基于定点突变的抗克百威单链抗体的亲和力成熟

为获得亲和力更高的抗克百威(CBF)单链抗体(scFv), 从抗CBF scFv氨基酸序列出发, 通过同源模建获得抗体模型, 找出抗体中的活性口袋区域, 进而将小分子药物与抗体进行分子对接, 发现疏水作用和氢键对于抗体亲和力具有重要作用. 进一步对口袋内亲水氨基酸残基HArg40和LHis38进行模拟替换, 再进行分子对接分析, 发现当以亮氨酸为突变氨基酸时, 对接评分最高. 在此基础上, 通过构建突变scFv基因及可溶性表达, 采用ELISA法对进化后的单链抗体(evoscFv)进行了鉴定. 结果表明, evoscFv对CBF的IC₅₀值为18.11 μg/L, 低于野生型抗体的27.25 μg/L, 亲和解离常数K_d为4.06×10^-8 mol/L, 相对亲和力比野生型scFv提高了2.23倍, 说明通过分子对接分析及对抗体活性口袋中氨基酸残基进行替换, 获得了一个亲和力更高的突变体抗体.     

Polymers of 2-methacryloyloxyethyl phosphorylcholine truly work as cell membrane mimic?

We have prepared several types of polymers derived from 2-methacryloyloxyethyl phosphorylcholine (MPC) to evaluate whether polymers of MPC work as cell membrane mimic or not. We firstly applied capturing test of target proteins of 4-carboxybenzenesulfonamide (Sul) or ibuprofen (Ibu) as a probe. As the results, the rather hydrophilic polymers based on MPC were able to suppress non-specific binding proteins as expected. Additionally, some of the MPC based polymeric surface was able to capture greater amount of carbonic anhydrase II than those of other polymers, when Sul was utilized as probe. In contrary, all the polymers having PC groups and Ibu probe were unable to capture Cyclooxygenase-1 (COX-1), its target protein. These results suggested that the position of PC groups realized hydrophilic polymer surface, while MPC based polymer was not able to supply the suitable environment for COX-1 to interact with Ibu.     

Current status and prospects for development of research on the hydrophilicity of disperse systems

The current status of research on the hydrophilicity of disperse systems was discussed. The criterial values of the surface pressure of the adsorbed water film, heat and contact angle of wetting with water which separate the hydrophilic and hydrophobic surfaces were determined. An analysis of the thermodynamic characteristics showed that the boundary layers of water are more ordered near a hydrophilic surface (mica, kaolinite) and less ordered near a hydrophobic surface (organokaolinite, graphite) in comparison to liquid water. The biexponential character of the change in the structural component of the disjoining pressure with the thickness of the water film was demonstrated for disperse materials with a hydrophilic surface (kaolinite). The classification of the forms of water bound by hydrophilic disperse materials was discussed. The changes in the thermodynamic functions of vermiculite in adsorption of water were found with complex adsorptioncalorimetric and dilatometric measurements. This adsorbent undergoes a first-order phase transition during adsorption. The prospects for use of the adsorption-calorimetric method for determining the amount and energy characteristics of hydrophilic sites was demonstrated for ZSM-5 hydrophobic zeolite.Presented to the meeting of the Chemistry Section of the Academy of Sciences of the Ukraine on October 27–29, 1992.Translated from Teoreticheskaya i Eksperimental'naya Khimiya, Vol. 29, No. 2, pp. 100–115, March–April, 1993.     

Superior magnetic monodisperse particles for direct purification of immunoglobulin G under magnetic field

Monodisperse magnetic acrylate based particles (5.0 µm in diameter) containing histidine were synthesized using a modified suspension polymerization method for the purification of immunoglobulin G from human plasma in a magnetically stabilized fluidized bed. N-methacryloyl-(L)-histidine methyl ester (MAH) was used as pseudo-specific ligand/co-monomer. MAH content of the magnetic particles was calculated as 55.3 µmol MAH/g polymer using elemental analysis. Immunoglobulin G binding amount of the magnetic particles decreased with increase of the flow-rate. The maximum immunoglobulin G binding was observed at pH 7.4 (phosphate buffer). Immunoglobulin G binding amount onto the magnetic poly(ethylene glycol dimethacrylate) [mPEGDMA] particles was found to be almost negligible due to the hydrophilic polymer structure. High binding values were obtained from aqueous solutions (1646 mg/g). Higher immunoglobulin G binding was observed when human plasma was used (2169 mg/g). Purity of the separated immunoglobulin G from human plasma was found to be 87%. Magnetic PEGDMAH particles could be used many times without significant loss in protein binding amount.     

Effects of membrane hydrophilicity on the removal of a trihalomethane via micellar-enhanced ultrafiltration process

Micellar-enhanced ultrafiltration (MEUF) process was explored for obtaining pure water from an aqueous solution containing small amount of trihalomethanes (THMs). A homologous series of polyethylene glycol alkylether was used as nonionic surfactant. To understand effects of membrane hydrophilicity on the performance of MEUF process, membranes for the ultrafiltration were prepared from polysulfone blends containing various amount of a hydrophilic copolymer, poly(1-vinylpyrrolidone-co-acrylonitrile) (P(VP-AN)). An increase in the permeate flux was observed with an increase of the membrane hydrophilicity. The performance of MEUF process in removing THM and surfactant was shown to depend on the membrane characteristics, surfactant characteristics, and operating pressure. The rejections of THM and surfactant were increased with increasing hydrophobicity of surfactant and hydrophilicity of membrane. The rejections of THM examined with hydrophilic membranes were increased with increasing operating pressure, while those examined with hydrophobic membranes were decreased with increasing operating pressure. THM included in water could be removed up to 99% via MEUF process. The performance of MEUF examined with hydrophilic membranes could be explained with the rejection of micelles containing THM, while that examined with hydrophobic membranes could be explained with hydrophobic interactions between surfactant and membrane materials.     

Determination of ethoxylated surfactants by functional fragments using group methods of analysis with a common reference compound

A method was proposed for the determination of an unknown nonionic surfactant; the method consists in the combination of two group methods: potentiometric titration with sodium tetraphenylborate (TPB) and UV spectrophotometry with the use of a common reference material. This allows the identification and quantitative determination of a nonionic surfactant without data on its molecular mass and the stoichiometry of its binding with TPB. The proposed method has less limitation than the use of only one method for the determination of the hydrophilic or hydrophobic fragment of the nonionic surfactant, which is used for the elucidation of the number of ethoxy groups in surfactant samples. The main performance characteristics of the procedures were estimated. Examples of the analysis of model and real materials, detergent solutions, and waste waters are given.     

Modified blood-contacting polymers

New blood-contacting materials have been synthesized via graft copolymerization of a mixture of a hydrophilic monomer and the macromonomers hirudin and ovomucoid onto a synthetic polymer, either polyethylene, poly(ethylene terephthalate), or polydimethylsiloxane. It has been shown that the surface modification of the polymer via grafting of the hydrophilic monomer alone reduces the degree of denaturation of adsorbed proteins and that the introduction of hirudin and ovomucoid into the hydrophilic layer leads to a decrease in the amount of platelets adhering to the polymer surface and an increase in the clotting time on the polymer surface.     

Synthesis of branched PEG brushes hybrid hydrophilic magnetic nanoparticles for the selective enrichment of N-linked glycopeptides

Hybrid Fe(3)O(4)@SiO(2)@PEG-Maltose MNPs were synthesized by SI-ATRP of branched PEG brushes on the surface and subsequent functionalization with hydrophilic maltose group, and the multifunctional materials were utilized for selective enrichment of N-linked glycopeptides from biological samples with high specifity, high sensitivity, and large binding capacity.     

Gd(III)[15-metallacrown-5] recognition of chiral α-amino acid analogues

Chiral Ln(III)[15-metallacrown-5] complexes with phenyl side chains have been shown to encapsulate aromatic carboxylates reversibly in their hydrophobic cavities. Given the importance of selective guest binding for applications of supramolecular containers in synthesis, separations, and materials design, the affinity of Gd(III)[15-metallacrown(Cu(II), L-pheHA)-5] hosts for a series of chiral carboxylate guests with varying substitutions on the α-carbon (phenylalanine, N-acetyl-phenylalanine, phenyllactate, mandelate, methoxyphenylacetate) has been investigated. Differential binding of S- and R-phenylalanine was revealed by X-ray crystallography, as the S-enantiomer exclusively forms associative hydrogen bonds with oxygen atoms in the metallacrown ring. Selective guest binding in solution was assessed with isothermal titration calorimetry, which measures the sequential guest binding in the hydrophobic cavity first and the hydrophilic face of the host, and a cyclic voltammetry assay, which quantifies guest binding strength in the hydrophobic cavity of the host exclusively. In solution, the Gd(III)[15-metallacrown(Cu(II), L-pheHA)-5] hydrophobic cavity exhibits modest chiral selectivity for enantiomers of phenylalanine (K(S)/K(R) = 2.4) and mandelate (K(S)/K(R) = 1.22). Weak binding constants of ～100 M(-1) were measured for neutral and -1 charged carboxylates with hydrophilic functional groups (ammonium, N-acetyl, methyl ether). Weaker binding relative to the unsubstituted guests is attributed to unfavorable interactions between the hydrophilic functionalities of the guest and the hydrophobic cavity of the host. In contrast, binding constants greater than 2000 M(-1) were measured for α-hydroxy analogues phenyllactate and mandelate. The significantly increased affinity likely arises from the guests being bound as a -2 anion upon metal-assisted deprotonation in the Gd(III)[15-metallacrown(Cu(II), l-pheHA)-5] cavity. It is established that guest binding affinity in the hydrophobic cavity of the host follows the general trend of neutral zwitterion < monoanion < dianion, with hydrophilic functional groups decreasing the binding affinity. These results have broad implications for the development of metallacrowns as supramolecular catalysts or in chiral separations.     

New restricted access materials combined to molecularly imprinted polymers for selective recognition/release in water media

A new restricted access materials combined to molecularly imprinted polymers (RAM-MIP) with hydrophilic external layer were prepared and their applicability in p-acetaminophenol (AMP) selective recognition and release was evaluated. Glycidilmethacrylate (GMA) was used as pro-hydrophilic co-monomer, and GMA epoxide ring opening with perchloric acid was performed for the hydrophilic modification of polymeric surface. Morphological and hydrophilic properties by scanning electron microscopy and water content measurement were determined and recognition and selectivity properties of RAM-MIP were compared to the unmodified MIP. With RAM-MIP, both hydrophobic non-specific interactions between template or its analogues and polymeric matrices, and bovine serum albumin absorption were drastically reduced.     

聚乙二醇化壳聚糖的制备及其应用研究进展

聚乙二醇(PEG)是一种具有无毒、亲油亲水、高生物相容和无免疫原性等特点的化合物。将聚乙二醇结构引入壳聚糖(CTS)糖链中得到的聚乙二醇化壳聚糖,不但保持了CTS的天然性和优良生物降解等特性,还具有更好的水溶性和对有机化合物的结合能力。通过对CTS进行聚乙二醇化改性,可进一拓展其应用领域。本文结合近20年国内外PEG改性CTS的研究特点,围绕PEG改性CTS的制备及其在药物负载与控制释放、组织工程、抗菌材料、生物活性物传递和环境保护等领域的应用进行了总结,并展望其未来的发展趋势。     

Interface engineering of synthetic pores: towards hypersensitive biosensors

Hydrophilic anchoring is introduced as a promising strategy to constructively control the various interactions of synthetic pore sensors with the surrounding biphasic environment. Artificial rigid-rod beta barrels are selected as classical synthetic multifunctional pores and random-coil tetralysines are attached as hydrophilic anchors. The synthesis of this advanced pore is accomplished in 32 steps from commercially available starting materials. With regard to pore activity as such, the key impact of hydrophilic anchoring is a change from a Hill coefficient n<1 to n=4. This change confirms successful suppression of the competing self-assembly with precipitation from the aqueous phase as the origin of the accomplished increase in pore activity. The hydrophilic anchors do not interfere with the blockage of the synthetic pore sensors by anionic analytes. In the case of stoichiometric binding of blockers (K(D)=EC(50) of the pore; EC(50)=concentration needed to observe 50 % pore activity), however, the increase in pore activity achieved by hydrophilic anchoring results in improved pore blockage under high dilution conditions. Controls confirm that this increase does not occur with analytes that do not exhibit stoichiometric binding (K(D)>EC(50)). These results not only reveal stoichiometric binding as the expected origin of the sensitivity limit of synthetic pore sensors, they also provide promising solutions for this problem. The combination of hydrophilic anchoring with targeted pore formation emerges as a particularly promising strategy to further reduce effective pore concentrations. The scope and limitations of this approach are exemplified with pertinent analyte pairs that are essential for the sensing of sucrose, lactose, acetate, and glutamate with synthetic pores in samples from the supermarket.     

Synthesis of hydrophilic microspheres with LCST close to body temperature for controlled dual‐sensitive drug release

Novel stimuli‐responsive hydrophilic microspheres were prepared by free radical polymerization of hydroxyethyl methacrylate (HEMA) and methacrylic acid (MA), as hydrophilic monomers, and N‐isopropylacrylamide (NIPAAm) and N,N′‐ethylenebisacrylamide (EBA), as thermo‐sensitive monomer and crosslinker, respectively. Hydrophilic comonomers were introduced in the macromolecular network to synthesize materials with tunable thermal behavior. In addition, by introducing in the polymerization feed both a hydrophilic and a pH‐sensitive monomer, such as MA, dual stimuli‐responsive (pH and temperature) hydrogels were synthesized. The incorporation of monomers in the network was confirmed by infrared spectroscopy, while the network density and the shape of hydrogels was found to strictly depend on the concentration of monomers in the polymerization feed. Thermal analyses showed negative thermo‐responsive behavior with pronounced water affinity of microspheres at a temperature lower than lower critical solution temperature (LCST). In our experiment, the LCST values of the hydrogels were in the range 34.6–37.5°C, close to the body temperature, and the amount of hydrophilic moieties in the polymeric network allows to collect shrinking/swelling transition temperatures higher than the LCST of NIPAAm homopolymers. In order to test the preformed materials as drug carriers, diclofenac diethylammonium salt (DDA) was chosen and drug entrapment percent was determined. Drug release profiles, in media at different temperature and pH, depend on hydrogels crosslinking degree and drug–bead interactions. By using semi‐empirical equations, the release mechanism was extensively studied and the diffusional contribute was evaluated. Copyright © 2010 John Wiley & Sons, Ltd.     

Immobilization of binding proteins on nonporous supports

Four different nonporous particulate materials, nylon, polystyrene, soda-lime silicate glass, and fused silica glass, have been evaluated for their appropriateness as immobilization supports for immunoglobulins. A method of protein quantitation that is usually applied to solutions, the bicinchoninic acid (BCA) assay, was used successfully to directly measure ng amounts of protein immobilized on the supports. Two proteins, a monoclonal antibody to theophylline and the biotin binding protein avidin, were studied. Radioactive theophylline and radioactive biotin were used to measure the activity of the immobilized protein. Ligand binding capacity per mm2 of support was measured as a function of amount of protein immobilized. By measuring both the amount of protein immobilized and its ligand binding capacity, we have determined that antitheophylline antibody adsorbed on polystyrene balls loses almost 90% of its binding activity after 65 h, although little protein is lost from the balls over this time. Avidin retains nearly full activity for biotin on polystyrene. The binding activity of biotinyl-antibody conjugate immobilized on avidin-adsorbed polystyrene is stable, even when stored for over 22 wk. Antibody covalently immobilized on soda-lime silicate glass beads retains its binding activity over long-term storage, although only 0.1 mol of 3H-theophylline bind per mol of immobilized antibody. Using fused silica glass particles as the solid support, the same antibody binds approx 0.6 mol of ligand per mol of immobilized antibody protein. The structural "softness" of the immunoglobulin requires that interaction with the surface be prevented in order to maintain activity.     

Hydrophilic adamantane derivatives engineered β-cyclodextrin-based self-assembly materials for highly efficient enrichment of glycopeptides

β-Cyclodextrin (β-CD) based materials have attracted great attention in the separation of hydrophilic glycopeptides due to the abundant hydroxyl groups in its exterior. However, the current materials based on β-CD generally has complex synthesis process and harsh experimental conditions, on the other hand, the interior cavity of β-CD is hydrophobic and is harmful to capture glycopeptides. Herein, a novel hydrophilic material based on β-CD was engineered via a self-assembly process utilizing l-cysteine (l-Cys) or glutathione (GSH) derived adamantane for highly efficient glycopeptide enrichment. It is the first attempt to make use of the hydrophobic interior cavity of β-CD for hydrophilic glycopeptide capture. Taking advantages of strong hydrophilicity and superparamagnetism, the as-prepared materials possess low detection limit, high selectively, and excellent reusability when employed to glycopeptide enrichment. In addition, the feasibility of the hydrophilic material based on β-CD was verified by enriching glycopeptides from human serum and saliva samples. This study provides a heuristic strategy for the application of β-CD-based self-assembly materials in the enrichment of glycopeptides. Importantly, this strategy certified a possible that the change of glycopeptide enrichment sites through host-guest interaction between β-CD and adamantane derivatives with different functional groups.     

Mitigation of Inflammatory Immune Responses with Hydrophilic Nanoparticles

While hydrophobic nanoparticles (NPs) have been long recognized to boost the immune activation, whether hydrophilic NPs modulate an immune system challenged by immune stimulators and how their hydrophilic properties may affect the immune response is still unclear. To answer this question, three polymers, poly(ethylene glycol) (PEG), poly(sulfobetaine) (PSB) and poly(carboxybetaine) (PCB), which are commonly considered hydrophilic, are studied in this work. For comparison, nanogels with uniform size and homogeneous surface functionalities were made from these polymers. Peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) and an LPS‐induced lung inflammation murine model were used to investigate the influence of nanogels on the immune system. Results show that the treatment of hydrophilic nanogels attenuated the immune responses elicited by LPS both in vitro and in vivo. Moreover, we found that PCB nanogels, which have the strongest hydration and the lowest non‐specific protein binding, manifested the best performance in alleviating the immune activation, followed by PSB and PEG nanogels. This reveals that the immunomodulatory effect of hydrophilic materials is closely related to their hydration characteristics and their ability to resist non‐specific binding in complex media.     

Self-assembly in aqueous bile salt solutions

The salts of bile acids (“bile salts”) self-assemble in aqueous solution, similar to classical amphiphiles. The micellization is not only driven by the hydrophobic effect, but also hydrogen binding. Moreover, instead of a small, hydrophilic head and a flexible, hydrophobic tail, bile salts are rigid, almost flat molecules with weakly separated hydrophobic and hydrophilic faces. This results in a complex self-assembly behaviour with very distinct aggregate properties. Some characteristics resemble the behaviour of classical amphiphiles, while others are very different and reminiscent of other classes of molecules, for example low-molecular weight gelators or chromonic materials. We review the peculiar properties of bile salt aggregates, concentrating on general trends rather than specific values and comparing them to classical amphiphiles.     

A study of the ageing of the gel structure in a nonionic O/W cream by X-ray diffraction,differential scanning calorimetry and spin-lattice relaxation measurements

The time dependent changes of the lamellar gel structure in a nonionic O/W cream were studied. It appeared that the changes were connected with alterations in the hydrophilic layers of this lamellar gel structure. The structure of the hydrocarbon layers did not change. The alterations were induced by an increasing hydration of the surfactant molecules on cooling from the preparation temperature to room temperature. Ageing of the cream involves a decrease of the thickness of the hydrophilic layers and a change of the distribution of the surfactant molecules, resulting in, among other things, a decrease of the release rate of a hydrophilic drug. Ageing of the cream can be prevented by using the appropriate amount of starting materials or by the use of polymerizable surfactants. In the former case a cream, from which a drug is slowly released, is obtained. On the other hand, creams containing polymerized surfactants can release drugs at a relatively high rate.     

Thermal Stability of Silica-polyorganosiloxane Systems

Thermal stability of silica-polyvinylsiloxane systems with different ratio of hydrophilic and hydrophobic components and its forms with adsorbed dyes were studied using thermochemical method. It was shown that the maximum of endoeffect relating to dehydroxylation of sorbents surface is observed at 325°C. The maximum temperature of the second endoeffect depends on the organosilica sorbents composition and increases with a decrease of hydrophilic and hydrophobic sites ratio. It was found that the values of the summary thermal effect lowers upon the raise of the quantity of silanol groups. It was shown that thermal stability of composition materials is higher in comparison with initial organosilica sorbents. This revised version was published online in August 2006 with corrections to the Cover Date.