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.

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.

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.

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.

Biocompatibility of Nanocellulose-Reinforced PVA Hydrogel with Human Corneal Epithelial Cells for Ophthalmic Applications.

Transparent composite hydrogel in the form of a contact lens made from poly(vinyl alcohol) (PVA) and cellulose nanocrystals (CNCs) was subjected to in vitro biocompatibility evaluation with human corneal epithelial cells (HCE-2 cells). The cell response to direct contact with the hydrogels was investigated by placing the samples on top of confluent cell layers and evaluating cell viability, morphology, and cell layer integrity subsequent to 24 h culture and removal of the hydrogels. To further characterize the lens-cell interactions, HCE-2 cells were seeded on the hydrogels, with and without simulated tear fluid (STF) pre-conditioning, and cell viability and morphology were evaluated. Furthermore, protein adsorption on the hydrogel surface was investigated by incubating the materials with STF, followed by protein elution and quantification. The hydrogel material was found to have affinity towards protein adsorption, most probably due to the interactions between the positively charged lysozyme and the negatively charged CNCs embedded in the PVA matrix. The direct contact experiment demonstrated that the physical presence of the lenses did not affect corneal epithelial cell monolayers in terms of integrity nor cell metabolic activity. Moreover, it was found that viable corneal cells adhered to the hydrogel, showing the typical morphology of epithelial cells and that such response was not influenced by the STF pre-conditioning of the hydrogel surface. The results of the study confirm that PVA-CNC hydrogel is a promising ophthalmic biomaterial, motivating future in vitro and in vivo biocompatibility studies.

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.

Amorphisation of Free Acid Ibuprofen and Other Profens in Mixtures with Nanocellulose: Dry Powder Formulation Strategy for Enhanced Solubility.

The formulation of arylpropionic acid derivatives (profens), which are poorly soluble Biopharmaceutical Classification System (BCS) Type II drugs, has a strong impact on their therapeutic action. This article shows that heat-treated powder mixtures of free acid profens with high surface area Cladophora cellulose induces drug amorphization and results in enhanced solubility and bioavailability. Similar mixtures produced using conventional low surface area cellulose, i.e., microcrystalline cellulose, does not produce the same effect. The concept is thoroughly described and links the solid-state characterization data, such as differential scanning calorimetry, X-ray powder diffraction, and Fourier-transform infra-red spectroscopy, with in vitro dissolution in biorelevant media and in vivo pharmacokinetic analysis in rats. The concept is demonstrated for several substances from the profens group, including ibuprofen (main model drug), ketoprofen, flurbiprofen, and naproxen. The presented approach opens new ways to produce solid dosage forms of profen drugs in their free acidic form as alternatives to existing analogues, e.g., drug-salt conjugates or soft gel liquid capsules.

Immediate-Release Nifedipine Binary Dry Powder Mixtures with Nanocellulose Featuring Enhanced Solubility and Dissolution Rate.

Nifedipine (NIF) is a 1,4-dihydropyridine-based calcium channel blocker with poor solubility, whose bioavailability is highly dependent on the type of formulation. Dry powder mixtures of 20% w/w NIF with microcrystalline cellulose (MCC) and its high surface area nanocellulose analogue, which is namely Cladophora (CLAD) cellulose, were produced by heating at the melting temperature of the drug for 1 h. Non-heated samples were used as a reference. The solid-state properties of the mixtures were characterized by scanning electron microscopy, differential scanning calorimetry and X-ray diffraction. The drug release was studied in biorelevant media, including simulated gastric fluid (SGF), fasted-state simulated intestinal fluid (FaSIF) and fed-state simulated intestinal fluid (FeSIF). An enhanced apparent solubility and faster dissolution rate of NIF were observed in the heated mixture of NIF with CLAD-H in all tested biorelevant media (i.e., SGF, FaSIF and FeSIF), which was due to NIF amorphization in the high surface area nanocellulose powder. Ordinary MCC, which is essentially non-porous, did not produce an enhancement of a similar magnitude. The results of the study suggest that dry powder formulation using high surface area nanocellulose is a facile new strategy for formulating calcium channel blocker drugs, which could potentially be a viable alternative to currently used soft gel liquid capsules.

Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers.

Pore-size distribution (PSD) is the most critical parameter for size-exclusion virus removal filters. Yet, different dry- and wet-state porometry methods yield different pore-size values. The goal of this work is to conduct comparative analysis of nitrogen gas sorption (NGSP), liquid-liquid and cryoporometry with differential scanning calorimetry (CP-DSC) methods with respect to characterization of regular and cross-linked virus removal filter paper based on cellulose nanofibers, i.e. the mille-feuille filter. The filters were further characterized with atomic force and scanning electron microscopy. Finally, the removal of the worst-case model virus, i.e. minute virus of mice (MVM; 20 nm, nonenveloped parvovirus) was evaluated. The results revealed that there is no difference of the obtained PSDs between the wet methods, i.e. DSC and liquid-liquid porometry (LLP), as well as no difference between the regular and cross-linked filters regardless of method. MVM filtration at different trans membrane pressure (TMP) revealed strong dependence of the virus removal capability on applied pressure. It was further observed that cross-linking filters showed enhanced virus removal, especially at lower TMP. In all, the results of this study highlight the complex nature of virus capture in size-exclusion filters.

Significance of Brownian Motion for Nanoparticle and Virus Capture in Nanocellulose-Based Filter Paper.

Pressure-dependent breakthrough of nanobioparticles in filtration was observed and it was related to depend on both convective forces due to flow and diffusion as a result of Brownian motion. The aim of this work was to investigate the significance of Brownian motion on nanoparticle and virus capture in a nanocellulose-based virus removal filter paper through theoretical modeling and filtration experiments. Local flow velocities in the pores of the filter paper were modeled through two different approaches (i.e., with the Hagen⁻Poiseuille equation) and by evaluating the superficial linear flow velocity through the filter. Simulations by solving the Langevin equation for 5 nm gold particles and 28 nm ΦX174 bacteriophages showed that hydrodynamic constraint is favored for larger particles. Filtration of gold nanoparticles showed no difference in retention for the investigated fluxes, as predicted by the modeling of local flow velocities. Filtration of ΦX174 bacteriophages exhibited a higher retention at higher filtration pressure, which was predicted to some extent by the Hagen⁻Poiseuille equation but not by evaluation of the superficial linear velocity. In all, the hydrodynamic theory was shown able to explain some of the observations during filtration.

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.

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.

High-Performance Virus Removal Filter Paper for Drinking Water Purification.

Access to drinking water is one of the greatest global challenges today. In this study, the virus removal properties of mille-feuille nanocellulose-based filter papers of varying thicknesses from simulated waste water (SWW) matrix are evaluated for drinking water purification applications. Filtrations of standard SWW dispersions at various total suspended solid (TSS) content are performed, including spiking tests with 30 nm surrogate latex particles and 28 nm ΦX174 bacteriophages. Filter papers of thicknesses 9 and 29 µm are used, and the filtrations are performed at two different operational pressures, i.e., 1 and 3 bar. The presented data using SWW matrix show, for the first time, that a filter paper made from 100% nanocellulose has the capacity to efficiently remove even the smallest viruses, i.e., up to 99.9980-99.9995% efficiency, at industrially relevant flow rates, i.e., 60-500 L m-2 h-1, and low fouling, i.e., V max > 103-104 L m-2. The filter paper presented in this work shows great promise for the development of robust, affordable, and sustainable water purification systems.

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.

Nanoparticle-loaded hydrogels as a pathway for enzyme-triggered drug release in ophthalmic applications.

The aim of this study was to develop nanoparticle loaded hydrogel based contact lenses that could be used for ocular drug delivery. Two potential contact lens platforms for controlled ophthalmic drug delivery were developed by incorporating chitosan-poly (acrylic acid) nanoparticles into polyvinyl alcohol (PVA) hydrogels and in-situ gelled nanoparticles and cellulose nanocrystals (CNC) in PVA lenses. The nanoparticles were shown to disintegrate in a physiological 0.2 mM concentration of lysozyme resulting from the hydrolysis of the chitosan chains by lysozyme. An extended release over a 28-h period was demonstrated once the nanoparticles had been integrated into the composite lenses, with nanoparticle-CNC PVA lenses showing even greater potential for extended release. The platform shows great promise in developing enzyme-triggered ocular drug delivery systems.

On importance of impurities, potential leachables and extractables in algal nanocellulose for biomedical use.

Nanocellulose-based biomaterials for biomedical and pharmaceutical applications have been extensively explored. However, studies on different levels of impurities in the nanocellulose and their potential risks are lacking. This article is the most comprehensive to date survey of the importance and characterization of possible leachables and extractables in nanocellulose for biomedical use. In particular, the (1,3)-ß-d-glucan interference in endotoxin detection in algal nanocellulose was addressed. Potential lipophilic and hydrophilic leachables, toxic heavy metals, and microbial contaminants are also monitored. As a model system, nanocellulose from Cladophora sp. algae is investigated. The leachable (1,3)-ß-d-glucan and endotoxin, which possess strong immunogenic potential, from the cellulose were minimized to clinically insignificant levels of 4.7µg/g and 2.5EU/g, respectively. The levels of various impurities in the Cladophora cellulose are acceptable for future biomedical applications. The presented approach could be considered as a guideline for other types of nanocellulose.

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.

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.

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.