Päivi Laaksonen

Interfacial Engineering by Proteins: Exfoliation and Functionalization of Graphene by Hydrophobins

Graphene has attracted vast interest as a new material with many uses. 2] Two-dimensional, crystalline graphene has many advantageous properties, such as extremely high electric and thermal conductivity, high strength, and a large surface area. Many more useful properties can result from graphene assemblies and modification by different functionalities or additional molecules. One of the usual ways to functionalize graphene is chemical modification; however, attempts to modify the surface of graphene in a noncovalent, nondestructive way have also been successful. These methods typically involve the buildup of charge on the graphene surface to enable the stabilization and assembly of the graphene sheets on the basis of electrostatic interactions. In a further step towards more complex functionalities, we have now modified graphene with more specifically interacting coatings consisting of biomolecules. One of the main challenges in the production of graphene is the scalable, controllable, and safe processing and handling of individual graphene sheets. Methods for the fabrication of graphene in a dry environment include the micromechanical cleavage of graphene sheets from graphite and the epitaxial growth of graphene on certain substrates. 11] By these methods, very large entities of single-layer graphene can be produced, but the scalability and handling problems remain. High-yielding solution-based chemical methods that enable the handling of graphene in dispersed form have been proposed; however, they involve the direct oxidation of graphene, which may lower the conductivity of graphene dramatically. Recent reports on the exfoliation of graphene either in pure solvents or in the presence of surfactants offer promise for the production of graphene. The main benefits of solution methods are the better processability and increased safety of graphene when it is dispersed in a liquid instead of being used as a dry powder. The dispersion of graphene into aqueous solutions is especially attractive because of their nonvolatile nature. Herein, we present a method for the exfoliation and functionalization of graphene sheets by an amphiphilic protein. It is known that a microbial adhesion protein, HFBI (Figure 1a), which belongs to a class of proteins called hydrophobins, interacts strongly with hydrophobic surfaces, such as graphite and silicon. The protein has a strongly cross-linked fold containing four disulfide bridges. Its most striking feature is a patch of hydrophobic residues on one face of its structure. Thus, the protein resembles a typical surfactant with a hydrophilic and a hydrophobic part. In solution, hydrophobic interactions between individual proteins lead to the formation of dimers or tetramers. In the vicinity of the interface between water and air, however, assembly of the protein at the interface is strongly preferred, and the protein crystallizes as a 2D lattice. Lateral interactions between surface proteins at interfaces may lead

Genetic Engineering of Biomimetic Nanocomposites: Diblock Proteins, Graphene, and Nanofibrillated Cellulose

Nature has materials with extraordinary stiffness, strength, and toughness that is based on aligned, tailored self-assemblies. They have inspired biomimetic nanocomposites with drastically better properties than synthetic composites. Herein we show a new approach to making biomimetic nanocomposites based on the exfoliation of graphite into a matrix of genetically engineered proteins and native nanofibrillated cellulose. The protein was genetically engineered to incorporate a hydrophobin block, which binds to graphene, and a cellulose-binding block, which binds to nanofibrillated cellulose, thereby bringing about both the self-assembly and adhesion between the nanoscale components. The aligned co-assembly leads to remarkably good mechanical properties (modulus: 20.2 GPa, strength: 278 MPa, strain-to-failure: 3.1 %, and work-of-fracture 57.9 kJ m ). The bifunctional protein was crucial for the excellent mechanical properties. This concept shows how high-performance biomimetic composites can be built through the binding and self-assembly of advanced biomolecules that have been genetically tailored. Biology shows numerous composite materials wherein aligned hard and soft self-assembled components are bound together to result in excellent mechanical properties such as the combination of toughness, strength, and stiffness. Such materials are, for example, nacre, plant tissue, bone, silk, and tendon. Factors contributing to their advantageous properties include the chemical nature of the hard-reinforcing and soft-dissipating components, their molecular interactions, their mechanical interlocking, dimensions, and alignment, which contributes to the mechanics of crack propagation. The soft matrix is especially interesting as it acts as glue that keeps the hard components together and allows dissipation of fracture energy. Still, very little is known about, for example, how the matrix proteins of nacre function. A rational route towards a controlled interconnectivity between the self-assembled domains in biomimetic composites is suggested by the design principles of block copolymers, which are used in materials science, for example, to interface two different polymers in mixtures or to stabilize colloidal systems, even for responses or functions. In this work we show the feasibility of genetically engineered proteins having two well-defined binding blocks, denoted as diblock proteins, that bind and assemble the structural components for biomimetic composites. Previously we have shown that the adhesive surfactantlike proteins, hydrophobins, allow exfoliation of graphite to give singleor few-layer flakes of graphene in aqueous solutions. Here, the same route to disperse singleor fewlayer flakes of graphene using proteins in a cellulose matrix was employed to form biomimetic nanocomposite materials. The dispersions of the singleor few-layer flakes of graphene are referred to herein simply as graphene dispersions, although there may be a range of flake thicknesses present. A genetically modified hydrophobin was used to combine graphene and native nanofibrillated cellulose (NFC), also called nanocellulose or microfibrillated cellulose. The structure of the resulting composite resembles that of nacre where self-assembled, aligned platelet-like aragonite reinforcements are embedded in a protein matrix containing nanofibrillar chitin. By using engineered molecules that contain unusual combinations of binding abilities, it is possible to build composites from components that do not occur in natural materials. This technique allowed us to combine flakes of graphene, one of the strongest materials presently known, and nanofibrillated cellulose having a modulus approaching the one of steel 16] in a nanocomposite material. The protein was genetically engineered to connect graphene and NFC, so that it self-assembles at the interfaces, thus leading to cohesion and alignment (Figure 1 a). Binding to graphene was achieved by a hydrophobin, more specifically the class II hydrophobin HFBI, which self-assembles on various interfaces and surfaces, including graphene. Binding to cellulose was achieved by using a protein denoted as a cellulose-binding domain (CBD) found in cellulose[*] Dr. P. Laaksonen, J.-M. Malho, Prof. M. B. Linder Nanobiomaterials, VTT Technical Research Centre of Finland P.O. Box 1000, 02044 VTT (Finland) E-mail: paivi.laaksonen@vtt.fi Homepage: http://www.vtt.fi/research/technology/nanobiotechnology.jsp

Drug release from nanoparticles embedded in four different nanofibrillar cellulose aerogels

Highly porous nanocellulose aerogels prepared by freeze-drying from various nanofibrillar cellulose (NFC) hydrogels are introduced as nanoparticle reservoirs for oral drug delivery systems. Here we show that beclomethasone dipropionate (BDP) nanoparticles coated with amphiphilic hydrophobin proteins can be well integrated into the NFC aerogels. NFCs from four different origins are introduced and compared to microcrystalline cellulose (MCC). The nanocellulose aerogel scaffolds made from red pepper (RC) and MCC release the drug immediately, while bacterial cellulose (BC), quince seed (QC) and TEMPO-oxidized birch cellulose-based (TC) aerogels show sustained drug release. Since the release of the drug is controlled by the structure and interactions between the nanoparticles and the cellulose matrix, modulation of the matrix formers enable a control of the drug release rate. These nanocomposite structures can be very useful in many pharmaceutical nanoparticle applications and open up new possibilities as carriers for controlled drug delivery.

Intravenous Delivery of Hydrophobin-Functionalized Porous Silicon Nanoparticles: Stability, Plasma Protein Adsorption and Biodistribution

Rapid immune recognition and subsequent elimination from the circulation hampers the use of many nanomaterials as carriers to targeted drug delivery and controlled release in the intravenous route. Here, we report the effect of a functional self-assembled protein coating on the intravenous biodistribution of (18)F-labeled thermally hydrocarbonized porous silicon (THCPSi) nanoparticles in rats. (18)F-Radiolabeling enables the sensitive and easy quantification of nanoparticles in tissues using radiometric methods and allows imaging of the nanoparticle biodistribution with positron emission tomography. Coating with Trichoderma reesei HFBII altered the hydrophobicity of (18)F-THCPSi nanoparticles and resulted in a pronounced change in the degree of plasma protein adsorption to the nanoparticle surface in vitro. The HFBII-THCPSi nanoparticles were biocompatible in RAW 264.7 macrophages and HepG2 liver cells making their intravenous administration feasible. In vivo, the distribution of the nanoparticles between the liver and spleen, the major mononuclear phagocyte system organs in the body, was altered compared to that of uncoated (18)F-THCPSi. Identification of the adsorbed proteins revealed that certain opsonins and apolipoproteins are enriched in HFBII-functionalized nanoparticles, whereas the adsorption of abundant plasma components such as serum albumin and fibrinogen is decreased.

The mucoadhesive and gastroretentive properties of hydrophobin-coated porous silicon nanoparticle oral drug delivery systems

Impediments to intestinal absorption, such as poor solubility and instability in the variable conditions of the gastrointestinal (GI) tract plague many of the current drugs restricting their oral bioavailability. Particulate drug delivery systems hold great promise in solving these problems, but their effectiveness might be limited by their often rapid transit through the GI tract. Here we describe a bioadhesive oral drug delivery system based on thermally-hydrocarbonized porous silicon (THCPSi) functionalized with a self-assembled amphiphilic protein coating consisting of a class II hydrophobin (HFBII) from Trichoderma reesei. The HFBII-THCPSi nanoparticles were found to be non-cytotoxic and mucoadhesive in AGS cells, prompting their use in a biodistribution study in rats after oral administration. The passage of HFBII-THCPSi nanoparticles in the rat GI tract was significantly slower than that of uncoated THCPSi, and the nanoparticles were retained in stomach by gastric mucoadhesion up to 3 h after administration. Upon entry to the small intestine, the mucoadhesive properties were lost, resulting in the rapid transit of the nanoparticles through the remainder of the GI tract. The gastroretentive drug delivery system with a dual function presented here is a viable alternative for improving drug bioavailability in the oral route.

Immobilization of protein-coated drug nanoparticles in nanofibrillar cellulose matrices-Enhanced stability and release

Nanosizing is an advanced approach to overcome poor aqueous solubility of active pharmaceutical ingredients. One main problem in pharmaceutical nanotechnology is maintaining of the morphology of the nanometer sized particles during processing and storage to make sure the formulation behaves as originally planned. Here, a genetically engineered hydrophobin fusion protein, where the hydrophobin (HFBI) was coupled with two cellulose binding domains (CBDs), was employed in order to facilitate drug nanoparticle binding to nanofibrillar cellulose (NFC). The nanofibrillar matrix provides protection for the nanoparticles during the formulation process and storage. It was demonstrated that by enclosing the functionalized protein coated itraconazole nanoparticles to the external nanofibrillar cellulose matrix notably increased their storage stability. In a suspension with cellulose nanofibrils, nanoparticles around 100 nm could be stored for more than ten months when the specific cellulose binding domain was fused to the hydrophobin. Also freeze-dried particles in the cellulose nanofibrils matrix were preserved without major changes in their morphology. In addition, as a consequence of formation of the immobilized nanodispersion, dissolution rate of itraconazole was increased significantly, which also enhanced the in vivo performance of the drug.

Facile method for stiff, tough, and strong nanocomposites by direct exfoliation of multilayered graphene into native nanocellulose matrix.

Nanofibrillated cellulose (NFC) is a natural fibrillar material with exceptionally high mechanical properties. It has, however, been exceedingly difficult to achieve nanocomposites with drastically improved mechanical properties by dispersing NFC as random networks to polymer matrices, even using compatibilization. We show nanocomposites consisting of aligned assemblies of multilayered graphene and NFC with excellent tensile mechanical properties without any surface treatments. The optimum composition was found at 1.25 wt % graphene multilayers, giving a Youngs modulus of 16.9 GPa, ultimate strength of 351 MPa, strain of 12%, and work-of-fracture of 22.3 MJ m(-3). This combines high strength with relatively high toughness and is obtained by direct exfoliation of graphite within aqueous hydrogels of NFC where an optimum sonication power is described. The results suggest the existence of an attractive interaction between multilayered graphene flakes and cellulose. Aligned assemblies are obtained by removal of water by filtration. The concept can be beneficial for applications because it results in high mechanical properties by a simple and environmentally green process.

Self-assembly of cellulose nanofibrils by genetically engineered fusion proteins

One central problem for the function and manufacture of materials where performance relies on nanoscale structure is to control the compatibility and interactions of the building blocks. In natural materials, such as nacre, there are examples of multifunctional macromolecules that have combined binding affinities for different materials within the same molecule, thereby bridging these materials and acting as a molecular glue. Here, we describe the use of a designed multifunctional protein that is used for self-assembly of nanofibrillar cellulose. Recent advances in the production of cellulose nanofibrils have given inspiration for new uses of cellulosic materials. Cellulose nanofibrils have mechanical and structural features that open new possibilities for performance in composites and other nanoscale materials. Functionalisation was realised through a bi-functional fusion protein having both an ability to bind to cellulose and a second functionality of surface activity. The cellulose-binding function was obtained using cellulose-binding domains from cellulolytic enzymes and the surface activity through the use of a surface active protein called hydrophobin. Using the bi-functional protein, cellulose nanofibrils could be assembled into tightly packed thin films at the air/water interface and at the oil/water interface. It was shown that the combination of protein and cellulose nanofibrils resulted in a synergistic improvement in the formation and stability of oil-in-water emulsions resulting in emulsions that were stable for several months. The bi-functionality of the protein also allowed the binding of hydrophobic solid drug nanoparticles to cellulose nanofibrils and thereby improving their long-term stability under physiological conditions.

The role of hemicellulose in nanofibrillated cellulose networks

Cellulose nanofibrils show remarkable properties with applications in several fields of materials science, such as for composites, hydrogels, aerogels, foams, and coatings. Cellulose nanofibrils are typically produced by mechanical and enzymatic processing leading to fibrils having a width in the nanometer range and very high aspect ratios. The formation of percolating networks and interactions between fibrils lead to useful properties in for example gel formation and composites. In this work we studied how the residual xylan that is found in cellulose nanofibrils that have been produced from hardwood pulp affects these properties. We used enzymatic hydrolysis to specifically remove xylan and studied rheological properties, morphological features, and properties of paper-like films of cellulose nanofibrils. We found that removal of xylan enhances the formation of fibril networks, resulting in both stiffer gels and stronger films. However xylan also stabilizes the fibrils against flocculation. Also the history of processing of the preparations affects the results significantly.

Evaluation of drug interactions with nanofibrillar cellulose

Nanofibrillar cellulose (NFC) (also referred to as cellulose nanofibers, nanocellulose, microfibrillated, or nanofibrillated cellulose) has recently gotten wide attention in various research areas and it has also been studied as excipient in formulation of the pharmaceutical dosage forms. Here, we have evaluated the interactions between NFC and the model drugs of different structural characteristics (size, charge, etc.). The series of permeation studies were utilized to evaluate the ability of the drugs in solution to diffuse through the thin, porous, dry NFC films. An incubation method was used to determine capacity of binding of chosen model drugs to NFC as well as isothermal titration calorimetry (ITC) to study thermodynamics of the binding process. A genetically engineered fusion protein carrying double cellulose binding domain was used as a positive control since its affinity and capacity of binding for NFC have already been reported. The permeation studies revealed the size dependent diffusion rate of the model drugs through the NFC films. The results of both binding and ITC studies showed that the studied drugs bind to the NFC material and indicated the pH dependence of the binding and electrostatic forces as the main mechanism.