WO2013131079A1

WO2013131079A1 - Fabrication of 3-d articles with chitosan

Info

Publication number: WO2013131079A1
Application number: PCT/US2013/028847
Authority: WO
Inventors: Donald E. Ingber; Javier Gomez FERNANDEZ
Original assignee: President And Fellows Of Harvard College
Priority date: 2012-03-02
Filing date: 2013-03-04
Publication date: 2013-09-06

Abstract

The invention provides methods for fabricating 3-D articles, e.g. consumer products from chitosan by inducing a desired viscosity in a chitosan solution and fabricating the 3-D article from the solution. The invention also provides the 3-D articles fabricated using the method.

Description

FABRICATION OF 3-D ARTICLES WITH CHITOSAN

RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 61/605,999, filed March 2, 2012, the content of which is incorporated herein by reference in its entirety.

TECHNOLOGICAL FIELD

[0002] The present disclosure provides methods for fabricating 3-D articles, e.g. consumer products. The present disclosure also provides chitosan based 3-D articles obtained by the method disclosed herein.

BACKGROUND

[0003] Non-degradable plastics are a growing problem in the world. The United States produce over 10.5 million tons of plastic waste a year, but recycles only 1 or 2% of it.

[0004] Plastics are manufactured from petroleum. This brings a host of issues (destruction of habitat, extraction of crude oil, security issues from the volatile countries where oil is produced, processing of petroleum, chemical manipulation into various types of plastics). The manufacture involves many chemicals, many of which have not been sufficiently tested for their toxicological impact on humans or animals.

[0005] The final plastic product is often a chemical entity that in and of itself has had insufficient toxicological and ecotoxicological testing.

[0006] Recent controversies over plastic bottles (many toxicologists recommending not reusing plastic water bottles and not storing food in tupperware) have highlighted the potential risks. Exacerbating the problem is that science is only now advancing to where it can detect plastic components in human blood and then trace concentrations and link them to human ailments and diseases.

[0007] The plastics industry itself often spins plastic and related plastic chemicals into a variety of products, some of which are hazardous and controversial (Teflon, PVC, Polyethylene, polystyrene, various silicones in body and hair care).

[0008] Plastics often leech component chemicals, including hazardous chemicals, through common temperature changes. It is for this reason that toxicologists do not recommend storing very cold foods in plastics or heating foods (microwaving especially) in plastics.

[0009] Plastics are durable materials. Thus, they are hard to eliminate once used and create tremendous waste. While some common plastics can be recycled (#1 and #2 plastics used in common soda and milk bottles), the vast majority cannot. They take up a lot of space in landfills and create air pollution when incinerated. Their accumulation in the third world countries is not only a source of pollution, but they also create stagnant pools which can become a breeding ground for malarial mosquitoes; and they suffocate or disrupt the digestion of animals that accidentally consume them.

[0010] Because most plastics require between 50 and 1 million years to decompose

(depending also on the thickness), nearly every piece of plastic ever made still exists today. Much of this plastic ends up in the ocean, where it is estimated that about 14 billion pounds of trash per year, much of it plastic, is being dumped. As a consequence, 1 million sea creatures die every year.

[0011] As most existing plastic come from non renewable sources, this problem grows with the expanding population and as we consolidate our development in disposable technologies, where consumer products are made for a limited use.

[0012] Hence, there is an important need for replacements for synthetic plastic that are strong, resistant to wear yet fully biodegradable and inexpensive.

[0013] Chitin is the second most abundant polymer on earth after cellulose, it is common waste in seafood factories and (as a natural polymer) it is biodegradable. However its processing in the laboratory for fabrication produce a hydrogel material with poor mechanical properties.

[0014] Chitin (CsH^OsN),, is a long-chain polymer of a N-acetylglucosamine, a derivative of glucose, and is found in many places throughout the natural world. It is the main component of the cell walls of fungi, the exoskeletons of arthropods such as crustaceans (e.g., crabs, lobsters and shrimp) and insects, the radulas of mollusks, and the beaks of cephalopods, including squid and octopuses. Chitin can also be obtained from a wide variety of other sources including arthropod exoskeletons and the cell walls of different forms of fungi.

[0015] In terms of structure, chitin can be compared to the polysaccharide cellulose and, in terms of function, to the protein keratin. Chitin has also proven useful for several medical and industrial purposes.

[0016] Chitin is insoluble in water and most commonly used solvents. The insolubility of chitin makes it difficult to process and makes formation of fibers, films, and other products therefrom problematical using prior art processing methodologies.

[0017] Chitosan is the product of deacetylation of chitin, and processes the formula:

It is an amorphous solid which is more soluble in water, having a pH below 6, than chitin, but it usually requires the use of aqueous organic acids to attain solubility. Chitosan is of nearly identical structure to chitin, except that it is de-acetylated. The chemical structure of chitosan is as follows:

[0018] Because chitosan is more easily solubilized and processable, a great deal of researchers and industrialists have experimented with and/or used chitosan coatings in a wide variety of applications. Consequently, more common is the reshaping and regenerating of chitosan than the one of chitin. Chitosan regeneration always involves treatment with alkali so as to neutralize carboxylic acid anions, and this produces chitosan which continues to be soluble in mild acids. Chitin, by contrast, is insoluble in all common solvents, with the exception of dimethyl acetamide anhydrous ca. 8% lithium chloride, DMAc/LiCl.

[0019] U.S. Pat. No. 4,309,534 to Austin describes a process for preparing renatured chitosan having particular optical rotation characteristics. Chitosan is aged in aqueous acid for a period of weeks. During this time period chitosan partial acetate is formed, and the salt slowly loses the acetic acid during aging. Thereafter, the chitosan/partially deacetylated chitin is poured out as a film and immersed in dilute base, such as sodium bicarbonate, to neutralize the acetic acid and regenerate the chitosan film as the free base.

SUMMARY

[0020] One aspect of the invention provides a method for fabricating an article using chitosan. In some embodiments, the method comprising obtaining a desired viscosity in a chitosan solution. In some embodiments, the method comprises using a chitosan solution comprising a filler material. The method also comprises using the chitosan solution to fabricate the article using any techniques used for processing polymeric materials into articles. After the chitosan solution has been processed into the desire article, article can be treated to induce a conformational change in the chitosan. This method can be repeated until obtaining the desired thickness of the chitosan.

[0021] In some embodiments, the article is fabricated using injection molding.

[0022] In another aspect, the disclosure provides a composition comprising chitosan and a filler material. Generally, the filler material is dispersed homogeneously in the chitosan matrix. In some embodiments, the filler material is selected from the group consisting of sand, wood flour, sodium carbonate, and coconut-based soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figs. 1 and 2 show some exemplary chitosan-based articles made by an embodiment of the process disclosed herein.

[0024] Fig. 3 shows encapsulation and extraction of molecules (e.g., dyes) from chitosan based articles.

[0025] Fig. 4 shows a dye containing chitosan film after a month in water.

[0026] Figs. 5A-5I show characterization of molecular arrangement. Fig. 5A - Films produced by different approaches. Left sequence is the brittle result of a coagulation process (data not shown). Right sequence is the characteristics of the film after the evaporation of the solvent (data not shown). Fig. 5B - Example of Mechanical test of the chitosan films by basic precipitation (black) and by evaporation (red). Fig. 5C - FTIR analysis of evaporated films after fabrication (red), stretched (green) and coagulated films (black). Fig. 5D - X-ray diffraction pattern of the same samples. Notable is the absence of the shoulder at 21.5°, differencing tendon (crab) and L-2 (shrimp) chitosan. Fig. 5E - 3D reproduction of the two fold helical structure of chitin chains. Grey atoms are C, red O, blue N and white H. Fig. 5F- Detail of the interaction between adjacent chains. Dashed lines represent water mediated bonds. Hydrogen atoms have been removed for clarity. Fig. 5G- Birefringence measurements of a coagulated film. Different colors represent different molecular alignment, characterized by the orientation of the slow axis of the material. Fig. 5H- Same analysis of a evaporated film, showing much larger domains. Fig. 51- Rearrangement of the chains after a unidirectionally stretch the film.

[0027] Figs. 6A-6C show rheological characterization of chitosan solutions. Fig. 6A - Variation of the G7G" (i.e. Tag da) with respect the concentration of polymer. Fig. 6B - Variation of the G7G" ratio of a 3% solution of chitosan with the temperature. The percentage indicates the stage of the crystallization process using amount of solvent removed as indicator. Fig. 6C - Images of a sphere of polymer solution at different temperatures after the removal of 60% of the solvent.

[0028] Figs. 7A-7T shows fabrication of 3D objects with chitosan. Fig. 7A - Diagram of the fabrication process. An initial solution of 3% chitosan is concentrated until reach the viscosity necessary to be molded. To cast the solution, it is warmed up to decrease the viscosity and poured over the mold. The viscosity increase at room temperature helps to keep the polymer on the walls of the mold, where the rest of the solvent is evaporated. The final crystallized form of chitosan is separated from the mold after the evaporation of the remaining solvent. In the case of injection molding the polymer (by its own or mixed with a filler) is concentrated to a plastic state. This solid is warmed up at 80°C to be injected in the mold. Just after the injection the mold is open and the fabricated objects removed. Fig. 7B - Examples of geometries fabricated by casting. Fig. 7C - Examples of articles fabricated using embodiment of the method disclosed herein. The chitosan solution used for fabrication included water soluble dyes (i.e Tartrazine, Allura Red, Brilliant Blue FCF and Fast Green FCF). Fig. 7D - Encapsulation of molecules (e.g., dye moleucles) at different pH's. Insets represent the capture and removal of spheres, representing dye molecules, depending on the polymer conformation. Fig. 7E - Mechanical properties of the material before and after 12h under water when treated with different organic waterproof coatings (i.e. bee wax and parylene, data not shown). Fig. 7F - Example of chess pieces made by injection molding using wood flour as fillers. Queen is 41.08mm tall. Fig.7G - Example of three chess pieces made of chitosan just after being casted in a single epoxy mold. Fig. 7H - The queen after fully removing the solvent. It shrinks to about 2/3 of the original volume. Fig. 71 - Injection of the chitosan matrix with sand grains just after the casting. Fig. 7 J - A close-up view of the horse after the evaporation of all the solvent. Figs. 7K and 7L - Same images as Fig. 71 and Fig. 7J using calcium carbonate as filler. Figs. 7M and 7N - SEM images of the mineral/organic composite employed in injection molding. Using fillers the definition of the features is determined by the grain size. Objects fabricated with sand (grain size of about 300μιη) contrast with the smooth surfaces in Fig. 7K and 7L produced by pure calcium carbonate, made of the about 15 μιη size crystals shown in Figs. 70 and 7P. Fig. 7Q - Closer look of the colorless egg carton of Fig. 7C. Fig. 7R - Closer view of a wood based piece. Fig. 7S - Three different pieces based in wood. Fig. 7T- An example of three different fillers together.

[0029] Figs. 8A and 8D show the recycling process of the polymer. Fig. 8A - the recycling process: Cycle Step A is the main recycling process, where a film (up) is immersed in an acidic solution (down), where it is dissolved (middle) and reused. If some colorant is added to the solution the colored polymer (middle) it can be casted in a colored film (Cycle B up) which can be redisolved again in an acidic solution (Cycle B down). Both cycles (A and B) are connected by the process Cycle C, where the colored polymer is precipitated in a basic solution (Cycle C, bottom right) and the color is separated from the polymer (Cycle C, bottom left). The former can be casted again (Cycle A). Fig. 8B shows strength of the polymer after each recycling process (Cycle A in Fig. 8A). Each cycle comprise the casting of a film, the removal of the remaining acetic acid, the re-dissolution of the film and its casting again.

[0030] Fig. 9 shows an example of crystal domains measurements. A slow axis measurement picture in combination with a color analysis of the result. In the image (particularly complicate because the presence of a nucleation point at the top left), we have traced a white contour of the domains found as connected areas with an maximum dispersion of the slow axis alignment of 15°. The area of all the domains is measured as the number of pixels included in it.

[0031] Fig. 10 shows an example of the FTIR characterization. FTIR analysis of the samples: chitosan films (red), stretched chitosan films (green) and coagulated films (black) where the symmetric and asymmetric stretching modes of the carboxilated anions have been marked.

[0032] Figs. 11A and 11B shows chitosan foams. Fig. 11A - Example of chitosan foam made by beating a solution. Fig. 11B - Example of chitosan foam based in the reaction of the acetic acid solvent with sodium bicarbonate.

DETAILED DESCRIPTION

[0033] The present invention discloses a method for fabrication a chitosan based articles, such as consumer products. While consumer products are discussed herein, it is to be understood that the fabricated article can be used for industrial or commercial purposes also. Discussion of consumer product is in no way intended to limit the type, shape, or use of the article fabricated using the methods and materials described herein.

[0034] Inventors have found inter alia that inducing a liquid-crystal state in a chitosan solution allows easy fabrication into various 3-D articles using commonly used techniques for fabrication of large size structures such as consumer products. Without wishing to be bound by a theory, chitosan in a liquid-crystal form can be easily fabricated into various articles, such as consumer products, using fabrication techniques known in the art and available to the artisan. Accordingly, one aspect of the invention relates to a method for fabricating a consumer product, the method comprising obtaining a desired viscosity in a solution of chitosan. After fabrication of an article, the article can be treated to induce a conformational change in chitosan.

[0035] The inventors have also discovered that shrinkage of chitosan based fabricated articles can be alleviated, inhibited, or reduced using a chitosan solution comprising a filler material. When a filler material is used, the chitosan can be acting as a binder to hold the filler material together. Thus, in some embodiments, the chitosan solution for fabrication of a 3-D article comprises a filler material. Without wishing to be bound by a theory, it is believed that the chitosan forms a solid network around the filler material, which resists compression when the solvent evaporates. Without limitations, any material can be used as the filler material.

Exemplary filler materials include, but are not limited to, sand; sodium carbonate; coconut-based soil; wood flour; cellulose; minerals; glass;, inorganic oxides such as aluminum oxide (AI₂O₃), silicon dioxide (Si0₂), magnesium oxide (MgO), calcium oxide (CaO), zinc oxide (ZnO) and titanium dioxide (Ti0₂); carbon black (also known as furnace black); silicates such as clays, talc, wollastonite (CaSiC^), magnesium silicate (MgSC^) anhydrous aluminum silicate, and feldspar (KAlSi₃0s); sulfates such as barium sulfate and calcium sulfate; metallic powders such as aluminum, iron, copper, stainless steel, and nickel; carbonates such as calcium carbonate (CaC0₃) and magnesium carbonate (MgCo₃); mica; silica (natural, fumed or precipitated); and nitrides and carbides, such as silicon carbide (SiC) and aluminum nitride (A1N). [0036] Without limitations, the filler material can be distributed homogeneously or heterogeneously within the chitosan matrix. As used herein, a homogenous distribution of the filler material means the filler material is randomly and/or uniformly distributed within the chitosan matrix. A heterogeneous distribution of the filler material means that the filler material is not randomly and/or uniformly distributed within the chitosan matrix. Thus, a heterogeneous distribution includes filler material distributed in one or more patterns in the matrix material. Without wishing to be bound by theory, a non-random distribution allows different regions of the fabricated article to have different physical or mechanical properties.

[0037] Without limitations, the filler material can be present in virtually any form, such as powder, pellet, fiber, sphere or bead. In some embodiments, the filler material is in the form of a particle, e.g., microparticels or nanoparticles. The term "particle" includes spheres; rods; shells; and prisms; and these particles can be part of a network or an aggregate. Without limitations, the filler material can have any size from nm to millimeters. As used herein, the term

"microparticle" refers to a particle having a particle size of about 1 μιη to about 1000 μιη. As used herein, the term "nanoparticle" refers to particle having a particle size of about 0.1 nm to about 1000 nm. Inventors have discovered that filler material particles of smaller sizes provide smooth surfaces in the fabricated articles. Thus, based on the desired surface morphology of the fabricated article, filler material of a particular size can be used. In some embodiments, the filler material has a particle size of about 50 μιη to about 10 mm.

[0038] It will be understood by one of ordinary skill in the art that particles usually exhibit a distribution of particle sizes around the indicated "size." Unless otherwise stated, the term "particle size" as used herein refers to the mode of a size distribution of particles, i.e., the value that occurs most frequently in the size distribution. Methods for measuring the particle size are known to a skilled artisan, e.g., by dynamic light scattering (such as photocorrelation

spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other techniques (such as rheology, and light or electron microscopy).

[0039] In some embodiments, the particles can be substantially spherical. What is meant by "substantially spherical" is that the ratio of the lengths of the longest to the shortest perpendicular axes of the particle cross section is less than or equal to about 1.5. Substantially spherical does not require a line of symmetry. Further, the particles can have surface texturing, such as lines or indentations or protuberances that are small in scale when compared to the overall size of the particle and still be substantially spherical. In some embodiments, the ratio of lengths between the longest and shortest axes of the particle is less than or equal to about 1.5, less than or equal to about 1.45, less than or equal to about 1.4, less than or equal to about 1.35, less than or equal to about 1.30, less than or equal to about 1.25, less than or equal to about 1.20, less than or equal to about 1.15 less than or equal to about 1.1. Without wishing to be bound by a theory, surface contact is minimized in particles that are substantially spherical, which minimizes the undesirable agglomeration of the particles upon storage. Many crystals or flakes have flat surfaces that can allow large surface contact areas where agglomeration can occur by ionic or non-ionic interactions. A sphere permits contact over a much smaller area.

[0040] In some embodiments, the particles have substantially the same particle size. Particles having a broad size distribution where there are both relatively big and small particles allow for the smaller particles to fill in the gaps between the larger particles, thereby creating new contact surfaces. A broad size distribution can result in larger spheres by creating many contact opportunities for binding agglomeration. The particles described herein are within a narrow size distribution, thereby minimizing opportunities for contact agglomeration. What is meant by a "narrow size distribution" is a particle size distribution that has a ratio of the volume diameter of the 90th percentile of the small spherical particles to the volume diameter of the 10th percentile less than or equal to 5. In some embodiments, the volume diameter of the 90th percentile of the small spherical particles to the volume diameter of the 10th percentile is less than or equal to 4.5, less than or equal to 4, less than or equal to 3.5, less than or equal to 3, less than or equal to 2.5, less than or equal to 2, less than or equal to 1.5, less than or equal to 1.45, less than or equal to 1.40, less than or equal to 1.35, less than or equal to 1.3, less than or equal to 1.25, less than or equal to 1.20, less than or equal to 1.15, or less than or equal to 1.1.

[0041] Geometric Standard Deviation (GSD) can also be used to indicate the narrow size distribution. GSD calculations involved determining the effective cutoff diameter (ECD) at the cumulative less than percentages of 15.9% and 84.1%. GSD is equal to the square root of the ratio of the ECD less than 84.17% to ECD less than 15.9%. The GSD has a narrow size distribution when GSD<2.5. In some embodiments, GSD is less than 2, less than 1.75, or less than 1.5. In one embodiment, GSD is less than 1.8.

[0042] Amount of the filler material in the chitosan solution can range from 0.01% to about 99%. In some embodiments, amount of the filler material in the chitosan solution ranges from about 1% to about 95%, from about 5% to about 90%, from about 10% to about 85%, from about 15% to about 80%, from about 20% to about 75%, from about 25% to about 70%, from about 30% to about 65%, or from about 35% to about 60%, from about 40% to about 55%. The amount of the filler material can be based on the weight or moles of the filler material to the moles, volumes or weight of chitosan in the solution, or the total volume or total weight of the chitosan solution. Thus, the % amount of the filler material can be stated as mole/mole, weight/weight, mole/weight, weight/mole, volume/volume, or weight/volume. Generally, the article fabricated from a given chitosan solution will comprise the different components in the same ratio as in the chitosan solution used. In other words, filler material/chitosan ratio in the fabricated article is about the same as in the solution used for fabrication. In some embodiments, the chitosan/filler ratio is about 1:5 by weight.

[0043] Inventors have discovered that chitosan solution of about 5% to about 40% (e.g., 10% to about 30%, or about 15% to about 25%) chitosan/filler material weight concentration are easily amenable to injection molding procedures. Accordingly, in some embodiments, chitosan solution used for fabricating an article using injection molding is about 20% chitosan/filler material weight concentration.

[0044] While the method and compositions herein are described using chitosan, the method and compositions can be carried using other molecules such as polymers, degradable polyesters such as poly(lactide-co-glycolide), proteins, nucleic acids, carbohydrates, or any other biocompatible biodegradable molecules.

[0045] Generally, the fabricating step comprises a step of introducing the chitosan solution into a mold (i.e. conforming the chitosan solution to a mold or casting the chitosan solution into/onto a mold); and removing at least a part of the solvent from the chitosan solution in the mold. In some embodiments, the article can be removed from the mold after a part of the solvent is removed from the solution and the remaining solvent removed outside the mold. Without limitations any method known to one of skill in the art can be used for removing the solvent. Exemplary methods for removing the solvent are described below.

[0046] The mold can be of any size and shape. In some embodiments, the molds are molds of consumer products. In some embodiments, the molds are molds for bottles, catering products, closures, container, cutlery, drink cups, food packaging, lids, overcap, prescription containers, or tubes. In some embodiments the molds are molds for agricultural film, building products, corrosion protection, custom films, flexible intermediate bulk containers (FIBCs), flexible packaging, food and film packaging, medical films, personal care film, sheeting, drop cloths, stretch film, grocery and cutterbox film, tapes, or trash bags.

[0047] In some embodiments, the process comprises, dissolving chitosan in an acetic solution; removing the solvent until a chitosan solution of a desired viscosity is obtained;

conforming the viscous chitosan to a mold; and removing at least a part of the solvent from the chitosan solution in the mold. In some embodiments, the article can be removed from the mold after a part of the solvent is removed from the solution and the remaining solvent removed outside the mold.

[0048] Without limitations, any art known method of forming 3-D articles from a solution can be employed in the process described herein. For example, as an artisan is well aware, polymeric materials can be processed into an article using a number of conventional techniques including, but are not limited to, solvent casting, melt molding, blow molding, compression molding, transfer molding, injection molding, and the like.

[0049] In the solvent casting, a liquid body in which individual components are dissolved or dispersed in a solvent is cast on a support, and then the solvent is dried, either partially or fully. Solvent casting can be repeated until the thickness of the article body is of desired thickness.

[0050] Examples of the melt molding include melt extrusion such as T-die, inflation molding, and the like, a calendar method, thermal pressing, injection molding, and the like.

[0051] In a typical blow molding process, a parison (an essentially cylindrical polymeric sleeve) is extruded and positioned within a mold, while still being moldable. Pressurized gas can be introduced into the interior of the parison which causes it to expand against walls of the mold. A variety of articles can be produced using blow molding techniques including bottles, containers, cases, automotive parts, toys and panels.

[0052] In some embodiment, the 3-D article is fabricated by a layer-by-layer process. For example, the mold can be coated with a first layer of chitosan. Such coated mold can then be coated with a second layer of chitosan of similar or not viscosity. This process can be repeated until the total chitosan reaches the proper thickness. After each coating with chitosan, the chitosan-layer can be optionally treated to induce a change in the conformation as described above.

[0053] Thickness of each chitosan layer can independently range from a few angstroms to millimeters, e.g., from about 1 A to about 5 mm. In some embodiments, thickness of the each chitosan layer can range from about 1 to about 250 μιη. In some embodiments, thickness of each chitosan layer is selected independently from the group consisting of from about 1 to about 100 μιη, from about 1 to about 75 μιη, from about 1 to about 50 μιη, from about 1 to about 40 μιη, from about 1 to about 30 μιη, from about 1 to about 25 μιη, from about 1 to about 20 μιη, from about 1 to about 15 μιη, from about 1 to about 10 μιη, and from about 1 to about 5 μιη. In some embodiments, all chitosan layers have the same thickness. In some embodiments, at least two layers have different thickness.

[0054] Generally, total thickness of the chitosan layers can range from about 1 to about 500 μιη. In some embodiments, total thickness of the chitosan layers can range from about 1 to about 250 μιη, from about 1 to about 150 μιη, from about 1 to about 100 μιη, from about 1 to about 75, from about 1 to about 50 μιη, from about 1 to about 25 μιη, from about 1 to about 20 μιη, from about 1 to about 15 μιη, from about 1 to about 10 μιη, or from about 1 to about 5 μιη.

[0055] In some cases it can be useful to first coat the mold surface with a low viscosity solution. This then can be followed by a second layer fabricated from a higher viscosity solution. This can be useful when fabricating complex structures. The low viscosity solution can allow the solution to cover the mold surface and the higher viscosity then providing the desired physical and mechanical properties. The process could be repeated as many times as necessary. As used herein, "low viscosity" means a viscosity of 50 Pa.s, 10 Pa.s, 1 Pa.s, 100 mPa.s, 50 mPa.s, 25 mPa.s, 20 mPa.s, 15 mPa.s, 10 mPa.s, 9 mPa.s, 8 mPa.s, 7 mPa.s, 6 mPa.s, 5 mPa.s, 4 mPa.s, 3 mPa.s, 2 mPa.s, 1 mPa.s or lower.

[0056] The ratio of viscosity of low viscosity solution to high viscosity solution can be 10 or higher, i.e., viscosity of the high viscosity solution is at least 10 times higher than that of the low viscosity solution. The viscosity ratio can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 750, or at least 1000.

[0057] In some embodiments, at least two layers of chitosan have the same viscosity. In some embodiments, at least two layers of chitosan have different viscosity. In some

embodiments, each layer has different viscosity. In some embodiments, all layers have the same viscosity.

[0058] The different layers can be made with different concentration chitosan solutions. In some cases it can be useful to first coat the mold surface with a low concentration chitosan solution. This then can be followed by a second layer fabricated from a higher concentration solution. This can be useful when fabricating complex structures. The low concentration solution can allow the solution to cover the mold surface and the higher concentration then providing the desired physical and mechanical properties. The process could be repeated as many times as necessary.

[0059] The ratio of low concentration to high concentration can be 1.1 or higher, i.e., the higher concentration solution is 1.1 or more times concentrated than the low concentration solution. The concentration ratio can be at least 1.1, at least 1.2, at least 1.4, at least 1.4, at least 1.5, at least 2, at least 2.5, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100.

[0060] In some embodiments, the layer-by-layer method comprises: (i) coating a mold surface with a chitosan solution to form a coating layer comprising chitosan; and (ii) repeating step (i) until the chitosan layer is of desired total thickness.

[0061] In some embodiments, the method comprising inducing a conformational change in the chitosan coating layer before the next layer is coated on. Thus, in some embodiments, the method comprises: (i) coating a mold surface with a chitosan solution to form a coating layer comprising chitosan; (ii) inducing a conformational change in the chitosan coating layer; and (iii) optionally repeating steps (i) and (ii) until the chitosan layer is of desired total thickness. [0062] In some embodiments, the process is repeated at least twice. For example, the coating process is repeated 2, 3, 4, 5, 6, 7, 8, or 10 times. In some embodiments, the process is repeated at least ten times. In some embodiments, the process is repeated at least a hundred times. For example, the coating process is repeated at least 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more times. In some embodiments, the process is repeated at least a thousand times. In some embodiments, the coating process is repeated before inducing the conformational change. In some embodiments, a conformational change is induced in the layer before the next coating is applied.

[0063] In one embodiment, the method comprises: (i) inducing a liquid-crystal state in a solution of chitosan; (ii) coating a mold surface with a chitosan solution to form a coating layer comprising chitosan; (iii) optionally inducing a conformational change in the chitosan coating layer; and (iv) optionally repeating steps (ii) and (iii) until the chitosan layer is of desired thickness.

[0064] In some embodiments, the coating layer is multilaminar. In some embodiments, the multilaminar chitosan layer comprises at least two chitosan layers. In some embodiments, the multilaminar chitosan layer comprises at least ten chitosan layers. In some embodiments, the multilaminar chitosan layer comprises at least a hundred chitosan layers. In some embodiments, the multilaminar chitosan layer comprises at least a thousand chitosan layers. When the coating layer is multilaminar, different layers can be fabricated using chitosan solutions of different temperatures.

[0065] When the coating layer is multilaminar, viscosity or concentration of the chitosan solution used for different layers can be the same or different. For Example, at least one layer in the coating can be fabricated from a chitosan solution that has viscosity or concentration that is different from chitosan solution used for at least one other layer in the coating. In some embodiments, all layers in the coating are fabricated from chitosan solutions having the same viscosity or concentration. This can be accomplished by using the same chitosan solution for fabrication of layers in the coating.

[0066] When the coating layer is multilaminar, the coating layer can comprise one or more layers fabricated from a material other than chitosan. For example, such a material can be a biodegradable or biocompatible material. Layers fabricated from material other than chitosan can be next to each other or separated by one or more chitosan layer.

[0067] In some embodiments, the chitosan-based article of the invention can be prepared by preparing alternating layers of carbohydrate and protein-layers on a suitable surface. A carbohydrate based substrate, such as a film, can first be prepared by drying a carbohydrate solution (e.g. a carbohydrate polymer solution).

[0068] The chitosan layer then can be coated with a layer of protein. This can be accomplished by dipping it in a protein solution. Alternatively, the chitosan layer can be coated with a layer of protein by depositing a layer of protein solution on it. Generally, any ratio of protein to chitosan, e.g., weight ratio or mole ratio, can be used in formatting the composite material. In some embodiment, the chitosan to protein ratio is 10: 1 to 1: 10. In some

embodiments, chitosan to protein ratio is 5: 1 to 1:5, 2.5: 1 to 1:2.5, 1.15: 1 to 1: 1.15. In some embodiments, the ratio is 1: 1.5 to 1:2.5. In some embodiments, the ratio is 1:2. The ratio can be based on dry weight or moles of chitosan and protein added to the solution for forming the respective layers.

[0069] In one aspect of the invention, a chitosan solution is obtained or prepared. The chitosan solution can be prepared by adding chitosan to a solvent or a solvent to chitosan. In some embodiments, the starting chitosan solution comprises chitosan and a solvent. In some embodiments, the chitosan solution further comprises an acid. Chitosan is commercially available from a wide variety of sources including VANSON of Redmond, Wash., and Protan of

Woodinville, Wash. The chitosan starting material can theoretically be substituted or unsubsituted at the ring hydroxy moiety or the hydroxy methyl moiety. The important feature is that the chitosan has a majority of free, primary amine groups along its polymeric backbone to form ionic complexes with dilute organic acids. Preferably the chitosan has an average molecular weight ranging from 10⁴ Da to 10⁶ Da however, the molecular weight can vary considerably depending on the properties of the film or fiber product desired.

[0070] In some embodiments, the chitosan has an average molecular weight greater than lkDa, greater than 5kDa, greater than lOkDa, greater than 20kDa, greater than 30kDa, greater than 40kDa, greater than 50kDa, greater than lOOkDa, greater than 500kDa, greater than lOOOkDa, greater than 5000kDa, or greater than lOOOOkDa.

[0071] In some embodiments, the chitosan has an average molecular weight lower than lOOOOkDa, lower than lOOOkDa, lower than 5000kDa, lower than lOOOkDa, lower than 500kDa, lower than lOOkDa, lower than 50kDa, lower than 40kDa, lower than 30kDa, molecular weight lower than 20kDa, lower than 19kDa, lower than 18kDa, lower than 17kDa, lower than 16kDa, lower than 15kDa, lower than 14kDa, weight lower than 13kDa, lower than 12kDa, lower than 1 lkDa, lower thanlOkDa, or lower than 7kDa.

[0072] In some embodiments, the chitosan starting material has an average molecular in the range with a lower limit of 5kDa, lOkDa, 20kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, lOOkDa, 500kDa, lOOOkDa, or 5000kDa, and an upper limit of lOkDa, 20kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, lOOkDa, 500kDa, lOOOkDa, 5000kDa, lOOOkDa, or lOOOOkDa.

[0073] In some embodiments, the chitosan starting material has the following chemical formula:

where n is equal or greater to 1 , and R is hydrogen or a one to twelve carbon ether or ester.

[0074] In some embodiments, the chitosan starting material is chitin.

[0075] In some embodiments, the solvent is an organic solvent. The term "organic solvent" is an art recognized term and generally refers to a solvent which belongs to the group of organic compounds and is generally used for the dissolution of organic materials. Organic solvents include, but are not limited to, hydrocarbons, aromatic hydrocarbon, esters, ethers,

halohydrocarbons, amines, amides, alkanolamides, ureas, alcohols, glycols, polyhydric alcohols, glycol ethers, glycol ether esters, and mixed solvents of two or more thereof. Exemplary organic solvents include , but are not limited to, without limitation, 1-butanol, 2-butanol, 2-butanone, Acetamide MEA (Witco Corporation, Greenwich, Conn.), acetone, acetonitrile, and n-methyl pyrrolidone, benzene, carbon tetrachloride, chlorobenzene, chloroform, cycloheptane, cyclohexane, cyclopentane, decane, dibutyl ether, dichlorobenzenes, dichloroethanes, 1,2- dichloroethane, dichloromethane (DCM), diethanolamine, diethylene glycol, diethylene glycol monomethyl ether, diglyme (diethylene glycol dimethyl ether), diglycerol, 1 ,2-dimethoxy-ethane (glyme, DME), dimethylether, dimethylsulfoxide (DMSO), dioxane, dipropylene glycol monomethyl ether, dodecane, ethanolamine, ethyl acetate, ethyl propionate, ethylene glycol, ethylene glycol monophenyl ether, formic acid, glycerin, glycerol, heptane,

Hexamethylphosphoramide (HMPA), hexamethylphosphorotriamide (HMPT), hexane, isopropanol, methanol, methyl acetate, methul t-butyl ether (MTBE), methyl ethyl ketone (MEK), methyl propionate, N -methyl-2-pyrrolidinone (NMP), Ν,Ν,Ν',Ν'-tetramethylurea, N,N- dimethylformamide (DMF), nitromethane, n-butanol, octane, pentane, petroleum ether (ligorine), polyethylene glycol, polypropylene glycol, 1-propanol, 2-propanol, pyridine, propylene glycol, propylene glycol monomethyl ether, propylene glycol monomethyl ether acetate, Schercomid AME-70 (Scher Chemicals, Inc., Clifton, N.J.), sorbitol, squalane, diethyl ether, t-butyl alcohol, tetrachloroethanes, tetrahydrofuran, tetrahydrofuran (THF), thiourea, toluene, trichloroethanes, triethanolamine, triethylene glycol, triglycerol, urea, xylene (o-, m- or p-), γ-butyrolactam, and mixture of two or more thereof. Other examples of organic solvents can be found, for example, in McCutcheon's Volume 2: Functional Materials, North American Edition (The Manufacturing Confectioner Publishing Co., 2006) and Vogel's Practical Organic Chemistry (Prentice Hall, 5th ed., 1996), content of both which is incorporated herein by reference in their entirety.

[0076] In some embodiments, the solvent is an alcohol. In some embodiments, the alcohol is methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, or t-butanol.

[0077] In some embodiments, the solvent comprises water. In some embodiments, the solvent comprises an organic acid. In some embodiments the solvent is an organic acid. In some embodiments, the organic acid is formic acid. In some embodiments, the organic acid is acetic acid. In some embodiments, the organic acid is malic acid. In some embodiment, the solvent comprises an inorganic acid.

[0078] The chitosan solution in the concentrated form can be extruded to form films, fibers, filaments, or the like, or it can be coated on a mold to form a variety of articles or coated directly on an item to be coated. Any procedure for applying the concentrated chitosan in the form of a solution, solvent, liquid, gel, or hydrogel to the surface of an article could be used in the practice of this invention.

[0079] In some embodiments, the starting solution of chitosan has a chitosan concentration of at least 1%. In some embodiments, the starting solution of chitosan has a chitosan

concentration of at least 2%. In some embodiments, the starting solution of chitosan has a chitosan concentration of at least 3%. In some embodiments, the starting solution of chitosan has a chitosan concentration of at least 4%. In some embodiments, the starting solution of chitosan has a chitosan concentration of at least 5%.

[0080] In some embodiments, the starting solution of chitosan has a chitosan concentration lower than 5%. In some embodiments, the starting solution of chitosan has a chitosan

concentration lower than 4%. In some embodiments, the starting solution of chitosan has a chitosan concentration lower than 3%. In some embodiments, the starting solution of chitosan has a chitosan concentration lower than 2%.

[0081] One aspect of the invention focuses on obtaining a desired viscosity. In some embodiments, the desired viscosity refers to the lowest viscosity that the chitosan solution can have to be moldable. In some embodiments, the lowest viscosity is a viscosity just above a viscosity that would not allow for the chitosan solution to remain on the mold.

[0082] In some embodiments, the desired viscosity is obtained when the solution becomes malleable. In some embodiments, the desired viscosity is obtained when the solution flow "but not so much as to be very thin and not too think that it cannot fully coat the mold surface before hardening into a solid".

[0083] In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 0.1 milliPascal.second (mPa.s) to about 10⁹ mPa.s at room temperature. In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 1 mPa.s to about 10³ mPa.s, from about 10 mPa.s to about 750 mPa.s, from about 20 mPa.s to about 500 mPa.s, from about 30 mPa.s to about 400mPa.s, from about 40 mPa.s to about 300 mPa.s, or from about 50 mPa.s to about 250 mPa.s, In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 10⁴ mPa.s to about 10⁵ mPa.s at room temperature. In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 10⁶ mPa.s to about 2xl0⁵ mPa.s at room temperature. In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 10⁵ to about 10⁷ mPa.s at room temperature. In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 5xl0⁷ mPa.s to about 10⁸ mPa.s at room temperature. In some embodiments, the desired viscosity is obtained when the chitosan solution is from about 100 Pa.s to about 400Pa.s at room temperature

[0084] In some embodiments, the obtaining the desired viscosity comprises inducing a liquid-crystal state of the chitosan in the solution of chitosan. Liquid-crystal state refers to a state of matter that has properties between those of a conventional liquid and those of a solid crystal. As used herein, "liquid-crystal state" comprises any mesophases of the liquid crystal state, and also includes any type of crystal resulting from liquid-crystal state, for example, thermotropic liquid crystals, lyotropic liquid crystals, and metallotropic liquid crystals. Liquid-crystal state encompasses gel, and hydrogel. In some embodiments, inducing a liquid-crystal state of the chitosan in the solution of chitosan makes the chitosan solution coatable.

[0085] In order to obtain a desired viscosity, the concentration of chitosan in the starting solution is increased. In some embodiment, the concentration of chitosan in the resulting solution is greater than 10%, greater than 20%, greater than 30%, greater than 35%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 85%.

[0086] In some embodiment, the concentration of chitosan in the resulting solution is lower than 85%, lower than 80%, lower than 70%, lower than 60%, lower than 50%, lower than 40%, lower than 35%, lower than 30%, lower than 25%, lower thanl5%, lower than 10%, or lower than 5%.

[0087] In some embodiments, the concentration of chitosan in the resulting solution is in the range with a lower limit of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 85% and an upper limit of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%.

[0088] In some embodiments, the concentration of chitosan in the resulting solution is between 10-85%, between 15-80%, between 20-75%, between 25-70%, between 25-65%, between 25-60%, between 25-55%, between 25-50%, between 30-75%, between 30-70%, between 30-65%, between 30-60%, between 30-55%, between 30-45%, between 40-60%, or between 45-55%.

[0089] In some embodiments, the concentration of chitosan in the starting solution is increased by removal of a portion of the solvent. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% but not 100% of the solvent is removed. Generally, at least some of the solvent remains in the solution after evaporation. Generally, the chitosan solution is dried to remove as much of the solvent as possible so that the chitosan remains cohesive, but without removing so much solvent that the chitosan hardens and begin losing malleability or otherwise lacks cohesiveness.

[0090] In some embodiments, after concentration, at least 1% of solvent remains. In some embodiments, after concentration, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of solvent remains.

[0091] Any method known to one of skill in the art can be used for increasing the

concentration of chitosan in the solution. Exemplary methods include, but are not limited to, adding more chitosan to the solution, removal of solvent, increasing the effective concentration of chitosan by adding an agent to the solution, and changing the temperature of the solution.

Without limitations, solvent can be removed by evaporation, osmotic concentration, filtration, etc...

[0092] In some embodiments, the solvent is partially removed by evaporation of the solvent. This can be accomplished by heating the solution and letting the solvent evaporate. For example, the solution can be heated to a temperature from about 45°C to about 100°C. In some embodiments, the solution can be heated to a temperature from about 50°C to about 80°C. In addition, the solution can be heated for at least 15 minutes. For example, the solution can be heated for from about 15 minutes to about 6 hours. In some embodiments, the solution is heated in a closed environment at 80°C for 15-30 minutes.

[0093] In some embodiments, the solvent can be removed at about 37°C

[0094] In some embodiments, the solvent can be partially removed by addition of an agent.

Without wishing to be bound by a theory, addition of an agent can increase the effective concentration of chitosan in the solution by "salt-out" effect. The agent can be a polymer or a salt.

[0095] In some embodiments, the solvent can be partially removed by osmotic concentration. In some embodiment, osmotic concentration comprises moving solvent molecules through a selectively permeable membrane into a region of higher solute concentration without input of energy. In some embodiments, the solvent is partially removed by centrifugal concentration. In some embodiments, centrifugal concentration is effected with a centrifugal concentrator. In some embodiments, the solvent is partially removed by lyophilization. In some embodiments, lyophilization comprises freezing the solution or material and then reducing the

surrounding pressure to allow the frozen water in the material or solution to sublimate directly from the solid phase to the gas phase. In some embodiments, the solvent is partially removed by altering temperature. In some embodiments, the temperature is above 25°C. In me embodiments, the temperature is below 25°C.

[0096] In some embodiments, the solvent is partially removed using an energy source. In some embodiments, the energy source is light. In some embodiments, the energy source is radiation. In some embodiments, the energy source is electricity. In some embodiments, the energy source is flow. In some embodiments, the energy source is a mechanical force, e.g., beating. In some embodiments, the energy source is another energy source.

[0097] In some embodiments, the liquid-crystal state is induced by the preparation of a chitosan melt. Methods for preparing melt of polymers and proteins are known in the art. See for example, Perriman A. W. et al Angew. Chem. Int. Ed. 2009, 48, 6242-6246 ("Solvent -free protein liquids and liquid crystals" Perriman A. W. et al Angew. Chem. Int. Ed. 2009, 48, 6242- 6246, content of which is incorporated herein by reference.

[0098] Generally, fabrication with chitosan can be carried out at any temperature. For example, the concentrated chitosan solution can be heated or cooled to a temperature from about 4°C to a 90°C. In some embodiments, the chitosan solution temperature can be in a range with a lower limit of about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, or about 80°C and upper limit of about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, or about 90°C. In some embodiments, the chitosan solution temperature can be from about 50°C to about 90°C.

[0099] After fabrication using a chitosan solution at elevated temperatures, the chitosan layer can be cooled down to room temperature, e.g., about 20°C to about 30°C before optionally inducing a conformational change in the chitosan layer.

[00100] One aspect of the invention focuses on inducing a conformational change in the chitosan layer. In some embodiments, the conformational change is induced by treating the chitosan layer. In some embodiments, treating comprises partially removing the solvent. In some embodiments, treating comprises heating, alcohol treatment, and drying. In some embodiments, the conformational change is induced under high pressure. In some embodiments, the pressure ranges from about 10 bar to about 1000 bar. In some embodiments, the pressure ranges from about 50 bar to about 100 bar.

[00101] In some embodiments, the chitosan layer is treated with heat. In some embodiments, the temperature is greater than 300°C. In some embodiments, the temperature is from about 400°C to about 500°C. In some embodiments, the temperature is about 300°C. In some embodiments, the temperature is below 300°C. In some embodiments, the temperature is about 250°C. In some embodiments, the temperature is about 200°C. In some embodiments, the temperature is about 150°C. In some embodiments, the temperature is about 100°C.

[00102] In one embodiment, the resulting chitosan article has no solvent content.

[00103] The chitosan based article can be modified to contain at least one molecule selected from the group consisting of filler materials, small molecules, polymers, proteins, peptides, peptidomimimetics, nucleic acids, organic compounds, inorganic compounds, crystalline compounds, biological compounds, biologically active compounds, compounds having biological activity, and a biological, a pharmaceutical or a therapeutic agent, and any combinations thereof. If the article comprises a multilaminar chitosan layer, the molecule can be present in one or more layers.

[00104] The molecule can be present in the chitosan solution used for fabricating the article. For example, the molecule can be mixed with the chitosan solution prior to forming the liquid crystal state, or loaded into liquid crystal sate or a portion thereof after it is formed. The molecule can also be covalently linked with the chitosan. For covalent linking, the molecule can be linked with the chitosan before formation of the liquid crystal state. Alternatively, the molecule can be covalently linked to the chitosan layer after formation of the liquid crystal state. Accordingly, the molecule can be directly linked with the chitosan with a bond or through an intermediate linker.

[00105] In some embodiments, the molecule is an active agent. The variety of active agents that can be used in conjunction with the chitosan liquid crystal of the present invention is vast and can include small molecules, polymers, proteins, peptides, peptidomimimetics, nucleic acids, organic compounds, inorganic compounds, biological compounds, biologically active compounds, compounds having biological activity. For example, the active agent can be a therapeutic agent or biological material, such as cells (including stem cells), proteins, peptides, nucleic acids (DNA, RNA, plasmids, siRNA, antisense oligonucleotides, decoy oligonucleotides, microRNA, aptamers, and ribozymes), nucleic acid analogues, nucleotides, oligonucleotides or sequences, peptide nucleic acids, antibodies, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, or enzymes, antibiotics, viruses, antivirals, toxins, prodrugs, chemotherapeutic agents, small molecules, drugs and combinations thereof.

[00106] As noted, one or more active agents can be used to modify the concentrated chitosan solution. Accordingly, when using the concentrated chitosan solution of the present invention as a platform to support biological material such as cells, it can be desirable to add other materials to promote the growth of the agent, promote the functionality of the agent after it is released from the composite, or increase the agent's ability to survive or retain its efficacy during the processing period. Exemplary materials known to promote cell growth include, but not limited to, cell growth media, such as Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), non-essential amino acids and antibiotics, and growth and morphogenic factors such as fibroblast growth factor (e.g., FGF 1-9), transforming growth factors (TGFs), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), insulinlike growth factor (IGF-I and IGF-II), bone morphogenetic growth factors (e.g., BMPs 1-7), bone morphogenetic -like proteins (e.g., GFD-5, GFD-7, and GFD-8), transforming growth factors (e.g., TGF-a, TGF-β I-III), nerve growth factors, and related proteins. Growth factors are known in the art, see, e.g., Rosen & Thies, Cellular & Mol. Basis Bone Formation & Repair (R.G.

Landes Co.).

[00107] In some embodiments, the active agent is an antimicrobial agent. In some

embodiments, the antimicrobial is an antibiotic. In some embodiments, the antimicrobial is an antifungal. In some embodiments, the antimicrobial is an antiviral. In some embodiments, the antimicrobial is an antiparasitic. In some embodiments, the antimicrobial is an antileprotic.In some embodiments, the antimicrobial is an antitumor. In some embodiments, the agent is from a natural source. In some embodiments, the antimicrobial agent is a synthetic agents. In some embodiments, the agent is a beta-lactam antibiotic (for e.g. penicillins, cephalosporins), a protein synthesis inhibitor (for e.g. aminoglycosides, macrolides, tetracyclines, chloramphenicol, polypeptides).

[00108] In some embodiments, the active agent is an antibacterial agent. In some

embodiments, the antibacterial agent is an aminoglycoside. In some embodiments, the antibacterial agent is an ansamycin. In some embodiments, the antibacterial agent is a

carbacephem. In some embodiments, the antibacterial agent is a carbapenem. In some

embodiments, the antibacterial agent is a cephalosporin. In some embodiments, the antibacterial agent is a glycopeptide. In some embodiments, the antibacterial agent is a lincosamide. In some embodiments, the antibacterial agent is a lipopeptide. In some embodiments, the antibacterial agent is a macrolide. In some embodiments, the antibacterial agent is a monobactam. In some embodiments, the antibacterial agent is a nitrofuran. In some embodiments, the antibacterial agent is penicillin. In some embodiments, the antibacterial agent is a polypeptide. In some embodiments, the antibacterial agent is a quinolone. In some embodiments, the antibacterial agent is a sulfonamide. In some embodiments, the antibacterial agent is a tetracycline. In some embodiments, the antibacterial agent is a drug against mycobacteria. In some embodiments, the antibacterial agent is an antibacterial not encompassed by the previously listed categories.

[00109] Antibacterial agents comprise but are not limited to: Amikacin, Gentamicin,

Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Geldanamycin, Herbimycin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cefalotin (or Cefalothin), Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime,

Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin,

Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spectinomycin, Aztreonam, Furazolidone, Nitrofurantoin, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin,

Sparfloxacin, Temafloxacin, Mafenide, Sulfonamidochrysoidine(archaic), Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanamide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim-Sulfamethoxazole(Co-trimoxazole) (TMP-SMX), Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,

Pyrazinamide, Rifampicin (Rifampin in US), Rifabutin, Rifapentine, Streptomycin,

Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Linezolid, Metronidazole,

Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Rifaximin, Thiamphenicol, Tigecycline, and Tinidazole.

[00110] In some embodiments, the chitosan based article comprises an outer waterproof coating. As used herein, the term "waterproof refers to a barrier against both liquid and gaseous water (i.e., against both liquid water and water vapor). Generally, the waterproof coating has a permeability of less than 1 as determined by the Water Vapor Transmission Test ASTME96. Exemplary water-repelling materials include, but are not limited to, parylene,

polydimethylsiloxane, polyethylene, polyvinyl, polypropylene, polyester, latex, oils, organic solvents, waxes, lipids, esters of fatty acids, esters of sterols, long chain alcohols, myricyl palmitate, cetyl palmitate, lanolin, candelila wax, ouricury wax, sugarcane wax, retamo wax, jojoba oil, paraffin, and any combinations thereof.

[00111] Another aspect of the invention discloses an article produced by a method described herein. In one embodiment, article is a consumer product. In some embodiments, the consumer products are bottles, catering products, closures, container, cutlery, drink cups, food packaging, lids, overcap, prescription containers, or tubes. In some embodiments the consumer products are agricultural film, building products, corrosion protection, custom films, flexible intermediate bulk containers (FIBCs), flexible packaging, food and film packaging, medical films, personal care film, sheeting, drop cloths, stretch film, grocery and cutterbox film, tapes, or trash bags. In some embodiments, the consumer product is a medical product.

[00112] In some aspects of the invention, the chitosan-based article comprises a dye within the matrix of the chitosan. For example, a dye can be added to the chitosan solution before, during or after obtaining the desired viscosity. Further, a dye molecule can be covalently linked to a chitosan molecule.

[00113] As used herein and in the claims, the term "static dyes" means a dye that does not substantially change color upon exposure to (or being shaded from) ultraviolet (UV) light. The term "photochromic dyes" as used herein and the claims means dyes (or substances) that reversibly change color upon exposure to UV light, as is known to the skilled artisan. Typically, upon exposure to a particular wavelength of UV light, a photochromic dye will be converted into an open or activated form which is colored (within a particular portion of the visible spectrum). Upon removal of the UV light source, the open/activated photochromic dye returns to a closed/inactivated form which is not colored, or which is at least less colored than the activated form.

[00114] Static dyes that can be present in the chitosan-based article include, for example, fabric dyes and disperse dyes as well as dyes that are known in the art as being suitable for tinting plastic articles, such as thermoplastic polycarbonate articles.

[00115] Examples of suitable disperse dyes include, but are not limited to, Disperse Blue #3, Disperse Blue #14, Disperse Yellow #3, Disperse Red #13 and Disperse Red #17. The classification and designation of the static dyes are recited herein in accordance with "The Colour Index", 3rd edition published jointly by the Society of Dyes and Colors and the American Association of Textile Chemists and Colorists (1971), which is incorporated herein by reference. Dyestuffs can generally be used either as a sole dye constituent or as a component of a dye mixture depending upon the color desired. Thus, the term static dye as used herein includes mixtures of static dyes. [00116] The static dye class known as "solvent Dyes" can be present in the chitosan-based article. Solvent dye examples include, but are not limited to, Solvent Blue 35, Solvent Green 3 and Acridine Orange Base.

[00117] Further suitable static dyes include, for example, water-insoluble azo, diphenylamine and anthraquinone compounds, acetate dyes, dispersed acetate dyes, dispersion dyes and dispersol dyes, such as are disclosed in Colour Index, 3rd edition, vol. 2, The Society of Dyers and Colourists, 1971, pp. 2479 and pp. 2187-2743, respectively all incorporated herein by reference. In some embodiments, dispersed dyes include Dystar's Palanil Blue E-R150

(anthraquinone/Disperse Blue) and DIANIX Orange E-3RN (azo dye/Cl Disperse Orange 25).

[00118] Another class of suitable static dyes includes non-migratory static dyes. A particular class of non-migratory static dyes can be represented by the following formula,

R5-(polymeric constituent- Y)_t wherein R5 represents an organic dyestuff radical (or chromophore radical); the polymeric constituent is selected independently for each (t) from homopolymers, copolymers and block-copolymers of poly(C₂-C4 alkylene oxides), e.g., homopolymers of polyethylene oxide and polypropylene oxide, poly(ethylene oxide-propylene oxide) copolymers, and di- or higher block copolymers of ethylene oxide and propylene oxide; (t) can be an integer from 1 to 6; and (Y) is selected independently for each (t) from hydroxyl, primary amine, secondary amine and thiol groups. The polymeric constituent can have a molecular weight of from, for example, 44 to 1500. Dyestuff radicals from which (Y) can be selected include, but are not limited to, nitroso, nitro, azo (e.g., monoazo, diazo and triazo), diarylmethane, triarylmethane, xanthene, acridene, methine, thiazole, indamine, azine, oxazine and anthraquinone dyestuff radicals. Non-migratory static dyes represented by formula IV are described in further detail in U.S. Pat. Nos. 4,284,729; 4,640,690; and 4,812,141.

[00119] Photochromic dyes that can be present in the chitosan-based article include those known to the skilled artisan. Classes of suitable photochromic dyes include, but are not limited to: spiro(indoline)naphthoxazines and spiro(indoline)benoxazines (e.g., as described in U.S. Pat. No. 4,818,096); and chromenes, such as benzopyrans and naphthopyrans (e.g., as described in U.S. Pat. No. 5,274,132), and benzopyrans having substituents at the 2-position of the pyran ring and an optionally substituted heterocyclic ring, such as a benzothieno or benzofurano ring fused to the benzene portion of the benzopyran (e.g., as described in U.S. Pat. No. 5,429,774). Further classes of photochromic dyes include, for example organo-metal dithizonates, such as (arylazo)- thioformic arylhydrazidates, e.g., mercury dithizonates (e.g., as described in U.S. Pat. No.

3,361,706); fulgides and fulgimides, such as 3-furyl and 3-thienyl fulgies and fulgimides (e.g., as described in U.S. Pat. No. 4,931,220).

[00120] Photochromic dyes or mixtures thereof can be used alone or in combination with one or more static dyes in the chitosan-based article in the method of the present invention. In some embodiments presence of photochromic dyes into chitosan-based articles will result in the formation of a dyed chitosan-based article having photochromic properties.

[00121] Propylene glycol based dyes can be used alone or in combination with other types of dyes in the chitosan-based article in the method of the present invention. In some embodiments, the propylene glycol based dyes are retained in the crystal chitosan but diffuse through the amorphous chitosan, enabling the release of the dye and the recovery of the raw material.

[00122] The invention can be defined by any of the following numbered paragraphs:

1. A method for fabricating a consumer product comprising:

a. providing a desired viscosity in a solution of chitosan; and

b. fabricating a 3-D article using the chitosan solution.

2. The method of paragraph 1 , wherein said providing the desired viscosity in the chitosan solution comprises inducing a liquid-crystal state of chitosan in the chitosan solution.

3. The method of paragraph 1 or 2, wherein the chitosan solution comprises a filler material.

4. The method of paragraph 3, wherein the filler material is present in an amount from

0.01% to 99% (w/w) of chitosan in the chitosan solution.

5. The method of any of paragraphs 3-4, wherein the filler material is selected from the group consisting of sand; sodium carbonate; coconut-based soil; wood flour; carbon fibers; carbon nanotubes; fiberglass; cellulose; minerals; glass;, inorganic oxides; carbon black (also known as furnace black); silicate; sulfates; metallic powders; carbonates; mica; silica; nitrides; carbides; and any combinations thereof.

6. The method of paragraph 5, wherein the filler material is in form of microparticles or nanoparticles.

7. The method of any of paragraph 3-6, wherein the filler material is homogenously

dispersed in the chitosan solution.

8. The method of any of paragraphs 1-7, wherein said fabrication comprises coating a mold surface, blow molding, compression molding, transfer molding; injection molding; layer- by-layer assembly; or any combination thereof.

9. The method of any of paragraphs 1-7, wherein the chitosan solution has an elevated

temperature.

10. The method of paragraph 8, wherein said elevated temperature is from about 30°C to about 90°C.

11. The method of paragraph 8 or 9, wherein said elevated temperature is about 80°C.

12. The method of any of paragraphs 1-10, wherein said desired viscosity is from about 1 pascal-second (Pa.s) to about 1000 Pa.s. The method of any of paragraphs 1-12, wherein said desired viscosity is obtained by partial evaporation of solvent, addition of a polymer, addition of a salt, by osmotic concentration, and any combinations thereof.

The method of any of paragraphs 1-13, wherein the chitosan solution of the desired viscosity has a chitosan concentration of at least about 30% before fabricating the article. The method of any of paragraphs 1-14, further comprising inducing a conformation change in chitosan after the fabricating step.

The method of paragraph 15, wherein said inducing a conformation comprising at least partially removing remaining solvent.

The method of any of paragraphs 15 or 16, wherein said inducing a conformation comprising applying heat to the article to at least partially remove any remaining solvent. The method of any of paragraphs 1-17, wherein said fabricating step comprising casting the chitosan solution in a mold; and partially removing solvent from the chitosan solution c as ted in the mold.

The method of paragraph 18, further comprising removing the partially dried article from the mold and further removing solvent from the article.

The method of any of paragraphs 1-19, further comprising treating the fabricated article with a basic solution.

The method of paragraph 20, wherein the basic solution comprises NaOH.

The method of any of paragraphs 1-21, further comprising coating the fabricated article with a waterproof coating layer.

The method of paragraph 22, wherein said waterproof coating layer comprises one or more of parylene, polydimethylsiloxane, polyethylene, polyvinyl, polypropylene, polyester, latex, oils, organic solvents, waxes, lipids, esters of fatty acids, esters of sterols, long chain alcohols, myricyl palmitate, cetyl palmitate, lanolin, candelila wax, ouricury wax, sugarcane wax, retamo wax, jojoba oil, and paraffin.

The method of any of paragraphs 1-23, wherein the article comprises a multilaminar layer comprising at least one layer of chitosan.

The method of any of paragraphs 1-24, wherein the article comprises a multilaminar layer comprising at least two layers of chitosan.

The method of any of paragraphs 1-25, wherein said fabricating comprises (i) coating a mold surface with the solution of chitosan to form a coating layer of chitosan on the surface; and (ii) repeating step (i) until a chitosan layer of desired thickness is obtained. The method of paragraph 26, further comprising inducing a conformational change in the coating layer before coating the next coating layer. 28. The method of paragraph 26 or 27, wherein viscosity of chitosan solution used for at least one coating layer is different from viscosity of chitosan solution used for at least one other coating layer.

29. The method of any of paragraphs 26-28, wherein viscosity of chitosan solution used for first coating layer is low relative to viscosity of chitosan solution used for the second coating layer.

30. The method of paragraph 29, wherein the low viscosity chitosan solution has a chitosan concentration of about 1% to about 10%.

31. The method of any of paragraph 29 or 30, wherein higher viscosity chitosan solution has a chitosan concentration of about 20% to about 80%.

32. The method of any of paragraphs 26-31, wherein the desired thickness is from about lnm to about 10 cm.

33. The method of any of paragraphs 1-32, wherein the article further comprises a molecule selected from the group consisting of small molecules; dyes; polymers; proteins; peptides; peptidomimimetics; nucleic acids; organic compounds; inorganic compounds; crystalline compounds; biological compounds; biologically active compounds; compounds having biological activity; a pharmaceutical agent; a therapeutic agent; and any combinations thereof.

34. The method of paragraph 33, wherein the molecule is an antibacterial agent or an

antimicrobial agent.

35. An article prepared by a method of any of paragraphs 1-34.

36. The article of paragraph 35, wherein the article is a composite material.

37. The article of paragraph 35 or 36, wherein the article is a consumer product.

38. The consumer product of paragraph 37, wherein the consumer product is a bottle, a

catering product, a closure, a container, a piece of cutlery, a drink cup, a food packaging, a lid, an overcap, a prescription container, a toy, an electronic device, or a tube.

39. The consumer product of paragraph 38, wherein the consumer product is an agricultural film, a building product, a corrosion protection, a custom film, a flexible intermediate bulk container (FIBC), a flexible packaging, a food and film packaging, a medical film, a personal care film, a sheeting, a drop cloth, a stretch film, a grocery film, a cutterbox film, a tape, or a trash bag.

Some definitions

[00123] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00124] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to the invention, yet open to the inclusion of unspecified elements, whether useful or not. The terms "comprising" and "comprises" include the terms "consisting of and "consisting essentially of."

[00125] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00126] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00127] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[00128] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[00129] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00130] The terms "decrease", "reduced", "reduction", "decrease" or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, "reduced", "reduction" or "decrease" or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[00131] The terms "increased", "increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[00132] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[00133] As used herein, the terms "effective" and "effectiveness" includes both

pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment. "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects.

[00134] The term "sphere" means a particle having an aspect ratio of at most 3: 1. The term "aspect ratio" means the ratio of the longest axis of an object to the shortest axis of the object, where the axes are not necessarily perpendicular.

[00135] The term "longest dimension" of a particle means the longest direct path of the particle. The term "direct path" means the shortest path contained within the particle between two points on the surface of the particle. For example, a helical particle would have a longest dimension corresponding to the length of the helix if it were stretched out into a straight line.

[00136] The term "rod" means a particle having a longest dimension of at most 200 nm, and having an aspect ratio of from 3: 1 to 20: 1.

[00137] The term "prism" means a particle having at least two non-parallel faces connected by a common edge. [00138] The "length" of a particle means the longest dimension of the particle.

[00139] The "width" of a particle means the average of the widths of the particle; and the

"diameter" of a particle means the average of the diameters of the particle.

[00140] The "average" dimension of a plurality of particles means the average of that dimension for the plurality. For example, the "average diameter" of a plurality of spheres means the average of the diameters of the spheres, where a diameter of a single sphere is the average of the diameters of that sphere.

EXAMPLES

Example 1: Fabrication of bio-mimetic macroscale objects with the structural biopolymer.

[00141] Chitin Vs Chitosan: While the different nomenclature between chitin and chitosan are practical, rather than chemical, reasons, it is assumed correct the use of the term chitosan for chitin with more than 50% degree of deacetylation. Chitin occurrence in nature is more widespread with high degrees of acetylation, being very abundant in this form in the arthropods cuticles. For this reason the main source of chitosan (i.e. chitin with low acetylation degree) is the deacetylation of seafood factories waste in a sodium hydroxide bath, instead of it direct harvest form natural resources. The main advantage of chitosan is its capability to form water soluble salts with organic and inorganic acids. Here we will use the term "chitin" when we make reference to properties of the molecule with any degree of acetylation, while chitosan will be employed for exclusive properties of highly deacetylated chitin.

[00142] Crystal conformations of chitin: Chitin is a semicrystalline unbranched polymer and, despite all the polymorphoic forms reported (differing in packing density and water content), all have an extended two fold helical structure. Several crystal forms of chitosan have been reported, dubbed as "tendon" (i.e. hydrated, from crab shell), "L-2" (i.e. hydrated, from shrimp shells) and "annealed", all having a twofold helical structure but differing in their water content. The crystal units are composed by two (annealed) to four (hydrated) antiparallel polymer chains. Chains at the same level are bonded through the N2- 06 atoms.

[00143] XRD: Diffraction experiments where performed with a Scintag XDS2000 fixed sample position diffractometer with a Peltier- cooled silicon detector (Kevex Corp., USA) and a nickel filtered Cu Ka radiation (λ=1.5418Α). The diffraction angle (i.e. 2Θ) was measured in between 2° and 70° with a step of 0.02°.

[00144] Crystal domain size: Crystal domains are measured by the use of a fixed polarizer and a liquid crystal modulator (Hinds Instruments, Inc, USA) in the light pathway of an inverted microscope. The produced images characterize the slow and fast axis of a 1024x1392 pixels image of the sample, assigning a RGB color code to each orientation between -π/2 and π/2. The inventors divided the image in areas with dispersion in the orientation of p/6 and measure the size of those areas (Figure 9). In the case of most evaporated films (and all those stretched films) the size of the domains was too big for their characterization in a single image.

[00145] IR Spectrometry: IR spectra were obtained with a resolution of 2 cm^"1 between 4000 and 500 cm^"1 (Vertex 70, Bruker, Germany) and analyzed with Essential FTIR (Operant LLC, USA). While all the samples show a similar IR absorption, coagulated films present a small but significant absorption due to the presence of a carboxylate anion (Fig. 10). Because coagulation happens from the outside of the sample to the inside, some acetate ions are trapped inside of the sample, which explains this small difference in this kind of samples.

[00146] Molecular representation: Molecules are represented with the open source software RasMol 2.7, developed by Roger Sayle. The inventors used published data and their own characterization to represent the molecules.

[00147] Stress/Strain measurements: Material characterization studies were carried out by cutting films of chitosan in strips (1.5 cm wide x 8 cm long) and measuring stress-strain relationships with an Instron 3342 instrument (500N, Instron, USA). The thickness of the samples was measured by microscopy (Axio Observer, Zeiss, Germany) as the average of 5 different points of the film.

[00148] From the Stress/Strain measures, the inventors determined the ultimate stress as the maximum ordinate, and the associated strain was defined as the ultimate strain. The Young Modulus (E) was determined by the average slope between the origin and the ultimate strain. The area under the curve, obtained by integration using a Riemann sum approximation between the zero and the ultimate strain, was used to determine the modulus of toughness.

[00149] The characterization of the waterproof samples was performed on films coated with parylene-C and bee wax before and after 12h in water at room temperature. It is important to note that the stress depends on the thickness of the samples, which in these cases include the coating. In the case of bee wax samples, the production of a continuous coating was performed by the spray of wax microdroplets on the chitosan surface and a heat treatment at 58°C. The fabrication and water barrier of wax coatings were, by far and with the available tools, not as efficient as using Parylene-C, but its use, besides being the main component of the epicuticle, was motivated by the low cost and biological origin of this kind of coatings.

[00150] Rheological Measurements: Oscillatory shear measurements were carried out in an AR-G2 rheometer (TA Intruments, Italy). The inventors employed a cone-and-plate aluminum geometry with a diameter of 20mm and an angle of 2°, at a strain less than 0.02. The strain amplitude was chosen to ensure that all measurements were conducted within the linear viscoelastic regime, where the dynamic moduli are independent of the strain amplitude (a condition checked in every experiment). A frequency sweep extending from about 0.015 to 6 Hz was performed for each sample.

[00151] To avoid evaporation of solvent in the sample, the inventors encapsulated it in a closed cell. All the measurements, except the temperature ramp, were performed at a constant temperature of 25°C. The temperature ramp was performed in steps of 2°C/min.

[00152] Scanning electron Microscopy: The Scanning Electron Microscope images were taken with a Zeiss field emission Ultra55 SEM (Carl Zeiss SMT GmbH, Germany). Dry samples, immobilized on an aluminum holder, were introduced without modification in the chamber and examined under a 5 to 15KeV electron beam.

[00153] Molding: Medium molecular weight chitosan was dissolved in a 1 % (v/v) acetic acid water solution. For the casting of positive and/or hollow structures, 60% of the solvent was removed by it evaporation in a 37°C chamber (the process last about 6h) and stored at this concentration in a close container to prevent further evaporation. Before its use the solution is warmed up to about 40°C and poured on the mold at room temperature. The evaporation of the remaining solvent gives rise to a hard positive replica of the initial mold. The samples are then treated with a fast immersion in 4% (w/v) water solution of NaOH, which neutralize any remaining acetic acid and the associated "vinegar" smell.

[00154] The polymer can be used directly in an injection molding process. From the initial 3% (w/v) of chitosan solution 80% of the solvent is removed, giving rise to a plastic solid which is molded in bars for its storage upon use. The injection molding is performed by decreasing the viscosity of the material at 80°C, and the casted with a manual benchtop injection molder (Galomb Inc., USA) in an epoxy mold. The technique, based in a moderate pressure ensures also the absence of bubbles due to the elimination of the Harvey nuclei in the process [29]. The resulting negative replicas of the mold (Fig. 7) are removed and stored at 37°C for about 3-10h (depending of the size and geometry of the fabricated object), where the remaining solvent is evaporated.

[00155] The removal of the water has associated wit it a shrinking of the whole structure about 1/3 of its original volume. In structures requiring high precision in the final result, this effect can make the technique unviable. To compensate for this shrinkage, the inventors added calcium carbonate, wood flour or sand to the initial 3% solution of chitosan, while the rest of the injection molding process still being the same.

[00156] Despite the diversity of biological materials and structures with extraordinary mechanical characteristics as well as the urgent need for sustainable technologies, the

implementation of these biomaterials in modern engineering is still very limited. In stark contrast to their synthetic counterparts, structural biomaterials are based on a holistic composition of chemistry and hierarchical designs; features that are generally neglected in artificial materials design. Recent designs of certain bio-inspired surfaces (eg. Lotus leaves [1], gecko feet [2], shark skin [3]) or composites [4] have given rise to materials with outstanding properties. However, to achieve those properties, the ecological integration and the assimilated structures are always limited by the use of artificial components with known manufacture procedures.

[00157] Chitin and chitosan are a paradigm of our inability to assimilate natural mechanisms in material sciences. Referred as "the last biomass" [5], they are, after cellulose, the most abundant organic compound on earth. Like their plant counterpart, these polysaccharides are synthesized to produce structural scaffolds in different organisms, being responsible of some of the most remarkable biological materials, such as the arthropod cuticle, fungal walls or nacre. Paradoxically, these exceptional structural polymers are mainly discarded [6] or used in cosmetics, organic fertilizers, and dietary supplements [7].

[00158] In this study, the inventors demonstrate how to assimilate the physiological design of chitosan structures to fabricate large artificial components with superior mechanical properties, by a low cost and environmental friendly procedure. The inventors have also developed the coatings for their application in humid environments, including and ability to mix in and remove colors. The inventors further demonstrate the potential for these constructions to be recycled and composted.

[00159] Chitin has an estimated production of about 10¹¹ tons per year [8], with a very large occurrence in the form of a highly acetylated polymer. The low acetylated polymer (i.e. chitosan) can be produced at large scale by its extraction from Mucoralean fungi {Zygomycetes) cultures [9]. However, because the highly acetylated chitin is a common waste in sea food processing factories, nowadays the worldwide production of chitosan is mostly based in the transformation of that waste. In contrast with cellulose (which requires the life-threatening carbon disulfide to be regenerated), the existence of amine groups in chitosan enable its dissolution in low concentrations of carboxylic acids (e.g. acetic, formic, valeric...), where the protonation of the amine groups introduce a repulsive interchain force, strong enough to disperse the polymer in the solution [10]. Because this interaction is purely electrostatic, chitosan can be regenerated from the chitosonium form by the neutralization of the solvent in a basic bath (i.e. coagulation) or by the spontaneous evaporation of the solvent at room temperature in the case of small acid molecules (e.g. acetic acid) [11-13]. Both processes produce insoluble films, however coagulated films are opaque and brittle, while evaporated films are tough and transparent (Figs. 5A and 5B).

[00160] Although coagulated and evaporated films are obviously different at the macroscale, their molecular characterization does not show significant differences; the FTIR (Fig. 5C) and XRD (Fig. 5D) characterizations show that both the evaporated and coagulated samples of chitosan, are the L-2 allomorph (characteristic of chitosan from shrimp shells, in our case from the specie Pandalus Borealis), firstly characterized by Sakurai et al. in 1985 [14]. This hydrated polymorph forms sheets of molecules stabilized by water (Figs. 5E and 5F) [15]. However, as in any structural biomaterial, the mechanical properties of chitosan structures cannot be understood without taking into account the organization at different scales. The birefringent analysis of the evaporated chitosan samples show how the chitosan crystals organize in domains oriented in the same direction which, in most cases, reach several millimeters in size (Fig. 5H). These domains are separated by smooth transitions, where the polymer chains in between domains gradually rotate from the characteristics alignment of a domain to the next one. On the other hand, the domains of the coagulated samples are much smaller (49.17±24.69μηι2 ) and characterized by sharp boundaries, explaining the opacity of the films due to the internal light scattering (Figs. 5G and 9). Under stress, the polymer chains in the evaporated films align parallel to the force, giving rise to surfaces of isotropic alignment (Fig. 51). This effect under an external force contrast with the response of coagulated films where, the apparently independent domains, break without any appreciable conformational change. The reorientation of the same system under an external force has been previously observed in nature; in the arthropod cuticle there is a direct correlation between the stress and the chitin alignment, being the orientation of the polymer susceptible to change after the time of deposition [16].

[00161] Therefore, as observed in other structural biopolymers [17] and composites [18], the mechanical properties of the processed chitosan depend on the method employed rather than in its chemistry. As a result, one can ignore the large amount of chemical modifications attempted on chitosan [19] and explore the natural mechanical properties of the polymer by the reproduction of two levels of order: the molecular conformation by the use of a weak and volatile carboxylic acid (i.e. acetic acid) and a crystallographic distribution generated by a controlled evaporation of that solvent. However, it has been the main objective in the field to show if this kind of mechanical properties observed at small scales in biological systems can be reproduced in macroscopic materials [20].

[00162] The details of how the regeneration process affects the macroscale properties of the biopolymer are studied by monitoring the ratio between the storage (G') and loss (G") moduli of the material (magnitudes representative of the energy stored elastically (solid) and the energy dissipated as heat (liquid), respectively), which reveals that, while the solvent is removed, the initial liquid crystal solution of chitosan covers every state between the liquid and solid forms without any discontinuous phase transition (Fig. 6A). Additionally, while chitosan undergoes thermal degradation prior to melting, the properties of the solution, even at large concentrations, depend on temperature (Fig. 6B). As a result, the concentration of the polymer can be employed to produce a pliable material suitable for a specific fabrication process, while the temperature is suitable tool to produce a fast (but limited) change in the viscosity of a polymer solution where the intermolecular interactions characteristic of the crystal form are already present [21].

[00163] The inventors used this characteristic to concentrate an initially diluted solution of chitosan (i.e. p<30Kg/m³) so it can be employed at room temperature in a casting process or at even higher concentrations, (where the polymer shows a plastic behavior) in injection molding at about 80°C (Fig. 6C). The results show the suitability of the polymer to be manufactured in any geometry (Fig. 7A) by its casting in the appropriate mold. Similarly to the approach in natural cuticles, the use of a coating providing the waterproof properties that naturally supply the waxy epicuticle, enables the use of chitosan structures even in water based applications (Fig. 7E). In the case of voluminous constructions made by injection molding, the shrinking of the polymer is too great to keep the original shape. To avoid this effect we make use of the polymer in combination with different fillers, which prevents the shrinkage of the structure. The use of wood flour, a waste from wood processing and common filler in plastic industry, results in a thought composite of wood particles interconnected by a chitosan based organic matrix, which can be manufactured as regular wood (i.e. drilled, carved, polished...). It additionally has an even lower production cost and, while the final result is of lesser toughness than the pure organic matrix, the shrinkage due to solvent evaporation of this cheaper composite is greatly reduced (S=1.21±0.68% with a 20% chitosan/wood weight concentration), being more appropriate for injection molding procedures. The use of chitosan as binder has been also demonstrated with other common fillers such as sand or calcium carbonate (Figs. 7M-70); however, those objects are of lesser toughness due to the coagulation of the chitosan resulting from the increase of pH when mixed with a salt.

[00164] The data shown herein further demonstrate the production of chitosan objects in a variety of different colors (Fig. 7B). Current use of dyes in plastics has the drawback of a much difficult recycling process: objects made from a single type of plastic and color are relatively easy to be sorted however, for consumer products with many small parts consisting different types and colors of plastics, the resources it would take to separate the plastics far exceed their value, and the items are hence discarded. Chitosan can capture and retain other molecules (property exploited in remediation systems [22]) but is also suitable to produce a controlled release of those molecules under specific conditions (a characteristic that is being utilized in drug delivery systems [23, 24]). The inventors employ these properties to retain and program the controlled release of the colorant. Dye molecules are retained in the chitosan in moderate acid and basic solutions, while under more acidic environments the color molecules are released (Fig. 7D), a characteristic the inventor use to avoid the labor-intensive sorting of the plastic object by colors before the recycling process. This process enables the recycle of non-dyed (Fig. 8A, Cycle A) and colored (Fig. 8A, Cycle B) dyed pieces. However, differently from other polymer where the colorant is covalently bonded to the structure, in this case the dye is introduced and discarded in each recycling step (Fig. 8B, Cycle C), enabling the reuse of the polymer independently of the coloration. Additionally, because then on existence of chemical modifications during the process, the objects made of chitosan do not show a significant degradation of their mechanical properties after recycling (Fig 8B).

[00165] While chitosan based objects are suitable for recycling, nowadays less that 5% of the plastic produced worldwide capitalize on this process [25]. The rest of it is incinerated or accumulates in oceans and ground. In contrast with most of these synthetic polymers, biomaterials are produced and assimilated in one or several natural cycles. Chitosan, for example, has been demonstrated to be a growth enhancer in plants [26].

[00166] As shown herein, the objects fabricated with chitosan are strong and suitable for manufacturing and recycling. Additionally, the natural relevance of the polymer in some of the most exceptional structural biomaterials enables the use of the technology proposed here for the exploration, on its own or in combination with other components, of a branch in materials sciences and sustainable engineering based on the reproduction and application of those biological structures.

[00167] Chitosan based foams: The formation of chitosan foams by lyophilization has been previously reported; however, while this technique is employed in high cost applications (i.e. medical devices), its use for the manufacture of consumable products is unfeasible due to the high cost and large production time. In the case of lyophilization, the pores are made through sublimation of the solvent, which leaves behind the matrix of polymer. However, in the case of chitosan, several other approaches can be employed and optimized depending on the nature of the solvent in which chitosan is dissolved. Specifically, if chitosan is dissolved in low concentration (<1% V/V) solutions of acetic acid in water, the acetic acid can be removed relatively easily by evaporation or reaction.

[00168] The inventors have discovered that that the energy provided by applying a mechanical force to a chitosan solution (e.g., by beating the solution) is sufficient to separate solvent and polymer in different phases, giving rise to strong foams (Fig. 11A). Similar results were also obtained by injecting air into a concentrated chitosan solution, which enables the production of chitosan foams in a similar process than that used for production of regular expanded foam. Additionally, the chemical reactivity of the acetic acid solution can be employed to generate a gas produce when it is neutralized (e.g. use of calcium bicarbonate gives rise to C0₂). This reaction produce foams in a fast and cheap method. The solution of chitosan is mixed with variables amounts of Calcium bicarbonate that neutralize the acid and precipitates the polymer, while the C0₂ "bubbles" it produces gives rise to the porous structure (Fig. 11B). This method has been disclosed previously, however its application inside molds for the formation of shaped foams hasn't been explored yet. Accordingly, while random shaped foams with sodium bicarbonate and acetic acid has been explored, the formation foams shaped as the mold has not been reported previously. The formation of the foam in a mold results in a foam shaped as the mold. The molding occurs because the expansion of the material against the wall when the foam is forming (C0₂ is being produced).

[00169] Production of composites (i.e. use of chitosan as binder): Chitosan has a high reactivity and also, as demonstrated previously, it is suitable for the construction of strong objects and polymer networks. The fabrication of these objects involves the evaporation of a solvent, but this process results in significant shrinking of the material (Fig. 11). To avoid shrinking of chitosan-based constructions, the inventors employed the use of a filler (i.e. solid particles added to a "binder" material), which has proven to be an inexpensive and reliable method. This is because the solid network forms around the particles of the filler, which resist compression when the solvent evaporates and the network tends to shrink. So far, the inventors have successfully used sand, sodium carbonate, coconut-based soil and wood flour as fillers with chitosan with similar success. However, the technique is suitable for use with other fillers, such as wastes from other industries (e.g. cellulose or mineral based materials).

[00170] In the case of salts (calcium carbonate and sand), the resulting structures were clearly of lesser toughness than those based on wood. This is because, when mixed with the salt, the chitosan solution raises it pH, precipitating the chitosan in small and disconnected domains. In the case of material not affecting the pH, the polymer crystallizes in bigger and stronger domains.

[00171] This method can be employed for injection molding chitosan; however, these composites also can be manufactured in many other ways. Wood based pieces have mechanical properties similar to the natural wood, and they can be modified and machined (i.e. drilled, carved, polished, sanded, etc...) in a similar way. In general, chitosan solutions can be employed as an ecological and cheap substitute to other binders and resins, such as natural and synthetic plastics, gums and resins (e.g. epoxy resin, polyurethane or Urea-formaldehyde).

[00172] The strength of the components fabricated and the versatility of the injection molding approach will enable its use for many applications. An example could be its use in self- constructed furniture (i.e. IKEA style), where all the functional small plastic pieces can be substituted by this eco friendly component.

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[00173] All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00174] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein.

Claims

CLAIMS What is claimed is

1. A method for fabricating a consumer product comprising:

a. providing a desired viscosity in a solution of chitosan; and

b. fabricating a 3-D article using the chitosan solution.

2. The method of claim 1 , wherein said providing the desired viscosity in the chitosan

solution comprises inducing a liquid-crystal state of chitosan in the chitosan solution.

3. The method of claim 1 or 2, wherein the chitosan solution comprises a filler material.

4. The method of claim 3, wherein the filler material is present in an amount from 0.01% to 99% (w/w) of chitosan in the chitosan solution.

5. The method of any of claims 3-4, wherein the filler material is selected from the group consisting of sand; sodium carbonate; coconut-based soil; wood flour; carbon fibers; carbon nanotubes; fiberglass; cellulose; minerals; glass;, inorganic oxides; carbon black (also known as furnace black); silicate; sulfates; metallic powders; carbonates; mica; silica; nitrides; carbides; and any combinations thereof.

6. The method of claim 5, wherein the filler material is in form of microparticles or

nanoparticles.

7. The method of any of claim 3-6, wherein the filler material is homogenously dispersed in the chitosan solution.

8. The method of any of claims 1-7, wherein said fabrication comprises coating a mold surface, blow molding, compression molding, transfer molding; injection molding; layer- by-layer assembly; or any combination thereof.

9. The method of any of claims 1-7, wherein the chitosan solution has an elevated

temperature.

10. The method of claim 8, wherein said elevated temperature is from about 30°C to about 90°C.

11. The method of claim 8 or 9, wherein said elevated temperature is about 80°C.

12. The method of any of claims 1-10, wherein said desired viscosity is from about 1 pascal- second (Pa.s) to about 1000 Pa.s.

13. The method of any of claims 1-12, wherein said desired viscosity is obtained by partial evaporation of solvent, addition of a polymer, addition of a salt, by osmotic

concentration, and any combinations thereof.

14. The method of any of claims 1-13, wherein the chitosan solution of the desired viscosity has a chitosan concentration of at least about 30% before fabricating the article.

15. The method of any of claims 1-14, further comprising inducing a conformation change in chitosan after the fabricating step.

16. The method of claim 15, wherein said inducing a conformation comprising at least

partially removing remaining solvent.

17. The method of any of claims 15 or 16, wherein said inducing a conformation comprising applying heat to the article to at least partially remove any remaining solvent.

18. The method of any of claims 1-17, wherein said fabricating step comprising casting the chitosan solution in a mold; and partially removing solvent from the chitosan solution casted in the mold.

19. The method of claim 18, further comprising removing the partially dried article from the mold and further removing solvent from the article.

20. The method of any of claims 1-19, further comprising treating the fabricated article with a basic solution.

21. The method of claim 20, wherein the basic solution comprises NaOH.

22. The method of any of claims 1-21, further comprising coating the fabricated article with a waterproof coating layer.

23. The method of claim 22, wherein said waterproof coating layer comprises one or more of parylene, polydimethylsiloxane, polyethylene, polyvinyl, polypropylene, polyester, latex, oils, organic solvents, waxes, lipids, esters of fatty acids, esters of sterols, long chain alcohols, myricyl palmitate, cetyl palmitate, lanolin, candelila wax, ouricury wax, sugarcane wax, retamo wax, jojoba oil, and paraffin.

24. The method of any of claims 1-23, wherein the article comprises a multilaminar layer comprising at least one layer of chitosan.

25. The method of any of claims 1-24, wherein the article comprises a multilaminar layer comprising at least two layers of chitosan.

26. The method of any of claims 1-25, wherein said fabricating comprises (i) coating a mold surface with the solution of chitosan to form a coating layer of chitosan on the surface; and (ii) repeating step (i) until a chitosan layer of desired thickness is obtained.

27. The method of claim 26, further comprising inducing a conformational change in the coating layer before coating the next coating layer.

28. The method of claim 26 or 27, wherein viscosity of chitosan solution used for at least one coating layer is different from viscosity of chitosan solution used for at least one other coating layer.

29. The method of any of claims 26-28, wherein viscosity of chitosan solution used for first coating layer is low relative to viscosity of chitosan solution used for the second coating layer.

30. The method of claim 29, wherein the low viscosity chitosan solution has a chitosan

concentration of about 1% to about 10%.

31. The method of any of claim 29 or 30, wherein higher viscosity chitosan solution has a chitosan concentration of about 20% to about 80%.

32. The method of any of claims 26-31, wherein the desired thickness is from about lnm to about 10 cm.

33. The method of any of claims 1-32, wherein the article further comprises a molecule

selected from the group consisting of small molecules; dyes; polymers; proteins; peptides; peptidomimimetics; nucleic acids; organic compounds; inorganic compounds; crystalline compounds; biological compounds; biologically active compounds; compounds having biological activity; a pharmaceutical agent; a therapeutic agent; and any combinations thereof.

34. The method of claim 33, wherein the molecule is an antibacterial agent or an

antimicrobial agent.

35. An article prepared by a method of any of claims 1-34.

36. The article of claim 35, wherein the article is a composite material.

37. The article of claim 35 or 36, wherein the article is a consumer product.

38. The consumer product of claim 37, wherein the consumer product is a bottle, a catering product, a closure, a container, a piece of cutlery, a drink cup, a food packaging, a lid, an overcap, a prescription container, a toy, an electronic device, or a tube.

39. The consumer product of claim 38, wherein the consumer product is an agricultural film, a building product, a corrosion protection, a custom film, a flexible intermediate bulk container (FIBC), a flexible packaging, a food and film packaging, a medical film, a personal care film, a sheeting, a drop cloth, a stretch film, a grocery film, a cutterbox film, a tape, or a trash bag.