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WO2009018484A1 - Compositions for delivery of biologically active agents to surfaces - Google Patents

Compositions for delivery of biologically active agents to surfaces Download PDF

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Publication number
WO2009018484A1
WO2009018484A1 PCT/US2008/071825 US2008071825W WO2009018484A1 WO 2009018484 A1 WO2009018484 A1 WO 2009018484A1 US 2008071825 W US2008071825 W US 2008071825W WO 2009018484 A1 WO2009018484 A1 WO 2009018484A1
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WO
WIPO (PCT)
Prior art keywords
binding
sbp
biological substrate
peptide
drug delivery
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2008/071825
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French (fr)
Inventor
Paul Theodore Hamilton
Benjamin Marcus Buehrer
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Affinergy Inc
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Affinergy Inc
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Filing date
Publication date
Application filed by Affinergy Inc filed Critical Affinergy Inc
Publication of WO2009018484A1 publication Critical patent/WO2009018484A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6955—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a plaster, a bandage, a dressing or a patch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6957—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a device or a kit, e.g. stents or microdevices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • compositions and methods for targeted delivery of biologically active agents to a surface of a non-biological substrate More particularly, the invention is directed to compositions comprising and methods of using surface-binding peptides to target a drug delivery vehicle, carrying biologically active agent, to one or more surfaces of a non-biological substrate, and preferably a non-biological substrate to be introduced, or introduced, into a biological environment.
  • Liposomes and microspheres have been used widely as carriers to deliver a variety of therapeutic agents. Encapsulation of the therapeutic agent into a drug carrier can act to provide controlled release of the therapeutic agent as the drug carrier dissolves or biodegrades in a biological environment (e.g., in vivo). However, the efficiency of the delivery of therapeutic agent to a desired cell population to be treated with therapeutic agent has been constrained by the lack of a means for targeting the drug carrier to cells, so that the therapeutic agent can be released in the vicinity of the target cell population to be treated. Peptides have been conjugated to lipids and incorporated into liposome bilayers to improve stability of the liposome (see, e.g., US Patent 6,339,069).
  • Peptides have also been used to target cells in delivering an agent.
  • a peptide having binding specificity for cancer cells has been labeled with a radiopharmaceutical, and used to target human breast cancer cells (see, e.g., Askoxylakis et al., Clin. Cancer Res., 2005, 1 1 :6705- 12); peptides having tumor-binding specificity have been used to direct therapeutic agents to tumor cells (see, e.g., Ruoslahati et al., Curr. Pharm. Des. 2005, 1 1 :3655-60; Lee et al., Cancer Res., 2004, 64:8002-08; Shaddi et al., FASEB J., 2003, 17:256-8).
  • drugs have been conjugated to cell- penetrating peptides to facilitate internalization of the conjugated drug by a cell.
  • Medical devices including implants and prostheses for medical or dental use
  • Medical devices are increasingly utilized for treatment of a variety of disease conditions and in preventative procedures.
  • Medical devices are often intended to provide beneficial functions for extended periods of time, ranging from weeks to years. The general trend is that the longer a device can be utilized without replacement or repair, the better for the individual (e.g., fewer invasive procedures, lower medical costs, etc.).
  • the presently disclosed subject matter provides a composition for coating a non- biological substrate, wherein the composition comprises a drug delivery vehicle (DV) coupled (directly or via a linker) to surface-binding peptide (SBP), and wherein the drug delivery vehicle is configured to carry biologically active agent (BA).
  • DV drug delivery vehicle
  • SBP surface-binding peptide
  • BA biologically active agent
  • the composition can be contacted with a surface of a non- biological substrate to coat the surface of the substrate such that the biologically active agent, carried by the drug delivery vehicle, is delivered and localized at the surface.
  • Surface-binding peptide is used to target the drug delivery vehicle carrying biologically active agent to a non-biological substrate desired to be targeted.
  • the composition can also comprise a pharmaceutically acceptable carrier.
  • the presently disclosed subject matter provides a composition for coating a non-biological substrate, wherein the composition comprises: a drug delivery vehicle (DV) carrying a biologically active agent (BA); a first surface-binding peptide (SBP-i) that binds non- covalently to the non-biological substrate; and, optionally, a second surface- binding peptide (SBP 2 ) having binding affinity to a surface of the drug delivery vehicle (DV).
  • the drug delivery vehicle (DV) can comprise a shell (e.g., outer layer) comprised of a synthetic component (e.g., one or more of a synthetic polymer, synthetic surfactant, synthetic lipid and the like that is not found in nature).
  • the first surface-binding peptide (SBP 1 ) binds has binding affinity for and binds non-covalently to the non-biological substrate.
  • a non-biological substrate can be a device, container, medical device, array, etc., to be coated for localization and retention of the drug delivery vehicle (DV) carrying the biologically active agent (BA).
  • the second surface-binding peptide (SBP 2 ) is coupled, covalently or non-covalently, to the synthetic component of the shell of the drug delivery vehicle.
  • a biologically active agent can be delivered at the surface of the non-biological substrate, wherein the first surface-binding peptide is coupled (either directly or via a linker) to the second surface-binding peptide.
  • the composition can also comprise a pharmaceutically acceptable carrier.
  • Another aspect of the presently disclosed subject matter provides for a delivery system for biologically active agent that is capable of facilitating the delivery, localization, and retention of biologically active agent to a non- biological substrate such as a medical device prior to positioning or once in situ.
  • SBP is, when present, a surface-binding peptide of 8 to 60 amino acids
  • SBP 1 is the SBP surface-binding peptide comprising a surface-binding peptide domain having binding affinity for a surface comprising a non-biological substrate
  • SBP 2 can be present or absent and is the SBP surface-binding peptide having binding affinity for a synthetic component of a drug delivery vehicle (DV)
  • L can be present or absent and comprises a linker
  • the DV comprises a drug delivery vehicle that carries a BA, wherein the BA is one or more biologically active agents; wherein in the presence of the SBP 2 , the DV is noncovalently coupled to the SBP 2 ; wherein in the absence of the SBP 2 and in the presence of the L, the SBPi is coupled to the DV either covalently or nocovalently; and wherein in the absence of both the SBP 2 and the L, the SBPi is coupled directly to the DV either covalently or noncovalently.
  • DV is a drug delivery vehicle that carries BA, wherein BA is one or more biologically active agents ("biologically active agent").
  • BA is one or more biologically active agents ("biologically active agent").
  • DV can carry BA by one or more processes selected from the group consisting of coupling BA and DV together (e.g., to, in, on, within, or a combination thereof), encapsulation of BA by DV, and a combination thereof.
  • Coupling can be by one or more of covalent and/or non-covalent attachment of BA and DV (the latter including but not limited to trapping, embedding and a combination thereof, and can be via one or more of hydrophobic and electrostatic interactions and can be via the structure of DV (e.g., outer layer or surface (e.g., shell), or internal lattice, whether physical, chemical, or a combination thereof).
  • BA and DV the latter including but not limited to trapping, embedding and a combination thereof, and can be via one or more of hydrophobic and electrostatic interactions and can be via the structure of DV (e.g., outer layer or surface (e.g., shell), or internal lattice, whether physical, chemical, or a combination thereof).
  • L and SBP 2 are absent
  • SBP 1 and DV are directly (e.g., without use of a linker) coupled together either covalently or non-covalently.
  • SBP 1 and SBP 2 are covalently coupled together.
  • non-biological substrates are provided having a surface coated with a composition according to the presently disclosed subject matter.
  • FIG. 1 shows a digital image comparing a non-biological substrate onto which has been formed a composition according to the presently disclosed subject matter carrying a biologically active agent comprising a fluorescent dye (Panel A), with a non-biological substrate to which has been contacted with fluorescent dye in a drug delivery vehicle (Panel B).
  • a biologically active agent comprising a fluorescent dye (Panel A)
  • Panel B a drug delivery vehicle
  • FIG. 2 shows a graph comparing the mean density of fluorescence of a non- biological substrate onto which has been formed a composition according to the presently disclosed subject matter carrying a biologically active agent comprising a fluorescent dye ("SBP-[DV(BA)]”), with a non-biological substrate to which has been applied fluorescent dye in a drug delivery vehicle ("control").
  • FIG. 3 shows an image comparing a non-biological substrate comprising a metal onto which has been applied a composition according to the presently disclosed subject matter having binding affinity for metal and carrying a biologically active agent comprising a fluorescent dye (Panel A), with a non- biological substrate comprising a metal to which has been applied fluorescent dye in a drug delivery vehicle (Panel B).
  • FIG. 4 shows an image comparing a non-biological substrate comprising a polymer onto which has been applied a composition according to the presently disclosed subject matter having binding affinity for polymer and carrying a biologically active agent comprising a fluorescent dye (Panel A), with a non-biological substrate comprising a polymer to which has been applied fluorescent dye in a drug delivery vehicle (Panel B).
  • FIG. 5 is a graph showing extended release of antimicrobial activity from surface-binding peptide-delivered microparticles. Disks were coated and challenged with plasma and bacteria. At each time point (days 1 through 4), disks were re-challenged with plasma and bacteria. Bacterial growth was inhibited in the wells with the surface-binding peptide (AFF-5061 (SEQ ID NO:
  • AFF-5103 SEQ ID NO: 125
  • FIG. 6 shows an image of TSA plates displaying the antimicrobial effects of surface-binding peptide-delivered vancomycin microparticles.
  • Metal pins were coated with a composition comprising surface-binding peptide AFF-5061
  • the metal pins were then incubated with bacteria and removed pins were rolled over the TSA plates and pin culture broth dilutions were plated.
  • the image illustrates the antimicrobial effects of the coating composition against both the adherent (on pin) and suspended bacteria (broth dilutions).
  • FIG. 7 is a graph showing the in vivo antimicrobial effects of a coating composition comprising metal surface-binding peptide AFF-5061 (SEQ ID NO: 1
  • Metal pins were coated with a composition comprising surface-binding peptide AFF-5061 (SEQ ID NO: 124) and microparticles carrying the antibiotic vancomycin and the coated pins were tested against S. Aureus in a rat tibia infection model.
  • compositions comprising a surface-binding peptide coupled to a drug delivery vehicle carrying biologically active agent, methods of coating surfaces of non- biological substrates with a coating composition, and non-biological substrates coated with the compositions.
  • a drug delivery vehicle for delivery of biologically active agent to a surface of a non-biological substrate, wherein the drug delivery vehicle comprises a microparticle carrying biologically active agent, and a peptide coupled to the microparticle; wherein the peptide has binding affinity for and binds non-covalently to the surface of the non- biological substrate.
  • a drug delivery vehicle for delivery of biologically active agent to a surface of a non- biological substrate, wherein the drug delivery vehicle comprises: (a) a shell (or surface-exposed outer layer) comprised of a synthetic component, and wherein the drug delivery vehicle carries biologically active agent; (b) a first surface-binding peptide that binds specifically and noncovalently to the non- biological substrate; and (c) a second surface-binding peptide having binding affinity for, and noncovalently coupled to, the synthetic component of the shell of the drug delivery vehicle; wherein the first and second surface-binding peptides are covalently coupled together either directly or via a linker.
  • first and second are used herein for purposes of the specification and claims for ease of explanation in differentiating between two different molecules, and are not intended to be limiting the scope of the presently disclosed subject matter, nor imply a spatial, sequential, or hierarchical order unless otherwise specifically stated.
  • non-biological substrate is used herein for purposes of the specification and claims to mean a substrate that is not a quality or component of a living system.
  • a non-biological substrate can comprise any form suitable to its intended use including but not limited to a container, reactor, device, array, medical device, particle, or the surface of a non- biological substrate contained in a liquid.
  • Representative non-biological substrates include, but are not limited to, plastic, silicone, synthetic polymer, metal (including mixed metal alloys), metal oxide (e.g., glass), non-metal oxide, ceramic, carbon-based materials (e.g., graphite, carbon nanotubes, carbon "buckyballs", and metallo-carbon composites), and combinations thereof.
  • non-biological substrates that can benefit from the presently disclosed subject matter include, but are not limited to, (a) medical supplies, such as medical surgical gowns, diapers, incontinence apparel, drapes, bandages, dressings, sponges, covers, and the like; (b) laboratory equipment, such as bioreactors, fermentors, test tubes, assay plates, arrays, culture containers, and the like; (c) packaging or product protection (e.g., packaging materials, coverings (such as wraps)), such as applied to perishables such as foods, drugs, and medical devices; (d) cleaning supplies (e.g., for use in one or more of hospitals, industries, and households) such as sanitary wipes, cloths, wet and dry wipes, paper-based tissues, and sponges; and hygienic coatings for use with table tops, counter tops, door knobs, door handles, fixtures, and the like.
  • medical supplies such as medical surgical gowns, diapers, incontinence apparel, drapes, bandages, dressings, sponge
  • metal is used herein for purposes of the specification and claims to mean one or more compounds or compositions comprising a metal represented in the Periodic Table (e.g., a transition metal, alkali metals, and alkaline earth metals, each of these comprise metals related in structure and function, as classified in the Periodic Table), and can further refer to a metal alloy, a metal oxide, a silicon oxide, and bioactive glass.
  • a metal alloy e.g., a transition metal, alkali metals, and alkaline earth metals, each of these comprise metals related in structure and function, as classified in the Periodic Table
  • metal alloy e.g., a metal oxide, a silicon oxide, and bioactive glass.
  • preferred metals include, but are not limited to, titanium, titanium alloy, stainless steel, aluminum, zirconium alloy metal substrate (e.g., OxiniumTM), cobalt chromium alloy, gold, silver, rhodium, zinc, tungsten, platinum, rubidium, and copper.
  • polymer is used herein for purposes of the specification and claims to mean a molecule or material comprised of repeating structural units (a structural unit typically referred to as a monomer) connected by covalent chemical bonds.
  • a polymer can be biodegradable (e.g., one or more of self-dissolving, or bioresorbable, or degradable in vivo) or non-biodegradable; or synthetic (manufactured, and not found in nature) or natural (found in nature, as made in living tissues of plants and/or animals).
  • Non-limiting examples of suitable synthetic polymers described as being biodegradable include: poly-amino acids; polyanhydhdes including maleic anhydride polymers; polycarboxylic acid; some polyethylenes including, but not limited to, polyethylene glycol, polyethylene oxide; polypropylenes, including, but not limited to, polypropylene glycol, polypropylene fumarate; one or more of polylactic acid or polyglycolic acid (and copolymers and mixtures thereof, e.g., poly(L-lactic acid) (PLLA), poly(D,L,-lactide), poly(lactic acid-co-glycolic acid), 50/50 (DL-lactide-co-glycolide)); polyorthoesters; polydioxanone; polyphosphazenes; polydepsipeptides; one or more of polycaprolactone (and co-polymers and mixtures thereof, e.g., poly(D,L- lactide- co-caprolact
  • Representative natural polymers described as being biodegradable include macromolecules (such as polysaccharides, e.g., alginate, starch, chitosan, cellulose, or their derivatives (e.g., hydroxypropylmethyl cellulose); proteins and polypeptides, e.g., gelatin, collagen, albumin, fibrin, fibrinogen); polyglycosaminoglycans (e.g. hyaluronic acid, chondroitin sulfate); and mixtures, combinations, and copolymers of any of the foregoing.
  • macromolecules such as polysaccharides, e.g., alginate, starch, chitosan, cellulose, or their derivatives (e.g., hydroxypropylmethyl cellulose); proteins and polypeptides, e.g., gelatin, collagen, albumin, fibrin, fibrinogen); polyglycosaminoglycans (e.g. hyaluronic acid,
  • Non-limiting examples of suitable synthetic polymers described as being non-biodegradable include: inert polyaryletherketones, including polyetheretherketone ("PEEK”), polyether ketone, polyetherketoneketone, and polyetherketoneetherketoneketone; polyurethanes; polystyrene, and styrene- ethylene/butylene-styrene block copolymers; polyisobutylene copolymers and styrene-isobutylene-styrene block copolymers; polyvinylpyrrolidone; polyvinyl alcohols; copolymers of vinyl monomers; polyvinyl ethers; polyvinyl aromatics; polyethylene oxides; polyesters including polyethylene terephthalate; some polyamides; polyacrylamides; polyethers including polyether sulfone; polyalkylenes including polypropylene, polyethylene; copolymers of ethylene and polypropylene; some polycarbonates, silicone and
  • ceramic is used herein for purposes of the specification and claims to mean inorganic non-metallic materials whose formation is due to the action of heat.
  • Suitable ceramic materials include but are not limited to silicon oxides, aluminum oxides, alumina, silica, hydroxyapatites, glasses, quartz, calcium oxides, calcium phosphates, indium tin oxide (ITO), polysilanols, phosphorous oxide, and combinations thereof.
  • the term "effective amount” is used herein, in referring to a composition according to the presently disclosed subject matter and for purposes of the specification and claims, to mean an amount sufficient of the composition so as to (a) mediate binding of the composition to at least one surface of the non-biological substrate; and (b) promote attachment of a drug delivery vehicle, carrying a biologically active agent, to at least one surface of the non-biological substrate.
  • the term "effective amount” is used herein, in referring to biologically active agent in a composition according to the presently disclosed subject matter and for purposes of the specification and claims, to mean an amount of the biologically active agent effective for modulating, preventing, ameliorating, or treating the condition and/or disease intended by administration of the biologically active agent.
  • biologically active agent refers to one or more agents selected from the group consisting of a therapeutic agent, an agent having biological activity, a diagnostic agent, a prophylactic agent, a chemical catalyst, and a combination thereof.
  • Representative biologically active agents include, but are not limited to, growth factor, cells, biologically active drug, hormone, vitamin, nucleic acid molecule encoding any of the foregoing, nucleic acid molecules having biological activity.
  • Representative hormones include, but are not limited to sex hormones (e.g., estrogen, progesterone, testosterone), thyroid hormones, insulin, adrenal cortical and pituitary hormones, and growth hormones.
  • Representative cells include, but are not limited to, one or more cells or cell types, and preferably cells of human origin, such as stem cells, osteoprogenitor stem cells, mesenchymal stem cells, osteocytes, osteoblasts, osteoclasts, periosteal stem cells, endothelial cells, stromal cells, hematopoietic progenitor cells, adipose tissue precursor cells, cord blood stem cells, myoblasts, Schwann cells, oligodendrocytes, insulin producing cells (e.g., beta cells), neuroprogenitor cells, and a combination thereof.
  • stem cells such as stem cells, osteoprogenitor stem cells, mesenchymal stem cells, osteocytes, osteoblasts, osteoclasts, periosteal stem cells, endothelial cells, stromal cells, hematopoietic progenitor cells, adipose tissue precursor cells, cord blood stem cells, myoblasts, Schwann cells, oli
  • Representative vitamins include any one or more of fat-soluble vitamins (A, D, E and K), and water-soluble (8 B vitamins and vitamin C), and their derivatives (e.g., vitamin D derivatives include 1 , 25-di hydro xyvitamin D3, 1 ⁇ - hydroxyvitamin D2).
  • Diagnostic agents include, but are not limited to, radiolabels, radiopaque compounds, colohmethc reagents, dyes, fluorophores, fluorescent molecules, fluorescent nanocrystals, luminescent molecules, chromophores, and the like.
  • Catalysts can be selected from the group consisting of heterogeneous catalysts, homogeneous catalysts, biocatalysts (e.g., enzymes in metabolic or biological pathways), electrocatalysts (e.g., metal-rich catalysts used in fuel cells, or energy generation), organocatalysts (simple organic molecules used as catalysts in chemical reactions), as known to those skilled in the art.
  • biocatalysts e.g., enzymes in metabolic or biological pathways
  • electrocatalysts e.g., metal-rich catalysts used in fuel cells, or energy generation
  • organocatalysts simple organic molecules used as catalysts in chemical reactions
  • Growth factor is a term used to refer to one or more growth factors or cytokines.
  • Representative growth factors can include, but are not limited to, bone morphogenetic protein (BMP, including the family of BMPs, such as BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-1 1 , BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, and BMP-18), transforming growth factor beta (TGF-beta), transforming growth factor alpha (TGF-alpha), vascular endothelial cell growth factor (VEGF, including its variants), epidermal growth factor (EGF), fibroblast growth factor (e.g., basic fibroblast growth factor, acidic fibroblast growth factor, FGF-1 to FGF-23), epidermal growth factor (EGF), insulin-like growth factor (I or II), interleukin-l, interfer
  • a biological analog has an amino acid sequence having from about 1 % to about 25% of the amino acids substituted, as compared to the amino acid sequence of the peptide growth factor from which the analog was derived.
  • a biologically active analog thereof has between 1 and 10 amino acid changes, as compared to the amino acid sequence of the peptide from which the analog was derived.
  • antibiotics classes of antibiotics are known to include, but are not limited to, penicillins (e.g., penicillin G, penicillin V, ampicillin, methicillin, oxacillin, amoxicillin, amoxicillin-clavulanate, ticarcillin, nafcillin, cloxacillin, piperacillin-tazocbactam, and dicloxacillin), cephalosporins and cephams (e.g., cefazolin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefaclor, cefprozil, loracarbef, ce
  • time sufficient for binding generally refers to a temporal duration sufficient for non-covalent and specific binding of a surface-binding peptide described herein to a non-biological substrate for which the peptide has binding affinity, as known to those skilled in the art.
  • a time sufficient for binding a composition according to the presently disclosed subject matter to a non-biological substrate ranges from about 5 minutes to no more than 60 minutes.
  • composition is used herein, in reference to a composition of the presently disclosed subject matter and for purposes of the specification and claims, to refer to a composition comprising formula I: A composition of the formula: SBP 1 - L -SBP 2 [DV(BA)], wherein
  • SBP is, when present, a surface-binding peptide of 8 to 60 amino acids
  • SBP 1 is the SBP surface-binding peptide comprising a surface-binding peptide domain having binding affinity for a surface comprising a non-biological substrate
  • SBP 2 can be present or absent and is the SBP surface-binding peptide having binding affinity for a synthetic component of a drug delivery vehicle (DV)
  • L can be present or absent and comprises a linker
  • the DV comprises a drug delivery vehicle that carries a BA, wherein the BA is one or more biologically active agents; wherein in the presence of the SBP 2 , the DV is noncovalently coupled to the SBP 2 ; wherein in the absence of the SBP 2 and in the presence of the L, the SBPi is coupled to the DV either covalently or nocovalently; and wherein in the absence of both the SBP 2 and the L, the SBPi is coupled directly to the DV either covalently or noncovalently.
  • DV is a drug delivery vehicle that carries BA, wherein BA is one or more biologically active agents ("biologically active agent").
  • BA is one or more biologically active agents ("biologically active agent").
  • DV can carry BA by one or more processes selected from the group consisting of coupling BA and DV together (e.g., to, in, on, within, or a combination thereof), encapsulation of BA by DV, and a combination thereof.
  • Coupling can be by one or more of covalent and/or non-covalent attachment of BA and DV (the latter including but not limited to trapping, embedding and a combination thereof, and can be via one or more of hydrophobic and electrostatic interactions and can be via the structure of DV (e.g., outer layer or surface (e.g., shell), or internal lattice, whether physical, chemical, or a combination thereof).
  • BA and DV the latter including but not limited to trapping, embedding and a combination thereof, and can be via one or more of hydrophobic and electrostatic interactions and can be via the structure of DV (e.g., outer layer or surface (e.g., shell), or internal lattice, whether physical, chemical, or a combination thereof).
  • composition according to the invention can comprise drug delivery vehicle carrying biologically active agent, wherein drug delivery vehicle comprises carrying one biologically active agent, or carrying two or more biologically active agents.
  • a composition can comprise drug delivery vehicle comprising two or more types of drug delivery vehicles, each type of drug delivery vehicle differing from another type of drug delivery vehicle in biologically active agent carried (and can also differ by a property selected from the group consisting of composition of drug delivery vehicle, release profile, stability, surface-binding peptide coupled thereto, and a combination thereof).
  • a composition can comprise one or more of either or both the first and second surface-binding peptides according to the following formula: SBP 1 — L -SBP 2 [DV(BA)] previously described herein above.
  • a composition can be "homogeneous" and comprise only first and/or second surface-binding peptides having the same amino acid sequence.
  • a composition can comprise, for example, two or more first surface-binding peptides SBP 1 that differ in amino acid sequence but bind specifically to the same non-biological substrate.
  • a composition can comprise in another embodiment, for example, two or more first surface-binding peptides SBP 1 that differ in amino acid sequence and bind different non-biological substrates.
  • a composition can comprise, for example, two or more second surface-binding peptides SBP 2 having different amino acid sequences and binding the same drug delivery vehicle DV.
  • a composition can comprise in another embodiment, for example, two or more second surface- binding peptides SBP 2 having different amino acid sequences and binding different drug delivery vehicles DV.
  • a composition can comprise any combination of the foregoing examples of first and second surface-binding peptides having different amino acid sequences and binding either the same of different non-biological substrates and/or drug delivery vehicles.
  • a surface-binding peptide used in accordance with the presently disclosed subject matter can also comprise an oligomer (e.g., dimer, multimer) of the same peptide amino acid sequence or of two or more different amino acid sequences.
  • an oligomer e.g., dimer, multimer
  • two or more surface- binding peptides are coupled together (e.g., by one or more of non-chemical physical bonds, a chemical bond either directly or through a linking or other chemical modifier group, (via chemical synthesis or recombinant expression) in such a way that each retains its respective function to bind to the respective non-biological substrate for which it has binding affinity.
  • Such coupling can include forming a multimeric molecule having two or more peptides having binding affinity to the same non-biological substrate, two or more peptides having binding affinity for different non-biological substrates and/or different drug delivery vehicles, and combinations thereof.
  • two peptides can be coupled via a side chain-to-side chain bond (e.g., where each of the peptides has a side chain amine (e.g., such as the epsilon amine of lysine)), a side chain-to-N terminal bond (e.g., coupling the N-terminal amine of one peptide with the side chain amine of the other peptide), a side chain-to-C-terminal bond (e.g., coupling the C-terminal chemical moiety (e.g., carboxyl) of one peptide with the side chain amine of the other peptide), an N-terminal-to-N-terminal bond, an N-terminal to C- terminal bond, a C-terminal to C-terminal bond, or a combination thereof.
  • a side chain-to-side chain bond e.g., where each of the peptides has a side chain amine (e.g., such as the epsil
  • two or more peptides can be coupled directly to a peptide by synthesizing or expressing the two or more peptides as a single peptide.
  • the coupling of two or more peptides can also be via a linker to form surface-binding peptide used in the composition according to the presently disclosed subject matter (e.g., see, for example, SEQ ID NOs:1 15 & 1 16 which are oligomers of SEQ ID NO:1 13).
  • a linking compound or moiety can be used that acts as a molecular bridge to couple at least two different molecules, for example, to couple at least one surface-binding peptide to drug delivery vehicle or to couple a first surface- binding peptide to a second surface-binding peptide.
  • the linking moiety can couple the at least two different molecules by either a covalent or a non- covalent bond.
  • coupling at least one surface-binding peptide to drug delivery vehicle can involve one portion of the linker binding to at least one surface-binding peptide having binding affinity for a non-biological substrate and another portion of the linker binding to at least one drug delivery vehicle.
  • two different molecules can be coupled to the linker in a step-wise manner, or can be coupled simultaneously to the linker.
  • the linker There is no particular size or content limitations for the linker so long as it can fulfill its purpose as a molecular bridge, and that the binding affinity of a surface-binding peptide in a coating composition is substantially retained.
  • Linkers are known to those skilled in the art to include, but are not limited to, chemical compounds (e.g., chemical chains, compounds, reagents, and the like).
  • the linkers can include, but are not limited to, homobifunctional linkers and heterobifunctional linkers.
  • Heterobifunctional linkers well known to those skilled in the art, contain one end having a first reactive functionality (or chemical moiety) to specifically link a first molecule, and an opposite end having a second reactive functionality to specifically link to a second molecule.
  • bifunctional or polyfunctional reagents both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, III.), amino acid linkers (typically, a short peptide of between 3 and 15 amino acids, and often containing amino acids such as glycine, and/or serine), and polymers (e.g., polyethylene glycol) can be employed as a linker with respect to the presently disclosed subject matter.
  • amino acid linkers typically, a short peptide of between 3 and 15 amino acids, and often containing amino acids such as glycine, and/or serine
  • polymers e.g., polyethylene glycol
  • representative peptide linkers comprise multiple reactive sites to be coupled to a binding domain (e.g., polylysines, polyornithines, polycysteines, polyglutamic acid and polyaspartic acid) or comprise substantially inert peptide linkers (e.g., lipolyglycine, polysehne, polyproline, polyalanine, and other oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl amino acid residues.
  • a binding domain e.g., polylysines, polyornithines, polycysteines, polyglutamic acid and polyaspartic acid
  • substantially inert peptide linkers e.g., lipolyglycine, polysehne, polyproline, polyalanine, and other oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl amino acid residues.
  • Suitable polymeric linkers are known in the art, and can comprise a synthetic polymer or a natural polymer.
  • Representative synthetic polymers include but are not limited to polyethers (e.g., poly(ethylene glycol) (“PEG”)), polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA)), polyamines, polyamides (e.g., nylon), polyurethanes, polymethacrylates (e.g., polymethylmethacrylate; PMMA), polyacrylic acids, polystyrenes, polyhexanoic acid, flexible chelators such as EDTA, EGTA, and other synthetic polymers which preferably have a molecular weight of about 20 daltons to about 1 ,000 kilodaltons.
  • Natural polymers include but are not limited to hyaluronic acid, alginate, chondroitin sulfate, fibrinogen, fibronectin, albumin, collagen, calmodulin, and other natural polymers which preferably have a molecular weight of about 200 daltons to about 20,000 kilodaltons (for constituent monomers).
  • Polymeric linkers can comprise a diblock polymer, a multi-block copolymer, a comb polymer, a star polymer, a dendritic or branched polymer, a hybrid linear-dendritic polymer, a branched chain comprised of lysine, or a random copolymer.
  • a linker can also comprise a mercapto(amido)carboxylic acid, an acrylamidocarboxylic acid, an acrlyamido- amidotriethylene glycolic acid, 7-aminobenzoic acid, and derivatives thereof.
  • the linkers of the presently disclosed subject matter can be fatty acids.
  • the fatty acids of the presently disclosed subject matter include saturated and unsaturated fatty acids such as but not limited to butyric acid, caproic acid, caprylic acid, capric acid, undecanoic acid (AUD), lauric acid, myhstic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, eicosanoic acid, docosanoic acid, tetracosanoic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.
  • saturated and unsaturated fatty acids such as but not limited to butyric acid, caproic acid, caprylic acid, capric acid, undecanoic acid (AUD), lauric acid, myhstic acid, palmitic acid,
  • the fatty acid linkers are used as a linking group between the first surface-binding peptide and the second surface-binding peptide.
  • the fatty acid molecules of the presently disclosed subject matter can also be used in various other embodiments to modify one or both surface-binding peptides.
  • the fatty acids are used to modify one or both ends of either or both the first and second surface-binding peptides.
  • a single surface-binding molecule is modified at one or both ends with a fatty acid molecule (i.e., the case where SBP 2 is absent and, optionally, L is also absent).
  • Linkers can also utilize copper-catalyzed azide-alkyne cycloaddition (e.g., "click chemistry") or any other methods well known in the art.
  • Linkers are known in the art and include linkers that can be cleaved (e.g., by heat, by natural enzymes found in or on the body of an individual, by pH sensitivity), and linkers that can be made reactive toward other molecular moieties or toward themselves, for cross-linking purposes.
  • pH-sensitive materials useful as linkers can include, but are not limited to, cellulose acetate phthalate, cellulose acetate thmellitate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.
  • the linker can vary in length and composition for optimizing such properties as preservation of biological function, stability, resistance to certain chemical and/or temperature parameters, and of sufficient stereo-selectivity or size.
  • the linker should not significantly interfere with the ability of a composition according to the presently disclosed subject matter to sufficiently bind specifically, with appropriate avidity for the purpose, to a non-biological substrate, or the ability of a drug delivery vehicle carrying biologically active agent to deliver biologically active agent.
  • a preferred linker can be a molecule with activities that enhance or complement the effect of a composition of the presently disclosed subject matter.
  • a preferred linker can be used in the presently disclosed subject matter to the exclusion of a linker other than the preferred linker.
  • binding affinity and like terms used herein, are used for the purposes of the specification and claims, to refer to the ability of a peptide (as described herein) to have a binding affinity that is greater for one target molecule or surface material (i.e., the non-biological substrates of the presently disclosed subject matter) over another non-target molecule or surface material.
  • a target molecule or surface material i.e., the non-biological substrates of the presently disclosed subject matter
  • an affinity for a given non-biological substrate in a heterogeneous population of other substrates that is greater than, for example, that attributable to non-specific adsorption.
  • a surface- binding peptide of the presently disclosed subject matter has binding affinity for metal or non-biological polymer or another non-biological substrate of the presently disclosed subject matter when the peptide demonstrates binding to the non-biological substrate characterized by an EC50 of 10 ⁇ M or less.
  • binding affinity can be dependent upon the presence of a particular conformation, structure, amino acid sequence, amino acid composition and/or charge on or within the peptide and/or material for which it has binding affinity.
  • a surface-binding peptide that binds specifically to a particular surface, material or composition binds at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or a higher percentage, tighter than the surface-binding peptide binds to an appropriate control such as, for example, a different material or surface, or a protein typically used for such comparisons such as bovine serum albumin.
  • binding affinity can determined by an assay in which a signal is quantified (e.g., fluorescence, or colohmethc) that represents the relative amount of binding between a peptide and a non-biological substrate.
  • a surface-binding peptide has a binding affinity that is characterized by a relative binding affinity as measured by an EC50 of 10 ⁇ M or less, preferably less than 1 ⁇ M, and more preferably less than 0.1 ⁇ M.
  • the EC50 can be determined using any number of methods known in the art, such as by generating a concentration response curve from a binding assay in which the concentration of the peptide is titered with a known amount of the non-biological substrate for which the peptide has binding (see, for example, methods described in Example 2 herein). In such case, the EC50 represents the concentration of peptide producing 50% of the maximal binding observed for that peptide in the assay.
  • surface-binding peptide is used herein for the purposes of the specification and claims to refer to an amino acid chain of no less than about 7 amino acids and no more than about 100 amino acid residues in length, wherein the amino acid chain can include naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non- genetically encoded amino acids, and combinations thereof; however, specifically excluded from the scope and definition of "surface-binding peptide" herein is an antibody.
  • a surface-binding peptide used in accordance with the presently disclosed subject matter can be produced by chemical synthesis, recombinant expression, biochemical or enzymatic fragmentation of a larger molecule, chemical cleavage of larger molecule, a combination of the foregoing or, in general, made by any other method in the art, and preferably isolated.
  • isolated means that the surface-binding peptide is substantially free of components which have not become part of the integral structure of the surface-binding peptide itself; for example, such as substantially free of cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized or produced using biochemical or chemical processes.
  • Surface-binding peptides can include L-form amino acids, D-form amino acids, or a combination thereof.
  • Representative non-genetically encoded amino acids include but are not limited to 2-aminoadipic acid; 3- aminoadipic acid; ⁇ -aminopropionic acid; 2-aminobutyhc acid; 4-aminobutyhc acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2- aminoisobutyhc acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4- diaminobutyric acid; desmosine; 2,2'-diaminopimelic acid; 2,3- diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo- hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo- isoleucine; N-methylglycine
  • Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups can be derivatized to form O- acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.
  • the at least one surface-binding peptide having binding affinity for non-biological substrate can be modified, such as having an N-terminal amino acid, a C-terminal amino acid, or a combination thereof, wherein such amino acid is a non-genetically encoded amino acid that enhances the binding avidity (strength of binding interactions) of the peptide to a non-biological substrate.
  • amino acids can be incorporated into a peptide by standard methods known in the art for solid phase and/or solution phase synthesis.
  • a hydroxy-amino acid e.g., one or more of hydroxylysine, allo-hydroxylysine, hydroxyproline, and the like
  • a hydroxy-amino acid e.g., one or more of hydroxylysine, allo-hydroxylysine, hydroxyproline, and the like
  • the peptide is used in the coating composition according to the presently disclosed subject matter for enhancing the strength of the binding interactions (e.g., via electrostatic or ionic interactions) between the coating composition and the non-biological substrate to be coated.
  • a surface-binding peptide according to the presently disclosed subject matter can be modified, such as by addition of chemical moieties to one or more amino acid termini, and side chains; or substitutions, insertions, and deletions of amino acids; where such modifications provide for certain advantages in its use, and provided that the surface-binding function of the surface-binding peptide is sufficiently retained to be useful for the compositions and methods of the presently disclosed subject matter.
  • the term "peptide” encompasses any of a variety of forms of peptide derivatives including, for example, amides, conjugates with proteins, cyclone peptides, polymerized peptides, conservatively substituted variants, analogs, fragments, chemically modified peptides, and peptide mimetics. Any peptide derivative that has desired binding characteristics of the family of surface- binding peptides according to the presently disclosed subject matter can be used in the practice of the presently disclosed subject matter.
  • a chemical moiety, added to the N-terminal amino acid of a synthetic peptide to block chemical reactivity of that amino terminus of the peptide comprises an N-terminal group.
  • N-terminal groups for protecting the amino terminus of a peptide are well known in the art, and include, but are not limited to, lower alkanoyl groups, acyl groups, sulfonyl groups, and carbamate forming groups.
  • Preferred N-terminal groups can include acetyl, Fmoc, and Boc.
  • a chemical moiety, added to the C-terminal amino acid of a synthetic peptide to block chemical reactivity of that carboxy terminus of the peptide comprises a C-terminal group.
  • Such C-terminal groups for protecting the carboxy terminus of a peptide are well known in the art, and include, but are not limited to, an ester or amide group. Terminal modifications of a peptide are often useful to reduce susceptibility by proteinase digestion, and to therefore prolong a half-life of peptides in the presence of biological fluids where proteases can be present.
  • a peptide, as described herein can comprise one or more amino acids that have been modified to contain one or more chemical moieties (e.g., reactive functionalities such as fluorine, bromine, or iodine) to facilitate linking the peptide to a linker molecule.
  • peptide also encompasses a peptide wherein one or more of the peptide bonds are replaced by pseudopeptide bonds including but not limited to a carba bond (CH 2 -CH 2 ), a depsi bond (CO-O), a hydroxyethylene bond (CHOH-CH 2 ), a ketomethylene bond (CO-CH 2 ), a methylene-oxy bond (CH 2 -O), a reduced bond (CH 2 -NH), a thiomethylene bond (CH 2 -S), an N-modified bond (-NRCO-), and a thiopeptide bond (CS-NH).
  • pseudopeptide bonds including but not limited to a carba bond (CH 2 -CH 2 ), a depsi bond (CO-O), a hydroxyethylene bond (CHOH-CH 2 ), a ketomethylene bond (CO-CH 2 ), a methylene-oxy bond (CH 2 -O), a reduced bond (CH 2 -NH), a thiomethylene bond (
  • Surface-binding peptides that are useful in a composition or method according to the presently disclosed subject matter also include peptides having one or more substitutions, additions and/or deletions of residues relative to the sequence of an exemplary surface-binding peptide disclosed in Tables I-V, and SEQ ID NOs: 1-124 herein, so long as the binding properties of the original exemplary peptide are substantially retained.
  • the presently disclosed subject matter includes surface-binding peptides that differ from the exemplary sequences disclosed herein by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (depending on the length of the exemplary peptide disclosed herein), and that share sequence identity with the exemplary sequences disclosed herein of at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity.
  • Sequence identity can be calculated manually or it can be calculated using a computer implementation of a mathematical algorithm, for example, GAP, BESTFIT, BLAST, FASTA, and TFASTA, or other programs or methods known in the art. Alignments using these programs can be performed using the default parameters.
  • a peptide having an amino acid sequence substantially identical to a sequence of an exemplary surface-binding peptide disclosed herein can have one or more different amino acid residues as a result of substituting an amino acid residue in the sequence of the exemplary surface-binding peptide with a functionally similar amino acid residue (a "conservative substitution"); provided that peptide containing a conservative substitution will substantially retain the binding affinity of the exemplary surface-binding peptide not containing the conservative substitution.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one aromatic residue such as tryptophan, tyrosine, or phenylalanine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue such as aspartic acid or glutamic acid for another.
  • one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another
  • the substitution of one aromatic residue such as tryptophan, tyrosine, or phenylalanine for another
  • the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine,
  • a surface-binding peptide can be described herein as comprising a peptide consisting essentially of a peptide (and/or its amino acid sequence) useful in the presently disclosed subject matter.
  • the terminology “consisting essentially of refers to a peptide which includes the amino acid sequence of the surface-binding peptides described herein, and a peptide having at least 70% identity thereto, and preferably at least 95% identity thereto, (as described herein), along with additional amino acids at the carboxyl and/or amino terminal ends (e.g., ranging from about 1 to about 20 additional amino acids at one end or at each of both ends) which maintains the primary activity of the surface-binding peptide as a binding domain described herein.
  • a surface-binding peptide "consisting essentially of any one of the amino acid sequences illustrated as SEQ ID NOs:1-124 will possess the activity of binding a non- biological substrate with binding affinity, as provided herein; and will not possess any characteristics which constitute a material change to the basic and novel characteristics of the peptide to function as a surface-binding peptide (e.g., thus, in the foregoing example, a full length naturally occurring polypeptide, or a genetically engineered polypeptide, which has a primary activity other than as a binding domain described herein, and which contains the amino acid sequence of a surface-binding peptide described in the presently disclosed subject matter, would not constitute a peptide "consisting essentially of a peptide described in the presently disclosed subject matter).
  • pharmaceutically acceptable carrier when used herein for purposes of the specification and claims, means a carrier medium that does not significantly alter the biological activity of the composition according to the presently disclosed subject matter to which it is added.
  • a carrier medium include, but are not limited to, aqueous solutions, aqueous or non-aqueous solvents, suspensions, emulsions, gels, pastes, and the like.
  • a suitable pharmaceutically acceptable carrier can comprise one or substances, including but not limited to, water, buffered water, medical parenteral vehicles, saline, 0.3% glycine, aqueous alcohols, isotonic aqueous buffer; and can further include one or more substances such as alginic acid, water-soluble polymer, glycerol, glycols (e.g., polyethylene glycol), polyols (e.g., glycerin, sorbitol, etc.), oils, salts (such as sodium, potassium, magnesium and ammonium, phosphonates), esters (e.g., carbonate esters, ethyl oleate, ethyl laurate, etc.), fatty acids, carbohydrates, polysaccharides, starches, glycoproteins (for enhanced stability), buffering agents (e.g., magnesium hydroxide, aluminum hydroxide, and the like), excipients, wetting agents, and preservatives (
  • medical device refers to a structure (a) that is positioned or positionable into or onto an individual's body to prevent, treat, modulate or ameliorate damage, repair or restore a function of a damaged tissue, or to provide a new function; and (b) comprises at least one surface that is a non- biological substrate to be coated by a composition according to the presently disclosed subject matter.
  • Representative medical devices include, but are not limited to: hip endoprostheses, artificial joints, jaw or facial implants, dental implants, tendon and ligament replacements, skin replacements, metal replacements and metal screws, metal nails or pins, metal graft devices, polymer-containing grafts, vascular prostheses, heart pacemakers, artificial heart valves, blood filters, closure devices (e.g., for closure of wounds, incisions, or defects in tissues, including but not limited to skin and other organs (heart, stomach, liver, etc.)), sutures, breast implants, penile implants, stents, catheters, shunts, nerve growth guides, leads for battery-powered medical devices, intraocular lenses, wound dressings, tissue sealants, aneurismal coils, prostheses (e.g., cochlear implants, visual prostheses (including, but not limited to, contact lenses, and other visual aid devices), neurostimulators, muscular stimulators, joint prosthesis, dental prosthesis, etc.), ophthalmic devices (glaucoma
  • drug delivery vehicle when used herein for purposes of the specification and claims, means a carrier for one or more biologically active agents; preferably, comprising a microparticle, liposome, polymer, or combination thereof, and generally in the size range of nanometers to microns.
  • the drug delivery vehicle has a size in the range of from 1 nanometer to 1000 microns, and preferably in a range of from 10 nm to 200 microns, and more preferably in a the range of 0.05 microns to 10 microns; the size depending on factors such as the nature and amount of biologically active agent carried by the drug delivery vehicle, the composition of the drug delivery vehicle, the intended route of administration, and desired pharmacokinetic parameters (e.g., release profile, biological half life, etc.).
  • a drug delivery vehicle is biodegradable and biocompatible (e.g., substantially non-toxic).
  • Microparticle is used herein to mean particles including, but not limited to, microspheres, microcapsules, nanospheres, nanoparticles, solid lipid nanoparticles, gas-filled microparticles (particularly useful for loading lipophilic biologically active agents), and solid phase porous microspheres; and can be solid, porous, hollow or have an internal lattice-type structure (e.g., a sponge-like structure, a honeycomb structure, etc.).
  • a microparticle can be entirely formed of biologically active agent.
  • microparticles can be produced using methods known in the art such as, for example, spray drying.
  • the microparticles entirely formed of biologically active agent are linked to surface-binding peptide.
  • Microparticles can be spherical or non-spherical in shape, and can be a shape selected from the group comprising spherical, oval, non-spherical, rod-like, acicular (needle-like), columnar, flake, disc-like, cubical, lamellar, blade-like, polygonal, and a combination thereof.
  • a microparticle can be comprised of one or more materials, including but not limited to, polymer, lipid, peptide, protein, carbohydrate (saccharides, polysaccharides, etc.), pharmaceutically acceptable salt thereof, and a combination thereof.
  • Representative proteins include, but are not limited to, gelatin, collagen, albumin, and whey.
  • Representative carbohydrates include, but are not limited to, dextran, saccharide, modified polysaccharide, cellulose esters, chitin, chitosan, cellulose, starch, hyaluronic acid, and pectin.
  • Representative synthetic polymers can include, but are not limited to, one or more of: poly(lactide-co-glycolide) (e.g., PLGA); polylactide-polyglycolide polymer; lactide/glycolide copolymer; polyurethane; aliphatic polyester (e.g., poly-glycolic acid, polylactic acid, and the like); poly-amino acid; polyanhydride; polyhydroxylbutrate-related copolymer; acrylic polymers (e.g., polyacrylic acid, polymethylmethacrylate, polybutyl methacrylate, and the like); polyethylene oxide; polyvinyl pyrrolidone; polypropylene oxide; polyethylene glycol; polypropylene glycol; block polymer of polyethylene oxide and polypropylene oxide; acrylate; acrylamide; methacrylate; poly(ortho esters); cyanoacrylate; polyacrylic acid; polyorthoesters; polydioxanone; polypho
  • a microparticle is comprised of synthetic polymer; and more preferably, comprises biodegradable synthetic polymer.
  • Typical methods known in the art for producing microparticles include, but are not limited to, a spray-drying method, and a phase separation method.
  • Liposomes are vesicles formed from one or more of lipids, fatty acids, phospholipids, non-ionic surfactant ("niosomes"), and polymer-conjugated lipids ("polymerosomes”), using methods well known in the art.
  • liposomes can comprise single lipid bilayer ("unilamellar") vesicles or multiple lipid bilayer (“multilamellar”) vesicles with sizes ranging from about 0.05 micron to 10 microns.
  • a liposome useful in the presently disclosed subject matter is preferably biodegradable and non-toxic.
  • Representative lipids include, but are not limited to, amphipathic lipids, phospholipids, lecithin, synthetic lipids, or dehvatized (e.g., charged) lipids, and a combination thereof.
  • Representative synthetic lipids include, but are not limited to, 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC) and 1 ,2-dioleoylphosphatidylcholine (DOPC), polymer-conjugated lipids, (e.g., poly(ethylene glycol)-diacylglycerol, N-[methoxy-(poly(ethylene glycol)diacylphosphatidylethanolamine, poly(ethylene glycol)-ceramide), and synthetic phospholipids and/or cationic lipids (e.g., 1 ,2,-diacyl-3-thmethylammonium-propane (DOTAP), dimethyldioctadecylammoni
  • a pre-formed liposome comprised of lipid and/or phospholipids can be dehvatized with a coating of polymer (such as to improve stability or provide a chemical moiety along a polymer chain to be reacted with and coupled to a chemical moiety of a surface-binding peptide having binding affinity for non-biological substrate).
  • a drug delivery vehicle to be used in producing a composition according to the presently disclosed subject matter, considered are factors such as the mode of administration, site of application, the indication or condition to be addressed by administration, biodegradability, physiochemical properties (stability, biocompatibility, relative to the site of treatment in facilitating delivery of biologically active agent), and mechanical properties (e.g., density, solubility, viscosity, and the like).
  • Illustrated in this example are various methods for utilizing phage display technology to produce a surface-binding peptide having binding affinity for non-biological substrate or a synthetic component of a drug delivery vehicle.
  • Phage display technology is well-known in the art, and can be used to identify additional peptides for use as binding domains in the compositions according to the presently disclosed subject matter.
  • a library of diverse peptides can be presented to a target substrate, and peptides that specifically bind to the substrate can be selected for use as binding domains. Multiple serial rounds of selection, called "panning," can be used.
  • any one of a variety of libraries and panning methods can be employed to identify a binding domain that is useful in a composition according to the presently disclosed subject matter. Panning methods can include, for example, solution phase screening, solid phase screening, or cell-based screening. Once a candidate binding domain is identified, directed or random mutagenesis of the sequence can be used to optimize the binding properties (including one or more of affinity and avidity) of the binding domain.
  • phage display libraries were screened for peptides that bind to a selected target non-biological substrate of the presently disclosed subject matter.
  • the non-biological target substrate was either bound to or placed in (depending on the selected substrate) a container (e.g., wells of a 96 well microtiter plate, or a microfuge tube).
  • a container e.g., wells of a 96 well microtiter plate, or a microfuge tube.
  • BSA bovine serum albumin
  • the containers were then washed 5 times with a buffer containing buffered saline with TweenTM 20 ("buffer-T").
  • Each library was diluted in buffer-T and added at a concentration of 10 10 pfu/ml in a total volume of 100 ⁇ l. After incubation (in a range of from 1 to 3 hours) at room temperature with shaking at 50 rpm, unbound phage were removed by multiple washes with buffer-T. Bound phage were used to infect E. coli cells in growth media. The cell and phage-containing media was cultured by incubation overnight at 37° C in a shaker at 200 rpm. Phage-containing supernatant was harvested from the culture after centrifuging the culture. Second and third rounds of selection were performed in a similar manner to that of the first round of selection, using the amplified phage from the previous round as input.
  • enzyme-linked immunosorbent (ELISA-type) assays were performed using an anti-phage antibody conjugated to a detector molecule, followed by the detection and quantification of the amount of detector molecule bound in the assay.
  • the DNA sequences encoding peptides from the phage that specifically bind to the selected target non-biological substrate were then determined; i.e., the sequence encoding a particular peptide was located as an insert in the phage genome, and was sequenced to yield the corresponding amino acid sequence displayed on the phage surface.
  • titanium and stainless steel were used as substrates for performing phage selection using several different libraries of phage. Titanium beads and stainless steel beads of approximately 5/32-inch diameter were individually prepared for selections by sequentially washing the beads with 70% ethanol, 40% nitric acid, distilled water, 70% ethanol and, finally, acetone, to remove any surface contaminants. After drying, one metal bead was placed per well of a 96-well polypropylene plate. Non-specific binding sites on the metal beads and the surface of the polypropylene plate were blocked with 1 % bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The plate was incubated for 1 hour at room temperature with shaking at 50 rpm. The wells were then washed 5 times with 300 ⁇ L of buffer-T.
  • BSA bovine serum albumin
  • PBS phosphate-buffered saline
  • Each library was diluted in buffer-T and added at a concentration of 10 10 pfu/mL in a total volume of 100 ⁇ l_. After 3 hours of incubation at room temperature and shaking at 50 rpm, unbound phage were removed by 5 washes of buffer-T. The phage were added directly to E. coli DH5 ⁇ F' cells in 2xYT media, and the phage-infected cells were transferred to a fresh tube containing 2xYT media and incubated overnight at 37 0 C in a shaker incubator. Phage supernatant was harvested by centhfugation at 8500xg for 10 minutes.
  • Second and third rounds of selection were performed in a similar manner to the first round, using the amplified phage from the previous round as input.
  • Each round of selection was monitored for enrichment of metal binding peptides using ELISA-like assays performed using an anti-M13 phage antibody conjugated to horseradish-peroxidase, followed by the addition of chromogenic agent ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and determining a read-out at 405 nm.
  • Libraries that showed enrichment of phage displaying metal binding peptides were plated on a lawn of E. coli cells, and individual plaques were picked and tested for binding to metals (e.g., titanium, stainless steel, etc.).
  • relative binding strengths of the phage were determined by testing serial dilutions of the phage for binding to metal substrate in an ELISA. For example, serial dilutions of the display-selected clones were exposed to titanium or steel in an ELISA. The higher dilutions represent more stringent assays for affinity; therefore, phage that yield a signal at higher dilutions represent peptides with higher relative affinity for the particular target metal. Primers against the phage vector sequence that flank the insertion site were used to determine the DNA sequence encoding the peptide for the phage in each group. The sequence encoding the peptide insert was translated to yield the corresponding amino acid sequence displayed on the phage surface. Similar procedures were used to develop surface-binding peptides that have binding affinity for polymers.
  • peptides useful in the presently disclosed subject matter can also comprise, in their amino acid sequence, such phage amino acids adjoining the peptide at the N- terminus and at the C-terminus (e.g., denoted as ss and sr in Table II).
  • Relative binding strengths (affinities) of the surface-binding peptides to non-biological substrate were determined by testing serial dilutions of the surface-binding peptides for binding to a target non-biological substrate (e.g., non-biological substrates comprising either metal or polymer). The absorbance observed across the concentration range for each peptide sequence was plotted to yield a binding curve of the peptide to its target non- biological substrate. The binding curves were used to determine an EC50 value (i.e., the concentration of peptide that gives 50% of the maximum signal in the binding curve). The EC50 was used as an estimate of the affinity of the peptide for the target non-biological substrate.
  • compositions according to the presently disclosed subject matter are surface- binding peptides that bind to the selected target non-biological substrate with binding affinity of preferably an EC50 of less than or equal to about 1 ⁇ M, and more preferably, in the nanomolar range (e.g., ⁇ 0.1 ⁇ M).
  • a typical assay for measuring the affinity of a surface-binding peptide for a non-biological substrate comprising titanium was performed according to the following procedure. Briefly, 5/32-inch diameter Grade 200 titanium beads were washed by sonication in acetone for 15 minutes, and the beads were allowed to dry. One bead was added to each well of a 96-well polypropylene plate. Two hundred fifty (250) ⁇ l_ of 1 % BSA in PBS was added to each well of the plate. The surface of the wells and the beads were blocked by incubation for 1 hour at 20 0 C with shaking at 500 rpm. The plate was washed three times with 250 ⁇ l_ of buffer-T per well.
  • a 1 :3 dilution series of each of the peptides was prepared using PBS as a diluent, starting at a peptide concentration of 20 ⁇ M, and going down to 0.0001 ⁇ M.
  • a 200 ⁇ l_ sample of each dilution was added to wells of the plate. The plate was incubated for 1 hour at 20 0 C with shaking at 500 rpm. The beads were washed three times with 250 ⁇ l_ of buffer-T per well.
  • streptavidin AP streptavidin-alkaline phosphatase
  • the plate was incubated for 30 minutes at room temperature.
  • the beads were washed three times with 250 ⁇ l_ of buffer- T per well.
  • Two hundred (200) ⁇ l_ of color development reagent (PNPP, p- nitrophenol phosphate) was added to each well. After color had developed (10 minutes), the samples were transferred to a clear 96-well plate and the absorbance at 405nm determined.
  • a binding curve was generated by plotting the absorbance at 405 nm against the peptide concentration ( ⁇ M).
  • non- biological substrates can be substituted for titanium in the foregoing example to determine EC50's for other surface-binding peptides and other non- biological substrates of the presently disclosed subject matter.
  • the particular assay conditions e.g., amounts and dilutions of surface binding peptides will depend on the binding affinity of the particular surface-binding peptide being studied.
  • Table I illustrates exemplary surface-binding peptides that can be used in the methods and compositions according to the presently disclosed subject matter to bind to non-biological substrate polymers.
  • the surface-binding peptides shown in Table I were isolated for binding to a polymeric non- biological substrate with an EC50 of 10 ⁇ M or less.
  • SEQ ID NOs: 1-22 were isolated for binding to polystyrene; SEQ ID NO:23 was isolated for binding to polyurethane; SEQ ID NOs: 24-37 were isolated for binding to polyglycolic acid; SEQ ID NOs: 38-43 were isolated for binding to polycarbonate; SEQ ID NOs: 44-52 were isolated for binding to nylon; and SEQ ID NOs: 53 and 54 were isolated for binding to teflon.
  • Peptides identified according to the presently disclosed subject matter, such as those listed in Table I can be used as surface-binding peptides to bind to a non- biological substrate comprising a polymer to which the peptides have binding affinity.
  • such peptides can be used to bind to a non-biological substrate that is a drug delivery vehicle DV that comprises a synthetic polymer (e.g., the peptide has binding affinity and binds non- covalently to the synthetic polymeric outer layer or shell of the DV).
  • a drug delivery vehicle DV that comprises a synthetic polymer
  • the peptides of SEQ ID NOs: 1-22 that have binding affinity for polystyrene can be used as illustrated in the Examples herein, to bind to a drug delivery vehicle DV comprising a microparticle comprised of polystyrene and carrying a biologically active agent BA.
  • Table Il illustrates exemplary surface-binding peptides that can be used in the methods and compositions according to the presently disclosed subject matter having binding affinity for a metal (including a metal alloy, a metal oxide, or a non-metal oxide).
  • the surface-binding peptides shown in Table Il were isolated for binding to a metal non-biological substrate with an EC50 of 10 ⁇ M or less.
  • SEQ ID NOs: 55-82 have binding affinity to titanium and SEQ ID NOs: 83-102 have binding affinity to stainless steel.
  • Peptides identified according to the presently disclosed subject matter can be used as surface- binding peptides to bind to a non-biological substrate comprising a metal to which the peptides have binding affinity.
  • a peptide can be used as a first surface-binding peptide (SBP 2 ) to bind to a non-biological substrate such as, for example, a medical device comprising a metal.
  • SBP 2 first surface-binding peptide
  • the peptides of SEQ ID NOs: 55-102 that have binding affinity for metals can be used as illustrated, for example, in Examples 8-10 herein, to bind to a non-biological substrate comprising a metal for delivery of a drug delivery vehicle DV carrying a biologically active agent BA to the surface of the non-biological substrate.
  • exemplary peptide sequences are disclosed herein, one skilled in the art will appreciate that the binding properties conferred by those sequences can be attributable to only some of the amino acids comprised by the sequences.
  • a peptide which comprises only a portion of an exemplary amino acid sequence disclosed herein can have substantially the same binding properties as the exemplary peptide comprising the full-length amino acid sequence.
  • also useful as surface-binding domains in the coating compositions according to the presently disclosed subject matter are peptides that comprise only 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 of the amino acids in a particular exemplary sequence provided herein.
  • Such amino acids can be contiguous or non-contiguous as long as the desired property (e.g., substantially retaining binding affinity for the selected material) of the surface- binding domain is retained, as determined by an appropriate assay (described herein and/or as known to those skilled in the art).
  • Such amino acids can be concentrated at the amino-terminal end of the exemplary peptide (for example, 4 amino acids can be concentrated in the first 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 amino acids of the peptide), at the carboxy-terminal end of the exemplary peptide, or they can be dispersed throughout the exemplary peptide (e.g., acting as specific contact points, with the material for which the peptide has binding affinity, spaced apart from each other).
  • surface-binding peptides consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:101 , and SEQ ID NO:102 have an amino acid motif of SEQ ID NO:103 according to the formula as follows:
  • a preferred surface-binding peptide for use in a composition according to the presently disclosed subject matter and having binding affinity for a non-biological substrate comprising a metal comprises a peptide consisting essentially of an amino acid sequence illustrated by SEQ ID NO:103.
  • Second-Generation Metal Surface-Binding Peptides In another example, based on the surface-binding peptides consisting essentially of an amino acid sequences illustrated by SEQ ID NOs: 75-82 in Table II, a series of synthetic, second-generation peptides were synthesized to further define the elements involved in metal binding, including varying the number (ranging from 0 to 3) of triplets of positively charged amino acids, and the amino acid sequence of triplets of positively charged amino acids. Each peptide was synthesized with an amino acid linker (GSSGK portion of SEQ ID NOs: 104-1 13) to facilitate biotinylation at the C-terminal lysine residue, and detection and quantification in the binding assay.
  • an amino acid linker GSSGK portion of SEQ ID NOs: 104-1 13
  • the binding assay was performed using the methods as previously outlined herein. Shown in Table III are the second-generation peptide sequences, all of which displayed binding affinities to metal of an EC50 of less than 1 ⁇ M, with some of the peptide sequences having an EC50 of less than 0.10 ⁇ M.
  • SEQ ID NO:1 14 amino acid motif, comprising a metal binding domain, illustrated by SEQ ID NO:1 14 as follows:
  • Xaa is an amino acid, for example, one of the 20 naturally occurring amino acids found in proteins in either the L or D form of chiral amino acids or a modified amino acid, except that Xaa is an amino acid other than lysine or histidine when occurring between two Z (e.g., Xaa of the amino acid sequence Z 1 (Xaa) j Z 2 is not lysine or histidine); Z is a triplet of amino acids consisting of at least one histidine residue and at least one lysine residue, no other amino acids other than histidine and lysine residues, but no more than two histidine residues or no more than two lysine residues (e.g., KHK, HKH, KKH, HKK, KHH), and most preferably, at least one of Z (e.g., either Zi or Z 2 , or both of Zi and Z 2 , in the amino acid sequence Zi (Xaa) j Z 2 ) is KHK.
  • Z is a
  • Another preferred surface-binding peptide for use in a composition according to the presently disclosed subject matter and having binding affinity for a non- biological substrate comprising a metal comprises a peptide consisting essentially of an amino acid sequence illustrated by SEQ ID NO:1 14.
  • EXAMPLE 4 Surface-Binding Peptide Oligomers Several oligomers of different surface-binding peptides were synthesized. Briefly, the oligomers were built on a lysine MAP core and comprised of two and four peptide modules, respectively, of a surface-binding peptide. In an illustrative example, this core matrix was used to generate a peptide dimer and peptide tetramer using, in each branch, a monomeric peptide consisting essentially of the amino acid sequence of SEQ ID NO:1 13. The oligomers were synthesized sequentially using solid phase chemistry on a peptide synthesizer.
  • the synthesis was carried out at a 0.05 mmol scale which ensures maximum coupling yields during synthesis.
  • the biotin reporter moiety was placed at the C-terminus of the molecule, and was appended by a short linker containing glycine and serine residues to the lysine core.
  • Standard Fmoc/ t-Bu chemistry was employed using AA/HBTU/ HOBt/NMM (1 :1 :1 :2) as the coupling reagents (AA is amino acid; HOBt is O-Pfp ester/1 - hydroxybenzothazole; HBTU is N-[1 H-benzotriazol-1-yl)(dimethylamino) methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; NMM is N-methylmorpholine). Amino acids were used in 5-10 fold excess in the synthesis cycles, and all residues were doubly, triply or even quadruply coupled depending upon the complexity of residues coupled.
  • HPLC high performance liquid chromatography
  • the polymers were also further analyzed by mass spectrometry for before subjecting each to final purification by HPLC.
  • the fractions containing the desired product were pooled and lyophilized to obtain a fluffy white powder (> 98% purity).
  • the illustrated oligomers can be represented by the following sequences.
  • Various truncations of the metal surface-binding peptide SEQ ID NO: 101 were synthesized. The resulting peptides were tested for binding affinity to stainless steel balls in comparison with the full-length parent sequence (SEQ ID NO: 101 ) and a poor metal-binding sequence as a negative control. Binding affinity was determined according to the following procedure. The wells of a 96-well polypropylene plate were blocked with 350 ⁇ l BSA 1 % in PBS for 30 mins at 2O 0 C with 500 rpm shaking. Freshly cleaned 3/32" stainless steel beads that had been sonicated in acetone were added to the wells of the plate.
  • Sequential 1/3 dilutions of each of the peptides in PBS starting at 10 ⁇ M were prepared directly in the wells of the polypropylene plate containing the beads. The final volume in each well was 200 ⁇ l. The plate was incubated for 1 h at 2O 0 C with 500 rpm shaking to allow for the binding to occur. The beads were washed 3 times with 250 ⁇ l PBS.
  • a direct immunoassay was used to determine the truncated surface- binding peptide affinity for the non-biological metal substrate.
  • Two hundred ⁇ l of streptavidin AP at 1/200 in TBS + 1 % BSA was added to each bead. The plate was incubated at room temperature for 20 mins. The beads were washed 3 times with 250 ⁇ l TBS-Tween. The beads were transferred to a clean polypropylene plate. Two hundred ⁇ l of PNPP was added to all the beads. When color had developed, the solution in each well was transferred to the corresponding well of a clear plate and the OD405 nm was read.
  • Table V shows an alignment of the metal surface-binding peptide SEQ ID NO:101 with truncation variants SEQ ID NOs:1 17-120 (from Table IV where the binding affinity was not significantly reduced), along with metal surface-binding peptide SEQ ID NO:102 and the second-generation metal surface-binding peptides SEQ ID NOs:104-1 12, and, in addition, aligned with metal surface-binding peptide sequences isolated using phage display SEQ ID NOs:68, 71-73, 77, 80-81 and 97-99.
  • the surface-binding peptides shown in Table V each have an EC50 of ⁇ 1 ⁇ M for binding to metal surfaces.
  • the "GSSGK” sequence functioned as a linker sequence for attachment of a biotin molecule to enable detection of the surface-binding peptides in the EC50 experiments. Accordingly, the C-terminal "K” residue was not charged in the EC50 determinations and, thus, not included in the count of positively charged residues.
  • an amino acid sequence domain comprising at least 8 amino acid residues, wherein at least 5 or 6 of the residues are positively charged residues (i.e. K, R or H), wherein in the case of a total of 5 positively charged residues, 3 of the charged residues are lysine, and in the case of 6 or more positively charged residues, at least 2 of the charged residues are lysine. Further, the distance between (and not including) the first and the fifth or sixth of the positively charged residues in the consensus sequence is at least 6 amino acids.
  • the first and the fifth or sixth of the positively charged residues in the consensus sequence are highlighted in black in the sequences listed in Table V.
  • the distance between the first and the fifth or sixth positively charged residue in the consensus metal surface-binding peptide domain sequence can be 6, 7, 8, 9, 10, 1 1 or more amino acid residues.
  • a preferred metal surface-binding peptide sequence of the presently disclosed subject matter comprises a metal-surface binding domain that conforms to the foregoing consensus rules derived from the sequences in Table V.
  • the consensus metal-surface binding domain can preferably range from 8 - 13 amino acid residues in length.
  • additional amino acid residues or other modifying groups can be present at either or both the N-terminal and C-terminal sides of the consensus metal binding domain sequence without affecting the surface-binding activity of the metal surface-binding peptide.
  • the amino acids of the metal surface-binding peptides can be either the L or D form of chiral amino acids or a modified amino acid as described in the presently disclosed subject matter.
  • the surface-binding peptides of the presently disclosed subject matter were modified at the N-terminus according to the following procedure.
  • the peptide SEQ ID NO: 101 was modified with fatty acid aminoundecanoic acid (AUD) repeats.
  • the modified peptide was synthesized using standard FMOC chemistry with t-butyl protecting groups and the AUD molecules were incorporated into the synthesis process directly on solid support (resin).
  • the peptide was synthesized by solid-phase peptide synthesis techniques on a Rainin Symphony Peptide Synthesizer using standard Fmoc chemistry. N-a-Fmoc-amino acids were purchased from Novabiochem. After all residues were coupled, simultaneous cleavage and side chain deprotection was achieved by treatment with a trifluoroacetic acid (TFA) cocktail.
  • TFA trifluoroacetic acid
  • Fmoc-AUD-C0 2 H (Peptide International) was activated using 0.2 M HOBt solution in NMP and was manually coupled sequentially at the N-terminus of the peptide resin using TBTU/NMM method. Following each coupling, the Fmoc group was removed using 20% piperidine in DMF and the resin subsequently coupled further with FmOC-AUD-CO 2 H untill completion of the reaction as judged by ninhydhn test. The terminal Fmoc was removed before subjecting the peptide resin to full cleavage using TFA cocktail.
  • the crude linear peptide was cyclized using the iodine oxidation method and the crude cyclic peptide was purified by prep RP- HPLC on a C-18 Kromasil column. The final product was further characterized by electrospray mass spectrometry.
  • Methods for producing microparticles can include, but are not limited to, solvent precipitation, solvent evaporation, spray drying, crystallization, melt extrusion, compression molding, hot melt encapsulation, and phase inversion encapsulation, using techniques well known in the art.
  • Methods for producing liposomes can include, but are not limited to, reverse-phase evaporation, hydration of dried lipids, solvent or detergent removal, double emulsion preparation, fusion, freeze-thawing, and lyophilization, using techniques well known in the art.
  • liposomes such as those comprising oil in water emulsions, are typically formed from amphipathic lipids or phospholipids having hydrophobic and polar head group moieties, and which can form spontaneously into bilayer vesicles in water.
  • Liposomes can be fabricated according to standard techniques well known in the art. One way of forming liposomes involves suspending a suitable lipid in an aqueous medium, followed by sonication of the mixture.
  • the hydrophobic tail of the surface-binding peptide is trapped between the hydrophobic chains of the amphipathic lipid (i.e., in the shell-like bilayer or multilayer of the vesicle formed), with the remaining portion of the surface- binding peptide (which is more hydrophilic than the hydrophobic tail) being orientated toward the exterior, polar surface of the vesicle formed, and available for binding specifically to non-biological substrate.
  • Hydrophobic tails of the surface-binding peptides of the presently disclosed subject matter include, for example, but are not limited to fatty acid molecules including lauric acid, myhstic acid, palmitic acid and undecanoic acid.
  • a biologically active agent e.g., with a hydrophobic portion
  • Carrying a biologically active agent can comprise encapsulation of the biologically active agent within the drug delivery vehicle, biologically active agent impregnated in the structure of the drug delivery vehicle (e.g., all or a portion of biologically active agent is dispersed within the layer(s) forming the matrix of the drug delivery vehicle), biologically active agent coupled (covalently or noncovalently) to the drug delivery vehicle (e.g., surface, layer, or internal structure), or a combination thereof.
  • a biologically active agent can be carried within the drug delivery vehicle such as by encapsulation.
  • Encapsulation into a drug delivery vehicle can be accomplished by using methods known in the art, such as by: passive entrapment of a water-soluble compound comprising a biologically active agent by hydrating a lipid film or solvent with an aqueous solution containing biologically active agent, in the process of forming drug delivery vehicle; passive entrapment of a hydrophobic or lipophilic compound comprising a biologically active agent by hydrating a lipid film or solvent containing the biologically active agent; or other methods, including, but not limited, to reverse evaporation phase preparation of a drug delivery vehicle, and spray drying.
  • surface-binding peptides having a hydrophobic tail can be incorporated onto drug delivery vehicles comprising microparticles with the hydrophobic tail absorbed onto, and the surface-binding peptide extending from, the surface of the microparticle for availability to bind non-biological substrate.
  • one or more biologically active agents to be loaded in the microparticle, surface-binding peptide, and polymer for forming the microparticle are prepared using the appropriate amounts of biologically active agent-to-surface-binding peptide-to-polymer weight ratios (polymer usually in the range of from about 75% to 98% w/w; biologically active agent is typically 5% to 20% w/w; surface-binding peptide can be 0.01 % to 10% w/w).
  • a water/oil emulsion is prepared by dissolving the polymer in an appropriate organic solvent, and adding biologically active agent and surface- binding peptide (e.g., the biologically active agent and surface-binding peptide in an aqueous solution can be added dropwise to the polymer solution under stirring with a homogenizer. Alternately, surface-binding peptide can be dissolved with the polymer in the appropriate organic solvent, and an aqueous solution containing biologically active agent is added dropwise with mixing). The drop size of the emulsion can be monitored and analyzed by optical microscopy according to methods known in the art.
  • biologically active agent and surface- binding peptide e.g., the biologically active agent and surface-binding peptide in an aqueous solution can be added dropwise to the polymer solution under stirring with a homogenizer. Alternately, surface-binding peptide can be dissolved with the polymer in the appropriate organic solvent, and an aqueous solution containing biologically active agent is
  • microparticles are then obtained by spraying the solutions through the nozzle of a spray-dryer using parameters (inlet air temperature, nozzle size, spray rate feed, etc.) to get microparticles of the appropriate size and shape, using methods known in the art.
  • This procedure can be varied using methods known in the art (e.g., such as altering the ratio of the components, methods of mixing, etc.) to obtain the desired amount of peptide absorbed to the surface of the microparticles.
  • the solvent in the oil phase of the emulsion is evaporated off to provide microparticles.
  • the microparticles carrying biologically active agent and coupled to surface- binding peptide are recovered, washed, lyophilized, and can further be processed to remove residual water and organic solvent.
  • Suitable organic solvents for use in preparing microparticles includes, but are not limited to, cyclohexane, cycloheptane, dimethylsulfoxide, chloroform, methylene chloride, cyclooctane, dimethylformamide, dimethylacetamide, or mixtures thereof.
  • a biologically active agent in an aqueous solution (which can further comprise an emulsifying agent; e.g., polyvinyl alcohol) or as a solid dispersion is added to polymer in a suitable organic solvent ("polymer solution") in forming a primary emulsion.
  • a surfactant or emulsifying agent e.g., polyvinyl alcohol, protein (e.g., albumin, gelatin, and the like), lipophilic emulsifier (e.g., poly(ethylene oxide-co-propyelene oxide), or a combination thereof) can be optionally be added to stabilize the primary emulsion.
  • a emulsifying agent in an aqueous solution is added to and mixed with the primary emulsion to extract the polymer and biologically active agent into the aqueous phase, followed by removing the organic solvent from the mixture (e.g., by adding excess water, and/or under vacuum) and harden the microparticles.
  • the microparticles, carrying biologically active agent can then be recovered by filtration, centhfugation, and then lyophilization.
  • a non-solvent can be used to extract the organic solvent from a primary emulsion.
  • biologically active agent formulated as a powder, is suspended in a polymer phase dissolved in a suitable organic solvent. The suspension is then spray-dried, followed by removal of the organic solvent, and by recovering microparticles carrying biologically active agent.
  • a surface-binding peptide, having binding affinity for a non-biological substrate can be coupled covalently or non-covalently to drug delivery vehicle carrying the biologically active agent.
  • such surface-binding peptide can be impregnated into the structure of the drug delivery vehicle, coupled (covalently or noncovalently) to the surface of the drug delivery vehicle, or a combination thereof; provided, the surface-binding peptide is able to bind with sufficient affinity to the non-biological substrate.
  • the surface-binding peptide having binding affinity for a non-biological substrate can be coupled to the drug delivery vehicle without use of a linker, or can be coupled via a linker.
  • two or more components of a composition according to the presently disclosed subject matter can be covalently coupled (e.g., surface-binding peptide to linker; surface-binding peptide to drug delivery vehicle; surface-binding peptide to a peptide having binding affinity for a synthetic component of the drug delivery vehicle; linker to a peptide having binding affinity for a synthetic component of the drug delivery vehicle; a biologically active agent to a drug delivery vehicle, and a combination thereof).
  • Covalent coupling can be achieved by any means known in the art.
  • a component to be linked can comprise a reactive functionality comprising a free chemical group which can covalently bond with a chemical-reactive group (reactive with the free chemical groups).
  • Free chemical groups include, but are not limited to, a thiol, carboxyl, hydroxyl, amino, amine, sulfo, phosphate, or the like; whereas chemical-reactive groups include, but are not limited to, thiol-reactive group, carboxyl-reactive group, hydroxyl-reactive group, amino-reactive group, amine-reactive group, sulfo-reactive group, or the like.
  • a linker can have: a carboxyl group to form a bond with the first component, and a carboxyl group to form a bond with the second component; or a carboxyl group to form a bond with the first component, and an aldehyde group to form a bond with the second component; or a carboxyl group to form a bond with the first component, and a halide group to form a bond with the second component.
  • a drug delivery vehicle comprising molecules of phosphatidylsehne, phosphatidylethanolamine ("PE”), or dioleoyl PE, provides free amino groups for chemical reaction with one or more amino-reactive groups of a surface- binding peptide (e.g., a carboxyl group) in covalently coupling a surface- binding peptide to a drug delivery vehicle.
  • a surface- binding peptide e.g., a carboxyl group
  • a surface-binding peptide can be non- covalently coupled to the drug delivery vehicle.
  • surface-binding peptide can bind non-covalently with sufficient affinity to the drug delivery vehicle such that extended from the outer surface of the drug delivery vehicle is the portion of the surface-binding peptide that is able to bind to a non- biological substrate.
  • the surface-binding peptide is synthesized to comprise a hydrophobic tail. The hydrophobic tail of the surface-binding peptide is inserted in a hydrophobic layer of the drug delivery vehicle formed, with the remaining, more hydrophilic portion, of the surface- binding peptide extending from drug delivery vehicle for binding specifically to a non-biological substrate.
  • the water in oil in water (w/o/w) approach was used to generate microparticles carrying biologically active agent. This approach has been favored for the encapsulation of hydrophilic molecules (Norton et al., 2005; Farokhzad et al, 2006; Hachicha et al, 2006).
  • vancomycin was encapsulated to test surface-binding peptide-mediated microparticle delivery.
  • an aqueous solution (1 ml.) of drug 100 ⁇ g
  • PLGA poly(D,L-lactic-co-glycolic acid)
  • This mixture formed immiscible layers, which were then mixed by sonication to create an emulsion.
  • the first emulsion was then added to a 1 % solution of polyvinyl alcohol (PVA) in water (10 ml_). This mixture was then emulsified by mechanical stirring. The second emulsion was then added to 5% isopropanol (100 ml_), and the mixture was stirred to extract and evaporate the organic solvent. The microparticles were then isolated by centrifugation and freeze drying. Microparticles were analyzed through light microscopy and SEM to determine the distribution of particle sizes.
  • PVA polyvinyl alcohol
  • a composition according to the invention was formed by coupling a surface-binding peptide having binding affinity for a non-biological substrate to a drug delivery vehicle.
  • drug delivery vehicle comprised commercially prepared polystyrene microparticles (0.04 ⁇ M) carrying a biologically active agent comprising fluorescent dye (a "yellow-green" dye proprietary to a commercial vendor, the dye having excitation/emission maxima of 505/515).
  • a composition according to the presently disclosed subject matter was produced by coupling a biotinylated surface-binding peptide to streptavidin-functionalized drug delivery vehicle (peptide added at a 4:1 ratio to streptavidin-functionalized drug delivery vehicle).
  • a composition was formed using a linker comprising biotin and streptavidin to couple surface-binding peptide to drug delivery vehicle carrying biologically active agent.
  • a composition according to the presently disclosed subject matter was formed in situ.
  • a surface-binding peptide having binding affinity for a non-biological substrate comprising metal (a peptide comprising an amino acid sequence consisting essentially of SEQ ID NO:1 13) was first contacted with non-biological substrate comprising titanium sputter-coated silicon wafers ("titanium disks") to coat the disks with surface- binding peptide.
  • the titanium disks were contacted with a buffered solution containing surface-binding peptide at a concentration of 1 ⁇ M for 30 minutes at room temperature.
  • control disks As a control for non-specific binding, some titanium disks were contacted only with the buffer (not contacted with surface-binding peptide; "control disks") under the same conditions in the assay. The disks were washed with buffer and then contacted with streptavidin-functionalized drug delivery vehicle in PBS, and incubated at room temperature for 30 minutes. The disks were washed in PBS, and detection of biologically active agent on the surface of the metal substrate was visualized using epifluorescence microscopy and digital images using a digital camera. The relative fluorescence was quantified using commercial imaging software measuring mean fluorescence intensity of each sample.
  • the fluorescence intensity was compared between the non-biological substrate contacted only with drug delivery vehicle (no surface-binding peptide present; "control disks”) and the non-biological substrate on to which is formed a composition according to the presently disclosed subject matter in situ. As shown in FIGs. 1 & 2, significantly more fluorescence is detected with non-biological substrate onto which is formed a composition according to the presently disclosed subject matter (FIG. 1 , Panel A; FIG. 2, "SBP-[DV(BA)]”) than a non-biological substrate which lacks a composition according to the invention and is only contacted with untargeted (not coupled to surface- binding peptide) drug delivery vehicle carrying biologically active agent (FIG. 2, Panel B; FIG. 2, "control”).
  • composition according to the presently disclosed subject matter demonstrates the ability to bind, localize, and retain biologically active agent to the surface of non-biological substrate for which the surface-binding peptide component of the composition has binding affinity.
  • surface-binding peptide having binding affinity for a non-biological substrate comprising metal was coupled to the drug delivery vehicle in forming a composition according to the invention.
  • Non-biological substrate comprising titanium disks were contacted with a buffered solution containing the composition at a concentration of 1 ⁇ M for 30 minutes at room temperature.
  • control disks some titanium disks were contacted only with the streptavidin-coated fluorescent microparticles (drug delivery vehicle not coupled to surface-binding peptide; "control disks”) under the same conditions in the assay.
  • the disks were washed in PBS, and the non-biological substrate was visualized using epifluorescence microscopy and digital images using a digital camera.
  • surface-binding peptide having binding affinity for a non-biological substrate comprising polymer was coupled to the drug delivery vehicle in forming a composition according to the invention.
  • Non- biological substrate comprising polyethylene teraphthalate suture material was contacted with a buffered solution containing the composition at a concentration of 10 ⁇ M for 30 minutes at room temperature.
  • some suture material was contacted only with the streptavidin-coated fluorescent microparticles (drug delivery vehicle not coupled to surface-binding peptide; "control material”) under the same conditions in the assay.
  • the suture material was washed in PBS, and the non-biological substrate was visualized using epifluorescence microscopy and digital images using a digital camera.
  • the fluorescence intensity was compared between the control material and the suture material coated with the composition according to the presently disclosed subject matter.
  • the composition according to the presently disclosed subject matter (“Panel A”) showed the ability to bind, localize, and retain biologically active agent to the surface of the non-biological substrate by demonstrating significantly more fluorescence, as compared to the control (“Panel B").
  • EXAMPLE 9 Sustained Antimicrobial Activity of Vancomycin Released from Microparticles
  • a method for delivering a biologically active agent to a non-biological substrate using a composition according to the invention (b) a method of coating a surface of a non-biological substrate to provide biologically active agent to the surface in providing a process selected from the group consisting of delivery of biologically active agent to the coated surface, localizing a biologically active agent to the coated surface, retaining a biologically active agent to the coated surface for controlled delivery, and a combination thereof; and (c) a method of applying a composition according to the presently disclosed subject matter to a non-biological substrate.
  • coating a non-biological substrate with a composition according to the presently disclosed subject matter or applying a composition according to the presently disclosed subject matter to a non-biological substrate comprises contacting at least one surface of non-biological substrate with an effective amount of a composition according to the presently disclosed subject matter so that the composition binds specifically to the at least one surface of the non-biological substrate.
  • the composition can be pre-formed (formed prior to applying it to non-biological substrate).
  • the composition can be applied prior to placing a non-biological substrate in position (e.g., such as prior to applying a medical device to an individual in need of the medical device), or can be applied to a non-biological substrate in situ (e.g., such as applying to a medical device already positioned in an individual in need of the medical device).
  • a composition according to the presently disclosed subject matter can be formed directly on the non-biological substrate (in situ or prior to placement in its intended position), rather than pre-formed, by stepwise applying various components of the composition to form the composition on the non-biological substrate.
  • a first step of a process of applying a composition to a non-biological substrate applied to at least one surface of a non-biological substrate is a first component comprising surface- binding peptide having binding affinity for non-biological substrate so that the first component binds specifically to the at least one surface of the non- biological substrate.
  • the first component can comprise surface-binding peptide to be coupled directly (via a linker or without use of a linker) to drug delivery vehicle, or can comprise surface-binding peptide coupled (via a linker or without use of a linker) to a peptide having binding affinity for drug delivery vehicle (e.g., having binding affinity for the a synthetic component on the surface or outer layer of the drug delivery vehicle).
  • a subsequent step in the process of applying the composition to the non- biological substrate, is added drug delivery vehicle carrying biologically active agent.
  • a composition according to the presently disclosed subject matter directly on non-biological substrate by contacting and coupling an effective amount of a component comprising drug delivery vehicle carrying biologically active agent to a component comprising surface-binding peptide bound specifically to non-biological substrate, formed is a composition according to the presently disclosed subject matter directly on non-biological substrate.
  • an effective amount of a component comprising drug delivery vehicle carrying biologically active agent to a component comprising a peptide having binding affinity the drug delivery vehicle, such peptide coupled to surface-binding peptide bound specifically to non-biological substrate formed is a composition according to the presently disclosed subject matter directly on non-biological substrate.
  • compositions or a component of the composition, if forming the composition directly on the non-biological substrate
  • processes are known to include, but are not limited to, mixing, dipping, brushing, spraying, and vapor deposition.
  • a solution or suspension comprising the composition can be applied through the spray nozzle of a spraying device, creating droplets that contact at least one surface of non-biological substrate to which is to be coated with the composition.
  • the non-biological substrate having composition bound specifically thereto can be allowed to dry, and can then be further processed, if desired, prior to use or positioning or placement (e.g., washed in a solution (e.g., water or isotonic buffer) to remove excess composition not bound specifically to the non-biological substrate; by sterilization using any one or methods known in the art for sterilizing non- biological substrate; etc). Steps of this process can be repeated if the composition is being applied step-wise by its various components to form the composition directly on the non-biological substrate.
  • a solution e.g., water or isotonic buffer
  • the non-biological substrate is dipped into a liquid (e.g., solution or suspension, aqueous or solvent) containing the composition (or component thereof) in an amount effective to for the composition to bind specifically to the non-biological substrate.
  • a liquid e.g., solution or suspension, aqueous or solvent
  • the surface is dipped or immersed into a bath containing the composition.
  • Suitable conditions for applying the composition (or a component thereof) include contacting the surface with the liquid containing composition (or component thereof) for a suitable period of time (e.g., ranging from about 5 minutes to about 12 hours; more preferably, ranging from 15 minutes to 60 minutes), at a suitable temperature (e.g., ranging from 4 0 C to about 50 0 C; more preferably, ranging from room temperature to 37 0 C).
  • a suitable period of time e.g., ranging from about 5 minutes to about 12 hours; more preferably, ranging from 15 minutes to 60 minutes
  • a suitable temperature e.g., ranging from 4 0 C to about 50 0 C; more preferably, ranging from room temperature to 37 0 C.
  • the surface or non-biological substrate having composition applied thereon can then be further processed, as necessary for use (washing, sterilization, and the like).
  • the composition according to the presently disclosed subject matter is formulated in a dry powder (e.g., via air drying or lyophilizing the composition) which is then mixed with the non-biological substrate present in a form comprising a solid, powder, paste, filler, binder, gel, sponge, and a combination thereof.
  • a dry powder e.g., via air drying or lyophilizing the composition
  • the non-biological substrate present in a form comprising a solid, powder, paste, filler, binder, gel, sponge, and a combination thereof.
  • these illustrative processes for applying a composition to a non-biological substrate are not exclusive, as other coating and stabilization methods can be employed (as one of skill in the art will be able to select the compositions and methods used to fit the needs of the particular non-biological substrate and purpose).
  • Disks were thoroughly washed and transferred to a clean 96-well plate.
  • Human plasma (American Red Cross) was added to each well and inoculated with 10 4 CFU of S. aureus strain MZ100. Plates were sealed and incubated for 24h at 37 0 C. Bacterial supernatant was removed and the BacTiter-Glo Microbial Cell Viability assay used to quantify bacteria (Promega). The disks were re-challenged with fresh plasma and bacteria each day, for 4 days (FIG. 5).
  • bacterial growth was inhibited in the wells with the surface-binding peptide/microparticle- coated disks (SEQ ID NO: 123 and SEQ ID NO: 124, respectively) as compared to the disks that were coated with microparticles alone and uncoated control disks (FIG. 5).
  • the data indicate that a non-biological metal substrate such as an implant coated with a composition of the presently disclosed subject matter comprising a surface-binding peptide and drug delivery vehicle carrying biological agent such as the drug loaded microparticles in this example, can maintain prolonged antimicrobial efficacy for at least three days in human plasma.
  • metal surface-binding peptide (5 ⁇ M; SEQ ID NO: 123) and vancomycin-loaded microparticle was applied to titanium pins (as described in Example 9 above) and the pins were inserted into silicone tubing containing 30 ⁇ l_ of 10 6 cfu per ml. S. aureus (ATCC 49230). After incubating pins in cultured bacteria overnight, the pin was removed and rolled over TSA plates (see top "On pin” portion of FIG. 6). In addition the broth was aspirated and diluted 10X from left to right, beginning with undiluted culture broth from the tubing (see bottom "Broth dilutions" portion of FIG. 6). These data show very few, if any detectable bacteria remaining in broth or on microparticle + surface-binding peptide coated titanium pins.
  • a coating composition comprising metal surface-binding peptide (AFF- 5061 ; SEQ ID NO: 123) and vanc-microparticles was also tested in a rodent model.
  • the following coating compositions were applied to titanium pins: surface-binding peptide (5 ⁇ M) and microparticles, vancomycin microparticles without peptide (control), or PBS (bare metal control).

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Abstract

The presently disclosed subject matter relates to compositions for delivering biologically active agent to non-biological substrates, the compositions comprising a surface-binding peptide coupled to a drug delivery vehicle, wherein the surface-binding peptide has binding affinity for the non- biological substrate and the drug delivery vehicle carries the biologically active agent. Also provided are methods of applying the compositions by contacting a non-biological substrate with the composition such that the composition binds non-covalently to the non-biological substrate.

Description

COMPOSITIONS FOR DELIVERY OF BIOLOGICALLY ACTIVE AGENTS TO
SURFACES
RELATED APPLICATIONS The presently disclosed subject matter claims the benefit of U.S.
Provisional Patent Application Serial No. 60/952,872 filed July 31 , 2007; the disclosure of which is incorporated herein by reference in its entirety.
GOVERNMENT INTEREST This presently disclosed subject matter was made with U.S.
Government support under Grant No. 1 R43NS062471-01 awarded by the United States National Institutes of Health/National Institutes for Neurological Diseases and Stroke. Thus, the U.S. Government has certain rights in the presently disclosed subject matter.
FIELD OF THE INVENTION
The presently disclosed subject matter relates to compositions and methods for targeted delivery of biologically active agents to a surface of a non-biological substrate. More particularly, the invention is directed to compositions comprising and methods of using surface-binding peptides to target a drug delivery vehicle, carrying biologically active agent, to one or more surfaces of a non-biological substrate, and preferably a non-biological substrate to be introduced, or introduced, into a biological environment.
BACKGROUND OF THE INVENTION
Liposomes and microspheres ("drug carrier") have been used widely as carriers to deliver a variety of therapeutic agents. Encapsulation of the therapeutic agent into a drug carrier can act to provide controlled release of the therapeutic agent as the drug carrier dissolves or biodegrades in a biological environment (e.g., in vivo). However, the efficiency of the delivery of therapeutic agent to a desired cell population to be treated with therapeutic agent has been constrained by the lack of a means for targeting the drug carrier to cells, so that the therapeutic agent can be released in the vicinity of the target cell population to be treated. Peptides have been conjugated to lipids and incorporated into liposome bilayers to improve stability of the liposome (see, e.g., US Patent 6,339,069). Peptides have also been used to target cells in delivering an agent. For example, a peptide having binding specificity for cancer cells has been labeled with a radiopharmaceutical, and used to target human breast cancer cells (see, e.g., Askoxylakis et al., Clin. Cancer Res., 2005, 1 1 :6705- 12); peptides having tumor-binding specificity have been used to direct therapeutic agents to tumor cells (see, e.g., Ruoslahati et al., Curr. Pharm. Des. 2005, 1 1 :3655-60; Lee et al., Cancer Res., 2004, 64:8002-08; Shaddi et al., FASEB J., 2003, 17:256-8). Additionally, drugs have been conjugated to cell- penetrating peptides to facilitate internalization of the conjugated drug by a cell.
Medical devices (including implants and prostheses for medical or dental use) are increasingly utilized for treatment of a variety of disease conditions and in preventative procedures. Medical devices are often intended to provide beneficial functions for extended periods of time, ranging from weeks to years. The general trend is that the longer a device can be utilized without replacement or repair, the better for the individual (e.g., fewer invasive procedures, lower medical costs, etc.). Accordingly, there has also been an increased interest in site-specific drug delivery by coating medical devices with drug, such as by a polymer coating impregnated with drug, in attempts to do one or more of facilitate acceptance by and integration of the medical device to the site it is positioned, and inhibiting detrimental effects on health as a result of positioning the device (e.g., clotting, infection, and the like, depending on the device and site of positioning). A deficiency of some polymer coatings is that a majority of the drug is released over a relatively short period of time, resulting in what is known as "drug dumping". In other polymer coatings, only a small amount of the drug is released from the outer region of the coating, leaving a significant amount of drug in the coating for the lifetime of the medical device. In either case, an effective dose of the drug can be delivered at the site of the medical device for an undesirable period of time (shorter or longer than desired to achieve the full beneficial effect).
Thus, there remains a need to facilitate localization and retention of a biologically active agent on, as well as to modulate the delivery of biologically active agent from surfaces of non-biological substrates such as medical devices, prior to positioning, or once in situ.
SUMMARY OF THE INVENTION This invention relates to the field of coatings for surfaces of non- biological substrates, and preferably of devices. In one embodiment, the presently disclosed subject matter provides a composition for coating a non- biological substrate, wherein the composition comprises a drug delivery vehicle (DV) coupled (directly or via a linker) to surface-binding peptide (SBP), and wherein the drug delivery vehicle is configured to carry biologically active agent (BA). The composition can be contacted with a surface of a non- biological substrate to coat the surface of the substrate such that the biologically active agent, carried by the drug delivery vehicle, is delivered and localized at the surface. Surface-binding peptide is used to target the drug delivery vehicle carrying biologically active agent to a non-biological substrate desired to be targeted. The composition can also comprise a pharmaceutically acceptable carrier.
In another embodiment, the presently disclosed subject matter provides a composition for coating a non-biological substrate, wherein the composition comprises: a drug delivery vehicle (DV) carrying a biologically active agent (BA); a first surface-binding peptide (SBP-i) that binds non- covalently to the non-biological substrate; and, optionally, a second surface- binding peptide (SBP2) having binding affinity to a surface of the drug delivery vehicle (DV). The drug delivery vehicle (DV) can comprise a shell (e.g., outer layer) comprised of a synthetic component (e.g., one or more of a synthetic polymer, synthetic surfactant, synthetic lipid and the like that is not found in nature). The first surface-binding peptide (SBP1) binds has binding affinity for and binds non-covalently to the non-biological substrate. For example, such a non-biological substrate can be a device, container, medical device, array, etc., to be coated for localization and retention of the drug delivery vehicle (DV) carrying the biologically active agent (BA). The second surface-binding peptide (SBP2) is coupled, covalently or non-covalently, to the synthetic component of the shell of the drug delivery vehicle. Accordingly, in this embodiment of the presently disclosed subject matter, a biologically active agent can be delivered at the surface of the non-biological substrate, wherein the first surface-binding peptide is coupled (either directly or via a linker) to the second surface-binding peptide. The composition can also comprise a pharmaceutically acceptable carrier. One aspect of the presently disclosed subject matter provides methods and compositions for providing biologically active agent-releasing coatings for non-biological substrates such as medical devices.
Another aspect of the presently disclosed subject matter provides for a delivery system for biologically active agent that is capable of facilitating the delivery, localization, and retention of biologically active agent to a non- biological substrate such as a medical device prior to positioning or once in situ.
Accordingly, some embodiments of the presently disclosed subject matter can be described by a composition of the formula: SBP1 - L -SBP2 [DV(BA)], wherein
SBP is, when present, a surface-binding peptide of 8 to 60 amino acids; SBP1 is the SBP surface-binding peptide comprising a surface-binding peptide domain having binding affinity for a surface comprising a non-biological substrate; SBP2 can be present or absent and is the SBP surface-binding peptide having binding affinity for a synthetic component of a drug delivery vehicle (DV); L can be present or absent and comprises a linker; and the DV comprises a drug delivery vehicle that carries a BA, wherein the BA is one or more biologically active agents; wherein in the presence of the SBP2, the DV is noncovalently coupled to the SBP2; wherein in the absence of the SBP2 and in the presence of the L, the SBPi is coupled to the DV either covalently or nocovalently; and wherein in the absence of both the SBP2 and the L, the SBPi is coupled directly to the DV either covalently or noncovalently. DV is a drug delivery vehicle that carries BA, wherein BA is one or more biologically active agents ("biologically active agent"). DV can carry BA by one or more processes selected from the group consisting of coupling BA and DV together (e.g., to, in, on, within, or a combination thereof), encapsulation of BA by DV, and a combination thereof. Coupling can be by one or more of covalent and/or non-covalent attachment of BA and DV (the latter including but not limited to trapping, embedding and a combination thereof, and can be via one or more of hydrophobic and electrostatic interactions and can be via the structure of DV (e.g., outer layer or surface (e.g., shell), or internal lattice, whether physical, chemical, or a combination thereof). In an embodiment where L and SBP2 are absent, SBP1 and DV are directly (e.g., without use of a linker) coupled together either covalently or non-covalently. In an embodiment where L is absent and SBP2 is present, SBP1 and SBP2 are covalently coupled together.
In another aspect of the presently disclosed subject matter, methods are provided for coating a surface of a non-biological substrate with a composition of the presently disclosed subject matter. In another aspect of the presently disclosed subject matter, non-biological substrates are provided having a surface coated with a composition according to the presently disclosed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a digital image comparing a non-biological substrate onto which has been formed a composition according to the presently disclosed subject matter carrying a biologically active agent comprising a fluorescent dye (Panel A), with a non-biological substrate to which has been contacted with fluorescent dye in a drug delivery vehicle (Panel B).
FIG. 2 shows a graph comparing the mean density of fluorescence of a non- biological substrate onto which has been formed a composition according to the presently disclosed subject matter carrying a biologically active agent comprising a fluorescent dye ("SBP-[DV(BA)]"), with a non-biological substrate to which has been applied fluorescent dye in a drug delivery vehicle ("control").
FIG. 3 shows an image comparing a non-biological substrate comprising a metal onto which has been applied a composition according to the presently disclosed subject matter having binding affinity for metal and carrying a biologically active agent comprising a fluorescent dye (Panel A), with a non- biological substrate comprising a metal to which has been applied fluorescent dye in a drug delivery vehicle (Panel B). FIG. 4 shows an image comparing a non-biological substrate comprising a polymer onto which has been applied a composition according to the presently disclosed subject matter having binding affinity for polymer and carrying a biologically active agent comprising a fluorescent dye (Panel A), with a non-biological substrate comprising a polymer to which has been applied fluorescent dye in a drug delivery vehicle (Panel B).
FIG. 5 is a graph showing extended release of antimicrobial activity from surface-binding peptide-delivered microparticles. Disks were coated and challenged with plasma and bacteria. At each time point (days 1 through 4), disks were re-challenged with plasma and bacteria. Bacterial growth was inhibited in the wells with the surface-binding peptide (AFF-5061 (SEQ ID NO:
124) and AFF-5103 (SEQ ID NO: 125)) / microparticle-coated disks.
FIG. 6 shows an image of TSA plates displaying the antimicrobial effects of surface-binding peptide-delivered vancomycin microparticles. Metal pins were coated with a composition comprising surface-binding peptide AFF-5061
(SEQ ID NO: 124) and microparticles carrying the antibiotic vancomycin. The metal pins were then incubated with bacteria and removed pins were rolled over the TSA plates and pin culture broth dilutions were plated. The image illustrates the antimicrobial effects of the coating composition against both the adherent (on pin) and suspended bacteria (broth dilutions).
FIG. 7 is a graph showing the in vivo antimicrobial effects of a coating composition comprising metal surface-binding peptide AFF-5061 (SEQ ID
NO: 124) and microparticles carrying the antibiotic vancomycin. Metal pins were coated with a composition comprising surface-binding peptide AFF-5061 (SEQ ID NO: 124) and microparticles carrying the antibiotic vancomycin and the coated pins were tested against S. Aureus in a rat tibia infection model.
The y axis of the graph shows colony forming units (CFU) remaining following pin sonication after removal from the animals. (Bars at 50O=CFU ≥ 500).
Total N = 15 per group.
DETAILED DESCRIPTION OF THE INVENTION The presently disclosed subject matter provides compositions comprising a surface-binding peptide coupled to a drug delivery vehicle carrying biologically active agent, methods of coating surfaces of non- biological substrates with a coating composition, and non-biological substrates coated with the compositions.
In one embodiment, a drug delivery vehicle is provided for delivery of biologically active agent to a surface of a non-biological substrate, wherein the drug delivery vehicle comprises a microparticle carrying biologically active agent, and a peptide coupled to the microparticle; wherein the peptide has binding affinity for and binds non-covalently to the surface of the non- biological substrate. In another embodiment, a drug delivery vehicle is provided for delivery of biologically active agent to a surface of a non- biological substrate, wherein the drug delivery vehicle comprises: (a) a shell (or surface-exposed outer layer) comprised of a synthetic component, and wherein the drug delivery vehicle carries biologically active agent; (b) a first surface-binding peptide that binds specifically and noncovalently to the non- biological substrate; and (c) a second surface-binding peptide having binding affinity for, and noncovalently coupled to, the synthetic component of the shell of the drug delivery vehicle; wherein the first and second surface-binding peptides are covalently coupled together either directly or via a linker.
Definition Section While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the invention.
The terms "first" and "second" are used herein for purposes of the specification and claims for ease of explanation in differentiating between two different molecules, and are not intended to be limiting the scope of the presently disclosed subject matter, nor imply a spatial, sequential, or hierarchical order unless otherwise specifically stated.
The term "non-biological substrate" is used herein for purposes of the specification and claims to mean a substrate that is not a quality or component of a living system. A non-biological substrate can comprise any form suitable to its intended use including but not limited to a container, reactor, device, array, medical device, particle, or the surface of a non- biological substrate contained in a liquid. Representative non-biological substrates include, but are not limited to, plastic, silicone, synthetic polymer, metal (including mixed metal alloys), metal oxide (e.g., glass), non-metal oxide, ceramic, carbon-based materials (e.g., graphite, carbon nanotubes, carbon "buckyballs", and metallo-carbon composites), and combinations thereof. In addition to medical devices, as described more in detail herein, other non-biological substrates that can benefit from the presently disclosed subject matter include, but are not limited to, (a) medical supplies, such as medical surgical gowns, diapers, incontinence apparel, drapes, bandages, dressings, sponges, covers, and the like; (b) laboratory equipment, such as bioreactors, fermentors, test tubes, assay plates, arrays, culture containers, and the like; (c) packaging or product protection (e.g., packaging materials, coverings (such as wraps)), such as applied to perishables such as foods, drugs, and medical devices; (d) cleaning supplies (e.g., for use in one or more of hospitals, industries, and households) such as sanitary wipes, cloths, wet and dry wipes, paper-based tissues, and sponges; and hygienic coatings for use with table tops, counter tops, door knobs, door handles, fixtures, and the like.
The term "metal" is used herein for purposes of the specification and claims to mean one or more compounds or compositions comprising a metal represented in the Periodic Table (e.g., a transition metal, alkali metals, and alkaline earth metals, each of these comprise metals related in structure and function, as classified in the Periodic Table), and can further refer to a metal alloy, a metal oxide, a silicon oxide, and bioactive glass. Examples of preferred metals include, but are not limited to, titanium, titanium alloy, stainless steel, aluminum, zirconium alloy metal substrate (e.g., Oxinium™), cobalt chromium alloy, gold, silver, rhodium, zinc, tungsten, platinum, rubidium, and copper. A preferred type or composition of metal can be used in accordance with the presently disclosed subject matter to the exclusion of a type or composition of metal other than the preferred type or composition of metal.
The term "polymer" " is used herein for purposes of the specification and claims to mean a molecule or material comprised of repeating structural units (a structural unit typically referred to as a monomer) connected by covalent chemical bonds. Depending on its intended use, a polymer can be biodegradable (e.g., one or more of self-dissolving, or bioresorbable, or degradable in vivo) or non-biodegradable; or synthetic (manufactured, and not found in nature) or natural (found in nature, as made in living tissues of plants and/or animals).
Non-limiting examples of suitable synthetic polymers described as being biodegradable include: poly-amino acids; polyanhydhdes including maleic anhydride polymers; polycarboxylic acid; some polyethylenes including, but not limited to, polyethylene glycol, polyethylene oxide; polypropylenes, including, but not limited to, polypropylene glycol, polypropylene fumarate; one or more of polylactic acid or polyglycolic acid (and copolymers and mixtures thereof, e.g., poly(L-lactic acid) (PLLA), poly(D,L,-lactide), poly(lactic acid-co-glycolic acid), 50/50 (DL-lactide-co-glycolide)); polyorthoesters; polydioxanone; polyphosphazenes; polydepsipeptides; one or more of polycaprolactone (and co-polymers and mixtures thereof, e.g., poly(D,L- lactide- co-caprolactone) or polycaprolactone co-butylacrylate; polyhydroxybutyrate valerate and blends; some polycarbonates (e.g., tyrosine-derived polycarbonates and arylates), polyiminocarbonates, calcium phosphates; cyanoacrylate; some polyamides (including nylon); polyurethane; polydimethylthmethylcarbonates; synthetic cellulosic polymers (e.g, cellulose acetate, cellulose butyrate, cellophane); and mixtures, combinations, and copolymers of any of the foregoing. Representative natural polymers described as being biodegradable include macromolecules (such as polysaccharides, e.g., alginate, starch, chitosan, cellulose, or their derivatives (e.g., hydroxypropylmethyl cellulose); proteins and polypeptides, e.g., gelatin, collagen, albumin, fibrin, fibrinogen); polyglycosaminoglycans (e.g. hyaluronic acid, chondroitin sulfate); and mixtures, combinations, and copolymers of any of the foregoing.
Non-limiting examples of suitable synthetic polymers described as being non-biodegradable include: inert polyaryletherketones, including polyetheretherketone ("PEEK"), polyether ketone, polyetherketoneketone, and polyetherketoneetherketoneketone; polyurethanes; polystyrene, and styrene- ethylene/butylene-styrene block copolymers; polyisobutylene copolymers and styrene-isobutylene-styrene block copolymers; polyvinylpyrrolidone; polyvinyl alcohols; copolymers of vinyl monomers; polyvinyl ethers; polyvinyl aromatics; polyethylene oxides; polyesters including polyethylene terephthalate; some polyamides; polyacrylamides; polyethers including polyether sulfone; polyalkylenes including polypropylene, polyethylene; copolymers of ethylene and polypropylene; some polycarbonates, silicone and silicone rubber; siloxane polymers; polytetrafluoroethylene; expanded polytetrafluoroethylene (e-PTFE); nylons and related polyamide copolymers; nylon; fluohnated ethylene propylene; hexafluroropropylene, polymethylmethacrylate (PMMA); 2-hydroxyethyl methacrylate (PHEMA); polyimides; polyethyleneterephthalate; polysulfone, and polysulfides; and mixtures, combinations, and copolymers (including cross-linked copolymers) of any of the foregoing.
The term "ceramic" is used herein for purposes of the specification and claims to mean inorganic non-metallic materials whose formation is due to the action of heat. Suitable ceramic materials include but are not limited to silicon oxides, aluminum oxides, alumina, silica, hydroxyapatites, glasses, quartz, calcium oxides, calcium phosphates, indium tin oxide (ITO), polysilanols, phosphorous oxide, and combinations thereof. The term "effective amount" is used herein, in referring to a composition according to the presently disclosed subject matter and for purposes of the specification and claims, to mean an amount sufficient of the composition so as to (a) mediate binding of the composition to at least one surface of the non-biological substrate; and (b) promote attachment of a drug delivery vehicle, carrying a biologically active agent, to at least one surface of the non-biological substrate. The term "effective amount" is used herein, in referring to biologically active agent in a composition according to the presently disclosed subject matter and for purposes of the specification and claims, to mean an amount of the biologically active agent effective for modulating, preventing, ameliorating, or treating the condition and/or disease intended by administration of the biologically active agent.
The term "individual", as used herein for purposes of the specification and claims, refers to either a human or an animal.
The term "biologically active agent", as used herein for purposes of the specification and claims, refers to one or more agents selected from the group consisting of a therapeutic agent, an agent having biological activity, a diagnostic agent, a prophylactic agent, a chemical catalyst, and a combination thereof. Representative biologically active agents include, but are not limited to, growth factor, cells, biologically active drug, hormone, vitamin, nucleic acid molecule encoding any of the foregoing, nucleic acid molecules having biological activity. Representative hormones include, but are not limited to sex hormones (e.g., estrogen, progesterone, testosterone), thyroid hormones, insulin, adrenal cortical and pituitary hormones, and growth hormones. Representative cells include, but are not limited to, one or more cells or cell types, and preferably cells of human origin, such as stem cells, osteoprogenitor stem cells, mesenchymal stem cells, osteocytes, osteoblasts, osteoclasts, periosteal stem cells, endothelial cells, stromal cells, hematopoietic progenitor cells, adipose tissue precursor cells, cord blood stem cells, myoblasts, Schwann cells, oligodendrocytes, insulin producing cells (e.g., beta cells), neuroprogenitor cells, and a combination thereof. Representative vitamins include any one or more of fat-soluble vitamins (A, D, E and K), and water-soluble (8 B vitamins and vitamin C), and their derivatives (e.g., vitamin D derivatives include 1 , 25-di hydro xyvitamin D3, 1α- hydroxyvitamin D2). Diagnostic agents include, but are not limited to, radiolabels, radiopaque compounds, colohmethc reagents, dyes, fluorophores, fluorescent molecules, fluorescent nanocrystals, luminescent molecules, chromophores, and the like. Catalysts can be selected from the group consisting of heterogeneous catalysts, homogeneous catalysts, biocatalysts (e.g., enzymes in metabolic or biological pathways), electrocatalysts (e.g., metal-rich catalysts used in fuel cells, or energy generation), organocatalysts (simple organic molecules used as catalysts in chemical reactions), as known to those skilled in the art.
"Growth factor" is a term used to refer to one or more growth factors or cytokines. Representative growth factors can include, but are not limited to, bone morphogenetic protein (BMP, including the family of BMPs, such as BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-1 1 , BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, and BMP-18), transforming growth factor beta (TGF-beta), transforming growth factor alpha (TGF-alpha), vascular endothelial cell growth factor (VEGF, including its variants), epidermal growth factor (EGF), fibroblast growth factor (e.g., basic fibroblast growth factor, acidic fibroblast growth factor, FGF-1 to FGF-23), epidermal growth factor (EGF), insulin-like growth factor (I or II), interleukin-l, interferon, tumor necrosis factor, nerve growth factor, neurotrophins, platelet-derived growth factor (PDGF), heparin-binding growth factor (HBGF), hepatocytic growth factor, keratinocyte growth factor, macrophage colony stimulating factor, growth and differentiation factor (e.g., GDF4 to GDF8), isoforms thereof, biologically active analogs thereof, and a combination thereof. Typically, a biological analog has an amino acid sequence having from about 1 % to about 25% of the amino acids substituted, as compared to the amino acid sequence of the peptide growth factor from which the analog was derived. For peptides less than or equal to 50 amino acids in length, typically a biologically active analog thereof has between 1 and 10 amino acid changes, as compared to the amino acid sequence of the peptide from which the analog was derived.
Representative drugs (listed by standard classes) include, but are not limited to, analgesics/antipyretics (e.g., aspirin, morphine, oxycodone, codeine, ibuprofen); antibiotics (classes of antibiotics are known to include, but are not limited to, penicillins (e.g., penicillin G, penicillin V, ampicillin, methicillin, oxacillin, amoxicillin, amoxicillin-clavulanate, ticarcillin, nafcillin, cloxacillin, piperacillin-tazocbactam, and dicloxacillin), cephalosporins and cephams (e.g., cefazolin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefaclor, cefprozil, loracarbef, cefadroxil, cephalexin, cephradineze, cefepime, cefpirome, cefataxidime pentahydrate, ceftazadime, cefteram, cefotiam, cefamandole, cefotetan, cefalexin, cefaparin, cefbuperazone, cefcapene, cefditohn, ceftamet, cefnetrazole, cefminox, cefoperazone, ceforanide, cefotiam, cefoxitin, cefpimazole, cefpiramide, cefradine, cefroxadine, cefsulodin), aminoglycosides (e.g., amikacin, gentamycin, tobramycin, netromycin, streptomycin, kanamycin, paromomycin, neomycin), oxazolidinones (e.g., linezolid), streptogramins (e.g., quinupristin, dafopristin, synercid, phstinamycin), sulfonamides (e.g., co-trimoxazole, sulfamethoxasol, sulfadiazine, sulfadoxine, trimethoprim), tetracyclines (tetracycline, demeclocycline, minocycline, doxycycline), macrolides (erythromycin, clarithromycin, azithromycin, axithromycin, dirithromycin, troleandomycin, oleandomycin, roxithromycin, telithromycin), carbapenems (imipenem, meropenem, ertapenem, panipenem/ betamipron), ketolides, fluoroquinolones/ quinolones (e.g., ciprofloxacin, levofloxacin, ofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, pazufloxacin), and glycopeptides (e.g., vancomycin, teicoplanin, daptomycin, oritavancin), antimicrobial peptides (indolicidin, defensins (beta or alpha), cathelicidin, plectasin, caerin, maculatin, KIK peptides (so called because of the prevalence of lysine (K) and isoleucine (I) in their amino acid sequences), magainin, gramicidin, NK-2, dermaseptin, defensin, bacteriocin, pleurocidin, LL-37, halocidin, dermicidin, mucocidin, heparin-binding protein peptide, and the like), nubiotics (DNA or RNA-based antimicrobial agents); antifungal agents (e.g., ketoconazole, grisefulvin, amphotericin B, nystatin, etc.); antiviral agents (e.g., acyclovir, ribavirin, interferon, etc.); antimicrobial agents (e.g., thclosan); antihypertensive agents (e.g., propanolol, nifedipine, reserpine, thmethaphan, etc.); ant-inflammatohes (COX-2 inhibitors (e.g, celecoxib, lumiracoxib, etoricoxib, etc.), cortisone, indomethicin, flubriprofen, dexamethasone, , prednisone, etc.); antineoplastics/ antiproliferative (e.g., doxorubicin, cyclophosphamide, inhibitors of VEGF, inhibitors of EGFR (epidermal growth factor receptor), cisplatin, tamoxifen, taxanes (e.g., paclitaxel, docetaxel, etc.), rapamycin (sirolimus), campothecin, 5-fluorouracil, gemcitabine, vinblastine, bleomycin, etoposide, methotrexate, etc.); antiarrhythmics (e.g., verapamil, digoxin, digitoxin, procainamide, quinidine sulfate, etc.); anticoagulants (low molecular weight heparin, warafin, thrombin (Factor Na) inhibitors, and Factor Xa inhibitors, etc.); antiplatelet agents (e.g., adenosine diphosphate receptor antagonists that include clopidogrel, ticlopidine, and the like, a glycoprotein llb/llla inhibitor, vitronectin receptor antagonists, etc.); immunomodulatory agent (e.g., alpha, beta, gamma interferons, and their derivatives (e.g., beta- seron), calcineuhn inhibitors, mTOR inhibitors, antiproliferatives, corticosteroids, etc.); antioxidants (polyphenols, resveratrol, probucol, vitamins C and E, coenzyme Q-10, glutathione, L-cysteine and N- acetylcysteine, etc.); a thrombolytic agent (e.g., urokinase, streptokinase, a tissue plasminogen activator, etc.), an antithrombogenic agent (e.g., hirudin, hirulog, argatroban, D-phenylalanyl-L-poly-L-arginyl chloromethyl ketone, etc.); calcium channel blockers (e.g., amilohde, verapamil, nifedipine, etc.); cell expression modulators (e.g., genes encoding biologically active proteins, antisense DNA, antisense RNA, microRNA (miRNA), silencing RNA (siRNA), interfering RNA (RNAi), gene modulating RNA, modulators of DNA methylation, etc.); hypoglycemic agents (insulin (human, synthetic, or animal), glyburide, chloropropamide, glipizide, tolbutamide, etc.), hypolipidemic agents (e.g., lovastatin, pravastatin, probucol, clofibrate, etc.), . A preferred biologically active agent can be used in accordance with the presently disclosed subject matter to the exclusion of a biologically active agent other than the preferred biologically active agent.
The term "time sufficient for binding" generally refers to a temporal duration sufficient for non-covalent and specific binding of a surface-binding peptide described herein to a non-biological substrate for which the peptide has binding affinity, as known to those skilled in the art. Based on the affinity/binding specificity of a surface-binding peptide used in a composition according to the presently disclosed subject matter, generally a time sufficient for binding a composition according to the presently disclosed subject matter to a non-biological substrate ranges from about 5 minutes to no more than 60 minutes.
The term "composition" is used herein, in reference to a composition of the presently disclosed subject matter and for purposes of the specification and claims, to refer to a composition comprising formula I: A composition of the formula: SBP1 - L -SBP2 [DV(BA)], wherein
SBP is, when present, a surface-binding peptide of 8 to 60 amino acids; SBP1 is the SBP surface-binding peptide comprising a surface-binding peptide domain having binding affinity for a surface comprising a non-biological substrate; SBP2 can be present or absent and is the SBP surface-binding peptide having binding affinity for a synthetic component of a drug delivery vehicle (DV); L can be present or absent and comprises a linker; and the DV comprises a drug delivery vehicle that carries a BA, wherein the BA is one or more biologically active agents; wherein in the presence of the SBP2, the DV is noncovalently coupled to the SBP2; wherein in the absence of the SBP2 and in the presence of the L, the SBPi is coupled to the DV either covalently or nocovalently; and wherein in the absence of both the SBP2 and the L, the SBPi is coupled directly to the DV either covalently or noncovalently. DV is a drug delivery vehicle that carries BA, wherein BA is one or more biologically active agents ("biologically active agent"). DV can carry BA by one or more processes selected from the group consisting of coupling BA and DV together (e.g., to, in, on, within, or a combination thereof), encapsulation of BA by DV, and a combination thereof. Coupling can be by one or more of covalent and/or non-covalent attachment of BA and DV (the latter including but not limited to trapping, embedding and a combination thereof, and can be via one or more of hydrophobic and electrostatic interactions and can be via the structure of DV (e.g., outer layer or surface (e.g., shell), or internal lattice, whether physical, chemical, or a combination thereof).
In an embodiment where L and SBP2 are absent, SBPi and DV are directly (e.g., without use of a linker) coupled together either covalently or non-covalently. In an embodiment where L is absent and SBP2 is present, SBP1 and SBP2 are covalently coupled together. It is understood that a composition according to the invention can comprise drug delivery vehicle carrying biologically active agent, wherein drug delivery vehicle comprises carrying one biologically active agent, or carrying two or more biologically active agents. For example, where two or more biologically active agents are carried by the drug delivery vehicle, they can be of the same class (e.g., 2 different antibacterial agents, such as vancomycin and gentamycin) or they can be of different classes (e.g., antiplatelet agent, and a growth factor (e.g., VEGF)) of biologically active agents. Likewise, a composition can comprise drug delivery vehicle comprising two or more types of drug delivery vehicles, each type of drug delivery vehicle differing from another type of drug delivery vehicle in biologically active agent carried (and can also differ by a property selected from the group consisting of composition of drug delivery vehicle, release profile, stability, surface-binding peptide coupled thereto, and a combination thereof).
Accordingly, it can be understood that a composition can comprise one or more of either or both the first and second surface-binding peptides according to the following formula: SBP1 — L -SBP2 [DV(BA)] previously described herein above. For example, a composition can be "homogeneous" and comprise only first and/or second surface-binding peptides having the same amino acid sequence. In another embodiment, a composition can comprise, for example, two or more first surface-binding peptides SBP1 that differ in amino acid sequence but bind specifically to the same non-biological substrate. Similarly, a composition can comprise in another embodiment, for example, two or more first surface-binding peptides SBP1 that differ in amino acid sequence and bind different non-biological substrates. In another embodiment, a composition can comprise, for example, two or more second surface-binding peptides SBP2 having different amino acid sequences and binding the same drug delivery vehicle DV. Similarly, a composition can comprise in another embodiment, for example, two or more second surface- binding peptides SBP2 having different amino acid sequences and binding different drug delivery vehicles DV. Further, a composition can comprise any combination of the foregoing examples of first and second surface-binding peptides having different amino acid sequences and binding either the same of different non-biological substrates and/or drug delivery vehicles. In addition, in some embodiments a surface-binding peptide used in accordance with the presently disclosed subject matter can also comprise an oligomer (e.g., dimer, multimer) of the same peptide amino acid sequence or of two or more different amino acid sequences. For example, in one embodiment of the presently disclosed subject matter, two or more surface- binding peptides are coupled together (e.g., by one or more of non-chemical physical bonds, a chemical bond either directly or through a linking or other chemical modifier group, (via chemical synthesis or recombinant expression) in such a way that each retains its respective function to bind to the respective non-biological substrate for which it has binding affinity. Such coupling can include forming a multimeric molecule having two or more peptides having binding affinity to the same non-biological substrate, two or more peptides having binding affinity for different non-biological substrates and/or different drug delivery vehicles, and combinations thereof.
For example, using standard reagents and methods known in the art of peptide chemistry, two peptides can be coupled via a side chain-to-side chain bond (e.g., where each of the peptides has a side chain amine (e.g., such as the epsilon amine of lysine)), a side chain-to-N terminal bond (e.g., coupling the N-terminal amine of one peptide with the side chain amine of the other peptide), a side chain-to-C-terminal bond (e.g., coupling the C-terminal chemical moiety (e.g., carboxyl) of one peptide with the side chain amine of the other peptide), an N-terminal-to-N-terminal bond, an N-terminal to C- terminal bond, a C-terminal to C-terminal bond, or a combination thereof. In synthetic or recombinant expression, two or more peptides can be coupled directly to a peptide by synthesizing or expressing the two or more peptides as a single peptide. The coupling of two or more peptides can also be via a linker to form surface-binding peptide used in the composition according to the presently disclosed subject matter (e.g., see, for example, SEQ ID NOs:1 15 & 1 16 which are oligomers of SEQ ID NO:1 13). In some embodiments of the presently disclosed subject matter, a linking compound or moiety can be used that acts as a molecular bridge to couple at least two different molecules, for example, to couple at least one surface-binding peptide to drug delivery vehicle or to couple a first surface- binding peptide to a second surface-binding peptide. The linking moiety can couple the at least two different molecules by either a covalent or a non- covalent bond. Thus, for example, coupling at least one surface-binding peptide to drug delivery vehicle can involve one portion of the linker binding to at least one surface-binding peptide having binding affinity for a non-biological substrate and another portion of the linker binding to at least one drug delivery vehicle. As apparent to those skilled in the art, and using methods known in the art, two different molecules can be coupled to the linker in a step-wise manner, or can be coupled simultaneously to the linker. There is no particular size or content limitations for the linker so long as it can fulfill its purpose as a molecular bridge, and that the binding affinity of a surface-binding peptide in a coating composition is substantially retained.
Linkers are known to those skilled in the art to include, but are not limited to, chemical compounds (e.g., chemical chains, compounds, reagents, and the like). The linkers can include, but are not limited to, homobifunctional linkers and heterobifunctional linkers. Heterobifunctional linkers, well known to those skilled in the art, contain one end having a first reactive functionality (or chemical moiety) to specifically link a first molecule, and an opposite end having a second reactive functionality to specifically link to a second molecule. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, III.), amino acid linkers (typically, a short peptide of between 3 and 15 amino acids, and often containing amino acids such as glycine, and/or serine), and polymers (e.g., polyethylene glycol) can be employed as a linker with respect to the presently disclosed subject matter. In one embodiment, representative peptide linkers comprise multiple reactive sites to be coupled to a binding domain (e.g., polylysines, polyornithines, polycysteines, polyglutamic acid and polyaspartic acid) or comprise substantially inert peptide linkers (e.g., lipolyglycine, polysehne, polyproline, polyalanine, and other oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl amino acid residues.
Suitable polymeric linkers are known in the art, and can comprise a synthetic polymer or a natural polymer. Representative synthetic polymers include but are not limited to polyethers (e.g., poly(ethylene glycol) ("PEG")), polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA)), polyamines, polyamides (e.g., nylon), polyurethanes, polymethacrylates (e.g., polymethylmethacrylate; PMMA), polyacrylic acids, polystyrenes, polyhexanoic acid, flexible chelators such as EDTA, EGTA, and other synthetic polymers which preferably have a molecular weight of about 20 daltons to about 1 ,000 kilodaltons. Representative natural polymers include but are not limited to hyaluronic acid, alginate, chondroitin sulfate, fibrinogen, fibronectin, albumin, collagen, calmodulin, and other natural polymers which preferably have a molecular weight of about 200 daltons to about 20,000 kilodaltons (for constituent monomers). Polymeric linkers can comprise a diblock polymer, a multi-block copolymer, a comb polymer, a star polymer, a dendritic or branched polymer, a hybrid linear-dendritic polymer, a branched chain comprised of lysine, or a random copolymer. A linker can also comprise a mercapto(amido)carboxylic acid, an acrylamidocarboxylic acid, an acrlyamido- amidotriethylene glycolic acid, 7-aminobenzoic acid, and derivatives thereof. In another embodiment, the linkers of the presently disclosed subject matter can be fatty acids. The fatty acids of the presently disclosed subject matter include saturated and unsaturated fatty acids such as but not limited to butyric acid, caproic acid, caprylic acid, capric acid, undecanoic acid (AUD), lauric acid, myhstic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, eicosanoic acid, docosanoic acid, tetracosanoic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. For example, in some embodiments, the fatty acid linkers are used as a linking group between the first surface-binding peptide and the second surface-binding peptide. In addition to their use as linkers, the fatty acid molecules of the presently disclosed subject matter can also be used in various other embodiments to modify one or both surface-binding peptides. For example, in other embodiments the fatty acids are used to modify one or both ends of either or both the first and second surface-binding peptides. In some embodiments, a single surface-binding molecule is modified at one or both ends with a fatty acid molecule (i.e., the case where SBP2 is absent and, optionally, L is also absent).
Linkers can also utilize copper-catalyzed azide-alkyne cycloaddition (e.g., "click chemistry") or any other methods well known in the art. Linkers are known in the art and include linkers that can be cleaved (e.g., by heat, by natural enzymes found in or on the body of an individual, by pH sensitivity), and linkers that can be made reactive toward other molecular moieties or toward themselves, for cross-linking purposes. Examples of pH-sensitive materials useful as linkers can include, but are not limited to, cellulose acetate phthalate, cellulose acetate thmellitate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate. Depending on such factors as the molecules to be linked, and the conditions in which the linking is performed, the linker can vary in length and composition for optimizing such properties as preservation of biological function, stability, resistance to certain chemical and/or temperature parameters, and of sufficient stereo-selectivity or size. For example, the linker should not significantly interfere with the ability of a composition according to the presently disclosed subject matter to sufficiently bind specifically, with appropriate avidity for the purpose, to a non-biological substrate, or the ability of a drug delivery vehicle carrying biologically active agent to deliver biologically active agent. A preferred linker can be a molecule with activities that enhance or complement the effect of a composition of the presently disclosed subject matter. A preferred linker can be used in the presently disclosed subject matter to the exclusion of a linker other than the preferred linker.
The term binding "affinity", and like terms used herein, are used for the purposes of the specification and claims, to refer to the ability of a peptide (as described herein) to have a binding affinity that is greater for one target molecule or surface material (i.e., the non-biological substrates of the presently disclosed subject matter) over another non-target molecule or surface material. For example, an affinity for a given non-biological substrate in a heterogeneous population of other substrates that is greater than, for example, that attributable to non-specific adsorption. For example, a surface- binding peptide of the presently disclosed subject matter has binding affinity for metal or non-biological polymer or another non-biological substrate of the presently disclosed subject matter when the peptide demonstrates binding to the non-biological substrate characterized by an EC50 of 10 μM or less. Such binding affinity can be dependent upon the presence of a particular conformation, structure, amino acid sequence, amino acid composition and/or charge on or within the peptide and/or material for which it has binding affinity.
In some embodiments, a surface-binding peptide that binds specifically to a particular surface, material or composition binds at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or a higher percentage, tighter than the surface-binding peptide binds to an appropriate control such as, for example, a different material or surface, or a protein typically used for such comparisons such as bovine serum albumin. For example, binding affinity can determined by an assay in which a signal is quantified (e.g., fluorescence, or colohmethc) that represents the relative amount of binding between a peptide and a non-biological substrate. In a preferred embodiment, a surface-binding peptide has a binding affinity that is characterized by a relative binding affinity as measured by an EC50 of 10 μM or less, preferably less than 1 μM, and more preferably less than 0.1 μM. The EC50 can be determined using any number of methods known in the art, such as by generating a concentration response curve from a binding assay in which the concentration of the peptide is titered with a known amount of the non-biological substrate for which the peptide has binding (see, for example, methods described in Example 2 herein). In such case, the EC50 represents the concentration of peptide producing 50% of the maximal binding observed for that peptide in the assay.
The phrase "surface-binding peptide" is used herein for the purposes of the specification and claims to refer to an amino acid chain of no less than about 7 amino acids and no more than about 100 amino acid residues in length, wherein the amino acid chain can include naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non- genetically encoded amino acids, and combinations thereof; however, specifically excluded from the scope and definition of "surface-binding peptide" herein is an antibody. A surface-binding peptide used in accordance with the presently disclosed subject matter can be produced by chemical synthesis, recombinant expression, biochemical or enzymatic fragmentation of a larger molecule, chemical cleavage of larger molecule, a combination of the foregoing or, in general, made by any other method in the art, and preferably isolated.
The term "isolated" means that the surface-binding peptide is substantially free of components which have not become part of the integral structure of the surface-binding peptide itself; for example, such as substantially free of cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized or produced using biochemical or chemical processes.
Surface-binding peptides can include L-form amino acids, D-form amino acids, or a combination thereof. Representative non-genetically encoded amino acids include but are not limited to 2-aminoadipic acid; 3- aminoadipic acid; β-aminopropionic acid; 2-aminobutyhc acid; 4-aminobutyhc acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2- aminoisobutyhc acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4- diaminobutyric acid; desmosine; 2,2'-diaminopimelic acid; 2,3- diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo- hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo- isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline; norvaline; norleucine; ornithine; and 3-(3,4-dihydroxyphenyl)-L-alanine ("DOPA"). Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O- acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.
In another embodiment of a coating composition according to the presently disclosed subject matter, the at least one surface-binding peptide having binding affinity for non-biological substrate can be modified, such as having an N-terminal amino acid, a C-terminal amino acid, or a combination thereof, wherein such amino acid is a non-genetically encoded amino acid that enhances the binding avidity (strength of binding interactions) of the peptide to a non-biological substrate. Such amino acids can be incorporated into a peptide by standard methods known in the art for solid phase and/or solution phase synthesis. For example, in one embodiment, from about one to about three residues of DOPA , a hydroxy-amino acid (e.g., one or more of hydroxylysine, allo-hydroxylysine, hydroxyproline, and the like) or a combination thereof, is added as terminal amino acids of an amino acid sequence of a peptide during synthesis, wherein the peptide is used in the coating composition according to the presently disclosed subject matter for enhancing the strength of the binding interactions (e.g., via electrostatic or ionic interactions) between the coating composition and the non-biological substrate to be coated.
A surface-binding peptide according to the presently disclosed subject matter can be modified, such as by addition of chemical moieties to one or more amino acid termini, and side chains; or substitutions, insertions, and deletions of amino acids; where such modifications provide for certain advantages in its use, and provided that the surface-binding function of the surface-binding peptide is sufficiently retained to be useful for the compositions and methods of the presently disclosed subject matter. Thus, the term "peptide" encompasses any of a variety of forms of peptide derivatives including, for example, amides, conjugates with proteins, cyclone peptides, polymerized peptides, conservatively substituted variants, analogs, fragments, chemically modified peptides, and peptide mimetics. Any peptide derivative that has desired binding characteristics of the family of surface- binding peptides according to the presently disclosed subject matter can be used in the practice of the presently disclosed subject matter.
For example, a chemical moiety, added to the N-terminal amino acid of a synthetic peptide to block chemical reactivity of that amino terminus of the peptide, comprises an N-terminal group. Such N-terminal groups for protecting the amino terminus of a peptide are well known in the art, and include, but are not limited to, lower alkanoyl groups, acyl groups, sulfonyl groups, and carbamate forming groups. Preferred N-terminal groups can include acetyl, Fmoc, and Boc. A chemical moiety, added to the C-terminal amino acid of a synthetic peptide to block chemical reactivity of that carboxy terminus of the peptide, comprises a C-terminal group. Such C-terminal groups for protecting the carboxy terminus of a peptide are well known in the art, and include, but are not limited to, an ester or amide group. Terminal modifications of a peptide are often useful to reduce susceptibility by proteinase digestion, and to therefore prolong a half-life of peptides in the presence of biological fluids where proteases can be present. Optionally, a peptide, as described herein, can comprise one or more amino acids that have been modified to contain one or more chemical moieties (e.g., reactive functionalities such as fluorine, bromine, or iodine) to facilitate linking the peptide to a linker molecule. As used herein, the term "peptide" also encompasses a peptide wherein one or more of the peptide bonds are replaced by pseudopeptide bonds including but not limited to a carba bond (CH2-CH2), a depsi bond (CO-O), a hydroxyethylene bond (CHOH-CH2), a ketomethylene bond (CO-CH2), a methylene-oxy bond (CH2-O), a reduced bond (CH2-NH), a thiomethylene bond (CH2-S), an N-modified bond (-NRCO-), and a thiopeptide bond (CS-NH). Surface-binding peptides that are useful in a composition or method according to the presently disclosed subject matter also include peptides having one or more substitutions, additions and/or deletions of residues relative to the sequence of an exemplary surface-binding peptide disclosed in Tables I-V, and SEQ ID NOs: 1-124 herein, so long as the binding properties of the original exemplary peptide are substantially retained. Thus, the presently disclosed subject matter includes surface-binding peptides that differ from the exemplary sequences disclosed herein by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (depending on the length of the exemplary peptide disclosed herein), and that share sequence identity with the exemplary sequences disclosed herein of at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Sequence identity can be calculated manually or it can be calculated using a computer implementation of a mathematical algorithm, for example, GAP, BESTFIT, BLAST, FASTA, and TFASTA, or other programs or methods known in the art. Alignments using these programs can be performed using the default parameters.
A peptide having an amino acid sequence substantially identical to a sequence of an exemplary surface-binding peptide disclosed herein can have one or more different amino acid residues as a result of substituting an amino acid residue in the sequence of the exemplary surface-binding peptide with a functionally similar amino acid residue (a "conservative substitution"); provided that peptide containing a conservative substitution will substantially retain the binding affinity of the exemplary surface-binding peptide not containing the conservative substitution. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one aromatic residue such as tryptophan, tyrosine, or phenylalanine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue such as aspartic acid or glutamic acid for another. In yet another embodiment of the presently disclosed subject matter, a surface-binding peptide can be described herein as comprising a peptide consisting essentially of a peptide (and/or its amino acid sequence) useful in the presently disclosed subject matter. When used herein in reference to the presently disclosed subject matter and for purposes of the specification and claims, the terminology "consisting essentially of refers to a peptide which includes the amino acid sequence of the surface-binding peptides described herein, and a peptide having at least 70% identity thereto, and preferably at least 95% identity thereto, (as described herein), along with additional amino acids at the carboxyl and/or amino terminal ends (e.g., ranging from about 1 to about 20 additional amino acids at one end or at each of both ends) which maintains the primary activity of the surface-binding peptide as a binding domain described herein. Thus, as a non-limiting example, a surface-binding peptide "consisting essentially of any one of the amino acid sequences illustrated as SEQ ID NOs:1-124 will possess the activity of binding a non- biological substrate with binding affinity, as provided herein; and will not possess any characteristics which constitute a material change to the basic and novel characteristics of the peptide to function as a surface-binding peptide (e.g., thus, in the foregoing example, a full length naturally occurring polypeptide, or a genetically engineered polypeptide, which has a primary activity other than as a binding domain described herein, and which contains the amino acid sequence of a surface-binding peptide described in the presently disclosed subject matter, would not constitute a peptide "consisting essentially of a peptide described in the presently disclosed subject matter). The term "pharmaceutically acceptable carrier", when used herein for purposes of the specification and claims, means a carrier medium that does not significantly alter the biological activity of the composition according to the presently disclosed subject matter to which it is added. Examples of such a carrier medium include, but are not limited to, aqueous solutions, aqueous or non-aqueous solvents, suspensions, emulsions, gels, pastes, and the like. As known to those skilled in the art, a suitable pharmaceutically acceptable carrier can comprise one or substances, including but not limited to, water, buffered water, medical parenteral vehicles, saline, 0.3% glycine, aqueous alcohols, isotonic aqueous buffer; and can further include one or more substances such as alginic acid, water-soluble polymer, glycerol, glycols (e.g., polyethylene glycol), polyols (e.g., glycerin, sorbitol, etc.), oils, salts (such as sodium, potassium, magnesium and ammonium, phosphonates), esters (e.g., carbonate esters, ethyl oleate, ethyl laurate, etc.), fatty acids, carbohydrates, polysaccharides, starches, glycoproteins (for enhanced stability), buffering agents (e.g., magnesium hydroxide, aluminum hydroxide, and the like), excipients, wetting agents, and preservatives (including, but not limited to, antioxidants; e.g., ascorbic acid, cysteine hydrochloride, sodium bisulfite, ascorbyl palmitate, tocopherol, polyphenols), and/or stabilizers (to increase shelf-life or as necessary and suitable for manufacture and distribution of the composition).
The phrase "medical device" as used herein for purposes of the specification and claims, refers to a structure (a) that is positioned or positionable into or onto an individual's body to prevent, treat, modulate or ameliorate damage, repair or restore a function of a damaged tissue, or to provide a new function; and (b) comprises at least one surface that is a non- biological substrate to be coated by a composition according to the presently disclosed subject matter. Representative medical devices include, but are not limited to: hip endoprostheses, artificial joints, jaw or facial implants, dental implants, tendon and ligament replacements, skin replacements, metal replacements and metal screws, metal nails or pins, metal graft devices, polymer-containing grafts, vascular prostheses, heart pacemakers, artificial heart valves, blood filters, closure devices (e.g., for closure of wounds, incisions, or defects in tissues, including but not limited to skin and other organs (heart, stomach, liver, etc.)), sutures, breast implants, penile implants, stents, catheters, shunts, nerve growth guides, leads for battery-powered medical devices, intraocular lenses, wound dressings, tissue sealants, aneurismal coils, prostheses (e.g., cochlear implants, visual prostheses (including, but not limited to, contact lenses, and other visual aid devices), neurostimulators, muscular stimulators, joint prosthesis, dental prosthesis, etc.), ophthalmic devices (glaucoma shunts, ophthalmic inserts, intraocular lenses, overlay lenses, ocular inserts, optical inserts), and nebulizers. Medical devices can be comprised of one or more non-biological substrates including, but not limited to, metals, metal alloys, polymers, metal oxides, non- metal oxides, and the like.
The term "drug delivery vehicle", when used herein for purposes of the specification and claims, means a carrier for one or more biologically active agents; preferably, comprising a microparticle, liposome, polymer, or combination thereof, and generally in the size range of nanometers to microns. Typically, the drug delivery vehicle has a size in the range of from 1 nanometer to 1000 microns, and preferably in a range of from 10 nm to 200 microns, and more preferably in a the range of 0.05 microns to 10 microns; the size depending on factors such as the nature and amount of biologically active agent carried by the drug delivery vehicle, the composition of the drug delivery vehicle, the intended route of administration, and desired pharmacokinetic parameters (e.g., release profile, biological half life, etc.). Generally and preferably, a drug delivery vehicle is biodegradable and biocompatible (e.g., substantially non-toxic). "Microparticle" is used herein to mean particles including, but not limited to, microspheres, microcapsules, nanospheres, nanoparticles, solid lipid nanoparticles, gas-filled microparticles (particularly useful for loading lipophilic biologically active agents), and solid phase porous microspheres; and can be solid, porous, hollow or have an internal lattice-type structure (e.g., a sponge-like structure, a honeycomb structure, etc.). A microparticle can be entirely formed of biologically active agent. For example, in one embodiment microparticles can be produced using methods known in the art such as, for example, spray drying. In one embodiment, the microparticles entirely formed of biologically active agent are linked to surface-binding peptide. Microparticles can be spherical or non-spherical in shape, and can be a shape selected from the group comprising spherical, oval, non-spherical, rod-like, acicular (needle-like), columnar, flake, disc-like, cubical, lamellar, blade-like, polygonal, and a combination thereof.
A microparticle can be comprised of one or more materials, including but not limited to, polymer, lipid, peptide, protein, carbohydrate (saccharides, polysaccharides, etc.), pharmaceutically acceptable salt thereof, and a combination thereof. Representative proteins include, but are not limited to, gelatin, collagen, albumin, and whey. Representative carbohydrates include, but are not limited to, dextran, saccharide, modified polysaccharide, cellulose esters, chitin, chitosan, cellulose, starch, hyaluronic acid, and pectin. Representative synthetic polymers can include, but are not limited to, one or more of: poly(lactide-co-glycolide) (e.g., PLGA); polylactide-polyglycolide polymer; lactide/glycolide copolymer; polyurethane; aliphatic polyester (e.g., poly-glycolic acid, polylactic acid, and the like); poly-amino acid; polyanhydride; polyhydroxylbutrate-related copolymer; acrylic polymers (e.g., polyacrylic acid, polymethylmethacrylate, polybutyl methacrylate, and the like); polyethylene oxide; polyvinyl pyrrolidone; polypropylene oxide; polyethylene glycol; polypropylene glycol; block polymer of polyethylene oxide and polypropylene oxide; acrylate; acrylamide; methacrylate; poly(ortho esters); cyanoacrylate; polyacrylic acid; polyorthoesters; polydioxanone; polyphosphazenes; polypropylene fumarate; polydepsipeptides; one or more of polycaprolactone and co-polymers and mixtures thereof, for example, poly(D,L-lactide- co-caprolactone) or polycaprolactone co-butylacrylate; polycarbonates; polyvinyl alcohol; and copolymers thereof, or block copolymers thereof. In a preferred embodiment, a microparticle is comprised of synthetic polymer; and more preferably, comprises biodegradable synthetic polymer. Typical methods known in the art for producing microparticles include, but are not limited to, a spray-drying method, and a phase separation method.
Liposomes are vesicles formed from one or more of lipids, fatty acids, phospholipids, non-ionic surfactant ("niosomes"), and polymer-conjugated lipids ("polymerosomes"), using methods well known in the art. Typically, liposomes can comprise single lipid bilayer ("unilamellar") vesicles or multiple lipid bilayer ("multilamellar") vesicles with sizes ranging from about 0.05 micron to 10 microns. A liposome useful in the presently disclosed subject matter is preferably biodegradable and non-toxic. Representative lipids include, but are not limited to, amphipathic lipids, phospholipids, lecithin, synthetic lipids, or dehvatized (e.g., charged) lipids, and a combination thereof. Representative synthetic lipids include, but are not limited to, 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC) and 1 ,2-dioleoylphosphatidylcholine (DOPC), polymer-conjugated lipids, (e.g., poly(ethylene glycol)-diacylglycerol, N-[methoxy-(poly(ethylene glycol)diacylphosphatidylethanolamine, poly(ethylene glycol)-ceramide), and synthetic phospholipids and/or cationic lipids (e.g., 1 ,2,-diacyl-3-thmethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), and 1 ,2-diacyl-sn-glycero-3- ethylphospho-choline). Using methods known in the art, a pre-formed liposome comprised of lipid and/or phospholipids can be dehvatized with a coating of polymer (such as to improve stability or provide a chemical moiety along a polymer chain to be reacted with and coupled to a chemical moiety of a surface-binding peptide having binding affinity for non-biological substrate).
In choosing a drug delivery vehicle to be used in producing a composition according to the presently disclosed subject matter, considered are factors such as the mode of administration, site of application, the indication or condition to be addressed by administration, biodegradability, physiochemical properties (stability, biocompatibility, relative to the site of treatment in facilitating delivery of biologically active agent), and mechanical properties (e.g., density, solubility, viscosity, and the like).
EXAMPLE 1
Phage Screening and Selections
Illustrated in this example are various methods for utilizing phage display technology to produce a surface-binding peptide having binding affinity for non-biological substrate or a synthetic component of a drug delivery vehicle.
Phage display technology is well-known in the art, and can be used to identify additional peptides for use as binding domains in the compositions according to the presently disclosed subject matter. In general, using phage display, a library of diverse peptides can be presented to a target substrate, and peptides that specifically bind to the substrate can be selected for use as binding domains. Multiple serial rounds of selection, called "panning," can be used. As is known in the art, any one of a variety of libraries and panning methods can be employed to identify a binding domain that is useful in a composition according to the presently disclosed subject matter. Panning methods can include, for example, solution phase screening, solid phase screening, or cell-based screening. Once a candidate binding domain is identified, directed or random mutagenesis of the sequence can be used to optimize the binding properties (including one or more of affinity and avidity) of the binding domain.
For example, a variety of different phage display libraries were screened for peptides that bind to a selected target non-biological substrate of the presently disclosed subject matter. The non-biological target substrate was either bound to or placed in (depending on the selected substrate) a container (e.g., wells of a 96 well microtiter plate, or a microfuge tube). Nonspecific binding sites on the surfaces of the container were blocked with a buffer containing bovine serum albumin ("BSA"; e.g., in a range of from 1 % to 10%). The containers were then washed 5 times with a buffer containing buffered saline with Tween™ 20 ("buffer-T"). Each library was diluted in buffer-T and added at a concentration of 1010 pfu/ml in a total volume of 100 μl. After incubation (in a range of from 1 to 3 hours) at room temperature with shaking at 50 rpm, unbound phage were removed by multiple washes with buffer-T. Bound phage were used to infect E. coli cells in growth media. The cell and phage-containing media was cultured by incubation overnight at 37° C in a shaker at 200 rpm. Phage-containing supernatant was harvested from the culture after centrifuging the culture. Second and third rounds of selection were performed in a similar manner to that of the first round of selection, using the amplified phage from the previous round as input. To detect phage that specifically bind to the selected substrate, enzyme-linked immunosorbent (ELISA-type) assays were performed using an anti-phage antibody conjugated to a detector molecule, followed by the detection and quantification of the amount of detector molecule bound in the assay. The DNA sequences encoding peptides from the phage that specifically bind to the selected target non-biological substrate were then determined; i.e., the sequence encoding a particular peptide was located as an insert in the phage genome, and was sequenced to yield the corresponding amino acid sequence displayed on the phage surface.
As a specific illustrative example for developing surface-binding peptides having binding affinity for metal; titanium and stainless steel were used as substrates for performing phage selection using several different libraries of phage. Titanium beads and stainless steel beads of approximately 5/32-inch diameter were individually prepared for selections by sequentially washing the beads with 70% ethanol, 40% nitric acid, distilled water, 70% ethanol and, finally, acetone, to remove any surface contaminants. After drying, one metal bead was placed per well of a 96-well polypropylene plate. Non-specific binding sites on the metal beads and the surface of the polypropylene plate were blocked with 1 % bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The plate was incubated for 1 hour at room temperature with shaking at 50 rpm. The wells were then washed 5 times with 300 μL of buffer-T.
Each library was diluted in buffer-T and added at a concentration of 1010 pfu/mL in a total volume of 100 μl_. After 3 hours of incubation at room temperature and shaking at 50 rpm, unbound phage were removed by 5 washes of buffer-T. The phage were added directly to E. coli DH5αF' cells in 2xYT media, and the phage-infected cells were transferred to a fresh tube containing 2xYT media and incubated overnight at 370C in a shaker incubator. Phage supernatant was harvested by centhfugation at 8500xg for 10 minutes. Second and third rounds of selection were performed in a similar manner to the first round, using the amplified phage from the previous round as input. Each round of selection was monitored for enrichment of metal binding peptides using ELISA-like assays performed using an anti-M13 phage antibody conjugated to horseradish-peroxidase, followed by the addition of chromogenic agent ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and determining a read-out at 405 nm. Libraries that showed enrichment of phage displaying metal binding peptides were plated on a lawn of E. coli cells, and individual plaques were picked and tested for binding to metals (e.g., titanium, stainless steel, etc.).
In some cases, relative binding strengths of the phage were determined by testing serial dilutions of the phage for binding to metal substrate in an ELISA. For example, serial dilutions of the display-selected clones were exposed to titanium or steel in an ELISA. The higher dilutions represent more stringent assays for affinity; therefore, phage that yield a signal at higher dilutions represent peptides with higher relative affinity for the particular target metal. Primers against the phage vector sequence that flank the insertion site were used to determine the DNA sequence encoding the peptide for the phage in each group. The sequence encoding the peptide insert was translated to yield the corresponding amino acid sequence displayed on the phage surface. Similar procedures were used to develop surface-binding peptides that have binding affinity for polymers.
The DNA sequences encoding peptides isolated on either polymer substrates or metal substrates were determined (see Tables I & Il below). While typically phage amino acids adjoining the peptide displayed had no significant contribution to the binding affinity of the peptide, the peptides useful in the presently disclosed subject matter can also comprise, in their amino acid sequence, such phage amino acids adjoining the peptide at the N- terminus and at the C-terminus (e.g., denoted as ss and sr in Table II).
Table I. Surface-Binding Peptides for Polymeric Non-Biological Substrates
Figure imgf000033_0001
Figure imgf000034_0001
Table II. Surface-Binding Peptides for Metal-Comprising Non-Biological Substrates
Figure imgf000034_0002
Figure imgf000035_0001
EXAMPLE 2
Surface-Binding Peptide Affinity Characterizations Relative binding strengths (affinities) of the surface-binding peptides to non-biological substrate were determined by testing serial dilutions of the surface-binding peptides for binding to a target non-biological substrate (e.g., non-biological substrates comprising either metal or polymer). The absorbance observed across the concentration range for each peptide sequence was plotted to yield a binding curve of the peptide to its target non- biological substrate. The binding curves were used to determine an EC50 value (i.e., the concentration of peptide that gives 50% of the maximum signal in the binding curve). The EC50 was used as an estimate of the affinity of the peptide for the target non-biological substrate. Preferred for use in compositions according to the presently disclosed subject matter are surface- binding peptides that bind to the selected target non-biological substrate with binding affinity of preferably an EC50 of less than or equal to about 1 μM, and more preferably, in the nanomolar range (e.g., <0.1 μM).
A typical assay for measuring the affinity of a surface-binding peptide for a non-biological substrate comprising titanium was performed according to the following procedure. Briefly, 5/32-inch diameter Grade 200 titanium beads were washed by sonication in acetone for 15 minutes, and the beads were allowed to dry. One bead was added to each well of a 96-well polypropylene plate. Two hundred fifty (250) μl_ of 1 % BSA in PBS was added to each well of the plate. The surface of the wells and the beads were blocked by incubation for 1 hour at 200C with shaking at 500 rpm. The plate was washed three times with 250 μl_ of buffer-T per well. A 1 :3 dilution series of each of the peptides was prepared using PBS as a diluent, starting at a peptide concentration of 20 μM, and going down to 0.0001 μM. A 200 μl_ sample of each dilution was added to wells of the plate. The plate was incubated for 1 hour at 200C with shaking at 500 rpm. The beads were washed three times with 250 μl_ of buffer-T per well. Two hundred (200) μl_ of streptavidin-alkaline phosphatase ("streptavidin AP") reagent, at a dilution of 1 :2000 in buffer + 1 % BSA, was added to each well. The plate was incubated for 30 minutes at room temperature. The beads were washed three times with 250 μl_ of buffer- T per well. Two hundred (200) μl_ of color development reagent (PNPP, p- nitrophenol phosphate) was added to each well. After color had developed (10 minutes), the samples were transferred to a clear 96-well plate and the absorbance at 405nm determined. A binding curve was generated by plotting the absorbance at 405 nm against the peptide concentration (μM). In addition, it will be understood by one of ordinary skill in the art that different non- biological substrates can be substituted for titanium in the foregoing example to determine EC50's for other surface-binding peptides and other non- biological substrates of the presently disclosed subject matter. The particular assay conditions (e.g., amounts and dilutions of surface binding peptides) will depend on the binding affinity of the particular surface-binding peptide being studied.
Table I illustrates exemplary surface-binding peptides that can be used in the methods and compositions according to the presently disclosed subject matter to bind to non-biological substrate polymers. The surface-binding peptides shown in Table I were isolated for binding to a polymeric non- biological substrate with an EC50 of 10 μM or less. For example, SEQ ID NOs: 1-22 were isolated for binding to polystyrene; SEQ ID NO:23 was isolated for binding to polyurethane; SEQ ID NOs: 24-37 were isolated for binding to polyglycolic acid; SEQ ID NOs: 38-43 were isolated for binding to polycarbonate; SEQ ID NOs: 44-52 were isolated for binding to nylon; and SEQ ID NOs: 53 and 54 were isolated for binding to teflon. Peptides identified according to the presently disclosed subject matter, such as those listed in Table I, can be used as surface-binding peptides to bind to a non- biological substrate comprising a polymer to which the peptides have binding affinity. In one embodiment, for example, such peptides can be used to bind to a non-biological substrate that is a drug delivery vehicle DV that comprises a synthetic polymer (e.g., the peptide has binding affinity and binds non- covalently to the synthetic polymeric outer layer or shell of the DV). Specifically, for example, the peptides of SEQ ID NOs: 1-22 that have binding affinity for polystyrene, can be used as illustrated in the Examples herein, to bind to a drug delivery vehicle DV comprising a microparticle comprised of polystyrene and carrying a biologically active agent BA. Similarly, Table Il illustrates exemplary surface-binding peptides that can be used in the methods and compositions according to the presently disclosed subject matter having binding affinity for a metal (including a metal alloy, a metal oxide, or a non-metal oxide). The surface-binding peptides shown in Table Il were isolated for binding to a metal non-biological substrate with an EC50 of 10 μM or less. For example, SEQ ID NOs: 55-82 have binding affinity to titanium and SEQ ID NOs: 83-102 have binding affinity to stainless steel. Peptides identified according to the presently disclosed subject matter, such as those listed in Table II, can be used as surface- binding peptides to bind to a non-biological substrate comprising a metal to which the peptides have binding affinity. In one embodiment, for example, such a peptide can be used as a first surface-binding peptide (SBP2) to bind to a non-biological substrate such as, for example, a medical device comprising a metal. Specifically, for example, the peptides of SEQ ID NOs: 55-102 that have binding affinity for metals can be used as illustrated, for example, in Examples 8-10 herein, to bind to a non-biological substrate comprising a metal for delivery of a drug delivery vehicle DV carrying a biologically active agent BA to the surface of the non-biological substrate.
While these exemplary peptide sequences are disclosed herein, one skilled in the art will appreciate that the binding properties conferred by those sequences can be attributable to only some of the amino acids comprised by the sequences. Thus, a peptide which comprises only a portion of an exemplary amino acid sequence disclosed herein can have substantially the same binding properties as the exemplary peptide comprising the full-length amino acid sequence. Thus, also useful as surface-binding domains in the coating compositions according to the presently disclosed subject matter are peptides that comprise only 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 of the amino acids in a particular exemplary sequence provided herein. Such amino acids can be contiguous or non-contiguous as long as the desired property (e.g., substantially retaining binding affinity for the selected material) of the surface- binding domain is retained, as determined by an appropriate assay (described herein and/or as known to those skilled in the art). Such amino acids can be concentrated at the amino-terminal end of the exemplary peptide (for example, 4 amino acids can be concentrated in the first 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 amino acids of the peptide), at the carboxy-terminal end of the exemplary peptide, or they can be dispersed throughout the exemplary peptide (e.g., acting as specific contact points, with the material for which the peptide has binding affinity, spaced apart from each other).
For example, surface-binding peptides consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:101 , and SEQ ID NO:102 have an amino acid motif of SEQ ID NO:103 according to the formula as follows:
X1-H-X-X-X2-X2-X2-K-XrXrX-K-X1-X1-N-K (SEQ ID NO:103); where X is any amino acid; Xi is K, N, or S, but preferably either K or N; and X2 is K, N, or H. Thus, a preferred surface-binding peptide for use in a composition according to the presently disclosed subject matter and having binding affinity for a non-biological substrate comprising a metal comprises a peptide consisting essentially of an amino acid sequence illustrated by SEQ ID NO:103.
EXAMPLE 3
Second-Generation Metal Surface-Binding Peptides In another example, based on the surface-binding peptides consisting essentially of an amino acid sequences illustrated by SEQ ID NOs: 75-82 in Table II, a series of synthetic, second-generation peptides were synthesized to further define the elements involved in metal binding, including varying the number (ranging from 0 to 3) of triplets of positively charged amino acids, and the amino acid sequence of triplets of positively charged amino acids. Each peptide was synthesized with an amino acid linker (GSSGK portion of SEQ ID NOs: 104-1 13) to facilitate biotinylation at the C-terminal lysine residue, and detection and quantification in the binding assay. The binding assay was performed using the methods as previously outlined herein. Shown in Table III are the second-generation peptide sequences, all of which displayed binding affinities to metal of an EC50 of less than 1 μM, with some of the peptide sequences having an EC50 of less than 0.10 μM.
Table III. Second-Generation Metal Surface-Binding Peptides
Figure imgf000039_0001
Surface-binding peptides consisting essentially of an amino acid sequence selected from the group consisting of any one of SEQ ID NOs:104- 1 13 have an amino acid motif, comprising a metal binding domain, illustrated by SEQ ID NO:1 14 as follows:
Z1 (Xaa), Z2 (SEQ ID NO:1 14);
wherein Xaa is an amino acid, for example, one of the 20 naturally occurring amino acids found in proteins in either the L or D form of chiral amino acids or a modified amino acid, except that Xaa is an amino acid other than lysine or histidine when occurring between two Z (e.g., Xaa of the amino acid sequence Z1 (Xaa)j Z2 is not lysine or histidine); Z is a triplet of amino acids consisting of at least one histidine residue and at least one lysine residue, no other amino acids other than histidine and lysine residues, but no more than two histidine residues or no more than two lysine residues (e.g., KHK, HKH, KKH, HKK, KHH), and most preferably, at least one of Z (e.g., either Zi or Z2, or both of Zi and Z2 , in the amino acid sequence Zi (Xaa)j Z2) is KHK. Thus, another preferred surface-binding peptide for use in a composition according to the presently disclosed subject matter and having binding affinity for a non- biological substrate comprising a metal comprises a peptide consisting essentially of an amino acid sequence illustrated by SEQ ID NO:1 14.
EXAMPLE 4 Surface-Binding Peptide Oligomers Several oligomers of different surface-binding peptides were synthesized. Briefly, the oligomers were built on a lysine MAP core and comprised of two and four peptide modules, respectively, of a surface-binding peptide. In an illustrative example, this core matrix was used to generate a peptide dimer and peptide tetramer using, in each branch, a monomeric peptide consisting essentially of the amino acid sequence of SEQ ID NO:1 13. The oligomers were synthesized sequentially using solid phase chemistry on a peptide synthesizer. The synthesis was carried out at a 0.05 mmol scale which ensures maximum coupling yields during synthesis. The biotin reporter moiety was placed at the C-terminus of the molecule, and was appended by a short linker containing glycine and serine residues to the lysine core. Standard Fmoc/ t-Bu chemistry was employed using AA/HBTU/ HOBt/NMM (1 :1 :1 :2) as the coupling reagents (AA is amino acid; HOBt is O-Pfp ester/1 - hydroxybenzothazole; HBTU is N-[1 H-benzotriazol-1-yl)(dimethylamino) methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; NMM is N-methylmorpholine). Amino acids were used in 5-10 fold excess in the synthesis cycles, and all residues were doubly, triply or even quadruply coupled depending upon the complexity of residues coupled. The coupling reactions were monitored by Kaiser ninhydhn test. The Fmoc deprotection reactions were carried out using 20% pipehdine in dimethyl- formamide. Peptide cleavage from the resin was accomplished using thfluoracetic acid (TFA: H20:Thisopropylsilane = 95: 2.5: 2.5) at room temperature for 4 hours. The crude product was precipitated in cold ether. The pellet obtained after centhfugation was washed thrice with cold ether and lyophilized to give a white solid as crude desired product. The crude products were analyzed by analytical high performance liquid chromatography (HPLC) on a C-18 column using mobile eluants (A =H2O / TFA (0.1 %TFA) and B = Acetonithle /TFA (0.1 %TFA). The polymers were also further analyzed by mass spectrometry for before subjecting each to final purification by HPLC. The fractions containing the desired product were pooled and lyophilized to obtain a fluffy white powder (> 98% purity).
The illustrated oligomers can be represented by the following sequences.
Figure imgf000041_0001
Figure imgf000041_0002
EXAMPLE 5
Truncation of Metal Surface-Binding Peptides
Various truncations of the metal surface-binding peptide SEQ ID NO: 101 were synthesized. The resulting peptides were tested for binding affinity to stainless steel balls in comparison with the full-length parent sequence (SEQ ID NO: 101 ) and a poor metal-binding sequence as a negative control. Binding affinity was determined according to the following procedure. The wells of a 96-well polypropylene plate were blocked with 350 μl BSA 1 % in PBS for 30 mins at 2O0C with 500 rpm shaking. Freshly cleaned 3/32" stainless steel beads that had been sonicated in acetone were added to the wells of the plate. Sequential 1/3 dilutions of each of the peptides in PBS starting at 10 μM were prepared directly in the wells of the polypropylene plate containing the beads. The final volume in each well was 200 μl. The plate was incubated for 1 h at 2O0C with 500 rpm shaking to allow for the binding to occur. The beads were washed 3 times with 250 μl PBS.
A direct immunoassay was used to determine the truncated surface- binding peptide affinity for the non-biological metal substrate. Two hundred μl of streptavidin AP at 1/200 in TBS + 1 % BSA was added to each bead. The plate was incubated at room temperature for 20 mins. The beads were washed 3 times with 250 μl TBS-Tween. The beads were transferred to a clean polypropylene plate. Two hundred μl of PNPP was added to all the beads. When color had developed, the solution in each well was transferred to the corresponding well of a clear plate and the OD405 nm was read.
The results of the binding affinity measurements for the metal surface- binding peptide truncation sequences of SEQ ID NO: 101 are shown below in Table IV. The results show that loss of up to 4 amino acid residues (including 2 charged residues) on either the N-terminal or C-terminal half of the peptide does not significantly affect binding affinity. Binding affinity is only decreased after 6 amino acids (including 4 charged residues) are removed from the C- terminus. Table IV. Relative Binding Affinity for Metal Surface-Binding Peptide Truncations
Figure imgf000043_0001
Table V shows an alignment of the metal surface-binding peptide SEQ ID NO:101 with truncation variants SEQ ID NOs:1 17-120 (from Table IV where the binding affinity was not significantly reduced), along with metal surface-binding peptide SEQ ID NO:102 and the second-generation metal surface-binding peptides SEQ ID NOs:104-1 12, and, in addition, aligned with metal surface-binding peptide sequences isolated using phage display SEQ ID NOs:68, 71-73, 77, 80-81 and 97-99. The surface-binding peptides shown in Table V each have an EC50 of < 1 μM for binding to metal surfaces.
Table V. Alignment of Metal Surface-Binding Peptide Sequences
Figure imgf000044_0001
* The "GSSGK" sequence functioned as a linker sequence for attachment of a biotin molecule to enable detection of the surface-binding peptides in the EC50 experiments. Accordingly, the C-terminal "K" residue was not charged in the EC50 determinations and, thus, not included in the count of positively charged residues.
The alignment of the sequences in Table V shows that a consensus metal surface-binding peptide domain sequence of the presently disclosed subject matter is provided as follows: an amino acid sequence domain comprising at least 8 amino acid residues, wherein at least 5 or 6 of the residues are positively charged residues (i.e. K, R or H), wherein in the case of a total of 5 positively charged residues, 3 of the charged residues are lysine, and in the case of 6 or more positively charged residues, at least 2 of the charged residues are lysine. Further, the distance between (and not including) the first and the fifth or sixth of the positively charged residues in the consensus sequence is at least 6 amino acids. The first and the fifth or sixth of the positively charged residues in the consensus sequence are highlighted in black in the sequences listed in Table V. As can be seen in the aligned sequences in Table V, the distance between the first and the fifth or sixth positively charged residue in the consensus metal surface-binding peptide domain sequence can be 6, 7, 8, 9, 10, 1 1 or more amino acid residues.
Accordingly, a preferred metal surface-binding peptide sequence of the presently disclosed subject matter comprises a metal-surface binding domain that conforms to the foregoing consensus rules derived from the sequences in Table V. For example, the consensus metal-surface binding domain can preferably range from 8 - 13 amino acid residues in length. However, additional amino acid residues or other modifying groups can be present at either or both the N-terminal and C-terminal sides of the consensus metal binding domain sequence without affecting the surface-binding activity of the metal surface-binding peptide. This is exemplified by the aligned sequences in Table V, and throughout the specification and experimental results described herein and, in particular, for example, from the truncation studies with SEQ ID NO: 101 and the activity of the metal surface-binding peptides described at EXAMPLES 8-10. In addition, the amino acids of the metal surface-binding peptides can be either the L or D form of chiral amino acids or a modified amino acid as described in the presently disclosed subject matter.
EXAMPLE 6 Modification of Surface-Binding Peptides
The surface-binding peptides of the presently disclosed subject matter were modified at the N-terminus according to the following procedure. In this example, the peptide SEQ ID NO: 101 was modified with fatty acid aminoundecanoic acid (AUD) repeats. The modified peptide was synthesized using standard FMOC chemistry with t-butyl protecting groups and the AUD molecules were incorporated into the synthesis process directly on solid support (resin).
Specifically, the peptide was synthesized by solid-phase peptide synthesis techniques on a Rainin Symphony Peptide Synthesizer using standard Fmoc chemistry. N-a-Fmoc-amino acids were purchased from Novabiochem. After all residues were coupled, simultaneous cleavage and side chain deprotection was achieved by treatment with a trifluoroacetic acid (TFA) cocktail. Crude peptide was then precipitated with cold diethyl ether and purified by high-performance liquid chromatography on a Shimadzu Analytical/ Semi-preparative HPLC unit on Vydac C18 silica column (preparative 10 mm, 250 mm X 22 mm) using a linear gradient of water/acetonithle containing 0.1 % TFA. Homogeneity of the synthetic peptides was evaluated by analytical RP-HPLC (Vydac C18 silica column, 10 mm, 250 mm x 4.6 mm) and the identity of the peptide was confirmed with MALDI-TOF-MS.
For AUD containing peptide synthesis, Fmoc-AUD-C02H (Peptide International) was activated using 0.2 M HOBt solution in NMP and was manually coupled sequentially at the N-terminus of the peptide resin using TBTU/NMM method. Following each coupling, the Fmoc group was removed using 20% piperidine in DMF and the resin subsequently coupled further with FmOC-AUD-CO2H untill completion of the reaction as judged by ninhydhn test. The terminal Fmoc was removed before subjecting the peptide resin to full cleavage using TFA cocktail. The crude linear peptide was cyclized using the iodine oxidation method and the crude cyclic peptide was purified by prep RP- HPLC on a C-18 Kromasil column. The final product was further characterized by electrospray mass spectrometry.
EXAMPLE 7 Drug Delivery Vehicles
In this example, methods are described for making drug delivery vehicles, for loading a drug delivery vehicle, and for making a composition according to the presently disclosed subject matter. Methods for producing microparticles can include, but are not limited to, solvent precipitation, solvent evaporation, spray drying, crystallization, melt extrusion, compression molding, hot melt encapsulation, and phase inversion encapsulation, using techniques well known in the art. Methods for producing liposomes can include, but are not limited to, reverse-phase evaporation, hydration of dried lipids, solvent or detergent removal, double emulsion preparation, fusion, freeze-thawing, and lyophilization, using techniques well known in the art.
In one example, liposomes, such as those comprising oil in water emulsions, are typically formed from amphipathic lipids or phospholipids having hydrophobic and polar head group moieties, and which can form spontaneously into bilayer vesicles in water. Liposomes can be fabricated according to standard techniques well known in the art. One way of forming liposomes involves suspending a suitable lipid in an aqueous medium, followed by sonication of the mixture. Thus, by including surface-binding peptides having a hydrophobic tail into the mixture used to form a liposome, the hydrophobic tail of the surface-binding peptide is trapped between the hydrophobic chains of the amphipathic lipid (i.e., in the shell-like bilayer or multilayer of the vesicle formed), with the remaining portion of the surface- binding peptide (which is more hydrophilic than the hydrophobic tail) being orientated toward the exterior, polar surface of the vesicle formed, and available for binding specifically to non-biological substrate. Hydrophobic tails of the surface-binding peptides of the presently disclosed subject matter include, for example, but are not limited to fatty acid molecules including lauric acid, myhstic acid, palmitic acid and undecanoic acid. Using similar methods, a biologically active agent (e.g., with a hydrophobic portion) can be trapped into a hydrophobic structure or layer of a drug delivery vehicle.
"Carrying" a biologically active agent can comprise encapsulation of the biologically active agent within the drug delivery vehicle, biologically active agent impregnated in the structure of the drug delivery vehicle (e.g., all or a portion of biologically active agent is dispersed within the layer(s) forming the matrix of the drug delivery vehicle), biologically active agent coupled (covalently or noncovalently) to the drug delivery vehicle (e.g., surface, layer, or internal structure), or a combination thereof. In one embodiment, a biologically active agent can be carried within the drug delivery vehicle such as by encapsulation. Encapsulation into a drug delivery vehicle can be accomplished by using methods known in the art, such as by: passive entrapment of a water-soluble compound comprising a biologically active agent by hydrating a lipid film or solvent with an aqueous solution containing biologically active agent, in the process of forming drug delivery vehicle; passive entrapment of a hydrophobic or lipophilic compound comprising a biologically active agent by hydrating a lipid film or solvent containing the biologically active agent; or other methods, including, but not limited, to reverse evaporation phase preparation of a drug delivery vehicle, and spray drying.
In one example, surface-binding peptides having a hydrophobic tail can be incorporated onto drug delivery vehicles comprising microparticles with the hydrophobic tail absorbed onto, and the surface-binding peptide extending from, the surface of the microparticle for availability to bind non-biological substrate. Basically, one or more biologically active agents to be loaded in the microparticle, surface-binding peptide, and polymer for forming the microparticle, are prepared using the appropriate amounts of biologically active agent-to-surface-binding peptide-to-polymer weight ratios (polymer usually in the range of from about 75% to 98% w/w; biologically active agent is typically 5% to 20% w/w; surface-binding peptide can be 0.01 % to 10% w/w).
A water/oil emulsion is prepared by dissolving the polymer in an appropriate organic solvent, and adding biologically active agent and surface- binding peptide (e.g., the biologically active agent and surface-binding peptide in an aqueous solution can be added dropwise to the polymer solution under stirring with a homogenizer. Alternately, surface-binding peptide can be dissolved with the polymer in the appropriate organic solvent, and an aqueous solution containing biologically active agent is added dropwise with mixing). The drop size of the emulsion can be monitored and analyzed by optical microscopy according to methods known in the art. The microparticles are then obtained by spraying the solutions through the nozzle of a spray-dryer using parameters (inlet air temperature, nozzle size, spray rate feed, etc.) to get microparticles of the appropriate size and shape, using methods known in the art. This procedure can be varied using methods known in the art (e.g., such as altering the ratio of the components, methods of mixing, etc.) to obtain the desired amount of peptide absorbed to the surface of the microparticles.
Alternatively, rather than using a spray drier, the solvent in the oil phase of the emulsion is evaporated off to provide microparticles. The microparticles carrying biologically active agent and coupled to surface- binding peptide are recovered, washed, lyophilized, and can further be processed to remove residual water and organic solvent. Suitable organic solvents for use in preparing microparticles includes, but are not limited to, cyclohexane, cycloheptane, dimethylsulfoxide, chloroform, methylene chloride, cyclooctane, dimethylformamide, dimethylacetamide, or mixtures thereof.
In another example for loading a microparticle with biologically active agent, a biologically active agent in an aqueous solution (which can further comprise an emulsifying agent; e.g., polyvinyl alcohol) or as a solid dispersion is added to polymer in a suitable organic solvent ("polymer solution") in forming a primary emulsion. A surfactant or emulsifying agent (e.g., polyvinyl alcohol, protein (e.g., albumin, gelatin, and the like), lipophilic emulsifier (e.g., poly(ethylene oxide-co-propyelene oxide), or a combination thereof) can be optionally be added to stabilize the primary emulsion. A emulsifying agent in an aqueous solution is added to and mixed with the primary emulsion to extract the polymer and biologically active agent into the aqueous phase, followed by removing the organic solvent from the mixture (e.g., by adding excess water, and/or under vacuum) and harden the microparticles. The microparticles, carrying biologically active agent, can then be recovered by filtration, centhfugation, and then lyophilization. Alternatively, a non-solvent can be used to extract the organic solvent from a primary emulsion. In yet another method, biologically active agent, formulated as a powder, is suspended in a polymer phase dissolved in a suitable organic solvent. The suspension is then spray-dried, followed by removal of the organic solvent, and by recovering microparticles carrying biologically active agent.
A surface-binding peptide, having binding affinity for a non-biological substrate, can be coupled covalently or non-covalently to drug delivery vehicle carrying the biologically active agent. For example, such surface-binding peptide can be impregnated into the structure of the drug delivery vehicle, coupled (covalently or noncovalently) to the surface of the drug delivery vehicle, or a combination thereof; provided, the surface-binding peptide is able to bind with sufficient affinity to the non-biological substrate. The surface-binding peptide having binding affinity for a non-biological substrate can be coupled to the drug delivery vehicle without use of a linker, or can be coupled via a linker.
In various embodiments of the presently disclosed subject matter, two or more components of a composition according to the presently disclosed subject matter can be covalently coupled (e.g., surface-binding peptide to linker; surface-binding peptide to drug delivery vehicle; surface-binding peptide to a peptide having binding affinity for a synthetic component of the drug delivery vehicle; linker to a peptide having binding affinity for a synthetic component of the drug delivery vehicle; a biologically active agent to a drug delivery vehicle, and a combination thereof). Covalent coupling can be achieved by any means known in the art. For example, a component to be linked can comprise a reactive functionality comprising a free chemical group which can covalently bond with a chemical-reactive group (reactive with the free chemical groups). Free chemical groups include, but are not limited to, a thiol, carboxyl, hydroxyl, amino, amine, sulfo, phosphate, or the like; whereas chemical-reactive groups include, but are not limited to, thiol-reactive group, carboxyl-reactive group, hydroxyl-reactive group, amino-reactive group, amine-reactive group, sulfo-reactive group, or the like.
As illustrative examples, to covalently couple a hydroxyl group of a first component with to an amino group of a second component, a linker can have: a carboxyl group to form a bond with the first component, and a carboxyl group to form a bond with the second component; or a carboxyl group to form a bond with the first component, and an aldehyde group to form a bond with the second component; or a carboxyl group to form a bond with the first component, and a halide group to form a bond with the second component. In another example, a drug delivery vehicle comprising molecules of phosphatidylsehne, phosphatidylethanolamine ("PE"), or dioleoyl PE, provides free amino groups for chemical reaction with one or more amino-reactive groups of a surface- binding peptide (e.g., a carboxyl group) in covalently coupling a surface- binding peptide to a drug delivery vehicle.
In another embodiment, a surface-binding peptide can be non- covalently coupled to the drug delivery vehicle. For example, surface-binding peptide can bind non-covalently with sufficient affinity to the drug delivery vehicle such that extended from the outer surface of the drug delivery vehicle is the portion of the surface-binding peptide that is able to bind to a non- biological substrate. In one illustration, the surface-binding peptide is synthesized to comprise a hydrophobic tail. The hydrophobic tail of the surface-binding peptide is inserted in a hydrophobic layer of the drug delivery vehicle formed, with the remaining, more hydrophilic portion, of the surface- binding peptide extending from drug delivery vehicle for binding specifically to a non-biological substrate.
In one example, the water in oil in water (w/o/w) approach was used to generate microparticles carrying biologically active agent. This approach has been favored for the encapsulation of hydrophilic molecules (Norton et al., 2005; Farokhzad et al, 2006; Hachicha et al, 2006). In the example, vancomycin was encapsulated to test surface-binding peptide-mediated microparticle delivery. First, an aqueous solution (1 ml.) of drug (100 μg) was added to a solution of 100 mg of poly(D,L-lactic-co-glycolic acid) (PLGA) in an organic solvent. This mixture formed immiscible layers, which were then mixed by sonication to create an emulsion. The first emulsion was then added to a 1 % solution of polyvinyl alcohol (PVA) in water (10 ml_). This mixture was then emulsified by mechanical stirring. The second emulsion was then added to 5% isopropanol (100 ml_), and the mixture was stirred to extract and evaporate the organic solvent. The microparticles were then isolated by centrifugation and freeze drying. Microparticles were analyzed through light microscopy and SEM to determine the distribution of particle sizes.
EXAMPLE 8
Delivery of Biological Agent to a Non-Biological Substrate In the following illustrative examples, a composition according to the invention was formed by coupling a surface-binding peptide having binding affinity for a non-biological substrate to a drug delivery vehicle. In this example, drug delivery vehicle comprised commercially prepared polystyrene microparticles (0.04 μM) carrying a biologically active agent comprising fluorescent dye (a "yellow-green" dye proprietary to a commercial vendor, the dye having excitation/emission maxima of 505/515). A composition according to the presently disclosed subject matter was produced by coupling a biotinylated surface-binding peptide to streptavidin-functionalized drug delivery vehicle (peptide added at a 4:1 ratio to streptavidin-functionalized drug delivery vehicle). Thus, a composition was formed using a linker comprising biotin and streptavidin to couple surface-binding peptide to drug delivery vehicle carrying biologically active agent.
In one example, a composition according to the presently disclosed subject matter was formed in situ. In this example, a surface-binding peptide having binding affinity for a non-biological substrate comprising metal (a peptide comprising an amino acid sequence consisting essentially of SEQ ID NO:1 13) was first contacted with non-biological substrate comprising titanium sputter-coated silicon wafers ("titanium disks") to coat the disks with surface- binding peptide. In this example, the titanium disks were contacted with a buffered solution containing surface-binding peptide at a concentration of 1 μM for 30 minutes at room temperature. As a control for non-specific binding, some titanium disks were contacted only with the buffer (not contacted with surface-binding peptide; "control disks") under the same conditions in the assay. The disks were washed with buffer and then contacted with streptavidin-functionalized drug delivery vehicle in PBS, and incubated at room temperature for 30 minutes. The disks were washed in PBS, and detection of biologically active agent on the surface of the metal substrate was visualized using epifluorescence microscopy and digital images using a digital camera. The relative fluorescence was quantified using commercial imaging software measuring mean fluorescence intensity of each sample. The fluorescence intensity was compared between the non-biological substrate contacted only with drug delivery vehicle (no surface-binding peptide present; "control disks") and the non-biological substrate on to which is formed a composition according to the presently disclosed subject matter in situ. As shown in FIGs. 1 & 2, significantly more fluorescence is detected with non-biological substrate onto which is formed a composition according to the presently disclosed subject matter (FIG. 1 , Panel A; FIG. 2, "SBP-[DV(BA)]") than a non-biological substrate which lacks a composition according to the invention and is only contacted with untargeted (not coupled to surface- binding peptide) drug delivery vehicle carrying biologically active agent (FIG. 2, Panel B; FIG. 2, "control"). As can be concluded from FIGs. 1 and 2, the composition according to the presently disclosed subject matter demonstrates the ability to bind, localize, and retain biologically active agent to the surface of non-biological substrate for which the surface-binding peptide component of the composition has binding affinity.
In another example, surface-binding peptide having binding affinity for a non-biological substrate comprising metal was coupled to the drug delivery vehicle in forming a composition according to the invention. Non-biological substrate comprising titanium disks were contacted with a buffered solution containing the composition at a concentration of 1 μM for 30 minutes at room temperature. As a control for non-specific binding, some titanium disks were contacted only with the streptavidin-coated fluorescent microparticles (drug delivery vehicle not coupled to surface-binding peptide; "control disks") under the same conditions in the assay. The disks were washed in PBS, and the non-biological substrate was visualized using epifluorescence microscopy and digital images using a digital camera. The fluorescence intensity was compared between the control disks and the disks coated with the composition according to the presently disclosed subject matter. As shown in FIG. 3, the composition according to the presently disclosed subject matter ("Panel A") showed the ability to bind, localize, and retain biologically active agent to the surface of the non-biological substrate by demonstrating significantly more fluorescence, as compared to the control ("Panel B").
In another example, surface-binding peptide having binding affinity for a non-biological substrate comprising polymer was coupled to the drug delivery vehicle in forming a composition according to the invention. Non- biological substrate comprising polyethylene teraphthalate suture material was contacted with a buffered solution containing the composition at a concentration of 10 μM for 30 minutes at room temperature. As a control for non-specific binding, some suture material was contacted only with the streptavidin-coated fluorescent microparticles (drug delivery vehicle not coupled to surface-binding peptide; "control material") under the same conditions in the assay. The suture material was washed in PBS, and the non-biological substrate was visualized using epifluorescence microscopy and digital images using a digital camera. The fluorescence intensity was compared between the control material and the suture material coated with the composition according to the presently disclosed subject matter. As shown in FIG. 4, the composition according to the presently disclosed subject matter ("Panel A") showed the ability to bind, localize, and retain biologically active agent to the surface of the non-biological substrate by demonstrating significantly more fluorescence, as compared to the control ("Panel B").
EXAMPLE 9 Sustained Antimicrobial Activity of Vancomycin Released from Microparticles In this example, illustrated are methods according to the presently disclosed subject matter: (a) a method for delivering a biologically active agent to a non-biological substrate using a composition according to the invention; (b) a method of coating a surface of a non-biological substrate to provide biologically active agent to the surface in providing a process selected from the group consisting of delivery of biologically active agent to the coated surface, localizing a biologically active agent to the coated surface, retaining a biologically active agent to the coated surface for controlled delivery, and a combination thereof; and (c) a method of applying a composition according to the presently disclosed subject matter to a non-biological substrate. In one example, coating a non-biological substrate with a composition according to the presently disclosed subject matter or applying a composition according to the presently disclosed subject matter to a non-biological substrate comprises contacting at least one surface of non-biological substrate with an effective amount of a composition according to the presently disclosed subject matter so that the composition binds specifically to the at least one surface of the non-biological substrate. Thus, the composition can be pre-formed (formed prior to applying it to non-biological substrate). The composition can be applied prior to placing a non-biological substrate in position (e.g., such as prior to applying a medical device to an individual in need of the medical device), or can be applied to a non-biological substrate in situ (e.g., such as applying to a medical device already positioned in an individual in need of the medical device).
In another example, a composition according to the presently disclosed subject matter can be formed directly on the non-biological substrate (in situ or prior to placement in its intended position), rather than pre-formed, by stepwise applying various components of the composition to form the composition on the non-biological substrate. Thus, in a first step of a process of applying a composition to a non-biological substrate, applied to at least one surface of a non-biological substrate is a first component comprising surface- binding peptide having binding affinity for non-biological substrate so that the first component binds specifically to the at least one surface of the non- biological substrate. The first component can comprise surface-binding peptide to be coupled directly (via a linker or without use of a linker) to drug delivery vehicle, or can comprise surface-binding peptide coupled (via a linker or without use of a linker) to a peptide having binding affinity for drug delivery vehicle (e.g., having binding affinity for the a synthetic component on the surface or outer layer of the drug delivery vehicle). In either case, in a subsequent step, in the process of applying the composition to the non- biological substrate, is added drug delivery vehicle carrying biologically active agent. In one embodiment, by contacting and coupling an effective amount of a component comprising drug delivery vehicle carrying biologically active agent to a component comprising surface-binding peptide bound specifically to non-biological substrate, formed is a composition according to the presently disclosed subject matter directly on non-biological substrate. In another embodiment, by contacting and coupling an effective amount of a component comprising drug delivery vehicle carrying biologically active agent to a component comprising a peptide having binding affinity the drug delivery vehicle, such peptide coupled to surface-binding peptide bound specifically to non-biological substrate, formed is a composition according to the presently disclosed subject matter directly on non-biological substrate.
Conventional processes known in the art can be used to apply the composition (or a component of the composition, if forming the composition directly on the non-biological substrate) according to the presently disclosed subject matter to non-biological substrate, such processes are known to include, but are not limited to, mixing, dipping, brushing, spraying, and vapor deposition. For example, a solution or suspension comprising the composition can be applied through the spray nozzle of a spraying device, creating droplets that contact at least one surface of non-biological substrate to which is to be coated with the composition. The non-biological substrate having composition bound specifically thereto can be allowed to dry, and can then be further processed, if desired, prior to use or positioning or placement (e.g., washed in a solution (e.g., water or isotonic buffer) to remove excess composition not bound specifically to the non-biological substrate; by sterilization using any one or methods known in the art for sterilizing non- biological substrate; etc). Steps of this process can be repeated if the composition is being applied step-wise by its various components to form the composition directly on the non-biological substrate. In another process for applying the composition to one or more surfaces of a non-biological substrate onto which the composition is to be applied, the non-biological substrate is dipped into a liquid (e.g., solution or suspension, aqueous or solvent) containing the composition (or component thereof) in an amount effective to for the composition to bind specifically to the non-biological substrate. For example, the surface is dipped or immersed into a bath containing the composition. Suitable conditions for applying the composition (or a component thereof) include contacting the surface with the liquid containing composition (or component thereof) for a suitable period of time (e.g., ranging from about 5 minutes to about 12 hours; more preferably, ranging from 15 minutes to 60 minutes), at a suitable temperature (e.g., ranging from 4 0C to about 50 0C; more preferably, ranging from room temperature to 37 0C). The surface or non-biological substrate having composition applied thereon can then be further processed, as necessary for use (washing, sterilization, and the like). In another process for applying the composition to non-biological substrate, the composition according to the presently disclosed subject matter is formulated in a dry powder (e.g., via air drying or lyophilizing the composition) which is then mixed with the non-biological substrate present in a form comprising a solid, powder, paste, filler, binder, gel, sponge, and a combination thereof. However, these illustrative processes for applying a composition to a non-biological substrate are not exclusive, as other coating and stabilization methods can be employed (as one of skill in the art will be able to select the compositions and methods used to fit the needs of the particular non-biological substrate and purpose). Extended antimicrobial efficacy was demonstrated for vancomycin- loaded microparticles immobilized by surface-binding peptides of the presently disclosed subject matter on a non-biological metal substrate. In this assay, stainless steel disks were coated with 10 μM of either metal surface- binding peptide
SSHRTNHKKNNPKKKNKTRGGSSGKSKSRASAAVFVFLAVALFSFSSRGSS GK (Biotin)-NH2 (SEQ ID NO: 123; AFF-5061 ) or metal surface-binding peptide (Aud)4-SSHRTNHKKNNPKKKNKTR-GSSG-K(biotin)-NH2 (SEQ ID NO: 124; AFF-5103) and 100 μL 0.5% solution PLGA-vancomycin microparticles (see FIG. 5). Two sets of control discs were prepared, microparticle-coated alone (FIG. 5, no peptide) and uncoated (FIG. 5, no peptide, no particles). Disks were thoroughly washed and transferred to a clean 96-well plate. Human plasma (American Red Cross) was added to each well and inoculated with 104 CFU of S. aureus strain MZ100. Plates were sealed and incubated for 24h at 370C. Bacterial supernatant was removed and the BacTiter-Glo Microbial Cell Viability assay used to quantify bacteria (Promega). The disks were re-challenged with fresh plasma and bacteria each day, for 4 days (FIG. 5). For the first two and three time points (two and three changes of plasma over two and three days), bacterial growth was inhibited in the wells with the surface-binding peptide/microparticle- coated disks (SEQ ID NO: 123 and SEQ ID NO: 124, respectively) as compared to the disks that were coated with microparticles alone and uncoated control disks (FIG. 5). The data indicate that a non-biological metal substrate such as an implant coated with a composition of the presently disclosed subject matter comprising a surface-binding peptide and drug delivery vehicle carrying biological agent such as the drug loaded microparticles in this example, can maintain prolonged antimicrobial efficacy for at least three days in human plasma.
EXAMPLE 10
Surface-Binding Peptide-Mediated Delivery of Antibiotic Loaded
Microparticles
In this example, metal surface-binding peptide (5 μM; SEQ ID NO: 123) and vancomycin-loaded microparticle was applied to titanium pins (as described in Example 9 above) and the pins were inserted into silicone tubing containing 30 μl_ of 106 cfu per ml. S. aureus (ATCC 49230). After incubating pins in cultured bacteria overnight, the pin was removed and rolled over TSA plates (see top "On pin" portion of FIG. 6). In addition the broth was aspirated and diluted 10X from left to right, beginning with undiluted culture broth from the tubing (see bottom "Broth dilutions" portion of FIG. 6). These data show very few, if any detectable bacteria remaining in broth or on microparticle + surface-binding peptide coated titanium pins.
A coating composition comprising metal surface-binding peptide (AFF- 5061 ; SEQ ID NO: 123) and vanc-microparticles was also tested in a rodent model. In this example, the following coating compositions were applied to titanium pins: surface-binding peptide (5 μM) and microparticles, vancomycin microparticles without peptide (control), or PBS (bare metal control). The compositions were allowed bind to the titanium pins for 30 min and washed with PBS prior to implantation of pins into Sprague-Dawley rat tibias (N=8 per group). A hole was drilled through cortical and cancellous bone, and the medullary cavity was injected with 3X103 cfu S. aureus (ATCC 49230). Pins were inserted into the drilled cavity and the wound was sutured. Pins were removed two weeks following implantation. Both pins and tibias (ground) were also sonicated to release bacteria, and the resulting extracts plated (see FIG. 7; (bone sonication data not shown)). The y axis of the graph shows colony forming units (CFU) remaining following pin sonication after removal from the animals. (Bars at 50O=CFU ≥ 500). Total N = 15 per group. The data for the pin and bone sonication both showed a robust infection was generated in the majority of animals receiving either bare metal or microparticle only pins and both measures show very few, if any, detectable bacteria remaining in animals receiving vancomycin microparticles + surface- binding peptide coated titanium pins.
The foregoing description of the specific embodiments of the presentlydisclosed subject matter have been described in detail for purposes of illustration. In view of the descriptions and illustrations, others skilled in the art can, by applying, current knowledge, readily modify and/or adapt the presently disclosed subject matter for various applications without departing from the basic concept of the presently disclosed subject matter; and thus, such modifications and/or adaptations are intended to be within the meaning and scope of the appended claims.

Claims

What is claimed is:
1. A composition of the formula:
SBP1 - L -SBP2 [DV(BA)], wherein SBP is a surface-binding peptide of 8 to 60 amino acids;
SBP1 is the SBP surface-binding peptide comprising a surface-binding peptide domain having binding affinity for a surface comprising a non-biological substrate;
SBP2 can be present or absent and is the SBP surface-binding peptide having binding affinity for a synthetic component of a drug delivery vehicle (DV);
L can be present or absent and comprises a linker; and the DV comprises a drug delivery vehicle that carries a BA, wherein the BA is one or more biologically active agents; wherein in the presence of the SBP2, the DV is noncovalently coupled to the SBP2; wherein in the absence of the SBP2 and in the presence of the L, the SBPi is coupled to the DV either covalently or nocovalently; and wherein in the absence of both the SBP2 and the L, the SBPi is coupled directly to the DV either covalently or noncovalently.
2. The composition of claim 1 , wherein the surface-binding peptide domain of SBPi is a metal surface-binding peptide domain and the domain comprises at least 8 amino acid residues; wherein at least 5 or 6 of the residues are positively charged lysine, arginine or histidine residues; wherein in the case of the 5 positively charged residues, 3 of the charged residues are lysine; wherein in the case of the 6 or more positively charged residues, at least 2 of the charged residues are lysine; and wherein a distance between (and not including) the 1 st and the 5th or the 6th of the positively charged residues is at least 6 amino acids and the distance can be 7, 8, 9, 10, or 1 1 or more amino acid residues.
3. The composition of claim 2, wherein the L is present or absent, the SBP2 is absent, the SBPi is modified with one or more fatty acid groups at the N- terminus and the SBPi is bound non-covalently to the DV.
4. The composition of claim 2, wherein the binding affinity of the SBPi for the surface comprising the non-biological substrate is an EC50 of 1 μM or less.
5. The composition of claim 2, wherein the DV comprises poly(D,L-lactic-co- glycolic acid) (PLGA).
6. The composition of claim 2, wherein the BA is an antibiotic.
7. A non-biological substrate to which is bound the composition of claim 1 .
8. The non-biological substrate of claim 7, wherein the surface of the non- biological substrate is selected from the group consisting of metal, metal oxide, non-metal oxide, ceramic, synthetic polymer, plastic, silicone, carbon-based materials, graphite, carbon nanotubes, metallo-carbon composites, and combinations thereof.
9. The non-biological substrate of claim 7, wherein the non-biological substrate is a medical device.
10. A drug delivery vehicle for delivery of a biologically active agent to a non- biological substrate, wherein:
(a) carried by the drug delivery vehicle is the biologically active agent;
(b) coupled to the drug delivery vehicle is a surface-binding peptide of 8-60 amino acids, wherein the surface-binding peptide comprises a surface-binding peptide domain having binding affinity for a non-biological substrate.
1 1. The drug delivery vehicle of claim 10, wherein the coupling of the surface- binding peptide to the drug delivery vehicle is via a linker.
12. The drug delivery vehicle of claim 10, wherein the coupling of the surface- binding peptide to the drug delivery vehicle is via non-covalent binding the surface-binding peptide to a synthetic component of the drug delivery vehicle.
13. The drug delivery vehicle of claim 10, wherein the surface-binding peptide domain comprises at least 8 amino acid residues; wherein at least 5 or 6 of the residues are positively charged lysine, arginine or histidine residues; wherein in the case of the 5 positively charged residues, 3 of the charged residues are lysine; wherein in the case of the 6 or more positively charged residues, at least 2 of the charged residues are lysine; and wherein a distance between (and not including) the 1 st and the 5th or the 6th of the positively charged residues is at least 6 amino acids and the distance can be 7, 8, 9, 10, or 1 1 or more amino acid residues.
14. A method of applying the composition of claim 1 to a non-biological substrate, the method comprising contacting at least one surface of the non- biological substrate with an effective amount of the composition so that the composition forms a non-covalent bond to the surface of the non-biological substrate.
15. The method of claim 14, wherein the SBP of the composition, in the presence or absence of the L, is coupled to the [DV(BA)] of the composition prior to applying it to the non-biological substrate.
16. The method of claim 15, wherein the SBP comprises the SBPi in the absence of the SBP2, and the SPBi is optionally modified with one or more fatty acid groups.
17. The method of claim 14, wherein the SBP of the composition, in the presence or absence of the L, is bound to the non-biological substrate by applying an effective amount of the SBP to the non-biological substrate; and applying the [DV(BA)] capable of being coupled to the SBP to non-biological substrate comprising the SBP, wherein the contact between the [DV(BA)] and the SBP results in coupling of the [DV(BA)] and the SBP.
18. The method of claim 17, wherein the SBP comprises the SBPi coupled to the SBP2 in the presence or absence of the L.
19. The method of claim 17, wherein the SBP comprises the SBPi in the absence of the SBP2, and the SPBi is optionally modified with one or more fatty acid groups.
20. The method of claim 14, wherein the surface of the non-biological substrate is selected from the group consisting of metal, metal oxide, non- metal oxide, ceramic, synthetic polymer, plastic, silicone, carbon-based materials, graphite, carbon nanotubes, metallo-carbon composites, and combinations thereof.
PCT/US2008/071825 2007-07-31 2008-07-31 Compositions for delivery of biologically active agents to surfaces Ceased WO2009018484A1 (en)

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