Patent Application
20250195856
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Mar. 1, 2023 having the file name “22-2197-WO.xml” and is 4,616 bytes in size.
Hierarchical structures in nature, such as nacre and bone, mainly consist of biopolymer matrices and biomineral inclusions that nucleate from ionic precursors. Inspired by the living structures, researchers have developed biomimetic strategies to utilize biological templates to control the nucleation, growth, and crystallization of inorganic crystals in nanoscale. However, the understanding of salt cations, as mineral precursors, interacting with biopolymers is limited to uncontrollable and disordered “salting out” processes. Therefore, little information is available about utilizing salt cations and their transformation into a mineral phase to direct the assembly of biopolymers to form predetermined nano- and micro-structures.
Tubular structures at the nanoscale possess significant advantages in a broad range of areas, such as catalysis, sensing, microencapsulation, selective mass transport, filtration and drug delivery, to name a few. While the fields of carbon nanotubes and nanotubes made of several non-carbon materials (e.g., metals, oxides, semiconductors) have been progressing rapidly, polymeric nanotubes are much less advanced.
This Summary introduces a selection of concepts in simplified form that are described further below in the Detailed Description. This Summary neither identifies key or essential features, nor limits the scope, of the claimed subject matter.
One aspect of the disclosure herein is a composition comprising a mixture of a soluble polymer in a solvent and an immiscible highly-soluble molecule in the same solvent, wherein the immiscible highly-soluble molecule substitutes inorganic ions, wherein the solubility of the immiscible highly-soluble molecule is more than 50 times higher than the solubility of the soluble polymer in the solvent, and wherein the soluble polymer can be cross linked to become insoluble during a material fabrication process.
In one embodiment of the composition disclosed herein, the solvent is an organic solvent selected from the group consisting of formic acid (FA), hexafluoroisopropanol (HFIP), and trichloroacetic acid/chloroform. In another embodiment, the solvent is water.
In one embodiment of the composition disclosed herein, the highly-soluble molecule is a polymer selected from the group consisting of polyglycolide (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), polyamide, polyacrylonitrile, polyacetal, polyester, and polyketone.
In one embodiment of the composition disclosed herein, the soluble polymer is selected from the group consisting of metacrilated silk, metacrilated collagen, tropoelastin, elastin, keratin, alginate, chitosan, chitin, cellulose and derivates, polyvinyl alcohol, chitosan with 1% acetic acid, and pectin. In one embodiment, the soluble polymer is silk fibroin.
In one embodiment of the composition disclosed herein, the immiscible highly-soluble molecule is an ionic salt.
In one embodiment of the composition disclosed herein, the composition comprises silk fibroin (SF) and an ionic salt mixed in water. In one embodiment, the SF is selected from the group consisting of larger silk fibroin (SFL), smaller silk fibroin (SFS), and a mixture thereof. In one embodiment, the SF is 5 wt %. In one embodiment, the ionic salt is selected from the group consisting of NaCl and CuSO4. In one embodiment, the ionic salt is 5 wt %.
In one embodiment of the composition disclosed herein, the weight ratio of immiscible highly soluble molecule:soluble polymer is from 1:100 to 10:100.
In one embodiment of the composition disclosed herein, the immiscible highly-soluble molecule is selected from the group consisting of a highly hydrophilic carbohydrate and a short peptide with less than five residues, or a short peptide which optionally can comprise more than five residues when the entire sequence consists of hydrophobic amino acids. In one embodiment, the hydrophobic amino acids are selected from the group consisting of W, L, I, F, M, V, and Y. In one embodiment, the highly hydrophilic carbohydrate is trehalose.
In one embodiment of the composition disclosed herein, the material fabrication process is selected from the group consisting of x-linking via UV, light, heat, and chemicals.
One aspect of the disclosure herein is a microneedle comprising the composition disclosed herein, wherein the microneedle comprises a hollow base and a penetrating tip.
In one embodiment of the microneedle disclosed herein, the microneedle and the tip are hollow, wherein the microneedle comprises one open channel from base to tip. In one embodiment, the microneedle comprises a hollow base and a porous tip. In one embodiment, the penetrating tip length is suitable for penetration of the microneedle to the xylem or phloem of a plant. In one embodiment, the penetrating tip diameter is capable of penetrating the xylem or phloem of a plant without disrupting the flow of material in the xylem or phloem. In one embodiment, the penetrating, porous tip is capable of sampling fluid in the xylem or phloem of a plant.
In one embodiment, the microneedle disclosed herein comprises at least one active agent selected from the group consisting of proteins, peptides, antigens, immunogens, vaccines, antibodies, or portions thereof, antibody-like molecules, enzymes, nucleic acids, siRNA, shRNA, aptamers, viruses, bacteria, small molecules, cells, hormones, antibiotics, therapeutic agents, diagnostic agents, and any combinations thereof.
One aspect of the disclosure herein is a phase front assembly method for manufacturing a hollow microneedle with a penetrating tip comprising: (a) mixing a soluble polymer in a solvent and an immiscible highly-soluble molecule in the same solvent, wherein the immiscible highly-soluble molecule substitutes inorganic ions, wherein the solubility of the immiscible highly-soluble molecule is more than 50 times higher than the solubility of the soluble polymer in the medium, and wherein the soluble polymer can be cross linked to become insoluble during the phase front assembly method; (b) placing droplets of the mixture in a negative polydimethylsiloxane (PDMS) mold, and (c) air drying the droplets to evaporate solvent from the mixture, resulting in homogenous nucleation and crystallization in the shape of hollow microneedles, thereby forming hollow microneedles.
In one embodiment of the method disclosed herein, the air drying occurs at room temperature and humidity. In one embodiment of the method disclosed herein, the imaging is performed under a microscope or by using a camera. In one embodiment the droplets are 5 μL.
In one embodiment, the method disclosed herein further comprises imaging the evaporation process to monitor needle formation.
In one embodiment, the method disclosed herein further comprises centrifuging the PDMS molds filled with the mixture and placing under vacuum to remove air bubbles, and peeling microneedles off from the mold after air drying.
One aspect of the disclosure herein is a method for delivering a payload to a locus in plant tissue comprising (a) providing a hollow microneedle comprising the composition of claim 1; (b) loading the microneedle device with the payload; and (c) contacting the plant tissue with the microneedle device under conditions sufficient to allow the payload to enter the locus.
One aspect of the disclosure herein is a method of sampling sap from plant tissue to detect the presence of agents in the sap, the method comprising (a) providing a hollow microneedle with a porous tip comprising the composition of claim 1; (b) contacting the plant tissue with the microneedle device under conditions sufficient to allow sap to flow out of the plant tissue and into the hollow microneedle, and (c) collecting the sap for sampling in a collector attached to the hollow microneedle. In one embodiment of the method disclosed herein, the agents to be detected are selected from the group consisting of Cd and As. One embodiment is a microneedle made by the method disclosed herein.
Another aspect of the disclosure is a method for making a silk protein nanostructure array, comprising: (a) infiltrating a nanoporous template with a silk protein solution; (b) material assembly in nanopores of the nanoporous component to produce a silk protein nanostructure array; and (c) removing the nanoporous template in a dissolving solvent. In one embodiment, the nanoporous template comprises polycarbonate (PC), anodic aluminum oxide, and/or macroporous silicon. In another embodiment, the silk protein solution comprises silk fibroin, such as silk fibroin extracted from Bombyx mori cocoons. In some embodiments, the silk fibroin is present in the solution at 2% to 20%, or 2% to 10%, by weight. In one embodiment, the silk protein solution comprises a silk-water suspension, including but not limited to silk suspended in formic acid (FA). In another embodiment, the infiltrating comprises placing the nanoporous template on a reservoir of the silk protein solution. In one embodiment, the substrate comprises a glass slide, coverslip, or silicon wafer. In a further embodiment, the methods comprise air drying the PC after infiltrating it with the silk protein solution to template the silk protein in an array of nanostructures within the PC nanopores. In one embodiment, removing the nanoporous template comprises: (a) immersing the glass slide into dichloromethane (DCM) to dissolve the PC; and (b) air drying the glass slide coated with the silk protein nanostructure array. In another embodiment, the silk protein nanostructures comprise silk protein nanotubes, wherein the method further comprises crystalizing inorganics in the silk protein nanotubes. In one embodiment, the nanoporous template comprises PC and the substrate comprises PDMS or polystyrene. In one such embodiment, the method comprises air drying the PDMS or polystyrene after being coated with the silk protein solution and PC to template the silk protein into an array of nanotubular structures on the PDMS or polystyrene. In a further embodiment, the method comprises removing the nanoporous template in a dissolving solvent comprises: (a) peeling the array of silk nanotubular structures off the PDMS or polystyrene, and immersing the array in DCM to dissolve the PC; and (b) air drying the free-standing silk nanotubular structure array. In one emboodiment, the silk protein solution comprises silk proteins, horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). In one such embodiment, the nanoporous template comprises a PC membrane, wherein infiltrating the silk protein solution to the PC membrane is conducted at about 99% humidity, for a time sufficient for the silk solution to fill up the entire pore volume of the PC membrane and undergo sol-gel transition catalyzed by HRP in the presence of H2O2 within pores of the PC membrane to form silk gel-PC composites. In a further embodiment, removing the nanoporous template in a dissolving solvent comprises successive dehydration of the silk gel-PC composites in increasing concentrations of ethanol followed by drying and dissolving of the PC template in DCM to generate porous silk pillars. In one embodiment, the nanoporous template comprises PC, and wherein the silk fibers are dissolved in FA with about 2 wt % CaCl2 to a final concentration of about 6 wt % silk to produce a silk/CaCl2—PC composite nanopillars. In a further such embodiment, the method further comprises air drying of the silk/CaCl2—PC composite followed by exposing the composite to (1) water vapor to induce silk nanopillar to silk nanotube conversion; or (2) vapor of a 80 v/v % EtOH/water solution to induce formation of mushroom-shaped silk nanopillars.
Another aspect of the disclosure provides silk nanostructure arrays as described herein. In one embodiment, the silk nanostructure array comprises aligned silk nanostructures across an area of at least one cm2. In another embodiment, one or more of the silk nanostructures on the array have an aspect ratio of between about 5 to about 60, or wherein a majority or all of the silk nanostructures on the array have an aspect ratio of between about 5 to about 60. In various embodiments, the silk nanostructures comprise silk nanotubes and/or silk nanopillars. In another embodiment, the silk protein nanostructure array is at least 2 cm×2 cm in size. In a further embodiment the silk protein nanostructures comprise silk protein nanotubes, wherein the silk protein nanotubes comprise crystalized inorganics in the silk protein nanotubes. In one embodiment, the crystalized inorganics comprise hydroxyapatite and/or CaCO3. In other embodiments, the silk nanostructures comprise silk fibroin, such as silk fibroin extracted from Bombyx mori cocoons.
The following Detailed Description references the accompanying drawings which form a part this application, and which show, by way of illustration, specific example implementations. Other implementations may be made without departing from the scope of the disclosure.
Reference numbers in brackets “[ ]” herein refer to the corresponding literature listed in the attached Bibliography which forms a part of this Specification, and the literature is incorporated by reference herein.
As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the words “herein,” “above” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.
All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise. As used herein, “about” means+/−5% of the recited value.
In one aspect, the disclosure provides compositions comprising a mixture of a soluble polymer in a solvent and an immiscible highly-soluble molecule in the same solvent, wherein the immiscible highly-soluble molecule substitutes inorganic ions, wherein the solubility of the immiscible highly-soluble molecule is more than 50 times higher than the solubility of the soluble polymer in the solvent, and wherein the soluble polymer can be cross linked to become insoluble during a material fabrication process. Any suitable solvent may be used as appropriate for an intended use. In some embodiments, the solvent is an organic solvent selected from the group consisting of formic acid (FA), hexafluoroisopropanol (HFIP), and trichloroacetic acid/chloroform. Any suitable highly-soluble molecule may be used as appropriate for an intended use. In some embodiments, the highly-soluble molecule is a polymer selected from the group consisting of polyglycolide (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid (PLGA), polyamide, polyacrylonitrile, polyacetal, polyester, and polyketone.
In one embodiment, the solvent is water. In one further such embodiment, the soluble polymer is selected from the group consisting of metacrilated silk, metacrilated collagen, tropoelastin, elastin, keratin, alginate, chitosan, chitin, cellulose and derivates, polyvinyl alcohol, chitosan with 1% acetic acid, and pectin. In a specific embodiment, the soluble polymer is silk fibroin.
Any suitable immiscible highly-soluble molecule may be used as appropriate for an intended use. In one embodiment, the immiscible highly-soluble molecule is an ionic salt. In a further embodiment, the composition comprises silk fibroin (SF) and an ionic salt mixed in water. As used herein, the term “silk fibroin” refers to silk fibroin protein or fragment thereof, whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Silk is naturally produced by various species, including, without limitation: Antheraea mylitta; Antheraca pernyi; Antheraca yamamai; Galleria mellonella; Bombyx mori; Bombyx mardarina; Galleria mellonella; Nephila clavipes; Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia; Arancus diadematus; Latrodectus geometricus; Araneus bicentenarius; Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus; Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and Nephila madagascariensis. (See U.S. Pat. No. 11,147,282) In some embodiments, silk fibroin is obtained from a solution containing a dissolved silkworm silk or spider silk. For example, in some embodiments, silkworm silk fibroins are obtained, from the cocoon of Bombyx mori. In some embodiments, spider silk fibroins are obtained, for example, from Nepbila clavipes. In the alternative, in some embodiments, silk fibroins suitable for use in the invention are obtained from a solution containing a genetically engineered silk or recombinantly produced silk harvested from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in its entirety.
Silk fibroin is a hydrophobic structural protein having amphiphilic properties. Silk fibroin heavy chain are made of amorphous and crystalline fractions. Beta-sheets of fibroin proteins stack to form crystals, whereas other segments form amorphous domains. See 11,147,282, incorporated by reference herein in its entirety. The protein secondary as well as tertiary structures can be further controlled due to the polymorphism of the protein, permitting control over the protein physical and mechanical properties. The molecular weight of regenerated silk fibroin varies depending on the processing conditions, such as boiling time.
Although different species of silk-producing organisms, and different types of silk, have different amino acid compositions, various fibroin proteins share certain structural features. A general trend in silk fibroin structure is a sequence of amino acids that is characterized by usually alternating glycine and alanine, or alanine alone. Such configuration allows fibroin molecules to self-assemble into a beta-sheet conformation. These “Ala-rich” and “Gly-rich” hydrophobic blocks are typically separated by segments of amino acids with bulky side-groups (e.g., hydrophilic spacers). See U.S. Pat. No. 11,147,282.
In some embodiments, core repeat sequences of the hydrophobic blocks of the silk fibroin are represented by the following amino acid sequences and/or formulae: (GAGAGS)5-15 (SEQ ID NO: 1); (GX)5-15 (X=V, I, A); GAAS (SEQ ID NO: 2); GX1-4 GGX (SEQ ID NO: 3); GGGX (X=A, S, Y, R, D V, W, R, D) (SEQ ID NO: 4); GLGGLG (SEQ ID NO: 5); GXGGXG (X=L, I, V, P) (SEQ ID NO:6); GPX (X=L, Y, I); (GP (GGX)1-4 Y) n (X=Y, V, S, A) (SEQ ID NO:7); GRGGAn (n is 1-10) (SEQ ID NO:8); GGXn (X=A, T, V, S; n is 1-10); GAG(A)6-7GGA (SEQ ID NO:9); and GGX GX GXX (X=Q, Y, L, A, S, R) (SEQ ID NO: 10).
In some embodiments, the silk fibroin contains multiple hydrophobic blocks, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 hydrophobic blocks within the peptide. In some embodiments, a fibroin peptide contains between 4-17 hydrophobic blocks.
In some embodiments, the silk fibroin comprises at least one hydrophilic spacer sequence (“hydrophilic block”) that is about 4-50 amino acids in length. Non-limiting examples of the hydrophilic spacer sequences include peptides at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
Silk fibroin proteins are characterized by modular units linked together to form high molecular weight, highly repetitive proteins. These modular units or domains, each with specific amino acid sequences and chemistries, are thought to provide specific functions. For example, sequence motifs such as poly-alanine (polyA) and poly-alanine-glycine (poly-AG) are inclined to be beta-sheet-forming; GXX motifs contribute to 31-helix formation; GXG motifs provide stiffness; and, GPGXX (SEQ ID NO: 18) contributes to beta-spiral formation (reviewed in Omenetto and Kaplan (2010) Science 329:528-531; see also WO 2011/130335 (PCT/US2011/032195), the contents of which are incorporated herein by reference.
In various embodiments, the silk fibroin has an average molecular weight of between about 5 kD and about 400 kD; about 5 kD and about 350 kD; about 5 kD and about 300 kD; about 5 kD and about 250 kD; about 5 kD and about 200 kD; about 5 kD and about 150 kD; about 5 kD and about 100 kD; about 50 kD and about 400 kD; about 50 kD and about 350 kD; about 50 kD and about 300 kD; about 50 kD and about 250 kD; about 50 kD and about 200 kD; about 50 kD and about 150 kD; about 50 kD and about 100 kD; about 100 kD and about 400 kD; about 100 kD and about 350 kD; about 100 kD and about 300 kD; about 100 kD and about 250 kD; about 100 kD and about 200 kD; or about 100 kD and about 150 kD.
In one embodiment, the SF is selected from the group consisting of larger silk fibroin (SFL), smaller silk fibroin (SFS), and a mixture thereof. As used herein, SF with average molecular weights of ˜270 kDa are SFL and ˜200 kDa are SFS. In one embodiment, the SF is present in the composition at 5 wt %. In another embodiment, the ionic salt is selected from the group consisting of NaCl and CuSO4. In a further embodiment, the ionic salt is present at 5 wt %. The weight ratio of immiscible highly soluble molecule:soluble polymer may be any as suitable for an intended purpose. In one embodiment, the weight ratio of immiscible highly soluble molecule:soluble polymer is from 1:100 to 10:100.
In another embodiment, the immiscible highly-soluble molecule is selected from the group consisting of a highly hydrophilic carbohydrate and a short peptide with less than five residues, or a short peptide which optionally can comprise more than five residues when the entire sequence consists of hydrophobic amino acids. In a further embodiment, the hydrophobic amino acids are selected from the group consisting of W, L, I, F, M, V, and Y. In one embodiment, the highly hydrophilic carbohydrate is trehalose.
The soluble polymer can be cross linked to become insoluble during any material fabrication process as suitable for an intended purpose. In one embodiment, the material fabrication process is selected from the group consisting of x-linking via UV, light, heat, and chemicals.
In another embodiment, the disclosure provides microneedles comprising the composition of any embodiment of combination of embodiments of this aspect, wherein the microneedle comprises a hollow base and a penetrating tip. In one embodiment, the microneedle and the tip are hollow, wherein the microneedle comprises one open channel from base to tip. In another embodiment, the microneedle comprises a hollow base and a porous tip.
The disclosure herein derives manipulated microneedle tips in solid, hollow, and porous structures by adding ionic salt in SF solutions (
In another aspect, the disclosure provides a phase front assembly method for manufacturing a hollow microneedle with a penetrating tip comprising: (a) mixing a soluble polymer in a solvent and an immiscible highly-soluble molecule in the same solvent, wherein the immiscible highly-soluble molecule substitutes inorganic ions, wherein the solubility of the immiscible highly-soluble molecule is more than 50 times higher than the solubility of the soluble polymer in the medium, and wherein the soluble polymer can be cross linked to become insoluble during the phase front assembly method; (b) placing droplets of the mixture in a negative polydimethylsiloxane (PDMS) mold, and (c) air drying the droplets to evaporate solvent from the mixture, resulting in homogenous nucleation and crystallization in the shape of hollow microneedles, thereby forming hollow microneedles. Herein disclosed is the simultaneous manipulation of polymer assembly, exemplified by silk fibroin (SF) assembly, and nucleation of salt crystals at their phase fronts to drive the formation of ordered nanoporous and hollow microstructures in micro-confinement. The phase front design methods of this aspect permit fabrication of new microneedles that bridge biotic/abiotic interfaces to promote sustainable food and agriculture systems. For example, the phase front assembly methods enable the formation of hollow microneedles disclosed herein, which in turn allows, for example, the delivery of large quantities of cargo molecules to plants by adding to their vasculature and also enables their translocation in fruits.
In one embodiment, the air drying occurs at room temperature and humidity. In a further embodiment, the method further comprises a step of imaging the evaporation process to monitor needle formation. Any suitable imaging may be used, including but not limited to microscopy or camera imaging. Any suitable size droplets of the mixture may be used. In one embodiment, the droplets are 5 μL in diameter. In another embodiment, the method further comprises (d) centrifuging the PDMS molds filled with the mixture and placing under vacuum to remove air bubbles, and (e) peeling microneedles off from the mold after air drying. In another embodiment, the disclosure provides a microneedle produced by the method of any embodiment of the disclosure.
The disclosure further provides methods for delivering a payload to a locus in plant tissue comprising (a) providing a hollow microneedle comprising the composition of any embodiment disclosed herein; (b) loading the microneedle device with the payload; and (c) contacting the plant tissue with the microneedle device under conditions sufficient to allow the payload to enter the locus. The disclosure also provides methods of sampling sap from plant tissue to detect the presence of agents in the sap, the method comprising (a) providing a hollow microneedle with a porous tip comprising the composition of any embodiment disclosed herein; (b) contacting the plant tissue with the microneedle device under conditions sufficient to allow sap to flow out of the plant tissue and into the hollow microneedle, and (c) collecting the sap for sampling in a collector attached to the hollow microneedle. The method can be used, for example, to detect an agent in the sap, including but not limited to Cd and As.
In another aspect, the disclosure provides methods for making a silk protein nanostructure array, comprising: (a) infiltrating a nanoporous template with a silk protein solution; (b) material assembly in nanopores of the nanoporous component to produce a silk protein nanostructure array; and (c) removing the nanoporous template in a dissolving solvent. As described in the examples, the methods of the disclosure are rapidly scalable and robust for fabricating large, well-aligned 1D nanostructures made of silk proteins. Benefitting from the polymorphic nature of silk, precise control over the size, density, aspect ratio, morphology (nanotubes versus nanopillars) and polymorphs of silk nanostructures are achieved, which then allows for programmable modulation of the end materials' functions and properties (e.g., hydrophobicity, oleophilicity and gas permeability). The silk nanotube arrays fabricated present great utility as anti-fouling coatings against marine algae, in oil extraction from oil-water mixtures, and as a packaging material with improved gas barrier property.
The silk protein nanostructure array may comprise any silk protein nanostructures (i.e., having their smallest dimension in the nm to sub-micron scale) or combinations thereof as deemed appropriate for an intended use. In some embodiments, the nanostructures may comprise silk nanotubes (hollow). In other embodiments, the nanostructures may comprise silk nanopillars (solid).
Any nanoporous template may be used as appropriate for an intended purpose. In some embodiments, the nanoporous template comprises polycarbonate (PC), anodic aluminum oxide, and/or macroporous silicon. In one embodiment, the nanoporous template comprises PC.
Any silk protein may be present in the silk protein solution as suitable for an intended purpose. In one embodiment, the silk protein comprise silk fibroin, as described above. Any embodiments or combination of embodiments of silk fibroin disclosed above are equally applicable to this aspect.
In various embodiments, the silk fibroin has an average molecular weight of between about 5 kD and about 400 kD; about 5 kD and about 350 kD; about 5 kD and about 300 kD; about 5 kD and about 250 kD; about 5 kD and about 200 kD; about 5 kD and about 150 kD; about 5 kD and about 100 kD; about 50 kD and about 400 kD; about 50 kD and about 350 kD; about 50 kD and about 300 kD; about 50 kD and about 250 kD; about 50 kD and about 200 kD; about 50 kD and about 150 kD; about 50 kD and about 100 kD; about 100 kD and about 400 kD; about 100 kD and about 350 kD; about 100 kD and about 300 kD; about 100 kD and about 250 kD; about 100 kD and about 200 kD; or about 100 kD and about 150 kD.
In one embodiment, the silk protein solution comprises silk fibroin, such as silk fibroin extracted from Bombyx mori cocoons.
The silk protein, such as silk fibroin, may be present in any concentration in the silk protein solution as appropriate for an intended use. In one embodiment, the silk fibroin is present in the solution at 2% to 20% by weight. In other embodiments, the silk fibroin is present in the solution at 2% to 15% or 2% to 10% by weight.
The silk protein solutions may differ dependent on the desired silk protein nanostructure array to be produced. In one embodiment, the silk protein solution comprises a silk-water suspension.
In another embodiment, the silk protein solution comprises silk suspended in formic acid (FA). In one such embodiment, the silk protein solution comprises degummed silk fibers suspended in FA and CaCl2). Degumming of silk fibers involves removal of sericin (a proteinaccous substance that covers the fiber) and random cleavage of peptide bonds of silk fibroin in a boiling alkaline solution (e.g., 0.02 M sodium carbonate). In another embodiment, the silk suspended in FA is obtained by lyophilizing a silk-water suspension and resuspending the lyophilized silk in FA.
Infiltrating the nanoporous template with the silk protein solution can be carried out in any way suitable for an intended use. In one embodiment, the infiltrating comprises placing the nanoporous template on a reservoir of the silk protein solution.
In another embodiment, the infiltrating comprises coating a solid substrate with a layer of silk protein solution and placing the nanoporous template on the layer of silk protein solution. Any solid substrate may be used in this embodiment as appropriate for an intended purpose. In one embodiment, the substrate comprises a glass slide, coverslip, or silicon wafer. In another embodiment, the nanoporous template comprises PC and the substrate comprises a glass slide.
The methods may comprise air drying the nanoporous template, such as PC, after infiltrating it with the silk protein solution to template the silk protein in an array of nanostructures within the nanoporous template.
In one embodiment, the nanoporous template comprises PC, and removing the nanoporous template comprises: (a) immersing the glass slide into dichloromethane (DCM) to dissolve the PC; and (b) air drying the glass slide coated with the silk protein nanostructure array.
In one embodiment, the silk protein solution comprises amorphous silk, wherein prior to immersing in DCM, the dried silk-PC composite is incubated in either pure water vapor or vapor from 80 v/v % EtOH/water for a time sufficient to induce conformational change of silk from amorphous to type II β-turns and/or antiparallel β-pleated sheets.
In another embodiment, the silk protein nanostructures comprise silk protein nanotubes, wherein the method further comprises crystalizing inorganics in the silk protein nanotubes. Any inorganics may be crystalized in the silk protein nanotube as appropriate for an intended purpose. In one embodiment, the inorganics comprise hydroxyapatite and/or CaCO3 in different crystalline phases. As described in the examples, crystallization can be pursued in the nanotube pores generating silk-CaCO3 composite with the possibility to control the phase composition of CaCO3 crystals (i.e. calcite and vaterite), as silk is able to interact with amorphous calcium carbonate and template its crystallization
In another embodiment, the nanoporous template comprises PC and the substrate comprises PDMS or polystyrene. In this embodiment, the method may further comprise air drying the PDMS or polystyrene after being coated with the silk protein solution and PC to template the silk protein into an array of nanotubular structures on the PDMS or polystyrene. In a further embodiment, removing the nanoporous template in a dissolving solvent comprises: (a) peeling the array of silk nanotubular structures off the PDMS or polystyrene, and immersing the array in DCM to dissolve the PC; and (b) air drying the free-standing silk nanotubular structure array.
In another embodiment, the silk protein solution comprises silk proteins, horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). This embodiment may be used, for example, in generating silk protein nanopillars. In this embodiment, the HRP and H2O2 may be present at any suitable concetration for catalyzing a sol-gel transition of the silk protein solution. In one embodiment, the HRP is present in the silk protein solution at a concentration of about 10 U/ml, and the hydrogen peroxide is present in the silk protein solution at a concentration of about 1.65 mM. In a further embodiment, the nanoporous template comprises a PC membrane, wherein infiltrating the silk protein solution to the PC membrane is conducted at about 99% humidity, for a time sufficient for the silk solution to fill up the entire pore volume of the PC membrane and undergo sol-gel transition catalyzed by HRP in the presence of H2O2 within pores of the PC membrane to form silk gel-PC composites. In one embodiment, removing the nanoporous template in a dissolving solvent comprises successive dehydration of the silk gel-PC composites in increasing concentrations of ethanol followed by drying and dissolving of the PC template in DCM to generate porous silk nanopillars. In another embodiment, increasing concentrations of ethanol comprise 50%, 75%, 90% and 98% ethanol for about 30 minutes each, and the drying comprises critical point drying.
In one embodiment, the silk protein solution comprises silk suspended in formic acid (FA) and the nanoporous template comprises PC. In one such embodiment, the silk fibers are dissolved in FA with about 2 wt % CaCl2 to a final concentration of about 6 wt % silk to produce silk/CaCl2—PC composite nanopillars. In another embodiment, the method further comprises air drying of the silk/CaCl2—PC composite followed by exposing the composite to (1) water vapor to induce silk nanopillar to silk nanotube conversion; or (2) vapor of an 80 v/v % EtOH/water solution to induce formation of mushroom-shaped silk nanopillars. In a further embodiment, removing the nanoporous template in a dissolving solvent comprises dissolving the PC template in DCM.
The resulting silk protein nanostructure array may be any size or dimension as suitable for an intended use. The methods of the disclosure are rapidly scalable and robust for fabricating large, well-aligned 1D nanostructures. The size may thus be any custom size, such as 20×20 cm2. In one embodiment, the silk protein nanostructure array is at least 1 cm×1 cm in size.
The nanoporous template may have pore sizes of any suitable size as appropriate for an intended use. In one embodiment, the nanoporous template has pore sizes ranging from about 10 nm to about 2 μm in diameter. The nanoporous template may have any thickness as appropriate for an intended use. In one embodiment, the nanoporous template has a thickness of about 6 μm to about 10 μm. In some embodiments, the silk protein nanostructures in the silk protein nanostructure array have an aspect ratio (defined by nanostructure length divided by nanostructure diameter) of about 5 to about 60.
In another aspect, the disclosure provides silk nanostructure arrays prepared by the method of any embodiment or combination of embodiments herein. In another aspect, the disclosure provides silk nanostructure arrays as described herein. The silk nanostructure arrays may comprise any embodiment or combination of embodiments or characteristics disclosed herein. In one embodiment, the array comprises aligned silk nanostructures across an area of at least one cm2. In one such embodiment, the array comprises aligned silk nanostructures within ±30° from the vertical across an area of at least one cm2. In another embodiment, one or more of the silk nanostructures on the array have an aspect ratio of between about 5 to about 60. In another embodiment, a majority (more than 50%) of the silk nanostructures on the array have an aspect ratio of between about 5 to about 60. In various further embodiments, 60%, 70%, 80%, 90% or more of the silk nanostructures on the array have an aspect ratio of between about 5 to about 60.
The array may comprise any type of silk nanostructure, or combinations of different types of silk nanostructures. In one embodiment, wherein the silk nanostructures comprise silk nanotubes. In another embodiment, the silk nanostructures comprise silk nanopillars. In a further embodiment, the silk nanostructures comprise silk nanotubes and silk nanopillars.
The arrays may be any size, as described herein. In some embodiments, the silk protein nanostructure array may be at least 2 cm×2 cm in size. In other embodiments, the silk protein nanostructure array may be at least 5 cm×5 cm; 10 cm×10 cm, or 20×20 cm in size.
In a further embodiment, the silk protein nanostructures may comprise silk protein nanotubes, wherein the silk protein nanotubes comprise crystalized inorganics in the silk protein nanotubes. In one embodiment, the crystalized inorganics comprise hydroxyapatite and/or CaCO3. In another embodiment, crystalized CaCO3 is present in different crystalline phases (calcite and vaterite).
The silk nanostructures in the array may comprise silk of any embodiment, including those disclosed herein. In one embodiment, the silk nanostructures comprise silk fibroin, such as silk fibroin extracted from Bombyx mori cocoons.
The regenerated silk fibroin was extracted from Bombyx mori silkworm cocoons following an established protocol [25]. Cocoons (available in Jeollanam-do, South Korea) were cut into small pieces (˜2×2 cm2) and boiled in 0.02 M sodium carbonate for ten min (SFL) and 30 min (SFS) to remove the sericin. The obtained degummed silk fibers were then washed with ultrapure water, dried overnight, and dissolved in 9.3 M lithium bromide for 4 h at 60° C. The dissolved silk fibroins were dialyzed against ultrapure water in a dialysis cassette (molecular weight cut-off: 3500 Da) for two days with frequent replacements. The resulting silk fibroin solutions were then centrifuged to remove impurities. The final silk fibroin solution was diluted with water to 5 wt %, and then stored at 4° C. before use. Size distribution of SFL and SFS were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad Laboratories) [22].
Fabrication and monitoring of silk films and needles. Silk fibroin solutions (5 wt %, SFL and SFS) ratio were mixed with ionic salts (5 wt %, NaCl and CuSO4) at varying ratios. Then, 5 μL droplets of each mixture were placed on flat PMDS (SYLGARDIM 184, Dow-Coming Corp.) surface and air dried at room temperature and humidity. Changes in droplet sizes and contact angles were monitored using a portable USB digital microscope (Jiusion). NaCl was chosen as a representative salt with high water solubility enabling the monitoring its crystallization behavior at a wide range of concentration. CuSO4 is also investigated as a highly soluble salt and its blue color allows us to monitor its phase separation more easily under microscope. The completed dried droplets were imaged under optical microscope (Nikon ECLIPSE™ TE2000-E).
The macroscale needles were prepared by casting the mixture of 5% SFS and 5% ionic CuSO4 solutions in a negative PDMS mold (0.6 cm base diameter and 0.9 cm long). The needle formation process was imaged every 30 minutes by a digital single-lens reflex camera (Canon EOS 5 MARK IV) with a macro lens (EF 100 mm f/2.8). The microneedle arrays were fabricated by casting the mixture of 2.0-3.6 mL silk fibroins (5 wt %, SFS and SFL) with varying volume of ionic salt solutions (5 wt %, NaCl and CuSO4) in negative PDMS molds (10×10 microneedles, 600 μm base diameter, 1,600 μm long, and 2 mm spacing between needles). The molds filled with the mixed solutions were centrifuged (4,000 rcf, 5 min) and placed under vacuum for 30 minutes to remove air bubbles entrapped between the molds and the solutions. The arrays were peeled off from the mold after air dry and cut into small pieces with desired number of microneedles for further experiments or characterization. As a post-treatment, microneedles were soaked in 80% ethanol for one day for silk crystallization. If needed, the crystallized silk microneedles were washed with deionized water or 0.1 mM hydrochloric acid for another day to remove NaCl and CuSO4 crystals precipitated in the microneedles.
The optical images of individual silk microneedles and were obtained with a microscope (Nikon ECLIPSE™ TE2000-E). The microneedles were sputter coated with gold for 10 nm for collecting SEM images (Zeiss MERLIN™ High-resolution SEM and JEOL JSM-6010LA), Line profiles of Cu/N weight ratios along the microneedles were collected by SEM/EDS (Zeiss MERLIN™ High-resolution SEM).
The porous microneedles were evaluated by a confocal Raman spectroscope system established in the Physical Optics and Electronics Group at MIT. Raman spectra along the microneedle from the tip end to the base at 100 μm were collected at a range of Raman shift (200-1900 cm−1) by using a 785 nm laser (4 mW power, integrating signals from 60 seconds×2 scans). The collected spectra were post-treated for the background subtraction, cosmic ray removal, and Savitzky-Golay filtering (order 2, frame length 11).
A hollow microneedle was scanned by the 3D X-ray micro-computed tomography system (ZEISS VERSA™ 520 micro-CT System) with the following parameters: source setting 80 kV and 7 W, source filter air, camera binning 1, and exposure time 5 seconds. The objective was 4× and the pixel size was around 1.5487 μm. 2401 projections were taken while the sample rotated 360°.
Residual Cu contents in the microneedles were analyzed by using an inductively coupled plasma-optical emission spectrometer (Agilent 5100 DVD). Samples were prepared by digesting three microneedles in 500 μL NaOH (3 N, 60° C.) for 5 h. The digested solutions were added to 4.5 mL nitric acid (5%) and filtered through 0.2 μL cellulose acetate syringe filter.
The tip breaking forces of the microneedles were evaluated by the static compression strength test by a DYNAMIC MECHANICAL ANALYSIS (TA instruments Q850) at a loading speed of 1 mm min−1.
Fourier-transform infrared spectroscopy (FTIR, Perkin Elmer SPECTRUM 65) was used to evaluate the effects of CuSO4 addition and post treatments silk materials on the secondary protein structure. For the evaluation, silk films were prepared with 400 μL SFS (5%) and 0-40 μL CuSO4 (5%) solutions in PMDS mold with rectangular wells (1×1×0.3 cm−3) that mimicked silk curing environment in the microneedle molds. FTIR spectra were collected at a wavelength range of 4,000 to 650 cm−1, with a resolution of 4 cm−1 and an accumulation of 64 scans. The relative fractions of β-sheet and random coil were determined by Fourier self-deconvolution of the amide I band (1705-1595 cm−1) and Gaussian curve-fitting of the deconvoluted spectra.
Tomato (Solanum lycopersicum) plant was chosen as a working model in this study because of their well-known vascular structure and importance in agriculture. They were grown in pots for 4-6 weeks from seeds with regular water and fertilizer feeding at a room temperature with 12 h photoperiod per day. Microneedles were injected into petioles of plants in pots, at approximately 10 cm above the root, for delivery and sampling of fluorescein (from sodium salt, Mw 376.27 g mol−1, pKa˜6.4, Sigma), used as a model biomolecule. The petioles with a dimeter around 2-2.5 mm were selected as injection spots so the microneedles can reach both xylem phloem located at the depths around 700 and 850 μm respectively [22]. For the delivery, a 2.5 μL droplet of 0.2% sodium fluorescein was placed on the backside of the microneedle that facings up, so the droplet can be absorbed into the microneedle or directly inside the petiole (in the case of hollow needle) with the gravitation force. A piece of tissue paper (0.3 cm×0.5 cm) was rolled and inserted into the well of the sampling microneedle from the backside as a collector of fluorescein delivered from another microneedle used as an injector.
Micro-Tom, a dwarf cultivar of tomato (Solanum lycopersicum L.) was used as the model to deliver Vitamin B12. They were grown in pots from seeds a plant room under long day (16 h light/8 h dark) at 80 μmol m−2 s−1, 70-90% relative humidity, day and night temperatures of 26° C. and 20° C., respectively. Plants were used for experiments when the growing fruits were around 1-3 g. Hollow microneedles were injected on stem, pedicel, and sepal. 1 mM Vitamin B12 solution (˜20 ul) was added continuously through the microneedle over 2 hours. The fruits were collected after 24 h of application and homogenized. 80% methanol was used to extract Vitamin B12 from the homogenized fruits at a volume weight ratio of 3:1. The mixture was sonicated for 30 minutes in an Ultrasonic Bath Sonicator, incubated for 24 hours at 4° C., and centrifuged at 20,000×g for 30 minutes. The supernatant was collected for LC/MS using an Agilent 6545 mass spectrometer coupled to an Agilent Infinity 1260 LC system with a ZORBAX Eclipse Plus™ C18 column (2.1×150 mm, 3.5 μm), Solvent A was 0.1% formic acid in water and solvent B was acetonitrile with 0.1% (v/v) Formic acid. The gradient conditions were 0% B (0-1.5 min), 0-40% B (1.5-10 min), 40-60% B (10-13 min), 60-99% B (13-14.5 min), 99-0% B (14.5-16 min) with constant flow 300 μl/min. Stoptime was 15 minutes and Posttime was 3 minutes. Acquisition parameters were set as Temperature 25° C., Source Dual AJS ESI, positive ion polarity, mode MS (Seg), mass range 100-3000 m/z, acquisition rate 2 spectra/s, and injection volume 5 μl. Vitamin B12 was calculated at 678.29. 678.79, and 679.29 m/z using the standard curve y=2.02×106× (adjusted R2=0.99987).
For the sampling of Cd and As, tomato plants (4-6 weeks) were transferred to 100 mL plant media (Hoagland's No. 2 Basal Salt Mixture) containing cadmium nitrate tetrahydrate (1 or 10 mg Cd L−1, Sigma-Aldrich) or sodium arsenite (1 mg As L−1, Spectrum Chemical) one day before the microneedle injection. The injected microneedle was digested in 500 μL NaOH (3 N, 60° C.) for 5 h, then transferred to 2 mL nitric acid (5%). The tissue paper collector attached to the microneedle was transferred to 1 mL nitric acid (5%) and heated at 60° C. for 5 h. After heating, additional 1.5 mL nitric acid (5%) were added for dilution. Bioaccumulated Cd and As in the plant leaves after sampling experiments were extracted in concentrated nitric acid and hydrogen peroxide by the microwave digestion system (Milestone ULTRAWAVE™). Solutions extracting Cd and As from the microneedle, tissue paper, and leaves were filtered through 0.2 μL cellulose acetate syringe filters and analyzed by a inductively coupled plasma-mass spectrometer (ICP-MS, Agilent 7900).
We first observed the crystallization of ionic sodium chloride (NaCl) and copper sulfate (CuSO4) by evaporating water droplets containing SF extracted from Bombyx mori cocoons. SF solutions with two average molecular weights of ˜270 kDa (larger SF, SFL) and ˜200 kDa (smaller SF, SFS) were used for comparison (
SF reduced the surface energy between the droplet and the PDMS surface, pinning the contact line during evaporation. The contact line kept drawing the solution from the droplet via capillary flow, accumulating the fibroins at the pinned contact line (known as the coffee-stain effect,
The salt formation patterns changed significantly when the initial fibroins concentration increased sufficiently to form a self-standing film (>1%). At these high concentrations, silk fibroins self-assemble into micelles that hydrophobic chains surround hydrophilic intermolecular spacers [32], repelling ionic precursors from the silk-accumulating contact line and forcing the particle formation close to the center of the film (e.g., NaCl:SFL=1:9 and CuSO4:SFS=1:20). The self-standing films became insoluble by increasing the β-sheet structure via ethanol-treatment [33], allowing selective removal of NaCl crystals and generating porous structures (
Using time-lapse photography, we monitored the salt crystallization and silk assembly in a PDMS mold, replicating a macroscale needle (
We applied these phenomena to fabricate hollow microneedle arrays using a small PDMS mold (10×10 needles, 600 μm base diameter and 1.6 mm long) with varying concentrations of NaCl and CuSO4 (
At all ratios, CuSO4 addition resulted in either hollow (
Considering the consistency in the fabrication, we chose three types of microneedles made of CuSO4 for further evaluating their properties for plant applications: SMN (CuSO4:SFS=0:1), HMN (CuSO4:SFS=1:100 and 1:50) and PMN (CuSO4:SFS=1:20). Scanning electron microscopy/energy dispersive X-ray spectrometry (SEM/EDS) analysis of the porous microneedle (CuSO4:SFS=1:20) shows the clear interface between the Cu-heavy porous tip and Cu-low smooth column regions (indicated as * and *,
Cu is one of the plant micronutrients with an adequate concentration of 600 ng per gram fresh weight [38,39] and has been widely used in antimicrobial compounds for crop protections more than a century [40]. Cu is also an essential mineral present in natural foods, such as beef, and also available as a dietary supplement with 900 μg of Daily Value for adults [41]. The ethanol-treated PMN (CuSO4:SFS=1:20) contains ˜10 μg Cu per mg of microneedle (
Mechanical testing of ethanol-treated microneedles showed that the initial breaking forces of HMN were statistically higher than other two types (1.40±0.39 vs. 0.64±0.26,
We applied droplets containing a model biomolecule, fluorescein on the backside of these microneedles injected into tomato petioles (
Using one HMN as a source of payload supply, we injected second HMN 0.5 cm downstream from the source for the sampling purpose (
These microneedles were also evaluated for sampling of Cd and As from plant sap because their bioaccumulation in agricultural products are major concerns even at lower concentrations in water sources [43]. A single SMN injection into petiole effectively extracted Cd (˜0.21±0.10 ng Cd per needle equivalent to ˜0.27 ng Cd per mg of needle) for a day from the tomato plant exposed to 1 mg L−1 Cd in a plant medium (
Vitamin B12 is crucial in many physiological activities, including DNA synthesis, red blood cell production, and neurologic function. However, the prevalence of Vitamin B12 deficiency is pretty high, particularly in developing countries. In addition, Vitamin B12 cannot be obtained from common vegetables, which raises a health concern for vegans. Therefore, we also selected Vitamin B12 as a model payload and delivered it to tomato fruits via HMNs injected on stem, pedicel, and sepal (
Monitoring toxic elements that are being bioaccumulated in agricultural products, such arsenic in rice, can enhance food safety reduce chemicals, energy, and water for growing unqualified products. Despite progress in the field, delivering large amount of payloads, such as macronutrients, and sampling targets at low concentration are still challenging. In this study, we presented a simple and straightforward strategy of fabricating degradable polymeric hollow microneedles that are stably operated in plant tissues for the continuous cargo delivery from external sources and accumulative collection. By controlling the kinetics and extents of formation of nanocrystals and the phase front silk fibroin assembly, we were able to manipulate tips into different structures, enabling target-specific application that can effectively tackle this challenge. Beyond the direct application for sampling and delivery, the structures of HMN and PMN with the large well space connected to plant tissues PERMIT coupling with electrochemical or optical sensors for in vivo signal detection [45,46].
Concentrations of metallic ions, such as K+ and Ca2+, vary significantly along with the silk glands at different spinning stages to regulate the storage condition of silk fibroins and the mechanical properties of silk fibers upon spinning [35,52]. Therefore, adding ionic salts in regenerated silk fibroin solution is an effective option to decorate the properties of silk-based materials. Typical silk fabrication processes involve water evaporation that drives the assembly of fibroins. Therefore, the added ions are concentrated and eventually nucleate salt crystals. However, little information is available regarding the influence of salt concentrations and their crystallization on the assembly of silk fibroins or vice versa, the influence of the silk fibroin assembly on salt crystal formation. The strategy of controlling microneedle structures using chemical reactions at phase front of silk assembly and inorganic nanocrystal nucleation expands microneedle application for more complex systems beyond the transdermal patches, contributing to designing new functional hybrid materials.
Tubular structures at the nanoscale possess significant advantages in a broad range of areas, such as catalysis, sensing, microencapsulation, selective mass transport, filtration and drug delivery, to name a few. While the fields of carbon nanotubes and nanotubes made of several non-carbon materials (e.g., metals, oxides, semiconductors) have been progressing rapidly, polymeric nanotubes is much more limited. Here, we integrate a template wetting approach with multiple silk-based solutions, and present a rapidly scalable and robust technology for fabricating large arrays (up to 20×20 cm) of well-aligned 1D nanostructures made of silk proteins. Benefitting from the polymorphic nature of silk, precise control over the size, density, aspect ratio, morphology (nanotubes versus nanopillars) and polymorphs of silk nanostructures are achieved, which then allows for programmable modulation of the end materials' functions and properties (e.g., hydrophobicity, oleophilicity and gas permeability). The silk nanotube arrays fabricated present great utility as anti-fouling coatings against marine algae, in oil extraction from oil-water mixtures, and as a packaging material with improved gas barrier property.
The preparation of nanoscopic tubular objects and more importantly their organization into macroscopic (at least centimeter scale) arrays is a nontrivial task, especially for soft polymeric materials that are more appropriate to work at the biotic/abiotic interface.
Here we describe a robust approach for fabricating centimeter-scale arrays of well-aligned 1D nanostructures made of silk proteins. The process starts with the employment of commercially available nanoporous polycarbonate membranes as sacrificial templates which can be infiltrated by silk solutions, followed by materials assembly in the confined space of nanopores. Through regulating the biopolymer formulation thereby changing its surface energy, infiltration of the silk-based solution results in either wetting of the pore walls by a mesoscopic precursor liquid film or complete filling of the pore interior, generating large arrays (up to 20×20 cm) of well-aligned silk nanotubes or nanopillars, respectively, after template removal in a polycarbonate-dissolving solvent (e.g. dichloromethane). Integration of the template wetting approach with the versatility of silk materials allows for precise control over the size, density, aspect ratio, morphology (single- and multi-walled tubes, solid and porous pillars, as well as mushroom-shaped structures) and polymorphs of silk nanostructures, which serves as the basis for programmably regulating the end materials' functions and properties (hydrophobicity, oleophilicity and gas permeability). Moreover, the silk nanotube/pillar arrays can either be grown directly on an external substrate (e.g. glass) as a coating material or be fabricated as a free-standing film, offering great flexibility in their utility. Together, the proposed technology represents a paradigm shift in nanofabrication with structural biopolymers, as it enables rapidly scalable manufacturing of nanostructured biopolymer arrays with programmable morphologies, properties and functions by design, which are unattainable with previous similar endeavors (e.g. electron-beam lithography to generate silk nanostructures).
The general silk nanotube/pillar fabrication procedure is shown in
In order to obtain pillar structures out of the PC templates, complete filling of the PC pore volume is required (
Another important merit of using this template-based approach is that the size of silk nanotube/pillar arrays fabricated can be extremely large, which in theory only depends on the size of the porous template available. Here, a self-standing silk nanotube array of size 20×20 cm2 is demonstrated as an example (
Inspired by the self-cleaning effects of lotus leaf, many studies have shown that micro-/nanostructured surfaces generally exhibit good water repellency, as their high surface roughness allows for air pockets entrapped between water droplets and the underlying substrate, which is well-captured by the Cassie-Baxter model.[19] Considering that the silk nanotube array obtained in this work possesses well-aligned sub-micron features and a large pore volume at the surface, it is expected that the silk nanotube array will be highly hydrophobic. To verify this assumption, contact angles (CA) of water droplets were measured on three types of nanotube arrays made of (1) silk dried from silk-FA suspension; (2) silk annealed in 80 v/v % EtOH/water vapor; and (3) silk annealed in water vapor (
In light of the good hydrophobicity (i.e. water repellency) of nanotube arrays made from silk-FA suspension, we further hypothesized that they might function as an anti-fouling coating against microorganisms when submerged under water. We then tested the anti-fouling performance of our silk nanotube arrays against a species of marine algae named Porphyridium cruentum which has a spherical shape of around 4 μm in diameter (
Superoleophilicity and Oil Extraction from Oil-Water Emulsions
Besides being close to superhydrophobic, the silk nanotube arrays are also found to be superoleophilic. Here, we chose dibutyl adipate (DA) as a model oil to characterize the wettability of silk nanotube arrays by general oily compounds. The choice of DA was based on its wide usage in agriculture, food and cosmetics industry (e.g. as a solvent for pesticides/herbicides/fungicides). From contact angle measurements (
Considering that the silk nanotube arrays are both highly hydrophobic and oleophilic, we then evaluated the capability of silk nanotube arrays to extract oil from oil-water mixtures. To this end, DA dyed with Sudan III was vigorously mixed with water to form a homogenous emulsion by using tip sonication. Upon adding an emulsion droplet on a silk nanotube array, the oil component spread quickly over the entire nanostructured silk array, leaving only a clear water droplet on top of the array in the end (
Apart from the studies on how silk nanotube arrays behave when brought into contact with liquids, we were also interested to know if the silk nanotube arrays fabricated as free-standing membranes interact with gases differently. This question was also motivated by potentially using these nanostructured silk membranes as a packaging material, as previous studies have shown that food coatings made of silk fibroin present good gas barrier properties.[24] To this end, we measured the water vapor permeability (WVP) of silk membranes fabricated from PC templates of different pore sizes and densities, by following a ASTM protocol (E96/E96M) with some modifications (see Supporting Information for more details).[24,25] Interestingly, we found that nanostructured silk membranes show lower WVP compared to bulk silk membranes of the same thickness, and the WVP decreases as the nominal pore sizes of the nanostructured silk membranes decrease (
To summarize, we have reported a simple and robust approach for fabricating large arrays of well-aligned 1D nanostructures made of silk proteins, by retrofitting a well-studied template wetting phenomenon. Through the use of commercially available and low-cost PC membranes with a diverse range of pore sizes and densities as sacrificial templates and by regulating the biopolymer formulation thereby changing its surface energy, wetting of the PC templates occur via either formation of a mesoscopic liquid film or complete filling of the pore interior, generating large arrays (up to 20×20 cm) of well-aligned silk nanotubes or nanopillars, respectively. Such silk nanotube/pillar arrays can either be grown directly on an external substrate as a coating material or be fabricated as a free-standing film, which are found to be both highly hydrophobic and superoleophilic. Benefiting from its high water-repellency, substrates coated with silk nanotube arrays present significantly reduced fouling by marine algae, compared to PEG-coated surfaces. Furthermore, the silk nanotube arrays can rapidly extract oil from oil-water emulsions due to its simultaneous hydrophobicity and superoleophilicity, which offers an effective solution in oil spill response. Lastly, effects of nanostructures on the gas barrier property of silk membranes are investigated, using water vapor as a model gas. The nanotubular-structured silk membranes are found to possess lower water vapor permeability (WVP) than bulk membranes, which may serve as a better material for food packaging. Together, by integrating the template wetting approach with the versatility of silk proteins, precise control over the size, density, aspect ratio, morphology and polymorphs of silk nanostructures can be achieved, which then allows for programmably regulating the functions and macroscopic properties of the end materials. We believe that the biodegradable, non-toxic and edible nature of silk proteins in combination with the capability to organize silk proteins into centimeter-scale arrays with well-aligned nanostructures will open up exciting opportunities for a broad range of applications, particularly in the AgroFood and environmental domain.
All PVP-coated polycarbonate (PC) membranes used in this study were purchased from Structure Probe, Inc. (USA), except the PVP-coated PC sheets used to fabricate the 20×20 cm2 silk arrays which were purchased from Sterlitech (USA), PEG-coated glass slides were purchased from MicroSurfaces, Inc. (USA). Liquid cultures of Porphyridium cruentum and the corresponding media kit (salts+nutrients) for algae culture were purchased from Algae Research and Supply (USA). Dibutyl adipate (99.0+%) was purchased from TCI America (USA). The refractive index oil (RI=1.550) was purchased from ENVIROPORE (USA). All other chemicals were purchased from Sigma Aldrich (USA).
Silk fibroin regeneration: Silk fibroin was extracted from Bombyx mori cocoons following established protocols.[26] In brief, chopped silk cocoons were degummed in a boiling 0.02 M sodium carbonate solution for 30 minutes to remove the sericin. The obtained silk fibers were then washed with Milli-Q™ water for several times followed by overnight drying. The dried silk fibers were dissolved in 9.3 M lithium bromide at 60° C. for 4 hours followed by dialysis against Milli-Q™ water for 2 days with constant changing of water. The resulting silk fibroin solution was then purified by two centrifugation cycles at 20,000×g for 30 minutes each, yielding a final silk fibroin solution of ˜7 wt %. The regenerated silk fibroin solution was then stored at 4° C. until use.
Fabrication of silk nanotube arrays: To fabricate nanotube arrays, two silk solutions of concentration 2-10 wt % were prepared: (1) silk-water suspension obtained right after silk fibroin regeneration from Bombyx mori cocoons; (2) silk foams lyophilized from the silk-water suspension and re-suspended in formic acid (FA) to form a silk-FA suspension. Wetting of the porous PC membranes by silk solution was simply achieved by putting a PC membrane on top of a silk solution reservoir. To coat glass slides with silk nanotube arrays, a thin layer of silk solution was first drop cast on an oxygen-plasma treated glass slide, followed by adding a PC membrane on top of the silk solution. The silk solution was then air dried with the PC membrane in place to template the top layer of silk into nanotubular structures. Finally, the whole glass slide was immersed in DCM for 1 min to dissolve away the PC membrane and then taken out and air dried, so that only the silk nanotube array was left on the glass slide. To fabricate a free-standing silk nanotube array with bulk silk as the base, the same process was repeated except that PDMS or polystyrene petri dish was used as the substrate for drop casting silk, so that the dried silk array can be easily peeled off from the substrate and then immersed in DCM to remove the PC followed by air dry to obtain a free-standing silk nanotube array.
For the amorphous silk nanotube array obtained from silk-water suspension, annealing of the dried silk-PC composites was performed by incubating them in either pure water vapor or vapor from 80 v/v % EtOH/water for 24 hours to induce conformational change of silk from amorphous to silk I or silk II structures, respectively, both of which render the silk nanotube array water-insoluble. The silk-PC composites were then immersed in DCM to remove the PC template, leaving only the annealed silk nanotube array in the end.
Fabrication of silk nanopillar arrays: To fabricate nanopillar arrays, two silk-based solutions were prepared: (1) A 2 wt % silk-water suspension was mixed with HRP (final concentration: 10 U/ml), followed by addition of H2O2 (final concentration: 1.65 mM) to initiate gelation. Immediately after mixing the three components, a PC membrane was placed on top of the solution, which was then incubated in a humid chamber for 2 hours to allow for complete gelation of silk in the PC nanopores. The silk-PC composite was then consecutively immersed in a series of ethanol/water solution with increasing ethanol concentration (i.e. 50%, 75%, 90% and 98% EtOH) for 30 minutes each, and finally immersed in pure ethanol overnight for complete dehydration before critical point drying. (2) Degummed silk fibers were dissolved in FA with 2 wt % CaCl2 to a final concentration of 6 wt % silk for 12 hours under stirring. The clear silk-FA/CaCl2 suspension was then aged for two weeks before use. Wetting of the porous PC membranes by silk-FA/CaCl suspension and the following air dry process were the same as described before. Annealing of the dried silk pillar-PC composites in pure water vapor for 3 hours was performed to induce pillar to tube transformation. Annealing of the dried silk pillar-PC composites in vapor from 80 v/v % EtOH/water for 3 hours was performed to induce formation of the mushroom-like pillar structure. Finally, all silk-PC composites were immersed in DCM to remove the PC template, leaving only the silk nanopillar array in the end.
Critical Point Drying (CPD): Most of the samples prepared in this study were air dried. For the samples that require CPD, they were first dehydrated by soaking them in a series of ethanol/water solution with increasing ethanol concentration as described before, and then dried in a critical point dryer (Tousimis Autosamdri-815B) following the manufacturer's recommended procedures.
Fabrication of CaCO3-silk nanotube composite: CaCO3 crystallization in silk nanotubes was performed following a published protocol with some modifications.[27] Calcium chloride (CaCl2)) was first added to a 6 wt % silk-water suspension to a final concentration of 30 mM. A thin layer of the silk-CaCl solution was then drop cast on a coverslip, followed by placing a PC membrane on top of the silk-CaCl2) solution to initiate wetting of the PC pores. Without letting the silk-CaCl2 solution dry out, the coverslip was immediately placed into a weigh boat sealed by a parafilm. The weigh boat was then transferred to a closed desiccator, where another weigh boat containing 5 g of solid ammonium carbonate sealed by a parafilm was placed at the bottom. To allow for slow diffusion of the gaseous decomposition products (i.e. carbon dioxide and ammonia) of ammonium carbonate into the silk-CaCl solution, the parafilms covering both weigh boats were punched with a 20-gauge needle to generate 4 holes. CaCO3 crystallization was then initiated under the action of carbon dioxide and ammonia and allowed to proceed for 48 hours in the closed desiccator, before taking the samples out and letting them dry out. The dried samples were then immersed in DCM for 1 minute to dissolve away the PC templates followed by air dry, leaving only the CaCO3-silk nanotube array on the coverslip.
Preparation of Dibutyl Adipate (DA)-water emulsion: DA was first dyed with Sudan III (a water-insoluble dye) for better visual differentiation from water during oil-water separation. 1 mL of dyed DA was then added to 1 mL water and vigorously mixed through tip-sonication (Branson SFX 550 Sonifier). The 2 mL DA-water mixture was sonicated in an ice bath for 3 minutes with an amplitude of 35% (165 W output power) and pulse durations of 5s on-time and 5s off-time to generate a homogenized emulsion. Following sonication, the DA-water emulsion was used immediately for oil-water separation tests.
Scanning Electron Microscopy (SEM): The morphology and microscopic view of various silk nanotube and nanopillar arrays as prepared were imaged with a Zeiss Merlin High-resolution SEM, under an acceleration voltage of 1 kV and a probe current of 80 pA.
Wide-angle X-ray Scattering (WAXS): WAXS measurements of free-standing silk nanotube/pillar membranes were performed on a SAXSLAB™ instrument in transmission mode with a Dectris Pilatus3R™ 300 K detector set at a distance of 109.1 mm from the sample and a Rigaku™ 002 microfocus X-ray source producing Cu Kat X-rays of wavelength 1.5409 Å. Each spectrum was collected for 5 minutes.
Contact Angle Measurements: Contact angle measurements were performed on a Ramé-Hart goniometer (Model 500-F1). For measurements with water, a water droplet of 3 μL was deposited on each sample through a microliter syringe (Hamilton) followed by immediate image capture, from which the static contact angle of water was measured by the DROPimage™ software (Ramé-Hart). For measurements with DA, a series of images were captured by setting up a method through the DROPimage™ software that specifies the total number of frames (set to be 501) and the time interval between adjacent frames (set to be 0.2 second). As the series of images were being captured, dynamic contact angles were measured simultaneously by the DROPimage™ software, generating a LOG file in the end that stores the contact angle measurements for all frames.
Algae Fouling Experiments: Liquid cultures of Porphyridium cruentum purchased from Algae Research and Supply were re-cultured in 750 mL cell culture flasks at room temperature (23° C.) with a lighting condition of 12-hours on, following the supplier's recommended algae culture protocol. Half of the total culture volume was replaced with fresh culture media every 3 days. 100 mL of freshly-cultured algae were then transferred to a petri dish (100×25 mm), followed by submerging the samples in the algae culture. The algae culture in petri dishes were also grown under the same condition, and half of the total culture volume (i.e. 50 mL) was replaced with fresh culture media every 3 days. The hydrostatic pressure on the testing samples were kept at ≈125 Pa.
Fluorescence Microscopy: Fluorescence microscopy images of algae distribution were captured by a Nikon TE2000-E inverted microscope equipped with a Texas Red™ filter cube set. All samples were gently flushed with 0.9 wt % NaCl solution to wash away loosely-bound algae and then sealed in between a glass slide and a coverslip with 0.9 wt % NaCl solution to ensure samples being hydrated and algae being alive during imaging.
Algae Coverage Quantification: Area fractions of algae coverage were quantified from the fluorescence microscopy images through ImageJ analysis.[28] A fluorescence micrograph (RGB color) was first converted to a 16-bit grayscale image, followed by setting an appropriate threshold to mask out the algae particles within the image. Fraction of the unmasked area (denoted as Abackground) was measured by ImageJ, and the area fraction of algae coverage then equals 1-Abackground.
Water Vapor Permeability (WVP) measurements: WVP of silk membranes (both nanostructured and bulk) were measured following the test cup method described in ASTM E96/E96M with some modifications.[25,29] Briefly, a circular silk membrane (Φ=12 mm) was sandwiched between a glass vial (VWR, Catalog Number: 66011-041) filled up with anhydrous CaCl2 pellets (Fisher Scientific) and a lid with a circular opening (Φ=7 mm, McMaster-Carr, Part Number: 1576N12). The silicone rubber septa that comes with the lid was punched to generate a Φ=7 mm hole in the middle and placed in between the silk membrane and the glass vial to make a better seal. After assembling the four parts (i.e. lid, silk membrane, septa, and glass via filled with desiccant, from top to bottom order) together by screwing down the lid to hand tight, several layers of parafilm were wrapped around the lid and glass vial interface to further strengthen the seal. The assembled glass vials were then placed in an airtight container that was equilibrated at 75% RH and room temperature (23° C.) by placing a bowl of saturated NaCl solution in the container. A hygrometer was also placed in the container to verify that the targeted humidity level (i.e. 75% RH) had been reached throughout the measurement period. The glass vials were weighed every 24 hours for a total period of 7 days, and the water vapor transmission rate (WVTR) was calculated by performing a linear fitting of the glass vial weight gain with respect to time. WVP for each sample was then calculated as follows:
where d is the thickness of the silk membrane, A is the permeation area (i.e. the area of the circular opening in the lid and septa), Sis the saturation vapor pressure of water at 23° C., RH1 and RH2 are the relative humidity inside the glass vials and in the container, respectively (i.e., RH1=0% and RH2=75%).
Thermogravimetric analysis (TGA): Quantification of residual water contents in silk arrays dried from different formulations were performed on a TA instruments Discovery TGA. For each measurement, ˜4 mg of samples were first equilibrated at 40° C. for 1 minute and then heated up to 600° C. with a 10° C./min ramp rate.
It should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific implementations described above. The specific implementations described above are disclosed as examples only.
This application claims priority to U.S. Provisional Patent Application Ser. Nos. 63/319,499 filed Mar. 14, 2022 and 63/481,633 filed Jan. 26, 2023, each incorporated by reference herein in their entirety.
This invention was made with government support under grant number N00014-21-1-2402 awarded by Office of Naval Research, grant number N000141912317 awarded by the Office of Naval Research, and grant number CMMI-1752172 awarded by the National Science Foundation. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/064110 | 3/10/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63481633 | Jan 2023 | US | |
| 63319499 | Mar 2022 | US |
