Polyvinyl alcohol/chitosan hydrogels with enhanced antioxidant and antibacterial properties induced by lignin nanoparticles.

Polyvinyl alcohol/chitosan (PVA/Ch) hydrogels containing 1 and 3wt% of lignin nanoparticles (LNPs) were prepared through a freezing-thaw procedure. Results from microstructural, thermal and mechanical characterization of LNPs based PVA/Ch demonstrated that the lowest amount of LNPs (1wt%) was beneficial, whereas the presence of agglomerates at higher LNP content limited the effect. Moreover, a different swelling behaviour was observed for hydrogels containing LNPs with respect of PVA/Ch, due to the formation of a porous honeycomb-like structure. A synergic effect of Ch and LNPs was revealed in terms of antioxidative response by DPPH (1,1-Diphenyl-2-picryl-hydrazyl) activity of migrated substances, whereas results from antimicrobial tests confirmed LNPs as effective against Gram negative bacteria (E. coli) when compared to Gram positive (S.aureus and S. epidermidis) strains. The obtained results suggested the possible use of produced PVA/Ch hydrogels incorporating LNPs in many different sectors, such as drug delivery, food packaging, wound dressing.

Cellulose nanocrystals as templates for cetyltrimethylammonium bromide mediated synthesis of Ag nanoparticles and their novel use in PLA films.

In the present paper, we reported how cellulose nanocrystals (CNC) from microcrystalline cellulose have the capacity to assist in the synthesis of metallic nanoparticles chains. A cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used as modifier for CNC surface. Silver nanoparticles were synthesized on CNC, and nanoparticle density and size were optimized by varying concentrations of nitrate and reducing agents, and the reduction time. The experimental conditions were optimized for the synthesis and the resulting Ag grafted CNC (Ag-g-CNC) were characterized by means of TGA, SEM, FTIR and XRD, and then introduced in PLA matrix. PLA nanocomposite containing silver grafted cellulose nanocrystals (PLA/0.5Ag-g-1CNC) was characterized by optical and thermal analyses and the obtained data were compared with results from PLA nanocomposites containing 1% wt. of CNC (PLA/1CNC), 0.5% wt. of silver nanoparticles (PLA/0.5Ag) and hybrid system containing CNC and silver in the same amount (PLA/1CNC/0.5Ag). The results demonstrated that grafting of silver nanoparticles on CNC positively affected the thermal degradation process and cold crystallization processes of PLA matrix. Finally, the antibacterial activity of the different systems was studied at various incubation times and temperatures, showing the best performance for PLA/1CNC/0.5Ag based nanocomposite.

Revalorization of sunflower stalks as novel sources of cellulose nanofibrils and nanocrystals and their effect on wheat gluten bionanocomposite properties.

Novel gluten based bionanocomposites reinforced with cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC) extracted from sunflower stalks by respectively a steam explosion treatment and a hydrolysis procedure, were prepared by casting/evaporation. The extracted cellulose nanomaterials, both CNC and CNF, were embedded in gluten matrix and their effect was investigated. Morphological investigations highlighted that gluten based bionanocomposites showed a homogenous morphology, the absence of visible cellulose nanoreinforcements, and the presence of holes for Gluten_CNF nanocomposites. Gluten_CNF showed a reduction of water vapour permeability coefficients but the values are higher respect to gluten reinforced with CNC. This behaviour could be related to the ability of CNC to increase the tortuous path of gas molecules. Moreover, the results from thermal, mechanical and barrier properties confirmed the strong interactions obtained between CNC and gluten matrix during the process. The study suggested the possibility to re-valorise agricultural wastes with potential applications as reinforcement in polymer matrix bionanocomposites.

Effect of cellulose and lignin on disintegration, antimicrobial and antioxidant properties of PLA active films.

This study reports the effects on antimicrobial, antioxidant, migration and disintegrability activities of ternary nanocomposite films based on poly(lactic acid) incorporating two biobased nanofillers, (cellulose nanocrystals (CNC) and lignin nanoparticles (LNP)), in two different amounts (1 and 3% wt.). Results from antimicrobial tests revealed a capacity to inhibit the Gram negative bacterial growth of Xanthomonas axonopodis pv. vesicatoria and Xanthomonas arboricola pv. pruni along the time, offering innovative opportunities against dangerous bacterial plant pathogens. LNP proved to be highly efficient in antioxidation activity, based on the disappearance of the absorption band at 517nm of the free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH) upon reduction by an antiradical compound; moreover the combination of LNP and CNC generates a synergistic positive effect in the antioxidation response of PLA ternary films. Furthermore, all the studied formulations showed a disintegrability value up to 90% after 15days of incubation in composting conditions. Migration results showed that the films can be considered suitable for application in food packaging field.

Bionanocomposite films based on plasticized PLA-PHB/cellulose nanocrystal blends.

Optically transparent plasticized poly(lactic acid) (PLA) based bionanocomposite films intended for food packaging were prepared by melt blending. Materials were plasticized with 15wt% of acetyl(tributyl citrate) (ATBC) to improve the material processability and to obtain flexibile films. Poly(hydroxybutyrate) (PHB) was used to increase PLA crystallinity. The thermal stability of the PLA-PHB blends was improved by the addition of 5 wt% of cellulose nanocrystals (CNC) or modified cellulose nanocrystals (CNCs) synthesized from microcrystalline cellulose. The combination of ATBC and cellulose nanocrystals, mainly the better dispersed CNCs, improved the interaction between PLA and PHB. Thus, an improvement on the oxygen barrier and stretchability was achieved in PLA-PHB-CNCs-ATBC which also displayed somewhat UV light blocking effect. All bionanocomposite films presented appropriate disintegration in compost suggesting their possible applications as biodegradable packaging materials.

Keratins extracted from Merino wool and Brown Alpaca fibres: thermal, mechanical and biological properties of PLLA based biocomposites.

Keratins extracted from Merino wool (KM) and Brown Alpaca fibres (KA) by sulphitolysis and commercial hydrolyzed keratins (KH) were used as fillers in poly(l-lactic) acid based biocomposites processed by solvent casting in chloroform. Different contents (1 wt.% and 5 wt.%) of keratins were considered and the morphological, thermal, mechanical, chemical and biological behaviours of the developed PLLA biocomposites were investigated. The results confirmed that surface morphologies of biocomposites revealed specific round-like surface topography function of different microsized keratin particles in different weight contents, such as the analysis of bulk morphologies which confirmed a phase adhesion strictly dependent by the keratin source. Transparency and thermal responses were deeply affected by the presence of the different keratins and their interaction with the PLLA matrix. Tensile test results underlined the possibility to modulate the mechanical behaviour of PLLA selecting the keratin type and content in order to influence positively the elastic and/or plastic response. It was demonstrated that surface characteristics of PLLA/KA systems also influenced the bovine serum albumin adsorption, moreover PLLA and PLLA biocomposites based on different kinds of keratins supported the culture of human bone-marrow mesenchymal stem cells, indicating that these biocomposites could be useful materials for medical applications.

Spin coated cellulose nanocrystal/silver nanoparticle films.

In this study, thin films of cellulose nanocrystals (CNC) and silver nanoparticles (Ag) were assembled on different substrates by spin coating. The effect of substrates, deposition parameters, and nanocrystal modification on the topographical and hydrophilic properties of the obtained layers was investigated. Dilute concentrations of pristine cellulose nanocrystals (CNC) and surfactant modified crystals (s-CNC) were used in order to evaluate the effect of modification and concentration on the uniformity of the spin coated cellulose/silver layers. Morphological investigations by field emission scanning electron microscopy and atomic force microscopy were performed in order to prove the uniformity of the obtained films, while the wettability of different surfaces were studied and correlated to the cellulose modification and content. The ability of s-CNC to form a stable dispersion in chloroform permits the formation of a uniform cellulose film on the substrate surfaces generating regular films during the spin coating process. Topographical investigations show, on the other hand, that the CNC/Ag suspension produces a non-uniform distribution. These effects can be mainly attributed to the surfactant action rather than to the chemical and electrical properties of the substrate surface. Finally, contact angle studies, underline the hydrophilic nature of s-CNC/Ag based films highlighting that the wettability properties are strongly influenced by the cellulose nanocrystal nature.

Multifunctional PLA-PHB/cellulose nanocrystal films: processing, structural and thermal properties.

Cellulose nanocrystals (CNCs) synthesized from microcrystalline cellulose by acid hydrolysis were added into poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB) blends to improve the final properties of the multifunctional systems. CNC were also modified with a surfactant (CNCs) to increase the interfacial adhesion in the systems maintaining the thermal stability. Firstly, masterbatch pellets were obtained for each formulation to improve the dispersion of the cellulose structures in the PLA-PHB and then nanocomposite films were processed. The thermal stability as well as the morphological and structural properties of nanocomposites was investigated. While PHB increased the PLA crystallinity due to its nucleation effect, well dispersed CNC and CNCs not only increased the crystallinity but also improved the processability, the thermal stability and the interaction between both polymers especially in the case of the modified CNCs based PLA-PHB formulation. Likewise, CNCs were better dispersed in PLA-CNCs and PLA-PHB-CNCs, than CNC.

PVA bio-nanocomposites: a new take-off using cellulose nanocrystals and PLGA nanoparticles.

The formation of a new generation of hybrid bio-nanocomposites is reported: these are intended at modulating the mechanical, thermal and biocompatibility properties of the poly(vinyl alcohol) (PVA) by the combination of cellulose nanocrystals (CNC) and poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) loaded with bovine serum albumin fluorescein isothiocynate conjugate (FITC-BSA). CNC were synthesized from microcrystalline cellulose by hydrolysis, while PLGA nanoparticles were produced by a double emulsion with subsequent solvent evaporation. Firstly, binary bio-nanocomposites with different CNC amounts were developed in order to select the right content of CNC. Next, ternary PVA/CNC/NPs bio-nanocomposites were developed. The addition of CNC increased the elongation properties without compromising the other mechanical responses. Thermal analysis underlined the nucleation effect of the synergic presence of cellulose and nanoparticles. Remarkably, bio-nanocomposite films are suitable to vehiculate biopolymeric nanoparticles to adult bone marrow mesenchymal stem cells successfully, thus representing a new tool for drug delivery strategies.

Nano-biocomposite films with modified cellulose nanocrystals and synthesized silver nanoparticles.

Ternary nano-biocomposite films based on poly(lactic acid) (PLA) with modified cellulose nanocrystals (s-CNC) and synthesized silver nanoparticles (Ag) have been prepared and characterized. The functionalization of the CNC surface with an acid phosphate ester of ethoxylated nonylphenol favoured its dispersion in the PLA matrix. The positive effects of the addition of cellulose and silver on the PLA barrier properties were confirmed by reductions in the water permeability (WVP) and oxygen transmission rate (OTR) of the films tested. The migration level of all nano-biocomposites in contact with food simulants were below the permitted limits in both non-polar and polar simulants. PLA nano-biocomposites showed a significant antibacterial activity influenced by the Ag content, while composting tests showed that the materials were visibly disintegrated after 15 days with the ternary systems showing the highest rate of disintegration under composting conditions.

Binary PVA bio-nanocomposites containing cellulose nanocrystals extracted from different natural sources: part I.

PVA bio-nanocomposites reinforced with cellulose nanocrystals (CNC) extracted from commercial microcrystalline cellulose (MCC) and from two types of natural fibres, Phormium tenax and Flax of the Belinka variety, were produced by solvent casting in water. Morphological, thermal, mechanical and transparency properties were studied while the respective efficiency of the extraction process of CNC from the three sources was evaluated. The effect of CNC types and content on PVA properties and water absorption capacity were also evaluated. Natural fibres offered higher levels of extraction efficiency when compared with MCC hydrolysis yield. Thermal analysis proved that CNC promotes the crystallization of the PVA matrix, while improving its plastic response. It was also clarified that all PVA/CNC systems remain transparent due to CNC dispersion at the nanoscale, while being all saturated after the first 18-24h of water absorption.

Ternary PVA nanocomposites containing cellulose nanocrystals from different sources and silver particles: part II.

Cellulose nanocrystals (CNC) extracted from three different sources, namely flax, phormium, and commercial microcrystalline cellulose (MCC) have been used in a polyvinyl alcohol (PVA) matrix to produce anti-bacterial films using two different amounts of silver nanoparticles (0.1 wt% and 0.5 wt%). In general, CNC confer an effect of reinforcement to PVA film, the best values of stiffness being offered by composites produced using phormium fibres, whilst for strength those produced using flax are slightly superior. This was obtained without inducing any particular modification in transition temperatures and in the thermal degradation patterns. As regards antibacterial properties, systems with CNC from flax proved slightly better than those with CNC from phormium and substantially better than those including commercial MCC. Dynamic mechanical thermal analysis (DMTA) has only been performed on the ternary composite containing 0.1 wt% Ag, which yielded higher values of Young's modulus, and as a whole confirmed the above results.

Production of nanocrystalline cellulose from lignocellulosic biomass: technology and applications.

The use of renewables materials for industrial applications is becoming impellent due to the increasing demand of alternatives to scarce and unrenewable petroleum supplies. In this regard, nanocrystalline cellulose, NCC, derived from cellulose, the most abundant biopolymer, is one of the most promising materials. NCC has unique features, interesting for the development of new materials: the abundance of the source cellulose, its renewability and environmentally benign nature, its mechanical properties and its nano-scaled dimensions open a wide range of possible properties to be discovered. One of the most promising uses of NCC is in polymer matrix nanocomposites, because it can provide a significant reinforcement. This review provides an overview on this emerging nanomaterial, focusing on extraction procedures, especially from lignocellulosic biomass, and on technological developments and applications of NCC-based materials. Challenges and future opportunities of NCC-based materials will be are discussed as well as obstacles remaining for their large use.

Effects of modified cellulose nanocrystals on the barrier and migration properties of PLA nano-biocomposites.

The aim of this paper is to report the impact of the addition of cellulose nanocrystals on the barrier properties and on the migration behaviour of poly(lactic acid), PLA, based nano-biocomposites prepared by the solvent casting method. Their microstructure, crystallinity, barrier and overall migration properties were investigated. Pristine (CNC) and surfactant-modified cellulose nanocrystals (s-CNC) were used, and the effect of the cellulose modification and content in the nano-biocomposites was investigated. The presence of surfactant on the nanocrystal surface favours the dispersion of CNC in the PLA matrix. Electron microscopy analysis shows the good dispersion of s-CNC in the nanoscale with well-defined single crystals indicating that the surfactant allowed a better interaction between the cellulose structures and the PLA matrix. Reductions of 34% in water permeability were obtained for the cast films containing 1 wt.% of s-CNC while good oxygen barrier properties were detected for nano-biocomposites with both 1 wt.% and 5 wt.% of modified and un-modified cellulose nanocrystals, underlining the improvement provided by cellulose on the PLA films. Moreover, the migration level of the studied nano-biocomposites was below the overall migration limits required by the current normative for food packaging materials in both non-polar and polar simulants.

PLGA/Ag nanocomposites: in vitro degradation study and silver ion release.

New nanocomposite films based on a biodegradable poly (DL-Lactide-co-Glycolide) copolymer (PLGA) and different concentration of silver nanoparticles (Ag) were developed by solvent casting. In vitro degradation studies of PLGA/Ag nanocomposites were conducted under physiological conditions, over a 5 week period, and compared to the behaviour of the neat polymer. Furthermore the silver ions (Ag(+)) release upon degradation was monitored to obtain information on the properties of the nanocomposites during the incubation. The obtained results suggest that the PLGA film morphology can be modified introducing a small percentage of silver nanoparticles that do not affect the degradation mechanism of PLGA polymer in the nanocomposite. However results clearly evinced the stabilizing effect of the Ag nanoparticles in the PLGA polymer and the mineralization process induced by the combined effect of silver and nanocomposite surface topography. The Ag(+) release can be controlled by the polymer degradation processes, evidencing a prolonged antibacterial effect.

Novel poly(L-lactide) PLLA/SWNTs nanocomposites for biomedical applications: material characterization and biocompatibility evaluation.

Poly(L-lactide) (PLLA)/single-walled carbon nanotubes (SWNTs) nanocomposite films were produced using the solvent casting method, and morphological, thermal and mechanical properties were investigated. Biocompatibility was evaluated by using human bone cells, performing adhesion and proliferation studies. The role of single-walled nanotube incorporation and functionalization on PLLA bio-polymers was investigated. Pristine (SWNTs) and carboxylated (SWNTs-COOH) carbon nanotubes were considered in order to control the interaction between PLLA and nanotubes. SWNTs and SWNTs-COOH showed a good dispersion in the polymer matrix and improved the PLLA crystallinity. Thermal, morphological and dynamic-mechanical analyses revealed that carboxylic groups on the tube sidewalls increased compatibility between PLLA and nanostructures. Mechanical properties demonstrated an enhancement related to introduction and functionalization of carbon nanotubes. Biological investigations showed osteoblasts cultured on PLLA/SWNTs-COOH nanocomposites has higher cell adhesion and proliferation than osteoblasts cultured on PLLA and PLLA/SWNTs nanocomposites. These studies suggest that combination of biodegradable polymers and SWNTs opens a new perspective in the self-assembly of nanomaterials and nanodevices for biomedical applications with tunable properties.

Human neutrophils switch to an activated phenotype after homing to the lung irrespective of inflammatory disease.

Systemic inflammation can be investigated by changes in expression profiles of neutrophil receptors. Application of this technology for analysis of neutrophil phenotypes in diseased tissues is hampered by the absence of information regarding the modulation of neutrophil phenotypes after extravasation to tissues under non-inflammatory conditions. To fill this gap we measured the expression of neutrophil receptors in bronchoalveolar lavage fluid (BALF) and in the peripheral blood of healthy volunteers, which included both smokers and non-smokers. Blood and BALF neutrophils were identified by CD16(bright)/CD45(dim) cells, and triple-stained with antibodies directed against integrins, chemokine- and Fc gamma-receptors. BALF neutrophils of healthy volunteers showed an activated phenotype characterized by Mac-1 (CD11b)(bright), L-selectin (CD62L)(dim), intercellular adhesion molecule 1 (ICAM-1) (CD54)(bright), Fc gamma RII (CD32)(bright), C5a receptor (CD88)(bright) and CD66b(bright). A similar phenotype was observed for BALF neutrophils of patients affected by sarcoidosis. Furthermore, our results demonstrate a modulated expression of C5a receptor (CD88) and ICAM-1 (CD54) in neutrophils of sarcoidosis patients. In conclusion, our data indicate that neutrophils found in the lung exhibit an activated phenotype under both homeostatic and inflammatory conditions.

Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids.

We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

A multi-domain protein for beta1 integrin-targeted DNA delivery.

The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR)4inv, that is designed to target plasmid DNA to beta1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid alpha-helical linker, and the C-terminal beta1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)4inv could be purified at high yields using a bacterial expression system. We show that (SPKR)4inv binds with high affinity to both plasmid DNA and beta1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for beta1 integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)4inv-plasmid complexes are not active in an in vitro transfection assay. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of beta1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows beta1 integrin-specific targeting of plasmid-carrier complexes, while avoiding inefficient and cumbersome coupling chemistry. The modular design of the expression vector allows production of similar multi-domain proteins with a different affinity. The further development of such complexes for use in vivo is discussed.

Efficacy of a peptide-based gene delivery system depends on mitotic activity.

We have developed and tested a transfection compound based on synthetic peptides. It consists of a 12 amino acid DNA binding peptide (P2) with an alkyl group added to the aminoterminus (P2lip) and a peptide derived from the hemagglutinin protein (HA). The components aggregate spontaneously to particles that proved to be an efficient, easy to use and chemically stable transfection compound. With this system we found a marked correlation between transfection efficiency and mitotic activity. Cells that are allowed to perform a mitosis after exposure to either DNA-P2lip/HA or DNA-cationic liposome complexes are transfected much more efficiently than cells arrested in the cell cycle. In search of an explanation for this phenomenon we studied transport of plasmid DNA across the nuclear membrane. Plasmid DNA injected into the cytoplasm of quiescent human fibroblasts is not expressed, in contrast to DNA injected into the nucleus. FISH analysis showed that the plasmid DNA is not transported into the nucleus efficiently. Similarly, DNA-P2lip/HA complexes are readily taken up by both proliferating and nonproliferating cells, but do not readily penetrate the nuclear membrane. We conclude that delivery of plasmid DNA to the cytoplasm is not sufficient for transfection of eukaryotic cells. The nuclear membrane is apparently an important barrier. This explains why a mitotic event is required for efficient transfection with the currently available transfection systems. The implications for the further development of transfection compounds for use in vivo, where nonproliferating cells are often the target, are discussed.