WO2023215909A2 - Couches moléculaires fonctionnalisées et échafaudages, leurs méthodes de préparation et d'utilisation - Google Patents

Couches moléculaires fonctionnalisées et échafaudages, leurs méthodes de préparation et d'utilisation Download PDF

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WO2023215909A2
WO2023215909A2 PCT/US2023/066713 US2023066713W WO2023215909A2 WO 2023215909 A2 WO2023215909 A2 WO 2023215909A2 US 2023066713 W US2023066713 W US 2023066713W WO 2023215909 A2 WO2023215909 A2 WO 2023215909A2
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scaffold
amphiphiles
head groups
tcd
functional
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WO2023215909A3 (fr
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Shelley CLARIDGE
Juan Arango
Luis Solorio
Sarah Libring
Anamika Singh
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Purdue Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/25—Peptides having up to 20 amino acids in a defined sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating

Definitions

  • the present disclosure relates to a nanoscale, patterned monolayer surface for placement on a substrate comprising a polymeric material, wherein the surface can be functionalized. Methods for functionalizing the monolayer surface are also provided, as well as methods for producing the monolayer, transferring the monolayer to a scaffold (e.g., a hydrogel), and adhering cells or other targets to a polymeric material.
  • a scaffold e.g., a hydrogel
  • Nanoscale control over surface functionality is important in applications ranging from nanoscale electronics to regenerative medicine.
  • multivalent interactions between carbohydrates (CHOs) and proteins can enable a broad range of selective interactions of critical biological importance.
  • controlling the chemistry of hydrogels can be beneficial in numerous applications ranging from chromatographic separations to the design of cell culture supports and implants for regenerative medicine.
  • Nichols et al. Nondenaturing polyacrylamide gradient gel electrophoresis, Methods Enzymology 128: 417-431 (1985-1986); Bjellqvist et al., A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale, Electrophoresis 14: 1357-1365 (1993); Yang et al., Spatially patterned matrix elasticity directs stem cell fate, Proc. Natl. Academy Science USA 113: 4439-4445 (2016). [0006] Despite its advantages, controlling structure at soft interfaces is particularly challenging, due to the amorphous, often porous structure of such materials.
  • Nanometer-resolution functional patterns are difficult to achieve in broadly used monolayer chemistries such as functional alkylthiol monolayers on gold or functional alkylsilanes on silicon. [0007] Consequently, high resolution chemical patterning of hard, crystalline surfaces is more common.
  • Molecular ordering relative to the inorganic substrate can be used to generate structure at some nanometer scales, while molecules can be delivered to surfaces in microscale patterns using microcontact printing, or in some cases nanoscopic patterns using scanning probe lithography.
  • Soft surfaces can benefit from high-resolution chemical patterning, such as ligand clustering, which can be important in cell adhesion and other applications.
  • Molecular patterns noncovalently adsorbed on HOPG can be assembled using an internal polymerizable group to ‘set’ the molecular pattern, then covalently transferred to a soft surface such as polydimethylsiloxane (PDMS) or polymeric material (e.g., polyacrylamide (PAAm)).
  • PDMS polydimethylsiloxane
  • PAAm polyacrylamide
  • the hydrogel comprises PAAm.
  • PAAm is a hydrogel that is widely used in protein and deoxyribonucleic acid (DNA) chromatography, cell culture, and other applications. Nichols et al. (1985-1986), supra; Pandey & Mann, Proteomics to study genes and genomes, Nature 405: 837-846 (2000); Engler et al., Matrix elasticity directs stem cell lineage specification, Cell 126: 677-689 (2006). Gel formation is typically achieved through radical- mediated polymerization of an acrylamide (Aam) monomer with a bis-acrylamide (Bis) crosslinker.
  • Aam acrylamide
  • Bis bis-acrylamide
  • Pore sizes, and thus mechanical properties, can be controlled by adjusting the ratio of acrylate to water (%T), and the ratio of crosslinker to monomer (%C) as shown in Fig. 1A.
  • PAAm networks exhibit significant hierarchical complexity at mesoscopic scales (ca. 200-500 nm); thus, exerting high-precision control over the surface chemistry of polyacrylamide and similar hydrogels has traditionally been challenging. [0010] Notwithstanding the challenges traditionally present with PAAm hydrogels, highly ordered arrays of striped polydiacetylenes (sPDAs) can be generated, with the relatively rigid PDA maintaining the chemical patterns at the surface of the soft material.
  • sPDAs striped polydiacetylenes
  • biocompatible substrates such as hydrogels
  • biocompatible substrates such as hydrogels
  • all living cells are decorated by a dense and complex array of glycans which are expressed on cell surfaces in various forms (e.g., free oligosaccharides, glycoproteins, glycolipids and/or proteoglycans).
  • Cell surface glycans are recognized by glycan binding proteins (GBPs), also referred to as lectins.
  • the glycan–lectin interaction mediates or modulates many cellular interactions (e.g., influenza A virus (IAV) infections, cell adhesion, differentiation and tumor metastasis).
  • IAV influenza A virus
  • understanding glycan– lectin interactions at the molecular level is important for the development of diagnostic and therapeutic tools.
  • One challenge in replicating glycan-lectin interactions occurring in vivo is the relatively weak binding (dissociation constants in the millimolar range) between glycan monomers (i.e. monosaccharides) and lectins.
  • Multivalent glycan–lectin interactions increase binding strength, shifting the binding dissociation constant from the millimolar to the nanomolar range, and improve selectivity for specific glycans.
  • multivalent interactions occur at the nm scale. For example, the diameter of a typical hemagglutinin lectin expressed on the IAV surface is ca. 14 nm; similarly, the targeted glycan length is ca. 2–20 nm.
  • Overeem et al. A dynamic, supramolecular view on multivalent interaction between influenza virus and host cells, Small 17 (2021).
  • the molecular layer can comprise polymerized amphiphiles organized in a striped pattern to form an exposed polymer backbone, wherein each of the polymerized amphiphiles comprise one or more exposed alkyl chains and one or more functional head groups covalently bonded with an exposed alkyl chain.
  • the one or more functional head groups can be in an orientation capable of binding a target and the alkyl chains and functional head groups can be organized in a repeating striped pattern comprising a width at a periodicity.
  • the polymerized amphiphiles of the molecular layer can be organized in a lying-down orientation (e.g., the striped pattern) substantially parallel to the surface of the support material, whereas the one or more exposed alkyl chains and the one or more functional head groups are not so restricted.
  • the support material can comprise an amorphous, soft, and/or porous substrate.
  • the support material can comprise a hydrogel.
  • the support material can comprise a hydrophilic hydrogel.
  • the support material can comprise a polyacrylate, polyacrylamide, or polyethylene glycol (PEG) hydrogel.
  • the support material can be compatible with a traditional cell culture support.
  • the molecular layer is, in certain embodiments, covalently bonded to the surface of the support material.
  • the molecular layer can comprise a monolayer.
  • the periodicity of the pattern can be, in certain embodiments, tunable by adjusting a length of the one or more alkyl chains.
  • Each of the one or more exposed alkyl chains can be a nonpolar alkyl chain.
  • the amphiphiles can be polymerized through polymerizable functional groups within the one or more alkyl chains.
  • the polymerizable functional groups can be or comprise polymerizable diacetylene (DA) groups.
  • DA polymerizable diacetylene
  • the one or more functional head groups can comprise chemically or biologically reactive or interactive head groups.
  • the one or more functional head groups can be polar head groups.
  • the one or more functional head groups can comprise carbohydrate (CHO) head groups, a carboxylate, an amine, an alcohol, a peptide, a substrate of an enzyme, or any combination of two or more of the foregoing.
  • the head groups comprise CHO
  • the CHO type can be selected, and the pattern can be designed, such that the CHOs act together as a component of an extracellular matrix (ECM).
  • ECM extracellular matrix
  • the one or more functional head groups can comprise a monosaccharide, a glycan, a N-acetyl-D- glucosamine (GlcNAc), a glucuronic acid (GlcA), a N-acetyl- D -neuraminic acid, or any combination of two or more of the foregoing.
  • the polymerized amphiphiles can comprise C5-C30 functionalized amphiphiles with an internal diyne.
  • the polymerized amphiphiles can comprise 10,12-tricosadiynamine (10,12-TCD- NH 2 ), 4,6-tricosadiynamine (4,6-TCD-NH 2 ), 10,12-pentacosadiynamine (10,12-PCD-NH 2 ), 4,6- pentacosadiynamine (4,6-PCD-NH 2 ), or any combination of the foregoing.
  • the amphiphiles can comprise 10,12-tricosadiynol (10,12-TCD-OH), 10,12-pentacosadiynol (10,12-PCD-OH), 10,12- tricosadiynoic acid (10,12-TCD-COOH), 10,12-pentacosadiynoic acid (10,12-PCD-COOH), 4,6- tricosadiynol (4,6-TCD-OH), 4,6-pentacosadiynol (4,6-PCD-OH), 4,6-tricosadiynoic acid (4,6- TCD-COOH), 4,6-pentacosadiynoic acid (4,6-PCD-COOH), 1,2-bis(10,12-tricosadiynoyl)-sn- glycero-3-phosphocholine (diyne PC), 1,2-bis(10,12-tricosadi
  • the repeating elements of the striped pattern can be linear and substantially parallel to each other.
  • the pattern of the one or more functional head groups can have a sub-10-nm scale.
  • the width of the pattern of functional head groups can be at or about 1 nm (e.g., 1 nm).
  • the pattern of functional head groups can be parallel lines.
  • the pattern of the one or more functional head groups can, in some instances, mimic properties of macromolecules or components of an ECM useful for modulating cell adhesion, cell proliferation, cell differentiation, and/or reprogramming of a cell.
  • the pattern of the one or more functional head groups can mimic properties of polysaccharide components.
  • the one or more functional head groups can comprise CHO head groups and the pattern of CHO head groups can mimic hyaluronic acid.
  • the molecular layer comprises a multi-valent binding surface.
  • the target can be a cell, a biomolecule, or a bioactive molecule.
  • the target can be a myoblast.
  • individual ordered regions of the polymerized amphiphiles have linear dimensions of greater than about 100 nm in length while maintaining nanoscale periodicity of the striped pattern of functional head groups.
  • the individual ordered regions of the polymerized amphiphiles can have linear dimensions of greater than about 10 ⁇ m (e.g., 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, or 15 ⁇ m) in length while maintaining nanoscale periodicity of the striped pattern of functional head groups.
  • the individual ordered regions of the polymerized amphiphiles have linear dimensions of greater than about 1 ⁇ m (e.g., 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, or 4 ⁇ m) in length while maintaining nanoscale periodicity of the striped pattern of functional head groups.
  • the periodicity can be about 5 nm to about 10 nm (e.g., 5-10 nm).
  • the periodicity can be about 5 nm to about 7 nm (e.g., 5-7 nm).
  • the support material can have a pore diameter from about 1 nm to about 200 nm (e.g., 1- 200 nm).
  • the exposed polymer backbone can be covalently bonded to an azide-functionalized molecule or a thiol-functionalized molecule in the absence of copper.
  • the azide-functionalized molecule or the thiol-functionalized molecule is a cell adhesion molecule.
  • the cell adhesion molecule can be cyclic arginine-glycine-aspartic acid (RGD).
  • a functionality of the molecular layer can comprises CHO, one or more peptides, a tripeptide sequence of RGD or a functional analog thereof, a matrisome component, or a combination of any of the foregoing.
  • the scaffolds and molecular layers hereof can be for use in cell culturing, chromatographic biomolecule separation, modulating microscale chemical environments, as a regenerative medicament or implant, or any combination of the foregoing.
  • Cell or tissue culture systems are also provided, such systems comprising any of the scaffolds described herein.
  • multivalent receptor assays comprising any of the scaffolds described herein.
  • the multivalent receptor assay is or comprises a lectin assay.
  • a method of preparing a hydrogel comprising a functionalized surface comprises: obtaining a first substrate comprising a molecular layer positioned thereon, the molecular layer comprising: polymerized amphiphiles organized in a lying-down orientation to form an exposed polymer backbone, each of the polymerized amphiphiles comprising one or more alkyl chains and one or more functional head groups covalently bonded with an alkyl chain, wherein the one or more functional head groups are in an orientation capable of binding a target and the alkyl chains and one or more functional head groups are organized in a repeating striped pattern comprising a width at a periodicity; and transferring the molecular layer from the first substrate to a surface of a hydrogel by covalent binding, whereupon the surface of the hydrogel comprises the molecular layer and is functionalized with the one or more functional head groups.
  • the pattern of the one or more functional head groups can be dictated by organization of the polymerized amphiphiles within the exposed polymer backbone.
  • the one or more alkyl chains can lie flat on the first substrate following polymerization, but can be exposed after transferring the molecular layer to the surface of the amorphous and/or soft support material.
  • the method further comprises exfoliating the hydrogel from the first substrate.
  • the hydrogel can be amorphous, soft and/or porous.
  • Transferring the molecular layer to a surface of an amorphous and/or soft support material can be performed concurrently with curing the amorphous and/or soft support material.
  • Transferring the molecular layer to a surface of an amorphous and/or soft support material can be performed subsequently to curing the amorphous and/or soft support material.
  • transferring the molecular layer to a surface of an amorphous and/or soft support material is performed concurrently with curing the amorphous and/or soft support material and the method further comprises: applying on top of the molecular layer an aqueous reaction mixture comprising a monomer, a crosslinker, and a radical initiator, wherein the aqueous reaction mixture can undergo free radical polymerization to form a hydrogel; and allowing the aqueous reaction mixture to cure by free-radical polymerization to form the hydrogel.
  • the hydrogel can comprise available reaction sites and/or a thin layer of crosslinker on the surface of the hydrogel to which the molecular layer is to be transferred.
  • Th aqueous reaction mixture can further comprises a radical stabilizer.
  • Obtaining a first substrate comprising a molecular layer positioned thereon can comprise: ordering polymerizable amphiphiles as a standing phase film on an aqueous subphase, each of the polymerizable amphiphiles comprising one or more alkyl chains comprising a polymerizable functional group and one or more functional head groups covalently bonded with an alkyl chain; contacting a first substrate to the film in a manner that reorders the polymerizable amphiphiles from the standing phase into ordered a repeating striped pattern on the first substrate, wherein the ordered pattern forms a polymer backbone in which the polymerizable amphiphiles are organized in a lying-down orientation and define the pattern of the one or more functional head groups;
  • the aqueous reaction mixture can comprise an acrylamide monomer or a diacrylamide monomer.
  • the crosslinker can be bisacrylamide
  • the radical initiator is ammonium persulfate
  • the radical stabilizer can be N,N,N’,N’-tetramethylethylenediamine.
  • the reaction mixture comprises from about 10% to about 50% total acrylamide (e.g., 10% to about 50%, about 10% to 50%, or 10% to 50%) and from about 1% to about 10% bisacrylamide (e.g., 1% to about 10%, about 1% to 10%, or 1% to 10%), as a percentage of total acrylamide in each instance.
  • the polymerizable functional groups can comprise DA groups and polymerization thereof with the DA groups of adjacent amphiphiles forms a polydiacetylene within the exposed polymer backbone.
  • the first substrate can be HOPG, graphene, or a layered material comprising MoS2 or WS2.
  • the method can further comprise: after applying the aqueous reaction mixture on top of the patterned layer of polymerized amphiphiles, applying a second substrate, which can be the same as or different from the first substrate, on top of the aqueous reaction mixture, whereupon after the aqueous reaction mixture is allowed to cure, the hydrogel is also covalently bonded to the second substrate.
  • the second substrate can comprise, on its surface, a layer of reactive functional groups or a patterned layer of polymerized amphiphiles, which can be the same as or different from the patterned layer of polymerized amphiphiles of the first substrate.
  • the second substrate can comprise glass or vinyl-functionalized glass.
  • the first and second substrates can comprise the same material selected from the group consisting of HOPG, graphene, and a layered material comprising MoS2 or WS2.
  • the layer of reactive functional groups can comprise one or more alkenes.
  • the surface of the second substrate comprises the layer of reactive functional groups or a patterned layer of polymerized amphiphiles faces the aqueous reaction mixture when the second substrate is applied on top of the aqueous reaction mixture.
  • the pattern of the functional head groups can be parallel lines.
  • the one or more functional head groups can be chemically or biologically reactive or interactive head groups.
  • the chemically or biologically reactive or interactive head groups can be cationic under physiological conditions.
  • the cationic head group is an amine.
  • the one or more functional head groups can comprise CHO head groups.
  • the periodicity can be about 5 nm to about 6 nm (e.g., about 5 nm to 6 nm, 5 nm to about 6 nm, or 5 nm to 6 nm).
  • the hydrogel can have a pore diameter from about 1 nm to about 200 nm (e.g., about 1 nm to 200 nm, 1 nm to about 200 nm, or 1 nm to 200 nm).
  • the CHO head groups can be GlcNAc or GlcA.
  • the amphiphiles can comprise C5-C30 functionalized amphiphiles with an internal diyne.
  • the amphiphiles can comprise TCD-NH 2 , PCD-NH 2 , or a combination of the foregoing.
  • the amphiphiles can comprise 10,12-TCD-GlcNAc, 4,6-TCD-GlcNAc, TCD-OH, PCD-OH, TCD- COOH, PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • the chemically or biologically reactive or interactive head groups can be neutral or zwitterionic under physiological conditions.
  • the neutral head group can be a hydroxyl or the zwitterionic head group can be phosphoethanolamine.
  • the chemically or biologically reactive or interactive head groups can be anionic under physiological conditions.
  • the anionic one or more head groups can be carboxylate.
  • the method can further comprise contacting the exposed polymer backbone on the surface of the hydrogel with a thiol-functionalized or an azide-functionalized molecule under conditions that promote a click reaction between a polydiacetylene of the exposed polymer backbone and a thiol of the thiol-functionalized molecule or between a polydiacetylene of the exposed polymer backbone and an azide of the azide-functionalized molecule.
  • the hydrogel can be polyacrylamide.
  • the thiol-functionalized molecule or the azide- functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule can be cyclic RGD.
  • Chromatography platforms are also provided, wherein such platforms comprise a scaffold hereof.
  • An implant is provided comprising a scaffold hereof.
  • a cell culture platform is provided comprising a scaffold hereof.
  • the cell culture platform is for in vitro cell culture and can be, for example, selected from the group consisting of a 3D cell culture platform, an organ-on-a-chip platform, an immune cell culture platform, and an induced pluripotent stem cell culture platform.
  • a method of adsorbing a biomolecule in a sample is also provided.
  • the method of adsorbing a biomolecule in a sample can comprise contacting, under adsorptive conditions, a sample with a surface of a scaffold hereof; whereupon the biomolecule in the sample is adsorbed onto the molecular layer of the scaffold.
  • the support material can be a polyacrylamide hydrogel.
  • the amphiphile can comprise an amine functional head group.
  • the amphiphile can be TCD-NH 2 or PCD-NH 2 .
  • the biomolecule can be DNA. [0051] Still further, a method of adhering cells to a polymeric material is provided.
  • such a method of adhering cells to a polymeric material can comprise contacting cells with a polymeric material comprising a scaffold hereof, wherein the exposed polymer backbone of the molecular layer comprises polydiacetylene covalently bonded to a thiol-functionalized or an azide-functionalized molecule in the absence of copper.
  • the azide-functionalized or thiol- functionalized molecule can beis a cell adhesion molecule.
  • the cell adhesion molecule can be a cyclic RGD.
  • the cells can be a mixture of cells.
  • the cells can form a tissue.
  • a method of further functionalizing the surface of a polymeric material on the surface of which is a patterned layer of polymerized amphiphiles comprises: obtaining a polymeric material comprising a patterned layer of polymerized amphiphiles, each of which comprises a functional head group (e.g., a chemically or biologically reactive or interactive head group) covalently bonded to an alkyl chain comprising a polymerizable diacetylene group, which polymerizes with another diacetylene group of an adjacent amphiphile to form a polydiacetylene, by which the amphiphiles are polymerized, wherein the alkyl chains lie flat on the first substrate and are positioned to form a pattern of the chemically or biologically reactive head groups about 1 nm in width at a periodicity from about 5 nm to about 10 nm, and contacting the polymeric material with a thiol-functionalized molecule under conditions that promote a click reaction between
  • a functional head group e
  • the pattern can be parallel lines.
  • the periodicity can be about 5 nm to about 7 nm.
  • the polymeric material can have a pore diameter from about 1 nm to about 200 nm.
  • the polymeric material can be polyacrylamide.
  • the amphiphile can be a C5-C30 amine- functionalized amphiphile with an internal diyne, the position of which can vary (see, e.g., USPN 10,889,669), such as 10,12-tricosadiynamine (10,12-TCD-NH 2 ), 10,12-pentacosadiynamine (10,12-PCD-NH 2 ), 10,12-tricosadiynol (10,12-TCD- OH), 10,12-pentacosadiynol (10,12-PCD- OH), 10,12-tricosadiynoic acid (10,12-TCD-COOH), 10,12-pentacosadiynoi
  • the thiol-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule can be cyclic arginine- glycine-aspartic acid (cRGD).
  • cRGD cyclic arginine- glycine-aspartic acid
  • the pattern can be parallel lines.
  • the periodicity can be about 5 nm to about 7 nm.
  • the polymeric material can have a pore diameter from about 1 nm to about 200 nm.
  • the polymeric material can be polyacrylamide.
  • the amphiphile can be 10,12-TCD-NH 2 , 10,12- PCD-NH 2 , 10,12-TCD-OH, 10,12-PCD-OH, 10,12-TCD-COOH, 10,12-PCD-COOH, 4,6-TCD- NH 2 , 4,6-PCD-NH 2 , 4,6- TCD-OH, 4,6-PCD-OH, 4,6-TCD-COOH, 4,6-PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • the thio-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule can be cRGD.
  • a cell culture support comprising an above-described polymeric material is also provided.
  • the amphiphile can be 10,12-TCD-NH 2 , 10,12-PCD-NH 2 , 10,12-TCD-OH, 10,12- PCD-OH, 10,12-TCD-COOH, 10,12-PCD-COOH, 4,6-TCD-NH 2 , 4,6-PCD-NH 2 , 4,6-TCD-OH, 4,6-PCD- OH, 4,6-TCD-COOH, 4,6-PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • the thiol-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule can be cRGD.
  • amphiphiles are polymerized, wherein the alkyl chains are positioned to form a pattern of the functional head groups (e.g., chemically or biologically reactive or interactive head groups) about 1 nm in width at a periodicity from about 5 nm to about 10 nm, and wherein the polydiacetylenes are covalently bonded to a thiol of a thiol-functionalized molecule, under conditions that promote adhesion of the cells with the polymeric material.
  • the pattern can be parallel lines.
  • the periodicity can be about 5 nm to about 7 nm.
  • the polymeric material can have a pore diameter from about 1 nm to about 200 nm.
  • the polymeric material can be polyacrylamide.
  • the amphiphile can be 10,12-TCD-NH 2 , 10,12-PCD-NH 2 , 10,12-TCD-OH, 10,12-PCD-OH, 10,12-TCD-COOH, 10,12-PCD-COOH, 4,6-TCD-NH 2 , 4,6-PCD-NH 2 , 4,6- TCD-OH, 4,6-PCD- OH, 4,6-TCD-COOH, 4,6-PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • the thiol-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule can be cRGD.
  • the cells can be a mixture of cells.
  • the cells can form a tissue.
  • FIG. 1A is a schematic of striped phase polydiacetylenes (sPDA) structure on a substrate 8 (e.g., highly ordered pyrolytic graphite (HOPG)) (top left); a schematic of functional pattern transfer of sPDAs from a substrate 8 to a scaffold 10 mesh (e.g., polydimethylsiloxane (PDMS) or polyacrylamide (PAAm)) (transfer layer identified as 12) (top right); and an illustration of PAAm compositional variables (%T and %C) that can impact transfer efficiency and the creation of 1-nm functional patterns with sub-10-nm pitch (bottom box).
  • sPDA phase polydiacetylenes
  • FIG. 1B is a schematic illustrating the use of amine sPDAs on PAAm to control adsorption of double stranded deoxyribonucleic acid (dsDNA).
  • Fig. 1C is a schematic showing secondary functionalization of a surface 12 of a scaffold 10 via copper-free azidoalkane click reaction of the cell-adhesion peptide cyclic arginine-glycine- aspartic acid (cRGD) to carboxylic acid (-COOH) head groups on amphiphiles laid down on first a substrate (not shown) and thereafter – following transfer – on a scaffold 10 (e.g., PDMS or sPDA).
  • cRGD cell-adhesion peptide cyclic arginine-glycine- aspartic acid
  • -COOH carboxylic acid
  • FIG. 1D is a schematic showing increased cell adhesion to polymeric material surface 12 functionalized with cRGD cell-adhesion peptides (functional head groups 50).
  • Fig. 1E is a schematic illustrating binding selectivity for wheat germ agglutinin (WGA) at striped interfaces based on choice of carbohydrate amphiphile used for striped phase assembly.
  • WGA wheat germ agglutinin
  • FIG. 1F is a schematic illustrating multivalent glycan recognition by WGA (PDB: 2UVO) showing four dimerized receptor sites with 1.3 nm spacing (top left); and a schematic of striped phase assembly and structure on HOPG (bottom left); a schematic of PDA transfer to PAAm in relation to PAAm polymerization architecture and extended glycopolymer display for selective multivalent binding (right, top and bottom).
  • Fig. 1G is a schematic illustrating the stochastic distribution of glycans in a typical standing-phase monolayer and showing the limited spatial and orientation control at 1-10 nm.
  • Fig. 2A are molecular models of unpolymerized and polymerized TCDGlcNAc monolayers on HOPG.
  • Fig. 2B is an atomic force microscopy (AFM) image (left) illustrating the lamellar and domain structures of a polymerized TCDGlcNAc monolayer, and a scanning electron microscopy (SEM) image illustrating the presence of microscale domain structure.
  • Fig. 2C is scanning electron microscopy (SEM) image illustrating the presence of microscopic vacancies.
  • Fig.2D is fluorescence spectra of TCD-GlcNAc and TCD-GlcA on PAAm.
  • FIG. 2E are the chemical structures of GlcNAc and GlcA (left panels) and molecular models illustrating head group interactions in striped phases of TCD-GlcNAc and TCD-GlcA on HOPG (left, center panel).
  • Fig. 2F shows AFM micrographs of TCD-GlcNAc and TCD-GlcA on HOPG (right, center panel) and fluorescence micrographs of TCD-GlcNAc and TCD-GlcA on PAAm (right panel).
  • Fig. 3A are schematics illustrating transfer of sPDA on HOPG to PAAm by in situ crosslinking, where the top schematic shows carbohydrate head groups.
  • Fig.3B is a fluorescence spectra image of 10,12-TCDGlcNAc and PAAm.
  • Figs. 3C and 3D are fluorescence micrographs of TCD-GlcNAc/PAAm and PAAm, respectively.
  • Figs. 4A-4D are fluorescence images of various monolayers after exposure to WGA (5 ⁇ g/mL), where Fig. 4A shows TCDGlcNA-functionalized PAAm, Fig. 4B shows TCDOH- functionalized PAAm, Fig. 4C shows tricosadiynamine (TCDA)-functionalized PAAm, and Fig. 4D shows TCDGlcA-functionalized PAAm.
  • WGA 5 ⁇ g/mL
  • Figs. 4E-4H show fluorescence images of monolayer on PAAm.
  • Fig. 4I shows fluorescence spectra data of monolayers of Figs. 4A-4D after exposure to WGA (5 ⁇ g/mL).
  • Fig.4J shows fluorescence spectra of monolayers on PAAm.
  • Fig.4K shows WGA binding to monolayers normalized for surface coverage.
  • Fig. 4L shows fluorescence spectra illustrating minimal increase in adsorption of rhodamine-labeled ConA (5 ⁇ g/mL) to TCD-GlcNAc/PAAm versus unfunctionalized PAAm.
  • Figs. 4M-4O show ⁇ CP patterned TCD-GlcNAc (Fig. 4M) on HOPG, Fig. 4N shows ater transfer to PAAm, and Fig. 4O shows after transfer to PAAm and exposure to WGA. Inset in Fig. 4N uses enhanced contrast to emphasize the square pattern.
  • Fig. 4M shows ⁇ CP patterned TCD-GlcNAc (Fig. 4M) on HOPG
  • Fig. 4N shows ater transfer to PAAm
  • Fig. 4O shows after transfer to PAAm and exposure to WGA.
  • Inset in Fig. 4N uses enhanced contrast to emphasize the square pattern.
  • Fig. 4N uses enhanced contrast to emphasize the square pattern.
  • FIG. 5A shows molecular models of head group regions in polymerized monolayers of 10,12-TCD-GlcNAc/HOPG (top panel) and 4,6-TCD-GlcNAc/HOPG (bottom panel), illustrating the differences in head group organization between molecules.
  • Fig. 5B shows models following 4 ns dynamics with HOPG removed from PDA frozen, to simulate conditions on sPDAs transferred to PAAm.
  • Fig. 5C shows fluorescence intensities of 4,6-TCD-GlcNAc/PAAm and 10,12-TCD- GlcNAc/PAAm surfaces allowed to equilibrate with specified concentrations of WGA (0.5, 10, or 15 ⁇ g/mL).
  • Fig. 5A shows molecular models of head group regions in polymerized monolayers of 10,12-TCD-GlcNAc/HOPG (top panel) and 4,6-TCD-GlcNAc/HOPG (bottom panel), illustrating the differences in head group organization between molecules.
  • FIG. 5D shows fluorescence intensity spectra data of 10,12-TCD-GlcNAc/PAAm and 4,6- TCD-GlcNAc/PAAm surfaces, before (top graph) and after (bottom graph) exposure to WGA (0.5 ⁇ g/mL).
  • Fig. 5E shows data related to the distribution of neighboring GlcNAc-GlcNAc distances along each sPDA. The gray tie line at 13.0 ⁇ represents the distance between dimerized WGA binding sites.
  • Fig. 6A shows the molecular structure of TCD-NH 2 displayed above molecular models of unpolymerized and polymerized TCD-NH 2 on HOPG (TCD-NH 2 /HOPG or TCD-NH 2 @HOPG).
  • Fig.6B is an AFM image of polymerized TCD-NH 2 /HOPG showing lamellar periodicity.
  • Figs. 6C and 6D are SEM images of microscale TCD-NH 2 /HOPG structure.
  • Fig. 6E is an AFM micrograph image illustrating nanoscale lamellar ordering in striped domains of TCD-NH 2 /HOPG.
  • Fig.6F are SEM images of TCD-NH 2 illustrating microscale domain structure.
  • Fig.7A shows a schematic of an experimental setup for sPDA transfer.
  • Fig.1A shows a schematic of an experimental setup for sPDA transfer.
  • FIG. 7B shows fluorescence images of LS-TCD-NH 2 (left) and ⁇ CP-TCD-NH 2 /PAAm (right).
  • Fig. 7C shows fluorescence spectra of sPDA-functionalized PAAm and polydimethylsiloxane (PDMS).
  • Fig.7D shows fluorescence spectra of ⁇ CP-TCD-NH 2 /PAAm.
  • Figs. 8A and 8B show fluorescence emission intensity of PAAm gels with varied %T (mass % of total acrylate in gel) (Fig. 8A) and varied %C (mass % of crosslinker vs. total acrylate) (Fig. 8B).
  • Fig. 8A shows fluorescence emission intensity of PAAm gels with varied %T (mass % of total acrylate in gel)
  • Fig. 8B shows varied %C (mass % of crosslinker vs. total acrylate)
  • Fig. 8B shows
  • FIG. 8C shows a graph of PAAm pore size diameters calculated from cryogenic electroni microscopy (cryoEM) image analysis.
  • Figs. 8D-8F show cryoEM images of pore structure and texture of an unfunctionalized PAAm gel with 10 %T and 5 %C (Fig. 8D), 30 %T and 5 %C (Fig. 8E), and 40 %T and 5 %C (Fig.8F).
  • Figs. 8G-8I show cryoEM images of pore structure and texture of TCD-NH 2 /PAAm gel with 10 %T and 5 %C (Fig.8G), 30 %T and 5 %C (Fig. 8H), and 40 %T and 5 %C (Fig.8I).
  • Fig. 9A is a schematic illustrating the probability of transfer in relationship to local PAAm network density.
  • Fig. 9B are fluorescence images of TCD-NH 2 /PAAm and PAAm gels with histograms of pixels at each hue, illustrating shift with %T.
  • Fig. 9C is an image of PAAm prepared at 50 %T with increased contrast, showing faint speckling.
  • Fig. 9D illustrates the relationship between heterogeneous PAAm network structure and variability in crosslinking to PDA.
  • Fig. 10A shows the structures of polymerizable amphiphiles tested for DNA binding on sPDA/PAAm surfaces.
  • Fig. 10A shows the structures of polymerizable amphiphiles tested for DNA binding on sPDA/PAAm surfaces.
  • FIG. 10B shows a molecular model illustrating the relative scales of dsDNA and surface- patterned amines in sPDA.
  • Fig. 10C shows fluorescence spectra of TCD-NH 2 /PAAm in the absence (green) and presence (red) of ethidium bromide-labeled DNA (EtBr-DNA).
  • Fig. 10D shows fluorescence emission of PAAm in the presence (red) and absence (gray) of EtBr-DNA.
  • Fig. 10E shows fluorescence spectra of TCD-NH 2 /PAAm in the presence and absence of EtBr-DNA.
  • Fig. 10C shows fluorescence spectra of TCD-NH 2 /PAAm in the presence and absence of EtBr-DNA.
  • FIG. 10F shows composite fluorescence spectra from sPDA/PAAm substrates with amine, carboxylic acid, or hydroxyl surface patterns in the presence of EtBr-DNA.
  • Fig. 10G shows spectra from Fig. 10F with subtraction of bare PAAm in the presence of EtBr-DNA.
  • Fig. 11A is a molecular model of unpolymerized pentacosadiynamine (PCD-NH 2 ) on HOPG and polymerized
  • Fig.11B is a molecular model of polymerized PCD-NH 2 on HOPG.
  • Fig.11C is an AFM image of unpolymerized PCD-NH 2 on HOPG.
  • Fig. 11D is an AFM image of polymerized PCD-NH 2 on HOPG showing lamellar periodicity.
  • Fig. 12A shows fluorescence emission spectra of sPDA/PAAm before and after incubation with the functional azide arginine-glycine-aspartic acid (RGD).
  • Fig. 12B shows fluorescence emission spectra of sPDA/PAAm before and after incubation with the functional azide Bn-N 3 (Bn-N 3 ) with PAAm background subtraction.
  • Fig. 12A shows fluorescence emission spectra of sPDA/PAAm before and after incubation with the functional azide Bn-N 3 (Bn-N 3 ) with PAAm background subtraction.
  • FIG. 12C shows fluorescence mission spectra of sPDA/PAAm before and after incubation with the functional azide Bn-N3 with PAAm background subtraction.
  • Fig. 12D shows fluorescence emission spectra of diyne phophoethanolamine (dPE) sPDA/PAAm before and after incubation with the functional azide Bn-N 3 .
  • Fig.13A is an optical image of C2C12 murine myoblasts cultured on bare PAAm.
  • Fig.13B is an optical image of C2C12 murine myoblasts cultured on TCDA/PAAm.
  • Fig.13A is an optical image of C2C12 murine myoblasts cultured on TCDA/PAAm.
  • FIG. 13C is an optical image of C2C12 murine myoblasts cultured on bare PAAm exposed to cRGD under click conditions.
  • Fig.13D is an optical image of C2C12 murine myoblasts cultured on TCDA/PAAm+cRGD.
  • DETAILED DESCRIPTION Controlling the chemistry of hydrogels and other substrates is important in many applications.
  • the present disclosure is predicated, at least in part, on a method of controlling the surface chemistry of a support material (e.g., a polymeric substrate such as, for example a hydrogel (e.g., a polyacrylamide (PAAm))) (Fig. 1B).
  • a support material e.g., a polymeric substrate such as, for example a hydrogel (e.g., a polyacrylamide (PAAm))
  • the method involves a polymerization process involving in- situ radical reaction of a small molecule monomer (i.e., an amphiphile) to generate an ordered polymer network that displays one or more ligands in a repeating striped pattern.
  • a small molecule monomer i.e., an amphiphile
  • covalently linked linear patterns of amines are transferred to the surface, they can also be used to control adsorption of deoxyribonucleic acid (DNA) at the interface, laying the groundwork for increasingly complex structure assembly.
  • DNA deoxyribonucleic acid
  • the present methods provide detailed control over placement and orientation of ligands and/or functional head groups (such as carbohydrates, for example) on a surface of a polymeric substrate over scales from 1 to 10 nm or even up to 100 nm.
  • ligand is a molecule, ion, or atom that is attached to the central atom or ion. Geometrically localized patterns can also be generated by the local application of molecules to the surface using microcontact printing.
  • Nanostructured Molecular Layers and Scaffolds [0125] Novel molecular layers and scaffolds comprising such molecular layers are provided.
  • a scaffold can comprise a molecular layer positioned on a surface of a support material.
  • the support material can be an amorphous and/or soft substrate.
  • the term “amorphous” means that the material does not exhibit diffraction Bragg peaks in the x-ray diffraction pattern that otherwise results from a repeating, long range periodic atomic or molecular ordering.
  • soft substrate means any material that is flexible, pliable, or malleable when exposed to external force. Other physical characteristics common to soft substrates suitable for use in the scaffolds hereof include linear elasticity and incompressibility. Generally, soft substrates can have a Young’s modulus in the range of about 1 to about 100,000 pascal (Pa).
  • the soft substrates hereof can include those that can be tunable to a stiffness of physiological tissues with a Young’s modulus of about 1,000 to about 100,000 Pa, about 1,000 to about 5,000 Pa, about 1,000 to about 10,000 Pa, about 5,000 to 10,000 Pa, about 5,000 to about 20,000 Pa, about 10,000 to about 20,000 Pa, about 10,000 to about 50,000 Pa, about 20,000 to about 50,000 Pa, about 30,000 to about 50,000 Pa, about 40,000 to about 50,000 Pa, about 50,000 to about 100,000 Pa, about 60,000 to about 100,000 Pa, about 70,000 to about 100,000 Pa, about 80,000 to about 100,000 Pa, about 90,000 to about 100,000 Pa.
  • the soft substrate is prepared from a single material. In other embodiments, the soft substrate is prepared from more than one material.
  • a soft substrate is prepared by layering one or more soft substrates, e.g., to mimic tissue layers in vivo.
  • the support material can be an amorphous, soft, and/or porous substrate.
  • a molecular layer comprises striped phase polydiacetylenes (sPDAs) with ordered patterns of functional groups thereon in nanometer resolution.
  • sPDAs are a type of conjugated polymer that exhibit unique phase behavior, characterized by alternating regions of different molecular conformations. These regions can be aligned in stripes (or domains) and the properties of sPDAs are highly dependent on their molecular structure and organization.
  • the molecular layers hereof are customizable and are readily transferrable to a support material to functionalize the surface thereof – even an amorphous support material or soft substrate comprising substantial heterogeneity on length scales (e.g., a hydrogel).
  • the molecular layers and scaffolds hereof can pattern interactions with DNA, for example; a capability of potential importance for preconcentration in chromatographic applications, as well as for the development of nanostructured hybrid materials and supports for cell culture.
  • such molecular layers and scaffolds can comprise nanometer-resolution carbohydrate patterns to allow for selective, multivalent interactions (KD ⁇ 40 nM) that can, for example, be used to design synthetic glycan-mimetic interfaces with features from molecular to mesoscopic scales, including soft scaffolds for cells.
  • KD ⁇ 40 nM nanometer-resolution carbohydrate patterns
  • the present disclosure is predicated, at least in part, on the surprising and unexpected discovery that sPDAs can participate in copper-free azidoalkyne “click” reactions, which typically require a strained alkyne (e.g., cyclooctyne) reaction partner.
  • the monolayer can be further functionalized with secondary surface chemistry (e.g., secondary functionalities) installed along the sPDA backbone.
  • secondary functionalities e.g., secondary functionalities
  • the molecular layers can be secondarily functionalized with peptides and the like, making it possible to achieve a high degree of complexity with respect to the interface.
  • nanometer-resolution functional patterns have been difficult to achieve in broadly used interface chemistries such as functional alkyl-thiol monolayers on gold or functional alkylsilanes on silicon.
  • 1-nm resolution functional patterns can be readily assembled on substrates such as highly oriented pyrolytic graphite (HOPG) using lying-down phases of simple diyne amphiphiles.
  • amphiphiles spontaneously assemble in a lying-down (or striped) orientation with the alkyl chains parallel to the substrate (e.g., HOPG) surface (see Fig. 1A) and are stabilized by the van der Walls interaction between adjacent alkyl chains and hydrogen-bonding between polar head groups.
  • This can generate a polymer backbone on the initial substrate comprising, for example, striped 1 nm-resolution functional patterns with a specific pitch (e.g., sub-10 nm pitch).
  • DA diacetylene
  • topochemical photopolymerization can be used to generate sPDAs (i.e.
  • the periodicity equates with about twice the molecular length of the alkyl chain. Accordingly, lengthening or shortening the alkyl chains can have a direct effect on at least the periodicity of the pattern. [0132] This type of patterning can be controlled over large areas and can influence both interfacial wetting and further assembly at the interface.
  • Polydiacetylenes can also be utilized in a reaction process that transfers the nm-resolution functional patterns to soft, biocompatible scaffolds such as PDMS and polyacrylamide hydrogels.
  • the present disclosure synthesizes polymerizable amphiphiles with various head groups using sPDA assembly to achieve nanometer-resolution patterning on a hard, crystalline material (e.g., the first substrate), and subsequently transferring the polymerized, patterned surface to a desired support material (e.g., a hydrogel or soft substrate) to form the scaffold.
  • a desired support material e.g., a hydrogel or soft substrate
  • the molecular layer is the portion comprising the functionalization that is formed on a first substrate and for placement on a support material to form the scaffold.
  • a molecular layer (e.g., for placement on a support material) is provided.
  • the molecular layer can be a monolayer.
  • a monolayer and “thin film” can be used interchangeably herein without distinctions and are defined as a layer of material ranging from less than about 1 nm to several thousand nanometers in thickness.
  • a monolayer is a two-dimensional (2D) material, generally having a layer thickness between one atomic layer and a few nanometers (nm).
  • a monolayer is a three-dimensional (3D) material.
  • a scaffold is provided comprising the molecular layer positioned on a surface of a support material.
  • the molecular layer can be, in some embodiments, covalently bonded to the surface of the support material (e.g., using the methods hereof).
  • the support material can comprise any structure upon which a functionalized surface comprising the molecular layer is desired.
  • the support material can be an amorphous material.
  • the support material can comprise a soft substrate.
  • the support material can comprise an amorphous and/or soft substrate.
  • the support material is an amorphous, porous substrate.
  • the support material comprises a hydrogel.
  • the hydrogel can be homopolymeric, copolymeric, or a multipolymer interpenetrating polymeric hydrogel (IPN), which is made of two independent cross-linked synthetic and/or natural polymer components contained in a network form.
  • IPN multipolymer interpenetrating polymeric hydrogel
  • the hydrogel can be semicrystalline or crystalline.
  • the hydrogel can be a matrix, a film or a microsphere.
  • the hydrogel can be nonionic, ionic (e.g., anionic or cationic), ampholytic (i.e., containing both acidic and basic groups), or zwitterionic (i.e., containing anionic and cationic groups in each structural repeating unit).
  • the hydrogel can be a hydrophilic hydrogel.
  • the hydrogel can comprise a polyacrylate, polyacrylamide, or polyethylene glycol (PEG) hydrogel.
  • the hydrogel can be any type of hydrogel suitable for use as a cell adhesion scaffold.
  • the hydrogel can be any type of hydrogel suitable for use as an implant.
  • the hydrogel can be any type of hydrogel suitable for use as a cell support scaffold.
  • the support material is compatible with a traditional cell culture support (e.g., such that the support material can be used to facilitate cell growth or adhesion).
  • the hydrogel can have any suitable pore diameter, such as from about 1 nm to about 200 nm. Typically, most hydrogels have a heterogeneous structure with pores of various sizes. For example, a hydrogel can have both small pores (e.g., about 1 to about 10 nm) and large pores (e.g., about 20 to about 200 nm).
  • the hydrogel comprises PAAm.
  • PAAm is a hydrogel that is widely used in protein and deoxyribonucleic acid (DNA) chromatography, cell culture, and other applications.
  • Gel formation is typically achieved through radical- mediated polymerization of an acrylamide (Aam) monomer with a bis-acrylamide (Bis) crosslinker. Pore sizes, and thus mechanical properties, can be controlled by adjusting the ratio of acrylate to water (%T), and the ratio of crosslinker to monomer (%C) as shown in Fig. 1A.
  • the molecular layer can comprise polymerized amphiphiles organized in a lying-down orientation to form an exposed polymer backbone.
  • Amphiphile means a chemical compound comprising hydrophilic and hydrophobic constituents.
  • Each of the polymerized amphiphiles can comprise one or more alkyl chains.
  • each of the polymerized amphiphiles can comprise one or more functional head groups covalently bonded with an alkyl chain.
  • the one or more functional head groups can be in an orientation capable of binding a target.
  • the alkyl chains and functional head groups can be organized in a repeating striped pattern (e.g., comprising a width at a periodicity). In certain embodiments, the repeating striped pattern comprises lines substantially parallel to each other.
  • the amphiphiles can spontaneously assemble in this lying-down (or striped) orientation and are stabilized by the van der Walls interaction between adjacent alkyl chains and hydrogen-bonding between polar head groups to generate the polymer backbone.
  • the polymer backbone can orient in, for example, 1 nm-resolution functional patterns with a specific pitch (e.g., sub-10 nm pitch).
  • the polymerized amphiphiles are organized in a lying-down (striped pattern) orientation that is substantially parallel to the surface of the support material, whereas the one or more exposed alkyl chains and the one or more functional head groups are not so restricted.
  • the amphiphile of the molecular layer can be any amphiphile suitable for inclusion in the molecular layer.
  • the polymer backbone comprises a single type of amphiphile.
  • the polymer backbone comprises two or more different amphiphiles. It will be appreciated that the types of amphiphile(s) used can be customized for the intended purpose of the resulting monolayer.
  • the amphiphile comprises a C5-C30 functionalized amphiphile with an internal diyne.
  • the amphiphile can comprise a functionalized amphiphile with an internal diyne that comprises more than 30 carbons.
  • the amphiphile can be or comprise 10,12-tricosadiynamine (10,12-TCD-NH 2 ).
  • the amphiphile be or can comprise 4,6-tricosadiynamine (4,6-TCD-NH 2 ).
  • the amphiphile can be or comprise 4,6-pentacosadiynamine (4,6-PCD-NH 2 ) or 10,12- pentacosadiynamine (10,12-PCD-NH 2 ).
  • the amphiphile is or comprises 10,12-tricosadiynol (10,12-TCD-OH), 10,12-pentacosadiynol (10,12-PCD-OH), 10,12- tricosadiynoic acid (10,12-TCD-COOH), and/or 10,12-pentacosadiynoic acid (10,12-PCD-COOH).
  • the amphiphile can be or comprise 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (diyne PC) or 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (diyne PE).
  • the amphiphiles comprises a C 5 -C 30 amine-functionalized amphiphiles with an internal diyne, such as, by way of non-limiting example, 10,12-TCD-NH 2 or 10,12-PCD-NH 2 .
  • the amphiphiles are or comprise 10,12-TCD-OH, 10,12- PCD-OH, 10,12-TCD-COOH, 10,12-PCD-COOH, 4,6-TCD-NH 2 , 4,6-PCD-NH 2 , 4,6-TCD-OH, 4,6-PCD-OH, 4,6-TCD-COOH, 4,6-PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • Each amphiphile can comprise one or more alkyl chains.
  • the one or more functional groups of the amphiphiles can be covalently bonded to the relevant alkyl chain.
  • the alkyl chains can be exposed.
  • the functional head groups can be exposed.
  • the polymer backbone can be exposed (e.g., for attachment). These exposures result, at least in part, from the lying-down orientation of the polymer backbone, with the amphiphiles positioned parallel to the interface (i.e., lying flat) and can be important for transfer (i.e., the ability for the polymer backbone to undergo additional functionalization reactions).
  • the polymer backbone can comprise polydiacetylene.
  • each amphiphile Prior to polymerization, each amphiphile can comprise a polymerizable functional group within the one or more alkyl chains. The amphiphiles can be polymerized through the polymerizable functional groups within the one or more alkyl chains.
  • the polymerizable functional groups can be positioned at any suitable position along the length of the alkyl chains.
  • the position of the polymerizable functional group on an alkyl chain can affect accessibility of the chemically or biologically reactive head group (i.e., functional head group) as described in U.S. Patent No. 10,889,669, which is hereby incorporated by reference for its teachings regarding the same.
  • the polymerizable functional groups can be polymerized by any suitable polymerization method. Examples of suitable polymerization methods are exposure to UV light (see, e.g., U.S. Patent No. 10,525,502), heat, and/or energetic electrons.
  • a device that can be used for in situ thermal control and transfer of the layer of polymerized amphiphiles onto the first substrate is described in U.S. Patent No. 11,031,268, which is incorporated herein by reference for its teachings regarding the same. See also U.S. Patent Application Publication No. 2021/0060604 for the use of an apparatus to perform noncovalent functionalization of a substrate using Langmuir-Schaefer conversion (U.S. Patent Application Publication No. 2021/0060604 is hereby incorporated by reference for its teachings regarding the same).
  • a polymerizable functional group can be a polymerizable DA group.
  • polymerization can be used to generate sPDAs (i.e. stripes of conjugated polydiacetylenes) that link together molecules (i.e., functional head groups) along the row or polymer backbone.
  • sPDAs i.e. stripes of conjugated polydiacetylenes
  • the alkyl chains can be utilized to control the length between the polymer backbone and the patterned functionality (i.e., functional head groups). Accordingly, selection of the alkyl segments can be used to further tune the patterned design and properties of the interface. Pursuant to the present disclosure, such patterns can then be transferred to a scaffold (such as a hydrogel) surface, even when such scaffolds are soft and/or amorphous.
  • the alkyl chains can be positioned such that the functional head groups coupled thereto are organized in a pattern comprising a width at a periodicity (e.g., a repeating striped pattern dictated by the underlying pattern of the polymer backbone).
  • the pattern can also comprise the functional head groups being positioned in an orientation capable of binding a target (e.g., a target at the interface such as a cell).
  • a target e.g., a target at the interface such as a cell.
  • These ordered molecular patterns can be polymerized (e.g., photopolymerized by ultraviolet (UV) irradiation) to “lock in” the pattern and generate striped arrays of PDAs with polymer backbones exposed at the interface for further reactions.
  • UV ultraviolet
  • nanometer- resolution functional patterns on a substrate such as HOPG that extend over large microscopic areas and include diverse pattern symmetries and head group chemistries that impact macroscopic and nanoscopic wetting and assembly of nano- and microscopic objects at the interface.
  • These molecular layers comprising functional patterns can then be transferred to a support material as previously described.
  • the one or more alkyl chains of each amphiphile can be any suitable alkyl chains.
  • Alkyl generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms (e.g., C 1 -C 15 alkyl). Disclosures provided herein of an “alkyl” are intended to include independent recitations of a saturated “alkyl,” unless otherwise stated.
  • An alkyl can comprise one to thirteen carbon atoms (e.g., C 1 -C 13 alkyl).
  • An alkyl can comprise one to eight carbon atoms (e.g., C1-C8 alkyl).
  • An alkyl can comprise one to five carbon atoms (e.g., C1-C5 alkyl).
  • An alkyl can comprise one to four carbon atoms (e.g., C1-C4 alkyl).
  • An alkyl can comprise one to three carbon atoms (e.g., C 1 -C 3 alkyl).
  • An alkyl can comprise one to two carbon atoms (e.g., C1-C2 alkyl).
  • An alkyl can comprise one carbon atom (e.g., C1 alkyl).
  • An alkyl can comprise about ten to thirty carbon atoms (e.g., C10-C30 alkyl).
  • An alkyl can comprise about ten to about twenty-five carbon atoms (e.g., C 10 -C 25 alkyl).
  • An alkyl can comprise about fifteen to about twenty-five carbon atoms (e.g., C15-C25 alkyl).
  • An alkyl can comprise about twenty to about twenty-five carbon atoms (e.g., C20-C25 alkyl).
  • An alkyl can comprise about twenty-three to about twenty-five carbon atoms (e.g., C 23 -C 25 alkyl).
  • the alkyl chain is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl).
  • An alkyl chain can be a nonpolar alkyl chain.
  • the alkyl chain can be attached to the rest of the molecule by a single bond.
  • the alkyl chains can be linear or branched. Linear alkyl chains can be preferrable in certain applications.
  • the one or more functional head groups can be covalently bonded with an exposed alkyl chain.
  • the functional head groups can comprise chemically or biologically reactive or interactive head groups. “Chemically or biologically reactive or interactive” is used herein to describe a functional head group on an amphiphile that can undergo a chemical or biological reaction or interaction with a component of a sample, such as a cell or a tissue, for example.
  • the head group can be cationic under physiological conditions and can be neutral during deposition of the patterned layer or assembly.
  • the one or more functional head groups can be polar head groups.
  • a functional head group can be an amine, a CHO, a carboxylate, an alcohol, a peptide a substrate of an enzyme, a nucleic acid, a lipid, or any combination of the foregoing.
  • a functional head group can be a CHO head group.
  • the CHO head group can be or comprise a monosaccharide, a glycan, a N-acetyl- D -glucosamine (GlcNAc), a glucuronic acid (GlcA), and/or a N-acetyl- D-neuraminic acid.
  • the functional head group can be neutral.
  • the functional head group is neutral and comprises a hydroxyl.
  • the head group can be a zwitterionic head group.
  • the functional head group is a zwitterionic head group and comprises a phosphoethanolamine.
  • the head group can be anionic under physiological conditions and can be neutral during deposition of the patterned layer or assembly.
  • the anionic head group can be a carboxylate.
  • the head groups comprise CHO head groups (e.g., N-acetyl-D- glucosamine (GlcNAc)) to achieve nm-resolution CHO patterning that mimics key aspects of cell- surface-glycan architectures.
  • the one or more functional head groups are organized in a pattern comprising a width at a periodicity and further in an orientation capable of binding a target (e.g., a cell, a biomolecule, a bioactive, and/or a myoblast).
  • the repeating elements of the striped pattern can be linear and substantially parallel to each other.
  • the pattern in which the functional head groups are organized can be any ordered pattern but such pattern is typically, at least in part, dictated by the organization of the polymer backbone.
  • the pattern comprises parallel lines.
  • the pattern of the one or more functional head groups is a nanoscale pattern (e.g., a pattern having a nanoscale).
  • the pattern of the one or more functional head groups can have a sub-10-nm scale.
  • the pattern of the one or more functional head groups can be organized to mimic certain desired properties, for example, of nature.
  • the pattern of the one or more functional head groups i.e., comprising CHO head groups
  • the CHO type can be selected, and the pattern of the one or more functional groups designed, such that, in use, the CHOs act together as a component of an extracellular matrix (ECM).
  • ECM extracellular matrix
  • the pattern of the one or more functional head groups mimics properties of macromolecules or components of an ECM (for example, as a means for modulating cell adhesion, spreading, proliferation or differentiation, or for cellular reprogramming).
  • ECM for example, as a means for modulating cell adhesion, spreading, proliferation or differentiation, or for cellular reprogramming.
  • the monolayer can be useful for modulating cell adhesion, cell proliferation, cell differentiation, and/or reprogramming of a cell.
  • one or more of the functional head groups can comprise a CHO.
  • the placement and orientation of the CHOs can be selected over scales from 1 to 10 nm for individual lectins and up to 100 nm or more when lectins themselves are clustered, as in a viral capsid.
  • the wheat germ agglutinin (WGA) protein exhibits binding specificity for GlcNAc and N-acetyl- D -neuraminic acid (sialic acid, Sia) with a 1.3 nm distance between receptor sites for GlcNAc (Fig. 1F) while concanavalin A (ConA) has a distance of about 6.5 nm between mannose binding sites.
  • Carbohydrate linkage and ring orientation are also important factors (e.g., ⁇ 2,3-linked Sia is associated with avian influenza viruses and ⁇ 2,6-linkages with human viruses). Such interactions can also span larger scales when lectins are clustered for recognition.
  • the diameter of a typical hemagglutinin lectin expressed on the influenza A virus (IAV) surface is ca. 14 nm, while the virus diameter is 100-150 nm; the targeted glycan length is ca. 2-20 nm, with multiple glycans binding to lectins across the IAV surface.
  • the distribution of glycans across a cell surface can lead to pattern formation over length scales from nanometers to micrometers, extending to even larger scales across tissues.
  • the molecular layers hereof can be used to display monosaccharides and glycans at interfaces, providing convenient readouts of binding interactions, for example, as well as the potential for longer-range ensemble interactions.
  • the molecular layers hereof can comprise a multi- valent binding surface.
  • the one or more functional head groups are held in an orientation capable of binding a target.
  • the target can be a cell (e.g., a myoblast).
  • the target can be a biomolecule.
  • the target can be a bioactive molecule.
  • the width of the pattern of the functional head groups is at or about 1 nm (such as 1 nm).
  • individual ordered regions of the polymerized amphiphiles have linear dimensions of greater than about 100 nm in length while maintaining nanoscale periodicity of the striped pattern of function head groups.
  • the ordered regions of the polymerized amphiphiles can have linear dimensions of greater than about 10 ⁇ m in length (such as 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 15 ⁇ m, or greater than 15 ⁇ m) while maintaining nanoscale periodicity of the striped pattern of functional head groups.
  • the ordered regions of the polymerized amphiphiles can have linear dimensions of greater than about 1 ⁇ m in length (such as 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, or greater than 4 ⁇ m) while maintaining nanoscale periodicity of the striped pattern of functional head groups.
  • the periodicity can be any suitable periodicity, such as about 5 nm to about 7 nm (such as 5 nm to about 7 nm or about 5 nm to 7 nm) or about 5 nm to about 10 nm (such as 5 nm to about 10 nm or about 5 nm to 10 nm).
  • the pattern of the one or more functional head groups can have a sub- 100-nm scale.
  • the width of the pattern of functional head groups can be at or about 1 nm (such as 1 nm). In certain embodiments, the width of a striped phase is at or about 1 nm (such as 1 nm).
  • the width of the pattern is about 1 nm to about 2 nm (such as about 1 nm to 2 nm, 1 nm to about 2 nm, or 1 nm to 2 nm).
  • the molecular layer hereof can be for placement on a support material, for example. In certain embodiments, the molecular layer is transferred to a surface of a support material. In certain embodiments, the molecular layer is covalently transferrable to the support material.
  • the polymer backbone (e.g., the exposed polymer backbone) of the molecular layer can optionally be secondarily functionalized.
  • the polymer backbone of the molecular layer can participate in copper-free azidoalkyne “click” reactions.
  • complex secondary surface functionalization moieties such as cyclic arginine-glycine-aspartic acid (cRGD) cell-adhesion peptides or fragments or functional analogs thereof, can be applied to the surfaces of polymeric materials.
  • cRGD cyclic arginine-glycine-aspartic acid
  • the molecular layer can provide a straightforward and structured display of a wide range of azide- or thiol-functionalized molecules on its surface, even where the support material is a soft, biocompatible material and in addition to any primarily functionalization afforded by the functional head groups.
  • the polymer backbone can be covalently bonded to an azide-functionalized or a thiol-functionalized molecule in the absence of copper.
  • the azide-functionalized molecule or the thiol-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule is cRGD.
  • the functionality of the molecular layer comprises CHO, one or more peptides, a tripeptide sequence of RGD or a functional analog or fragment thereof, a matrisome component, or a combination of any of the foregoing.
  • the scaffolds and/or molecular layers can be for use in cell culturing, chromatographic biomolecule separation, modulating microscale chemical environments, as a regenerative medicament or implant, or any combination of the foregoing.
  • the scaffold comprises a molecular layer comprise diyne amphiphiles with CHO head groups assembled in nm-resolution CHO patterns covalently bonded to a surface of a hydrogel.
  • transferred patterns of appropriate CHOs e.g., GlcNAc
  • WGA wheat germ agglutinin
  • model lectin that exhibits multivalent binding with appropriately spaced G1cNAc moieties.
  • the present scaffolds and/or molecular layers can also be used for multivalent receptor microarrays for screening molecular interactions, display geometries, spatial organization of ligands and/or other binding characteristics.
  • the multivalent receptor microarray can comprise a lectin assay for screening glycan-lectin interactions.
  • the molecular layer can control glycan display from molecular to microscopic scales, on both hard and soft substrates, which combines desirable aspects of polymer and surface-based approaches to bridge length scales.
  • such approach can be utilized as a potent screening and inhibitory tool for glycan binding proteins (GBPs).
  • GBPs glycan binding proteins
  • peptide- and protein-based scaffolds that provide elements of control over spacing require complex synthesis.
  • Developing a modular, biocompatible scaffold with precisely-controlled chemistries thereon can offer numerous advantages that are significant to various industries. Glycan density and orientation could lead to improved glycan screening array performance by more closely replicating multivalent binding geometries observed in vivo. Nanometer-resolution functional patterns are difficult to achieve in broadly used monolayer chemistries such as functional alkylthiol monolayers on gold or functional alkylsilanes on silicon.
  • a scaffold that comprises a surface upon or to which a molecular layer is applied and/or covalently transferred.
  • the scaffold can be a cell culture support.
  • the scaffold can be a cell support material compatible to be added to a cell culture support.
  • the scaffold can comprise a hydrogel.
  • the cell culture platform can comprise an in vivo cell culture platform (i.e., designed for use in vivo).
  • the cell culture platform can comprise an in vitro cell culture platform (i.e., designed for use in vitro).
  • an in vitro cell culture platform include a 3D cell culture platform, an organ-on-a-chip platform, an immune cell culture platform, and an induced pluripotent stem cell culture platform.
  • Receptors or multivalent lectin assay formats are also provided.
  • the receptor or multivalent lectin assay format comprises any of the hydrogels described herein.
  • a chromatography or biosensing platform, a cell culture support, or an implant for regenerative medicine can comprise any of the scaffolds hereof, on the surface of which is a patterned layer of polymerized amphiphiles.
  • the clustering of functional groups on the material can be used to induce selective binding of an analyte for purification and/or the surface display can be used to generate customized chromatographic stationary phases displaying complex molecules needed for bio-separation or to create multimodal stationary phases.
  • the functional head groups of the monolayer for such application can be head groups that bind DNA, for example.
  • the spatial arrangement and/or orientation and coupling of functional head groups can mimic structural elements of macromolecular components (e.g., polysaccharides, such as hyaluronic acid) of an ECM, such as one encoded by a matrisome.
  • the support can be used to culture any cell, such as one used to engineer a tissue or repair a tissue, e.g., a myoblast, in vitro or in vivo.
  • the amphiphile can be TCD-NH 2 , PCD-NH 2 , TCD-OH, PCD-OH, TCD-COOH, PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • the thiol-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule can be cRGD.
  • Implants are also provided.
  • the implant can comprise a molecular layer bonded (e.g., covalently bonded) to a surface of a support material such as a hydrogel.
  • the implant can be an implantable biological device.
  • the implant can be an implantable hardware.
  • the surface can be used to display peptides or proteins such as those that are components of biological basement membranes.
  • the monolayer and materials hereof can help to facilitate cell adhesion and/or growth in and around an implant displaying the monolayer and/or materials hereof.
  • the molecular layer can comprise polymerized amphiphiles organized in a lying-down orientation to form an exposed polymer backbone.
  • Each of the polymerized amphiphiles can comprise one or more alkyl chains and one or more functional head groups covalently bonded with an alkyl chain.
  • the one or more functional head groups can be in an orientation capable of binding a target (e.g., a cell, bioactive molecule, biomolecule, or the like) and the alkyl chains and functional head groups can be organized in a repeating striped pattern comprising a width and periodicity.
  • the one or more alkyl chains lie flat on the first substrate (at least following polymerization) and are positioned such that the functional head groups are organized in the pattern.
  • the pattern of the functional head groups can be, for example, any of the patterns described herein such as about 1 nm to about 2 nm in width (e.g., about 1 nm to 2 nm, 1 nm to about 2 nm, or 1 nm to 2 nm) at a periodicity from about 5 nm to about 10 nm (e.g., about 5 nm to 10 nm, 5 nm to about 10 nm, or 5 nm to 10 nm).
  • the pattern of the functional head groups can be, for example, parallel lines.
  • the one or more functional head groups can be chemically or biologically reactive or interactive head groups.
  • the one or more functional head groups can be chemically or biologically reactive or interactive head groups which are cationic under physiological conditions.
  • the cationic head group is an amine.
  • the one or more functional head groups comprises CHO head groups.
  • the functional head groups can be any of those described herein or would be reasonably known in the art in view of the present disclosure.
  • the functional head groups can be neutral or zwitterionic under physiological conditions.
  • the head groups can be a neutral head group such as hydroxyl.
  • An example of a zwitterionic head group is phosphoethanolamine.
  • the chemically or biologically reactive or interactive head groups can be anionic under physiological conditions.
  • An example of anionic head groups is carboxylate.
  • the polymerizable functional groups can, as previously described, comprise DA groups. In such instances, polymerization of the DA groups with DA groups of adjacent amphiphiles can form polydiacetylene within the exposed polymer backbone.
  • the amphiphiles can comprise C5-C30 functionalized amphiphiles with an internal diyne.
  • the amphiphiles the amphiphiles comprise TCD-NH 2 , PCD-NH 2 , or a combination of the foregoing.
  • the amphiphiles can comprise 10,12-TCD-GlcNAc, 4,6-TCD-GlcNAc, TCD-OH, PCD- OH, TCD-COOH, PCD-COOH, diyne PC, diyne PE, or any combination of the foregoing.
  • the first substrate comprising the molecular layer positioned thereon can be provided independently, it can optionally be generated.
  • obtaining the first substrate comprising the molecular layer comprises: (1) ordering polymerizable amphiphiles as a standing phase film on an aqueous subphase, each of the polymerizable amphiphiles comprising one or more alkyl chains comprising a polymerizable functional group and one or more functional head groups covalently bonded with an alkyl chain; (2) contacting the first substrate to the film in a manner that reorders the polymerizable amphiphiles from the standing phase into ordered a repeating striped pattern on the first substrate, wherein the ordered pattern forms a polymer backbone in which the polymerizable amphiphiles are organized in a lying-down orientation and define the pattern of the one or more functional head groups; and (3) polymerizing the polymerizable functional groups of the polymerizable amphiphiles with polymerizable functional groups of adjacent amphiphiles to stabilize the pattern and polymerize the amphiphiles.
  • the alkyl chains can lie flat on the first substrate and can be positioned such that the functional head groups are organized in the pattern.
  • the patterned layer of polymerized amphiphiles affixed to the first substrate is then transferred to a surface of a hydrogel/support material by covalent bonding such that the surface of the cured hydrogel comprises the patterned layer of polymerized amphiphiles comprising the one or more functional head groups.
  • the alkyl chains can be exposed following transfer of the molecular layer to the surface of the support material/hydrogel. Additionally, the polymer backbone and the head groups of the molecular layer are also exposed and available for binding/reactions.
  • the pattern of the one or more functional head groups can be dictated by the organization of the polymerized amphiphiles within the exposed backbone.
  • curing which can involve a cross-linking reaction between a vinyl-terminated base polymer and a cross-linker containing reactive Si-H bonds, for example.
  • Curing a scaffold such as a hydrogel in contact with the sPDA molecular layer hereof, then exfoliating, can result in transfer of the sPDA monolayer to the scaffold surface, thereby functionalizing the scaffold.
  • This surface functionalization can be leveraged to enable control over interactions with nanoscale objects (e.g., inorganic nanowires) and molecules (e.g., polyelectrolytes or CHOs) in the environment.
  • the first substrate can be any suitable substrate.
  • the substrate comprises graphene.
  • the substrate can comprise polydiacetylene.
  • the substrate can comprise PDMS.
  • the substrate can comprise polyacrylamide (PAAm).
  • PAAm polyacrylamide
  • the substrate comprises a layered material comprising MoS 2 or WS 2 .
  • the substrate can comprise pyrolytic graphite (HOPG).
  • the first substrate can comprise polydimethylsiloxane (PDMS; also known as dimethylpolysiloxane or dimethicone).
  • PDMS polydimethylsiloxane
  • the first substrate can have any suitable dimensions and can be 2D or 3D.
  • the first substrate can be of any suitable thickness.
  • the first substrate can comprise a multivalent binding surface (e.g., by virtue of the polymerized amphiphiles and one or more functional head groups thereof).
  • the first substrate has a pore diameter from about 1 nm to about 200 nm (such as 1 nm to about 200 nm, about 1 nm to 200 nm, or 1 nm to 200 nm).
  • the first substrate can be prepared using any suitable method known in the art, such as Langmuir-Schaefer (see, e.g., U.S.
  • the method comprises providing the first substrate comprising a patterned layer of polymerized amphiphiles thereon. Such patterned orientation of the polymerized amphiphiles can dictate the pattern of the one or more functional head groups.
  • an aqueous reaction mixture comprising a monomer, a crosslinker, and a radical initiator can be applied on top of the patterned layer of polymerized amphiphiles. In such case, the aqueous reaction mixture can cure by free-radical polymerization to form the hydrogel while in contact with the patterned layer.
  • the aqueous reaction mixture can be any suitable aqueous reaction mixture that can be polymerized into a hydrogel which, depending on the application, can desirably be biocompatible.
  • aqueous reaction mixtures include, without limitation, those that result in the production of polyacrylamide, poly-2-hydroxyethyl methacrylate, polyacrylic acid, polyacrylonitrile, cross-linkable polyethylene glycol (PEG), protein (e.g., collagen or gelatin), and/or polysaccharides (e.g., starch, alginate, or agarose).
  • the monomer in the aqueous reaction mixture can be any monomer that can be polymerized into a hydrogel, which, depending on the application, can desirably be biocompatible.
  • the aqueous reaction mixture can comprise a single type of monomer or a mixture of monomers.
  • each monomer can comprise one or more functional groups, such as acrylic acrylamide, allyl, PEG diacrylate, vinyl, methacrylic, alkenyl, and any combination thereof.
  • the reaction mixture comprises from about 10% to about 50% total acrylamide (e.g., 10% to about 50%, about 10% to 50%, or 10% to 50%) and from about 1% to about 10% bisacrylamide (e.g., 1% to about 10%, about 1% to 10%, or 1% to 10%), as a percentage of total acrylamide in each instance.
  • one or more degradation sites can be incorporated into the monomer.
  • the site can be selected to be degraded by hydrolysis, photolysis, microbes, or enzymes, for example.
  • degradation sites include, but are not limited to, polylactic acid and matrix-metalloproteinase sensitive (MMP-sensitive) peptide.
  • MMP-sensitive matrix-metalloproteinase sensitive
  • the crosslinker in the aqueous reaction mixture can be any crosslinker that can polymerize the monomer. Examples of crosslinkers include, but are not limited to, bisacrylamide (such as when the monomer is an acrylamide or diacrylamide), PEG dimethacrylate, and N,N’-methylene bisacrylamide.
  • the crosslinker is bisacrylamide
  • the radical initiator is ammonium persulfate
  • the radical stabilizer is N,N,N’,N’-tetramethylethylenediamine.
  • the radical initiator in the aqueous reaction mixture can be any radical initiator that can initiate free radicals for polymerization of the monomer. Examples of radical initiators include, but are not limited to, ammonium persulfate (such as when the monomer is an acrylamide or diacrylamide), azoisobutylnitrile, benzoylperoxide, or benzoin.
  • the aqueous reaction mixture can further comprise a radical stabilizer.
  • the radical stabilizer can be any radical stabilizer that can stabilize the free radicals for polymerization of the monomer.
  • radical stabilizers include, but are not limited to, N,N,N’,N’- tetramethylethylenediamine (such as when the monomer is an acrylamide or diacrylamide).
  • the reaction mixture can comprise from about 10% to about 50% total acrylamide and from about 1% to about 10% bisacrylamide (as a percentage of total acrylamide), such as about 30% total acrylamide and about 5% bisacrylamide (as a percentage of total acrylamide).
  • the reaction mixture can comprise about 10% PEG diacrylate, with appropriate amounts of ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) (such as about 0.1% by mass).
  • APS ammonium persulfate
  • TEMED N,N,N',N'-tetramethylethylenediamine
  • the aqueous reaction mixture is allowed to cure by an appropriate curing method depending on the components of the mixture.
  • An example of a curing method is free-radical polymerization.
  • Transferring the molecular layer to the surface of a support material can be performed concurrently or sequentially with curing the support material/hydrogel.
  • an aqueous reaction mixture can be applied on top of the molecular layer which is positioned over and/or adjacent to an uncured hydrogel.
  • the aqueous reaction mixture can comprise a monomer, a crosslinker, and a radical initiator such that the aqueous reaction can undergo free radical polymerization to form the hydrogel or otherwise crosslink and/or bond the molecular layer therewith.
  • the hydrogel can alternatively be cured prior to the transfer step.
  • a layer of crosslinker can be applied to the surface of the hydrogel to which the molecular layer is to be transferred such that such excess crosslinker facilitates the covalent bonding therewith.
  • the hydrogel can be formulated to comprise available reaction sites on the targeted surface such that the molecular layer can covalently bond therewith.
  • the hydrogel to the surface of which has been transferred by covalent bonding the patterned layer of polymerized amphiphiles, is exfoliated from the first substrate by any suitable method known in the art.
  • An example of an exfoliating method is gentle insertion of a sharp blade between the hydrogel and the first substrate at the edge of the first substrate. Exfoliation can be assisted through the introduction of water or other polar solvent that wets the hydrogel.
  • the hydrogel can be homopolymeric, copolymeric, or a multipolymer IPN, which is made of two independent cross-linked synthetic and/or natural polymer components contained in a network form.
  • the hydrogel can be amorphous, semicrystalline, or crystalline.
  • the hydrogel can be a matrix, a film or a microsphere.
  • the hydrogel can be nonionic, ionic (e.g., anionic or cationic), ampholytic (i.e., containing both acidic and basic groups), or zwitterionic (i.e., containing anionic and cationic groups in each structural repeating unit).
  • the hydrogel can have any suitable pore diameter, such as from about 1 nm to about 200 nm.
  • most hydrogels have a heterogeneous structure with pores of various sizes.
  • a hydrogel can have both small pores (e.g., about 1 to about 10 nm) and large pores (e.g., about 20 to about 200 nm).
  • Transfer of the patterned layer of polymerized amphiphiles can primarily occur in areas of the hydrogel with pore sizes below about 100 nm. For hydrogels made from collagen or gelatin, pore diameters may be larger, e.g., up to 5 ⁇ m.
  • the method can further comprise applying a second substrate, which can be the same as or different from the first substrate, on top of the aqueous reaction mixture.
  • the second substrate can comprise on its surface, which faces the aqueous reaction mixture, a layer of reactive functional groups, e.g., one or more alkenes, or a patterned layer of polymerized amphiphiles, which can be the same as or different from the patterned layer of polymerized amphiphiles of the first substrate.
  • reactive functional groups include, without limitation, a peptide (such as a peptide comprising the amino acid sequence Arg-Gly-Asp (RGD) or a fragment or functional analog thereof), a substrate of an enzyme, a nucleic acid, a lipid, and/or a component of an ECM (such as one encoded by a matrisome).
  • the one or more functional head groups comprises a CHO
  • the reactive functional group(s) comprises a tripeptide sequence of RGD (e.g., cyclic RGD) or a fragment or functional analog thereof, a matrisome component, or a combination of any of the foregoing.
  • RGD e.g., cyclic RGD
  • the hydrogel can also be covalently bonded to the second substrate.
  • An example of a different substrate is glass (e.g., vinyl- functionalized glass).
  • the first and second substrates comprise the same material selected from the group consisting of HOPG, graphene, and a layered material comprising MoS2 or WS2.
  • the method can further comprise contacting the patterned layer of polymerized amphiphiles on the surface of the hydrogel with a thiol-functionalized molecule or an azide-functionalized molecule under conditions that promote a click reaction between a polydiacetylene and a thiol of the thiol- functionalized molecule or between a polydiacetylene and an azide of the azide-functionalized molecule.
  • the thiol-functionalized or azide-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule is cRGD.
  • Methods are also provided for adsorbing a biomolecule in a sample.
  • the method can comprise contacting the sample with a hydrogel hereof (e.g., a polyacrylamide hydrogel), on the surface of which is a patterned layer of polymerized amphiphiles comprising a functional head group under adsorptive conditions, whereupon the biomolecule in the sample is adsorbed onto the surface of the hydrogel.
  • a hydrogel hereof e.g., a polyacrylamide hydrogel
  • the biomolecule can be DNA, for example.
  • the patterned layer of polymerized amphiphiles can be any of the embodiments of the same described or otherwise contemplated herein.
  • the amphiphile comprises an amine functional head group.
  • the amphiphile is TCD-NH 2 or PCD-NH 2
  • Methods of adsorbing a biomolecule in a sample e.g., a biological sample from a subject
  • a method of adsorbing a biomolecule in a sample can comprise, for example, contacting, under adsorptive conditions, a sample with a surface of any of the scaffolds hereof, whereupon the biomolecule in the sample is adsorbed onto the molecular layer of the scaffold. It will be appreciated that such applications can be used to detect DNA in a sample, for example.
  • the support material of the scaffold can be a polyacrylamide hydrogel.
  • the amphiphile comprises an amine functional head group.
  • the amphiphile can be TCD-NH 2 or PCD-NH 2 .
  • the biomolecule can be DNA.
  • Methods of adhering cells to a polymeric material comprises contacting cells with a polymeric material comprising a scaffold hereof (or, for example, a patterned layer of polymerized amphiphiles and one or more alkyl chains comprising a polymerizable DA group on a surface thereof, each of the polymerized amphiphiles comprising one or more functional head groups covalently bonded to the one or more alkyl chains and wherein: the one or more functional head groups are organized in a pattern comprising a width of about 1 nm at a periodicity of about 5 nm to about 10 nm, and positioned in an orientation capable of binding a target, and the DA groups are covalently bonded to an azide-functionalized or thiol-functionalized molecule in the absence of copper).
  • the exposed polymer backbone of the molecular layer can comprise polydiacetylene covalently bonded to a thiol-functionalized and/or an azide- functionalized molecule in the absence of copper (i.e., the molecular layer can be secondarily functionalized).
  • the azide-functionalized or thiol-functionalized molecule can be a cell adhesion molecule.
  • the cell adhesion molecule is a cRGD.
  • the cells can be a mixture of cells. The cells can form a tissue.
  • connection or link between two components Words such as attached, linked, coupled, connected, and similar terms with their inflectional morphemes are used interchangeably, unless the difference is noted or made otherwise clear from the context. These words and expressions do not necessarily signify direct connections but include connections through mediate components. It should be noted that a connection between two components does not necessarily mean a direct, unimpeded connection, as a variety of other components may reside between the two components of note.
  • connection does not necessarily mean a direct, unimpeded connection unless otherwise noted.
  • the disclosure is presented in this manner merely for explanatory purposes and the principles and embodiments described herein may be applied to monolayers and materials that have configurations other than as specifically described herein. Indeed, it is expressly contemplated that the components of the monolayers and materials hereof be tailored in furtherance of the desired application thereof (e.g., for use with implants, regenerative medicine, chromatography studies, etc.).
  • a compound/composition is substituted with “an” alkyl or aryl
  • the compound/composition is optionally substituted with at least one alkyl and/or at least one aryl.
  • ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included.
  • the term “about,” when referring to a number or a numerical value or range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., +/- 5 % to 15% of the recited value) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • Example 1 Materials and general methods
  • 10,12-tricosadiynoic acid (10,12-TCDA, 97 % purity) was purchased from GFS Chemicals (Powell, OH), dissolved in chloroform, and filtered using a 13-mm syringe filter with a Polytetrafluoroethylene (PTFE) membrane and 0.2- ⁇ m pores (VWR, Radnor, PA).
  • PTFE Polytetrafluoroethylene
  • 1,2-Bis(10,12- tricosadiynoyl)-sn- glycero-3-phosphoethanolamine was purchased from Avanti Polar Lipids (Alabaster, AL) and used as received.
  • Acrylamide Aam, ⁇ 99.9% purity
  • bisacrylamide Bis
  • ammonium persulfate APS
  • TEMED Bio- Rad Laboratories
  • Chloroform ( ⁇ 99.5% purity), ethidium bromide (EtBr, 10 mg/mL in H 2 O), 3- (trimethoxysilyl)propyl methacrylate (TMS-PMA), deoxyribonucleic acid sodium salt from salmon testes (dsDNA, 2000 bp), and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received.
  • EtBr ethidium bromide
  • TMS-PMA 3- (trimethoxysilyl)propyl methacrylate
  • dsDNA deoxyribonucleic acid sodium salt from salmon testes
  • PBS phosphate-buffered saline
  • Oxalyl chloride ( ⁇ 99% purity), lithium aluminum hydride (LiAlH4, 95% purity), anhydrous dichloromethane (DCM, ⁇ 99.8% purity), anhydrous diethyl ether ( ⁇ 99.7% purity), and ammonium hydroxide solution (28-30%, NH 3 basis) were also all purchased from Millipore Sigma (St. Louis, MO) and used as received.
  • Isopropanol (IPA, ⁇ 99.5% purity) and benzyl azide (Bn-N 3 , ⁇ 94% purity
  • Fisher Scientific Hampton, NH
  • Ethanol 200 Proof was purchased from Decon Laboratories, Inc. (King of Prussia, PA).
  • Anhydrous sodium sulfate (Na 2 SO 4 , ⁇ 99.0% purity), N,N-dimethylformamide (DMF, ⁇ 99.8% purity), sodium dodecyl sulfate (SDS), and sodium hydroxide were all purchased from Fisher Scientific (Hampton, NH) and used as received.
  • 10,12-tricosadiynoic acid (TCD-COOH, 97% purity) was purchased from GFS Chemicals (Powell, OH), dissolved in chloroform, and filtered using a 13-mm syringe filter with a PTFE membrane and 0.2 ⁇ m pores (VWR, Radnor, PA).
  • PELCO conductive liquid silver paint, standard SEM pin stub mounts, AFM specimen discs (alloy 430), and double-coated carbon conductive tape were purchased from Ted Pella (Redding, CA).
  • Cyclo[Arg-Gly-Asp-D-PheLys(Azide)] (cRGD) was purchased from Vivtide LLC (Gardner, MA). Milli-Q water ( ⁇ 18.2 M ⁇ ⁇ cm resistivity) was used whenever water was required in an experiment. Ultrahigh purity nitrogen (UHP N2, 99.999% purity) was purchased from Indiana Oxygen Company (Indianapolis, IN). Embedding medium for cryotomy was purchased from Sakura Finetek (Torrence, CA). [0239] Methods Generally.
  • the stage was utilized in conjunction with a MicroTrough XL Langmuir ⁇ Blodgett trough (Kibron Inc., Helsinki, Finland).
  • HOPG substrates were mounted on standard 12-mm diameter stainless-steel AFM specimen discs and positioned on a magnet recessed in the temperature- controlled stage.
  • Conductive carbon tape was used to affix the HOPG to the specimen disk surface to ensure temperature uniformity across the substrate surface. The temperature of the substrate was measured using a thermocouple prior to dipping.
  • MMA mean molecular area
  • HOPG substrates were freshly cleaved, mounted on the thermally controlled stage, heated to 70 °C for full coverage monolayers, then lowered (at 2 mm/min) into contact with the subphase. Contact was maintained for 2 minutes, and the HOPG was then lifted out of contact at a rate of 2 mm/min.
  • dPE films were created at the air-water interface by depositing 40 ⁇ L of 0.6 mg/mL chloroform solution of dPE on a subphase of Milli-Q water maintained at 30 oC. Following deposition, 30 minutes was allowed for the chloroform to evaporate. Trough barriers were then moved inward at 6.23 mm/min until a target surface pressure of 30 mN/m was achieved.
  • HOPG substrates were freshly cleaved, mounted on a the thermally controlled stage, and heated to 70 oC, then lowered (at 6 mm/min) into contact with the subphase. Contact was maintained for 2 minutes. The HOPG was then lifted out of contact at 2 mm/min.
  • LS conversion was performed using a MicroTrough XL Langmuir ⁇ Blodgett trough (Kibron Inc., Helsinki, Finland).
  • the film was assembled at the air–water interface by depositing chloroform solution of TCDA (0.75 mg/mL, 34 ⁇ L) or dPE (0.6 mg/mL, 41 ⁇ L) on a subphase of water ( ⁇ 18 M ⁇ cm) at 30 °C.
  • the chloroform was allowed to evaporate for 15 minutes before the trough barriers were slowly moved inward (6 mm/min barrier motion) until the target MMA (35 ⁇ 2 /molecule for TCDA) or target pressure (30 mN/m) was achieved.
  • a freshly cleaved HOPG substrate was mounted on an automated dipper attachment, with the HOPG oriented nearly parallel to the air–water interface (cleaved side facing down), then lowered horizontally at a constant rate of 6 mm/min.
  • the temperature of substrates was held at 45 °C for TCDA and 70 °C for dPE monolayer. After 2 minutes of contact with the interface, the HOPG was gently lifted out of contact with the liquid at the same speed. After contact with the interface was broken, the substrate was blown dry with UHP N 2 . [0248] AFM imaging.
  • Relative coverage was estimated by digital image analysis with ImageJ after scale normalization for quantification of 40 x fluorescence micrographs. A representative area of 20 ⁇ m 2 was identified for region of interest (ROI) analysis of pixel intensity values. For each fluorescence data point graphed, 3 samples were prepared and 6 images were analyzed per sample. Pixel intensity values were averaged, and corresponding standard deviations were determined and reported for the data point tabulated. [0250] SEM imaging. SEM imaging under high magnification was performed using a Teneo VS SEM (FEI Company, Hillsboro, Oregon) at a working distance of about 7 mm, using the segmented in-lens T3 secondary electron (SE) detector.
  • SE secondary electron
  • An image acquisition beam current of 0.4 nA was typically utilized with a 32- ⁇ m diameter aperature at an accelerating voltage of 5 kV. Substrates were mounted with conductive carbon tape to standard SEM pin stub specimen mounts. A small amount of colloidal silver paint (PELCO ® , Ted Pella, Inc.) was applied along the perimeter of the HOPG to enhance substrate-mount conductivity. All images were collected with a 1536 x 1024 pixel resolution and a 30- ⁇ s dwell time, unless otherwise specified. [0251] Molecular modeling. Software packages Maestro and Macromodel (Schrödinger, Cambridge, MA) were used, respectively, to visualize molecular structures and to perform force field minimizations.
  • a SYLGARD 184 silicone elastomer base and curing (crosslinking) agents were combined at a 10:1 m/m ratio and thoroughly mixed.
  • a 40- ⁇ m square Nickel mesh was cut into 1 cm 2 pieces and affixed, using double-sided tape, onto a glass Petri dish.
  • the PDMS mixture was poured over the Nickel mesh, then deaerated in a vacuum desiccator until no bubbles remained.
  • the PDMS was cured overnight at 60 oC; the underlying Nickel mesh pieces were then gently peeled from the PDMS, generating a micropatterned surface.
  • Micropatterned stamps were cleaned by sonication in a 1:1:1 (v/v/v) mixture of ethanol, methanol, and Milli-Q water for 1 hour, and subsequently placed in an oven for an additional 1 hour at 60 oC to allow residual polar solvent mixture to evaporate.
  • the micropatterned stamps were then soaked in hexanes for 6 hours, replacing the solvent with fresh hexanes every 2 hours. Finally, the micropatterned stamps were dried overnight at oC and stored covered in a petri dish, pattern side up, prior to use.
  • Microcontact printing ( ⁇ CP) of dPE Microcontact printing ( ⁇ CP) of dPE.
  • microcontact printed HOPG substrate was then exposed to UV (to induce diyne photopolymerization) in a UVP CL-1000 Ultraviolet crosslinker device (254 nm, 8 W) for 1 hour with about 10 cm between the lamp and substrate surface.
  • UVP CL-1000 Ultraviolet crosslinker device (254 nm, 8 W) for 1 hour with about 10 cm between the lamp and substrate surface.
  • Covalent transfer of PDA striped phases from HOPG to PAAm [0255] Preparation of vinyl functionalized coverslips. Surfaces were grafted with 3- (trimethoxysilyl)propyl methacrylate (TMS-PMA) using the procedures described in Yuk et al., Tough bonding of polymeric materials to diverse non-porous surfaces, Natural Materials 15: 190-196 (2016).
  • the glass coverslips were submerged in 40 mL or 100 mL of silane solution (100 mL water, 10 ⁇ L acetic acid (used to adjust the solution pH to 3.5), and 2 wt % TMS-PMA) and incubated for 2 hours or overnight (ca. 12 hours), respectively, at room temperature. Finally, the substrates were thoroughly rinsed with ethanol and water, in triplicates, then dried and stored in a low-humidity environment prior to use. [0257] Preparation of polymeric material precursor solution.
  • PAAm polyacrylamide
  • the polyacrylamide (PAAm) monomer mixture was prepared by following the protocol reported in Denisin & Pruitt, Tuning the range of polyacrylamide gel stiffness for mechanobiology applications, ACS Applied Materials & Interfaces 8(34): 21893-21902 (2016).
  • PAAm polymeric materials hydrogels
  • Tse & Engler Preparation of polymeric material substrates with tunable mechanical properties, Current Protocols in Cell Biology 47: 10.16.11-10.16.11 (2010).
  • acrylamide (AAm) and bis- acrylamide (Bis) were dissolved together in 5 mL of Milli-Q water, using compositions described herein based on the total concentration of AAm/Bis (w/v) in the gel mixture (%T), and based on the crosslinker-to-monomer ratio (%C): [0258] Solutions were ultrasonicated for at least 15 minutes (or until all the solid was dissolved) and then sealed with parafilm and stored at room temperature.
  • acrylamide (Aam) (3.910 g) and bisacrylamide (Bis) (0.205 g) were added to water (13.7 mL). Precursor solution was ultrasonicated until all the solid was dissolved.
  • Preparation of HOPG substrate for polymeric material gelation For PAAm curing and sPDA monolayer exfoliation, the monolayer (e.g., sPDA)/HOPG samples were first covered with rubber molds 2 cm in diameter and 2 mm thick (Ted Pella, Inc.) with an 0.5 cm ⁇ 0.5 cm square cut to fit the HOPG area.
  • Double-sided clear tape (3M Scotch) was used to create a temporary seal between the HOPG and the rubber mold, preventing polymeric material precursor solution from leaking.
  • APS ammonium persulfate
  • C2C12 cell culture C2C12 cells were passaged twice before substrate exposure. Substrates were seeded with 6,000 cells for 24 hours. Cells were then fixed with paraformaldehyde and blocked with 1 % BSA. Phalloidin staining was carried out using 2 ⁇ L of 4x Alexa fluor 568 (A568) in 800 ⁇ L 0.1 % BSA.
  • C2C12 cells were cultured in Advanced DMEM containing 10% FBS, 1X penicillin/streptomycin, and 1% glutagro supplement at 37 oC in 5% CO 2 . Cells were seeded on sterilized PDMS at 2.5 x 10 4 cells/mL and grown to 70% confluence before switching to a 2% horse serum differentiation mdedia.
  • the ratio of dsDNA sodium salt from salmon testes (2000 base pairs long, ⁇ 680 nm) to Eb was adjusted to produce a ratio of ca. 20 Eb molecules/DNA strand.
  • Eb dsDNA sodium salt from salmon testes
  • the ratio of dsDNA sodium salt from salmon testes (2000 base pairs long, ⁇ 680 nm) to Eb was adjusted to produce a ratio of ca. 20 Eb molecules/DNA strand.
  • 2 mL The ratio was sonicated for 1 hour, mixed thoroughly by vortex for at least 5 minutes, and allowed to further equilibrate overnight. This solution was stored at -20 oC when not in use.
  • Eb-intercalation in dsDNA The components were weighted together and diluted ot the corresponding concentration with Milli-Q water, and further sonicated for 1 hour.
  • a UMWB2 filter cube was utilized (460-490 nm excitation band-pass filter, dichroic filter wavelength of 500 nm, long-pass emission filter wavelength of 520 nm).
  • the dwell times for imaging were kept at 1 second for 10x and 40x images, and 4 seconds for 100x images, with 3 accumulations taken under integration capturing mode.
  • Image resolution was 1024 x 1024 pixels, unless otherwise specified.
  • Micrographs and corresponding spectra were obtained at a resolution of 512 x 512 pixels, a bit depth of 8 bits, a dwell time of 16.38 ⁇ s/pixel (using unidirectional scanning and averaging 16 times per line), and a pinhole set to 1 Airy unit (A.U.).
  • 16 of the 32 channels were used to collect data, with bins centered at values from 531-664 and a resolution (bin width) of 8.9 nm.
  • FIG. 2B-2D, 3B-3F, and 4B-4D show AFM and SEM images illustrating the lamellar and domain structure for stiped phase polydiacetylene (PDA) films.
  • FIGs. 3E and 3F show larger AFM and SEM images, respectively, illustrating nanoscale and microscale molecular ordering achieved using thermally regulated Langmuir-Schaefer conversion.
  • Fig. 3E AFM images of TCD-NH 2 exhibit striped phase domains with edge lengths > 100 nm. Also visible in the images are long linear features generally crossing the entire image from left to right, which correspond to HOPG step edges (an example is labeled in Fig. 3E).
  • Fig. 3E show AFM and SEM images illustrating the lamellar and domain structure for stiped phase polydiacetylene (PDA) films.
  • Figs. 3E and 3F show larger AFM and SEM images, respectively, illustrating nanoscale and microscale molecular ordering achieved using thermally
  • SEM images provide microscale information on assembled TCD-NH 2 striped phase domain morphology on HOPG.
  • Long linear defects evolve in ordered monolayers during polymerization due to conformational changes when the diacetylene rehybridizes to form the polydiacetylene; these defects are emphasized in the SEM electron beam.
  • Linear defect orientation can be used to infer molecular row orientation.
  • Example 2 Synthesis of amphiphiles [0275] Synthesis of 4,6-TCD-COOH. Synthesis was carried out using a modification of the procedures set forth in Mowery & Evans, The synthesis of conjugated diacetylene monomers for the fabrication of polymerized monolayer assemblies, Tetrahedron Letters 38(1): 11-14 (1997).
  • the reaction was allowed to warm to room temperature and stirred overnight. If the solution turned blue, additional aliquots of hydroxylamine hydrochloride were added to the reaction mixture.
  • the reaction was quenched with an aqueous solution of 30% sulfuric acid.
  • the solution was extracted with diethyl ether (3 ⁇ 15 mL) and washed with water (3 ⁇ 15 mL) and brine (3 ⁇ 15 mL), respectively.
  • the organic layer was dried over anhydrous Na 2 SO 4 .
  • the solvent was removed under reduced pressure.
  • the crude 4,6-tricosadiynoic acid (4,6-TCDCOOH) was recrystallized from heptane.
  • 10,12-TCD-GlcA was prepared by modification of the procedures set forth in Pitt et al., Synthesis of a glucuronic acid and glucose conjugate library and evaluation of effects on endothelial cell growth, Carbohydrate Research 339(11): 1873-1887 (2004) and Tosin & Murphy, Synthesis of alpha-glucuronic acid and amide derivatives in the presence of a participating 2-acyl protection group, Organic Letters 4921): 3675-3678 (2002).
  • D- glucuronic acid (GlcA, 1 equiv, 7.7 mmol) was added to acetic anhydride (23 mL), and the resulting suspension was stirred at 0 °C for 10 minutes.
  • Triphenylphosphine (1.3 equiv, 1.7 mmol) was dissolved in anhydrous DCM (5 mL), and the resulting solution was added to the reaction mixture dropwise. The reaction was stirred at room temperature for 15 hours. Subsequently, the mixture was diluted with CHCl 3 (20 mL), washed once with a saturated aqueous solution of NaHCO 3 (10 mL) and then with water (10 mL) until neutral pH was achieved. After drying over Na2SO4, the solution was filtered, then evaporated to dryness, to obtain 10,12-OMe-TCD-GlcA. [0280] A solution of 0.05 M lithium hydroxide was prepared in 2.5:1:0.5 methanol–water–THF.
  • 10,12-OMe-TCD-GlcA (1 equiv, 0.3 mmol) was dissolved in 0.05 M lithium hydroxide solution (6 equiv.), and the solution was stirred at 0 °C for 3 hours. The mixture was diluted with water and pH was adjusted to 3 with Amberlite ® IR 120 H. The Amberlite was removed by filtration and the solvent was removed under reduced pressure. The resulting aqueous solution was freeze dried to yield 10,12-TCD-GlcA as white solid.
  • Scheme 3 Synthesis of 10,12-TCD-GlcA [0281] Synthesis of 10,12-TCD-OH.
  • 10,12-pentacosadynamine (PCD- NH 2 ) and 10,12-tricosadiynamine (TCD-NH 2 ) were both prepared from the structurally analogous 10,12-diynoic acid (i.e., 10,12-pentacosadiynoic acid (PCD-COOH) and 10,12-tricosadiynoic acid (TCD-COOH) using a modification of the procedures reported in Howarth et al., Lipophilic peptide nucleic acids containing a 1,3-diyne function: synthesis, characterization and production of derived polydiacetylene liposomes, Tetrahedron 61: 8875-8887 (2005) and Lee et al., A reversibly mechanochromic conjugated polymer, Chemistry Communications 55: 9395-9398 (2019).
  • PCD-COOH 10,12-pentacosadiynoic acid
  • TCD-COOH 10,12-
  • 10,12-diynoic acid (1 eq) e.g., TCD-COOH or PCD-COOH, respectively
  • aqueous ammonium hydroxide 1.3 eq
  • 10,12-diynoyl chloride (1 eq) was dissolved in THF and the resulting solution was added to the ammonium hydroxide solution at 0 oC. The reaction mixture was stirred at room temperature for 6 hours. The product was extracted with DCM (3 x 50 mL) and the combined organic extract was dried over anhydrous Na2SO4. The DCM was evaporated under reduced pressure to yield 10,12- tricosadiynoyl amide as a white solid. 10,12-diynoyl amide (1 eq) was placed in a round-bottom flask. Anhydrous diethyl ether was added to the flask under N2 atmosphere, yielding a white suspension.
  • TCD-NH 2 was dissolved in chloroform and filtered using 13-mm syringe filters with PTFE membranes and 0.2- ⁇ m pores (VWR, Radnor, PA) prior to use.
  • VWR, Radnor, PA 13-mm syringe filters with PTFE membranes and 0.2- ⁇ m pores
  • sPDA striped polydiacetylene
  • Figs. 1A-1B and 6A-6D were prepared via L-S conversion using a custom-bult temperature-controlled transfer stage. Davis et al. (2016), supra; Bang et al. (2016), supra; and Hayes et al. (2017), supra. The stage was utilized in conjunction with a MicroTrough XL Langmuir-Blodgett trough (Kibron Inc., Helsink, Finland).
  • L-S conversion begins with assembly of a standing phase Langmuir film of amphiphiles on an aqueous subphase.
  • Freshly cleaved HOPG substrates were mounted on standard 12-mm diameter stainless steel atomic force microscopy (AFM) specimen discs and positioned on a magnet recessed in the temperature-controlled stage.
  • Conductive carbon tape was used to affix the HOPG to the specimen disk surface to ensure temperature uniformity across the substrate surface. The temperature of the substrate was measured using a thermocouple prior to dipping.
  • Films of each amphiphile were assembled at the air-water interface by depositing the appropriate amount of a 0.75 mg/mL amphiphile solution in chloroform, on a subphase comprised of 40 mM CaCl2 in Milli-Q water, maintained at 30 oC. Barriers were then swept inward at 6.23 mm/min to achieve the target MMA; the trough feedback mechanism was set to maintain the target MMA.
  • HOPG substrates were freshly cleaved, mounted on the thermally controlled stage, set at either 25 oC for transfer of monolayers containing circular vacancies, or 70 oC for full coverage monolayers, then lowered (at 2 mm/min) into contact with the standing phase monolayer (i.e., L-S film) and a subset of molecules on the L-S film, causing some molecules within the film to transfer to the HOPG surface and convert from the standing arrangement on the subphase, to a lying-down orientation on the HOPG, with their alkyl chains parallel to the substrate. Contact was maintained for 2 minutes, and the HOPG was then lifted out of contact at a rate of 2 mm/min.
  • the standing phase monolayer i.e., L-S film
  • HOPG substrates were blown dry with UHP N 2 .
  • the HOPG substrates were then placed under a hand-held ultraviolet (UV) lamp (254 nm, 8 W) for 1 hour with about 10 cm between the lamp and the substrate to induce diyne photopolymerization.
  • UV hand-held ultraviolet
  • Test Set 1 Amphiphile solution volume and target MMA for L-S transfer of each amphiphile.
  • sPDAs assembled from TCD-NH 2 (Fig. 6A, structure at top) were utilized. Molecules assembled head-to-head, generating lines of functional head groups about 1 nm in width.
  • Energy-minimized models (Fig. 6A, bottom) illustrate an edge-to-edge lamellar width of 5.3 mm (Fig. 6A, right) for the polymerized system, in good agreement with the 5.6 nm periodicity observed by AFM in Fig. 6B.
  • SEM scanning electron microscopy
  • amphiphiles comprised PCD-NH 2
  • the amphiphiles lying on HOPG ordered into a repeating striped (lamellar) pattern, which generated 1-nm-wide stripes of functional head groups embedded within the layer of exposed alkyl chains, with a periodicity of about 6.5 nm
  • Irradiation with UV light induced topochemical polymerization of the ordered diacetylene (DA) moieties Fig. 11B. Both striped pattern and polymerization were visible at nanoscopic scales in AFM images (Figs. 11C and 11D) and at larger scales using scanning electron microscopy (SEM) (Fig. 11E).
  • Patterns of GlcNAc for controlled multivalent binding were also prepared by generating monolayers of TCD-GlcNAc on HOPG (Fig. 3A) through L-S conversion. Briefly, the molecules were ordered as a standing phase Langmuir film on an aqueous subphase, and a heated HOPG substrate was then lowered horizontally into contact with the Langmuir film causing a subset of the molecules to reorder from the standing phase into a striped lamellar phase. This arrangement formed about 1.7 nm wide stripes of GlcNAc comprising two opposing rows of GlcNAc, with alky chains on both edges, ca. 7 nm in width (Figs. 2A and 2B).
  • HOPG substrates were freshly cleaved, mounted on a custom-built, thermally controlled dipping apparatus (Hayes et al. (2017), supra) with the HOPG oriented nearly parallel to the air- water interface (cleaved side facing down), then lowered horizontally at a constant rate of 6 mm/min. The temperature of substrates was held at 65 °C. After 2 minutes of contact with the interface, the HOPG was gently lifted out of contact with the liquid at the same speed. After contact with the interface was broken, the substrates were blown dry with UHP N2.
  • TCD-GlcNAc monolayers were prepared under L-S conditions that induced formation of microscopic vacancies. The procedure was significantly similar to those the present inventors demonstrated for other diyne amphiphiles. Davis et al., Hierarchically patterned noncovalent functionalization of 2D materials by controlled Langmuir-Schaefer conversion, Langmuir 34: 1353-1362 (2018). Briefly, to induce formation of circular vacancies in the monolayer, the dipper speed and substrate temperature were decreased to 2 mm/min and 25 °C, respectively.
  • PAAm was functionalized with amphiphiles having head groups representing the individual functional groups common in glycans: a hydroxyl group (TCD-OH) and a carboxylic acid (TCD-COOH or TCDA). Additionally, PAAm substrates were functionalized with sPDAs generated from a glucuronic acid (Glc) amphiphile (TCD-GlcA), with a carbohydrate head group that was structurally similar to the GlcNAc, but not a target for WGA binding. [0299] sPDA-functionalized PAAm surfaces were exposed to WGA (5 ⁇ g/mL).
  • Fluorescence emission intensity from TCDGlcNAc monolayers was substantially lower than that for TCDOH, TCDGlcA and TCDA monolayers.
  • the interfacial concentration of GlcNAc on PAAm was lower in comparison to other functional groups, by up to a factor of 10 in comparison with TCDA, and about five times in comparison with TCDOH and TCDGlcA.
  • maximum emission intensity (I584) of WGA-treated monolayers was divided by emission intensity at the PDA emission peak (I584).
  • Fig. 4K shows WGA binding normalized for surface coverage using this approach.
  • Example 5 Measurement of surface dissociation constant (KD) [0304]
  • KD surface dissociation constant
  • proximal chain length was evaluated with different proximal chain lengths: 4,6-TCD- GlcNAc (3-carbon proximal chain) and 10,12-TCD-GlcNAc (9-carbon proximal chain).
  • Minimized molecular models of polymerized 4,6-TCD-GlcNAc and 10,12-TCD-GlcNAc monolayers on HOPG illustrate the potential for restrictions on the trajectories of GlcNAc head groups based on the position of the PDA (highlighted in white).
  • the distance from the end of the GlcNAc to the PDA was about 1.0 nm for 4,6-GlcNAc, increasing to about 2.0 nm for the 10,12-TCD- GlcNAc.
  • sequential 1 ns molecular dynamics runs were performed in explicit water, with the HOPG substrate removed form the model and the PDA backbone frozen (Fig. 5B). This set of constraints was intended to mimic the periodic covalent linkage of the relatively stiff PDA (persistence length about 16 nm) to the PAAm mesh, while allowing for the motion of pendant alkyl chain segments and head groups.
  • TCD-GlcNAc/PAAm surfaces were exposed to increasing concentrations of rWGA, maintaining the surface density of GlcNAc in the sPDA constant.
  • the magnitude of emission intensity depends on the active interaction between GlcNAc and rWGA.
  • Polyacrylamide is generated through a free-radical polymerization of an acrylamide (AAm) monomer, bis(acrylamide) (Bis) crosslinker, ammonium persulfate (APS) radical initiator, and TEMED radical stabilizer.
  • AAm acrylamide
  • Bis(acrylamide) (Bis) crosslinker bis(acrylamide) (Bis) crosslinker
  • APS ammonium persulfate
  • TEMED radical stabilizer TEMED radical stabilizer.
  • the surface-templated glycopolymers were covalently transferred to PAAm using a covalent transfer strategy in which PAAm is cured in contact with the carbohydrate sPDA on HOPG. This process is effective in transferring sPDAs with simple carboxylic acid or amine head groups to PDMS or to PAAm. Davis et al. (2021), supra. Here, this strategy was leveraged to generate much higher-complexity functional patterns to generate selective multivalent binding on the amorphous hydrogel surface.
  • acrylamide (AAm) (8 % w/w) and bis-acrylamide (Bis) (2% w/w) were dissolved together in 5 mL of Milli-Q water, using compositions described herein based on the total concentration of AAm/Bis (w/v) in the gel mixture (%T), and based on the crosslinker-to-monomer ratio (%C):
  • the precursor solution was degassed by bubbling with UHP N2 for five minutes prior to gelation. 10 % w/v solution of the APS initiator was prepared in Milli-Q water.
  • confocal spectroscopy (Fig. 3B) and microscopy (Fig. 3C) illustrated surface morphological features and spectral emission features characteristic of sPDA transfer.
  • sPDA monolayers were assembled under conditions that resulted in circular vacancies (similar to those visible in the SEM image of Fig. 2C). Following transfer, similar surface features were visible in fluorescence emission.
  • Previously, the present inventors have shown that sPDA fluorescence emission can be used to quantify the extent of sPDA transfer to PAAm. Figs.
  • FIG. 7B and 7C illustrate this process using both full sPDA monolayers and ssquare patterns of TCD-NH 2 sPDAs assembled on HOPG by microcontact printing ( ⁇ CP) and then transferred.
  • Fluorescence emission spectra acquired over areas with transferred sPDAs (Fig. 7C) can be fitted to a primary peak near 548 nm, and a set of longer-wavelength peaks, similar to those previously observed for sPDAs transferred to PDMS. Shi et al. (2022), supra.
  • PAAm exhibits a peak near 584 nm (Fig. 7D, PAAm trace).
  • ⁇ CP-TCD-NH 2 fluorescence intensities are lower.
  • GlcNAc is known to bind to WGA, while GlcA does not. GlcNAc and GlcA differ at two substituents on the pyranose ring (Fig. 2E, left): at C2, GlcNAc has a bulky acetamido (-NHAc) group, while GlcA has a smaller OH; at C6, GlcNAc has an OH group, and GlcA has a COOH group.
  • -NHAc bulky acetamido
  • TCD-GlcNAc forms smaller ordered molecular domains (typical domain sizes 200-300 nm, Fig. 2F, top right) on HOPG in comparison with TCD-GlcA (700-900 nm, Fig. 2F, bottom right) and other amphiphiles with simpler head groups.
  • confocal spectroscopy and microscopy illustrate surface morphological features and spectral emission features characteristic of sPDA transfer, including a primary emission peak centered at 548 nm and sidebands extending up to about 640 nm. While the spectral characteristics for TCD-GlcNAc emission were similar to those previously observed for other sPDAs with simpler headgroup chemistries, emission intensity is somewhat lower, pointing to the possibility of lower transfer efficiency.
  • TCD-GlcA transferred more efficiently, as evidenced based on greater emission intensity, which is constent with previously work in which the data supported that monolayer chemistries that produce longer sPDAs also resulted in more efficient transfer.
  • Example 7 Impact of polyacrylamide gel structure on sPDA transfer [0324] A series of experiments were carried out to examine transfer efficiency in relation to PAAm composition. Recently, the relationship between sPDA polymer length and the efficiency of transfer to PDMS was examined, parameterizing the per-unit reaction probability with the PDA, and the number of linkages to the PDA required to exfoliate it with the bulk PDMS. Shi et al. (2022), supra. The previous models were developed by comparing transfer efficiency for molecules with known populations of polymer lengths.
  • PAAm polymerization is different than PDMS crosslinking, in that each AAm or Bis monomer can react with the PDA.
  • PAAm gel structure is typically controlled by varying the percentage of reactive acrylate (%T) in the reaction mixture, and the percentage of the acrylate that is crosslinker (%C) pursuant to the formula described in Example 3 above.
  • %T percentage of reactive acrylate
  • %C crosslinker
  • Each sample was then mounted on a flat specimen holder with optimal cutting temperature (OCT) cryo embedding media.
  • OCT optimal cutting temperature
  • Mounted samples in the specimen holder were clamped to an ALT118 brass Gatan plunger (Gatan, Oxford, UK) and immersed in a liquid nitrogen slush under ambient pressure until equilibrated.
  • Samples were then cryo-transferred into the Gatan Alto 2500 preparation chamber, preset to -180 °C.
  • Cross-sectional samples were prepared using a vise clamp holder and a surgical scalpel to fracture the sample at the base. Subsequently, the sample was transferred onto the Nova SEM cryo stage, which was pre- cooled to -85 °C for sublimation of ice.
  • Electrons were collected using an Everhart Thornley Detector for low magnification images, and an immersion- mode through-the-lens detector for high magnification images.
  • Analysis of cryoEM images estimates surface pore diameters at about 50 nm for 30 and 40 %T gels, which produce higher degrees of transfer (Fig. 8C).
  • CryoEM images of unfunctionalized PAAm (Figs. 8D-8F) and TCD-NH 2 /PAAm (Figs. 8G-8I) illustrate smoothing of the gel surface associated with the transfer of the TCD-NH 2 sPDA monolayer, particularly for PAAm compositions that result in high degrees of sPDA transfer based on fluorescence emission.
  • PAAm network heterogeneities could result in local strong crosslinking to the PDA monolayer in some regions, and more limited crosslinking in others.
  • Visual inspection of fluorescence images of TCD-NH 2 /PAAm illustrate speckling patterns that vary with %T (Fig. 9B). Unfunctionalized PAAm produced much weaker fluorescence emission overall; however, speckling was visible for 40 and 50 %T gels (Fig. 9C), at scales similar to those observed for functionalized surfaces. These observations may be consistent with the interpretation that one source of heterogeneities in sPDA transfer is heterogeneities in local PAAm gel structure (Fig. 9D).
  • Fig. 9E fluorescence intensities were separately extracted for high- and low-intensity pixels
  • Fig. 9F Models of transfer developed previously for PDMS were used (Figs. 9A and 9G) to relate these differences in PDA emission to differences in transfer and to estimate differences in sPDA crosslinking to the PAAm network (Figs.9H and 9I).
  • the models of the present disclosure for sPDA crosslinking and exfoliation onto a bulk polymer do not require knowledge of the chemical nature of the bulk polymer.
  • the model relates the per-PDA-unit probability of reaction with the bulk polymer (prxn) and number of crosslinks required for transfer (n) to the percentage of the sPDA monolayer projected to transfer (P transfer , Fig.
  • Fig. 10A Two amine sPDAs, as well as amphiphiles with carboxylic acid and hydroxyl head groups, were tested (structures shown in Fig. 10A).
  • the model in Fig. 10B illustrates the relative scale of a double-stranded DNA (dsDNA) helix and the sPDA surface pattern.
  • dsDNA double-stranded DNA
  • the capacity of the surfaces to control DNA adsorption was examined using long dsDNA isolated from salmon sperm, with helix lengths ranging up to 2 million base pairs. DNA was first incubated with the intercalating fluorophore ethidium bromide (EtBr), to produce fluorescent labeling along the contour length of each helix.
  • EtBr intercalating fluorophore ethidium bromide
  • FIG. 10C shows ⁇ CP-TCD-NH 2 /PAAm before (left) and after (right) exposure to EtBr-DNA, illustrating the appearance of red fluorescence in the amine-patterned areas.
  • FIG. 10E shows emission from TCD-NH 2 /PAAm without (green) and with (red) exposure to EtBr-DNA, with the same vertical scale used for the bare PAAm graph in Fig. 10D.
  • EtBr-DNA binding to a set of sPDA-functionalized PAAm surfaces was tested to assess the role of functional group type and density on the surface.
  • amine- functionalized surfaces TCD-NH 2 and PCD-NH 2
  • Fig. 10F amine- functionalized surfaces
  • Fig. 10F amine- functionalized surfaces
  • Fig. 10F amine- functionalized surfaces
  • TCD-OH hydroxyl
  • TCD-COOH carboxylic acid
  • Example 9 Secondary functionalization of sPDAs with azides [0341] Surface functionalized with sPDAs were then exposed to azides (which can undergo a click reaction with an alkyne) to produce an sPDA/PAAm surface with secondary functionalities installed along the sPDA backbone as shown in Fig. 1C.
  • TCDA/PAAm was first reacted with cyclic arginine-glycine-aspartic acid (RGD; a cell adhesion peptide) azide (cRGD-N 3 ) and with Bn- N 3 , a structurally simpler azide which limits other surface interactions. Following exposure to each azide for 4 hours, there was an about 50% decrease in the PDA fluorescence emission near ⁇ max (Figs.12A-12C), consistent with extensive reaction with the azide.
  • RGD cyclic arginine-glycine-aspartic acid
  • Bn- N 3 a structurally simpler azide which limits other surface interactions.
  • amine and phosphoethanolamine (PE) sPDAs increase myoblast adhesion, while carboxylic acid sPDAs do not.
  • cells were cultured with bare PAAm (Fig. 13A), TCDA/PAAm (Fig. 13B), bare PAAm exposed to cRGD under the click conditions described above (Fig. 13C), and TCDA/PAAm+cRGD (Fig. 13D). Out of these four conditions, only TCDA/PAAm+cRGD resulted in significant C2C12 myoblast adhesion.

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Abstract

L'invention concerne des couches moléculaires fonctionnalisées comprenant un squelette d'amphiphiles polymérisés destinés à être placés sur une surface d'un échafaudage, tel qu'un hydrogel. L'invention concerne également des méthodes de préparation de telles couches moléculaires et des hydrogels les comprenant, ainsi que leurs méthodes d'utilisation.
PCT/US2023/066713 2022-05-05 2023-05-05 Couches moléculaires fonctionnalisées et échafaudages, leurs méthodes de préparation et d'utilisation WO2023215909A2 (fr)

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