Three Dimensionally and Randomly Oriented Fibrous Structures

Abstract

A randomly-oriented 3-D fibrous structure and a method for making the same. The method involves electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant; to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a non-provisional application claiming the benefit of U.S. Provisional Patent Application 61/668,269, filed Jul. 5, 2012, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under a grant from the U.S. Dept. of Agriculture (NEB 37-037). The government has certain rights to this invention.

FIELD OF THE INVENTION

This invention is directed to electrospinning three-dimensionally (3D) and randomly oriented fibrous structures from various polymer sources, including plant proteins, animal proteins, and synthetic polymers.

BACKGROUND OF INVENTION

Ideal tissue engineering scaffolds should be capable of closely mimicking the topographies and spatial structures of native extracellular matrices (ECMs) to facilitate cells to grow and differentiate following the patterns similar to that found in native tissues and organs. Morphologies of ECMs vary according to functions of target tissues and cell types in the tissues. For example, in skin tissue, the top layer is formed by compact packing of epithelial cells on a two-dimensional (2D) fibrous ECM basement membrane. Three-dimensional spatial spreading of fibroblasts and immune cells occurs in the interior region of the skin tissue, and correspondingly the ECMs are constructed by stereoscopically and randomly oriented ultrafine protein fibers. Fibrous structures with 3D orientation and random distribution can also be found in native ECMs in breast, liver, bladder, lung, and many other organs and tissues. It has been reported that cells cultured on flat 2D substrates may differ considerably in morphology and differentiation pattern from those cultured in more physiological 3D environments. Therefore, it is reasonable to fabricate scaffolds with particular morphologies and structures according to categories and functions of original native tissues.

Due to its simplicity and high efficiency, electrospinning has been widely employed to fabricate tissue engineering scaffolds composed of nano- or submicrometer-fibers from numerous materials. However, conventional electrospun structures typically form 2D scaffolds with fibers aligned parallel to the collector and cells cultured on the conventional electrospun scaffolds could only develop into flat shapes. The functions and differentiation of many flattened cells could not resemble the native stereoscopic cells. Furthermore, small pore sizes, owing to the close arrangement of fibers, restricted access of cells to the interior of conventional electrospun scaffolds. Thus, on conventional electrospun scaffolds, cells could mainly spread and distribute within a shallow depth beneath the surface.

To date, many 3D electrospinning techniques have been developed to fabricate electrospun scaffolds with larger pores and higher porosity to improve cell accessibility of the scaffolds. Examples of such techniques include wet electrospinning, electrospinning with integration of coarse fibers, and electrospinning with porogens (e.g., dry ice, salt, or sucrose), which are based on the concept of including a “blocking agent” to increase the distances between electrospun fibers in order to lead to deeper penetration of cells into interior of scaffolds. Nonetheless, these techniques failed to change the planar orientations of the electrospun fibers and as a result the scaffolds tended to have parallel fibrous layer-by-layer structures. Additionally, the parallel fibrous layer-by-layer structures tended to not have pores that extend very far in the thickness direction as compared to in the planar directions. As a result, there was limited improvement in scaffold porosity and cells cultured thereon tended to have flattened morphologies rather than stereoscopically developed cells in many native tissues. Still further, there have been attempts to fabricate 3D electrospun scaffolds based on the electrostatic repulsion between as-spun fibers. Despite their fluffy appearances, such scaffolds still had a layer-by-layer structure of planar mats and parallel oriented fibers.

In view of the foregoing, a need still exists for randomly-oriented 3-D fibrous structures and a method for making the same via electrospinning.

SUMMARY OF INVENTION

In one embodiment, the present invention is directed to a method of making a randomly-oriented 3-D fibrous structure. The method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant; to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure.

In one embodiment, the present invention is directed to a method of making a randomly-oriented 3-D fibrous structure. The method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa, and wherein the polymer is selected from the group consisting of protein, synthetic polymer, and combinations thereof; and an anionic surfactant at about 5 to about 300 percent by weight of the polymer; to form one or more fibers of a fineness that is about 50 nm to about 100 μm and that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure that further comprises interconnected pores having sizes that are about 10 to 2000 μm and that has a porosity that is about 60 to about 99.9% by volume.

In one embodiment, the present invention is directed to a randomly-oriented 3-D fibrous structure comprising: one or more fibers that comprise a polymer-surfactant complex, wherein the fiber(s) have lengths that are at least about 100 nm and finenesses that are about 50 nm to about 100 μm, and are arranged randomly and evenly in three dimensions throughout the randomly-oriented 3-D fibrous structure; and interconnected pores having sizes that are about 10 to 2,000 μm, wherein the pores comprise about 60 to about 99.9% by volume of the randomly-oriented 3-D fibrous structure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains schematic diagrams of fibers being deposited via electrospinning in which the 2D column contains images (a), (b), and (c) showing the parallel deposition of conventional electrospun fibers and the 3D column contains images (d), (e), and (f) showing the deposition of fibers according to the present invention.

FIG. 2 contains photographic images of fibers being deposited via electrospinning in which the (a) column contains images 1, 2, and 3, which were taken at 0.125 second intervals showing the deposition of conventional, parallel-aligned electrospun fibers and the (b) column contains images 1, 2, and 3, which were taken at 0.125 second intervals showing the vertical deposition of fibers according to the present invention.

FIG. 3 is a photographic comparison of a zein electrospun randomly-oriented 3-D fibrous structure and a zein electrospun 2D fibrous structure; the structures are of the same weight.

FIG. 4 are images of a zein electrospun randomly-oriented 3-D fibrous structure: (a) is a SEM 70x top view image; (b) is a SEM 70x side view image; (c) is a CLSM 100x 45 degree front view image; and (d) is a CLSM 100x side view image.

FIG. 5 are CLSM 60x images of sequential sections of 2D [(a) 1, 2, 3, and 4] and randomly-oriented 3-D fibrous structures [(b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12] each taken at increasing 10 μm depths in which the lighter network is F-actin in NIH 3T3 cells stained with Phalloidin 633 after being cultured in the structures for 72 hours.

FIG. 6 is graph of MTS assay results of attachment (4 hours) and proliferation (24, 72, 120, and 168 hours) of NIH 3T3 fibroblase cells on 2D and 3D zein electrospun fibrous structures.

FIG. 7 are CLSM images showing the proliferation of NIH 3T3 cells on 2D and 3D zein electrospun fibrous structures about 48 hours after seeding (stained with Phalloidin 633 and Hoechst 33342). The three-dimensional reconstruction of the 2D fibrous structure is (a): x-y projection (I); x-z projection (II′); and y-z projection (III). The three-dimensional reconstruction of the 3D fibrous structures is (b): x-y projection (I′); x-z projection (II′); and y-z projection (III′).

FIG. 8 is a photographic comparison of an electrospun randomly-oriented 3-D PEG fibrous structure prepared with SDS and an electrospun 2D fibrous structure; the structures are of the same weight.

FIG. 9 is a photograph of a PEG electrospun randomly-oriented 3-D fibrous structure prepared with a nonionic surfactant.

FIG. 10 is a SDS-PAGE of soy protein, and wheat glutenin, wherein lane 1 shows standard protein markers, lane 2 is untreated soy protein, lane 3 is cysteine extracted soy protein, lane 4 is NaOH extracted soy protein, lane 5 is untreated wheat glutenin, and lane 6 is cysteine extracted wheat glutenin.

FIG. 11 is a photograph of a soy protein electrospun randomly-oriented 3-D fibrous structure.

FIG. 12 is are photographs of soy protein electrospun randomly-oriented 3-D fibrous structure, wherein (a) is before immersion in PBS and (b) is 3 days after immersion in PBS; scale bar is 20 μm.

FIG. 13 is a photograph of a feather keratin electrospun randomly-oriented 3-D fibrous structure.

FIG. 14 is a photograph of a wheat glutenin electrospun randomly-oriented 3-D fibrous structure.

FIG. 15 is a photograph of a wheat gliadin electrospun randomly-oriented 3-D fibrous structure.

FIG. 16 is a photograph of a casein electrospun randomly-oriented 3-D fibrous structure.

FIG. 17 is photograph of a peanut electrospun randomly-oriented 3-D fibrous structure.

FIG. 18 (a) is graph showing the relationship between specific pore volume of electrospun scaffolds and surface resistivity (the dashed line shows the simulated relation using power function) and FIG. 18 (b) is a graph showing the effect of sodium dodecyl sulfate (SDS) and NaCl on surface resistivity of PEG films based on the proportions of SDS to polymer (the molar concentration of NaCl was the same as that of SDS at each point).

FIG. 19 (a) are photographs of the deposition process of zein onto an insulator covered collector at time intervals of 0.125 seconds between each image (the spinning dope consisted of 25 wt % zein and 25 wt % SDS); FIG. 19 (b) is a photograph of the as-spun zein scaffold; and FIG. 19(c) is a CLSM 45° image at magnification of 100× of the above as-spun zein scaffold.

DETAILED DESCRIPTION OF INVENTION

An embodiment of the present invention is directed to a method of making a randomly-oriented 3-D fibrous structure. The method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant; to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure. Typically, the electrospinning is conducted at a temperature that is about 25 to about 70° C.

As used herein with respect to the present invention, the “randomly-oriented 3-D fibrous structure” is intended to mean fibrous structure in which the one or more fibers thereof are randomly oriented in all three dimensions rather than only being randomly oriented in two dimensions as can be found in conventional layer-by-layer, planar structures. The degree of random orientation of a fibrous structure may be quantified by, for example, identifying a cubic centimeter volume of said fibrous structure and identifying any particular imaginary plane of any orientation within said volume, identifying the number of fibers intersected or fiber intersections both of which are “intersections” with said plane, and identifying the number of said intersections in which said intersected fiber(s) are at angles greater than 30 degrees relative to said plane. In certain embodiments of the present invention, at least ½, ⅝, ⅔, and ¾ of the intersected fibers are at angles greater than 30 degrees with the plane.

As used herein the terms “scaffold” and “fibrous structure” are intended to have the same meaning and may be used interchangeably.

Mechanism

Without being bound to any particular theory, it is believed that the mechanisms of 2D electrospinning and an embodiment of 3D electrospinning (of the present invention produce) are depicted in FIG. 1. In both conventional and 3D electrospinning, at the beginning, the liquid droplet acquired negative charges and then was elongated into fibers. For conventional electrospinning, relatively few of the electrons are transferred to the collector at the moment the fiber ends hit the collector, owing to high surface resistivity of the fibers. The fibers with a relatively large amount of remaining electrons are strongly attracted to the positively charged collector. As a consequence, conventional 2D electrospun scaffolds have fibers oriented parallel to the collecting board and the fibrous structures are relatively tightly packed.

In contrast, the depicted 3D electrospinning embodiment is believed to be based on repulsive electrical force between fibers and the collector. More specifically, the method of the present invention, involves including a surfactant to decrease the surface resistivity of a fiber to thereby increase the transfer of charge from the fiber surface to the collector. When the fiber(s) strike the collector, surface static electricity transfers to the board in a faster manner, which results in less negative static electricity remaining on the fiber(s), and decreased attraction between fiber(s) and the collector. In some cases, the near portion of the fibers may even carry positive charges and may be repulsed by the collector, while the farther end of the fiber(s) is still attracted and moves towards the board. As a result, fiber(s) are collected onto the board in multiple orientations to form loose and fluffy 3D scaffolds with randomly-oriented fiber(s).

It is believed that the above-described mechanisms have been shown by experimental results shown in FIG. 2 in which FIG. 2a is conventional electrospinning and FIG. 2b is electrospinning in accordance with the present invention at 0.125 second intervals between sequential photographs with the the white objects PEG fibrous bulks.

Polymer

As indicated above, the spinning dope comprises a polymer, which may be any appropriate polymer such as protein, synthetic polymer, and combinations thereof. Proteins may be of particular interest because they are preferred in biomedical applications due to their molecular similarity to native ECMs, and tend to be widely available at low cost. For example, plant proteins, zein and soy protein, and animal proteins, such as keratin and collagen, have been proved to be supportive to cell growth in in vitro and in vivo studies. In addition, plant and animal proteins are widely available at a low cost and they are considered to be a renewable resource. The method of the present invention may be conducted using one or more proteins selected from the group consisting of plant protein, animal protein, and combinations thereof.

Many such proteins, however, are considered to be highly-linked and as a result tend to have limited solubility in water and organic or alcoholic solvents. In general, proteins with cysteine content higher than 1% in its amino acid composition are considered to be highly-crosslinked proteins. Exemplary highly-crosslinked proteins, including keratin, which has a cysteine content of about 7%, soy protein has a cysteine content of about 1.3%, and wheat glutenin a cysteine content of about 2%. In particular, electrospinning highly crosslinked proteins, such as soy protein, feather keratin and wheat glutenin, into fibrous structures via electrospinning has not been possible due to their insolubility in various solvents.

With reductant and denaturant in the spinning dope, coarse fibers from soy protein and wheat gluten via wet spinning have been produced. Reddy, N. and Y. Yang, Novel Protein Fibers from Wheat Gluten, Biomacromolecules, 2007, 8(2), p. 638-643; Reddy, N. and Y. Yang, Soy protein fibers with high strength and water stability for potential medical applications, Biotechnology Progress, 2009, 25(6), p. 1796-1802. However, electrospinning with pure protein required much better dissolution of proteins, and thus the spinning dope for wet spinning failed to be electrospun. Highly hydrolyzed soy protein with small molecules had been electrospun with PEG, which accounted for the spinability. Vega-Lugo, A. C. and L. Loong-Tak, Electrospinning of Soy Protein Isolate Nanofibers, Journal of Biobased Materials and Bioenergy, 2008, 2(3), p. 223-230. Hydrolyzed soy protein in its pure form, however, had not been electrospun.

The present invention of electrospinning may be practiced, however, with highly crosslinked protein by dissolving the protein in manner that preserves the protein subunits with appropriate molecular weights ranging from 5 to 150 kDa. Other methods of dissolving highly crosslinked proteins (e.g., U.S. Pat. Pub. No. 2006/0282958, Yang et al., entitled Process for the Production of High Quality Fibers from Wheat Proteins and Products Made From Wheat Proteins) allowed for the spinning of coarse fibers but were not suitable for electrospinning of relatively fine fibers. The electrospinning method of the present invention may be practiced with highly crosslinked proteins by using a reducing agent such a thiol, a sulfite, or a sulfide to break the disulfide bonds of the highly crosslinked proteins. For example, cysteine, an environmentally-benign thiol, may be used as a reducing agent to break the disulfide bonds and achieve dissolution of highly-crosslinked proteins. The amount of the reducing agent in the solution may be varied from about 1% to about 50% based on the weight of proteins. The solvent for the protein and cysteine was a 4-8 M urea solution with pH of 8 to 12, adjusted using 50 wt % sodium hydroxide solution. The weight ratio of protein-containing materials to urea solution may be varied within a range from, for example, 1:5 to 1:30. The temperature for the dissolution may be within a range from about 20° C. to about 90° C. Examples of additional appropriate thiols include methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, butanethiol, tert-butyl mercaptan, pentanethiols, thiophenol, thioacetic acid, coenzyme-A, glutathione, 2-mercaptoethanol, dithiothreitol, 2-mercaptoindole, 3-mercaptopropane-1,2-diol. Examples of appropriate sulfites and sulfides include sodium sulfite potassium sulfite, sodium bisulfite, potassium bisulfite, sodium sulfide, potassium sulfide, sodium metabisulfite, and potassium metabisulfite. The conditions for the reactions include the thiol/sulfite/sulfide at concentration(s) of about 0.5% to about 50% based on the weight of proteins, a pH from 3 to 12 for a duration of about 30 minutes to about 24 hours at a temperature of about 20° C. to about 90° C.

In view of the foregoing, the method of the present invention may, in certain embodiments, be conducted using appropriately dissolved highly-crosslinked plant protein, highly-crosslinked animal protein, and combinations thereof. When using proteins, including highly-crosslinked proteins, experimental results to date have shown the aging the dissolved protein at an aging temperature that is about 20° C. to about 90° C. for an aging duration that is about 0.5 to about 48 hours before conducting the electrospinning may be advantageous. For example, it has been found that such aging may provide a higher degree of disentanglement of the protein molecules to improve the spinnability of the protein spinning dope.

Exemplary plant proteins include wheat gluten, wheat gliadin, wheat glutenin, soy protein, camelina protein, peanut protein, canola protein, sorghum protein, rice protein, millet protein, sunflower seed protein, pumpkin seed protein, mung bean protein, red bean protein, chickpea protein, green pea protein, and combinations thereof;

Exemplary animal proteins include chicken feather, egg white, wool keratin, casein, silk, fibrin, collagen, gelatin, hair keratin, horn keratin, nail keratin, whey protein, and combinations thereof.

Exemplary synthetic polymers include polyethylene glycol (PEG), poly lactic acid (PLA), poly glycolic acid (PGA), polyhydroxyalkanoates (PHAs), poly(lactic-co-glycolic acid) (PLGA), poly-3-hydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and combinations thereof.

As indicated above, the solution may comprise one, two, or even more polymers. In certain embodiments with more than a single polymer, the polymer that is in the largest amount (usually more than half of the total polymer content) is often referred to as the “primary” polymer, component, or material and the additional polymers are often referred to as “secondary” polymer(s), component(s), or material(s) with concentrations that are from about 0.5% to about 50% by weight of the primary polymer.

Surfactant

As indicated above, the spinning dope comprises a surfactant, which may be any appropriate surfactant. The hydrophobic portions of surfactant bond with polymers through hydrophobic interaction to form a protein-surfactant complex. Without being bound to a particular theory, it is believed that the hydrophilic portions, including functional groups that carry positive or negative charges and polar uncharged groups, gather on the fiber surface and thus effectively increase the surface conductivity (or lower the surface resistivity) by introducing surface water layer, which facilitates delivery of charges on the surface.

The surfactant(s) may be of different hydrocarbon chain lengths and different electrical properties, including anionic surfactants, cationic surfactants, nonionic surfactants, zwitterionic surfactants. In an embodiment, combinations of surfactants may be used, as appropriate. In another embodiment, the surfactant is one or more anionic surfactants.

Typically, the spinning dope has a concentration of the surfactant that is about 5 to about 300 percent by weight of the polymer. In another embodiment, the spinning dope has a concentration of the surfactant that is about 50 to about 150 percent by weight of the polymer.

Exemplary anionic surfactants include sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, sodium lauryl sarcosinate, perflourobutanesulfonic acid, ammonium lauryl sulfate, sodium stearate, sodium pareth sulfate, dioctyl sodium sulfosuccinate, potassium lauryl sulfate, sodium laureth sulfate, sodium myreth sulfate, and combinations thereof.

Exemplary cationic surfactants include benzalkonium chloride, cetrimonium bromide, tetramethylammonium hydroxide, octenidine dihydrochloride cetyl trimethylammonium bromide, hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide (DODAB), and combinations thereof.

Exemplary nonionic surfactants include decyl glucoside, octyl phenol ethoxylated, polysorbate 80, polysorbate 20, and combinations thereof.

Exemplary zwitterionic surfactants include cocamidopropyl hydroxysultaine, cocamidopropyl betaine, lecithin, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and combinations thereof.

Solvent

As indicated above, the spinning dope comprises a solvent, which may be any appropriate solvent. Exemplary solvents include water, phosphate buffered saline (PBS), carbonate buffer, tris-glycine buffer, borate buffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, citric buffer, ethanol, chloroform, 1,4-dioxane, methanol, ethylene glycol, acetone, ethyl acetate, methyl acetate, hexane, petrol ether, citrus terpenes, diethyl ether, dichloromethane, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), formic acid, n-butanol, isopropanol (IPA), n-propanol, acetic acid, nitromethane, dichloromethane, and combinations thereof. In another embodiment, the solvent is selected from the group consisting of water, phosphate buffered saline (PBS), carbonate buffer, tris-glycine buffer, borate buffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, citric buffer, chloroform, and combinations thereof.

Additional Spinning Dope Components and Method Steps

In addition to the above-described components, the spinning dope may comprise one or more additional components such as a cross-linker, a sacrificial component, or a combination thereof. Alternatively or in addition to including such additional components in the spinning dope, the method may further comprise contacting the fibers with a second solution that comprises a second cross-linker, a surface decorator, or a combination thereof.

Cross-linker(s)

Exemplary cross-linkers include polycarboxylic acid which contains at least three carboxylic acid groups (such as citric acid, iso-citric acid, propane-1,2,3-tricarboxylic acid, trimesic acid, aconitic acid, mellitic acid, 1,2,3,4-Butane tetracarboxylic acid (BTCA)), oxysucrose, genepin, glutaraldehyde, oxaldehyde, NHS esters, maleimides, carbodiimide, isocyanate, and combinations thereof. If present in the spinning dope and/or in a cross-linking solution, the concentration of cross-linker is typically about 0.01 mol/L to about 10 mol/L. In an embodiment in which the spinning dope comprises a first cross-linker and the method further comprises contacting the fibers with a cross-linking solution that comprises a second cross-linker, the first and second cross-linkers may be independently selected and as such may be identical or different (e.g., in terms of material, concentration, or both). In an embodiment of the present invention, the first and second cross-linkers are independently selected from the group consisting citric acid, 1,2,3,4-Butane tetracarboxylic acid (BTCA), oxysucrose, genepin, glutaraldehyde, oxaldehyde, and combinations thereof.

Sacrificial Component

Exemplary sacrificial components include PEG, egg white protein, zein, wheat gliadin, and other materials that may be dissolved with the above-described polymer(s) before electrospinning at concentrations of about 0.5% to about 50% based on the weight of the polymer. The sacrificial component(s) may be removed from the spun fibrous structure via rinsing with water, organic solvents or alcoholic solvents.

Surface Decorator

Exemplary surface decorators of fibers include peptides such as Arg-Gly-Asp (RGD), Ile-Lys-Val-Ala-Val (IKVAV), Asn-Ser-Gly-Ala-Ile-Thr-Ile-Gly (NSGAITIG), and combinations thereof at concentrations of about 0.5% to about 10% based on the weight of the polymer.

Coagulation Solution

The method may further comprise contacting the fibers with a coagulation solution that comprises a coagulant to modify the water stability and mechanical properties of the fibers (e.g., tensile properties, compressive properties, abrasion properties, and bending properties). Exemplary coagulants include methanol, ethanol, sodium sulfate and acetic acid, acetone, sulfuric acid, hydrochloric acid, and combinations thereof. In an embodiment of the present invention, the coagulant is selected from the group consisting of methanol, ethanol, sodium sulfate and acetic acid, and combinations thereof. The amount of coagulant in the coagulant solution is such that the concentration of coagulant is typically about 1% to about 100%. Typically, the fibers are contacted with the coagulation solution for a duration of about 5 minutes to about 24 hours. Also, the coagulation solution is typically at a temperature that is from about 20° C. to about 100° C.

Fibers and Fibrous Structure

The above described method may be performed to make a randomly-oriented 3-D fibrous structure. Such a fibrous structure comprises: one or more fibers that comprise a polymer-surfactant complex, wherein the fiber(s) have lengths that are at least about 100 nm and finenesses that are about 50 nm to about 100 μm, and are arranged randomly and evenly in three dimensions throughout the randomly-oriented 3-D fibrous structure; and interconnected pores having sizes that are about 10 to 2,000 μm, wherein the pores comprise about 60 to about 99.9% by volume of the randomly-oriented 3-D fibrous structure. Advantageously, experimental results to date indicated that such randomly-oriented, three-dimensionally fibrous structures may be used to facilitate cells to grow and spread in three dimensions in a manner similar to that seen in native ECMs.

In another embodiment, the fibers have a fineness that is about 50 nm to about 100 μm and the randomly-oriented 3-D fibrous structure further comprises interconnected pores having sizes that are about 10 to 2000 μm and has a porosity that is about 60 to about 99.9% by volume.

In yet another embodiment, the fibers have a fineness that is about 50 nm to about 20 μm and the randomly-oriented 3-D fibrous structure further comprises interconnected pores having sizes that are about 10 to 1000 μm and has a porosity that is about 90 to about 99.9% by volume of the structure.

In an embodiment, the one or more fibers consist of the polymer-surfactant complex. In another embodiment, the randomly-oriented 3-D fibrous structure consists of the one or more fibers and the interconnected pores. In yet another embodiment, the randomly-oriented 3-D fibrous structure consists of one or more fibers and the interconnected pores, wherein the one or more fibers consist of the polymer-surfactant complex.

Heat Treating the Fibers

After the fibers/fibrous structure are formed it may be heat treated at a temperature that is about 70 to 150° C. to modify the water stability and mechanical properties of the fibers (e.g., tensile properties, compressive properties, abrasion properties, and bending properties).

EXAMPLES

General Procedures

Scaffold Preparation

Thirty 2D zein scaffolds were prepared by electrospinning 25 wt % zein (Freeman Industries LLC, Tuckahoe, N.Y.) in 70% v/v aqueous ethanol (EMD Chemicals Inc., Gibbstown, N.J.) solution. Three dimensional zein scaffolds were prepared by electrospinning aqueous solution containing 25 wt % zein and 25 wt % SDS. A concentration of 9 wt % (based on the weight of zein) citric acid (EMD Chemicals Inc., Gibbstown, N.J.) was added into both 2D and 3D spinning dopes for cross-linking. Different solvent systems were utilized since zein could not be dissolved in water. The 2D PEG scaffold was prepared by electrospinning 10 wt % PEG (50 kDa, Sigma-Aldrich, St. Louis, Mo.) aqueous solution. The 3D PEG scaffold was prepared by electrospinning 10 wt % PEG and 10 wt % SDS in aqueous solution. All the electrospinning parameters, including the extrusion speed of 2 mL/hr, voltage of 42 kV, and distance from the needle to the collecting board of 25 cm, were kept the same for all the samples. The needle was negatively charged, and the collecting board was positively charged.

Morphologies and Structures of Scaffolds

The 2D and 3D scaffolds were observed using a scanning electron microscope (S3000N, Hitachi Inc. Schaumburg, Ill.) and a Nikon A1 confocal laser scanning microscope (Nikon Inc., Melville, N.Y.).

Specific Pore Volume

Specific pore volume indicating volume of pore in unit mass of scaffolds as shown in the following equation was selected to evaluate fluffiness of the scaffolds:

$V_{\mathrm{sp}}=\frac{V_{\mathrm{pore}}}{m_{\mathrm{scaffold}}}=\frac{V_{\mathrm{scaffold}}}{m_{\mathrm{scaffold}}}-\frac{1}{{\rho }_{\mathrm{material}}}$

where V_sp is the specific pore volume, V_pore is the volume of pores encompassed in the scaffolds, m_scaffold is the mass of scaffolds, V_scaffold is the volume of the scaffolds after precise measurement of the length, width, and thickness of scaffolds, and ρ_material is the density of the material.

Surface Resistivity

Since the surface resistivity of ultrafine fibers is very difficult to test, films containing same polymer to surfactant/salt ratio with relevant electrospun fibers were prepared to measure the surface resistivity. The films were casted onto Teflon coated plates and dried at 20° C. and 65% relative humidity. Surface resistivity was measured by employing a surface resistivity tester (Monroe Electronics Inc., Lyndonville, N.Y.) according to ASTM D-257 standard.

Fiber Deposition Process

A CCD camera with a longworking-distance lens was used in capturing the moment photographs of fiber deposition and scaffold formation. The time interval for each consequential photograph was 0.125 seconds.

Cell Attachment and Proliferation

NIH 3T3 mouse fibroblast cells (ATCC CRL-1658, Manassas, Va.) were cultured to quantitatively estimate effects of 2D and 3D structures of zein scaffolds on cell attachment and proliferation. Cells were cultured in culture medium at 37° C. in a humidified 5% CO2 atmosphere. Electrospun 2D and 3D zein scaffolds were first rinsed in 60 wt % acetone (BDH, West Chester, Pa.) aqueous solution containing 5 wt % potassium chloride (Fisher Scientific, Fair Lawn, N.J.) to remove SDS, washed in distilled water three times, and then lyophilized. MTS assays were performed to quantitatively investigate cell viability at attachment and proliferation stages. Samples were prepared with same weight and then were subjected to sterilization at 120° C. for 1 hour. After sterilization, the scaffolds were placed in 48-well culture plates (TPP Techno Plastic Products, Switzerland). Fibroblast cells were seeded onto the scaffolds (1×10⁵ cells mL⁻¹, 500 μL well⁻¹) and then cultured at 37° C. in a humidified 5% CO₂ atmosphere for different time intervals. At each time point, the samples were washed with PBS, placed in new 48-well plates containing 450 μL well⁻¹ 20% MTS reagent (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promenade) in Dulbecco's modified Eagle's medium (DMEM) and incubated at 37° C. in a humidified 5% CO₂ atmosphere for 3 hours. After incubation, 150 μL of the solution from each well was pipetted into a 96-well plate and the optical densities were measured at 490 nm using a UV/vis multiplate spectrophotometer (Multiskan Spectrum, Thermo Scientific). The MTS solution in DMEM without cells served as the blank.

Cell Penetration and Spreading

To compare penetration ability of cells on 2D and 3D scaffolds, cells were stained by Phalloidin 633 solution (1:200 Alexa Fluor 633 Phalloidin, Invitrogen, Grand Island, N.Y.) and observed using a Nikon A1 confocal laser scanning microscope (Nikon Inc., Melville, N.Y.). Alexa Fluor 633 Phalloidin is a far red fluorescent dye that specifically bonds to F-actin in cells. This dye was selected since zein shows fluorescence across the full spectrum with the weakest signal in the far red range. To observe the spreading behaviors and stereoscopic morphologies of cells in 2D and 3D scaffolds, cells were stained by Phalloidin 633 solution for F-actin and Hoechst 33342 solution (Invitrogen, Grand Island, N.Y.) for the nuclei of cells.

Statistical Analysis

One-way analysis of variance with Tukey's pairwise multiple comparisons was employed to analyze the data. The confidence interval was set at 95%, and a P value less than 0.05 was considered to be a statistically significant difference. In the results, data labeled with different symbols were significantly different from each other.

3D Electrospun Zein Scaffolds

Ex. 1: Morphology of 3D Electrospun Zein Scaffolds

A piece of 3D zein scaffold was prepared by electrospinning aqueous solution containing 25 wt % of zein and 25 wt % of sodium dodecyl sulfate (SDS), and a piece of 2D zein scaffold was prepared by electrospinning 25 wt % zein in 70% v/v aqueous ethanol solution; 9 wt % of citric acid based on the weight of zein was added into both 2D and 3D spinning dopes for crosslinking. Different solvent systems were utilized because the zein, itself, could not be dissolved in water. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged.

As is shown in FIG. 3, the 3D electrospun zein scaffold has significantly higher porosity and fluffiness than its 2D counterpart with the same weight. FIG. 4 contains the microscopic morphologies of 3D electrospun zein scaffolds observed under scanning electron microscope (SEM, left) and confocal laser scanning microscope (CLSM, right), which show that the orientation of fibers in the 3D electrospun zein scaffold was random (from both the top surface and the side).

Ex. 2: In Vitro Cell Culture Study of 3D and 2D Electrospun Zein Scaffolds

NIH 3T3 fibroblast cells were cultured on both 2D and 3D electrospun zein scaffolds to evaluate the effects of scaffold architecture on cellular attachment, penetration and proliferation. Cell penetration was evaluated 48 hours after culturing the 2D and 3D scaffolds. Significantly higher cell accessibility of 3D zein electrospun scaffold compared with 2D scaffold is shown in FIG. 5. Cells cultured on the 3D electrospun zein scaffold were found at the depth of 120 μm from the surface, whereas cells could not be seen 40 μm below the surface of the 2D electrospun zein scaffold. It is believed that the “looser” structure of the 3D electrospun scaffold accounted for the much better cellular penetration in the 3D scaffold.

Methanethiosulfonate (MTS) assays were conducted to quantitatively investigate cell attachment and proliferation, the results of which are shown in FIG. 6. The amount of cells attached on the 3D zein scaffold 4 hours after seeding was 114% higher than that on the 2D scaffold. A remarkably higher proliferation rate was also found for cells cultivated on 3D zein scaffold. Cell proliferation on the 3D scaffold reached plateau 5 days after seeding and the cell density increased more than 4 times, while that on 2D scaffold reached plateau 3 days after seeding and the increase in cell density was less than twice. It is believed that the greater cyto-accessible space of the 3D scaffold may be the reason for higher cell accessibility and better proliferation.

3D zein scaffolds showed a great potential for tissue engineering applications. For example, FIG. 7b shows spheroid-shaped cells on 3D scaffolds whereas FIG. 7a shows cells with flattened morphologies on 2D scaffolds. More specifically, FIG. 7a.I shows he developed cytoskeletons of cells over the surface of 2D scaffold (the x-y projection). The thickness of the cells illustrated in the x-z projection (FIG. 7a.II) and the y-z projection (FIG. 7a.III) was much smaller than their planar sizes in the x-y projection (FIG. 7a.I). As can be seen, cells seeded on the 2D scaffolds developed into 2D planar morphologies. In contrast, the side views of 3D scaffolds in FIG. 7b.II′ and III′ show that the cells oriented in thickness direction rather than in planar x and y directions. It is believed that the 3D scaffolds facilitated the cells to develop into stereoscopic architectures that more closely mimic the cells in many native ECMs.

3D Electrospun PEG Scaffolds Using Anionic and Nonionic Surfactants

Ex. 3: Morphology of 3D Electrospun PEG Scaffolds Using Anionic Surfactant

PEG is a water soluble synthetic polymer, and could also be electrospun into 3D stereoscopic architecture. PEG (25 wt %) and SDS (25 wt %) were dissolved in water to prepare spinning dopes. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. As is shown in FIG. 8, the 3D electrospun zein scaffold has significantly higher porosity and fluffiness than its 2D counterpart with the same weight. The 3D PEG electrospun scaffold was composed of spatially randomly oriented fibers with diameter around 0.8 μm.

Ex. 4: Morphology of 3D Electrospun PEG Scaffolds Using Nonionic Surfactant

PEG (25 wt %) and TRITON X-100 (25 wt %), a nonionic surfactant, was dissolved in water for spinning. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. As is shown in FIG. 9, the 3D electrospun zein scaffold has significantly higher porosity and fluffiness than its 2D counterpart with the same weight. The 3D PEG electrospun scaffold was composed of spatially randomly oriented fibers with diameter around 0.8 μm.

3D Electrospun Scaffold From Water Insoluble Highly Crosslinked Proteins

Ex. 5: Extraction of Spinable Proteins from Soy Protein and Wheat Glutenin

Soy protein and wheat glutenin represent two types of highly crosslinked proteins that are high in molecular weights and insoluble in water. These proteins were treated in 8 M urea under mild alkaline condition with existence of cysteine, a common amino acid, which is also a nontoxic and environmentally benign reductant. After being treated at 70° C. for 24 hours, the soluble proteins were collected. SDS PAGE results of these extracted proteins showing molecular weights are shown in FIG. 10. Lane 1 showed standard protein markers. Lanes 2, 3, and 4 were untreated soy protein, cysteine extracted soy protein and soy protein treated with NaOH. The major bands (20 kDa, 37 kDa and 63 kDa) of soy protein remained in the extracted sample, while no obvious bands could be found in the NaOH treated sample. Lane 5 and 6 were wheat glutenin and cysteine extracted wheat glutenin. The highly similar bands of the two samples revealed the high efficient yet non-destructive manner of cysteine extraction.

Ex. 6: Morphology of 3D Electrospun Scaffold from Soy Protein

Soy protein extracted using protocol as mentioned in Example 4 (25 wt %) and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in FIG. 11. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. The diameter of the ultrafine fibers ranged from 1 to 3 μm.

Ex. 7: Water Stability of 3D Electrospun Soy Protein Scaffold

After post treatment of coagulation bath of 10% Na₂SO₄ and 10% acetic acid, the 3D electrospun soy protein scaffold was soaked in PBS at 50° C. for 3 days. As shown in FIG. 12, the scaffold retained fibers in their structures as the diameters of fibers increased from 1-3 μm to 2-5 μm after 3 days. The 3D electrospun soy protein scaffold showed fairly good water stability.

Ex. 8 Morphology of 3D Electrospun Scaffold from Feather Keratin

Feather keratin extracted using protocol as mentioned in Example 4 (25 wt %), 25 wt % of SDS and cysteine (10 wt % based on keratin) were dissolved in water and electrospun into 3D structures as shown in FIG. 13. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. The diameter of the ultrafine fibers ranged from 2 to 5 μm.

Ex. 9 Morphology of 3D Electrospun Scaffold from Wheat Glutenin

Wheat glutenin extracted using protocol as mentioned in Example 4 (25 wt %), 25 wt % of SDS and cysteine (10 wt % based on wheat glutenin) were dissolved in water and electrospun into 3D structures as shown in FIG. 14. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. The diameter of the ultrafine fibers around 2 μm.

3D Electrospun Scaffold From Other Proteins

Ex. 10: Morphology of 3D Electrospun Scaffold from Wheat Gliadin

Wheat gliadin (25 wt %), a prolamin in wheat, and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in FIG. 15. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. The diameter of fibers in 3D gliadin electrospun scaffold ranged from 1.5 to 3.5 μm.

Ex. 11: Morphology of 3D Electrospun Scaffold from Casein

Casein (25 wt %), the family of proteins commonly found in mammalian milk, and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in FIG. 16. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. The diameter of fibers in 3D casein electrospun scaffold ranged from 0.8 to 2.8 μm.

Ex. 12: Morphology of 3D Electrospun Scaffold from Peanut Protein

Peanut protein (25 wt %) and 25 wt % of SDS were dissolved in water and electrospun into 3D structures as shown in FIG. 17. The electrospinning process was conducted at an extrusion speed of 2 mL/hr, a voltage of 42 kV, and a distance from the needle to the collecting board of 25 cm. The needle was negatively charged and the collecting board was positively charged. The diameter of fibers in 3D peanut protein electrospun scaffold ranged from 0.9 to 2.9 μm.

Relationship between Specific Pore Volume and Surface Resistivity

A power function was used to simulate the relationship between the specific pore volume of electrospun scaffolds and corresponding polymer surface resistivity. As shown in FIG. 18a, the residual standard error of the model is 0.613, which is a reasonable number to indicate that the data could be well described by power function and suggests that there is a strong quantitative relationship between the specific pore volume and surface resistivity. The power function was constructed as y=ax^b where y represented specific pore volume, x represented surface resistivity, and a and b were coefficients varied with the type of materials. Here, for PEG, a equaled to 2.208×105 and b equaled to −0.5325. The specific pore volume decreased exponentially as surface resistivity increased. As surface resistivity decreased from 109 to 106 Ω/sq., the macrostructure of PEG scaffolds converted from 3D to 2D, and the specific pore volume decreased by about 20 times. It may be inferred that the increased surface conductivity increased fluffiness of scaffolds exponentially. It was found that surface resistivity of the polymer decreased with increasing SDS proportion. As shown in FIG. 18b, surface resistivity of PEG decreased as SDS content increased. Surface resistivity of pure PEG was higher than 109 Ω/sq. When the weight ratio of SDS to PEG was increased to 1:1, surface resistivity was reduced considerably to 106 Ω/sq. However, when NaCl was added into the polymer, the surface resistivity did not decrease as substantially as the same mole of SDS was added. This is because when water evaporated, SDS mainly distributed on the surface of polymer while NaCl may distribute more evenly in the polymer. The sulfate groups of SDS that concentrated on the surface of fiber oriented toward the outside and could induce formation of a surface water layer on the fibers. In the surface water layer of PEG fibers, free movement of dissociable sodium ions from SDS effectively decreased surface resistivity of PEG electrospun fibers. Whereas the evenly distributed NaCl would only decrease the volume resistivity but could not effectively decrease surface resistivity of fibers. In summary, by adding SDS, the polymer was converted from insulator to semiconductor, the capability of transferring static electricity of the fiber has been tremendously increased, and this correspondingly increased the fluffiness of scaffolds.

To further investigate the effect of electron transference on formation of 3D architectures, a solution with 25 wt % zein and 25 wt % SDS was electrospun onto the positively charged collecting board covered by a layer of insulator. Delivery of electrons was interrupted though positive potential still existed. Zein fibers with electrons on the surface were attracted by the positive collector and then hit the board vertically as shown in FIG. 19a. However, the electrons could not be transferred onto the collecting board and thus remained on the fibers. The highly negatively charged fibers attached onto the insulator tightly, owing to the strong electrical attraction, and consequently a traditional 2D electrospinning scaffold (FIGS. 19b and c) was formed.

Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles.

Although the materials and methods of this invention have been described in terms of various embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A method of making a randomly-oriented 3-D fibrous structure, the method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: to form one or more fibers that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure.

a solvent;

a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and

a surfactant;

2. The method of claim 1, wherein the polymer is selected from the group consisting of protein, synthetic polymer, and combinations thereof.

3. The method of claim of claim 2, wherein the protein is selected from the group consisting of plant protein, animal protein, and combinations thereof.

4. The method of claim 1, wherein the spinning dope has a concentration of the surfactant that is about 5 to about 300 percent by weight of the polymer.

5. The method of claim 1, wherein the surfactant is selected from the group consisting of anionic surfactant, cationic surfactant, nonionic surfactant, zwitterionic surfactant, and combinations thereof.

6. The method of claim 1, wherein the solvent is selected from the group consisting of water, phosphate buffered saline (PBS), carbonate buffer, tris-glycine buffer, borate buffer, acetate buffer, n-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, citric buffer, ethanol, chloroform, 1,4-dioxane, methanol, ethylene glycol, acetone, ethyl acetate, methyl acetate, hexane, petrol ether, citrus terpenes, diethyl ether, dichloromethane, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), formic acid, n-butanol, isopropanol (IPA), n-propanol, acetic acid, nitromethane, dichloromethane, and combinations thereof.

7. The method of claim 1, wherein:

the fibers have a fineness that is about 50 nm to about 100 μm;

the randomly-oriented 3-D fibrous structure further comprises interconnected pores having sizes that are about 10 to 2000 μm; and

the randomly-oriented 3-D fibrous structure has a porosity that is about 60 to about 99.9% by volume.

8. The method of claim 1, wherein:

the polymer consists of a primary polymer and one or more secondary polymers and the spinning dope further comprises a first cross-linker, a sacrificial component, or a combination thereof; or

the method further comprises contacting the fibers with a second solution that comprises a second cross-linker, a surface decorator, or a combination thereof; or

a combination thereof.

9. The method of claim 8, wherein the first cross-linker is at a concentration of about 0.01 mol/L to about 10 mol/L of the spinning dope, and

wherein the sacrificial component is at a concentration of about 0.5% to about 50% based on the weight of the polymer, and

wherein the second solution has a concentration of the second cross-linker that is about 0.01 mol/L to about 10 mol/L, and

wherein the surface decorator is at concentration of about 0.5% to about 10% based on the weight of the polymer.

10. The method of claim 9, wherein the first and second cross-linkers are independently selected from the group consisting of polycarboxylic acid which contains at least three carboxylic acid groups, oxysucrose, genepin, glutaraldehyde, oxaldehyde, NHS esters, maleimides, carbodiimide, isocyanate, and combinations thereof, and

wherein the sacrificial component is selected from the group consisting of PEG, egg white protein, zein, wheat gliadin, and combinations thereof, and

wherein the surface decorator is one or more peptides selected from the group consisting of Arg-Gly-Asp (RGD), Ile-Lys-Val-Ala-Val (IKVAV), and Asn-Ser-Gly-Ala-Ile-Thr-Ile-Gly (NSGAITIG).

11. The method of claim 1, wherein the polymer is a protein and the method further comprises aging the dissolved protein at an aging temperature that is about 20 to about 90° C. for an aging duration that is about 0.5 to about 48 hours before conducting the electrospinning.

12. The method of claim 1, further comprising contacting the fibers with a coagulation solution that comprises a coagulant to modify the water stability and mechanical properties of the fibers.

13. The method of claim 12, wherein the coagulant is selected from the group consisting of methanol, ethanol, sodium sulfate and acetic acid, acetone, sulfuric acid, hydrochloric acid, and combinations thereof.

14. A method of making a randomly-oriented 3-D fibrous structure, the method comprising electrospinning a spinning dope with an electrospinning apparatus, wherein the spinning dope comprises: to form one or more fibers of a fineness that is about 50 nm to about 100 μm and that comprise a polymer-surfactant complex and that arrange randomly and evenly in three dimensions when contacting a collecting board of the electrospinning apparatus thereby forming the randomly-oriented 3-D fibrous structure that further comprises interconnected pores having sizes that are about 10 to 2000 μm and that has a porosity that is about 60 to about 99.9% by volume.

a solvent;

a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 10 to about 50 kDa, and wherein the polymer is selected from the group consisting of protein, synthetic polymer, and combinations thereof; and

an anionic surfactant at about 5 to about 300 percent by weight of the polymer;

15. A randomly-oriented 3-D fibrous structure comprising:

one or more fibers that comprise a polymer-surfactant complex, wherein the fiber(s) have lengths that are at least about 100 nm and finenesses that are about 50 nm to about 100 μm, and are arranged randomly and evenly in three dimensions throughout the randomly-oriented 3-D fibrous structure; and

interconnected pores having sizes that are about 10 to 2,000 μm, wherein the pores comprise about 60 to about 99.9% by volume of the randomly-oriented 3-D fibrous structure.

16. The randomly-oriented 3-D fibrous structure of claim 15, wherein:

the fineness of the one or more fibers is about 50 nm to about 20 μm; and

the interconnected pores have sizes that are about 100 to 1,000 μm, wherein the pores comprise about 90 to about 99.9% by volume of the randomly-oriented 3-D fibrous structure.

17. The randomly-oriented 3-D fibrous structure of claim 15, wherein the polymer-surfactant complex is formed via electrospinning of a spinning dope that comprises a solvent; a polymer dissolved in the solvent, wherein the dissolved polymer is in subunits having molecular weights that are about 5 to about 150 kDa; and a surfactant.

18. The randomly-oriented 3-D fibrous structure of claim 17, wherein the surfactant is selected from the group consisting of anionic surfactant, cationic surfactant, nonionic surfactant, zwitterionic surfactant, and combinations thereof; and wherein the polymer is selected from the group consisting of protein, synthetic polymer, and combinations thereof.

19. The randomly-oriented 3-D fibrous structure of claim 18, wherein the protein is selected from the group consisting of plant protein, animal protein, and combinations thereof.

20. The randomly-oriented 3-D fibrous structure of claim 19, wherein:

the plant protein is selected from the group consisting of wheat gluten, wheat gliadin, wheat glutenin, soy protein, camelina protein, peanut protein, canola protein, sorghum protein, rice protein, millet protein, sunflower seed protein, pumpkin seed protein, mung bean protein, red bean protein, chickpea protein, green pea protein, and combinations thereof;

the animal protein is selected from chicken feather, egg white, wool keratin, casein, silk, fibrin, collagen, gelatin, hair keratin, horn keratin, nail keratin, whey protein, and combinations thereof; and

the synthetic polymer is selected from the group consisting of polyethylene glycol (PEG), poly lactic acid (PLA), poly glycolic acid (PGA), polyhydroxyalkanoates (PHAs), poly(lactic-co-glycolic acid) (PLGA), poly-3-hydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and combinations thereof.