Dissolution Behavior of Flufenamic Acid in Heated Mixtures with Nanocellulose.

Flufenamic acid (FFA) is a problem drug that has up to eight different polymorphs and shows poor solubility. Variability in bioavailability has been reported in the past resulting in limited use of FFA in the oral solid dosage form. The goal of this article was to investigate the polymorphism and amorphization behavior of FFA in non-heated and heated mixtures with high surface area nanocellulose, i.e., Cladophora cellulose (CLAD). As a benchmark, low surface area microcrystalline cellulose (MCC) was used. The solid-state properties of mixtures were characterized with X-ray diffraction, Fourier-transform infrared spectroscopy, and differential scanning calorimetry. The dissolution behavior of mixtures was studied in three biorelevant media, i.e., fasted state simulated gastric fluid, fasted state simulated intestinal fluid, and fed state simulated intestinal fluid. Additional thermal analysis and dissolution tests were carried out following 4 months of storage at 75% RH and room temperature. Heated mixtures of FFA with CLAD resulted in complete amorphization of the drug, whereas that with MCC produced a mixture of up to four different polymorphs. The amorphous FFA mixture with CLAD exhibited rapid and invariable fasted/fed state dissolution in simulated intestinal fluids, whereas that of MCC mixtures was highly dependent on the biorelevant medium. The storage of the heated FFA-CLAD mixture did not result in recrystallization or changes in dissolution profile, whereas heated FFA-MCC mixture showed polymorphic changes. The straightforward dry powder formulation strategy presented here bears great promise for reformulating a number of problem drugs to enhance their dissolution properties and reduce the fasted/fed state variability.

Virus removal filtration of chemically defined Chinese Hamster Ovary cells medium with nanocellulose-based size exclusion filter.

Sterility of bioreactors in biotherapeutic processing remains a significant challenge. Virus removal size-exclusion filtration is a robust and highly efficient approach to remove viruses. This article investigates the virus removal capacity of nanocellulose-based filter for upstream bioprocessing of chemically defined Chinese hamster ovary (CHO) cells medium containing Pluronic F-68 (PowerCHO™, Lonza) and supplemented with insulin-transferrin-selenium (ITS) at varying process parameters. Virus retention was assessed by spiking ITS-supplemented PowerCHO™ medium with small-size ΦX174 phage (28 nm) as a surrogate for mammalian parvoviruses. The nanocellulose-based size exclusion filter showed high virus retention capacity (over 4 log10) and high flow rates (around 180 L m-2 h-1). The filter had no impact on ITS supplements during filtration. It was further shown that the filtered PowerCHO™ medium supported cell culture growth with no impact on cell viability, morphology, and confluence. The results of this work show new opportunities in developing cost-efficient virus removal filters for upstream bioprocessing.

Spectroscopic and Physicochemical Characterization of Sulfonated Cladophora Cellulose Beads.

The work presents a full physicochemical characterization of sulfonated cellulose beads prepared from Cladophora nanocellulose intended for use in biological systems. 2,3-Dialdehyde cellulose (DAC) beads were sulfonated, and transformation of up to 50% of the aldehyde groups was achieved, resulting in highly charged and porous materials compared to the compact surface of the DAC beads. The porosity could be tailored by adjusting the degree of sulfonation, and a subsequent reduction of the aldehyde groups to hydroxyl groups maintained the bead structure without considerable alteration of the surface properties. The thermal stability of the DAC beads was significantly increased with the sulfonation and reduction reactions. Raman spectroscopy also showed to be a useful technique for the characterization of sulfonated cellulose materials.

Potentially Immunogenic Contaminants in Wood-Based and Bacterial Nanocellulose: Assessment of Endotoxin and (1,3)-ß-d-Glucan Levels.

Knowledge gaps in the biosafety data of the nanocellulose (NC) for biomedical use through various routes of administration call for closer look at health and exposure evaluation. This work evaluated the potentially immunogenic contaminants levels, for example, endotoxin and (1,3)-ß-d-glucan, in four representative NCs, that is, wood-based NCs and bacterial cellulose (BC). The hot-water extracts were analyzed with ELISA assays, HPSEC-MALLS, GC, and NMR analysis. Varying levels of endotoxin and (1,3)-ß-d-glucan contaminats were found in these widely used NCs. Although the ß-(1,3)-d-glucan was not detected from the NMR spectra due to the small extract samples amount (2-7 mg), the anomerics and highly diastereotopic 6-CH2 signals may suggest the presence of ß-(1,4)-linkages with ß-(1,6) branching in the polysaccharides of NCs' hot-water extracts, which were otherwise not detectable in the enzymatic assay. In all, the article highlights the importance of monitoring various water-soluble potentially immunogenic contaminants in NC for biomedical use.

Blood Compatibility of Sulfonated Cladophora Nanocellulose Beads.

Sulfonated cellulose beads were prepared by oxidation of Cladophora nanocellulose to 2,3-dialdehyde cellulose followed by sulfonation using bisulfite. The physicochemical properties of the sulfonated beads, i.e., high surface area, high degree of oxidation, spherical shape, and the possibility of tailoring the porosity, make them interesting candidates for the development of immunosorbent platforms, including their application in extracorporeal blood treatments. A desired property for materials used in such applications is blood compatibility; therefore in the present work, we investigate the hemocompatibility of the sulfonated cellulose beads using an in vitro whole blood model. Complement system activation (C3a and sC5b-9 levels), coagulation activation (thrombin-antithrombin (TAT) levels) and hemolysis were evaluated after whole blood contact with the sulfonated beads and the results were compared with the values obtained with the unmodified Cladophora nanocellulose. Results showed that neither of the cellulosic materials presented hemolytic activity. A marked decrease in TAT levels was observed after blood contact with the sulfonated beads, compared with Cladophora nanocellulose. However, the chemical modification did not promote an improvement in Cladophora nanocellulose hemocompatibility in terms of complement system activation. Even though the sulfonated beads presented a significant reduction in pro-coagulant activity compared with the unmodified material, further modification strategies need to be investigated to control the complement activation by the cellulosic materials.

Protein-Nanocellulose Interactions in Paper Filters for Advanced Separation Applications.

Protein-based pharmaceutics are widely explored for healthcare applications, and 6 out of 10 best-selling drugs today are biologicals. The goal of this work was to evaluate the protein nanocellulose interactions in paper filter for advanced separation applications such as virus removal filtration and bioprocessing. The protein recovery was measured for bovine serum albumin (BSA), γ-globulin, and lysozyme using biuret total protein reagent and polyacrylamide gel electrophoresis (PAGE), and the throughput was characterized in terms of flux values from fixed volume filtrations at various protein concentrations and under worst-case experimental conditions. The affinity of cellulose to bind various proteins, such as BSA, lysozyme, γ-globulin, and human IgG was quantified using a quartz crystal microbalance (QCMB) by developing a new method of fixing the cellulose fibers to the electrode surface without cellulose dissolution-precipitation. It was shown that the mille-feuille filter exhibits high protein recovery, that is, ∼99% for both BSA and lysozyme. However, γ-globulin does not pass through the membrane due to its large size (i.e., >180 kDa). The PAGE data show no substantial change in the amount of dimers and trimers before and after filtration. QCMB analysis suggests a low affinity between the nanocellulose surface and proteins. The nanocellulose-based filter exhibits desirable inertness as a filtering material intended for protein purification.

Strain-induced stiffening of nanocellulose-reinforced poly(vinyl alcohol) hydrogels mimicking collagenous soft tissues.

Soft tissues possess remarkable mechanical strength for their high water content, which is hard to mimic in synthetic materials. Here, we demonstrate how strain-induced stiffening in hydrogels plays a major role in mimicking the mechanical properties of collagenous soft tissues. In particular, nanocellulose reinforced polyvinyl alcohol (PVA) hydrogels of exceptionally high water content (90-93 wt%) are shown to exhibit collagen-like mechanical behavior typical for soft tissues. High water content and co-existence of both soft and rigid domains in the gel network are the main factors responsible for strain-induced stiffening. This observed effect due to the alignment of rigid components of the hydrogel is simulated through modeling and visualized through strain-induced birefringence experiments. Design parameters such as nanocellulose aspect ratio and solvent composition are also shown to be important to control the mechanical properties. In addition, owing to their transparency (90-95% at 550 nm) and hyperelastic properties (250-350% strain), the described hydrogels are promising materials for biomedical applications, especially in ophthalmology.

Cellulose Nanofibers Prepared via Pretreatment Based on Oxone® Oxidation.

Softwood sulfite bleached cellulose pulp was oxidized with Oxone® and cellulose nanofibers (CNF) were produced after mechanical treatment with a high-shear homogenizer. UV-vis transmittance of dispersions of oxidized cellulose with different degrees of mechanical treatment was recorded. Scanning electron microscopy (SEM) micrographs and atomic force microscopy (AFM) images of samples prepared from the translucent dispersions showed individualized cellulose nanofibers with a width of about 10 nm and lengths of a few hundred nm. All results demonstrated that more translucent CNF dispersions could be obtained after the pretreatment of cellulose pulp by Oxone® oxidation compared with the samples produced without pretreatment. The intrinsic viscosity of the cellulose decreased after oxidation and was further reduced after mechanical treatment. Almost translucent cellulose films were prepared from the dispersions of individualized cellulose nanofibers. The procedure described herein constitutes a green, novel, and efficient route to access CNF.

Preparation of Porous Cellulose Beads via Introduction of Diamine Spacers.

The current work presents a synthesis route based on the reductive amination of 2,3-dialdehyde cellulose beads with diamines to render micrometer-sized beads with increased specific surface area (SSA) and porosity in the mesoporous range. Specifically, the influence of the reductive amination of 2,3-dialdehyde cellulose (DAC) using aliphatic and aromatic tethered mono- and diamines on bead microstructure was investigated. Aliphatic and aromatic tethered monoamines were found to have limited utility for producing porous beads whereas the introduction of diamines provided beads with a porous texture and an SSA increasing from <1 to >30 m(2)/g. Both aliphatic and aromatic diamines were found to be useful in producing porous beads having a pore size distribution range of 10 to 100 nm, as verified by N2 gas adsorption and mercury intrusion porosimetry analyses. The true density of the functionalized DAC beads decreased to an average of about 1.36 g/cm(3) as compared to 1.48 g/cm(3) for the unfunctionalized, fully oxidized DAC beads. The total porosity of the beads was, according to mercury porosimetry, in the range of 54-64%. Reductive amination with 1,7-diaminoheptane provided beads that were stable under alkaline conditions (1 M NaOH). It was concluded that the introduction of tethered diamines into DAC beads is a facile method for producing mesoporous beads.

Transition from Bioinert to Bioactive Material by Tailoring the Biological Cell Response to Carboxylated Nanocellulose.

This work presents an insight into the relationship between cell response and physicochemical properties of Cladophora cellulose (CC) by investigating the effect of CC functional group density on the response of model cell lines. CC was carboxylated by electrochemical TEMPO-mediated oxidation. By varying the amount of charge passed through the electrolysis setup, CC materials with different degrees of oxidation were obtained. The effect of carboxyl group density on the material's physicochemical properties was investigated together with the response of human dermal fibroblasts (hDF) and human osteoblastic cells (Saos-2) to the carboxylated CC films. The introduction of carboxyl groups resulted in CC films with decreased specific surface area and smaller total pore volume compared with the unmodified CC (u-CC). While u-CC films presented a porous network of randomly oriented fibers, a compact and aligned fiber pattern was depicted for the carboxylated-CC films. The decrease in surface area and total pore volume, and the orientation and aggregation of the fibers tended to augment parallel to the increase in the carboxyl group density. hDF and Saos-2 cells presented poor cell adhesion and spreading on u-CC, which gradually increased for the carboxylated CC as the degree of oxidation increased. It was found that a threshold value in carboxyl group density needs be reached to obtain a carboxylated-CC film with cytocompatibility comparable to commercial tissue culture material. Hence, this study demonstrates that a normally bioinert nanomaterial can be rendered bioactive by carefully tuning the density of charged groups on the material surface, a finding that not only may contribute to the fundamental understanding of biointerface phenomena, but also to the development of bioinert/bioactive materials.

Susceptibility of Iα- and Iß-Dominated Cellulose to TEMPO-Mediated Oxidation.

The susceptibility of Iα- and Iß-dominated cellulose to TEMPO-mediated oxidation was studied in this work since the cellulose Iα-allomorph is generally considered to be thermodynamically less stable and therefore more reactive than the cellulose Iß-allomorph. Highly crystalline Cladophora nanocellulose, which is dominated by the Iα-allomorph, was oxidized to various degrees with TEMPO oxidant via bulk electrolysis in the absence of co-oxidants. Further, the Cladophora nanocellulose was thermally annealed in glycerol to produce its Iß-dominated form and then oxidized. The produced materials were subsequently studied using FTIR, CP/MAS (13)C NMR, XRD, and SEM. The solid-state analyses confirmed that the annealed Cladophora cellulose was successfully transformed from an Iα- to an Iß-dominated form. The results of the analyses of pristine and annealed TEMPO-oxidized samples suggest that Iα- and Iß-dominated cellulose do not differ in susceptibility to TEMPO-oxidation. This work hence suggests that cellulose from different sources are not expected to differ in susceptibility to the oxidation due to differences in allomorph composition.

Surface Chemistry of Nanocellulose Fibers Directs Monocyte/Macrophage Response.

The effect of surface functionalization of nanofibrillated cellulose (NFC) on monocyte/macrophage (MM) behavior is investigated to understand how the physicochemical properties of nanocelluloses influence the interactions of such materials with biological systems. Films of anionic (a-), cationic (c-), and unmodified (u-) NFC were synthesized and characterized in terms of surface charge. THP-1 monocytes were cultured on the surface of the films for 24 h in the presence and absence of lipopolysaccharide, and the cell response was evaluated in terms of cell adhesion, morphology, and secretion of TNF-α, IL-10, and IL-1ra. The results show that MMs cultured on carboxymethylated-NFC films (a-NFC) are activated toward a proinflammatory phenotype, whereas u-NFC promotes a mild activation of the studied cells. The presence of hydroxypropyltrimethylammonium groups on c-NFC, however, does not promote the activation of MMs, indicating that c-NFC closely behaves as an inert material in terms of MM activation. None of the materials is able to directly activate the MMs toward an anti-inflammatory response. These results may provide a foundation for the design of future NFC-based materials with the ability to control MM activation and may expand the use of NFC in biomedical applications.

Ultrafast all-polymer paper-based batteries.

Conducting polymers for battery applications have been subject to numerous investigations during the last two decades. However, the functional charging rates and the cycling stabilities have so far been found to be insufficient for practical applications. These shortcomings can, at least partially, be explained by the fact that thick layers of the conducting polymers have been used to obtain sufficient capacities of the batteries. In the present letter, we introduce a novel nanostructured high-surface area electrode material for energy storage applications composed of cellulose fibers of algal origin individually coated with a 50 nm thin layer of polypyrrole. Our results show the hitherto highest reported charge capacities and charging rates for an all polymer paper-based battery. The composite conductive paper material is shown to have a specific surface area of 80 m(2) g(-1) and batteries based on this material can be charged with currents as high as 600 mA cm(-2) with only 6% loss in capacity over 100 subsequent charge and discharge cycles. The aqueous-based batteries, which are entirely based on cellulose and polypyrrole and exhibit charge capacities between 25 and 33 mAh g(-1) or 38-50 mAh g(-1) per weight of the active material, open up new possibilities for the production of environmentally friendly, cost efficient, up-scalable and lightweight energy storage systems.

Would 20 nm Filtered Fetal Bovine Serum-Supplemented Media Support Growth of CHO and HEK-293 Cells?

Virus safety of fetal bovine serum (FBS) is a critical issue for cell culture and clinical applications of cell therapies. The size exclusion filtration of FBS-supplemented cell culture media through small-size virus retentive filter paper is presented to investigate its effect on cell culture. A substantial proportion of proteins (ca. 45%) was removed by nanofiltration, yet important transport proteins (albumin, fetuins, macroglobulins, transferrin) were unaffected. The cell viability of Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK-293) cells that were grown in media supplemented with nanofiltered FBS was surprisingly high, despite the observed protein losses. Protein depletion following nanofiltration resulted in detectable levels of autophagy markers.

Directly Compressed Tablets of Free Acid Ibuprofen with Nanocellulose Featuring Enhanced Dissolution: A Side-by-Side Comparison with Commercial Oral Dosage Forms.

We have previously reported that heated powder mixtures of ibuprofen (IBU) and high surface area nanocellulose exhibit an enhanced dissolution and solubility of the drug due to IBU amorphization. The goal of the present work was to further elaborate the concept and conduct side-by-side in vitro drug release comparisons with commercial formulations, including film-coated tablets, soft gel liquid capsules, and IBU-lysine conjugate tablets, in biorelevant media. Directly compressed tablets were produced from heated mixtures of 20% w/w IBU and high surface area Cladophora cellulose (CLAD), with 5% w/w sodium croscarmelose (AcDiSol) as superdisintegrant. The side-by side studies in simulated gastric fluid, fasted-state simulated intestinal fluid, and fed-state simulated intestinal fluid corroborate that the IBU-CLAD tablets show more rapid and less variable release in various media compared to three commercial IBU formulations. On the sidelines of the main work, a possibility of the presence of a new meta-crystalline form of IBU in mixture with nanocellulose is discussed.

Aggregate Removal Nanofiltration of Human Serum Albumin Solution Using Nanocellulose-Based Filter Paper.

This study is dedicated to the rapid removal of protein aggregates and viruses from plasma-derived human serum albumin (HSA) product to reduce the risk of viral contamination and increase biosafety. A two-step filtration approach was implemented to first remove HSA aggregates and then achieve high model virus clearance using a nanocellulose-based filter paper of different thicknesses, i.e., 11 µm (prefilter) and 22 µm (virus filter) at pH 7.4 and room temperature. The pore size distribution of these filters was characterized by nitrogen gas sorption analysis. Dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography (SE-HPLC) were performed to analyze the presence of HSA aggregates in process intermediates. The virus filter showed high clearance of a small-size model virus, i.e., log10 reduction value (LRV) > 5, when operated at 3 and 5 bar, but a distinct decrease in LRV was detected at 1 bar, i.e., LRV 2.65-3.75. The throughput of HSA was also dependent on applied transmembrane pressure as was seen by Vmax values of 110 ± 2.5 L m-2 and 63.6 ± 5.8 L m-2 at 3 bar and 5 bar, respectively. Protein loss was low, i.e., recovery > 90%. A distribution of pore sizes between 40 nm and 60 nm, which was present in the prefilter and absent in the virus filter, played a crucial part in removing the HSA aggregates and minimizing the risk of virus filter fouling. The presented results enable the application of virus removal nanofiltration of HSA in bioprocessing as an alternative to virus inactivation methods based, e.g., on heat treatment.

Two-Step Size-Exclusion Nanofiltration of Prothrombin Complex Concentrate Using Nanocellulose-Based Filter Paper.

Coagulation Factor IX-rich protrhombin complex concentrate (FIX-PCC) is a therapeutic biologic product that consists of a mixture of several human plasma-derived proteins, useful for treating hemophilia B. Due to its complex composition, FIX-PCC is very challenging to bioprocess through virus removing nanofilters in order to ensure its biosafety. This article describes a two-step filtration process of FIX-PCC using a nanocellulose-based filter paper with tailored porosity. The filters were characterized with scanning electron microscopy (SEM), cryoporometry with differential scanning calorimetry, and nitrogen gas sorption. Furthermore, in order to probe the filter's cut-off size rejection threshold, removal of small- and large-size model viruses, i.e., ΦX174 (28 nm) and PR772 (70 nm), was evaluated. The feed, pre-filtrate, and permeate solutions were characterized with mass-spectrometric proteomic analysis, dynamic light scattering (DLS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analytical size-exclusion high-performance liquid chromatography (SEHPLC). By sequential filtration through 11 µm pre-filter and 33 µm virus removal filter paper, it was possible to achieve high product throughput and high virus removal capacity. The presented approach could potentially be applied for bioprocessing other protein-based drugs.

Influence of the type of oxidant on anion exchange properties of fibrous Cladophora cellulose/polypyrrole composites.

The electrochemically controlled anion absorption properties of a novel large surface area composite paper material composed of polypyrrole (PPy) and cellulose derived from Cladophora sp. algae, synthesized with two oxidizing agents, iron(III) chloride and phosphomolybdic acid (PMo), were analyzed in four different electrolytes containing anions (i.e., chloride, aspartate, glutamate, and p-toluenesulfonate) of varying size.The composites were characterized with scanning and transmission electron microscopy, N2 gas adsorption,and conductivity measurements. The potential-controlled ion exchange properties of the materials were studied by cyclic voltammetry and chronoamperometry at varying potentials. The surface area and conductivity of the iron(III) chloride synthesized sample were 58.8 m2/g and 0.65 S/cm, respectively, while the corresponding values for the PMo synthesized sample were 31.3 m2/g and 0.12 S/cm. The number of absorbed ions per sample mass was found to be larger for the iron(III) chloride synthesized sample than for the PMo synthesized one in all four electrolytes. Although the largest extraction yields were obtained in the presence of the smallest anion (i.e., chloride) for both samples, the relative degree of extraction for the largest ions (i.e., glutamate and p-toluenesulfonate) was higher for the PMo sample. This clearly shows that it is possible to increase the extraction yield of large anions by carrying out the PPy polymerization in the presence of large anions. The results likewise show that high ion exchange capacities, as well as extraction and desorption rates, can be obtained for large anions with high surface area composites coated with relatively thin layers of PPy.

Ionic motion in polypyrrole-cellulose composites: trap release mechanism during potentiostatic reduction.

This work investigates the movement of anions during potentiostatic controlled reduction of novel composite materials consisting of high surface area cellulose substrates, extracted from the Cladophora sp. algae, coated with thin ( approximately 50 nm) layers of the intrinsically conducting polymer (ICP) polypyrrole. The coating was achieved by chemical polymerization of pyrrole on the cellulose fibers with iron(III) chloride and phosphomolybdic acid, respectively. The composites are in the form of paper sheets and can be directly immersed into an electrolyte solution for ion absorption/desorption. The motion of glutamate and aspartate anions during cathodic polarization was investigated as a function of preceding anodic polarization at various potentials. The composite was found to exhibit memory effect as the response to a cathodic polarization of constant magnitude produced different responses depending on the magnitude of the preceding anodic potential. After the application of a cathodic potential to the composite, the reduction current curvesgenerated by anions leaving the compositewere found to initially increase in magnitude followed by a monotonic decay. A similar response has not been described and analyzed for electrochemical reduction of anion containing ICP materials earlier. A theoretical model was developed to aid the analysis of the experimental data. The model accounts for both freely mobile anions and anions that may be temporarily trapped in a contracting PPy network during cathodic polarization. By fitting the recorded reduction current curves to this model, detailed information about the ionic movement in the composite could be obtained, which may be used to further optimize the materials properties of conducting polymer systems aimed for specific electrochemical ion exchange processes.