Microencapsulation of bovine hemoglobin with high bio-activity and high entrapment efficiency using a W/O/W double emulsion technique

This study focused on the influences of solvent removal method and wall polymer composition on microspheres characteristics in W/O/W double emulsion procedure. Monomethoxypoly(ethylene glycol)-b-poly- -lactide (PELA) microspheres containing bovine hemoglobin (BHb, a model protein) were prepared by four solvent removal methods, including solvent-evaporation at atmosphere, at reduced pressure, solvent-extraction and solvent-diffusion methods, where the last method used ethyl acetate (EA) as organic solvent and the others used methylene chloride (MC). The bio-activity of encapsulated BHb, encapsulation efficiency, particle size and surface morphology of microspheres were evaluated in relation to the influences of solvent removal method and PELA composition. BHb encapsulated by the W/O/W double emulsion–solvent diffusion method with EA as organic solvent displayed a bio-activity near to that of native BHb. The efficiency of BHb entrapment achieved by this method was much higher than those by other methods (ca. 90% versus 30%). When using this process, the copolymers with MPEG 2000 block (molecular weight of PEG block: 2000 g/mol) yielded much higher efficiencies of BHb entrapment than those with MPEG 5000 block (90% versus 36%). Copolymer composition had less impact on microsphere size, but had a pronounced effect on surface morphology of microspheres. This study suggests that the W/O/W double emulsion–solvent diffusion method with EA as organic solvent is an effective process to prepare microspheres containing therapeutic proteins, and that the PELA copolymers containing MPEG 2000 block are promising wall material for biodegradable microsphere protein delivery system.     

Magnetic Silica‐Coated Sub‐Microspheres with Immobilized Metal Ions for the Selective Removal of Bovine Hemoglobin from Bovine Blood

Magnetic silica‐coated magnetite (Fe₃O₄) sub‐microspheres with immobilized metal‐affinity ligands are prepared for protein adsorption. First, magnetite sub‐microspheres were synthesized by a hydrothermal method. Then silica was coated on the surface of Fe₃O₄ particles using a sol–gel method to obtain magnetic silica sub‐microspheres with core‐shell morphology. Next, the trichloro(4‐chloromethylphenyl) silane was immobilized on them, reacted with iminodiacetic acid (IDA), and charged with Cu²⁺. The obtained magnetic silica sub‐microspheres with immobilized Cu²⁺ were applied for the absorption of bovine hemoglobin (BHb) and the removal of BHb from bovine blood. The size, morphology, and magnetic properties of the resulting magnetic micro(nano) spheres were investigated by using scanning microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD), and a vibrating sample magnetometer (VSM). The measurements showed that the magnetic sub‐microspheres are spherical in shape, very uniform in size with a core‐shell, and are almost superparamagnetic. The saturation magnetization of silica‐coated magnetite (Fe₃O₄) sub‐microspheres reached about 33 emu g^?1. Protein adsorption results showed that the sub‐microspheres had a high adsorption capacity for BHb (418.6 mg g^?1), low nonspecific adsorption, and good removal of BHb from bovine blood. This opens a novel route for future applications in removing abundant proteins in proteomic analysis.     

运用离子选择性电极研究氟离子与蛋白质的相互作用

摘要 在醋酸-醋酸钠缓冲溶液中（pH = 5.68）, 运用氟离子选择性电极研究了288 K～308 K时F–与牛血清蛋白（BSA）、牛血红蛋白（BHb）和卵清蛋白(OVA)的相互作用。F–和三种蛋白质相互作用体系的实验数据通过Klotz方程进行处理,得到了很好的线性关系,根据拟合的直线得到了F–在三种蛋白分子上的束缚位点数以及相应的束缚常数。结果表明,F–在BSA和BHb分子上的束缚位点数随着温度的升高而增大,而在OVA分子上的束缚位点数随着温度的升高而减小。另外,F–与BSA和BHb作用的束缚常数随着温度的升高,先减小后增大;而与OVA的束缚常数则表现出相反的规律,即先增大后减小。根据蛋白质分子二级结构的差别,对上述现象进行了合理的解释。运用热力学方程计算出了不同温度下每个束缚过程的热力学函数（△G,△H,△S）,说明F–和蛋白质分子之间主要依靠静电作用力相结合。     

Analysis of mass transport models for protein adsorption to cation exchanger by visualization with confocal laser scanning microscopy

The mass transfer of bovine serum albumin (BSA) to a cation exchanger, SP Sepharose FF, has been studied by finite batch adsorption experiments. The uptake curve was simulated with three mass transport models (i.e., effective pore diffusion model, surface diffusion model and Maxwell-Stefan model) incorporating the particle size distribution of the adsorbent particles. All the three models can simulate the uptake curves reasonably well. However, how well these models could simulate the real concentration profile within the adsorbent particle cannot be verified by the fitness of the models to the uptake curve. Thus, confocal laser scanning microscopy (CLSM) was used to visualize protein uptake to the porous adsorbent particles during the batch experiments. Using a fluorescent dye-labeled bovine serum albumin (BSA) for the dynamic adsorption experiments, the radial concentration profiles of the labeled BSA molecules into individual adsorbent particles at different times were obtained from the CLSM images. The protein distribution profiles within various particle diameters at different time were compared with the radial protein distributions predicted from the models. It reveals that surface diffusion model describes the intraparticle protein concentration profiles better than the other two models.     

Encapsulation and release of biomolecules from Ca-P/PHBV nanocomposite microspheres and three-dimensional scaffolds fabricated by selective laser sintering

This study focused on the fabrication of calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite scaffolds loaded with biomolecules using the selective laser sintering (SLS) technique and their evaluation. Ca-P/PHBV nanocomposite microspheres loaded with bovine serum albumin (BSA) as the model protein were fabricated using the double emulsion solvent evaporation method. The encapsulation efficiency of BSA in PHBV polymer microspheres and Ca-P/PHBV nanocomposite microspheres were 18.06 ± 0.86% and 24.51 ± 0.60%, respectively. The BSA loaded Ca-P/PHBV nanocomposite microspheres were successfully produced into three-dimensional porous scaffolds with good dimensional accuracy using the SLS technique. The nanocomposite microspheres served as protective carriers and maintained the bioactivity of BSA during SLS. The effects of SLS parameters such as laser power and scan spacing on the encapsulation efficiency of BSA in the scaffolds and in vitro BSA release were studied. An initial burst release was observed, which was followed by a slow release of BSA. After 28-day release, The PHBV matrix was slightly degraded after 28-day in vitro release study. It was shown that nanocomposite scaffolds with controlled architecture obtained via SLS could be incorporated with biomolecules, enhancing them with more functions for bone tissue engineering application or making them suitable for localized delivery of therapeutics.     

Study of the interaction between terazosin and serum albumin: Synchronous fluorescence determination of terazosin

The interactions between terazosin and bovine serum albumin (BSA) were studied by spectrofluorimetry. The binding constants of terazosin with BSA were measured at different temperatures. The effects of various metal ions on the binding constants of terazosin with BSA were also studied. The optimum conditions of synchronous fluorometric determination of terazosin were studied and the method was successfully applied to the determination of terazosin added to serum and urine samples (3σ detection limit 0.21 mg l⁻¹).     

分子光谱法研究铝酞菁与牛血红蛋白的相互作用

利用紫外可见吸收光谱和荧光光谱研究了在生理pH条件下铝酞菁与牛血红蛋白(BHb)的相互作用.实验结果表明:铝酞菁分子与BHb发生反应生成基态复合物,导致BHb内源荧光的猝灭,该猝灭属于静态猝灭.测定了不同温度下该反应的表观结合常数、结合位点数及结合热力学参数,热力学参数的变化表明铝酞菁与BHb之间以静电和疏水作用力为主;根据Frster能量转移理论,测得供体与受体间结合距离r和能量转移效率E;并用同步荧光光谱法探讨了铝酞菁对BHb构象的影响.     

Interaction Between <Emphasis Type="Italic">Trans</Emphasis>-resveratrol and Serum Albumin in Aqueous Solution

The interaction between trans-resveratrol (TR) with bovine serum albumin (BSA) in aqueous solution was investigated by means of fluorescence, synchronous fluorescence and infrared spectroscopy. The fluorescence of BSA can be quenched remarkably by TR in aqueous solution. A notable red-shift of the maximum emission of BSA from 340 to 353 nm together with appearance of an isoemissive point at 395 nm were observed. The results indicate that TR binds to BSA, forming a TR–BSA complex. The TR–BSA binding distance was determined to be less than 7 nm, suggesting that energy transfer from BSA to TR may occur. The interaction process is spontaneous. Based on the obtained thermodynamic parameters, electrostatic forces may play a major role in this process. Both synchronous fluorescence and FT-IR spectra confirmed the interaction, and indicate the conformational changes of BSA.     

Organic–inorganic hybrid silica as supporting matrices for selective recognition of bovine hemoglobin via covalent immobilization

The synthesis of poly‐aminophenylboronic acid (APBA) imprinted hybrid silica‐based polymers for selective recognition of bovine hemoglobin (BHb) was described, where the mesoporous hybrid silica supporting matrices were prepared by a mild sol–gel process with tetraethoxysilane and 3‐aminopropyltriethoxysilane as two precursors. Covalent immobilization of BHb was adopted in order to create homogeneous recognition sites. After removal of the template, the resulting imprinted polymers showed high binding affinity toward BHb and the imprinting factor (α) reached 2.12. The specificity of the BHb recognition was evaluated with competitive experiments, indicating the imprinted polymers have a higher selectivity for the template BHb. The easy preparation protocol and good protein recognition properties made the approach an attractive solution to depletion of high‐abundance protein from bovine blood.     

包埋于PLGA膜中的BSA海藻酸钙微球的制备及其释放行为

在组织工程中，为了促进和调节细胞在细胞支架上的增殖和分化，一些特殊的生物活性分子(生长因子(Growth Factor，简称GF))，必须引入支架．这些生长因子是一类具有诱导和刺激细胞增殖、维持细胞存活等生物效应的多肽蛋白类物质，因此，在组织工程研究中，生长因子是三个重要因素之一，     

Preparation of Functional Fluorescent Microspheres Used for HTS and Their Interaction with Biomolecules

There is considerable interest in protein adsorption onto microspheres because of its importance in a wide range of biomedical applications, such as artificial tissues and organs, drug delivery systems, biosensors, solid-phase immunoassays, immunomagnetic cell separation and immobilized enzymes or catalyst. It has been well known that the interaction between proteins and microspheres plays important roles in this process. Major interaction involved in the adsorption can be classified as electrostatic, hydrophobic and hydrogen-bonding. Indeed, adsorption of proteins onto microspheres is a complex process and often can involve many dynamic steps, from the initial attachment of the protein on the surface of microspheres to the equilibrium. Also the conformation of proteins probably occurs to a certain degree of deformation or structural change due to the large area of contact. Recently, much interest has been shown in sulfonated microspheres, since sulfonate-group itself is one of components in bio-bodies, as well as is sensitive to the change of pH or ionic strength. Indeed, so far, scanty investigations have been performed in the full range. Also few researches have involved the data on adsorption rate and the maximum amount of protein adsorbed, or the reversibility of the process and conformational change of protein adsorbed as well.In present study, BSA (bovine serum albumin) was chosen as the model protein and sulfonated PMMA [poly(methyl methacrylate)] microspheres as the matrix to investigate the adsorption process.The purpose is to show some information especially the intrinsic information involved by the adsorption process Adsorption of BSA onto sulfonated microspheres (MS) has been investigated as a function of time, protein concentration and pH. The adsorption appears to be a reversible process and the presence of sulfonate groups can play important roles in the adsorption process, so as to increase the amount of protein adsorbed and influences the interaction of BSA molecules. Fig. 1 also shows that the reciprocation between unadsorbed and adsorbed BSA or rearrangement of adsorbed BSA molecules does not produce visible change in the properties of the adsorbed protein. Close to the isoelectric point of BSA (pI 4.7), the amount of protein adsorbed exhibits a maximum. A higher or lower pH results in the significant decrease of the adsorption amount. This is related to the dependence of BSA conformations at different pH conditions.     

Dynamic Behaviors and Morphology Change of Anionic Phospholipid DPPG Monolayer Caused by Bovine Serum Albumin at Air-Water Interface

The interaction between bovine serum albumin (BSA) and the anionic 1.2-dipalmitoyl-snglycero- 3-(phospho-rac-(1-glycerol)) (sodium salt) (DPPG) phospholipid at different subphase pH values was investigated at air-water interface through surface pressure measurements and atomic force microscopy (AFM) observation. By analyzing surface pressure-mean molecular area (π-A) isotherms, the limiting molecular area in the closed packing state-the concentration of BSA (A_lim-[BSA]) curves, the compressibility coefficient-surface pressure (C_S^-1-π) curves and the difference value of mean molecular area-the concentration of BSA (ΔA-[BSA]) curves, we obtained that the mean molecular area of DPPG monolayer became much larger when the concentration of BSA in the subphase increased at pH=3 and 5. But the isotherms had no significant change at different amount of BSA at pH=10. In addition, the amount of BSA molecules adsorbed onto the lipid monolayer reached a threshold value when [BSA]>5×10^-8 mol/L for all pHs. From the surface pressure-time (π-t) data, we obtained that desorption and adsorption processes occurred at pH=3, however, there was only desorption process occurring at pH=5 and 10. These results showed that the interaction mechanism between DPPG and BSA molecules was affected by the pH of subphase. BSA molecules were adsorbed onto the DPPG monolayers mainly through the hydrophobic interaction at pH=3 and 5, and the strength of hydrophobic interaction at pH=3 was stronger than the case of pH=5. At pH=10, a weaker hydrophobic interaction and a stronger electrostatic repulsion existed between DPPG and BSA molecules. AFM images revealed that the pH of subphase and [BSA] could affect the morphology features of the monolayers, which was consistent with these curves. The study provides an important experimental basis and theoretical support to understand the interaction between lipid and BSA at the air-water interface.     

BSA‐imprinted synthetic receptor for reversible template recognition

A novel approach to the manufacturing of protein‐responsive imprints on a home‐made chitosan substrate was established together with m‐aminophenylboronic acid (APBA) as a functional monomer. The produced polymers were characterized using both (1) equilibrium adsorption assays and (2) high performance liquid chromatography analysis. Results confirmed that the synthesized BSA‐MIP (molecularly imprinted polymer) has a high affinity towards its template compared to the determined control proteins. The produced BSA‐MIP featured largely in its good adsorption reversibility, especially in competitive binding assays, which is of great biological significance in separations. Non‐specific binding was reduced to almost zero in a BSA/BHb competitive binding event. An excellent HPLC profile of template recognition was found for BSA‐MIP, even under harsh mobile phase conditions. In the present work, the adopted trapped‐template‐release method permits recovery of bound BSA [1]. The strategy of making an artificial protein‐receptor with high adsorption affinity and reversibility is promising in on‐line isolation of target protein from complicated biological environments.     

Adsorption of bovine serum albumin to acrylamide–itaconic acid hydrogels

In this study, acrylamide–itaconic acid hydrogels containing different amounts of itaconic acid prepared by irradiating with γ radiation are discussed. They have been used in experiments of swelling, diffusion and bovine serum albumin (BSA) adsorption. Maximum and minimum swellings were observed with water (1520%) and BSA (890%), respectively. Diffusion of water, NaCl and BSA within hydrogels were found to be non-Fickian in character. In the experiments of BSA adsorption, type III adsorption was found. The hydrogel prepared with 60 mg itaconic acid and irradiated at 2.00 kGy was found to be the best adsorption system for BSA. The adsorption capacity of acrylamide–itaconic acid hydrogel was found to exceed that of acrylamide hydrogel by more than 80–100%.     

免疫亲和富集-荧光光谱法检测牛血清白蛋白的研究

本文通过戊二醛法合成免疫磁球(IMMs),采用荧光电镜、透射电镜和振动样品磁强计对其进行表征。合成的IMMs为单分散的球形结构,粒径约为510 nm,具有超顺磁性(饱和磁化强度为40.44 emu/g)。基于此,以牛血清白蛋白(BSA)作为蛋白质模型,建立了免疫亲和富集-荧光光谱法用于目标蛋白质的分离与检测。考察并优化了影响免疫亲和富集的因素。在最佳实验条件下,IMMs对目标蛋白质的最大吸附容量为107.7μg/g,可重复使用10次以上。将该方法应用于人血清基质中BSA的测定,加标回收率不低于89.3%,由基质辅助激光解吸电离飞行时间质谱仪测得分离后的BSA纯度较高。结果表明,该方法适用于人血清样品中蛋白质的选择性分离与检测。     

Investigation on Adsorption Dynamics of Protein/Tween‐20 Mixture at the Surface of Solution by Surface Pressure Measurement

The dynamic surface tension (γ_t) and apparent diffusion coefficient (D) of nonionic surfactant Tween‐20 in the presence and absence of bovine serum albumin (BSA) have been investigated via the measurements of surface pressure (π) at different time (t). The curves of γ_t～t are obtained from π～t isotherms. Results show that the γ_t～t relationships of Tween‐20 solution with or without BSA accord with the Ward‐Tordai equation in the region of initial adsorption. D value obtained from the γ_t～t^1/2 curves shows that the diffusion of Tween‐20 slows down with the increase of the concentration of Tween‐20 (c _Tween‐20). And D value of Tween‐20 in the presence of BSA is almost the same as that of the system without BSA when c _Tween‐20 is constant, suggesting that the interaction between Tween‐20 and BSA is weak.     

Protein Templated Au-CuO Bimetallic Nanoclusters toward Neutral Glucose Sensing

In this study, the application of bovine serum albumin (BSA) as a carrier to glucose-sensitive materials for the detection of glucose was proposed. Au-CuO bimetallic nanoclusters (Au-CuO/BSA) were prepared using BSA as a template, the new sensing material (Au-CuO/BSA/MWCNTs) was synthesized by mixing with multi-walled carbon nanotubes (MWCNT) and applied to non-enzymatic electrochemical sensors to detect glucose stably and effectively under neutral condition. The scanning electron microscopy was used to investigate the morphology of the synthesized nanocomposite. The electrochemical properties of the sensor were studied by cyclic voltammetry. Glucose detection experiments show that Au-CuO/BSA/MWCNTs/Au electrode has good glucose detection ability, stability, accuracy, repeatability, and high selectivity in neutral environment. Unlike existing glucose-sensitive materials, due to the use of BSA, the composite material is firmly fixed to the electrode surface without a Nafion solution, which reduces the current blocking effect on the modified electrode. The composite materials can be effectively preserved for extremely long periods, higher than 80% activity is maintained at room temperature in a closed environment for 3 to 4 months, due to the special effects of BSA. In addition, the feasibility of using BSA in glucose-sensitive materials is confirmed.     

牛血清蛋白对二硬脂酰基磷脂酰乙醇胺单层膜结构的影响

利用Langmuir-Blodgett(LB)技术结合原子力显微镜(AFM),研究了牛血清蛋白(BSA)在气/液界面上对二硬脂酰基磷脂酰乙醇胺(DSPE)单层膜结构的影响.通过改变亚相的pH值和BSA浓度,获得了不同条件下DSPE单层膜的等温线、吸附曲线和压缩循环曲线等.实验结果表明,亚相中BSA的存在对DSPE单层膜的压缩性、稳定性以及相变行为产生了较大的影响.吸附动力学结果表明,DSPE单层膜对BSA分子的吸附量存在一定的阈值,且该阈值的大小与亚相pH值相关.通过分析实验数据可知,当亚相pH=3时,BSA的疏水残基几乎全部暴露在外面,2种分子之间的相互作用最强;而pH=7时,BSA仅有少量的疏水残基暴露在外面,2种分子之间的相互作用最弱.原子力显微镜观测到的单层膜形态变化特点与曲线分析结果一致.该研究为了解牛血清蛋白与磷脂分子之间的相互作用机理提供了重要的实验基础和理论依据.     

Spectroscopic Study on the Interaction between Bovine Serum Albumin and Tween‐20

The interaction between nonionic surfactant Tween-20 and bovine serum albumin (BSA) is studied in Tris‐HCl buffer solution by spectroscopic methods. The intrinsic fluorescence of BSA is quenched by the addition of Tween‐20. The UV‐visible absorption spectra and the synchronous fluorescence spectra show that the addition of Tween‐20 changes the polarity of the environment around tryptophan (trp) residues of BSA. The fraction of trp residues on the surface of BSA with and without Tween- 20 is calculated via I^? quenching experiments.     

磁性壳聚糖微球对牛血清白蛋白的吸附性能

在微乳液体系中, 以戊二醛为交联剂制备了磁性壳聚糖纳米粒子(Fe₂O₃-CS). 采用透射电子显微镜(TEM)、红外光谱(IR)、振动样品磁强计(VSM)等手段对纳米粒子进行表征. 结果表明, 纳米粒子的粒径在40 nm左右, 分散性良好, 具有较好的磁响应性能. 以碳二亚胺(EDC)为活化剂, 研究了Fe₂O₃-CS纳米粒子对牛血清白蛋白(BSA)分子的吸附性能, 并使用原子力显微镜(AFM)、紫外分光光度计(UV)进行表征. Fe₂O₃-CS粒子对BSA 的吸附大致符合Langmuir吸附模型, 298 K时饱和吸附量约为250 mg·g^-1, 吸附常数为0.007 L·mg^-1. 将BSA-粒子分散在不同pH的缓冲溶液中, 研究BSA-粒子复合物的稳定性. 用聚丙烯酰氨凝胶电泳(SDS-PAGE)对结果进行表征, 发现在碱性条件下BSA分子能从磁性粒子表面脱附下来.