HNO3/H3PO4–NANO2 mediated oxidation of cellulose — preparation and characterization of bioabsorbable oxidized celluloses in high yields and with different levels of oxidation

The reaction of cellulose with a mixture of HNO₃/H₃PO₄–NaNO₂ (2:1:1.4, v/v/%w) at room temperature for different time intervals has been investigated to produce oxidized cellulose (OC), a biocompatible and bioresorbable polymer. The results revealed an increase in carboxyl content of OC with increasing reaction time, corresponding to about 8.0, 13.4, 17.4 and 18.4% carboxyl content after 12, 24, 36, and 48 h, respectively. The yield of OC ranged between 75 and 81%. The use of different ratios of HNO₃ and H₃PO₄, (11:1, 4:1, 2:1, 1:1, 1:2, and 1:4; v/v), in the reaction had no significant effect on the carboxyl content and yield of the OC products. All products, as produced, were low crystallinity (27–35%) fibrous materials. The length of fibers decreased with increasing reaction time. After ball milling for 24 h, the length of fibers further decreased and products converted into a fine powder consisting of small fibers and aggregated non-fibrous particles. The degrees of polymerization (DP) of the OC products produced after 12, 24, and 48 h of reaction duration were 81, 63, and 53, respectively. After ball milling for 24 h, the corresponding values changed to 57, 51 and 46. However, no significant change in the crystallinity of the products was noted after ball milling. The TGA results showed the OC products to be less thermally stable than cellulose. The degradation temperature appears to decrease with increasing carboxyl content. In conclusion, the results show that the low crystallinity OC products can be successfully prepared in high yields and with different levels of carboxyl content from cellulose by treatment with a mixture of HNO₃/H₃PO₄–NaNO₂.     

Examination of aqueous oxidized cellulose dispersions as a potential drug carrier. I. Preparation and characterization of oxidized cellulose-phenylpropanolamine complexes

Partially neutralized aqueous dispersions of oxidized cellulose (OC) (COOH content 24.2%; degree of neutralization [DN] 0.22-0.44; solid content 14.4% wt/wt), a biocompatible biodegradable polymer, were prepared and their use to entrap an amine drug was demonstrated. Phenylpropanolamine hydrochloride (PPA.HCl) was used as a model drug. OCA-PPA complexes were prepared by adding the drug solution to the OC dispersion. Light microscopy, powder x-ray diffractometry (PXRD), and Fourier-transform infrared (FT-IR) spectroscopy were used to characterize hydrated and dried OC and the OC-PPA complexes. Drug loading and drug-loading efficiency were calculated from high-performance liquid chromatography. Light microscopy revealed the partially neutralized OC to exist as swollen fibers in the dispersion. The degree of swelling increased with increasing DN of the OC. All dispersions, irrespective of DN, showed a pseudo-plastic flow. The drug loading (12.6%-26.7%) and drug-loading efficiency (30%-48%) increased linearly with increasing DN and drug concentration. The PXRD of the OC-PPA complexes showed no diffraction peaks due to PPA, suggesting that the drug exists in the amorphous state. The FT-IR spectra of the complexes revealed the presence of an ionic linkage between OC and PPA. In conclusion, the results show that the aqueous OC dispersions can be used to molecularly entrap amine drugs to produce an OC-drug complex linked via an ionic linkage.     

Relationship between length and degree of polymerization of TEMPO-oxidized cellulose nanofibrils

The influence of 2,2,6,6-tetrametylpiperidine-1-oxyl (TEMPO)-mediated oxidation of wood cellulose and the mechanical disintegration of oxidized cellulose in water on degree of polymerization determined by viscosity measurement (DP(v)) and the apparent length of the TEMPO-oxidized cellulose nanofibrils (TOCNs) was investigated. DP(v) values decreased from 1270 to 500-600 with increasing addition of NaClO in the TEMPO-mediated oxidation stage. The DP(v) values were further decreased by mechanical fibrillation in water. There is a linear relationship between the average fibril length and DP(v); the lengths of TOCNs can be approximated from DP(v) using 0.5 M copper ethylenediamine as a solvent of both the cellulose and oxidized celluloses in TOCNs. Based on the cellulose fibril models and TEMPO oxidation mechanism, the depolymerization behavior of TOCNs is tentatively explained in terms of distribution of disordered regions in wood cellulose fibrils and formation of C6-aldehydes in cellulose fibrils during TEMPO-mediated oxidation.     

A mechanistic study of the histochemical reactions between aldehydes and Basic Fuchsin in acid alcohol used as a simplified substitute for Schiff's reagent

Synopsis For the identification of polysaccharides after periodic acid oxidation or of DNA after acid hydrolysis, a solution of 0.5% w/v Basic Fuchsin in acid alcohol (water-ethanol-concentrated hydrochloric acid 80:20:1 by volume) may be used instead of Schiff's reagent. Sections are stained in the Fuchsin solution for 20 min, after which the unreacted dye is washed off with ethanol. Except for its yellower colour the Fuchsin staining is almost indistinguishable from Schiff's reagent staining.Histochemical blocking studies indicated that the Fuchsin stain, like Schiff's reagent, reacts with aldehyde groups or subsequent oxidation products. The results of studies of model systems (cellulose film oxidized by periodic acid and also of aqueous formaldehyde solution) in which infra-red spectroscopy and, where appropriate, chromatography were used are consistent with the initial coloured products being azomethines which may react further to produce coloured secondary amine derivatives.     

Electrospun polyethylene oxide/cellulose nanocrystal composite nanofibrous mats with homogeneous and heterogeneous microstructures

An electrospinning process was successfully used to fabricate polyethylene oxide/cellulose nanocrystal (PEO/CNC) composite nanofibrous mats. Transition of homogeneous to heterogeneous microstructures was achieved by tailoring the concentration of PEO/CNC mixture in the solution from 5 to 7 wt %. Morphology investigation of the obtained nanofibers demonstrated that rod-shaped CNCs were well-dispersed in the as-spun nanofibers and highly aligned along the nanofiber long-axis. PEO/CNC nanofibers became more uniform and smaller in diameter with increased CNC-loading level. The heterogeneous composite mats were composed of rigid-flexible bimodal nanofibers. Results of structure characterization indicated that the incorporated CNCs interacted strongly with the PEO matrix through hydrogen bonding. Mechanical properties of both types of mats were effectively improved by using CNCs, with heterogeneous mats being stronger than their homogeneous counterparts for all compositions (0-20 wt % CNC contents). When a smaller diameter needle was used to form homogeneous mats, enhanced thermal and mechanical properties were obtained.     

Superhydrophobicity of cellulose triacetate fibrous mats produced by electrospinning and plasma treatment

Nano/micro-fibrous cellulose triacetate (CTA) mats were prepared by electrospinning a fixed concentration of CTA with different methylene chloride (MC)/ethanol (EtOH) ratios and with various concentrations of CTA at a fixed MC/EtOH 80/20 (v/v) ratio. All of the electrospun CTA mats had a high water contact angle (WCA) compared to the CTA cast film. At a solvent composition of 80/20 (v/v) and 5 wt.% CTA concentration, the CTA mat without plasma treatment had good surface roughness and electrospinning processability, and its WCA was 142°. To further improve its hydrophobicity, the CTA fibrous mat electrospun from the 5 wt.% solution of CTA was treated with a CF₄ plasma for various times. Superhydrophobicity could be obtained after the CF₄ plasma treatment. The WCA of the CTA mat reached as high as 153° after plasma treatment for 60 s.     

Osteoblastic phenotype expression of MC3T3-E1 cultured on electrospun polycaprolactone fiber mats filled with hydroxyapatite nanoparticles

Electrospun (e-spun) fiber mats of polycaprolactone (PCL; Mn = 80 000 g mol-1) with or without the presence of hydroxyapatite (HAp) nanoparticles (at 1% w/v based on the volume of the PCL solution) were successfully fabricated. The potential for use of these e-spun fiber mats as bone scaffolds was assessed by mouse calvaria-derived pre-osteoblastic cells, MC3T3-E1, in terms of attachment, proliferation, differentiation, and mineralization. Despite the lower number of cells attached at early time points, both the fibrous scaffolds supported the proliferation of MC3T3-E1 at similar levels to tissue-culture polystyrene plate (TCPS), with the cells growing on the PCL/HAp fiber mat (i.e., PCL/HAp-FS) showing the greatest proliferation rate on day 3 after the initial attachment period of 16 h. Alkaline phosphatase (ALP) activity of the cells grown on TCPS was the greatest on day 3 after cell culturing, while that of the cells grown on PCL/HAp-FS reached a maximum on day 5. On the other hand, the ALP activity of the cells grown on the neat PCL fiber mat (i.e., PCL-FS) was the lowest at any given time point. MC3T3-E1 cultured on the surface of PCL/HAp-FS expressed the greatest amount of osteocalcin (OC) gene on day 14 after cell culturing and OC protein on day 21 after cell culturing, respectively, when compared with those cultured on the surfaces of PCL-FS and TCPS. This corresponded to the greatest extent of mineralization for the cells grown on the surface of PCL/HAp-FS on day 21, followed by that for the cells grown on PCL-FS and TCPS, respectively.     

Biocomposite hydrogels with carboxymethylated, nanofibrillated cellulose powder for replacement of the nucleus pulposus

Biocomposite hydrogels with carboxymethylated, nanofibrillated cellulose (c-NFC) powder were prepared by UV polymerization of N-vinyl-2-pyrrolidone with Tween 20 trimethacrylate as a cross-linking agent for replacement of the native, human nucleus pulposus (NP) in intervertebral disks. The swelling ratios and the moduli of elasticity in compression of neat and biocomposite hydrogels were evaluated in dependence of c-NFC concentration (ranging from 0 to 1.6% v/v) and degree of substitution (DS, ranging from 0 to 0.23). The viscoelastic properties in shear and the material relaxation behavior in compression were measured for neat and biocomposite hydrogels containing 0.4% v/v of fibrils (DS ranging from 0 to 0.23), and their morphologies were characterized by cryo-scanning electron microscopy (cryo-SEM). The obtained results show that the biocomposite hydrogels can successfully mimic the mechanical and swelling behavior of the NP. In addition, the presence of the c-NFC shows lower strain values after cyclic compression tests and consequently creates improved material relaxation properties compared with neat hydrogels. Among the tested samples, the biocomposite hydrogel containing 0.4% v/v of c-NFC with a DS of 0.17 shows the closest behavior to native NP. Further investigation should focus on evaluation and improvement of the long-term relaxation behavior.     

Preparation and characterization of novel oxidized cellulose acetate methyl esters

In this paper, we report the preparation of oxidized cellulose acetate methyl esters (OCAM) from OCA (OC14A: carboxylic acid content 10.6% (w/w), degree of acetyl group substitution: 1.89; OC21A: carboxylic acid content 15.7% (w/w), degree of acetyl group substitution: 1.70) by treatment with methanol at room temperature using 4-dimethylaminopyridine (DMAP) as a catalyst and dicyclohexylcarbodiimide (DCC) as a coupling agent. The new polymers were characterized by Fourier-transform infrared (FT-IR) and (1)H and (13)C nuclear magnetic resonance spectroscopies, carboxylic acid content determination, moisture sorption isotherms, intrinsic viscosity, and powder X-ray diffractometry. The new polymers are amorphous powders. It is practically insoluble in water but show solubility in a range of organic solvents.     

Photoelectron spectroscopic studies of cellulose, starch and their oxidation products, in powdered form

The use of X-ray photoelectron spectroscopic techniques (XPS or ESCA) in the characterization of cellulose, starch and their oxidation products is discussed. The ESCA results obtained from these materials in the finely powdered state are presented and compared to the results obtained with celluloses in the fibrous form. A far greater amount of surface impurities were found to be present for the powdered polysaccharides due to their greater surface area. Also, it has been shown that a high full-width at half-maximum (f.w.h.m.) value for the C_1s signal is obtained with 100% periodate oxidized maize starch, while a smaller value was found for 11% periodate oxidized cellulose powder. Finally, cellulosic powders seem to produce the O_1s signal 0·8 eV downfield (i.e. higher binding energy value) as compared to the starches and this may be related to the rigid, crystalline and linear structure of the cellulose molecule as compared to the more amorphous and branched structure of the starches.     

Preparation of cellouronic acids and partially acetylated cellouronic acids by TEMPO/NaClO oxidation of water-soluble cellulose acetate

Water-soluble cellulose acetates with a degree of substitution (DS) of 0.5, prepared by partial deacetylation of cellulose acetate of DS=2.5, were oxidized with catalytic amount of 2,2,6,6,-tetramethyl-1-piperidinyloxy radical (TEMPO), sodium hypochlorite, and sodium bromide to provide useful cellouronic acids. The oxidation was conducted at a constant pH of 10 and at 2 degrees C to avoid the occurrence of side products. Whereas only the primary hydroxyl groups of cellulose acetate were oxidized, a variable degree of oxidation (DO) resulted in a range of 0.33 to 1.0, depending on the concentration in sodium hypochlorite. Thus, polyglucuronic acid as well as partially acetylated cellouronic acid, having a range of DO were obtained.     

Determination of phosphatidylcholine and disaturated phosphatidylcholine content in lung surfactant by high performance liquid chromatography

A rapid isocratic method for determining the total phosphatidylcholine and disaturated phosphatidylcholine levels in lung surfactant preparations by high performance liquid chromatography (HPLC) is described. The analysis was performed on a 3.9 x 300 mm mu-Porasil column with detection by refractive index. The lipids were eluted with a solvent system of chloroform-acetonitrile-methanol-water-85% phosphoric acid 650:650:500:130:2 (v/v/v/v/v). A 4.6 x 30 mm silica guard column was used in place of an injector loop which served as a sample concentrator and purifier. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol, all known components of lung surfactants, were eluted from the loop column and were prevented from reaching the analytical column. Sphingomyelin and lysophosphatidylcholine elute later than the phosphatidylcholines on the analytical column. The method was developed so that phosphatidylcholines elute as a single peak regardless of the fatty acid chain length (C12-C20). When the sample was first oxidized with a potassium permanganate-potassium metaperiodate solution, and potentially interfering oxidation products were removed by extraction into a basic aqueous phase, then only the disaturated phosphatidylcholines were analyzed.     

Stimulatory effect of oxidized cellulose on cellulase formation by Trichoderma reesei

Crystalline cellulase has been electrochemically oxidized to yield preparations containing various different percentages of oxidized end-groups. These celluloses have been used as carbon sources for growth and cellulase production by Trichoderma reesei . A low content of oxidized end groups in the celluloses (0.1–0.65%) stimulated cellulase production but not growth, whereas higher contents (> 1%) where inhibitory to both. The cellulolytic enzyme system secreted under stimulated conditions contained the same proportion of individual cellulase enzymes (cellobiohydrolase I and II, endoglucanase I) as the control, indicating a general stimulatory effect of oxidized cellulose. Activity of cellulases against oxidized celluloses in vitro was not stimulated, and only slightly inhibitory at high degrees of oxidation. The data support a potential role of cellulose oxidation in regulating cellulase formation by T. reesei .     

Anaerobic sulfide oxidation with nitrate by a freshwater Beggiatoa enrichment culture

A lithotrophic freshwater Beggiatoa strain was enriched in O2-H2S gradient tubes to investigate its ability to oxidize sulfide with NO3- as an alternative electron acceptor. The gradient tubes contained different NO3- concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O2, H2S, pH, and NO3- were determined with microsensors. The more NO3- that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO3- to oxidize sulfide at depths below the depth that O2 penetrated. In the presence of NO3- Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO3- was added. These thick mats spatially separated O2 and sulfide but not NO3- and sulfide, and therefore NO3- must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O2 flux and a twofold-higher sulfide flux into the NO3- -exposed mats compared to the fluxes for controls without NO3-. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S0 with NO3- as the electron acceptor.     

Silk–PVA Hybrid Nanofibrous Scaffolds for Enhanced Primary Human Meniscal Cell Proliferation

In this study, silk fibroin nanofibrous scaffolds were developed to investigate the attachment and proliferation of primary human meniscal cells. Silk fibroin (SF)–polyvinyl alcohol (PVA) blended electrospun nanofibrous scaffolds with different blend ratios (2:1, 3:1, and 4:1) were prepared. Morphology of the scaffolds was characterized using atomic force microscopy (AFM). The hybrid nanofibrous mats were crosslinked using 25 % (v/v) glutaraldehyde vapor. In degradation study, the crosslinked nanofiber showed slow degradation of 20 % on weight after 35 days of incubation in simulated body fluid (SBF). The scaffolds were characterized with suitable techniques for its functional groups, porosity, and swelling ratio. Among the nanofibers, 3:1 SF:PVA blend showed uniform morphology and fiber diameter. The blended scaffolds had fluid uptake and swelling ratio of 80 % and 458 ± 21 %, respectively. Primary meniscal cells isolated from surgical debris after meniscectomy were subcultured and seeded onto these hybrid nanofibrous scaffolds. Meniscal cell attachment studies confirmed that 3:1 SF:PVA nanofibrous scaffolds supported better cell attachment and growth. The DNA and collagen content increased significantly with 3:1 SF:PVA. These results clearly indicate that a blend of SF:PVA at 3:1 ratio is suitable for meniscus cell proliferation when compared to pure SF-PVA nanofibers.     

Enhancement of the nanofibrillation of wood cellulose through sequential periodate-chlorite oxidation

Sequential regioselective periodate-chlorite oxidation was employed as a new and efficient pretreatment to enhance the nanofibrillation of hardwood cellulose pulp through homogenization. The oxidized celluloses with carboxyl contents ranging from 0.38 to 1.75 mmol/g could nanofibrillate to highly viscous and transparent gels with yields of 100-85% without clogging the homogenizer (one to four passes). On the basis of field-emission scanning electron microscopy images, the nanofibrils obtained were of typical widths of approximately 25 ± 6 nm. All of the nanofibrillar samples maintained their cellulose I crystalline structure according to wide-angle X-ray diffraction results, and the crystallinity index was approximately 40% for all samples.     

Oxidation of n-Tetradecane and 1-Tetradecene by Fungi

Cunninghamella blakesleeana (minus strain) and a Penicillium species were grown in a mineral-salts medium containing either n-tetradecane or 1-tetradecene as substrate, and ether extracts of the mycelial mats were analyzed for oxidation products. Extracts from Cunninghamella revealed tetradecanoic acid and 13-tetradecenoic acid from the oxidation of n-tetradecane and 1-tetradecene, respectively, thereby indicating that these hydrocarbons were subject to methyl group oxidation. In contrast to Cunninghamella, the Penicillium oxidized the two substrates by subterminal attacks on methylene rather than methyl groups. This was evidenced by tentative identifications of the following alcohols and ketones from oxidation of the hydrocarbons: tetradecan-2-ol, dodecan-1-ol, tetradecan-2-one, and tetradecan-4-one from n-tetradecane, and tetradecen-4-ol, 13-tetradecen-4-ol, tetradecen-3-ol, 13-tetradecen-4-one, and tetradecen-3-one from 1-tetradecene. A pathway for hydrocarbon oxidation is proposed for subterminal oxidation at the methylene alpha to the methyl group.     

Derivatization-free gel permeation chromatography elucidates enzymatic cellulose hydrolysis

Background

The analysis of cellulose molecular weight distributions by gel permeation chromatography (GPC) is a powerful tool to obtain detailed information on enzymatic cellulose hydrolysis, supporting the development of economically viable biorefinery processes. Unfortunately, due to work and time consuming sample preparation, the measurement of cellulose molecular weight distributions has a limited applicability until now.

Results

In this work we present a new method to analyze cellulose molecular weight distributions that does not require any prior cellulose swelling, activation, or derivatization. The cellulose samples were directly dissolved in dimethylformamide (DMF) containing 10-20% (v/v) 1-ethyl-3-methylimidazolium acetate (EMIM Ac) for 60?minutes, thereby reducing the sample preparation time from several days to a few hours. The samples were filtrated 0.2?μm to avoid column blocking, separated at 0.5?mL/min using hydrophilic separation media and were detected using differential refractive index/multi angle laser light scattering (dRI/MALLS). The applicability of this method was evaluated for the three cellulose types Avicel, α-cellulose and Sigmacell. Afterwards, this method was used to measure the changes in molecular weight distributions during the enzymatic hydrolysis of the different untreated and ionic liquid pretreated cellulose substrates. The molecular weight distributions showed a stronger shift to smaller molecular weights during enzymatic hydrolysis using a commercial cellulase preparation for cellulose with lower crystallinity. This was even more pronounced for ionic liquid-pretreated cellulose.

Conclusions

In conclusion, this strongly simplified GPC method for cellulose molecular weight distribution allowed for the first time to demonstrate the influence of cellulose properties and pretreatment on the mode of enzymatic hydrolysis.

     

Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase

To effectively convert complex and recalcitrant biomass carbohydrates to simple platform sugars useful for fuel and chemicals production, mechanical or chemical pre-treatments are often required to make the carbohydrates more accessible for enzymatic hydrolysis. Due to their harsh conditions, some pre-treatments might negatively affect enzymatic hydrolysis because of events such as cellulose oxidation. To study how oxidative modification may impact cellulose's reactivity toward hydrolysis by cellulases, we prepared three cellulose substrates by cupric ion and hypochlorite oxidations, and subjected the derived celluloses to hydrolysis by various cellobiohydrolases from glycoside hydrolase families 6 and 7, and one cellulolytic Hypocrea jecorina extracellular enzyme mixture. We observed a profound decrease of enzymatic hydrolysis on the oxidized celluloses. The effect was attributed to the interference, from oxidized functional groups in cellulose, on its binding/activation in the active pocket/tunnel of cellobiohydrolases. Potential implication of the observed effect from cellulose oxidation on pre-treatment optimization and cellulase improvement was discussed.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10'-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.