Classifications

• • 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
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/501—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/02—Medicinal preparations containing materials or reaction products thereof with undetermined constitution from inanimate materials
• • 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
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/5021—Organic macromolecular compounds
  • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present application generally relates to tissue engineering and administration of therapeutic compounds.
• Biodegradable microspheres have been widely used as carriers for controlled release of drug molecules, including small molecules (DeF ail et al. 2006), DNA (Jang and Shea, 2003), and proteins (Yang et al. 2001). Although these carriers have become prevalent in biomedical applications ranging from injectable drug delivery (Pandy and Khuller, 2007) to manipulation of stem cell differentiation (Newman and McBurney, 2004; Ferreira et al.
• Hybrid materials composed of organic polymers coated with inorganic minerals have attracted much attention in biology and medicine due to their combination of advantageous properties.
• Polymeric materials are a desirable base material for biomedical applications, as they can be processed into a variety of sizes and geometries, and can be designed to bioresorb in a controllable timeframe. Therefore, polymeric biomaterials have been featured in a variety of applications, including medical devices, tissue engineering scaffolds, and drug delivery systems.
• Calcium phosphate based mineral coatings represent desirable surfaces for biomedical applications, as they are similar in composition to bone tissue, and have been shown to promote favorable interactions with natural bone, a property termed "bioactivity".
• hydroxyapatite - the major inorganic component of bone mineral- is osteoconductive (Ducheyne et al., 1999), and may also be capable of inducing new bone formation in vivo (Habibovic et ⁇ /., 2006).
• the mechanism for mineral nucleation and growth on these materials is based on the interaction of carboxylate and hydroxyl groups on the hydrolyzed surface with calcium- and phosphate-rich nuclei in solution, creating a driving force for heterogeneous nucleation and mineral growth (Murphy and Mooney, 2002).
• This coating process is particularly suitable for biodegradable polymers, as it can be carried out at physiological temperature and pH (Tanahashi et al., 1994), and the mild processing conditions also suggest that it is possible to incorporate biologically active molecules such as polypeptides and polynucleotides, during the coating process.
• microspheres can be produced that have a calcium-containing mineral coating. See Examples. These microspheres are useful, at least for providing slow release of therapeutic compounds combined therewith.
• the application is directed to a microsphere comprising a bead coated with a first calcium-containing mineral.
• the application is also directed to a method of producing a microsphere.
• the method comprises incubating a bead in a physiological saline solution comprising carbonate, calcium, and phosphate such that a first calcium-containing mineral layer coating forms on the bead.
• the bead with the mineral layer coating is the microsphere.
• the application is directed to a method of administering a compound to a vertebrate.
• the method comprises administering the above microsphere to the vertebrate.
• FIG. 1 is a schematic and micrographs of PLG microspheres before and after mineral coating.
• FIG. 2 are graphs of XRD and FTIR spectra.
• Panel A shows a representative
• Panel B shows a representative FTIR spectrum showing phosphate peaks (1087, 1035, 950, 560 cm “1 ) and carbonate peaks (1456 and
• FIG. 3 is graphs and a micrograph relating to protein binding on mineral-coated microspheres.
• Panel A is a representative FT-IR spectrum of protein bound onto mineral-coated microspheres showing phosphate peaks (1087, 1035, 950, 560 cm “1 ), carbonate peaks (1456 and
• Panel C shows binding curves of bovine serum albumin (BSA) and Cytochrome c (Cyt c) on the mineral-coated microspheres surface.
• FIG. 4 is graphs and micrographs relating to release of protein from mineral- coated microspheres.
• Panel A is a comparison of cumulative release of BSA bound to mineral- coated PLG microspheres and BSA encapsulated in PLG microspheres. Both approaches show sustained release, with the total amount of bound BSA release significantly higher than the encapsulated BSA after 30 days.
• Panels B and C are SEM images of mineral-coated microspheres (B); and PLG microspheres (C) after the 30 day release period.
• Panel D shows cumulative release of Cyt c from mineral-coated PLG microspheres at pH 4 and pH 7.4.
• FIG. 5 is SEM images of a PLG microsphere (A) and mineral-coated microsphere after incubation in mSBF for 7 days.
• FIG. 6 Panels A-D are SEM images of mineral-coated microspheres after a 7 day incubation in mSBF solution, 0.25% w/v (A), 0.50% w/v (B), 0.75% w/v (C), and 1.00% (D) w/v.
• Panel E is a graph showing the relationship between the microsphere concentration in solution during mineral growth, and the size of mineral-coated microsphere aggregates.
• FIG. 7 is a graph showing the ⁇ potential of PLG microspheres in buffers PBS, mSBF, and mSBF + 0.1% (v/v) Tween 20TM (Panel A) and nonhydrolyzed and hydrolyzed PLG films in comparison with PLG microspheres (Panel B).
• FIG. 8 is graphs showing X-ray diffraction analysis of mineral-coated microspheres and hydroxyapatite powder (included for comparison) (Panel A), and Fourier transform infrared analysis of mineral-coated microspheres (Panel B). Peaks associated with carbonate are denoted by *, and peaks associated with phosphate are denoted by f .
• FIG. 9 shows an EDS spectrum of mineral-coated microspheres after a 7 day incubation in mSBF.
• FIG. 10 is SEM images showing the process of mineral nucleation and growth on
• FIG. 11 is a graph showing the percentage of aggregated microspheres suspended for 1, 3 and 7 days in PBS, mSBF and mSBF+Tween20TM.
• FIG. 12 is graphs and SEM images of mineral dissolution of mineral-coated microspheres.
• Panel A shows cumulative dissolution of Ca 2+ and PO 4 3" during a 25 day incubation in tris-buffered saline (TBS).
• TBS tris-buffered saline
• Panel B shows cumulative dissolution of PO 4 " during a
• Panels C is SEM images of mineral-coated microspheres after the
• Panel D is SEM images of mineral-coated microspheres after the 25 day
• FIG. 13 is SEM images and a graph showing the effect of surfactant (T ween
• Panel E shows an FTIR spectrum of PLG microspheres coated with mineral via a 28 day mSBF incubation in the presence of 0.1 %v/v Tween 20TM. A spectrum of commercial HA powder is included for comparison.
• FIG. 14 is SEM images showing nanometer-scale mineral morphology on the surface of microspheres formed in the presence (A) or absence (B) of 0.1% v/v Tween 20TM.
• the inventors have developed methods for producing novel microspheres that have a calcium-containing mineral coating. They have also characterized these microspheres and established that they have advantageous properties useful for utilizing the microspheres to deliver therapeutic compounds to tissues. See Examples.
• the application is directed to a microsphere comprising a bead coated with a first calcium-containing mineral.
• a microsphere comprising a bead coated with a first calcium-containing mineral.
• the Examples describe exemplary methods for producing these microspheres using a modified simulated body fluid (mSBF). By adjusting the mineral composition, and/or concentration of the mSBF, the composition of the mineral precipitated on the microspheres can be manipulated. See also U.S. Patent Application
• Inorganic minerals suitable for producing a calcium-containing mineral coating include various bone mineral ions, such as, but not limited to calcium and phosphate and combinations of bone mineral ions, such as calcium-phosphates.
• the calcium-containing mineral coating can comprise, e.g., hydroxyapatite (HAP), ⁇ -tricalcium phosphate ( ⁇ -TCP), ⁇ - tricalcium phosphate ( ⁇ -TCP), amorphous calcium phosphate, dicalcium phosphate, octacalcium phosphate or calcium carbonate.
• the calcium-containing mineral coating can comprise a plurality of layers, e.g., separate layers having distinct dissolution profiles.
• solubility of calcium phosphate species adhere to the following trend: amorphous calcium phosphate>dicalcium phosphate>octacalcium phosphate> ⁇ -TCP>HAP.
• a dicalcium phosphate mineral will typically have a dissolution rate that is more than fifty times higher than that of HAP. Therefore, creation of a matrix with distinct calcium phosphate layers allows for a broad range of dissolution patterns.
• the bead can be formed of any suitable material known in the art. The selection of the bead material for any particular application can be made without undue experimentation.
• the bead comprises a negative charge, which can promote the deposition of the calcium containing material.
• the negative charge could be provided by any moiety present on the bead, for example a carboxylate group, as is present in poly(D,L-lactide- co-glycolide) (PLG).
• PLG poly(D,L-lactide- co-glycolide)
• the bead is made of a polymer, for example a synthetic polymer. In various aspects of these embodiments, the polymer is bioabsorbable.
• Nonlimiting examples of suitable bead materials include, for example, a collagen gel, polyvinyl alcohol, a marine adhesive protein, a PLG fiber matrix, a polyglactin fiber, a calcium alginate gel, a polyglycolic acid, polyester (e.g., poly-(L-lactic acid) or a poly anhydride), a polysaccharide (e.g.
• alginate chitosan, polyphosphazene, polyacrylate, polyethylene oxide- polypropylene glycol block copolymer, fibrin, collagen, and fibronectin, polyvinylpyrrolidone, hyaluronic acid, poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates), polyurethanes, polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof), polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonated polyolifms, polyethylene oxide, polyvinyl alcohol, teflon®, nylon, and analogs, mixtures, combinations and
• the bead is made of a polymer that comprises polar oxygen groups.
• polymers include polycarboxylates, polyanhydrides, poly( ⁇ - hydroxyesters), poly(ethylene terephthalates), poly(carbonates), poly(amides), poly(lactones), a poly(saccharides) and poly(acrylates).
• the bead is made of a PLG, for example at a ratio of about 85:15 lactide:glycolide ("85:15 PLG").
• 85:15 PLG copolymers is advantageous as a decrease in the lactide/glycolide ratio of the copolymer is believed to increase the rate of surface hydrolysis.
• the first calcium-containing mineral is a carbonated-substituted calcium-deficient hydroxyapatite and the bead is PLG, wherein the PLG is about 85:15 lactide:glycolide.
• the microsphere further comprises a component adhering to the first calcium-containing mineral, wherein the component introduces a functional group to the first calcium-containing mineral. Introduction of such a functional group allows covalent binding of any additional materials (e.g. therapeutic compounds) to the microspheres.
• Nonlimiting examples of functional groups that can be introduced on the component is a carboxylate, an amine, a carbonyl, a nitro, a hydroxyl, an aldehyde, or an ester.
• the component comprises a poly(aspartic acid), a poly(glutamic acid), or a bisphosphonate. See e.g., Murphy et ah, 2007.
• Other nonlimiting examples of components useful in these embodiments are the oligopeptides AAAAEPRREVAEL or
• AAAA ⁇ EPRR ⁇ EVA ⁇ EL where ⁇ E is carboxyglutamate.
• the microsphere further comprises a first compound adhering to the first calcium-containing mineral or the component.
• the first calcium-containing mineral is a carbonated-substituted calcium-deficient hydroxyapatite and the bead is poly(D,L-lactide-co-glycolide) (PLG), wherein the PLG is about 85:15 lactide:glycolide.
• PLG poly(D,L-lactide-co-glycolide)
• the first compound can be an organic compound less than 2000 MW, or less than 1000 MW, or less than
• Nonlimiting examples include antibiotics, corticosteroids and statins. More specific examples include cefazolin, cefuroxime, clindamycin, vancomycin and dexamethasone.
• the first compound can be an oligopeptide or polypeptide.
• an oligopeptide comprises a linear chain of 30 or less amino acids.
• a polypeptide comprises more than 30 amino acids.
• examples of oligopeptides are GGRGDSP (a cell adhesion peptide derived from f ⁇ bronectin), GGIKVAV (a cell adhesion peptide derived from laminin), GGYIGSR (a cell adhesion peptide derived from laminin), GGDGEA (a cell adhesion/signaling peptide derived from type I collagen), GGKIPKASSVPTELSAISTLYL (a peptide derived from bone morphogenetic protein-2), AAAAEPRREVAEL (a modified peptide derived from osteocalcin - some affinity for hydroxyapatite mineral), AAAA ⁇ EPRR ⁇ EVA ⁇ EL, where ⁇ E is carboxyglutamate (a modified peptide derived from osteocalcin - high affinity for hydroxyapatite mineral),
• the first compound is a polypeptide, for example a cytokine, an enzyme, or a protein comprising an antibody binding site (e.g., an antibody).
• polypeptides that could be included in the microspheres are virtually any hormone, neurotransmitter, growth factor, growth factor receptor, interferon, interleukin, chemokine, cytokine, colony stimulating factor and/or chemotactic factor protein or polypeptide.
• transcription or elongation factors include transcription or elongation factors, cell cycle control proteins, kinases, phosphatases, DNA repair proteins, oncogenes, tumor suppressors, angiogenic proteins, anti- angiogenic proteins, immune response stimulating proteins, cell surface receptors, accessory signaling molecules, transport proteins, enzymes, anti-bacterial and/or anti-viral proteins or polypeptides, and the like, depending on the intended use of the ultimate composition.
• GH growth hormone
• PTH parathyroid hormone
• BMPs bone morphogenetic proteins
• BMPs bone morphogenetic proteins
• TGF- ⁇ transforming growth factor- ⁇
• FGF fibroblast growth factor
• GMCSF epidermal growth factor
• EGF platelet derived growth factor
• IGF insulin-like growth factor
• LIF leukemia inhibitory factor
• VEGF vascular endothelial growth factor
• bFGF basic fibroblast growth factor
• PDGF hepatocyte growth factor/scatter factor
• G-CSF hepatocyte growth factor/scatter factor
• the polypeptide is a BMP-2, a BMP-7, a VEGF, an FGF-2, a PDGF, a TGF- ⁇ , an interleukin, or a human GH.
• the first compound can also be a nucleic acid.
• Non-limiting examples include a microRNA, an antisense nucleic acid, and a vector.
• the first compound is a vector, any vector known or later discovered can be included here.
• the vector comprises a sequence encoding a therapeutic protein, such as any of the proteins discussed above.
• the first compound can noncovalently adhere to the microsphere.
• the microsphere can further comprise a component adhering to the first calcium-containing mineral and introducing a functional group to the microsphere, to which the compound is covalently attached. See, e.g., Murphy et al., 2007.
• the first compound is at more than one level of the first calcium-containing mineral. These microspheres generally release the compound over a longer period of time than where the compound is only at one level (for example the outer surface of the first calcium-containing mineral.
• the first compound is modified to change the rate at which the compound is released from the microsphere.
• the first compound further comprises a moiety that increases the strength of binding of the compound to the first calcium- containing mineral, the compound would be released more slowly than if the moiety is not present.
• moieties include amino acid sequences rich in glutamic acid, aspartic acid or phosphoserine, which interact directly with calcium ions in mineralized extracellular matrices.
• Other examples include AAAAEPRRE V AEL or AAAA ⁇ EPRR ⁇ EVA ⁇ EL, where ⁇ E is carboxyglutamate.
• the microsphere further comprises a second compound adhering to the first calcium-containing mineral or the component.
• the microspheres are not limited as to the nature of the second compound.
• the second compound can be for example an organic compound less than 2000 MW or 1000 MW or 500 MW.
• the second compound can be an oligopeptide or polypeptide, or a nucleic acid.
• the first compound and the second compound can be on the same or different levels of the first calcium-containing minerals. Having the two compounds on different levels is useful when it is desired that the two compounds are released at different times.
• the microsphere can also further comprise a third, fourth, fifth, etc. compound as desired.
• the microsphere comprises a living cell.
• the living cell can be from any organism, including an Archaea, a prokaryote, or a eukaryote.
• the cell is a mammalian cell.
• the cell can be naturally occurring or, alternatively, can be transformed to express a recombinant molecule, e.g., a protein or nucleic acid (such as an miRNA).
• the first calcium-containing mineral is a carbonated-substituted calcium-deficient hydroxyapatite and the bead is poly(D,L-lactide-co-glycolide) (PLG), wherein the PLG is about 85:15 lactide:glycolide.
• PLG poly(D,L-lactide-co-glycolide)
• the cell can be adhered to the microsphere by any known means.
• the microsphere comprises a first binding agent that binds to a second binding agent on the cell.
• Nonlimiting examples of such agents are a receptor and a ligand of the receptor, complementary nucleic acids, or a cell adhesion peptide and a ligand of the cell adhesion peptide.
• Suitable cell adhesion peptides are GGRGDSP, GGIKVAV, GGYIGSR or GGDGEA.
• These peptides or any other first binding agent can be part of a larger molecule, for example a molecule that binds to the first calcium-containing mineral as discussed above.
• the microsphere comprises a cell as well as a compound
• the cytokine is advantageously close to the cell, such that the compound is likely to contact and thus interact with the cell.
• the microsphere further comprises a coating of a second calcium-containing mineral.
• the second coating can be a mineral that has a different degradation rate than the first calcium-containing mineral.
• the microsphere comprising the two coatings can further comprise one or more than one compound. Either of the two coatings can be, for example, any of hydroxyapatite (HAP), ⁇ -tricalcium phosphate ( ⁇ -TCP), ⁇ -tricalcium phosphate ( ⁇ -TCP), amorphous calcium phosphate, dicalcium phosphate, octacalcium phosphate or calcium carbonate.
• HAP hydroxyapatite
• ⁇ -TCP ⁇ -tricalcium phosphate
• ⁇ -TCP ⁇ -tricalcium phosphate
• amorphous calcium phosphate dicalcium phosphate
• octacalcium phosphate or calcium carbonate amorphous calcium phosphate
• the first calcium-containing mineral can be hydroxyapatite and further comprises a first therapeutic compound
• the second calcium-containing mineral can be ⁇ -TCP and is coated above (i.e., closer to the surface of the microsphere) the first calcium containing mineral and further comprises a second therapeutic compound.
• the second calcium-containing mineral would degrade first, releasing the second therapeutic compound before the first therapeutic compound is released.
• a compound is present in the microsphere, its release depends on a number of factors, for example (a) how strongly the compound adheres to the calcium-containing mineral or component, (b) how far away from the surface of the microsphere the compound is (for example if a layer of calcium-containing mineral is deposited on the microsphere after the compound is added), (c) the degradation/bioabsorption rate of the calcium-containing mineral, (d) whether the compound is covalently bound to a component, and if so, (e) how strongly the component is bound to the mineral, (f) how strongly the covalent bond is between the compound and the component, and (g) whether there are enzymes present, e.g., from a tissue in which the microsphere is implanted, that will break the covalent bond and release the compound.
• each of these factors may have more or less influence on the release of the compound, depending on the configuration of the microsphere.
• the component binds to the calcium-containing material strongly, and there are no enzymes to break the covalent bond, then the main factor influencing the release of the component is the rate of degradation of the calcium-containing mineral.
• the compound is noncovalently adhering directly to the calcium-containing mineral, then all of the above factors (a) - (c) will have an influence in the rate of release of the compound.
• the compound is in the bead. In these embodiments, the compound would likely be released upon degradation of the bead, after the degradation of the calcium-containing mineral.
• the various microspheres discussed herein generally have a diameter between about 0.5 ⁇ m and about 500 ⁇ m. In other embodiments, the microsphere has a diameter between about 0.5 ⁇ m and about 100 ⁇ m. In additional embodiments, the microsphere has a diameter between about 2 ⁇ m and about 6 ⁇ m.
• the microspheres tend to aggregate, particularly if they are produced by incubation in mSBF at a high concentration of microspheres or for an extended period of time (see Examples).
• the application is directed to a plurality of any of the above-described microspheres, aggregated.
• the first calcium-containing mineral is a carbonated-substituted calcium-deficient hydroxyapatite and the bead is poly(D,L-lactide-co- glycolide) (PLG), wherein the PLG is about 85:15 lactide:glycolide.
• PLG poly(D,L-lactide-co- glycolide)
• a first compound, as described above adheres to the first calcium-containing mineral.
• the application is also directed to a method of producing a microsphere.
• the method comprises incubating a bead in a physiological saline solution comprising carbonate, calcium, and phosphate such that a first calcium-containing mineral layer coating forms on the bead, where the bead with the mineral layer coating is the microsphere.
• the solution comprises NaCl, KCl, MgCl 2 , MgSO 4 , NaHCO 3 , Tris, CaCl 2 , and KH 2 PO 4 .
• the solution comprises about 100-200 mM NaCl, about 1-8 mM KCl, about 0.1-2 mM MgSO 4 , about 0.2-5 mM MgCl 2 , about 1-100 mM NaHCO 3 , about 2-20 mM CaCl 2 , and about 0.5-10 mM KH 2 PO 4 .
• the solution comprises about 141 mM NaCl, about 4.0 mM KCl, about 0.5 mM MgSO 4 , about 1.0 mM MgCl 2 , about 4.2 mM NaHCO 3 , about 5.0 mM CaCl 2 , and about 2.0 mM KH 2 PO 4 .
• the solution also comprises a surfactant, which can change the morphology of the calcium-containing mineral layer, and reduce aggregation of the microspheres. See Example 2. Any surfactant now known or later discovered may be used here. In some embodiments, the surfactant is Tween 20TM.
• the mineral coating described herein is developed by incubating the constituents in the above solution, which can be called a "simulated body fluid” (SBF) or a “modified simulated body fluid” (mSBF), for five days or more at a pH of about 6.8 to about 7.4 and at a temperature of about 37 0 C.
• SBF simulated body fluid
• mSBF modified simulated body fluid
• the procedure produces a calcium- deficient, carbonate-containing apatite material on alginate and on poly-( ⁇ -hydroxy esters). See U.S. Pat. No. 6,767,928, incorporated herein by reference.
• mSBF includes elevated calcium and phosphate. In general, an increase in pH favors hydroxyapatite growth, while a decrease in pH favors octacalcium phosphate mineral growth.
• conditions favorable for hydroxyapatite formation include a pH between about 5.0 and about 8.0 and a calcium concentration multiplied by a phosphate concentration between about 10 5 and about 10 8 M.
• conditions favorable for octacalcium phosphate formation include a pH between about 6.0 and about 8.0 and a calcium concentration multiplied by a phosphate concentration between about 10 5 and about 10 7'5 M.
• conditions favorable for dicalcium phosphate dehydrate formation include a pH between about 6.0 and about 8.0 and a calcium concentration multiplied by a phosphate concentration between about 10 4 and about 10 6 M.
• the bead Prior to deposition of the first calcium-containing mineral, the bead may be surface-functionalized to allow increased mineral deposition by utilizing chemical pre-treatment to achieve surface hydrolysis, e.g., using an NaOH solution. Surface degradation by this technique causes an increase in the amount of polar oxygen functional groups on the surface of the material. The functionalized surface is then incubated in a mineral-containing solution.
• the method further comprises adding a component that adheres to the first calcium-containing mineral layer to the microsphere. In these embodiments, the component introduces a functional group to the first calcium-containing mineral layer.
• nonlimiting examples of such components include peptides comprising a poly(aspartic acid) sequence, a poly(glutamic acid) sequence, AAAAEPRREVAEL and AAAA ⁇ EPRR ⁇ EVA ⁇ EL, where ⁇ E is carboxyglutamate.
• the method further comprises incubating the microsphere with a first compound such that the first compound adheres to the microsphere.
• the first compound can be any chemical compound, such as an organic compound less than 2000 MW, an oligopeptide or polypeptide (e.g., a cytokine, an enzyme, or a protein comprising an antibody binding site), or a nucleic acid (e.g., a microRNA, an antisense nucleic acid, or a vector).
• the first compound is incubated with the microsphere such that the first compound is non-covalently bound to the microsphere.
• a component that introduces a functional group to the microsphere is adhered to the microsphere, the first compound is covalently attached to the functional group.
• the first compound is incubated with the bead in the physiological saline solution such that the first compound is deposited on the bead along with the mineral layer coating.
• Such microspheres generally have the first compound deposited throughout the mineral layer coating, whereas if the compound is bound to the bead after the mineral layer is coated onto the bead, the compound would only be present on the surface of the mineral layer. The first compound would be expected to be released over a longer period of time in the former case than in the latter case.
• the bead is incubated in the physiological saline solution both before and after the first compound is adhered to the microsphere, after which additional first compound is adhered to the microsphere.
• the first compound would be internal to the surface of the mineral layer and would be released after a first compound deposited on the surface.
• the method can further comprise incubating the microsphere with a second compound such that the second compound adheres to the microsphere.
• the bead is incubated in the physiological saline solution before and after the first compound is adhered, after which the second compound is adhered to the microsphere, such that the first compound and the second compound are on different layers of the first calcium- containing mineral layer. Such a microsphere would release the second compound before the first compound.
• the application is further directed to a method of administering a compound to a vertebrate.
• the method comprises administering any of the microspheres disclosed above to the vertebrate, where the microsphere further comprises the compound.
• the microsphere is in a pharmaceutically acceptable material.
• pharmaceutically acceptable it is meant a material that (i) is compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition.
• pharmaceutically acceptable carriers include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, microemulsions, and the like.
• microspheres can be formulated without undue experimentation for administration to a vertebrate, including humans, as appropriate for the particular application. Additionally, proper dosages of the microspheres can be determined without undue experimentation using standard dose-response protocols. [0073] Accordingly, the microspheres may be enclosed in gelatin capsules.
• the microspheres of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
• the microspheres can alternatively be administered parenterally such as for example, by intravenous, intramuscular, intrathecal or subcutaneous injection.
• administration can be systemic, for example if the microspheres were administered by injection into the blood stream.
• the administration can be local, e.g., an injection of the microspheres directly onto an area where there is a tissue defect, where the compound is a cytokine that stimulates filling in the defect.
• Parenteral administration can be accomplished by incorporating the microspheres into a suspension.
• Such suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents.
• Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of osmolarity such as sodium chloride or dextrose may also be added.
• the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
• Rectal administration includes administering the microspheres, in a pharmaceutical composition, into the rectum or large intestine. This can be accomplished using suppositories or enemas.
• Suppository formulations can easily be made by methods known in the art.
• suppository formulations can be prepared by heating glycerin to about 120 0 C, dissolving the composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.
• Transdermal administration includes percutaneous adsorption of the microspheres to the skin.
• Transdermal formulations include patches (such as the well-known nicotine patch), ointments, creams, gels, salves and the like.
• the present invention includes nasally administering to the vertebrate a therapeutically effective amount of the microspheres.
• nasally administering or nasal administration includes administering the compound to the mucous membranes of the nasal passage or nasal cavity of the patient.
• pharmaceutical compositions for nasal administration of the compound include therapeutically effective amounts of the compound prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the compound may also take place by applying the microspheres in a nasal tampon or nasal sponge.
• these methods can be used to treat any vertebrate, including wild or domesticated mammals or birds, including farm animals and pets.
• the vertebrate is a human.
• the vertebrate has a condition that is treatable by administering the compound.
• conditions include cancer, diabetes, and others.
• the condition is a tissue defect.
• the tissue defect can be, for example, in a bone, a soft tissue, or an internal organ.
• the defect may be caused by disease or trauma, or it may be congenital.
• the application is also directed to the use of any of the above-described microspheres for the manufacture of a medicament for treating a vertebrate with a compound.
• the vertebrate is a mammal, e.g., a human.
• the application is directed to the use of any of the above-described microspheres for the treatment of a vertebrate with a compound.
• the vertebrate is a mammal, e.g., a human.
• Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
• Example 1 Mineral-coated polymer microspheres for controlled protein binding and release.
• the work described herein evaluates the hypothesis that protein-mineral interactions could be used as an alternative mechanism to create biodegradable micro-carriers for controlled protein binding and release.
• the calcium phosphate mineral hydroxyapatite is employed as a substrate for protein binding and release, as it has been used for over 50 years in chromatographic protein separations based on its ability to bind and release both acidic and basic proteins under particular solution conditions.
• biodegradable polymer microspheres can be coated with an inorganic hydroxyapatite layer, and that this biodegradable coating can be used as a substrate for binding and sustained release of acidic and basic proteins.
• This approach involves nucleation and growth of inorganic calcium phosphate mineral coatings on the surface of organic, biodegradable polymer microspheres at near physiologic temperature and pH.
• This mineral growth process mimics natural biomineralization processes (Mann, 2001), and results in a mineral coating that is similar in structure (platelike nanostructure) and composition (carbonated-substituted, calcium-deficient hydroxyapatite phase) to human bone mineral, as detailed previously on macroscopic polymer films (Murphy and Mooney, 2002).
• protein-releasing, mineral-coated polymer microspheres were prepared here via a two-step process involving: i) formation of biodegradable PLG microspheres using a standard water-oil-water double emulsion process (Meng et ah, 2003); and ii) coating of PLG microspheres with an inorganic, bone-like mineral (BLM) film via incubation in a modified simulated body fluid (mSBF), an aqueous solution which contains the ionic constituents of blood plasma with 2-fold higher concentrations of calcium and phosphate ions (Murphy and Messersmith, 2000) (FIG. 1).
• mSBF modified simulated body fluid
• X-ray diffraction and FTIR spectra indicate that the mineral grown on PLG microspheres is an apatite mineral (FIG. 2).
• Scanning electron microscopy (SEM) indicates that the mineral film is continuous on the microsphere surface and has a plate-like nanostructure. Therefore, the mineral layer grown on biodegradable polymer microspheres is similar in composition and morphology to bone mineral, as described previously Murphy and Mooney, 2002).
• BSA bovine serum albumin
• Cyt c a basic protein
• albumin is one of the most abundant proteins found in blood plasma and has been shown to promote formation of bone tissue (Yamaguchi et al., 2003), while cytochrome c serves as a model protein for several basic growth factors, such as fibroblast growth factor-2, bone morphogenic proteins, and transforming growth factor ⁇ .
• cytochrome c serves as a model protein for several basic growth factors, such as fibroblast growth factor-2, bone morphogenic proteins, and transforming growth factor ⁇ .
• the FTIR spectrum shows phosphate peaks (1087, 1035, 950, and 560 cm “1 ) and carbonate peaks (1456 and 1417 cm 1 ) corresponding to carbonate-substituted hydroxyapatite mineral coatings, consistent with previous studies of BLM coatings formed on biodegradable polymer substrata (Murphy and Mooney, 2002).
• the FTIR spectrum also shows amide peaks (1653 and 1558 cm "1 ) corresponding to the presence of BSA bound to the coating surface. SEM analysis corroborates the FTIR analysis, and shows bound protein deposited on mineral-coated PLG microspheres (FIG. 3B).
• BSA binding on the mineral coating was next quantified by incubating mineral-coated microspheres for 4 hours in PBS with varying BSA concentrations.
• the results show that the amount of bound BSA on the microspheres increased linearly in the range of 0-200 ⁇ g/ml and reached a plateau at 400 ⁇ g/ml (FIG. 3C).
• This binding is consistent with a typical Langmuir isotherm, and corroborates previous studies of BSA binding to hydroxyapatite powder (Hughes Wassell et al., 1995). Cyt c showed a binding curve similar to that of BSA, and it is noteworthy that Cyt c bound to the mineral more efficiently than BSA at the same solution protein concentrations.
• mSBF modif ⁇ ed-simulated body fluid
• BSA cytochrome c
• Cyt c cytochrome c
• Five mg of mineral-coated microspheres were immersed in 1.5 ml solutions containing variable protein concentrations (0- 800 ⁇ g/ml, 4 hr, 37 0 C). The solution was centrifuged to sediment the microspheres, and the amount of protein in the supernatant was measured. The centrifuged microspheres were washed with distilled water and freeze dried prior to SEM and FTIR spectroscopy.
• Five mg of mineral-coated microspheres were immersed in 1.5 ml of protein solutions (200 ⁇ g/ml, 4 hr, 37 0 C) as described above, to produce protein-containing, mineral- coated microspheres.
• Example 2 Fabrication and Characterization of Mineral-Coated Poly(lactide-co-glycolide) Microspheres.
• Mineral-coated microspheres were prepared via a bioinspired, heterogeneous nucleation process at physiologic temperature.
• Poly(DL-lactide-co-glycolide) (PLG) microspheres were fabricated via a water-in-oil-in- water emulsion method and were mineral- coated via incubation in a modified simulated body fluid (mSBF).
• mSBF modified simulated body fluid
• X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy with associated energy dispersive X-ray spectroscopy confirmed the presence of a continuous mineral coating on the microspheres.
• the mineral grown on the PLG microsphere surface was a carbonate-containing hydroxyapatite, and the mineral shows a porous structure of plate-like mineral crystals at the nanometer scale. Aggregation of mineral-coated microspheres was observed when microsphere concentrations above 0.50 mg/mL were incubated in mSBF for 7 days, and the size of aggregates was dependent on the microsphere concentration in solution. In vitro mineral dissolution studies performed in Tris-buffered saline confirmed that the mineral formed was resorbable.
• a surfactant additive (T ween 20TM [PEG(20)sorbitan monolaurate]) was incorporated into mSBF to prevent microsphere aggregation during the mineral growth process, and Tween 20TM not only prevented aggregation, but also influenced the characteristics of the mineral formed on the surface of PLG microspheres.
• hydroxyapatite is the ability to bind to biological molecules.
• hydroxyapatite is commonly used as a resin for chromatographic purification of proteins and plasmid DNA (Colman et al., 1978; Schroder et al., 2003), as the mineral surface contains both positive (Ca 2+ ) and negative (PO 4 3" ) ions capable of interacting electrostatically with basic and acidic molecules, respectively.
• PLG microspheres were fabricated by water-in-oil-in-water (W/O/W) double emulsion technique as reported elsewhere (Berchane et ah, 2006). Briefly, the organic phase consisted of 5% (w/v) PLG in 1 ml ethyl acetate. The aqueous phase consisted of 0.1 ml phosphate buffered saline (PBS). The aqueous and organic phases were mixed and sonicated using Sonif ⁇ er 250 (VWR International, Inc., West Chester, PA) for 15 s.
• VWR International, Inc. West Chester, PA
• the resulting first emulsion was added immediately into 1 ml of aqueous 1% (w/v) PVA in 7% (v/v) ethyl acetate that was being mechanically vortexed for 15 s to form a second emulsion.
• the resulting solution was then added to a beaker containing 200 ml of 0.3% PVA in 7% ethyl acetate and further rigorously stirred for 4 hr to allow for organic solvent evaporation.
• the resulting microspheres were collected by filtration through 0.22 ⁇ m filter, washed three times with de -ionized water, and resuspended in de-ionized water. The microspheres were lyophilized for a minimum of 48 hr and were stored at -20 0 C in the presence of a desiccant.
• mSBF was prepared by dissolving 141 mM NaCl, 4.0 mM KCl, 0.5 mM MgSO 4 , 1.0 mM MgCl 2 , 4.2 mM NaHCO 3 , 5.0 mM CaCl 2 , and 2.0 mM KH 2 PO 4 in distilled water, buffered to pH 6.8, and was held at 37 0 C for the duration of the incubation period.
• 0.1% of Tween 20TM (Sigma- Aldrich, St. Louis, MO) was added to the mSBF to prevent the aggregation of microspheres.
• EQUINOX 55 spectrometer (Bruker AXS Inc., Madison, WI). Samples were examined in transmission mode in the 400 - 4000 cm "1 range and data were analyzed by OPUS software. [00104] The morphology and composition of the coated mineral on the microsphere surface was analyzed using scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS). Microspheres before and after mSBF incubation were mounted on aluminum stubs with double sided carbon tape, sputtered with gold for 30s at 45mA and characterized using a LEO DSM 1530 field emission SEM, operating at 2kV for SEM and 1OkV for EDS.
• SEM scanning electron microscopy
• EDS energy-dispersive X-ray spectroscopy
• a working AAM (acetone-acid-molybdate) solution was prepared by mixing 2 parts acetone with 1 part 5 N sulfuric acid, and 1 part 10 mM ammonium molybdate solution.
• the assay was performed in a 96-well plate by adding lOO ⁇ l a freshly made working solution to 100 ⁇ l sample.
• the amount of phosphate complex was quantitatively detected by measuring the absorbance at 405 nm on a SynergyTM HT Multidetection Microplate Readers (Bio-Tek Instruments, Inc., UK) and comparing to a set of standards with known phosphate concentrations.
• Micrographs of the microspheres incubated in mSBF for 7 days show continuous mineral coatings on individual microspheres incubated at 0.25% and 0.50% (w/v) (FIG. 6A, B). Microsphere aggregation was observed as microspheres concentration increased, and mineral coatings were observed on the surface of microsphere aggregates (FIG. 6B, C, D).
• the peak areas in the XRD spectrum of mineral-coated microspheres are broader than that of hydroxyapatite powder, and this may be due to the small crystal size of the mineral deposited on the PLG microsphere surfaces.
• FTIR peaks observed in the 1600-400 cm “1 region can be attributed to carbonate-substituted hydroxyapatite, including phosphate peaks at 570, 950, 1046, and 1098 cm “1 , and carbonate peaks at 870, 1410, and 1456 cm “1 (FIG. 8B).
• microspheres were incubated in three different solutions: (1) a IxPBS solution (calcium-deficient), (2) mSBF, and (3) mSBF in the presence of Tween 20TM. Each incubation was performed at 37 0 C with daily solution changes for 7 days. Results showed that 35% and 42% microspheres aggregated after the first day of incubation in PBS and mSBF, respectively. The percentage of aggregated microspheres increased to 87% in PBS and 90% in mSBF after 7 days of incubation.
• Dissolution and re -precipitation are key characteristics of hydroxyapatite coatings, particularly in orthopedic implant design and drug delivery applications. Some forms of hydroxyapatite mineral have been shown to degrade slowly or incompletely over extended timeframes, and the permanent presence of these materials in vivo is undesirable in applications that call for material degradation over time ⁇ e.g. tissue engineering, drug delivery). In addition, mineral dissolution can influence release of biological molecules from these coatings in drug delivery applications, as described in Example 1. Therefore, in this study dissolution of mineral coatings in Tris-buffered saline (TBS) and in DMEM was characterized for 25 days. Ca 2+ and PO 4 " were gradually released from mineral coatings over time in TBS (FIG.
• the morphology of the mineral coating after incubation in DMEM was similar to the morphology of the mineral prior to incubation in DMEM, which indicates that if re-precipitation is occurring, it is resulting in growth of a new mineral phase that is similar to the mineral phase grown initially.
• Tween 20TM was added to the mSBF solution.
• Tween 20TM is a common surfactant used as a stabilizer in studies which measure drug release from PLG microspheres in vitro (He et al., 2005; Raman et al., 2005).
• the rate of mineral formation on the surface of microspheres in the presence of Tween 20TM (FIG. 10A-D) is clearly slower than in the absence of surfactant (FIG. 5, 6).
• FTIR spectra of the mineral formed in the presence of surfactant FIG.
• a rough surface with a relatively well-distributed mineral coating has an increased surface area, and the corresponding increase in available binding sites could result in higher protein binding (Example 1; LeGeros, 2002).
• Laurencin et al. showed that mineral coating of a sintered PLG microsphere scaffold in mSBF increase the protein adsorption capacity and decreased the initial burst release of protein from the polymer scaffold when compare to non-mineralized scaffolds (Jabbarzadeh et al., 2007).
• Mineral-coated PLG microsphere have been fabricated by a simple and inexpensive two-step process involving microsphere fabrication via double emulsion and coating of those microspheres with mineral by immersing in mSBF solution.
• XRD and FTIR spectra indicated that the coatings comprised a carbonated-substituted hydroxyapatite mineral with a porous, plate-like nanoscale morphology.
• the size of the mineral-coated microspheres or microsphere aggregates can be controlled by varying the microsphere concentration in the mSBF solution.
• Hydroxyapatite coatings were degradable in tris-buffered saline, and the quantitative analysis of calcium and phosphate release from the coatings indicate that the Ca/P molar ratio in the mineral is consistent with that of carbonated-substituted hydroxyapatite.
• the presence of a surfactant during the mineral growth process delayed the formation of mineral, and also significantly affected the morphology of the mineral.
• these findings indicate the feasibility of processing mineral-coated PLG microspheres in a controlled fashion using a bioinspired process. This material may be useful in a variety of applications that may benefit from the bulk properties of polymer microspheres and the surface properties of hydroxyapatite minerals, including tissue engineering, drug delivery, and biomolecule purification.
• Green, D. W.; Mann, S.; Oreffo, R. O. C Mineralized polysaccharide capsules as biomimetic microenvironments for cell, gene, and growth factor delivery in tissue engineering.
• Urist M. R., Y. K. Huo, A. G. Brownell, W. M. Hohl, J. Buyske, A. Lietze, P.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Epidemiology (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Zoology (AREA)
• Inorganic Chemistry (AREA)
• Biophysics (AREA)
• Microbiology (AREA)
• Plant Pathology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Physics & Mathematics (AREA)
• Physical Education & Sports Medicine (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicinal Preparation (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Related Parent Applications (1)

Related Child Applications (3)

Publications (1)

Family

ID=42060103

Family Applications (1)

Country Status (5)

Cited By (13)

Families Citing this family (11)

Citations (12)

Family Cites Families (4)

• 2009
  • 2009-09-25 JP JP2011529272A patent/JP2012503673A/ja active Pending
  • 2009-09-25 EP EP21213679.0A patent/EP4011363B1/en active Active
  • 2009-09-25 WO PCT/US2009/058419 patent/WO2010036919A1/en not_active Ceased
  • 2009-09-25 EP EP09816920.4A patent/EP2349212B1/en active Active
  • 2009-09-25 CA CA2740633A patent/CA2740633C/en active Active
  • 2009-09-25 US US13/879,178 patent/US11607391B2/en active Active

Patent Citations (13)

Non-Patent Citations (63)

Also Published As

Similar Documents

Legal Events