Possible Action of Transition Divalent Metal Ions at the Inter-Pentameric Interface of Inactivated Foot-and-Mouth Disease Virus Provide A Simple but Effective Approach to Enhance Stability.

The structural instability of inactivated foot-and-mouth disease virus (FMDV) hinders the development of vaccine industry. Here we found that some transition metal ions like Cu2+ and Ni2+ could specifically bind to FMDV capsids at capacities about 7089 and 3448 metal ions per capsid, respectively. These values are about 33- and 16-folds of the binding capacity of non-transition metal ion Ca2+ (about 214 per capsid). Further thermodynamic studies indicated that all these three metal ions bound to the capsids in spontaneous enthalpy driving manners (ΔG<0, ΔH<0, ΔS<0), and the Cu2+ binding had the highest affinity. The binding of Cu2+ and Ni2+ could enhance both the thermostability and acid-resistant stability of capsids, while the binding of Ca2+ was helpful only to the thermostability of the capsids. Animal experiments showed that the immunization of FMDV bound with Cu2+ induced the highest specific antibody titers in mice. Coincidently, the FMDV bound with Cu2+ exhibited significantly enhanced affinities to integrin ß6 and heparin sulfate, both of which are important cell surface receptors for FMDV attaching. Finally, the specific interaction between capsids and Cu2+ or Ni2+ was applied to direct purification of FMDV from crude cell culture feedstock by the immobilized metal affinity chromatography. Based on our new findings and structural analysis of the FMDV capsid, a "transition metal ion bridges" mechanism that describes linkage between adjacent histidine and other amino acids at the inter-pentameric interface of the capsids by transition metal ions coordination action was proposed to explain their stabilizing effect imposed on the capsid.IMPORTANCE How to stabilize the inactivated FMDV without affecting virus infectivity and immunogenicity is a big challenge in vaccine industry. The electrostatic repulsion induced by protonation of a large amount of histidine residues at the inter-pentameric interface of viral capsids is one of the major mechanisms causing the dissociation of capsids. In the present work, this structural disadvantage inspired us to stabilize the capsids through coordinating transition metal ions with the adjacent histidine residues in FMDV capsid, instead of removing or substituting them. This approach was proved effective to enhance not only the stability of FMDV, but also enhance the specific antibody responses; thus, providing a new guideline for designing an easy-to-use strategy suitable for large-scale production of FMDV vaccine antigen.

A Novel Particulate Delivery System Based on Antigen-Zn2+ Coordination Interactions Enhances Stability and Cellular Immune Response of Inactivated Foot and Mouth Disease Virus.

The interactions between antigen and adjuvant were among the most significant factors influencing the immunogenicity of vaccines, especially for unstable antigens like inactivated foot and mouth disease virus (iFMDV). Here we propose a novel antigen delivery pattern based on the coordination interaction between transition metal ions Zn2+ chelated to chitosan nanoparticles and iFMDV, which is known to be rich in histidine. The zinc chelated chitosan particles (CP-PEI-Zn) were prepared by cross-linking chitosan particles (CP) with sodium tripolyphosphate (TPP), modifying with metal chelator polyethylenimine (PEI), and subsequent chelating of Zn2+. The coordination interaction was confirmed by analyzing the adsorption and desorption behavior of iFMDV on CP-PEI-Zn by high-performance size exclusion chromatography (HPSEC), while the CP-PEI without chelating Zn2+ loads iFMDV mainly through electrostatic interactions. The iFMDV loaded on CP-PEI-Zn showed better thermal stability than that on CP-PEI, as revealed by a slightly higher transition temperature (Tm) related to iFMDV dissociation. After subcutaneous immunization in female Balb/C mice, antigens loaded on CP-PEI and CP-PEI-Zn all induced higher specific antibody titers, better activation of B lymphocytes, and more effector-memory T cells proliferation than the free antigen and iFMDV adjuvanted with ISA 206 emulsion did. Moreover, CP-PEI-Zn showed superior efficacy to CP-PEI in promoting the proliferation of effector-memory T cells and secretion of cytokines, indicating a more potent cellular immune response. In summary, the CP-PEI-Zn stabilized the iFMDV after loading and promoted both humoral and cellular immune responses, thus reflecting its potential to be a promising adjuvant for the iFMDV vaccine and other unstable viral antigens.

Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A Case Study for the Site-Specific Attachment of Polyethylene Glycol (PEGylation) of Recombinant Human Ciliary Neurotrophic Factor.

In this paper, we reported a novel strategy for the site-specific attachment of polyethylene glycol (PEGylation) of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was monomethoxy-polyethylene glycol-maleimide (mPEG-MAL). PEGylation with mPEG (40 kDa)-MAL at pH 7.0 under normal pressure for 5 h achieved a less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: (1) high-hydrostatic-pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG-MAL), while the conventional process needs a much-higher ratio. (2) Short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. (3) The reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by a factor of three from pH 7.0 to 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software, and we found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was the facilitation of target-site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated that the HHPP product was comparable to the PEGylated rhCNTF prepared through a conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules.

Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with ß-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

Reversible protection of Cys-93(ß) 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(ß) 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(ß) can protect Lys-95(ß), 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.

Superior intratumoral penetration of paclitaxel nanodots strengthens tumor restriction and metastasis prevention.

Recently discovered intratumoral diffusion resistance, together with poor solubility and nontargeted distribution of chemotherapeutic drugs, has significantly impaired the performance of cancer treatments. By developing a well-designed droplet-confined/cryodesiccation-driven crystallization approach, we herein report the successful preparation of nanocrystallites of insoluble chemotherapeutic drug paclitaxel (PTX) in forms of nanodots (NDs, ≈10 nm) and nanoparticles (NPs, ≈70 nm) with considerably high drug loading capacity. Superficially coated Pluronic F127 is demonstrated to endow the both PTX nanocrystallites with excellent water solubility and prevent undesired phagocyte uptake. Further decoration with tumor-penetrating peptide iRGD, as expected, indiscriminatively facilitates tumor cell uptake in traditional monolayer cell culture model. On the contrary, distinctly enhanced performances in inward penetration and ensuing elimination of 3D multicellular tumor spheroids are achieved by iRGD-NDs rather than iRGD-NPs, revealing the significant influence of particle size variation in nanoscale. In vivo experiments verify that, although efficient tumor enrichment is achieved by all nanocrystallites, only the iRGD-grafted nanocrystallites of ultranano size realize thorough intratumoral delivery and reach cancer stem cells, which are concealed inside the tumor core. Consequently, much strengthened restriction on progress and metastasis of orthotopic 4T1 mammary adenocarcinoma is achieved in murine model, in sharp contrast to commercial PTX formulation Taxol.

Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein.

SDS-PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein-containing conjugates, mostly pegylated proteins. PEG-maleimide (MAL) is frequently used to site-specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C-S linkage between protein and PEG-MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein was observed. It is a thiol-independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG-MAL modified conjugates although PEG-vinylsulfone conjugates to thiol-containing proteins also through a C-S linkage. Cleavage kinetics of PEG40k-MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide-thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG-MAL modified protein/peptide by SDS-PAGE analysis.

A hydrophobic interaction chromatography strategy for purification of inactivated foot-and-mouth disease virus.

A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold purification, with 92% recovery. The columns were coupled through an intermediate ultrafiltration unit to concentrate the virus. The entire process was completed in about 3.5h, with 75% final FMDV recovery, and 247-fold purification. The final product had purity above 98%, with over 99.5% of host cell DNA removed. High-performance size exclusion chromatography (HPSEC), Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM) indicated that the purified virus contained the required antigen, and was structurally intact with a spherical shape and a particle size of 28 nm.

Microcosmic mechanism of dication for inhibiting acylation of acidic Peptide.

PURPOSE: For long-effective peptide formulation based on poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, acylation often leads to peptide instability during its release and reduced drug efficacy. Among the reported solving strategies, adding dication such as Ca(2+) and Mn(2+) in the formulation was the most convenient method for inhibiting basic peptide acylation. However, the strategies for the acidic peptide still remain unexplored, possibly due to the peptide's changeable charge state in acid environment within degraded PLGA microspheres. Moreover, the previous studies mainly focusing on the macroscopical adsorption of peptide to PLGA cannot demonstrate the inhibition mechanism. METHODS: Acylation inhibition for acidic peptide (exenatide) by dications (Ca(2+), Mn(2+) and Zn(2+)) was studied for the first time, and Quartz Crystal Microbalance with Dissipation (QCM-D) was innovatively employed to analyze microcosmic mechanism of the inhibition. RESULTS: These dications played different roles in acylation inhibition of acidic peptide. The effects of dications on acylation outside or inside PLGA microspheres indicated that Ca(2+) did not work, Mn(2+) played a weak role, and Zn(2+) possessed the greatest inhibition. CONCLUSIONS: Zn(2+) was the most effective dication for the acylation inhibition because of the complex formation and its steric-hindrance effect, which was a new function for this dication.

Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres.

Lipase (EC 3.1.1.3) is a versatile enzyme which has been widely used in ester-reaction industries. We have previously discovered that gigaporous polystyrene (PST) microspheres can be used as a novel immobilization carrier for lipase. In this work, a series of gigaporous microspheres with different densities of epoxy group including poly(glycidyl methacrylate) (PGMA) and poly(styrene-co-glycidyl methacrylate) [P(ST-GMA)] were evaluated as lipase immobilization carriers, which were also compared with gigaporous PST microspheres and the commercial immobilized lipase Novozym 435. Lipase immobilized in gigaporous PGMA microspheres showed the highest activity yield, reusability, and stability as well as the best affinity for the substrate. The characterizations of adsorption curves, the change of epoxy group amounts, and hydrophobic-hydrophilic properties of the microspheres were carried out to investigate the interaction between lipase molecules and carriers. It was found that covalent binding played a key role in improving the properties of lipase immobilized in gigaporous PGMA microspheres.

Comparison of site-specific PEGylations of the N-terminus of interferon beta-1b: selectivity, efficiency, and in vivo/vitro activity.

PEGylation, including nonspecific and site-directed approaches, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics together with a reduction in immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification; however, the generation of multimodified products is inevitable because of lysine chemistry, which always leads to difficulties in purification and quantification. In this study, we developed a specific PEGylation strategy through the periodation of the N-terminus of interferon beta-1b (IFN-ß-1b) followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of the PEG-hydrazide-modified protein indicated the development of an effective process for improving the pharmacology and immunogenicity of IFN-ß-1b. We further conducted comparisons on the selectivity, velocity, yield, and pharmacokinetics of the two methods. The results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate but also generated heterogeneous multimodified products in the aldehyde-based process. In addition, a better PEGylation yield was found for the hydrazide conjugation compared with that of the aldehyde strategy. These investigations supply a practical approach for the site-specific modification of proteins with an N-terminal serine or threonine to achieve improved homogeneity of the conjugates as well as enhanced pharmacological properties.

Enhanced humoral and cell-mediated immune responses generated by cationic polymer-coated PLA microspheres with adsorbed HBsAg.

Surface-engineered particulate delivery systems for vaccine administration have been widely investigated in experimental and clinical studies. However, little is known about charge-coated microspheres as potential recombinant subunit protein antigen delivery systems in terms of adsorption and related immune responses. In the present study, cationic polymers, including chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI), were used to coat PLA microspheres to build positively charged surfaces. Antigen adsorption capacity was enhanced with increased surface charge of coated microspheres. In macrophages, HBsAg adsorbed on the surface of cationic microspheres specifically enhanced antigen uptake and augmented CD86, MHC I, and MHC II expression and IL-1ß, IL-6, TNF-α, and IL-12 release. Antigens were more likely to localize independent of lysosomes after phagocytosis in antigen-attached cationic microsphere formulations. After intraperitoneal immunization, cationic microsphere-based vaccine formulations generated a rapid and efficient humoral immune response and cytokine release as compared with aluminum-adsorbed vaccine and free antigens in vivo. Moreover, microspheres coated with cationic polymers with relatively high positive charges and higher antigen adsorption exhibited strong stimulation of the Th1 response. In conclusion, PLA microspheres coated with cationic polymers may be a potential recombinant antigen delivery system to induce strong cell and humoral immune responses.

Comparative studies on the influences of primary emulsion preparation on properties of uniform-sized exenatide-loaded PLGA microspheres.

PURPOSE: It is well known that primary emulsion (W1/O) preparation process (by ultrasonication or homogenization) plays an important role in the properties of drug-loaded microspheres, such as encapsulation efficiency, release behavior and pharmacodynamics. However, its involved mechanism has not been intensively and systematically studied, partly because that broad size distribution of the resultant particles prepared by conventional preparation can greatly disturb the analysis and reliability of the results. Here, we focused on the relevant studies. METHODS: In order to eliminate the disturbance caused by broad size distribution, uniform-sized exenatide-loaded poly(DL-lactic-co-glycolic acid) (PLGA) microspheres were prepared by Shirasu Porous Glass (SPG) premix membrane emulsification. The properties of microspheres whose W1/O was formed by ultrasonication (UMS) and homogenization (HMS) were compared including in vitro release, pharmacology and so forth. RESULTS: HMS exhibited fast release rate and hyperglycemic efficacy within first 14 days, but declined afterwards. Comparatively, UMS showed slower polymer degradation, more acidic microclimate pH (µpH) in vitro, and stable drug release with sustained efficacy during 1 month in vivo. CONCLUSIONS: HMS was desirable for the 2-week-sustained release in vivo, while UMS was more appropriate for the longer time release (about 1 month). These comparative researches can provide guidance for emulsion-microsphere preparation routs in pharmaceutics.

"Ready-to-use" hollow nanofiber membrane-based glucose testing strips.

A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose oxidase and horseradish peroxidase in the core-phase solution, and a chromogenic agent either in the core solution (in which case 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was used) or in the shell-phase solution (in which case o-dianisidine was used) for co-axial electrospinning, in situ co-encapsulation of the two enzymes within the hollow nano-chamber and incorporation of chromogenic agents either inside the nano-chamber or in the shell of the hollow nanofibers was realized. Such unique "all-in-one" feature enabled the prepared hollow nanofiber membrane-based test strips to be applied either as colorimetric sensors in solution or as an optical biosensor operated in the "dip-and-read" mode. When used as a colorimetric biosensor in solution, the test strip with o-dianisidine as chromogenic agent shows an excellent linear response range between 0.01 mM to 20 mM and a high apparent lumped activity recovery of 62.1% as compared to the reaction rate of the free bi-enzyme system. While the activity recovery of the test strip with ABTS as chromogenic agent is only 18.0%, and the test strip is found to be unstable due to spontaneous-oxidation of the ABTS. The o-dianisidine test strip was also applied as an optical biosensor, visible rufous color was quickly developed on the surface of the membrane upon dropping 10 µL of glucose sample, and an excellent correlation between differential diffusive reflectance of the test strip at 440 nm and glucose concentration was obtained in the range of 0.5-50 mM. The test strips also exhibited excellent long-term storage stability with a half-life at 25 °C as long as four months.

Integration of Zn2+, ATP, and bFGF to Nanodressing with Core-Shell Structure Fabricated by Emulsion Electrospinning for Wound Healing.

Basic fibroblast growth factor (bFGF) plays an important role in active wound repair. However, the existing dosage forms in clinical applications are mainly sprays and freeze-dried powders, which are prone to inactivation and cannot achieve a controlled release. In this study, a bioactive wound dressing named bFGF-ATP-Zn/polycaprolactone (PCL) nanodressing with a "core-shell" structure was fabricated by emulsion electrospinning, enabling the sustained release of bFGF. Based on the coordination and electrostatic interactions among bFGF, ATP, and Zn2+, as well as their synergistic effect on promoting wound healing, a bFGF-ATP-Zn ternary combination system was prepared with higher cell proliferation activity and used as the water phase for emulsion electrospinning. The bFGF-ATP-Zn/PCL nanodressing demonstrated improved mechanical properties, sustained release of bFGF, cytocompatibility, and hemocompatibility. It increased the proliferation activity of human dermal fibroblasts (HDFs) and enhanced collagen secretion by 1.39 and 3.45 times, respectively, while reducing the hemolysis rate to 3.13%. The application of the bFGF-ATP-Zn/PCL nanodressing in mouse full-thickness skin defect repair showed its ability to accelerate wound healing and reduce wound scarring within 14 days. These results provide a research basis for the development and application of this bioactive wound dressing product.

Metagenomics reveals the influence of small microplastics on microbial communities in coastal sediments.

The ecological impact of microplastics (MPs) in coastal environments has been widely studied. However, the influence of small microplastics in the actual environment is often overlooked due to measurement challenges. In this study, Hangzhou Bay (HZB), China, was selected as our study area. High-throughput metagenomic sequencing and micro-Raman spectrometry were employed to analyze the microbial communities and microplastics of coastal sediment samples, respectively. We aimed to explore the ecological impact of MPs with small sizes (≤ 100 µm) in real coastal sediment environments. Our results revealed that as microplastic size decreased, the environmental behavior of MPs underwent alterations. In the coastal sediments, no significant correlations were observed between the detected MPs and the whole microbial communities, but small MPs posed potential hazards to eukaryotic communities. Moreover, these small MPs were more prone to microbial degradation and significantly affected carbon metabolism in the habitat. This study is the first to reveal the comprehensive impact of small MPs on microbial communities in a real coastal sediment environment.

Pore-blocking steric mass-action model for adsorption of bioparticles.

The steric mass-action (SMA) model has been widely reported to describe the adsorption of proteins in different types of chromatographic adsorbents. Here in the present work, a pore-blocking steric mass-action model (PB-SMA) was developed for the adsorption of large-size bioparticles, which usually exhibit the unique pore-blocking characteristic on the adsorbent and thus lead to a fraction of ligands in the deep channels physically inaccessible to bioparticles adsorption, instead of being shielded due to steric hindrance by adsorbed bioparticles. This unique phenomenon was taken into account by introducing an additional parameter, Lin, which is defined as the inaccessible ligand densities in the physically blocked pore area, into the PB-SMA model. This fraction of ligand densities (Lin) will be deducted from the total ligand (Lt) for model development, thus the steric factor (σ) in the proposed PB-SMA will reflect the steric shielding effect on binding sites by adsorbed bioparticles more accurately than the conventional SMA model, which assumes that all ligands on the adsorbent have the same accessibility to the bioparticles. Based on a series of model assumptions, a PB-SMA model was firstly developed for inactivated foot-and-mouth disease virus (iFMDV) adsorption on immobilized metal affinity chromatography (IMAC) adsorbents. Model parameters for static adsorption including equilibrium constant (K), characteristic number of binding sites (n), and steric factor (σ) were determined. Compared with those derived from the conventional SMA model, the σ values derived from the PB-SMA model were dozens of times smaller and much closer to the theoretical maximum number of ligands shielded by a single adsorbed iFMDV, indicating the modified model was more accurate for bioparticles adsorption. The applicability of the PB-SMA model was further validated by the adsorption of hepatitis B surface antigen virus-like particles (HBsAg VLPs) on an ion exchange adsorbent with reasonably improved accuracy. Thus, it is considered that the PB-SMA model would be more accurate in describing the adsorption of bioparticles on different types of chromatographic adsorbents.

Propylbenzmethylation at Val-1(α) markedly increases the tetramer stability of the PEGylated hemoglobin: a comparison with propylation at Val-1(α).

BACKGROUND: Hemoglobin (Hb)-based oxygen carriers (HBOCs) are potential pharmaceutical agents that can be used in surgery or emergency medicine. PEGylation can modulate the vasoactivity of Hb and is a widely used approach to develop HBOCs. However, PEGylation can significantly enhance the tetramer-dimer dissociation of Hb, which may perturb the structure of Hb and increase its observed adverse effect. Thus, it is necessary to increase the tetramer stability of the PEGylated Hb. METHODS: Propylbenzmethylation at Val-1(α) of HbA was carried out to stabilize the Hb tetramer. The propylbenzmethylated Hb at Val-1(α) (PrB-Hb) was used as the starting material for site-specific PEGylation at Cys-93(ß) of Hb using maleimide PEG. Structural and functional properties, autoxidation rate and thermal stability of the resultant product (PEG-PrB-Hb) were measured. RESULTS: Propylbenzmethylation at Val-1(α) led to 25-fold and 24-fold decreases in the tetramer-dimer dissociation constant of HbA and PEG-Hb, respectively. The increased tetramer stability is due to the enhanced hydrophobicity of the area around Val-1(α) and the increased polar interaction of Hb upon propylbenzmethylation. Thus, the structural and functional properties of PEG-Hb were improved, and its autoxidation rate and thermal denaturation were decreased. CONCLUSION: Propylbenzmethylation at Val-1(α) showed higher ability than propylation at Val-1(α) to improve the structural and functional properties and decrease the side effect of PEG-Hb. GENERAL SIGNIFICANCE: Our study can facilitate the biotechnological development of stable PEGylated Hb as more advanced HBOC. Our study is also expected to improve the stability of the tetrameric or dimeric proteins (e.g., uric oxidase) by propylbenzmethylation at their N-terminus.

Phenyl linker-induced dense PEG conformation improves the efficacy of C-terminally monoPEGylated staphylokinase.

PEGylation can improve the protein efficacy by prolonging serum half-life and reducing proteolytic sensitivity and immunogenicity. However, PEGylation may decrease the bioactivity of a protein by interfering with binding of its substrate or receptors. Here, staphylokinase (SAK), a thrombolysis agent for therapy of myocardial infarction, was mono-PEGylated at the C-terminus of SAK far from its bioactive domain. Phenyl, propyl, and amyl moieties were used as linkers between SAK and polyethylene glycol (PEG), respectively. Flexible propyl and amyl linkers lead to loose conformation. In contrast, rigid and hydrophobic phenyl linker induces dense PEG conformation that can extensively shield most domains adjacent to C-terminus (e.g., the antigen epitopes and proteolytic sites) of SAK and inefficiently shield its bioactive domain. As compared with loose PEG conformation, dense PEG conformation is more efficient to maintain the bioactivity, increase the plasma half-life, and decrease the proteolytic sensitivity and immunogenicity of the PEGylated SAK.

Enzymatic synthesis of oleic acid-based epoxy monomer for the production of value added polymers.

An oleic acid-based epoxy monomer was synthesized by reacting 2-hydroxyethyl acrylate with epoxy stearic acid and an immobilized lipase. NMR, electrospray ionization mass spectrometry and gel permeation chromatography were used to characterize the intermediates and products. 2-(Acryloyloxy) ethyl epoxy stearate was synthesized with a yield of 87 % w/w. After free radical polymerization, epoxy stearic acid-grafted epoxy polymer with molecular weight of 15,150 g/mol was obtained; the final yield was 81 % w/w.