Immobilization of protein on cellulose hydrogel

A technique of immobilizing an enzyme/antibody was developed using cellulose hydrogel prepared from an aqueous alkali-urea solvent. Partial oxidation by sodium periodate activated the cellulose gel for introducing aldehyde groups. Proteins were covalently introduced to cellulose gel by a Schiff base formation between the aldehyde and the amino groups of proteins, and stabilized by a reduction of imines. Coloring reactions confirmed the high activity of the immobilized enzymes. The activity of the immobilized enzymes increased with aldehyde content, but the effect leveled off at a low degree of oxidation, at approximately 8.1 of oxidized glucose/100 glucose unit. The amount of immobilized peroxidase calculated from the activity was 8.0 ng/g for an aldehyde content of 0.18 mmol/g: 14.6 ng/g for both 0.46 mmol/g and 1.04 mmol/g. The same method could be applied to the peroxidase antibody. Thus, various active proteins could be immobilized on cellulose gels by mild and facile processing. Owing to high mechanical and chemical stability of cellulose, this technique and resulting materials are potentially useful in biochemical processing and sensing technologies.     

Hydrogel Films Based on Chitosan and Oxidized Carboxymethylcellulose Optimized for the Controlled Release of Curcumin with Applications in Treating Dermatological Conditions

Cross-linked chitosan (CS) films with aldehyde groups obtained by oxidation of carboxymethyl cellulose (CMC) with NaIO₄ were prepared using different molar ratios between the CHO groups from oxidized carboxymethyl cellulose (CMCOx) and NH₂ groups from CS (from 0.25:1 to 2:1). Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy demonstrated the aldehyde groups’ presence in the CMCOx. The maximum oxidation degree was 22.9%. In the hydrogel, the amino groups’ conversion index value increased when the -CHO/-NH₂ molar ratio, cross-linking temperature, and time increased, while the swelling degree values decreased. The hydrogel films were characterized by scanning electron microscopy (SEM) and FTIR analysis. The curcumin encapsulation efficiency decreases from 56.74% to 16.88% when the cross-linking degree increases. The immobilized curcumin release efficiency (R_Ef%) and skin membrane permeability were evaluated in vitro in two different pH solutions using a Franz diffusion cell, and it was found to decrease when the molar ratio -CH=O/NH₂ increases. The curcumin R_Ef% in the receptor compartment was higher at pH = 7.4 (18%- for the sample with a molar ratio of 0.25:1) than at pH = 5.5 (16.5%). The curcumin absorption in the skin membrane at pH = 5.5 (47%) was more intense than at pH = 7.4 (8.6%). The curcumin-loaded films’ antioxidant activity was improved due to the CS presence.     

基于席夫碱键的可注射糖肽水凝胶的制备及性能

以氧化葡聚糖(ODEX)和聚赖氨酸-聚乙二醇-聚赖氨酸(PLL₂₄-PEG-PLL₂₅)三嵌段聚合物为前驱体, 通过ODEX中的醛基与PLL中的氨基之间的席夫碱键反应, 制备了ODEX/PLL₂₄-PEG-PLL₂₅水凝胶. 研究了其凝胶强度、 降解时间及对阿霉素(DOX)释放量的影响. 结果表明, 随着ODEX中醛基密度的增加, 凝胶强度逐渐增大, 最大强度为3100 Pa. 流变学研究结果表明, 由于ODEX中的醛基与DOX中的氨基存在席夫碱键作用, 导致凝胶强度从2160 Pa降至1730 Pa. 降解实验结果表明, ODEX/PLL₂₄-PEG-PLL₂₅水凝胶具有较长的降解时间, 最长时间达到29 d. 药物释放结果表明, ODEX/PLL₂₄-PEG-PLL₂₅水凝胶具有酶促降解释放药物的性能. 在Elastase溶液中, ODEX/PLL₂₄-PEG-PLL₂₅水凝胶所载DOX累积释放量达到最大值74.35%. 结果表明, ODEX/PLL₂₄-PEG-PLL₂₅水凝胶具有进一步应用于体内局部药物传输的潜力.     

Wood cellulose nanofibrils prepared by TEMPO electro-mediated oxidation

A softwood bleached kraft pulp (SBKP) was subjected to electro-mediated oxidation in water with TEMPO or 4-acetamido-TEMPO without any chlorine-containing oxidant. Solid recovery ratios of water-insoluble fractions of the oxidized SBKPs were more than 80%, and C6-carboxylate contents increased up to approximately 1 mmol g⁻¹ after oxidation for 48 h. Significant amounts of C6-aldehyde groups (0.17–0.38 mmol g⁻¹) were also formed in the oxidized SBKPs. The degree of polymerization decreased from 2,200 to 520 and 1,400 by the oxidation for 48 h with TEMPO at pH 10 and 4-acetamido-TEMPO at pH 6.8, respectively. The original cellulose I crystal structure and crystallinity of SBKP were maintained after the oxidation, indicating that all C6-oxidized groups were selectively formed on crystalline cellulose microfibril surfaces. The oxidized SBKPs with carboxylate contents of more than 0.9 mmol g⁻¹ were convertible to individual cellulose nanofibrils in yields of more than 80% by disintegration in water.     

The Design of Rapid Self-Healing Alginate Hydrogel with Dendritic Crosslinking Network

Self-healing alginate hydrogels play important roles in the biological field due to their biocompatibility and ability to recover after cracking. One of the primary targets for researchers in this field is to increase the self-healing speed. Sodium alginate was oxidized, generating aldehyde groups on the chains, which were then crosslinked by poly(amino) amine (PAMAM) via Schiff base reaction. The dendritic structure was introduced to the alginate hydrogel in this work, which was supposed to promote intermolecular interactions and accelerate the self-healing process. Results showed that the hydrogel (ADA-PAMAM) formed a gel within 2.5 min with stable rheological properties. Within 25 min, the hydrogel recovered under room temperature. Furthermore, the aldehyde degree of alginate dialdehyde with a different oxidation degree was characterized through gel permeation chromatograph aligned with multi-angle laser light scattering and ultraviolet (UV) absorption. The chemical structure of the hydrogel was characterized through Fourier transform infrared spectroscopy and UV-vis spectra. The SEM and laser scanning confocal microscope (CLSM) presented the antibiotic ability of ADA-PAMAM against both S. aureus and E. coli when incubated with 10⁻⁷ CFU microorganism under room temperature for 2 h. This work presented a strategy to promote the self-healing of hydrogel through forming a dendritic dynamic crosslinking network.     

纤维素固定化血红蛋白研究

纤维素是一种重要的化工原料,由于来源广泛、价廉以及良好的再生性、反应性和通用性,被广泛用做载体材料．在酶的分离纯化,蛋白质分离纯化,亲合色谱固定相,拆分手性化合物等应用领域,往往采用球形纤维素或膜状纤维素形式．而在水吸附剂,分离回收金属离子,从海水中...     

A resorbable calcium-deficient hydroxyapatite hydrogel composite for osseous regeneration

It was previously discovered that the unique structure and chemistry of bacterial cellulose (BC) permits the formation of calcium-deficient hydroxyapatite (CdHAP) nanocrystallites under aqueous conditions at ambient pH and temperature. In this study, BC was chemically modified via a limited periodate oxidation reaction to render the composite degradable and thus more suitable for bone regeneration. While native BC does not degrade in mammalian systems, periodate oxidation yields dialdehyde cellulose which breaks down at physiological pH. The composite was characterized by tensile testing, X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. X-ray diffraction showed that oxidized BC retains its structure and could biomimetically form CdHAP. Degradation behavior was analyzed by incubating the samples in simulated physiological fluid (pH 7.4) at 37 °C under static and dynamic conditions. The oxidized BC and oxidized BC-CdHAP composites both lost significant mass after exposure to the simulated physiological environment. Examination of the incubation solutions by UV–Vis spectrophotometric analysis demonstrated that, while native BC released only small amounts of soluble cellulose fragments, oxidized cellulose releases carbonyl containing degradation products as well as soluble cellulose fragments. By entrapping CdHAP in a degradable hydrogel carrier, this composite should elicit bone regeneration then resorb over time to be replaced by new osseous tissue.     

固定化血红蛋白氧载体的研究(Ⅱ)——氧化纤维素共价键联血红蛋白

以纤维素为载体,经高碘酸钠氧化后共价键联血红蛋白,制备氧载体.研究了氧化条件对血红蛋白固定量的影响.纤维素只有经过预氧化、碱处理和再氧化三步活化反应,才能使血红蛋白大量固定化,每克纤维素固定血红蛋白量达1.0g.氧载体稳定性好,血红蛋白不脱落.经铁氰化钾氧化,氧载体放氧效率为33.1%.     

Selective oxidation of cellulose catalyzed by NHPI/Co(OAc)2 using air as oxidant

With NHPI/Co(OAc)₂ as catalyst and air as oxidant, carboxylic group functionalized cellulose was prepared by oxidation of cellulose in acetic acid. Fourier transform infrared spectroscopy was utilized to detect the generation of carboxylic group and the acid amount was determined by acid–base titration method. The present results revealed that C6 primary hydroxyl groups on glucose units were partly converted to carboxylic groups during the catalytic oxidation process. The degree of polymerization of oxidized cellulose, which was determined by viscosity measurement, decreased slightly as compared with its parent. The structure of cellulose was characterized by X-ray diffraction and scanning electron microscopy, and it was almost unchanged.     

Surface functionalization of polymer latex particles. III. A convenient method of producing ultrafine poly(methylstyrene) latexes with aldehyde groups on the surface

A convenient method of preparing ultrafine poly(methylstyrene) (PMS) latex particles with aldehyde groups on the surface is developed. PMS latexes in the size range 33–81 nm were prepared via microemulsion polymerization, using cetyltrimethylammonium bromide (CTAB) as surfactant. The surface of the PMS latexes was oxidized in the presence of tert-butyl hydroperoxide catalyzed by copper(II) chloride. As the degree of oxidation increased up to 6 h, the amounts of aldehyde group increased. Bigger particles were found to have a slightly higher rate of oxidation than small ones under the same oxidation conditions. The particle size underwent little change during oxidation; thus, the amounts of functional groups and the particle size could be controlled concurrently. Dialysis study of the oxidized PMS microlatexes indicated that the instability of highly oxidized PMS microlatexes was caused by the reduction of total surface charge density due to the presence of carboxylic acid groups. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 2103–2109, 1998     

Immobilization of lipase onto cellulose ultrafine fiber membrane for oil hydrolysis in high performance bioreactor

Practical application of biphasic enzyme-immobilized membrane bioreactors (EMBR) requires efficient loading of the enzyme with retention of enzymatic activity. Here, we report a method to fabricate an ultrafine fiber membrane conjugated to lipase with high levels of enzyme loading and activity retention. A cellulose acetate (CA) non-woven ultrafine fiber membrane was prepared with 200 nm nominal fiber diameter by electrospinning, followed by alkaline hydrolysis to obtain regenerated cellulose (RC). The RC ultrafine fiber membrane was oxidized by exposure to NaIO₄, simultaneously generating aldehyde groups to couple with pentaethylenehexamine (PEHA) as a spacer for lipase immobilization. A biphasic EMBR was assembled with the PEHA-modified and lipase-immobilized membranes. The effect of operation variables, namely aqueous-phase system, reaction pH, accelerant (sodium taurocholate) content, reaction temperature, and membrane usage on the performance of this bioreactor was investigated with the hydrolysis of olive oil. A bioreactor activity as high as 9.83 × 10⁴ U/m² was obtained under optimum operational conditions.     

Water dispersion of cellulose II nanocrystals prepared by TEMPO-mediated oxidation of mercerized cellulose at pH 4.8

Mercerized wood cellulose was oxidized by 4-acetamide-TEMPO/NaClO/NaClO₂ system at 60 °C and pH 4.8 for 1–5 days. Mostly individual nanocrystals 4–7 nm in width and 100–200 nm in length were obtained by ultrasonication of the oxidized product in water. The nanocrystals had the cellulose II structure, and carboxylate contents of 2.0–2.4 mmol/g, indicating that these carboxylate groups were selectively formed on the cellulose II crystallite surfaces in mercerized cellulose. Moreover, the original wood cellulose and mercerized cellulose were acid-hydrolyzed, and then subjected to the TEMPO-mediated oxidation under the same conditions at pH 4.8 to prepare reference samples. TEM images, light transmittance and rheological properties of water dispersions showed that the nanocrystals prepared from mercerized cellulose by the TEMPO oxidation and sonication in water had the highest dispersibility of individual nanocrystals with less amounts of bundles in water, resulting from the highest carboxylate contents.     

Novel poly-glutamic acid functionalized microfiltration membranes for sorption of heavy metals at high capacity

Various sorbent/ion exchange materials have been reported in the literature for metal ion entrapment. We have developed a highly innovative and new approach to obtain high metal pick-up utilizing poly-amino acids (poly-l-glutamic acid, 14,000 MW) covalently attached to membrane pore surfaces. The use of microfiltration (0.2–0.6 μm) membrane-based sorbents containing multiple functional groups is a novel technique to achieve high metal sorption under convective flow conditions. For our studies, both commercial membranes and laboratory prepared cellulose membranes containing aldehyde groups were used for the attachment of poly-amino acids. Cellulose membranes were prepared by converting cellulose acetate microfiltration membranes to cellulose (using alkali treatment), subsequent oxidation of hydroxyl groups to aldehyde using sodium periodate, and attachment of poly-l-glutamic acid via Schiff base chemistry. Extensive experiments (pH 3–6) were conducted (under convective flow mode) with the derivatized membranes involving the heavy metals: lead, cadmium, nickel, copper, and selected mixtures with calcium in aqueous solutions. Metal sorption results were found to be a function of derivatization (aldehydes) density of membranes and degree of attachment of the polyfunctional groups, number of functional groups per chain, membrane surface area, and the type of metals to be sorbed. We have obtained metal sorption capacities as high as 1.5 g metal/g membrane. Of course, depending on the desired goals the membrane containing metal could be regenerated or stabilized for appropriate disposal.     

Morphological changes of sterically stabilized nanocrystalline cellulose after periodate oxidation

Periodate oxidation breaks the C2–C3 bond in the glucose repeat units of cellulose, forming two vicinal aldehyde groups. When the cellulose is partially oxidized, three products were generated after periodate oxidation: fibrous cellulose, sterically stabilized nanocrystalline cellulose (SNCC) and dialdehyde modified cellulose. Thus, by periodate oxidation alone, we can produce nanocellulose. SNCCs were produced after 26, 42 and 84 h periodate oxidation. Their morphologies were examined by transmission electron microscopy, which show that the three SNCCs have similar diameters (5–10 nm). In contrast, the average length of SNCC decreases with aldehyde content: from approximately 590 nm after 26 h of oxidation to 100 nm for an oxidation period of 84 h. It indicates that the morphology of SNCC can be well controlled by the degree of periodate oxidation, which depends on the amount of periodate and the reaction time. Equivalent spherical diameters of SNCCs were also examined by dynamic light scattering, and the results correspond closely to the ones observed by TEM. The viscosities of SNCCs were measured by an Ubbelohde viscometer and compared with theory. Because the length of SNCC particles gradually reduces while their diameters remain almost the same, we propose that periodate reacts preferentially with the amorphous region of cellulose. After most of the amorphous regions have reacted, the reaction proceeds at the boundary of amorphous and crystalline regions, creating a reaction front that advances towards the crystalline regions, thus continually shortening them. Dynamic light scattering experiments on SNCC suspensions when adding cosolvents into them proved that SNCCs were sterically stabilized in water.     

Oxidation of wood cellulose using 2-azaadamantane N-oxyl (AZADO) or 1-methyl-AZADO catalyst in NaBr/NaClO system

A wood cellulose was oxidized with catalytic amounts of 2-azaadamantane N-oxyl (AZADO) or 1-methyl-AZADO, in an NaBr/NaClO system, in water at pH 10. The oxidation efficiency, carboxylate/aldehyde contents, and degree of polymerization (DP_v) of the oxidized celluloses thus obtained were evaluated in terms of the amount of AZADO or 1-methyl-AZADO catalyst added, in comparison with those prepared using the TEMPO/NaBr/NaClO system. When the AZADO/NaBr/NaClO and 1-methyl-AZADO/NaBr/NaClO oxidation systems were applied to wood cellulose using the same molar amount of TEMPO, the oxidation time needed for the preparation of oxidized celluloses with carboxylate contents of 1.2–1.3 mmol/g was reduced from ≈80 to 10–15 min. Moreover, the molar amounts of AZADO and 1-methyl-AZADO that had to be added for the preparation of oxidized celluloses with carboxylate contents of 1.2–1.3 mmol/g were reduced to 1/32 and 1/16 of the amount of TEMPO added, respectively. The DP_v values for the AZADO- and 1-methyl-AZADO-oxidized celluloses after NaBH₄ treatment were in the range of 600–800. This indicated that not only C6-carboxylate groups but also C2/C3 ketones were formed to some extent on the crystalline cellulose microfibril surfaces during the AZADO- and 1-methyl-AZADO-mediated oxidation. When the AZADO-oxidized wood cellulose, which had a carboxylate content of 1.2 mmol/g, was mechanically disintegrated in water, an almost transparent dispersion consisting of individually nano-dispersed oxidized cellulose nanofibrils was obtained, with a nanofibrillation yield of 89 %.     

Biocompatible hydrogel for cartilage repair with adjustable properties

Synthesis of hydrogel at mild conditions is considered one most important challenge, especially if the hydrogel will be used for hosting bioactive materials or drugs. The procedure of hydrogel preparation should have no effect on the properties of the hosted materials. Hyaluronic acid (HA) was modified by adding dialdehyde groups to its structure to facilitate formation of hydrogel at very mild conditions. Dialdehyde HA (DHA) was prepared through oxidation of HA using sodium metaperiodate as oxidizing agent. The prepared DHA was characterized by Fourier‐transform infrared (FTIR) spectroscopy and X‐ray diffraction (XRD) and aldehyde content. A hydrogel was prepared using different chitosan/DHA molar ratio and fixed amount of glutaraldehyde at 25°C. The prepared hydrogel has tunable properties and pores size depending on the chitosan/DHA molar ratio. Sodium diclofenac was loaded on the hydrogel as a model drug. The hydrogel was characterized by FTIR spectroscopy, swelling rate, gel fraction, drug release profile, and cytotoxicity. The results obtained indicated that the properties of the prepared hydrogel, including gelling time, gel fraction, swelling, pores size, and drug release profile are highly tuned depending on the chitosan/DHA molar ratio. The drug loading efficiency was in the range of 70% to 85%. The cytotoxicity results reveal that the prepared hydrogel has a very low toxicity in presence and absence of sodium diclofenac.     

Preparation and controlled degradation of oxidized sodium alginate hydrogel

Degradation is often a critical property of materials utilized in tissue engineering. Although alginate, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form hydrogels, its slow and uncontrollable degradation can be an undesirable feature. In this study, the degradation behavior of hydrogel based on oxidized sodium alginate (OSA) crosslinked with Ca²⁺ was studied in phosphate buffer solution (PBS, pH = 7.4) and Tris-(hydroxymethyl) aminomethane–HCl (Tris–HCl, pH = 7.4) at 37 °C. The degradation behavior of OSA hydrogels with different degrees of oxidation was evaluated as a function of degradation time by monitoring the changes of molecular weight and weight loss. It was found that the degradation behavior relied heavily on the degree of oxidation and the surrounding medium. This result indicates that the degradation rates of OSA hydrogels can be controlled by changing the degree of oxidation.     

Effect and Modeling of Glucose Inhibition and In Situ Glucose Removal During Enzymatic Hydrolysis of Pretreated Wheat Straw

The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw with the cellulolytic enzyme system, Celluclast? 1.5 L, from Trichoderma reesei, supplemented with a β-glucosidase, Novozym? 188, from Aspergillus niger. Addition of glucose (0–40 g/L) significantly decreased the enzyme-catalyzed glucose formation rates and final glucose yields, in a dose-dependent manner, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48–72 h of reaction recovered from 20–40% to become ≈70% of the rate recorded during 6–24 h of reaction. Although Michaelis–Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis–Menten inhibition models without great significance of the inhibition mechanism. Moreover, the experimental in situ removal of glucose could be simulated by a Michaelis–Menten inhibition model. The data provide an important base for design of novel reactors and operating regimes which include continuous product removal during enzymatic hydrolysis of lignocellulose.     

离子液体再生纤维素水凝胶的形成机理及其在凝胶电泳中的应用

以离子液体为溶剂,将天然纤维素溶解后,以水作为溶剂浴可再生出一种新型纤维素水凝胶.元素分析和红外光谱测试结果表明得到的纤维素水凝胶中只含有纤维素和水.纤维素水凝胶具有很高的透明度,2mm厚的水凝胶在650 nm处的透光率可以达到80%.应力应变试验结果表明纤维素水凝胶具有可接受的力学强度,能满足一般的应用要求.凝胶电泳的初步实验结果表明纤维素水凝胶可用做凝胶电泳支持物,可成功分离染料甚至蛋白质等目标分子.讨论了再生纤维素水凝胶的形成机理.多种溶剂均可制备相应的纤维素凝胶.研究发现,溶剂的性质将决定相应的纤维素凝胶是否可以形成.如果溶剂分子能够与离子液体混溶,而且溶剂分子中含有活泼氢,则该溶剂将可能在溶剂/纤维素体系中通过氢键构筑出三维网络结构,从而有助于该溶剂纤维素凝胶的形成.     

三维结构和氧化度对C6位氧化再生纤维素止血材料性能影响

C6位氧化再生纤维素作为可吸收止血材料,其止血性能受材料理化性质的影响。通过比较氧化再生纤维素的吸水率、凝胶化速率、溶解速率、降解速率、抗老化性能和止血效果,研究三维结构和氧化度对氧化再生纤维素性能影响。结果发现,氧化度增高,氧化再生纤维素吸水率增强,凝胶化速率加快,溶解速率和降解速率变快,抗老化性能变差,产品有效期变短。纤丝松散网状多孔结构比纱布致密结构更有利于提高吸水率,加快凝胶化速率和溶解速率;同时降低氧化剂用量,提高抗老化性能。同一氧化度条件下,纤丝止血速率优于纱布,止血效果更好。