WO2012162555A2 - Nano-amas d'héparine - Google Patents

Nano-amas d'héparine Download PDF

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Publication number
WO2012162555A2
WO2012162555A2 PCT/US2012/039459 US2012039459W WO2012162555A2 WO 2012162555 A2 WO2012162555 A2 WO 2012162555A2 US 2012039459 W US2012039459 W US 2012039459W WO 2012162555 A2 WO2012162555 A2 WO 2012162555A2
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composition
heparin
vegf
growth factor
nanocluster
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PCT/US2012/039459
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WO2012162555A3 (fr
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Tatiana Segura
Sean Anderson
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The Regents Of The University Of California
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Publication of WO2012162555A2 publication Critical patent/WO2012162555A2/fr
Publication of WO2012162555A3 publication Critical patent/WO2012162555A3/fr

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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A61K47/6903—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 semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • 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
    • A61K47/6921—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
    • A61K47/6939—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 a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin

Definitions

  • the present invention relates generally to the field of regenerative medicine.
  • the present invention relates to methods and materials used to deliver heparin- binding factors (e.g., growth factors) to cells in human tissue and organ systems.
  • heparin- binding factors e.g., growth factors
  • the invention relates to nanoparticles composed of heparin conjugated to growth factors and the interaction of such with cell surface receptors and extracellular matrix proteins.
  • the invention provides a composition including nanoclusters that include heparin and a heparin-binding factor, or a fragment thereof.
  • the heparin-binding factor is covalently bound to the nanocluster at an average density greater than 200 molecules per nanocluster.
  • the nanoclusters include nanoparticles that include heparin and a heparin-binding factor, or a fragment thereof.
  • the nanoparticles can include heparin immobilized on support.
  • the nanoparticles can be formed from heparin.
  • the heparin-binding factor can be a growth factor or an extracellular matrix protein.
  • Illustrative growth factors include angiogenic growth factors, such as, e.g., vascular endothelial growth factor.
  • the heparin-binding factor is covalently bound to the nanocluster at an average density greater than 400, 600, 800, or 900 molecules per nanocluster. In some embodiments, the average density is less than 1800, 1600, 1400, 1200, or 1100 molecules per nanocluster.
  • the composition includes greater than 10, 15, 20 ⁇ g heparin-binding factor per mg heparin. In some embodiments, the composition includes less than 45, 35, or 30 ⁇ g heparin-binding factor per mg heparin.
  • the nanoclusters have an average characteristic dimension that is less than 500, 200, or 100, nm. In illustrative embodiments, the nanoclusters have an average diameter that is in the range of approximately 10 nm to 220 nm, e.g., in the range of approximately 50 nm to 70 nm.
  • the composition are encapsulated in a biodegradable shell.
  • the biodegradable shell is susceptible to degradation by a protease, such as a matrix metalloproteinase.
  • the biodegradable shell includes a polyethylene glycol, a carbohydrate, a peptide, or a combination thereof.
  • the composition additionally includes a soluble heparin-binding factor or a fragment thereof, which can be the same as, or different from, the heparin-binding factor covalently bound to the nanocluster.
  • the soluble heparin- binding factor can be, for example, a growth factor or an extracellular matrix protein.
  • Illustrative growth factors include angiogenic growth factors, such as, e.g., vascular endothelial growth factor.
  • the nanoclusters are present in a gel, such as a fibrin gel.
  • the gel additionally includes a soluble heparin-binding factor or a fragment thereof, which can be the same as, or different from, the heparin- binding factor covalently bound to the nanocluster.
  • the soluble heparin-binding factor can be, for example, a growth factor or an extracellular matrix protein.
  • Illustrative growth factors include angiogenic growth factors, such as, e.g., vascular endothelial growth factor.
  • the invention also provides, in certain embodiments, a method of administering a heparin-binding factor to a cell, tissue, or subject, wherein the method includes administering an effective amount of any of the nanocluster compositions described herein to the cell, tissue, or subject, wherein the effective amount is an amount effective to produce a desired effect, and the effective amount is lower than the effective amount of the heparin-binding factor in soluble form.
  • the invention further provides, in certain embodiments, a method of providing an enhanced biological response to a heparin-binding factor in a cell, tissue, or subject, wherein the method includes administering an effective amount of any of the nanocluster compositions described herein to the cell, tissue, or subject, wherein the effective amount is an amount effective to produce a biological response that is greater than the biological response to the same amount of the heparin-binding factor in soluble form.
  • the invention provides methods useful in promoting various aspects of angiogenesis.
  • Such method entails, in certain embodiments, a method of enhancing endothelial branching or endothelial tube length or thickness, wherein the method includes administering an effective amount of any of the nanocluster compositions described herein to an endothelial cell or a tissue, wherein the effective amount is sufficient to induce endothelial branching or endothelial tube length or thickness, respectively, at a higher level than in the absence of said composition.
  • the endothelial cell or tissue can be in vitro or present in an organism.
  • the composition is administered to a damaged or diseased site and or at the site of an implant.
  • FIG. 1 VEGF modified particle synthesis.
  • Heparin coated polystyrene particles are fabricated by first oxidizing heparin to generate aldehyde groups. These groups are used to bind the photoactive crosslinker (ABH) and facilitate attachment to the amine- functionalized polystyrene particles. Once the particle is coated with heparin, the bind-and- lock strategy is employed. First VEGF interacts with heparin and forms its specific electrostatic interaction. Then, UV light activates the crosslinker, which non-specifically binds to the closest amine.
  • ABS photoactive crosslinker
  • the heparin-binding domain of VEGF has many available amines on the lysine groups that interact with the sulfate groups on heparin.
  • the heparin polymer chain is modified with the photoactive crosslinker and a dihydrazide through EDC chemistry. The dihydrazide is reacted in a large enough molar ratio to saturate binding and avoid unwanted crosslinking.
  • the modified polymer is purified, it is combined with surfactants into a hexane solution for sonication.
  • radical initiators are added to the solution to generate radical polymerization.
  • the formed nanoparticles are purified in a liquid-liquid extraction process and then bound to VEGF in a similar fashion as the heparin coated polystyrene particles.
  • C High density and low density binding are utilized, where distribution of VEGF in the gel is varied by maintaining constant growth factor
  • the particles are introduced to a fibrin gel and analyzed for induction of branching (arrow heads) and sprouts (arrows) from endothelial cell-coated cytodex beads. Tube length (line) and tube thickness (double-headed arrow) are also quantified. Tube lengths are summed to give total network length.
  • Figure 2 Particle physical characterization.
  • A DLS measurements of heparin-coated polystyrene nanoparticles show functionalization at each step. Each layer increases the diameter, but also the polydispersity, as expected.
  • Figure 3 Particle binding characterization.
  • A Release profiles of heparin coated polystyrene particles in free heparin wash shows stability of VEGF covalent binding to particle.
  • B Generation of high density and low density conditions for both particles with and without covalent binding indicates 1000 VEGF molecules/particle for high density condition, and 200 VEGF molecules/particle for low density condition.
  • FIG. 4 Particle activity characterization.
  • B
  • FIG. 5 VEGFR-2 phosphorylation assay. Binding of VEGF to particles enriches Y1214 signaling for all particles in all binding densities. Activation of Yl 175 is slightly decreased. Each band has the blot intensity background subtracted from the band intensity, and then the intensity of the bands from the phospho- species are divided by the intensities from total VEGFR-2.
  • FIG. 7 Tube formation assay with polystyrene particles.
  • B High density covalent binding leads to a significant increase in branching points for endothelial tubes (Vc high over Vc low (**p ⁇ 0.01), Vc high over soluble (**p ⁇ 0.01), Ve high over soluble (***p ⁇ 0.001)). The low density binding represents a more homogeneous distribution of the growth factor compared to high density binding.
  • C Total network length quantification shows high density VEGF heparin nanoparticles lead to a significant increase in the size of the vessel network over Vs (***p ⁇ 0.001).
  • D D
  • Figure 8 Tube formation assay with heparin nanoparticles.
  • B High density covalent binding leads to a significant increase in branching points for endothelial tubes (hNP high over hNP low (**p ⁇ 0.01), hNP high over soluble with (**p ⁇ 0.01) and without (***p ⁇ 0.001) unloaded hNP). Unloaded heparin nanoparticles refreshed with soluble VEGF showed a similar effect to the low density condition.
  • the low density binding represents a more homogeneous distribution of the growth factor compared to high density binding.
  • C Total network length quantification shows high density covalent VEGF heparin nanoparticles lead to a significant increase in the size of the vessel network (hNP high over Vs (***p ⁇ 0.001), over Vs-hNP (*p ⁇ 0.05); Vs-hNP over Vs (***p ⁇ 0.001)).
  • D Quantification of the number of sprouts emanating from the cytodex beads is not statistically different between conditions.
  • FIG. 9 CAM micrographs of fibrin implant and surrounding tissue show
  • FIG 11 FfUVEC migration induced by VEGF-heparin coated polystyrene particles.
  • Polystyrene particles of 10 ⁇ and 3 ⁇ diameters were coated with heparin and either electrostatically or covalently conjugated to VEGF.
  • Figure 12 VEGF-heparin coated particles internalized by HUVECs.
  • Polystyrene particles of 10 ⁇ diameters were coated with heparin and either
  • VEGF coated 10 ⁇ diameter polystyrene particles lead to specific
  • Figure 13 Particle diameter and polydispersity index (PDI) of heparin nanoparticles synthesized via different chemistries.
  • Michael addition particles were fabricated by combining two separate modified heparin polymers during inverse emulsion.
  • the first heparin polymer was modified with p-azidobenzoyl hydrazide (ABH, optional) and either a combination of adipic acid dihydrazide (ADH) and N-hydroxysuccinimide (NHS)-acrylate or N-(3-aminopropyl) methacrylamide (APMA) through l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) mediated chemistry.
  • ABS p-azidobenzoyl hydrazide
  • ADH adipic acid dihydrazide
  • NHS N-hydroxysuccinimide
  • APMA N-(3-aminopropyl) methacryl
  • the second heparin polymer was oxidized with sodium periodate and then conjugated to cystamine. Upon reduction, the second heparin polymer has sulfhydryl functional groups at the polymer ends. During the inverse emulsion, these sulfhydryl groups bind with the acrylate groups from the first polymer.
  • Acrylate particles were fabricated by polymerizing a modified heparin polymer with a radical chain reaction during inverse emulsion.
  • the heparin polymer was modified with ABH (optional) and a combination of ADH and NHS -acrylate through EDC chemistry.
  • Methacrylate particles were fabricated by polymerizing a modified heparin polymer with a radical chain reaction during inverse emulsion.
  • the heparin polymer was modified with ABH (optional) and APMA through EDC chemistry.
  • FIG. 14 VEGF-hNP dose response curve.
  • VEGF alone and VEGF conjugated to heparin nanoparticles were exposed to HUVECs at varying concentrations of VEGF.
  • VEGF-hNP led to greater cell growth at a concentration of 0.1 ng/ml and greater after 2 days of cell growth.
  • This dose response curve demonstrates the ability of particle conjugation to lower the effective dose of growth factors.
  • Figure 15 Cellular growth curves of HUVECs treated with 0.1 ng/ml and
  • Figure 16 Formation of protease degradable shell around VEGF coated heparin nanoparticle. The formation and degradation of the shell were assayed by amount of VEGF exposed. Beginning with a 1 ng/ml sample, the shell was cast and then degraded. This data demonstrates the proof-of-concept of encapsulated growth factor-heparin nanoparticles. The protease-degradable shell prevents antibodies from binding to VEGF during this ELISA. After the shell is degraded, VEGF is exposed. The mock shell condition denotes presence of shell monomers without radical polymerization initiators. The shell is degraded with treatment by trypsin.
  • the present invention relates to nanoclusters composed of heparin conjugated to a heparin-binding factor, such as a growth factor, or fragment thereof, and the interaction of such with cell surface receptors and/or extracellular matrix proteins.
  • the present invention further relates to reducing the effective dose of heparin-binding factors (e.g., growth factors) by conjugating them to heparin nanoparticles.
  • the invention relates to enhancing the cell signaling activity of heparin-binding factors (e.g., growth factors) by conjugating them to heparin nanoparticles.
  • the invention relates to heparin-binding factor-heparin nanoparticles (e.g., growth factor-heparin nanoparticles) encased in a degradable shell, which are useful in tissue engineering applications.
  • any heparin-binding factor can be conjugated to heparin in the form of a nanocluster.
  • Exemplary nanoclusters include those containing one or more growth factor(s), or fragment(s) thereof and/or one or more ECM proteins or fragments thereof.
  • the nanoclusters are illustrated herein using vascular endothelial growth factor (VEGF) as the heparin-binding factor to form nanoclusters useful in promoting angiogenesis.
  • VEGF vascular endothelial growth factor
  • covalent, high-density clustering of any heparin-binding factor useful in tissue engineering applications provides advantages (e.g., lowering the factor's effective dose and/or increasing the biological response to a given dose of factor) in such applications.
  • advantages e.g., lowering the factor's effective dose and/or increasing the biological response to a given dose of factor
  • aspects of the invention e.g., factor density in nanoclusters, nanocluster size, use of heparin immobilized on a nanoparticle support of a different material or use of nanoparticles formed from heparin, encapsulation of nanoclusters within a shell, use of nanoclusters with soluble factors and/or clustered, non-covalently bound factors, incorporation into a gel, etc.
  • growth factor-heparin nanoclusters are described below with respect to growth factor-heparin nanoclusters, but these aspects also apply equally to nanoclusters containing heparin- binding factors other than growth factors.
  • the present invention provides a composition of nanoclusters, such as, e.g., nanoparticles, that includes heparin in combination with a growth factor that can be used to promote angiogenesis.
  • the composition can be used to promote the formation of a larger and/or more branched network of vessel that, in some embodiments, are thicker, relative to that observed in the absence of the composition.
  • the composition is useful, in certain embodiments, to promote vascularization in ischemic wound healing.
  • a gel such as a fibrin gel, with suspended growth factor, e.g., VEGF-heparin nanoparticles, can be applied to wound site to promote re-vascularization and healing of wound.
  • the invention entails the formation of heparin nanoparticles. On the outside surface of the particle are growth factors covalently bound to the heparin. In particular embodiments, the particles are used to promote tissue
  • the particles can be suspended in a degradable fibrin hydrogel.
  • the infiltrating blood vessels will degrade the hydrogel and interact with the VEGF-heparin nanoparticles.
  • Clustered, covalently bound VEGF promotes blood vessel branching and perfusion of the hydrogel implant.
  • surrounding cells can inhabit the matrix and grow without constraints to oxygen and nutrient delivery. Because the growth factor is covalently bound to the particle, the whole particle can be internalized into the cell.
  • compositions discribed herein include that the
  • the growth factor will be in the correct orientation since heparin is used to bind the growth factor. When binding growth factors non-specifically, the orientation of the molecule is not controlled and leads to loss of activity.
  • the growth factor is presented in a clustered form. Signaling and morphology studies that show that covalent VEGF in clusters promotes endothelial cell migration, which leads to blood vessel branching and capillary formation. When building tissue from a scaffold, perfusion is key to supplying growing cells with oxygen and nutrients. Having only a few large blood vessels is not sufficient. The development of a vascularature is the first step to building tissue within an engineered implant. When the blood vessel infrastructure is complete, other cells can begin to fill in the area and degrade the matrix. Two tissue types that need to be well vascularized are heart tissue and adipose tissue.
  • Clustered covalently bound VEGF worked better at inducing perfused vessels in an in vivo chick embryo model than clustered electrostatic bound VEGF and soluble VEGF.
  • Clustered covalently bound VEGF is most effective at enhancing perfusion in vivo and inducing blood vessel branching in vitro if a high density of VEGF is used.
  • Clustered covalently bound VEGF and clustered electrostatic bound VEGF facilitated particle internalization in to HUVECs to a greater extent than particles without bound VEGF.
  • Heparin nanoparticles fabricated by different chemistries result in particles with similar dimensions and size distribution.
  • VEGF bound to heparin nanoparticles lowers the effective dose of the growth factor and results in greater cell growth rate.
  • VEGF bound to heparin nanoparticles can be encapsulated in a protective polymer shell that can be degraded by treatment with an enzyme.
  • a “nanoparticle” is any particle that has an average characteristic dimension, such as diameter, that is less than 1000 nm.
  • a “nanocluster” of heparin in combination with a growth factor is a cluster of these components that has an average characteristic dimension, such as diameter, that is less than 1000 nm.
  • heparin-binding factor refers to any moiety that has binding affinity for heparin.
  • the term includes, but is not limited to, factors that bind heparin with a Kd of less than 1 ⁇ .
  • Heparin-binding factors encompass any polypeptide, or fragment thereof, that includes a heparin-binding domain.
  • Heparin- binding factors useful in the methods and compositions described herein can have at least one other function, such as, e.g., promoting growth or providing structural support.
  • Exemplary heparin-binding factors include growth factors and extracellular matrix proteins, and fragments thereof.
  • growth factor includes angiogenic growth factors, such as Angiogenin, Angiopoietin-1, Del-1, Fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin, Granulocyte colony-stimulating factor (G-CSF), Hapatocyte growth factor (HGF)/ scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine, Placental growth factor, Platelet-derived endothelial cell growth factor (PD-ECGF), Platelet-derived growth factor-BB (PDGF-BB), Pleiotrophin (PTN), Proliferin, Transforming growth factor- alpha (TGF-alpha), Transforming growth factor-beta (TGF-beta), Tumor necrosis factor- alpha (TNF-alpha), and Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF).
  • angiogenic growth factors such as Angiogenin, Angiopoietin-1, Del-1,
  • angiogenic growth factors include Heparin-binding EGF- like growth factor, Interferon-gamma (IFN-gamma), Platelet factor-4 (PF-4), Macrophage inflammatory protein- 1 (MIP-1), Interferon-g-inducible protein- 10 (IP- 10), and HIV-Tat transactivating factor.
  • growth factor can also include non-angiogenic growth factors such as interleukin-2 (IL-2), nerve growth factor (NGF), bone morphogenic protein (BMP), heat shock protein (HSP), and epidermal growth factor (EGF).
  • IL-2 interleukin-2
  • NGF nerve growth factor
  • BMP bone morphogenic protein
  • HSP heat shock protein
  • EGF epidermal growth factor
  • ECM protein extracellular matrix protein
  • collagen includes collagen, fibronectin, laminin, vitronectin, fibrin, and the like.
  • fragment is used herein with reference to a polypeptide to describe a portion of a larger molecule.
  • a polypeptide fragment can lack an N- terminal portion of the larger molecule, a C-terminal portion, or both.
  • Fragments useful in the methods and compositions described herein typically have binding affinity for heparin, e.g., in the form of a heparin-binding domain.
  • Illustrative fragments typically include another functional domain, such as, e.g., one or more domains that stimulate cell growth (e.g., from a growth factor) or that provide mechanical structure (e.g., from an ECM protein).
  • Useful fragments can include, e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the full-length polypeptide.
  • Useful fragments can also include any percentage of the full-length polypeptide that falls within a range bounded by any of these values (e.g. 45-90%>).
  • peptide is used herein to refer to fragments of polypeptides, as well as short polypeptides (i.e., those that are short, but not necessarily a fragment of a larger polypeptide).
  • support includes: natural polymeric
  • carbohydrates and their synthetically modified, crosslinked, or substituted derivatives such as agar, agarose, cross-linked alginic acid, chitin, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins, and keratins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers, such as vinyl polymers, including
  • porous inorganic materials such
  • compositions Comprising Growth Factor-Heparin Nanoclusters
  • compositions including heparin-binding factor- (e.g., growth factor)-heparin nanoclusters can include nanoclusters that are encapsulated in a biodegradable shell, non- encapsulated nanoclusters, or a combination of both.
  • heparin-binding factor- e.g., growth factor
  • the compositions include a gel, e.g., a hydrogel, including encapsulated and/or non-encapsulated nanoclusters with optional soluble heparin-binding factor(s).
  • the invention provides a composition that includes nanoclusters of heparin and a growth factor.
  • Heparin is one of the most intensively studied glycosaminoglycans (GAGs) as a result of its anticoagulant properties.
  • GAGs glycosaminoglycans
  • Natural heparin is a mixture of linear anionic polysaccharides having 2-O-sulfo- a-L-iduronic acid, 2-deoxy-2- sulfamino-6-O-sulfo-a-D-glucose, ⁇ -D-glucuronic acid, 2-acetamido-2-deoxy- a D-glucose, and a -L-iduronic acid as major saccharide units.
  • LMW low molecular weight
  • heparins any heparin that can be formed into nanoclusters as described below can be used in various embodiments of the invention.
  • the heparin is preferably in a biocompatible form and retains its capacity to bind at least one growth factor.
  • Heparin-binding angiogenic growth factors include, for example, Fibroblast growth factors (FGFs), Hepatocyte growth factor (HGF), Heparin-binding EGF-like growth factor, HIV -Tat transactivating factor, Interferon- gamma (IFN-gamma), Interferon-g-inducible protein- 10 (IP- 10), Interleukin-8 (IL-8), Macrophage inflammatory protein- 1 (MIP-1), Placental growth factor (P1GF), Platelet- derived growth factor (PDGF), Platelet factor-4 (PF-4), Pleiotrophin, Transforming growth factor-beta (TGF-beta), and Vascular endothelial growth factor (VEGF).
  • FGFs Fibroblast growth factors
  • HGF Hepatocyte growth factor
  • HGF Heparin-binding EGF-like growth factor
  • HIV -Tat transactivating factor Interferon- gamma
  • IFN-gamma Interferon-g-inducible protein- 10
  • the growth factor is covalently bound to the nanocluster at an average density of greater than 200 molecules per nanocluster.
  • the average density in greater than 400, 600, 800, or 900 molecules per nanocluster.
  • the average density can have an upper limit of 1800, 1600, 1400, 1200, or 1100 molecules per nanocluster.
  • the average density of the growth factor can fall within any range bounded by any of these values, e.g., 200-1800 molecules per nanocluster, 200-600 molecules per nanocluster, or 1100-1600 molecules per nanocluster.
  • the concentration of the growth factor relative to the heparin can, in various embodiments, be greater than 10, 15, or 20 ⁇ g growth factor per mg heparin.
  • This relative concentration can, variously, be less than 45, 35, or 30 ⁇ g growth factor per mg heparin. Accordingly, the concentration of the growth factor relative to the heparin can fall within any range bounded by any of these values, e.g., 10-45 ⁇ g growth factor per mg heparin, 10-20 ⁇ g growth factor per mg heparin, or 30-35 ⁇ g growth factor per mg heparin.
  • Nanoclusters can be produced in any desired size, which can vary, depending on the specific application.
  • a nanocluster of heparin in combination with a growth factor has an average characteristic dimension, such as diameter, that is less than 1000 nm.
  • the average characteristic dimension can be less than 500, 300, 250, 220, 200, 180, 160, 140, 120, 100, 80, or 60 nm.
  • the average characteristic dimension can be more than 10, 20, 30, 40, or 50 nm, for example, in the range of approximately 50 nm to 70 nm. Sizes falling within any range bounded by any of these values, e.g., 50-1000 nm, 10-220 nm, 50-200 nm, or 200-500 nm, are also contemplated.
  • the nanoclusters comprise nanoparticles including heparin and a growth factor.
  • the growth factor can be covalently bound to the nanocluster via the heparin molecules.
  • the nanoparticles can include heparin immobilized on a support (e.g., a bead) made of a different material.
  • a support e.g., a bead
  • Suitable support materials for most tissue engineering applications are generally biocompatible and preferably biodegradable. Examples of suitable biocompatible and biodegradable supports include: natural polymeric carbohydrates and their
  • synthetically modified, crosslinked, or substituted derivatives such as agar, agarose, cross- linked alginic acid, chitin, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross- linked or modified gelatins, and keratins; vinyl polymers such as poly(ethylene glycol)- acrylate/methacrylate, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes; and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a pre-existing natural polymer.
  • biocompatible and biodegradable polymers are available for use in therapeutic applications; examples include: polycaprolactione, polyglycolide, polylactide, poly(lactic-co-glycolic acid) (PLGA), and poly-3-hydroxybutyrate. Methods for making nanoparticles in from such materials are well-known.
  • Methods for immobilizing heparin on a support will vary depending upon the nature of the support.
  • the heparin can present during nanoparticle formation so that it becomes incorporated into the nanoparticle, as long as sufficient heparin is displayed on the surface of the resulting nanoparticle to permit growth factor binding at the desired density.
  • heparin can be added to the support after nanoparticle formation.
  • embodiments in which the heparin is immobilized by forces stronger than simple electrostatic binding, e.g., covalent binding are preferred. Because heparin immobilized on surfaces is known to improve blood compatibility and biocompatibility, there is a wealth of experience with various chemistries for immobilizing heparin on various surfaces. See Murugesan et al. (2008) Immobilization of Heparin: Approaches and Applications, Current Topics in Medicinal Chemistry 8:80-100 for a review of commonly used methods.
  • Example 1 described an illustrative embodiment in which heparin is conjugated to amine functionalized polystyrene particles.
  • nanoparticles are formed from heparin. Any method that produces appropriately sized heparin nanoparticles that can bind growth factor can be employed.
  • heparin is first oxidized with sodium periodate and functionalized with a hydrazide photoactive crosslinker, such as p-azidobenzoyl hydrazide, to form heparin- ABH. This molecule should be kept in the dark for later use of the photoactive crosslinker.
  • the heparin- ABH is reacted with a bi-functional crosslinker, such as cystamine. At this stage, before undergoing reduction, the cystamine has an amine on each end and a disulfide in the middle.
  • heparin undergoes reduction to reduce the aldehyde-amine bond to a more stable bond and to expose the sulfer groups of the cystamine.
  • the heparin- ABH-cystamine is then reacted with an acid labile polymer, such as diacrylate, in a reverse phase emulsion process.
  • an acid labile polymer such as diacrylate
  • the sulfhydryl groups and acrylate groups undergo Micheal addition to form a bond, and the molecules are forced into nanoparticles because of the reverse phase emulsion.
  • the size of the particles can be controlled during the emulsion process.
  • the reaction mixture is centrifuged or dialyzed, and the result is heparin nanoparticles functionalized with a photoactive crosslinker. See Example 1 and 3.
  • Nanoclusters are formed by binding growth factor to the clustered heparin, e.g., nanoparticles bearing or formed from heparin. Any method that produces nanoclusters with bioactive growth factor can be employed.
  • a heparin- binding growth factor is incubated with the heparin nanoparticles in the dark at 4°C overnight. This incubation allows the growth factor to orient itself with respect to the heparin binding domain. Then, the particles are exposed to UV light at 365 nm wavelength for 10 minutes while on ice. This covalently binds the correctly oriented growth factors to the heparin nanoparticles.
  • the ABH undergoes ring expansion and binds to a nearby amine, which are readily available on the lysines of the heparin binding domain. See Example 1.
  • compositions including growth factor-heparin nanoclusters can have some or all of the nanoclusters encapsulated in a biodegradable shell.
  • a composition containing non-encapsulated and encapsulated nanoclusters can provides multi-phasic growth factor activity, with the non-encapsulated nanocluster providing immediate activity and the encapsulated nanoclusters providing activity after degradation of the shell by proteases.
  • tissue engineering applications for example, such a composition can take the form of a biocompatible, biodegradable hydrogel including the non-encapsulated and encapsulated nanoclusters.
  • hydrogel Application or implantation of the hydrogel to, or into, as subject leads to infiltration of the hydrogel by vessels from the host vasculature.
  • Degradation of the hydrogel exposes the infiltrating vessels to non- encapsulated VEGF and subsequent degradation of the encapsulated nanoclusters exposes the developing vessels to additional VEGF. See Example 5.
  • the biodegradable shell is one that is susceptible to degradation by a protease, e.g, a protease that is typically found in the environment in which the encapsulated nanoclusters will be used.
  • a protease e.g, a protease that is typically found in the environment in which the encapsulated nanoclusters will be used.
  • the protease can be a matrix metalloproteinase.
  • Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that typically degrade extracellular matrix. A large number of these enzymes have been identified and classified based on substrate specificity (e.g., collagenases, gelatinases, stromelysins).
  • the biodegradable shell includes one or more of poly(ethylene glycol), carbohydrate, peptide polyacrylamide, methacrylamide, or a combination thereof. See Example 5.
  • the shell is susceptible to degradation by
  • MMP-2 For example, nanoclusters prepared as described above can be coated with a charged monomer, 2-aminoethyl methacrylate.
  • a MMP-2 sensitive peptide diacrylate can be incubated with the particles, along with acrylamide, ammomium persulate and ⁇ , ⁇ , ⁇ ', ⁇ '-tetramethylethylenediamine in 10 mM pH 8.5 sodium bicarbonate buffer. These last two components are initiators of a polymerizing reaction that results in a protective shell around the nanocluster.
  • the MMP-2 sensitive peptide allows the shell to be digested by MMP-2 and expose the growth factor surface. See Example 5.
  • compositions can include encapsulated and/or non-encapsulated nanoclusters together with one or more soluble growth factor(s).
  • the soluble growth factor(s) can be the same as, or different from, growth factor(s) present in the nanoclusters.
  • the implant includes a soluble angiogenic growth factor, such as VEGF, which will initially attract host blood vessels to the implant.
  • the implant can optionally include heparin nanoparticles with non-covalently bound growth factor, which can be the same as, or different from, the covalently bound growth factor (e.g., VEGF), to provide an additional source of growth factor activity.
  • the implant will also contain nanoclusters that include a covalently bound angiogenic growth factor to promote endothelial branching and/or tube length and/or thickness.
  • a covalently bound angiogenic growth factor to promote endothelial branching and/or tube length and/or thickness.
  • the blood vessels Upon interaction with the clustered, covalently bound VEGF, the blood vessels will branch into capillaries and perfuse the implant matrix. If present, nanoclusters protected by shells will be degraded by advancing endothelial cells secreting MMP-2, and the thus revealed growth factor (e.g., VEGF) will act downstream kinetically. See Example 1.
  • the nanocluster-containing composition is a gel.
  • the gel is biocompatible and, in particular embodiments,
  • Biodegradable Biocompatible and biodegradable hydrogels, for example, find particular application in tissue engineering, where the hydrogel forms a matrix with properties sufficiently similar to extracellular matrix to permit cell and vessel migration into the matrix.
  • Hyaluronic acid, poly(ethylene glycol), and fibrin form suitable hydrogels.
  • Hyaluronic acid-based hydrogels can be formed from hyaluronic acid engineered, e.g., with sulfhydryl groups undergoing Michael addition with MMP-sensitive peptide diacrylates in a manner analagous to that described above. See also Example 1, describing the formation of fibrin gels.
  • Nanoclusters can be suspended in hydrogel and/or attached to the hydrogel matrix backbone. A variety of chemistries can be used to attach the nanoclusters to the matrix backbone including enzymatic reactions such as factor XHIa, carbodiimide chemistry, Michael addition chemistry, and radical initiated reactions. See Example 1.
  • the nanocluster-containing gel can also include:
  • the soluble growth factor(s) can be the same as, or different from, growth factor(s) present in the nanoclusters, as can the clustered, non- covalently bound growth factor(s).
  • the nanocluster-containing gel serves as the implant, which can be injected, or otherwise implanted into the body.
  • the effectiveness of heparin-binding factors can be enhanced by binding such factors with heparin nanoparticles to form nanoclusters.
  • the effective dose of the factor can thus be reduced and/or the biological response to a given dose of factor can be enhanced.
  • the enhanced biological response can, in some embodiments, be used to achieve effects (e.g., therapeutic benefits) that could not be achieved with soluble (non-clustered) factors alone.
  • the angiogenic compositions described herein are useful, for example, for enhancing endothelial branching in angiogenesis, as well as for enhancing endothelial tube length and thickness.
  • Nanoclusters and/or nanocluster compositions may administered in vitro to endothelial cells or a tissue containing them or may act on endothelial cells or tissues that contain them in an organism.
  • the composition is administered to a damaged or diseased site.
  • the site may be the site of an implant, and the compositions described herein may make up, or be part of, the implant.
  • the compositions described herein are also useful to a wide range of tissue engineering applications for regenerative medicine. This may include, but not limited to, bone regeneration, nerve regeneration, heart regeneration, stem cell differentiation, skin renewal, and cosmetic purposes.
  • VEGF Vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • fibrin hydrogels which contained HUVECs bound to cytodex beads.
  • Fibroblast cells are plated on top of the fibrin gel to further mimic a physiologic environment.
  • CAM assay to determine the role of VEGF presentation on angiogenesis in vivo.
  • VEGF bound in high density and low density to study differences between growth factor presentation in heterogeneous nanodomains and homogenous distribution.
  • VEGF covalently bound to nanoparticles at high density led to an increase in HUVEC tube branching, thickness, and total vessel network length compared to soluble VEGF. While VEGF bound
  • Tissue regeneration involves the growth of specific tissue types for replacement of damaged tissue that the body is incapable of regenerating[l].
  • a vascular supply is typically required[2].
  • Infiltration of blood vessels into the implant is not enough to guarantee adequate blood supply, nutrient delivery, and waste removal for the cells inhabiting the implant.
  • perfusion of the implant by branched capillaries is needed to provide a feasible infrastructure upon which the new tissue can mature [3].
  • Research over the past two decades has led to the development of biomaterials that support vascular formation within a tissue implant.
  • Encapsulation of growth factors that rely on non-specific release and diffusion to the target receptors is one method of supplementing a biomaterial scaffold with cell instructive molecules[4-6].
  • a more sophisticated method involves covalent incorporation of growth factors with genetically engineered domains that allow release upon secretion of proteases by migrating cells participating in natural wound healing— a method termed cell demanded release[7, 8].
  • Electrostatic binding of VEGF to synthetic and natural polymers including PLGA and heparin can extend the release kinetics of the growth factor[9-12]. Instead of distributing these VEGF -binding polymers homogeneously throughout the matrix, VEGF can be sequestered to particles composed of these polymers leading to heterogeneity within the matrix[13].
  • VEGF has also been covalently bound to the polymer backbone of a biomaterial without an engineered release mechanism.
  • a natural release mechanism is found in VEGF- 165 between the receptor binding domain and the
  • extracellular matrix binding domain [14].
  • a ten amino acid sequence located in this region can be cleaved by specific matrix metalloproteinases secreted into the environment by infiltrating endothelial cells[15]. Binding VEGF in this fashion has led to formation of branched, stable vessel structures capable of perfusion[16, 17].
  • VEGF is bound in low density and high density forms, where the low density form has less VEGF molecules bound per particle.
  • the low density form represents a more homogenous distribution of the growth factor in the gel.
  • the particles were characterized for binding, release kinetics, and activity, both on a cellular level and a molecular level.
  • the particles were embedded into a fibrin gel and combined with HUVECs in a tube formation assay [19] to study the effect of VEGF presentation on tube branching.
  • the particle-fibrin gels are introduced to the CAM of a chicken embryo and assayed for angiogenic potential.
  • heparin nanoparticles composed of a modified heparin polymer are bound to VEGF in the same approach and analyzed concurrently.
  • the particles offer an alternative approach to the polystyrene particles for use in future investigations into in vivo applications.
  • VEGF Vascular Endothelial Growth Factor
  • VEC LLC
  • Polystyrene particles were purchased from Spherotech (Lake Forest, IL). Fibrinogen was purchased from Enzyme Research Laboratories (South Bend, IN). Cytodex beads were purchased from Sigma- Aldrich (St. Louis, MO). Fertilized eggs were purchased from Kendor farms (Lake Balboa, CA). All other reagents and products were purchased from Fisher Scientific unless noted otherwise.
  • HUVECs were cultured in EGM-2 complete medium (Lonza, Walkersville,
  • the HUVECs were first obtained and cultured to passage 2. Tube formation experiments were conducted while the cells were at passage 2. In order to provide enough cells for all of the other experiments, the cells were expanded and frozen at passage 7. For each experiment, the cells were thawed and grown for 2 days in a T75 flask (Corning, Corning, NY), before being plated onto a 6 well dish. Fibroblast cells were a kind gift from Dr. Arispe, and these cells were cultured in EGM-2 complete medium.
  • Heparin was oxidized by dissolving 62.5 mg/ml heparin in 200 mM sodium periodate in 100 mM sodium acetate pH 4 for 30-60 minutes. The reaction was quenched with addition of glycerol, then diluted to 3 mg/ml heparin and adjusted to pH 7 with PBS. Heparin became photoactive by addition of azido-benzyl hydrazide (ABH, Pierce,
  • VEGF vascular endothelial growth factor
  • the resultant nanoparticles were purified via liquid-liquid extraction in hexane. In the final stage of the extraction process, bubbling nitrogen gas into the nanoparticle solution evaporated off excess hexane. The particles were then dialyzed in 100 kD MWCO dialysis units for several days and stored until use. The amount of heparin in the solution was determined by lyophilizing a small aliquot of the solution. Similar to the heparin-coated polystyrene particles, VEGF was incubated at 4°C at 100 ⁇ g/ml, followed by 365 nm wavelength UV light activation for 10 minutes to lock VEGF covalently to the surface. Excess VEGF was removed by dialysis in 100 kD MWCO dialysis units.
  • nanoparticles were collected and quantified for amount
  • TMB substrate Cell Signaling, Boston, MA
  • HUVECs were grown to confluency in a 6 well plate and then scratched with a pipet tip to create a wound. Phase micrographs captured the initial size of the wound created by the pipet tip for each condition. Soluble VEGF or VEGF nanoparticles at 2 ng/ml were added to the wells. After 18 hours, phase micrographs were taken again. ImageJ software was used to quantify the percent wound closure for each condition. Photographs were acquired using a Zeiss Observer microscope.
  • HUVECs were grown to confluency in a 6 well plate, and then serum starved for 6 hours. Prior to growth factor treatment, the cells were treated with 0.1 mM sodium vanadate for 5 minutes. The cells were then treated with 2 ng/ml of either soluble or bound VEGF at 37°C for 5 minutes. The cells were rinsed twice with ice cold PBS supplemented with 0.2 mM sodium vanadate.
  • lysis buffer 1% Non-idet, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 2.1 mM sodium orthovanadate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 ⁇ g/ml of aprotinin
  • Insoluble cell material was removed by centrifugation at 4°C for 10 min at 14,000 rpm (Beckman Coulter Microcentrifuge 22R).
  • Equal amounts of cell lysate were diluted in 5X loading buffer (1 M Tris-HCL, pH 6.8, 20% SDS, 50% glycerol) supplemented with 5% (v/v) ⁇ -mercaptoethanol, boiled for 10 min at 70°C, separated by SDS-PAGE (8% resolving, 2 h at 130 V), and transferred to nitrocellulose membranes (2 h at 400 mA).
  • the membranes were incubated in blocking buffer (5% milk in 0.1% Tween-20 in TBS) for 1 h at room temperature before overnight incubation with primary antibodies.
  • Phosphorylated proteins were detected by immunoblotting using anti- phosphotyrosine antibodies (p VEGFR-2/ 1175 Cell Signaling, pVEGFR-2/1214 Invitrogen, in blocking buffer) followed by secondary antibodies coupled with horseradish peroxidase (200 ng/ml, Invitrogen, 1 h at room temperature) and visualized by chemifluorescence (ECL detection reagents, GE Healthcare) using a Typhoon scanner (GE, Amersham Biosciences). Protein-loading control was assessed by Western blot using anti- VEGFR-2 (Cell Signaling Technology). Typhoon images were analyzed and normalized with ImageJ software.
  • HUVECs were grown in a T25 flask until confluency. Meanwhile, cytodex beads were autoclaved and then coated with fibronectin (Millipore, Temecula, CA) in an incubation solution of 10 ⁇ g/ml at 37°C for 2 hours. The cells were trypsinized and combined with the cytodex beads at a ratio of 1 million cells per 1200 beads for 4 hours at 37°C with occasional agitation. The HUVEC-coated cytodex beads were cultured overnight in a T25 flask, and then combined in the pre-gel solution at a concentration of 500 beads/ml.
  • fibronectin Millipore, Temecula, CA
  • Fibrinogen was diluted from its stock to 2 mg/ml and supplemented with aprotinin.
  • VEGF nanoparticles at 200 ng/ml were combined with the fibrinogen and cytodex bead/HUVEC solution.
  • Fibrin gel formation was initiated by adding 1.25 U/ml of thrombin in a 10% v/v ratio. The gels were allowed to stand for 5 minutes at room temperature, and then incubated at 37°C for 15 minutes. Meanwhile, fibroblast cells were trypsinized and plated at 40,000 cells/condition. The cells were cultured in VEGF withdrawn EGM-2 media for 9 days. The soluble VEGF condition was refreshed every other day with new soluble VEGF (200 ng/ml).
  • Phase micrographs captured the tube formation, and quantification was completed in ImageJ. For each condition, 10 beads were analyzed. Branching points were considered where two tubes grew out of a single tube. Sprouts were measured as tubes originating from the cytodex bead. Total network length was calculated by measuring the distance from the bead to the end of the sprout, and summing for all the sprouts on the bead. Thickness was measured across the vessel away from its base (interface with the bead).
  • Fertilized eggs were purchased from Kendor farms (Lake Balboa, CA) and stored in a humidified chamber for 2 days at 38°C. Air was released from the egg to flatten the embryo by inserting the tip of a 32 1 ⁇ 2 gauge needle into the broad side of the egg. After 2 additional hours of storage in the chamber, the eggs were opened and the embryo was transferred into a petri dish. The embryos continued to grow in the petri dishes within a humidified incubator at 38°C for 6 days. Fibrin gels were prepared as previously mentioned, with the exception of cytodex bead incorporation, and a VEGF dosage increase to 2 ⁇ g/ml.
  • the fibrin gels were grafted onto regions of the CAM located at a distance from the embryo and major vessels. After 2 days of incubation in the incubator, the vessels were perfused with FITC-dextran and allowed to circulate for 5 minutes. The gels and CAM surrounding the gels were removed and fixed in 4% paraformaldehyde. The embryos were then sacrificed. Micrographs were captured on a small Zeiss microscope. The fluorescent images were captured on a Zeis Observer using the 488 nm filter. The fibrin gel and the area surrounding the fibrin gel were photographed in order to determine the presence of vessels within the implant, and the morphology of vessels surrounding the implant.
  • Nanoparticles composed of only heparin were also developed using an inverse emulsion process. Heparin was modified via EDC/NHS chemistry to introduce photoreactive and acrylate functionalities. First, ABH was reacted in excess of carboxylic acid groups to ensure remaining binding sites for the next conjugation step. Then, adipic dihydrazide (ADH) was incubated with heparin in large molar excess to ensure efficient conjugation to the remaining carboxylic acid groups and reduce the occurrence of unintended crosslinking. Next, N-acryloxysuccinimide (NHS-acrylate) was conjugated to the hydrazide groups.
  • ADH adipic dihydrazide
  • NHS-acrylate N-acryloxysuccinimide
  • the polymer solution was then added to a ten- fold volume of hexane with surfactants Tween-80 and Span-80, and sonicated with addition of radical initiators N,N,N',N'-tetramethyl-ethane-l,2-diamine (TEMED) and ammonium persulfate (APS).
  • TEMED radical initiators N,N,N',N'-tetramethyl-ethane-l,2-diamine
  • APS ammonium persulfate
  • the heparin nanoparticles formed from this radical polymerization in the template of the nanoemulsion generated during the sonication treatment. Similar to the polystyrene particles, the heparin nanoparticles were incubated with VEGF, and then exposed to UV light to induce covalent attachment of the growth factor to the nanoparticle ( Figure 1).
  • Dynamic light scattering (DLS) measurements confirmed modification of the polystyrene particles, with significant increases in diameter from heparin coating and VEGF loading ( Figure 2A). As expected, with the addition of each layer, the PDI increased from below 0.1 to 0.2. For the heparin nanoparticles, DLS measurements indicated that the Z- average size was 58.36 ⁇ 0.42 nm, with a PDI of 0.46 ( Figure 2B).
  • VEGF was loaded onto the heparin-coated polystyrene particles and then monitored over several days to characterize the release kinetics of the growth factor from the nanoparticles.
  • heparin-coated polystyrene without the photoactive crosslinker, and polystyrene particles without heparin were included in the analysis. Release of covalently bound VEGF was significantly less than that released from heparin-coated particles without the photoactive crosslinker and un-coated polystyrene particles (Figure 3 A). Direct binding of VEGF to the heparin-coated polystyrene particles was investigated next for high binding and low binding densities.
  • VEGF molecules per particle After converting the ELISA readings to molecules of VEGF, and calculating the number of particles, the data was presented as VEGF molecules per particle. For both the covalent and electrostatic binding conditions, the high binding density was 1000 VEGF molecules per particle and the low binding density was 200 VEGF molecules per particle ( Figure 3B). For later experiments, these solutions were normalized to equivalent VEGF concentrations.
  • VEGF content With each successive wash, the amount of VEGF decreased until remaining steady (Figure 3C).
  • the heparin nanoparticles were analyzed for VEGF content, and the amount was normalized to mg of heparin to show high binding and low binding densities.
  • the high binding heparin nanoparticle had 23 ⁇ g VEGF/mg heparin while the low binding heparin nanoparticle had 10 ⁇ g VEGF/mg heparin ( Figure 3C).
  • the VEGF nanoparticle solutions were equalized relative to VEGF concentration before use in the subsequent experiments.
  • VEGF modified nanoparticles induce wound closure and VEGFR-2
  • VEGFR-2 phosphorylation, and cdc42 activation were studied.
  • a scratch was introduced to a confluent monolayer of human umbilical vein endothelial cells (HUVECs) and VEGF coated nanoparticles were incubated with the cells at a VEGF concentration of 2 ng/ml for all conditions.
  • the closure of the wound was monitored over 18 hours and the percent wound closure was quantified (Figure 4A).
  • VEGF covalently bound to both polystyrene and heparin particles led to wound closure rates comparable to the soluble VEGF control ( Figure 4B).
  • VEGF modified nanoparticles were able to phosphorylate VEGFR-2 at two different tyrosine residues, Yl 175 and Y1214 (Figure 5).
  • the intensity of receptor phosphorylation was different between Yl 175 and Y1214 for cells exposed to soluble or VEGF coated particles, with Yl 175 being more phosphorylated relative to Y1214 when cells were exposed to soluble VEGF and Y1214 being more phosphorylated relative to Yl 175 when cells were exposed to VEGF coated nanoparticles.
  • No differences were observed between low and high density VEGF nanoparticles.
  • a sprouting bead assay was used in which cytodex beads are coated with endothelial cells and the beads are placed inside a fibrin hydrogel scaffold, while fibroblast cells are seeded on top of the hydrogel ( Figure 7-8A)[19, 20].
  • cytodex beads coated with endothelial cells and the beads are placed inside a fibrin hydrogel scaffold, while fibroblast cells are seeded on top of the hydrogel ( Figure 7-8A)[19, 20].
  • fibrin hydrogel formation either 200 ng/ml VEGF modified nanoparticles, unmodified nanoparticles, or 200 ng/ml soluble VEGF were encapsulated within the hydrogel.
  • the HUVEC tubes from the cytodex beads were then quantified for branching points, sprouts, thickness, and total vessel network length (Figure ID, Figure 7-8).
  • PS-Vei 0W there was a statistically significant difference for PS-Vc (p ⁇ 0.001) but not for PS-Ve (p > 0.05), further indicating that the presentation of VEGF affected branching. No difference was observed between PS- Vc hlgh and PS-Ve hlgh (p > 0.05).
  • the total network length was quantified by measuring the length of individual sprouts and then summing for each bead (Figure 7C).
  • the total network length for PS-Vchigh and PS-Vehigh was statistically higher than for that observed for Vs (p ⁇ 0.001).
  • lowering the amount of VEGF displayed per particle, PS-Vci 0W and PS- Vei ow resulted in no significant increase in network length over Vs for PS-Vci 0W (p > 0.05) and a statistically significant increase in network length for PS-Vei 0W (p ⁇ 0.001), indicating that the presentation of the VEGF inside the fibrin hydrogel modulated the extent of the vascular network formed.
  • VEGF vascular endothelial growth factor
  • VEGF modified heparin nanoparticles were synthesized and similar analysis as those done with PS nanoparticles were performed ( Figure 8).
  • the number of branching points for hNP i gh was statistically significantly higher from the number of branching points observed in Vs (p ⁇ 0.001) and the number of branching points for hNPi 0W was not statistically significant from Vs (p > 0.05, Figure 8B).
  • Preloading of hNP with VEGF did result in a significant difference between hNP h i gh (p ⁇ 0.05) and Vs-hNP, but not hNPi 0W and Vs-hNP.
  • the presence of hNP alone with Vs led to a significant increase in vessel network formation for Vs-hNP v. Vs (p ⁇ 0.001).
  • hNP did not change the number of sprouts to a significant extent over Vs (Figure 8D).
  • One difference between VEGF modified PS nanoparticles and hNP was that tube thickness for hNP h i gh and Vs-hNP was significantly higher compared to Vs ( Figure 8E). No other differences were observed between the conditions.
  • Covalently bound VEGF induces blood vessel infiltration into fibrin gel
  • Fibrin gels with VEGF nanoparticles were placed on the chorioallantoic membrane (CAM) of embryonic chicken. After two days, the implants were removed and analyzed for induction of angiogenesis (Figure 9). The presence of VEGF had a noticeable increase in blood vessel density surrounding the implant. VEGF release from the implants was noted by the appearance of radial blood vessels originating from the fibrin gel protruding outward.
  • CAM chorioallantoic membrane
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • fibrin hydrogels which contained HUVECs bound to cytodex beads.
  • Fibroblast cells are plated on top of the fibrin gel to further mimic a physiologic environment[20].
  • VEGF bound electrostatically or covalently and in high density and low density formats to study differences between growth factor presentation in heterogeneous nanodomains and homogenous distribution.
  • VEGF modified nanoparticles were synthesized using either a polystyrene core (PS) or a heparin core.
  • PS polystyrene core
  • heparin core a polystyrene core
  • the surface of the nanoparticles contained amines, which were used to immobilize heparin.
  • heparin modified with a photoreactive group or unmodified heparin As we have previously described[18].
  • heparin nanoparticles were first generated using an inverse emulsion of water in hexane[22]. The heparin was modified with the same photoreactive group used above to covalently bind VEGF to the surface of the nanoparticle.
  • the first variable, matrix affinity, was modulated by inclusion of a photoactive crosslinker during nanoparticle synthesis.
  • the release kinetics show that when VEGF is covalently bound to the nanoparticle, less release over time is observed, indicating that the covalent bond reduces the release of VEGF from the nanoparticle ( Figure 3).
  • the second variable, distribution was controlled by incubating VEGF with different amounts of particles. The result is particles with different binding densities of VEGF. In order to normalize the amount of VEGF supplied to the cells during the analysis, more particles with low VEGF binding density are required. This leads to a situation where the growth factor is more homogeneously distributed throughout the gel relative to the high binding condition.
  • the control for both matrix affinity and distribution is soluble VEGF. It is provided freely diffusible during hydrogel formation and in the media changes.
  • nanoparticles or soluble VEGF were compared.
  • the migration rates are comparable to the soluble treatment ( Figure 4), indicating that the VEGF remains active throughout the process of particle modification.
  • VEGFR-2 phosphorylation at two sites, Yl 175 and Y1214 was investigated to ensure that the immobilized VEGF could phosphorylate its receptor.
  • Covalently bound VEGF to nanoparticles appears to enhance activation of Y1214 compared to the soluble condition ( Figure 5).
  • the same is observed for VEGF bound to bulk matrices where bound VEGF results in enhanced Y1214 phosphorylation over that observed with Vs [23](Anderson et al, submitted).
  • the particles appear to be slightly less effective in activation of Yl 175.
  • Phosphorylation at Yl 175 results in the activation of the AKT pathway and cellular proliferation[24-26], while
  • phosphorylation at Y1214 results in the activation of the p38 pathway and cellular migration[27, 28].
  • phosphorylation at Y1214 leads to the activation of cdc42 [27], which is involved in branching.
  • a GLISA assay was performed. Heparin nanoparticles modified with VEGF at high density were the most effective in activating cdc42 ( Figure 6). As mentioned, this GTPase is directly involved with processes at the cell membrane that lead to filopodia formation[29], which eventually result in branching orchestrated by the leading tip cell[30- 32].
  • Vs- hNP had significantly more branching than Vs (p ⁇ 0.05), indicating that the presence of hNP alone with Vs was able to affect change in branching behavior.
  • pre-loading of VEGF onto hNP in high density significantly increased branching over Vs-hNP (p ⁇ 0.01).
  • Vs-hNP also led to a larger vessel network with thicker vessels, but still was not as efficient at inducing more network size as pre-loaded hNP in high density.
  • extracellular matrix are not homogeneously dispersed, but rather clustered into these reservoirs. While the high density binding of VEGF does not lead to a larger network compared to low density binding (except for PS-Vc), it does induce more branching (except PS-Ve). Branching of endothelial tubes is important in creating a capillary network capable of perfusion in the new tissue implant[3].
  • Covalently binding VEGF does not allow VEGF to easily release and diffuse out of the gel. Electrostatic binding may not be adequate in the complex environment to retain VEGF and facilitate infiltration of the vessels into the matrix ( Figure 10). Previous work in our own laboratory has found that when VEGF DNA is delivered to cells, the VEGF that is produced leaves the gel and results in the radial vessel morphology observed in the soluble condition[36]. Production of VEGF in the gel does not guarantee it is retained within the gel. Covalently bound VEGF can release via action by MMP's[15], but the advantage of covalent binding can be seen in the CAM assay where blood vessel infiltration is observed.
  • Electrostatically binding VEGF is secure in a non-competitive environment, but in a complex solution other molecules with affinity for heparin can displace the bound VEGF, releasing it[ 18].
  • the high density and low density binding conditions did not show much difference at the molecular level, but did show observable differences in the tube formation experiment and the CAM assay. Further, between the high density cases, matrix affinity does not lead to significant changes in the tube branching.
  • the choice to include the covalent bond in the heparin nanoparticles is made based on the evidence generated here that the covalent bond does not hinder VEGF activity or downstream activation, and it has a stabilizing effect, particularly in a biomaterial setting.
  • Heparin coated polystyrene and heparin derived nanoparticles were covalently conjugated to VEGF through a bind-and-lock approach at different binding densities.
  • the particles were characterized for amount VEGF bound and activity, both at a cellular and molecular level.
  • Increasingly clustered growth factor nanodomains resulted in more endothelial tube branch points than more homogeneously distributed bound growth factor and soluble VEGF.
  • the high density VEGF nanodomains also led to a larger total network length over VEGF presented in the freely diffusible form.
  • the clustered, covalently bound VEGF nanoparticles successfully guided host vasculature into the implant, leading to a perfused capillary network within the fibrin gel.
  • Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor. Biomacromolecules 2007 May;8(5): 1607-1614.
  • VEGF -A Processing of VEGF -A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J Cell Biol 2005 May 23, 2005;169(4):681-691.
  • VEGFR2 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of
  • VE-PTP vascular endothelial-protein tyrosine phosphatase
  • VEGF-heparin coated particles a 10 ⁇ particle with a polystyrene core was used as a proxy to visualize internalization into human umbilical vein endothelial cells (HUVECs).
  • a wound was created with a pipet tip on a confluent monolayer of HUVECs in a 6-well dish. The particles were administered for 18 hours to track the wound closure. The migration of the HUVECs into the void created by the pipet scratch indicated VEGF activity while bound to the polystyrene particles. See Figure 11.
  • HUVECs were exposed to the functionalized particles for 20 min and 1 hour to analyze the ability of HUVECs to internalize the particles.
  • the cell monolayer was washed with an acid solution to remove cell surface bound particles.
  • the number of particles internalized per unit area was quantified by counting.
  • Figure 12 shows that VEGF-heparin coated 10 ⁇ diameter polystyrene particles are internalized by HUVECs after one hour of exposure.
  • Heparin was oxidized by dissolving 62.5 mg/ml heparin in 200 mM sodium periodate in 100 mM sodium acetate pH 4 for 30-60 minutes. The reaction was quenched with addition of glycerol, then diluted to 3 mg/ml heparin and adjusted to pH 7 with PBS. Heparin became photoactive by addition of azido-benzyl hydrazide (ABH, Pierce,
  • N is the number of particles
  • W is the weight of the polymer (g)
  • P is the density of the polymer (g/cm 3 )
  • D is the diameter ( ⁇ ).
  • HUVECs were grown to confluency in a 6 well plate and then scratched with a pipet tip to create a wound. Phase micrographs captured the initial size of the wound created by the pipet tip for each condition. Soluble VEGF or VEGF conjugated to heparin coated polystyrene particles (either covalently or electrostatically) at 2 ng/ml were added to the wells. After 18 hours, phase micrographs were taken again. ImageJ software was used to quantify the percent wound closure for each condition. Photographs were acquired using a Zeiss Observer microscope.
  • Confluent cell monolayers were exposed to 10 and 3 ⁇ polystyrene particles coated with heparin and VEGF (covalent and electrostatic) for 20 minutes and 1 hour. After the treatment, the cells were washed with an acid solution to disrupt the binding between the cell surface and the coated particles. This was done to differentiate between particles that were internalized and particles that were adhered to the cell surface.
  • heparin and VEGF covalent and electrostatic
  • Micrographs were acquired by the Zeiss Observer microscope and the particles internalized were counted using ImageJ.
  • EGFR epidermal growth factor receptor
  • MRBLs Modified epidermal growth factor receptor-bearing liposomes
  • Heparin nanoparticles can be synthesized via radical polymerization during inverse emulsion from reaction of both acrylate functional groups and methacrylate functional groups. hNP can also be synthesized via Michael addition from reaction of acrylate groups and sulfhydryl groups. These different chemistries result in nanoparticles of similar size and polydispersity (Figure 13).
  • the powder was dissolved in sodium acetate, pH 4, at 100 mg/ml and combined with Tween-80 and Span-80 (8% HLB).
  • the solution was placed in a ten- fold volume of hexane and combined with oxidized heparin modified with reduced cystamine.
  • the amount of sulfhydryls was quantified by Ellman's reagent following manufacturer's protocol (Pierce, Rockford, IL). After sonication, the resultant nanoparticles were purified via liquid-liquid extraction in hexane. In the final stage of the extraction process, bubbling nitrogen gas into the nanoparticle solution evaporated off excess hexane.
  • VEGF was incubated at 4°C at 100 ⁇ g/ml, followed by 365 nm wavelength UV light activation for 10 minutes to lock VEGF covalently to the surface. Excess VEGF was removed by dialysis in 100 kD MWCO dialysis units.
  • the resultant nanoparticles were purified via liquid-liquid extraction in hexane. In the final stage of the extraction process, bubbling nitrogen gas into the nanoparticle solution evaporated off excess hexane. The particles were then dialyzed in 100 kD MWCO dialysis units for several days and stored until use. The amount of heparin in the solution was determined by lyophilizing a small aliquot of the solution. Similar to the heparin-coated polystyrene particles, VEGF was incubated at 4°C at 100 ⁇ g/ml, followed by 365 nm wavelength UV light activation for 10 minutes to lock VEGF covalently to the surface. Excess VEGF was removed by dialysis in 100 kD MWCO dialysis units.
  • the powder was dissolved in sodium acetate, pH 4, at 100 mg/ml and combined with Tween-80 and Span-80 (8% HLB).
  • the solution was placed in a ten-fold volume of hexane and combined with N,N,N',N-tetramethyl-ethane-l,2-diamine (TEMED) and ammonium persulfate (APS) during sonication to initiate radical polymerization.
  • TEMED N,N,N',N-tetramethyl-ethane-l,2-diamine
  • APS ammonium persulfate
  • the resultant nanoparticles were purified via liquid-liquid extraction in hexane. In the final stage of the extraction process, bubbling nitrogen gas into the nanoparticle solution evaporated off excess hexane. The particles were then dialyzed in 100 kD MWCO dialysis units for several days and stored until use.
  • heparin in the solution was determined by lyophilizing a small aliquot of the solution. Similar to the heparin-coated polystyrene particles, VEGF was incubated at 4°C at 100 ⁇ / ⁇ 1, followed by 365 nm wavelength UV light activation for 10 minutes to lock VEGF covalently to the surface. Excess VEGF was removed by dialysis in 100 kD MWCO dialysis units.
  • VEGF was conjugated to heparin nanoparticles at a ratio of 10 ⁇ g VEGF to
  • heparin nanoparticle 1 mg heparin nanoparticle.
  • Human umbilical vein endothelial cells (HUVECs) were plated at a density of 10,000 cells per well, and were grown in minimal media supplemented with either soluble VEGF or VEGF conjugated to heparin nanoparticles (VEGF-hNP). The cell growth was monitored over four days.
  • VEGF-hNP led to increased cell growth for concentrations of 0.1 ng/ml and greater ( Figure 14). Over the four day growth, VEGF-hNP treated cells reached cell growth saturation before VEGF treated cells, indicating that nanoparticle binding increased effectiveness of the growth factor ( Figure 15).
  • the resultant nanoparticles were purified via liquid-liquid extraction in hexane. In the final stage of the extraction process, bubbling nitrogen gas into the nanoparticle solution evaporated off excess hexane. The particles were then dialyzed in 100 kD MWCO dialysis units for several days and stored until use. The amount of heparin in the solution was determined by lyophilizing a small aliquot of the solution. Similar to the heparin-coated polystyrene particles, VEGF was incubated at 4°C at 100 ⁇ g/ml, followed by 365 nm wavelength UV light activation for 10 minutes to lock VEGF covalently to the surface. Excess VEGF was removed by dialysis in 100 kD MWCO dialysis units.
  • Human umbilical vein endothelial cells were cultured and passaged as previously described. Cells were plated in 96-well cell culture wells at 10,000 cells per well in minimal media. After two hours of plating, the cell culture media was exchanged with minimal media supplemented with either soluble VEGF or VEGF conjugated to heparin nanoparticles at concentrations of 0 ng/ml, 0.001 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, and 10 ng/ml. After two days, half the cells were analyzed for cell growth using the DNA CyQuant assay (Life Technologies). For the cells that continued growth, the cell culture media was refreshed with soluble and particle bound VEGF at the same concentrations. On day four, all the cells were analyzed for cell growth using the DNA CyQuant assay.
  • VEGF-hNP can be protected by formation of a shell around the particle.
  • the shell is formed by radical polymerization of acrylamide and acrylated peptides.
  • the peptides can be digested by proteases.
  • the peptide is hydrolyzed by treatment with trypsin.
  • the amount of VEGF exposed was measured by ELISA ( Figure 16). The results demonstrate that the shell was formed and then degraded.
  • Heparin nanoparticles with bound VEGF were diluted in 10 mM phosphate buffer (pH 8.5) and combined with 1% N-(3-aminopropyl) methacrylamide (positive charged monomer), 1% acrylamide (neutral monomer), and 1% plasmin degradable peptide (acrylated) in a small vial with a stir bar stirring at a low rate in order to prevent bubble formation.
  • the positively charged polymer electrostatically binds to negatively charged regions of VEGF.
  • a protease degradable shell formed around the VEGF molecules bound to the heparin nanoparticle.
  • the reaction proceeded for 2.5 h at 4°C before being dialyzed with 3500 MWCO tubing against PBS.
  • the resultant particles were subjected to a protease treatment (plasmin) for 30 min at room temperature. Exposure of VEGF was quantified by testing the samples in an ELISA.

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Abstract

La présente invention concerne une composition de nanoparticules comprenant de l'héparine en association avec un facteur de liaison à l'héparine. Ladite composition peut être utilisée pour favoriser l'angiogenèse et la régénération tissulaire (nerf, cœur, os, tissu adipeux, cartilage, tendons, ligaments, muscle, peau etc.), et pour procurer des protéines de matrice extracellulaire (fibronectine, collagène, laminine, vitronectine etc.).
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WO2017077066A1 (fr) * 2015-11-06 2017-05-11 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Composition contenant un polymère biocompatible et biodégradable, des nanosupports et un médicament, et procédés de fabrication et d'utilisation associés
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TWI698241B (zh) * 2017-09-05 2020-07-11 國立成功大學 用於治療缺血的肝素組合物
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WO2019050893A1 (fr) * 2017-09-05 2019-03-14 Excel Med, Llc Composition d'héparine pour le traitement de l'ischémie
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US20210220388A1 (en) * 2018-05-10 2021-07-22 The Regents Of The University Of California Therapeutic hydrogel material and methods of using the same
CN112843244A (zh) * 2021-01-11 2021-05-28 中国药科大学 一种尺寸可变的智能化载药纳米簇系统及其制备方法和应用
CN112843244B (zh) * 2021-01-11 2023-05-12 中国药科大学 一种尺寸可变的智能化载药纳米簇系统及其制备方法和应用

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