Patent Application
20170291361
The mechanical properties of fibrous composite materials are strongly affected by the volume and orientation of individual fibers. In many biological composite materials, such as insect cuticle, the orientation of nanofibers is controlled on a micro- or even nanoscale. This allows to grow materials with highly controlled and “local” mechanical properties. By gradually varying the orientation or volume of the fibers even very smooth and controlled mechanical gradients can be created within a single bulk material. Such properties are highly desirable for artificial composite materials in many fields of application.
However, with the methods for preparing biopolymer nanofibers and fibrous composite materials it is not possible to fulfill all the requirements for obtaining such artificial composite materials with locally controlled mechanical properties.
The most important requirements are:
Thus, main objects of the present invention are to provide methods for preparing and orientating polymer, in particular biopolymer, nanofibers which overcome the drawbacks of the prior art and enable to fabricate improved composite materials with locally controlled mechanical properties.
These objects are achieved according to the present invention by providing the methods of claims 1 and 7 as well as the composite material of claim 18. More specific embodiments of the invention are the subject of further claims.
The method of the invention for preparing nanofibers comprises at least the following steps:
Preferably, the polymer used in the method of the present invention is a protein or a polysaccharide.
The term “protein” as used herein encompasses any sequence of more than about 10 amino acids, typically a sequence of about 10 to 1000 amino acids.
The term “polysaccharide” as used herein encompasses any sequence of more than about 10 monosaccharides, typically a sequence of 10 to 1000 monosaccharides (which may be different or identical). The monosaccharide basic units may comprise 3-9 carbon atoms, preferably 5-7 carbon atoms. The monosaccharide units may be, e.g., selected from the group comprising glucose, galactose, glucosamine, galactamine, gluconic acid, galacturonic acid, acetyl glucosamine, arabinose, fructose, fucose, mannose, rhamnose, sialic acid and derivatives thereof.
Specific, but not limiting examples of the polymer are fibronectin, elastin, fibrinogen, collagen, myosin, actin, BSA, α-actinin, laminin, chondroitin sulfate, hyaluronan, chitin-derivatives (e.g. chitosan) and mixtures thereof.
According to the present invention, a nanoporous material is used to produce nanofibers by a template-assisted extrusion process. Typically, the nanoporous material is a membrane or mesh, preferably a membrane.
The nanoporous material may be, e.g., anodic aluminum oxide (AAO), titanium dioxide, silicone dioxide, polycarbonate (PCTE), or a zeolite.
Typically, the nanoporous material has a mean pore size in the range from 4 nm to 900 nm, preferable from 100 nm to 200 nm, and a thickness in the range from 10 μm to 100 μm, preferably from 30 μm to 50 μm.
In an especially preferred embodiment, the membrane is an anodic aluminum oxide (AAO) membrane.
Nanoporous AAO membranes are chemically stable, bioinert and biocompatible and have highly ordered, self-organized nanochannels with regular pore size, uniform pore density and high porosity over a large scale. Pore diameters between 4 nm and several hundred nanometer can be achieved using an efficient, low-cost anodisation process with polyprotic acids, such as sulphuric or oxalic acid (e.g. A. Huczko, in Appl. Phys. α-Mater 70, 365-76).
Ordered AAO nanopores have been used as template materials to prepare vertical nanowires and nanoparticle arrays from various materials such as metals, semiconductors or synthetic polymers (e.g. G. Schmidt in J. Mater. Chem. 12, 1231-1238).
According to the present invention, a polymer, such as a protein or polysaccharide or a mixture thereof, is dissolved in a suitable physiological or non-physiological organic or inorganic solvent.
The solvent is not critical and a suitable solvent for a specific polymer can be easily selected by the skilled artisan using his general knowledge and/or routine experiments.
More specifically, the solvent may be selected from the group comprising acetic acid or ionic liquids (in particular for polysaccharides) or physiological buffers (in particular for proteins). Additional components (proteins, polysaccharides, nanoparticles, fluorescent or magnetic labels, etc.) can be added to the main component.
The polymer-solvent mixture is pressed or drawn (sucked) through the nanoporous material, preferably a membrane (such as anodic aluminum oxide, AAO) using controlled speed and pressure and nanofibers form at the pores of the membrane and are extruded.
For further processing, the nanofibers are usually separated from the solvent (by means of evaporation, centrifugation, sedimentation or any other suitable method of the art) and, if desired, can be further functionalised or purified.
According to the method of the invention for preparing a composite material with orientated nanofibers, the nanofibers, in particular nanofibers obtained with a method as described above, are first mixed with a matrix and converted into a desired form, such as into a 3D printable form (filament, powder, gel, etc.).
The nanofibers used for preparing the composite material typically have a length in the range from 100 nm to several millimeters, preferably from 1 μm to 5 mm and a diameter between typically 5 nm and 500 nm. Bundles of nanofibres used for preparing the composite material typically have a length in the range from 100 nm to several millimetres, preferably from 1 μm to 5 mm and a diameter between typically 1 μm and 10 μm. Such nanofibers and bundles thereof are obtainable by the extrusion method of the present invention.
Principally, the matrix material is not especially limited and may be any material, in particular any polymer, which allows to disperse the nanofibers therein.
More specifically, the matrix may be a material commonly used for 3D printing.
In particular, the matrix material may be selected from the group comprising polylactic acid (PLA), poly(lactic-co-glycolic acid) (PGLA), polyethylen glycol (PEG), polyethylen oxide (PEO), acrylnitril-butadien-styrol (ABS), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polycarbonate (PC), nylon, acrylnitril-styrol-acrylester (ASA), silicone.
An electric and/or magnetic field is used to locally and independently orientate the nanofibers within the matrix in 2D or 3D.
In a next step, the nanofibers-matrix mixture with the orientated or partially orientated nanofibers is deposited onto a substrate surface.
The material of the substrate surface is not critical and may be selected from a wide range of organic and inorganic materials, including metals, Si, SiO2, metal oxides, glass, polymers etc.
The deposition of the nanofibers-matrix-mixture may be affected by any method known in the art which allows to deposit the respective form of mixture, such as gel powder etc., precise and effectively on a desired area of a substrate surface.
In a preferred embodiment of the invention, the deposition of the nanofibers-matrix mixture onto a substrate surface is affected by a deposition means (e.g. a nozzle) of a printing device. Any known method of printing, in particular 3D printing, may be used, including polygraphic techniques and multi-jet-modeling.
The method of the present invention is particularly advantageous in that different nanofibers-matrix mixtures or nanofibers-matrix mixtures with varying orientation of the nanofibers can be deposited simultaneously or subsequently on various areas of the primary substrate surface or the substrate surface already covered with a layer of the nanofibers-matrix mixture.
Thus, it is possible to orientate the nanofibers locally different in various areas of the composite material resulting in a composite material with locally independent mechanical properties. Further, this approach facilitates controlled crosslinking of the matrix on a small scale, if desired.
In a preferred embodiment of the invention, a multi-layered hierarchical composite material is printed layer by layer (3D printing) onto a substrate surface and the nanofibers are oriented locally different in various layers of the composite material and/or in various areas of one layer of the composite material.
The method of the invention may further comprise a step of heating the nanofibers-matrix before and/or while orientating the nanofibers, e.g. within the printing nozzle.
Further, the method of the invention may include a step of curing the deposited (e.g. printed) composite material for preserving the material's structure. The curing may involve a crosslinking step, typically induced by a stimulus such as electromagnetic radiation, in particular UV light, or chemical crosslinking (for polymers, etc.). For this purpose, the nanofibers-matrix mixture may comprise crosslinkable components or functional groups as known in the art.
Summarizing, the method of the invention provides several important advantages over the prior art:
1. Length and diameter of the nanofibers are fully controllable using the extrusion setup. The method allows to produce longer and thicker nanofibers on a large scale. Additional components (such as polysaccharides, proteins, metallic nanoparticles and/or labels) can be added to the polymer-solvent blend to produce labelled composite-nanofibers. The nanoporous membrane can be cleaned with solvents and be re-used many times. The extrusion method allows to continuously produce nanofibers. Depending on the material (such as AAO) the pore diameter of the membrane can be easily controlled. The pore formation within the membrane is self-organized, simplifying the production process.
2. The mechanical properties of the composite material (crosslinking, thickness) are fully controllable by the choice of matrix, fibers and the printing parameters and process.
3. The orientation of the nanofibers is independently and locally controllable throughout the material. This allows to manufacture a composite material with locally independent mechanical properties.
A further, closely related aspect of the present invention relates to a composite material, in particular a composite material with locally independent mechanical properties which is obtainable by the methods of the present invention.
Typically, this composite material will comprise a polymer matrix and orientated nanofibers, wherein the nanofibers are oriented locally different in various areas of the composite material.
More specifically, the composite material is a multi-layered 3-dimensional composite material and the nanofibers are oriented locally different in various layers of the composite material and/or in various areas of one layer of the composite material.
The following non-limiting examples illustrate the present invention in more detail.
Using AAO membranes, nanofibers from several different proteins, polysaccharides and a new variety of nanofibers protein composites could be reproducible extruded. The resulting nanostructures were characterised by scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM) and confocal laser scanning microscopy.
1. Materials and Methods
1.1 Chemicals
Fibrinogen from human plasma was provided by Calbiochem (San Diego, Calif.) and fibrinogen from human plasma labelled with Alexa Fluor 647 was supplied by Life Technologies (Darmstadt, Germany). Collagen type I from calf skin, elastin from bovine neck ligament, laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, chondroitin sulphate sodium salt from shark cartilage and hyaluronic acid sodium salt from Streptococcus equi were purchased from Sigma Aldrich (Munich, Germany). Albumin bovine Fraction V and paraformaldehyde were provided from Serva Electrophoresis GmbH (Heidelberg, Germany).
Phosphate buffered saline (PBS) tablets were provided from Life Technologies. Ethanol was purchased from Carl Roth. G-buffer with pH 7.5 was prepared from 2.0 mM Tris-HCl (Carl Roth, Karlsruhe, Germany), 0.2 mM CaCl2 (Carl Roth), 0.2 mM Adenosine-5′-triphosphate.Na2-salt (ATP, Serva), 0.02% NaN3(Alfa Aesar, Karlsruhe, Germany) and 0.2 mM Dithiothreitol (Serva). D-Buffer at pH 6.5 contained 0.6 mM KCl (Carl Roth) and 50 mM K2HPO4 (Carl Roth). A-buffer at pH 7.4 was prepared from 1 mM KHCO3 (AppliChem, Darmstadt, Germany) and 0.02% NaN3. Tris buffered saline solution (TBS) at pH 7.5 was prepared from 150 mM NaCl (Roth) and 50 mM Tris-HCl. All solutions were prepared with nanopure water from a TKA GenPure system (TKA, Germany).
1.2 Protein Purification
Fibronectin was purified from human plasma by gel filtration and affinity chromatography over a Sepharose CL-4B column (Sigma), followed by a gelatin Sepharose column from GE Healthcare (Munich, Germany). Subsequently, fibronectin was eluted by 6 M urea (Sigma) in PBS and dialyzed against PBS before use.
Actin was isolated from an acetone powder of rabbit skeletal muscle in G-buffer by modifying the protocol of Spudich and Watt (in Journal of Biological Chemistry 246, 4866 ff). Actin was polymerized by adding 50 mM KCl and 2 mM MgCl2 (Carl Roth). Subsequently, KCl and MgCl2 were removed by dialysis with G-buffer, and the depolymerized actin was purified by gel filtration with a Superdex 200 column (GE Healthcare). According to the protocol of Margossian and Lowey (in Methods in Enzymology 85, 55-71) also isolated was myosin II from rabbit skeletal muscle using centrifugation and salting out. The purified myosin was diluted in D-buffer.
α-actinin was isolated from chicken gizzard following the protocol of Craig et al. (in Methods in Enzymology 85,316-321). After extraction with 1 mM KHCO3 α-actinin was salted out with (NH4)2SO4 (Carl Roth) and purified with ion exchange chromatography over a DEAE column (GE Healthcare) and gel filtration with a Superdex 200 column. Isolated α-actinin was stored in A-buffer.
1.3 Anodic Alumina Membranes
Nanoporous AAO membranes with pore diameters dAAO of 21 and 450 nm were prepared by anodization in a home-built setup according to Raoufi et al. (in Langmuir 28, 10091-69). Both sides open anodic alumina membrane were obtained by removing the underlying aluminum substrate (in a solution containing 3.5 g of CuCl2.H2O (Alfa Aesar), 100 mL of HCl (37 wt %, Carl Roth), and 100 mL of H2O) followed by chemical etching of the barrier layer (0.5 M aqueous phosphoric acid (Carl Roth) at 30° C.). Commercial Whatman® Anodisc membranes with a diameter of 200 nm were purchased from Sigma.
1.4 Extrusion of Nanofibers
For the preparation of various nanofibers, a customized extrusion setup was designed (see
1.5 Microscopic Analysis and Cell Culture
After extrusion, the protein and composite fibers were coated with approximately 7 nm gold and analyzed with scanning electron microscopy (SEM) using a Zeiss Ultra 55cv device (Zeiss, Oberkochen, Germany). All measurements were performed with an operation voltage of 3 to 5 kV. The software Image J (1.44 p) was used to analyses the SEM images. The inventors statistically analyzed the average fiber diameter from at least 30 fibers and standard deviation as error.
Rat embryonic fibroblasts stably transfected with paxillin fused to yellow fluorescent protein (REF-YFP-paxillin) were a kind gift of Benjamin Geiger (Weizmann Institute of Science, Rehovot, Israel). REF-YFP-paxillin cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/ml penicillin-streptomycin (all from Gibco Laboratories, Eggenstein, Germany) at 37° C. and 5% CO2. Before seeding cells onto the protein nanofibers substrates, REF-YFP-paxillin cells were trypsinized with trypsin-EDTA 2.5% solution (Gibco Laboratories) for 3 min. Cells were seeded at a density of 5×105 per substrate in DMEM containing 1% FBS. Live cell phase contrast microscopy investigation was performed with 10 ×/0.25 Ph1 A-Plan objective (Zeiss, Jena, Germany) using an AxioVert 40 CFL microscope (Zeiss, Jena, Germany). To characterize focal adhesion formation 63×/1.25 Ph2 Plan Semi Apo Phase objectives (Zeiss, Jena, Germany) were used.
2. Results
The method of the present invention enabled to fabricate nanofibers from a large variety of biopolymers under physiological conditions. Nanoporous aluminum oxide templates were used to extrude various ECM and intracellular proteins as well as polysaccharides and composites thereof into nanofibers with different hierarchical assemblies.
2.1 ECM Proteins
Using the customized setup (compare
The cross-linking of extruded protein nanofibers with PFA or other agents like carbodiimide or genipin could increase their mechanical properties, which can be beneficial for the development of novel durable biomaterials.
Furthermore, fluorescent fibrinogen labelled with Alexa 647 were extruded through 200 nm pores at a concentration of 10 μg/ml. In this extrusion, nanofibers with an average diameter of 34 nm were fabricated, which is in good agreement with the extrusion of unlabeled fibrinogen. The confocal microscopy image of a fluorescent fibrinogen fiber bundle in PBS solution shows that the fluorescent label was still functionally active after the protein solution was extruded into nanofibers (see
For collagen and fibronectin diluted in PBS, it was investigated how the diameter of extruded nanofibers depends on the concentration of the protein solution and the diameter of the nanoporous AAO membrane. Using pore diameters of 20 and 200 nm and varying the protein concentration between 10 and 1000 μg/ml it was possible to reproducibly control the nanofibers dimensions (see
For both collagen and fibronectin, it could be shown that the nanofibers diameter increased from approximately 10 nm at 10 μg/ml to 17 and 18 nm at 1000 μg/ml, respectively, when a pore diameter of 20 nm was used. With pore diameters of 200 nm the collagen and fibronectin fiber diameters increased from 29 and 32 nm at 10 μg/ml to 144 and 151 nm at 1000 μg/ml. Thus, for low protein concentrations the fiber diameter stayed below the diameter of the template nanopores and reached the dimension of the pore diameter when the protein concentration was increased.
These results clearly indicate that the diameter of extruded protein nanofibers can be tailored by adjusting the pore diameter and the protein concentration. In the novel extrusion approach the advantage of physiological buffers is combined with precise control of the nanofibers dimensions, which could not be achieved with the previously presented flow processing technique, which also utilized physiological solutions (Lai et al., in Regenerative Medicine 7, 649-691).
To assess the biocompatibility of extruded ECM proteins, the growth of REF-YFP-paxillin cells on nanofibers of collagen type I was studied. The nanofibers with an average diameter of 34±4 nm were deposited on glass slides with PLL coating and arranged into mesh-like mats as shown in
These results indicate that nanofibers meshes of ECM proteins are biocompatible. The large-scale fabrication of nanofibers ECM protein assemblies could lead to a novel class of tissue engineering scaffolds with defined porosity and density. Furthermore, different hierarchical nanofibers assemblies with varying stiffness could be used to specifically control cell adhesion and alignment or to induce stem cell differentiation.
2.2 Intracellular Proteins
In its natural environment, the intracellular protein actin also assembles into filamentous structures, which are interconnected by α-actinin, thus forming the cellular cytoskeleton. The actin-based cell motility is driven by myosin, a molecular motor, which binds to the actin filaments and converts ATP into mechanical energy. Therefore, the present inventors have also analyzed the extrudability of these intracellular proteins to find out whether cellular protein fiber networks can be reconstructed with the new approach.
Actin was diluted in G-buffer, myosin II was prepared in D-buffer, and α-actinin was diluted in A-buffer. All proteins were extruded with the standard setting of 200 nm pore diameter and a protein concentration of 10 μg/ml. This process reproducibly yielded nanofibers assemblies with average diameters of single nanofibers ranging from 31 to 37 nm (see
For actin, extrusions through 200 nm pores with 100 μg/ml were also performed, which yielded a fiber diameter of 64±6 nm (see
With the extrusion process the dimensions as well as the buffer conditions can be well controlled to mimic the natural environment of intracellular proteins more closely.
2.3 Protein Composites
The natural ECM consists of nanofibers from various ECM proteins, which are surrounded by an aqueous solution of long-chain polysaccharides, such as hyaluronan and chondroitin sulphate. To design novel biomaterials, which mimic the natural cellular environment more closely, the present inventors also prepared nanofibers composites from different ECM proteins and ECM proteins blended with polysaccharides. Furthermore, it was possible to extrude blended solutions of intracellular proteins and pure polysaccharides into nanofibers composites. All solutions were extruded with a total protein or blend concentration of 10 μg/ml using 200 nm large pores and the physiological buffers listed in Table 3. Thus, different nanofibers arrangements with single fiber diameters ranging from 28 to 38 nm were obtained.
A blend of collagen and fibronectin was successfully extruded into micron-sized bundles of blended nanofibers (see
The inventors also blended collagen with the polysaccharides hyaluronan and chondroitin sulphate, respectively, and were able to extrude composite nanofibers, which were assembled into expanded assemblies (
Such nanofibers composites containing different ECM proteins and/or polysaccharides could find application as tailored tissue engineering scaffolds which closely mimic a specific tissue in vitro.
Furthermore, extruding a blend of the intracellular proteins actin and myosin with the standard setting yielded 2-dimensional arrangements of nanofibers (se
Summarizing, it was possible to fabricate nanofibers of various biopolymers, including polysaccharide fibers as well as protein fibrils made of, e.g., fibronectin, fg, actin, collagen, myosin, BSA, α-actinin and laminin. The same principal approach is applicable for different type of polymers with different concentrations in different buffers.
The precise control over nanofibers geometry and alignment which is possible with this approach is advantageous for a wide range of applications in nanofabrication and tissue engineering. In an especially advantageous application, these nanofibers can be further processed to fabricate novel composite materials with improved properties as described above and in Example 2.
A composite material with locally controlled mechanical properties (which may mimic the 3D orientation of chitin fibers found within natural arthropod cuticle) can be prepared from mixtures of chitosan nanofibers bundles in a polylactide matrix.
The nanofibers and nanofibers bundles can be prepared using the methods described above. Chitosan (Sigma Aldrich, no. 448877) with a concentration of 1 mg/ml is dissolved under permanent stirring in acetic acid (1%) over a period of 24 h. The chitosan-acid mixture is extruded through an AAO membrane with a pore diameter of 200 nm. The nanofibers-solvent mixture is carefully centrifuged at low speeds to sediment the fibers. PLA powder is melted (approx. 210° C), mixed with the sedimented fibers and pressed into a filament form, compatible with a commercially available 3D printer (such as Makerbot Replicator, 1.75 mm filament diameter). The printer is equipped with a custom-designed printing head, including a temperature-controlled nozzle and perpendicularly orientated electrodes. A customized printing software is used to generate printing code, compatible with the customized printer. The code includes standard printing information (x,y,z position of the printing nozzle, filament extrusion and nozzle temperature) as well as the orientation and strength of an electric field. The filament is loaded into the printing nozzle and heated up. Once the filament is melted within the nozzle, the electric field and the dipole moment of the chitosan fibers are used to orientate the fibers. The melted, oriented filament is then printed layer by layer onto a desired substrate. When cooling down, the filament orientation within the printed filament is preserved.
Nanofibers with embedded iron oxide nanoparticles can be prepared by extrusion to facilitate fiber orientation in an external magnetic field.
The nanofibers and nanofibers bundles were prepared using the methods described above. Chitosan (Sigma Aldrich, no. 448877) with a concentration of 0.5 mg/ml was dissolved under permanent stirring in acetic acid (1%) over a period of 24 h.
Iron oxide nanoparticles with a diameter between 10 and 50 nm were added to the acidic chitosan solution with a final concentration of 0.1 mg/ml. This composite solution was extruded through an AAO membrane with a pore diameter of 200 nm using a constant flow rate of 500 μl/min.
The extruded fibers with embedded particles were placed in a custom-built observation chamber with adjustable magnetic field (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 14003414.1 | Oct 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/001942 | 10/2/2015 | WO | 00 |
