1,11-sCT. The optimized reaction resulted in the high production yield of Lys18-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys18-PEG-sCT conjugate showed improved biological stability without the loss in the in vitro and in vivo biological activity by PEGylation. Consequently, this site-specific PEGylation using an FMOC protection/deprotection strategy showed great usefulness in the production of the most promising Lys18-PEG-sCT conjugate with a high yield.

The method of surface PEGylation influences leukocyte adhesion and activation

The influence of different surface modifications with poly(ethyleneglycol) (PEG) layers on the adsorption of fibrinogen and the adhesion and activation of macrophage-like human leukocytes was investigated. Poly(ethylene terephthalate) (PET) was modified using pulsed AC plasma polymerization with two types of starting monomers to generate: 1) a reactive acid surface using maleic anhydride (MAH) as monomer, and 2) a PEG-like surface using diethyleneglycol methyl vinyl ether (DEGVE) as monomer. The MAH surface was used as a reactive platform to graft linear chains of non-fouling mPEG via an intermediate layer of poly(ethyleneimine) (PEI) under lower critical solution temperature (LCST) conditions of the mPEG. The DEGVE monomer is used to create PEG-like layers by use of low power plasma conditions. The ability of the surfaces to resist protein adsorption was investigated quantitatively using (125)I-radiolabeled human fibrinogen, and the conformation of the adsorbed protein was tested using an anti-fibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. The results showed that PEGylated surfaces adsorbed significantly less (up to 90% less) fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mPEG layers than on the PEG-like plasma polymer surfaces. Adhesion of in-vitro differentiated macrophage-like U937 cells was reduced on both the PEG-like plasma polymer surfaces and the linear mPEG layers compared to the unmodified PET surface, but cells adhering to the PEG-like plasma polymer surfaces secreted less tumor necrosis factor-alpha (TNF-alpha) than cells adhering to the linear mPEG layers. In conclusion, the method for preparing non-fouling surfaces for long-term implanted devices influence surface-induced cellular responses of the host.