Regenerated protein fibers. I. Research and development of a novel solvent for silk fibroin

An alcoholic–aqueous solution of LiBr·H₂O is a useful solvent for silk fibroin, if the ratio of alcohol content to water content therein is 1 or more. The mixed solvents consisting of ethanol, water, and LiBr·H₂O showed a higher solubility if they contained a lesser amount of water. They further proved able to easily dissolve more than 20% by weight of silk fibroin. Solvents poorer in water content and richer in LiBr·H₂O showed a higher value of [η]; Huggins' coefficient of said solutions varied within a range 0.5–1.0, the minimum appearing at a water content of about 10% weight. Fibroin molecules remained stable in those solutions. The novel solvents for silk fibroin that have been developed by us are named “MU solvents,” by taking the initials in the names of Matumoto/Uejima. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 1949–1954, 1997     

Salt-catalyzed addition reaction of monoepoxides with philosamia cynthia ricini and bombyx mori silk fibroins

The heterogeneous addition reaction of various monoepoxides with silk fibroins of Philosamia cynthia ricini and Bombyx mori was investigated at 45–75°C by use of aqueous solutions of various salts as padding catalysts. The effects of salt on the epoxide–silk fibroin reactions were attributed mainly to the nucleophilicity of the anions and also to the acidity or the electronegativity of the cations. The effect of the substituent of the epoxide on the add-ons was elucidated by the modified Taft equation, (log W ? log W₀)/σ* = ρ_p + ρ_sE_s/σ*, where W₀ and W are the add-ons for the reaction of a given compound and of its substituted derivatives, σ* and E_s are the polar and the steric substituent constants, ρ_p and ρ_s are the polar and the steric reaction constants, respectively. Histidine, lysine, arginine, tyrosine, serine, and acidic amino acids were found to react. The reactivity difference between Philosamia cynthia ricini and Bombyx mori fibroins towards the epoxide was discussed in the light of the observed phenomena.     

Green Process to Prepare Silk Fibroin/Gelatin Biomaterial Scaffolds

A new all‐aqueous and green process is described to form three‐dimensional porous silk fibroin matrices with control of structural and morphological features. Silk‐based scaffolds are prepared using lyophilization. Gelatin is added to the aqueous silk fibroin solution to change the silk fibroin conformation and silk fibroin–water interactions through adjusting the hydrophilic interactions in silk fibroin–gelatin–water systems to restrain the formation of separate sheet like structures in the material, resulting in a more homogenous structure. Water annealing is used to generate insolubility in the silk fibroin–gelatin scaffold system, thereby avoiding the use of organic solvents such as methanol to lock in the β‐sheet structure. The adjusting of the concentration of gelatin, as well as the concentration of silk fibroin, leads to control of morphological and functional properties of the scaffolds. The scaffolds were homogeneous in terms of interconnected pores, with pore sizes ranging from 100 to 600 µm, depending on the concentration of silk fibroin used in the process. At the same time, the morphology of the scaffolds changed from lamellar sheets to porous structures based on the increase in gelatin content. Compared with salt‐leaching aqueous‐derived scaffolds and hexafluoroisopropanol (HFIP)‐derived scaffolds, these freeze‐dried scaffolds had a lower content of β‐sheet, resulting in more hydrophilic features. Most of gelatin was entrapped in the silk fibroin–gelatin scaffolds, without the burst release in PBS solution. During in vitro cell culture, these silk fibroin–gelatin scaffolds had improved cell‐compatibility than salt‐leaching silk fibroin scaffolds. This new process provides useful silk fibroin‐based scaffold systems for use in tissue engineering. Furthermore, the whole process is green, including all‐aqueous, room temperature and pressure, and without the use of toxic chemicals or solvents, offering new ways to load bioactive drugs or growth factors into the process.

     

Preparation and Characterization of Natural Silk Fibroin Hydrogel for Protein Drug Delivery

In recent years, hydrogels have been widely used as drug carriers, especially in the area of protein delivery. The natural silk fibroin produced from cocoons of the Bombyx mori silkworm possesses excellent biocompatibility, significant bioactivity, and biodegradability. Therefore, silk fibroin-based hydrogels are arousing widespread interest in biomedical research. In this study, a process for extracting natural silk fibroin from raw silk textile yarns was established, and three aqueous solutions of silk fibroin with different molecular weight distributions were successfully prepared by controlling the degumming time. Silk fibroin was dispersed in the aqueous solution as “spherical” aggregate particles, and the smaller particles continuously accumulated into large particles. Finally, a silk fibroin hydrogel network was formed. A rheological analysis showed that as the concentration of the silk fibroin hydrogel increased its storage modulus increased significantly. The degradation behavior of silk fibroin hydrogel in different media verified its excellent stability, and the prepared silk fibroin hydrogel had good biocompatibility and an excellent drug-loading capacity. After the protein model drug BSA was loaded, the cumulative drug release within 12 h reached 80%. We hope that these investigations will promote the potential utilities of silk fibroin hydrogels in clinical medicine.     

Seeking Solvation: Exploring the Role of Protein Hydration in Silk Gelation

The mechanism by which arthropods (e.g., spiders and many insects) can produce silk fibres from an aqueous protein (fibroin) solution has remained elusive, despite much scientific investigation. In this work, we used several techniques to explore the role of a hydration shell bound to the fibroin in native silk feedstock (NSF) from Bombyx mori silkworms. Small angle X-ray and dynamic light scattering (SAXS and DLS) revealed a coil size (radius of gyration or hydrodynamic radius) around 12 nm, providing considerable scope for hydration. Aggregation in dilute aqueous solution was observed above 65 °C, matching the gelation temperature of more concentrated solutions and suggesting that the strength of interaction with the solvent (i.e., water) was the dominant factor. Infrared (IR) spectroscopy indicated decreasing hydration as the temperature was raised, with similar changes in hydration following gelation by freezing or heating. It was found that the solubility of fibroin in water or aqueous salt solutions could be described well by a relatively simple thermodynamic model for the stability of the protein hydration shell, which suggests that the affected water is enthalpically favoured but entropically penalised, due to its reduced (vibrational or translational) dynamics. Moreover, while the majority of this investigation used fibroin from B. mori, comparisons with published work on silk proteins from other silkworms and spiders, globular proteins and peptide model systems suggest that our findings may be of much wider significance.     

Dissolution of Silk Fibroin in N-methylmorpholine-N-oxide and Its Mixtures with Organic Solvents

Preparation of concentrated solutions of natural silk fibroin in N-methylmorpholine-N-oxide and its mixtures with protonic and aprotic solvents was experimentally studied. Solutions with the fibroin concentration of 6 wt % and more were prepared.     

甘油对丝素蛋白膜水溶性和结构的影响

探讨了甘油的加入对丝素蛋白溶解过程的结晶结构及结晶度的影响. 以甘油为添加剂用流延法于室温制备一系列丝素共混膜, 测试了其含水率、溶失率及结构和机械性能. 结果表明, 随着甘油加入量的增加, 丝素蛋白的溶失率逐渐降低; 当甘油/丝素质量分数超过10%时, 共混膜呈现水不溶性. 当甘油加入量较少时, 丝素蛋白呈现少量的Silk Ⅱ结晶, 而Silk Ⅰ结构不明显. 随着甘油含量的不断增加, Silk Ⅰ结晶逐渐增加而Silk Ⅱ结晶逐渐减少. 当甘油加入量达到不溶点(10%)时, 丝素蛋白主要转变为Silk Ⅰ结晶, 而几乎没有Silk Ⅱ结晶. 甘油的加入可使共混膜的柔韧性显著提高, 并促使丝素蛋白结晶度提高以及促使丝素蛋白向Silk Ⅰ结晶转变, 从而降低丝素蛋白膜的水溶性.     

环氧化合物与丝素蛋白化学交联凝胶的结构

对丝素蛋白水溶液与环氧化合物交联剂(PGDE)反应制备的丝素凝胶(CFG)的结构进行了研究.用位相差显微镜观察到CFG具有整体均一的形态结构,而未经PGDE交联的纯丝素凝胶(FG)为颗粒聚集结构.CFG的红外吸收光谱在1104cm^-1处出现PGDE分子中烷醚的吸收峰,而没有出现PGDE两端环醚的特征吸收峰,显示PGDE已经开环交联在丝素蛋白上.氨基酸分析结果显示,酪氨酸、组氨酸和赖氨酸的含量与反应前相比明显减少,PGDE在这些位点与丝素蛋白发生了交联反应.固体¹³CNMR谱观察到酪氨酸羟苯基上的碳发生了化学位移,进一步证实了酪氨酸残基与PGDE发生了化学交联.研究表明,PGDE与丝素蛋白交联形成了分子间交联网络结构,从而使CFG成为较为匀质的凝胶,并有较好的透明度和柔韧性.     

丝素纳米颗粒的制备及应用于L-天冬酰胺酶的固定化

丝素蛋白纤维溶于高浓度中性盐溴化锂溶液或氯化钙-乙醇-水三元溶剂中, 经过透析和纯化可以制成3种液态丝素. SDS-PAGE分析结果表明, 其分子量分布范围明显不同. 应用能与水混溶的有机溶剂如丙酮等可将这种丝素制成丝素纳米颗粒, 用SEM观察到丝素纳米颗粒粒径分布范围为50~120 nm. 以戊二醛为交联剂, 将治疗急性淋巴性白血病常用酶制剂L-天冬酰胺酶共价结合在丝素纳米颗粒上. 酶活性分析结果表明, 由肽链断裂较少的丝素制备的纳米颗粒更适合于酶的生物结合. 酶动力学研究结果表明, 这种固定化酶活性回收率为44%, 热稳定性较游离酶有明显提高, 最适pH值范围加宽为6.0~8.0, 最适反应温度提高10 ℃; 抗胰蛋白酶水解能力明显增强. 结果表明, 丝素纳米颗粒与丝素蛋白膜一样, 是一种酶固定化的良好载体, 在药物缓释系统方面具有潜在的研究和开发价值.     

蚕腺体内和再生丝素蛋白水溶液的性能研究

用偏光显微镜观察了蚕腺体内和再生丝素蛋白水溶液的流动状态和各向异性现象,用乌氏粘度计测试了其流出时间,并用HAKKE流变仪测试了其粘度。结果发现蚕腺体内和再生丝素蛋白水溶液的性质差别非常大,认为丝素蛋白水溶液在蚕腺体内存在一个逐步熟成的过程。随着丝素蛋白水溶液在腺体内的前移,丝素蛋白分子逐渐沿移动方向取向而呈有序态,最终成为粘度非常大的各向异性的凝胶体,而再生丝素蛋白水溶液是粘度非常低的各向同性溶液。这些差异表明,丝素蛋白水溶液在蚕腺体内的熟成过程是蚕能够吐出优良蚕丝的关键步骤,要想制备出高性能的纤维,在“仿生纺丝”之前,首先要“仿生制备纺丝液”。     

光谱法研究pH值对再生桑蚕丝素蛋白在水溶液中结构的影响

通过一系列光谱实验手段研究了再生桑蚕(Bombyx mori)丝素蛋白在水溶液中的构象转变情况. 由于丝素蛋白含有较多带电荷的氨基酸残基, 因此环境pH值对丝素蛋白的结构有着一定的影响: 酸性越强, 丝素蛋白越容易发生从无规线团到β-折叠结构转变; 相对而言, 碱性条件则更有利于丝素蛋白以无规线团结构稳定存在. 特别是当pH在4附近时, 丝素蛋白的无规结构最易发生改变; 而pH为6左右时, 丝素蛋白的结构则较为稳定. 这种变化趋势与沿着成熟蚕腺体中丝素蛋白所处的环境及其状态相当吻合, 由此表明pH值的调节是蚕在生物体中控制其丝素蛋白状态的一个相当重要的手段. 这一结果对人工纺制动物丝条件的调控有着极其重要的现实意义. 同时我们还发现, 在相当宽的pH范围内, 丝素蛋白的二级结构存在着中间体形态, 表明丝素蛋白的变性过程不符合简单的二态机制.     

Novel synthesis of polyethers by the polyaddition of bis(epoxide)s with bis(phosphinate)s

The polyaddition of bisphenol A diglycidyl ether with bis[4‐(P,P‐diphenylphosphinyloxy)phenyl] sulfone catalyzed by quaternary onium salt, such as tetrabutylammonium chloride afforded a new phosphorus‐containing polyether with good solubility in common organic solvents. Having studied various factors affecting the reaction, such as temperature, catalyst concentration, reaction time, etc., an appropriate polyaddition condition was suggested as using 5 mol % of suitable quaternary ammonium or phosphonium salt in polar solvent at 150°C within 25 h in an ampule for producing high molecular weight polymer. A number of polyethers bearing pendent phosphinate ester groups from the polyaddition of certain bis(epoxide)s and bis(phosphinate)s were synthesized under the above condition and characterized by GPC, IR, and NMR. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 1009–1016, 1999     

QUENCHING OF PHOTOEXCITED 4,4'-DICARBOXY-2,2'-BIPYRIDINEBIS(2,2'-BIPYRIDINE)RUTHENIUM(II) BY OXYGEN IN AQUEOUS SOLUTION AND IN SILK FIBROIN MEMBRANE

Abstract— Photoluminescence and quenching of 4,4'-dicarboxy-2,2'-bipyridinebis(2,2'-bipyridine)-ruthenium(II) complex (Ru(DCbpy)(bpy)²₂⁺) in an aqueous solution as well as in a silk fibroin membrane were studied. Emission quenching by oxygen in an aqueous solution showed a linear relationship with respect to oxygen concentration. When the complex was incorporated into a silk fibroin membrane by adsorption from an aqueous solution into a preformed membrane, the photoexcited state of the complex was not quenched by oxygen in an aqueous phase. However, when the complex was incorporated into a silk fibroin membrane by casting a mixture of the complex and silk fibroin, the photoexcited complex was quenched by oxygen in an aqueous phase. In this case the Stern-Volmer plots showed a downward-deviating curve indicating heterogeneity of the probe site. Emission intensity decreased with an increase of the water content in the silk fibroin membrane.     

Structure and solubility of natural silk fibroin

The solubility of silk fibroin in aqueous-salt, aqueous-organic, and organic media is analyzed. Factors affecting the formation of the secondary structural organization of fibroin in solutions and in the solid state after the recovery from solutions are analyzed.     

Silk fibroin solution properties related to assembly and structure

The impact of physiological factors on silk fibroin solution properties was studied. Specifically, the impact of fibroin concentration, protein purity, cation type and concentration, and pH on aqueous solution viscosity, shear behavior, and surface tension were assessed in the context of silk protein assembly. The results demonstrate that in vitro results could be correlated to in vivo processing events during silk spinning. Rheological properties with reference to the amphiphilic block structure of the protein are described, pH dependency of shear response was quantitatively correlated to the predicted pI values of the fibroin protein, and cooperativity among environmental factors such as pH and salts was identified. Stabilization of silk fibroin solution states by calcium was identified as a mode to control shear sensitivity of the fibroin solution. The cooperativities identified suggest tight control of fibroin aqueous solution rheological properties to gain a window of protection against premature crystallization of the fibroin during processing, assuring safe storage, transport, and finally successful fiber spinning.     

Characterization of silk fibroin based films loaded with rutin–β-cyclodextrin inclusion complexes

In this study, cyclodextrin inclusion complexes with rutin were prepared via co-precipitation method. Stability constant and solubility energy of beta-cyclodextrin complex were calculated as 262 M^?1 and 1,737 kJ mol^?1, respectively. Aqueous solubility of rutin was increased with inclusion complex of beta-cyclodextrin. The effect of temperature on both aqueous solubility of free rutin, and its inclusion complex was also studied. Characterization of cyclodextrin complexes were conducted with UV–Vis spectrophotometry, Fourier transform infrared spectroscopy, X-ray diffractometry, differential scanning calorimetry, thermal gravimetric analysis, nuclear magnetic resonance spectroscopy and scanning electron microscopy techniques. Characterization results supported formation of inclusion complexes. Dissolution profiles of rutin, physical mixture and inclusion complex of rutin were observed at 37 °C. Dissolution results proved the effect of cyclodextrin addition on solubility rate of rutin. After loading rutin and its complexes into silk fibroin based films, release tests were performed at 37 °C in neutral pH conditions for 24 h. Most of the rutin were released from silk fibroin films within the first 5 h and the rest of it was released slowly (sustained release). Electron microscope analyses showed that films had homogenous and dense morphologies. These results revealed that silk fibroin is useful for preparing bioactive films loaded with natural compounds and for modifying their release behaviour at physiological conditions.     

Structural changes of silk fibroin membranes induced by immersion in methanol aqueous solutions

Structural changes of native and regenerated silk fibroin membranes were induced by immersion in water-methanol solutions and examined as a function of immersion time and methanol concentration. X-ray diffractometry and infrared spectroscopy data showed that transition from random coil to β-sheet structure occurred favorably when both native and regenerated silk fibroin membranes were immersed in water-methanol solutions, regardless of the different immersion time. Only native silk membrane, treated for 2 min with pure methanol, maintained its original amorphous structure, as demonstrated by differential scanning calorimetric (DSC) curves. The degree of displacement, measured by thermomechanical analysis (TMA), was much greater for regenerated than for native silk fibroin membranes. SDS-PAGE pattern showed that native silk fibroin has a molecular weight of 350, while the regenerated sample is formed by a large number of polypeptides in the range of 200-50 KD. The amount of acidic and basic amino acids decreased slightly in regenerated silk fibroin. Physical properties of silk membranes treated with water-methanol solutions are discussed in terms of membrane structure, treatment conditions, and chemical structure of starting material. © 1994 John Wiley & Sons, Inc.     

Kinetics of 1,3‐dipolar cycloaddition reaction between C,N‐diphenylnitrone and dimethyl fumarate in various solvents and aqueous solutions

Second‐order rate constants and activation parameters of 1,3‐dipolar cycloaddition reaction between C,N‐diphenylnitrone and dimethyl fumarate were obtained in various solvents and aqueous solutions at 65°C. Second‐order rate constants of the reaction in water and ethylene glycol are approximately 33 and 8 times faster than those expected from solvent polarity, respectively. Increase of the reaction rate in aqueous solutions of ethanol is higher than that of propan‐1‐ol. A multiparameter correlation of log k₂ vs Sp and E_T^N in various solvents and aqueous solutions of ethanol shows that solvophobicity and solvent polarity parameter are important factors in occurrence of the reaction. © 2000 John Wiley & Sons, Inc. Int J Chem Kinet 32: 431–434, 2000     

Biomaterial coatings by stepwise deposition of silk fibroin

A completely aqueous, stepwise deposition process with Bombyx mori silk fibroin for the assembly of nanoscale thin film coatings is reported the first time. The focus of this work was to develop an understanding of the control of this deposition process and to characterize the films formed from a physicochemical perspective. The deposition process was monitored by UV spectrophotometry and research quartz crystal microbalance. Both absorbance and film thickness correlated linearly with the number of silk fibroin layers deposited, analogous to multilayered materials fabricated from conventional polyelectrolytes. The polymer adsorption process was stable and reproducible, with control of a single layer thickness ranging from a few to tens of nanometers, determined by the concentrations of silk fibroin, salt concentration in the dipping solution, and method of rinsing. The driving force for the assembly of silk fibroin onto the substrate was primarily hydrophobic interactions, while some electrostatic interactions were also involved. The difference with this approach from traditional polyelectrolyte layer-by-layer techniques is that an intervening drying step is used to control the structure and stability of the self-assembled silk fibroin. The assembled films were stable under physiological conditions and supported human bone marrow stem cell adhesion, growth, and differentiation. This approach offers new options to engineer biomaterial coatings as well as bulk materials with control of both interfacial properties conducive to specific cellular or tissue responses and the potential to entrap and deliver labile molecules or other components due to the all-aqueous process described.     

Improved solubility and stability of trialkylammonium selenocarboxylate in organic solvents for efficient amidation with azides

Trialkylammonium selenocarboxylate, formed in situ from the reaction of diacyl diselenide with piperidine in the presence of diisopropylethylamine, was found to react readily at room temperature with electron-deficient azides to form amides in excellent yields. The trialkylammonium selenocarboxylate salt formed has good solubility and stability in organic solvents. The enhanced stability allowed mild heating to improve the amidation yields with electron-rich azides.