Avidin-Conjugated Nanofibrillar Cellulose Hydrogel Functionalized with Biotinylated Fibronectin and Vitronectin Promotes 3D Culture of Fibroblasts.

The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization via biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications.

Pulmonary effects of nanofibrillated celluloses in mice suggest that carboxylation lowers the inflammatory and acute phase responses.

We studied if the pulmonary and systemic toxicity of nanofibrillated celluloses can be reduced by carboxylation. Nanofibrillated celluloses administered at 6 or 18 µg to mice by intratracheal instillation were: 1) FINE NFC, 2-20 µm in length, 2-15 nm in width, 2) AS (-COOH), carboxylated, 0.5-10 µm in length, 4-10 nm in width, containing the biocide BIM MC4901 and 3) BIOCID FINE NFC: as (1) but containing BIM MC4901. FINE NFC administration increased neutrophil influx in BAL and induced SAA3 in plasma. AS (-COOH) produced lower neutrophil influx and systemic SAA3 levels than FINE NFC. Results obtained with BIOCID FINE NFC suggested that BIM MC4901 biocide did not explain the lowered response. Increased DNA damage levels were observed across materials, doses and time points. In conclusion, carboxylation of nanofibrillated cellulose was associated with reduced pulmonary and systemic toxicity, suggesting involvement of OH groups in the inflammatory and acute phase responses.

Nanofibrillated cellulose causes acute pulmonary inflammation that subsides within a month.

Nanofibrillated cellulose (NFC) is a renewable nanomaterial that has beneficial uses in various applications such as packaging materials and paper. Like carbon nanotubes (CNT), NFCs have high aspect ratio and favorable mechanical properties. The aspect ratio also rises a concern whether NFC could pose a health risk and induce pathologies, similar to those triggered by multi-walled CNT. In this study, we explored the immunomodulatory properties of four NFCs in vitro and in vivo, and compared the results with data on bulk-sized cellulose fibrils and rigid multi-walled CNT (rCNT). Two of the NFCs were non-functionalized and two were carboxymethylated or carboxylated. We investigated the production of pro-inflammatory cytokines in differentiated THP-1 cells, and studied the pulmonary effects and biopersistence of the materials in mice. Our results demonstrate that one of the non-functionalized NFCs tested reduced cell viability and triggered pro-inflammatory reactions in vitro. In contrast, all cellulose materials induced innate immunity response in vivo 24 h after oropharyngeal aspiration, and the non-functionalized NFCs additionally caused features of Th2-type inflammation. Modest immune reactions were also seen after 28 days, however, the effects were markedly attenuated as compared with the ones after 24 h. Cellulose materials were not cleared within 1 month, as demonstrated by their presence in the exposed lungs. All effects of NFC were modest as compared with those induced by rCNT. NFC-induced responses were similar or exceeded those triggered by bulk-sized cellulose. These data provide new information about the biodurability and pulmonary effects of different NFCs; this knowledge can be useful in the risk assessment of cellulose materials.

Inhibition of Skin Wound Contraction by Nanofibrillar Cellulose Hydrogel.

BACKGROUND: Although wound contraction is an essential part of healing, excessive contraction can compromise healing through induction of scarring and fibrosis. This in turn leads to development of wound contractures that limit elasticity and function. Major research efforts have focused on development of novel therapeutic approaches to gain inhibitory control over wound contraction. Despite these efforts, the need for cost-effective, clinically feasible, and effective agents to inhibit wound contraction remains. METHODS: In this study, the authors investigated the effect of nanofibrillar cellulose hydrogel on wound contraction both in vitro and in vivo. Two different porcine full-thickness wounds (8-mm punch-biopsy wounds and 4 × 4-cm wounds covered with a 1:3-meshed split-thickness skin graft) were treated with or without nanofibrillar cellulose or carboxymethylcellulose (Purilon hydrogel), which was used as a reference treatment. Wound contraction was observed macroscopically, and histologic sections were taken at 14-day follow-up. RESULTS: Nanofibrillar cellulose hydrogel inhibited 70 percent of punch-biopsy wound contraction, whereas the carboxymethylcellulose hydrogel was ineffective. Importantly, application of nanofibrillar cellulose on split-thickness skin grafts did not inhibit epithelialization of the interstices or cell migration from the graft. CONCLUSION: The authors' results, although preliminary, indicate a potential for nanofibrillar cellulose hydrogel as a novel material for controlling excessive wound contraction.

Activation of TEMPO by ClO2 for oxidation of cellulose by hypochlorite-Fundamental and practical aspects of the catalytic system.

Bromide-free TEMPO-catalyzed oxidation of the primary alcohols by sodium hypochlorite (NaOCl) does not proceed without a prior activation of the catalyst. Here were demonstrate an immediate in situ activation of the catalyst with an equimolar addition of chlorine dioxide (ClO2) relative to TEMPO. Sodium bromide (NaBr) had a similar role in activating the catalyst although NaBr was needed in excess and the activation took several minutes depending on the dosage of NaBr. The activation method, or the concentration of NaBr, did not affect the bulk oxidation rate. The selectivity of the ClO2 initiated oxidation remained high up to NaOCl addition of 3mol/kg bleached birch kraft pulp after which additional loss in yield and depolymerization of cellulose were emphasized with negligible increase in carboxylate content. A carboxylate content of 0.8-1mol/kg, sufficient for easy mechanical fibrillation of the pulp, was achieved under mild conditions with NaOCl addition of 2-2.5mol/kg pulp.

Visualization of Nanofibrillar Cellulose in Biological Tissues Using a Biotinylated Carbohydrate Binding Module of ß-1,4-Glycanase.

Nanofibrillar cellulose is a very promising innovation with diverse potential applications including high quality paper, coatings, and drug delivery carriers. The production of nanofibrillar cellulose on an industrial scale may lead to increased exposure to nanofibrillar cellulose both in the working environment and the general environment. Assessment of the potential health effects following exposure to nanofibrillar cellulose is therefore required. However, as nanofibrillar cellulose primarily consists of glucose moieties, detection of nanofibrillar cellulose in biological tissues is difficult. We have developed a simple and robust method for specific and sensitive detection of cellulose fibers, including nanofibrillar cellulose, in biological tissue, using a biotinylated carbohydrate binding module (CBM) of ß-1,4-glycanase (EXG:CBM) from the bacterium Cellulomonas fimi. EXG:CBM was expressed in Eschericia coli, purified, and biotinylated. EXG:CBM was shown to bind quantitatively to five different cellulose fibers including four different nanofibrillar celluloses. Biotinylated EXG:CBM was used to visualize cellulose fibers by either fluorescence- or horse radish peroxidase (HRP)-tagged avidin labeling. The HRP-EXG:CBM complex was used to visualize cellulose fibers in both cryopreserved and paraffin embedded lung tissue from mice dosed by pharyngeal aspiration with 10-200 µg/mouse. Detection was shown to be highly specific, and the assay appeared very robust. The present method represents a novel concept for the design of simple, robust, and highly specific detection methods for the detection of nanomaterials, which are otherwise difficult to visualize.

Crystal morphology modification by the addition of tailor-made stereocontrolled poly(N-isopropyl acrylamide).

The use of additives in crystallization of pharmaceuticals is known to influence the particulate properties critically affecting downstream processing and the final product performance. Desired functionality can be build into these materials, e.g. via optimized synthesis of a polymeric additive. One such additive is the thermosensitive polymer poly(N-isopropyl acrylamide) (PNIPAM). The use of PNIPAM as a crystallization additive provides a possibility to affect viscosity at separation temperatures and nucleation and growth rates at higher temperatures. In this study, novel PNIPAM derivatives consisting of both isotactic-rich and atactic blocks were used as additives in evaporative crystallization of a model compound, nitrofurantoin (NF). Special attention was paid to possible interactions between NF and PNIPAM and the aggregation state of PNIPAM as a function of temperature and solvent composition. Optical light microscopy and Raman and FTIR spectroscopy were used to investigate the structure of the NF crystals and possible interaction with PNIPAM. A drastic change in the growth mechanism of nitrofurantoin crystals as monohydrate form II (NFMH-II) was observed in the presence of PNIPAM; the morphology of crystals changed from needle to dendritic shape. Additionally, the amphiphilic nature of PNIPAM increased the solubility of nitrofurantoin in water. PNIPAMs with varying molecular weights and stereoregularities resulted in similar changes in the crystal habit of the drug regardless of whether the polymer was aggregated or not. However, with increased additive concentration slower nucleation and growth rates of the crystals were observed. Heating of the crystallization medium resulted in phase separation of the PNIPAM. The phase separation had an influence on the achieved crystal morphology resulting in fewer, visually larger and more irregular dendritic crystals. No proof of hydrogen bond formation between PNIPAM and NF was observed, and the suggested mechanism for the observed dendritic morphology is related to the steric hindrance phenomenon. PNIPAM can be used as a crystallization additive with an obvious effect on the growth of NF crystals.

Gold nanoparticles protected with pH and temperature-sensitive diblock copolymers.

Aqueous dispersions of gold nanoparticles protected with a stimuli-sensitive diblock copolymer were studied as a function of pH and temperature. Poly(methacrylic acid)-block-poly(N-isopropylacrylamide), PMAA-b-PNIPAM, copolymer was synthesized using the RAFT technique. A one-pot method utilizing the dithiobenzoate functionalized polymer was used to prepare gold nanoparticles protected with PMAA-b-PNIPAM. The gold nanoparticles coated with block copolymers, with the PNIPAM block bound to the particle surface and PMAA as an outer block form stimuli-sensitive aggregates in water. The changes in the absorption maxima of the surface plasmon resonance, SPR, of the gold particles and in the size of the aggregates were investigated as a function of pH and temperature. pH was observed to affect the size of the aggregates, whereas the effect of temperature was moderate. However, a blue shift in the SPR was observed both with decreasing pH and increasing temperature. Whereas the PMAA blocks control the colloidal stability of the particles and their aggregates, the thermo-sensitive PNIPAM blocks have a noticeable effect on the polarity of the immediate surroundings of the particles.

Optical properties of thermally responsive amphiphilic gold nanoparticles protected with polymers.

Amphiphilic thermally responsive gold nanoparticles have been prepared by protecting the particles with both polystyrene, PS, and poly(N-isopropylacrylamide), PNIPAM, chains. Particles form a monolayer on a water surface in a Langmuir trough, and according to in situ spectroscopic measurements, the surface plasmon resonance, SPR, band undergoes a blue-shift during the monolayer compression. The compression-induced blue-shift is related to a change in the conformation of tethered PNIPAM chains; the phenomenon is discussed on the basis of Mie-Drude theory. In contrast, a red-shift in the SPR of the multilayers of the same nanoparticles transferred at different temperatures has been observed with increasing the deposition cycle, attributed to the presence of a weak interparticle coupling in the multilayer.

Characterisation of poly(N-isopropylacrylamide) by asymmetrical flow field-flow fractionation, dynamic light scattering, and size exclusion chromatography.

Asymmetrical flow field-flow fractionation (AsFIFFF) was used to determine the hydrodynamic particle sizes, molar masses, and phase transition behaviour of various poly(N-isopropylacrylamide) (PNIPAM) samples synthesised by reversible addition--fragmentation chain transfer (RAFT) and conventional free radical polymerisation processes. The results were compared with corresponding data obtained by dynamic light scattering (DLS) and size exclusion chromatography (SEC). Agreement between the three methods was good except at higher molar masses, where the molar mass averages obtained by SEC were much lower than those obtained by AsFIFFF and light scattering. The aggregation of the polymers, which are thermally sensitive, was studied by DLS and AsFIFFF at various temperatures. In deionised water there was an abrupt change in the particle size due to phase separation at approximately equal to 32-35 degrees C. The critical temperatures determined by AsFIFFF were 3-5 degrees C higher than those obtained by DLS.

Two phase transitions of poly(N-isopropylacrylamide) brushes bound to gold nanoparticles.

The thermally induced phase transition of the poly(N-isopropylacrylamide) (PNIPAM) brush covalently bound to the surface of the gold nanoparticles was studied using high-sensitivity microcalorimetry. Two types of PNIPAM monolayer protected clusters (MPCs) of gold nanoparticles were employed, denoted as the cumyl- and the cpa-PNIPAM MPCs, bearing either a phenylpropyl end group or a carboxyl end group on each PNIPAM chain, respectively. The PNIPAM chains of both MPCs exhibit two separate transition endotherms; i.e., the first transition with a sharp and narrow endothermic peak occurs at lower temperature, while the second one with a broader peak occurs at higher temperature. With increase of the MPC concentration, the transition temperature corresponding to the first peak only slightly changes but the second transition temperature strongly shifts to lower temperature. The calorimetric enthalpy change in the first transition is much smaller than that in the second transition. The ratio of the calorimetric enthalpy change to the van't Hoff enthalpy change indicates that in the first transition PNIPAM segments show much higher cooperativity than in the second one. The investigation of pH dependence of two-phase transitions further indicates the PNIPAM brush reveals two separate transitions even with a change in interchain/interparticle association. The observations are tentatively rationalized by assuming that the PNIPAM brush can be subdivided into two zones, the inner zone and the outer zone. In the inner zone, the PNIPAM segments are close to the gold surface, densely packed, less hydrated, and undergo the first transition. In the outer zone, on the other hand, the PNIPAM segments are looser and more hydrated, adopt a restricted random coil conformation, and show a phase transition, which is dependent on both concentration of MPC and the chemical nature of the end groups of the PNIPAM chains. Aggregation of the particles, which may also affect the phase transition, is briefly discussed.