Analysis of monoPEGylated human galectin-2 by small-angle X-ray and neutron scattering: concentration dependence of PEG conformation in the conjugate

Protein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility, and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two monosubstituted subunits per hGal-2 homodimer. Small angle X-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties. SAXS data gave a constant radius of gyration (R(g) = 21.5 ?) for the conjugate at different concentrations and provided no evidence for an alteration of homodimeric structure or hGal-2 ellipsoidal shape upon PEGylation. In contrast, SANS data revealed a concentration dependence of R(g) for the conjugate, with the value decreasing from 31.5 ? at 2 mg/mL to 26 ? at 14 mg/mL (based on hGal-2 concentration). Scattering data have been successfully described by the model of the ellipsoidal homogeneous core (hGal-2) attached with polymer chains (PEG) at the surface. Evidently, the PEG conformation of the conjugate strongly depends on conjugate concentration and PEG's radius of gyration decreases from 24.5 to 15 ?. An excluded volume effect, arising from steric clashes between PEG molecules at high concentration, was quantified by estimating the second virial coefficient, A(2), of PEGylated hGal-2 from the SANS data. A positive value of A(2) (6.0 ± 0.4 × 10(-4) cm(3) mol g(-2)) indicates repulsive interactions between molecules, which are expected to protect the PEGylated protein against aggregation.     

Optimization of PEGylation Conditions for BSA Nanoparticles Using Response Surface Methodology

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.     

Site-specific PEGylation of bone morphogenetic protein-2 cysteine analogues

Three cysteine analogues of bone morphogenetic protein (BMP)-2, BMP2A2C, BMP2N56C, and BMP2E96C, were generated in order to enable the attachment of SH-reactive poly(ethylene glycol) (PEG) at specific sites. Three different approaches (Ap) were used for SH-specific PEGylation: (Ap1) reaction of glutathione activated proteins with thiol PEG; (Ap2) reaction of DTT reduced proteins with orthopyridyl disulfide PEG; (Ap3) reaction of DTT reduced proteins with maleimide PEG. Non-, mono-, and di-PEGylated BMP-2 analogues could be separated by RP-HPLC. Trypsin digestion of PEGylated proteins and Trypsin and GluC double-digestion of N-ethylmaleimide-labeled proteins confirmed that the modifications were site-specific. Surface plasmon resonance analysis of type I and type II receptor binding of the PEGylated BMP-2 analogues revealed that all three PEGylation approaches were equivalent. PEGylation at positions 2 and 96 caused a similar decrease in receptor affinity. PEGylation at position 56 resulted in a larger decrease in affinity for both types of receptors. Mono-PEGylated BMP-2 analogues exhibited intermediate affinities in comparison with unmodified and di-PEGylated proteins. However, the biological activity of the PEGylated BMP-2 analogues as measured in alkaline phosphatase assay was higher than BMP-2 wild-type for the PEGylated BMP2A2C, slightly reduced for the BMP2N56C, and strongly reduced for the BMP2E96C. These results taken together indicate that specific attachment of PEG at engineered sites of BMP-2 is possible and that the attachment site is critical for biological activity. Furthermore, the biological activity of PEGylated BMP-2 analogues in cell culture seems to be determined not only by receptor affinity, but also by other factors such as protein solubility and stability. It is also discussed that the attached PEG interferes with the binding of BMP-2 to modulator proteins, co-receptors, or heparinic sites of proteoglycans in the extracellular matrix.     

Disulfide–bridging PEGylation during refolding for the more efficient production of modified proteins

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide‐bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG‐mono‐sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide‐bridging conjugation to give the PEGylated proteins. Interferon‐β1b (IFN‐β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging‐conjugation during refolding. The PEG‐IFN‐β1b was isolated by ion‐exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN‐β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN‐β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.     

Preparation and characterization of active site protected poly(ethylene glycol)–avidin bioconjugates

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin–PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin–PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin–PEG 10 and 20 kDa prepared in the presence of imminobiotin–PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79–85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin–PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.     

Oil-encapsulating PEO-PPO-PEO/PEG shell cross-linked nanocapsules for target-specific delivery of paclitaxel

PEO-PPO-PEO/PEG shell cross-linked nanocapsules encapsulating an oil phase in their nanoreservoir structure was developed as a target-specific carrier for a water-insoluble drug, paclitaxel. Oil-encapsulating PEO-PPO-PEO/PEG composite nanocapsules were synthesized by dissolving an oil (Lipiodol) and an amine-reactive PEO-PPO-PEO derivative in dichloromethane and subsequently dispersing in an aqueous solution containing amine-functionalized six-arm-branched poly(ethylene glycol) by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell architecture with an average size of 110.7 +/- 9.9 nm at 37 degrees C, as determined by dynamic light scattering and transmission electron microscopy. Paclitaxel could be effectively solubilized in the inner Lipiodol phase surrounded by a cross-linked PEO-PPO-PEO/PEG shell layer. The paclitaxel-loaded nanocapsules were further conjugated with folic acid to achieve folate receptor targeted delivery. Confocal microscopy and flow cytometric analysis revealed that folate-mediated targeting significantly enhanced the cellular uptake and apoptotic effect against folate receptor overexpressing cancer cells. The present study suggested that these novel nanomaterials encapsulating an oil reservoir could be potentially applied for cancer cell targeted delivery of various water-insoluble therapeutic and diagnostic agents.     

PEGylation of human serum albumin: reaction of PEG-phenyl-isothiocyanate with protein

Successful and cost-effective PEGylation protocols require pure functionalized PEG reagents, which can be synthesized by simple and efficient procedures, exhibit high stability against hydrolysis, and maintain a level of reactivity with protein functional groups under mild reaction conditions. PEG-phenyl-isothiocyanate (PIT-PEG) is a new functionalized PEG having these characteristics, and has been synthesized by condensation of the bifunctional reagent 4-isothiocyanato phenyl isocyanate with monomethoxy PEG (mPEG). The data of (1)H NMR and colormetric analysis of the new PEG reagent establish that the mPEG has been quantitatively functionalized. The t 1/4 values for the hydrolysis of PIT-PEG5K in 100 mM phosphate solution at pH 6.5 and 9.2 are about 95 and 40 h, respectively. Incubation of human serum albumin (HSA, 0.5 mM) with a 10-fold molar excess of PIT-PEG (3K or 5K) at pH 6.5 and 9.2 generated PEG-HSA conjugates with average of 3.5 and 6.0 PEG chains per HSA molecule, respectively. The circular dichroism spectra of the conjugates showed that PEGylation of HSA has little influence on the secondary structure of HSA. The hexaPEGylated HSA, (TCP-PEG5K) 6-HSA, exhibited very high hydrodynamic volume, and the molecular radius of HSA increased from 3.95 to 6.57 nm on hexaPEGylation. The hexaPEGylation also increased the viscosity of 4% HSA from 1.05 to 2.10 cP, and the colloid osmotic pressure from 15.2 to 48.0 mmHg. The large increase in the hydrodynamic volume and the solution properties of (TCP-PEG5K) 6-HSA suggest that it could be a potential candidate as a plasma volume expander. PIT-PEG is a useful addition to the spectrum of functionalized PEG reagents available for surface decoration of proteins with PEG.     

Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation.

PEG modification (PEGylation) has been shown to reduce immunogenicity and prolong circulating half-life of proteins. In the present study, site-directed PEGylation was used to reduce immunogenicity and prolong plasma half-life of trichosanthin (TCS). Four TCS mutants, i.e. S7C, Q219C, K173C and [K173C,Q219C] (KQ), were constructed by site-directed mutagenesis. PEG modifications were done by reacting PEG5k-maleimide or PEG20k-maleimide reagent with the newly introduced cysteine residue of the mutants. The plasma clearance rate of PEGylated TCS mutants decreased up to 100-fold and the decrease was inversely proportional to the effective molecular size. The in vitro activities such as ribosome-inactivating activity and cytotoxicity were also decreased. However, the in vivo abortifacient activity was, slightly decreased, unchanged, or even enhanced in some preparations. PEG5k modification had little effect on immunogenicity. However, PEG20k modification significantly reduced immunogenicity. All PEG20k modified TCS mutants induced lower level IgG and IgE antibodies. In particular, PEG20k-KQ and PEG20k-K173C induced weaker systemic anaphylaxis reaction in guinea pigs. In conclusion, the present results suggest that PEG20k is better than PEG5k for reducing immunogenicity and prolonging plasma half-life. The conjugate can become a better therapeutic agent.     

Identification of adenovirus serotype 5 hexon regions that interact with scavenger receptors

Most of an intravenous dose of species C adenovirus serotype 5 (Ad5) is destroyed by liver Kupffer cells. In contrast, another species C virus, Ad6, evades these cells to mediate more efficient liver gene delivery. Given that this difference in Kupffer cell interaction is mediated by the hypervariable (HVR) loops of the virus hexon protein, we genetically modified each of the seven HVRs of Ad5 with a cysteine residue to enable conditional blocking of these sites with polyethylene glycol (PEG). We show that these modifications do not affect in vitro virus transduction. In contrast, after intravenous injection, targeted PEGylation at HVRs 1, 2, 5, and 7 increased viral liver transduction up to 20-fold. Elimination or saturation of liver Kupffer cells did not significantly affect this increase in the liver transduction. In vitro, PEGylation blocked uptake of viruses via the Kupffer cell scavenger receptor SRA-II. These data suggest that HVRs 1, 2, 5, and 7 of Ad5 may be involved in Kupffer cell recognition and subsequent destruction. These data also demonstrate that this conditional genetic-chemical mutation strategy is a useful tool for investigating the interactions of viruses with host tissues.     

The inhibitory potencies of monoclonal antibodies to the macrophage adhesion molecule sialoadhesin are greatly increased following PEGylation

PEGylation of antibodies is known to increase their half-life in systemic circulation, but nothing is known regarding whether PEGylation can improve the inhibitory potency of antibodies against target receptors. In this paper, we have examined this question using antibodies directed to Sialoadhesin (Sn), a macrophage-restricted adhesion molecule that mediates sialic acid dependent binding to different cells. Anti-Sn monoclonal antibodies (mAbs), SER-4 and 3D6, were conjugated to PEG 5 kDa or and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. Following purification of PEGylated mAbs by anion exchange chromatography, it was shown that PEGylation had little or no effect on antigen binding activity but led to a dramatic increase in inhibitory potency that was proportional to both the size of the PEG and the degree of derivatization. Thus, PEGylation of antibodies directed to cell surface receptors could be a powerful approach to improve the therapeutic efficacy of antibodies, not only by increasing their half-life in vivo, but also by increasing their inhibitory potency for blocking receptor-ligand interactions.     

Inhibitory Effect of Supercritical Extracts from Arctium lappa L. on the Lectin Pathway of the Complement System

The complement system participates in host defense by eliminating microorganisms and triggering inflammation. However, insufficient control or exacerbated complement activation contributes to inflammatory diseases. Since promising antioxidant and anti‐inflammatory activities have been identified in Arctium lappa L. extracts, this study aims to explore the effect of A. lappa extracts on the lectin pathway (LP) of complement activation. Four extracts were obtained by supercritical extraction using scCO₂ with or without ethanol as co‐solvent, at different temperatures and pressures (E1: 2.2 mg/mL, E2: 2.6 mg/mL and E3: 2.0 mg/mL, E4: 1.5 mg/mL). To evaluate the effect of A. lappa extracts on the LP activation, an ELISA assay using mannose binding lectin pathway of complement was carried out with C4 detection. All extracts showed a concentration‐dependent inhibitory effect on the activation of complement by the LP. The following IC₅₀ were observed for E1, E2, E3 and E4: 179.4 μg/mL, 74.69 μg/mL, 119.1 μg/mL and 72.19 μg/mL, respectively. Our results suggest that A. lappa extracts are potential candidates for the treatment of inflammatory disorders that are complement‐related.     

Site-specific PEGylation for high-yield preparation of Lys(21)-amine PEGylated growth hormone-releasing factor (GRF) (1-29) using a GRF(1-29) derivative FMOC-protected at Tyr(1) and Lys(12)

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.     

An acetal-based PEGylation reagent for pH-sensitive shielding of DNA polyplexes

p-Piperazinobenzaldehyde methoxy poly(ethylene glycol) (mPEG, 5 kDa) acetal was synthesized by the Buchwald-Hartwig coupling reaction from piperazine and p-bromobenzaldehyde mPEG acetal. Introduction of a maleimide moiety yielded a novel acetal-based PEGylation reagent (PEG-acetal-MAL) for pH-sensitive conjugation of PEG to thiol-functionalized biomolecules. For reversible shielding of polyplexes, PEG-acetal-MAL was conjugated to polyethylenimine (PEI). At 37 degrees C, the PEG-acetal-PEI conjugate had a half-life of 3 min at endosomal pH 5.5 and 2 h at physiological pH 7.4, respectively. PEI polyplexes containing PEG-acetal-PEI had a zeta potential of +3 mV and were stable to salt-induced aggregation for 2 h at pH 7.4. In contrast, at endosomal pH, the particles were deshielded and aggregated within 0.5 h. Epidermal growth factor or transferrin receptor-targeted polyplexes shielded with the pH-sensitive PEG-acetal mediated enhanced luciferase gene expression in receptor-expressing target cells (Renca-EGFR or K562) as compared to stably shielded control polyplexes. Thus, the novel PEG-acetal-MAL reagent may present a versatile tool for drug and gene delivery formulations when pH-sensitive PEGylation is preferred.     

Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.     

Heat treatment increases the bioactivity of C-terminally PEGylated staphylokinase

PEGylation can effectively improve the therapeutic potential of staphylokinase (SAK), a thrombolysis agent for therapy of myocardial infarction. However, polyethylene glycol (PEG) can sterically shield SAK and drastically decrease its bioactivity. In the present study, N-terminally PEGylated SAKs (5 and 20 kDa PEG), C-terminally PEGylated SAKs with phenyl linker and the ones with amyl linker (5 and 20 kDa PEG) were prepared. The effects of the PEG length, the PEGylation site and linker chemistry on the bioactivity of the heat-treated PEGylated SAK were investigated. Heat treatment at 70 °C for 2 h can improve the bioactivity of the C-terminally PEGylated SAKs, where the one with amyl linker and 20 kDa PEG showed the highest increase extent (27%) in the bioactivity. Thus, our study can advance the development of long-acting pharmaceutical protein with high bioactivity.     

A method to optimize PEG-coating of red blood cells

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. A proposed method to overcome donor-recipient blood group incompatibility is to mask the blood group antigens by the covalent attachment of poly(ethylene glycol) (PEG) to the red blood cell (RBC) membrane. Despite much work in the development of PEG-coating of RBCs, there is a paucity of data on the optimization of the PEG-coating technique; it is the aim of this study to determine the optimum conditions for PEG coating using a cyanuric chloride reactive derivative of methoxy-PEG as a model polymer. Activated PEG of molecular mass 5 kDa was covalently attached to human RBCs under various reaction conditions. Inhibition of binding of a blood-type specific antiserum (anti-D) was employed to evaluate the effect of the PEG-coating, quantified by hemocytometry and flow-cytometry. RBC morphology was examined by light and scanning electron microscopy. Statistical analysis of experimental design together with microscopy results showed that the optimum PEGylation conditions are pH = 8.7, temperature = 14 degrees C, and reaction time = 30 min. An optimum concentration of reactive PEG could not be determined. At high polymer concentrations (>25 mg/mL) a predominance of type III echinocytes was observed, and as a result, a concentration of 15 mg/mL is the highest recommended concentration for a linear PEG of molecular mass 5 kDa.     

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.     

Intrapulmonary potential of polyethylene glycol-modified glucagon-like peptide-1s as a type 2 anti-diabetic agent

The pulmonary delivery of anti-diabetic peptide drugs can improve diabetic patient compliance. In this study, we tried to improve the pulmonary pharmacokinetic properties of glucagon-like peptide-1(7-36) (GLP-1) using a PEGylation approach. Initially, three types of site-specific (Lys(34)) PEGylated GLP-1 analogs were synthesized using PEGs of 2, 5, and 10 kDa, respectively. Their pharmacokinetic profiles were then examined in endotracheally cannulated rats. The results obtained show that all pharmacokinetic parameters (AUC(inf), C(max), t(1/2), V/F, and Cl/F etc.) of PEGylated GLP-1s were greatly improved by increasing PEG Mw. Specifically, the t(1/2) values of PEGylated GLP-1s (PEG Mw: 2, 5, 10 kDa) increased to 23.1+/-6.2, 41.6+/-12.3, and 81.7+/-11.7 min, respectively, vs. 8.9+/-2.9 min for intratracheally administered GLP-1. Also, PEGylated GLP-1s were found to have substantially greater C(max) values (7.4-7.8 ng/ml) than GLP-1 (4.0+/-2.4 ng/ml). Moreover, these PEGylated GLP-1s were found to have 10-20 fold more resistance to rat lung enzyme and plasma dipeptidyl peptidase IV (DPP IV). These findings indicate the dual-pharmacokinetic enhancements that PEGylated GLP-1s better survives proteolytic breakdown in the lungs than GLP-1s and better enters the systemic circulation, and that these analogs are more resistant to DPP IV-induced proteolysis and are much less rapidly removed from the systemic circulation. In conclusion, this study demonstrates the pharmaceutical usefulness of PEGylation in the context of the pulmonary delivery of GLP-1. These results show that PEGylated GLP-1s should be considered potential components of anti-diabetic inhalant preparations.     

Hepatocyte viability and protein expression within hydrogel microstructures

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.     

Effects of oestrus induction with progestagens or prostaglandin analogues on ovarian and pituitary function in sheep

The aim of the current study was to determine possible differences in ovarian and pituitary features explaining lower fertility rates in sheep with oestrus induced with intravaginal progestagens or prostaglandin analogues (group FGA and PGF, n=8 in both) when compared to a control group (group C, n=8). The growth profiles and the mean individual sizes of preovulatory follicles were similar between groups; however, the number of preovulatory follicles per ewe and, consequently, the number of ovulations were higher in groups FGA and PGF (2.3±0.3 and 2.0±0.1, respectively) than in group C (1.4±0.1, P<0.05). However, plasma oestradiol concentrations were similar between groups suggesting a defective function in some preovulatory follicles of groups FGA and PGF. In group FGA, the basal LH levels during the follicular phase were lower (0.21±0.0 ng/mL, P<0.005) than in groups C (0.41±0.1 ng/mL) and PGF (0.55±0.1 ng/mL); the onset of preovulatory discharge being later (21.0±2.3h vs. 12.8±1.5 in C and 14.5±1.5 in PGF; P<0.05 for both). Finally, luteal activity was also found to be affected in group FGA; the rate of progesterone secretion per total luteal tissue was lower (range: 0.46-0.65 ng/mL/cm(2)) than in ewes treated with cloprostenol (2.1-3.3 ng/mL/cm(2)) and control sheep (2.0-3.4 ng/mL/cm(2)).