HEPARIN NANOCLUSTERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application no.

61/519,569, filed May 24, 2011, which is hereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under grant nos.

EB007730, GM067555, and 1R01NS079691 awarded by The National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of regenerative medicine.

Specifically, 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. In particular embodiments, 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.

SUMMARY OF THE INVENTION

[0004] In certain embodiments, 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. In particular embodiments, the nanoclusters include nanoparticles that include heparin and a heparin-binding factor, or a fragment thereof. For example, the nanoparticles can include heparin immobilized on support. Alternatively, the nanoparticles can be formed from heparin.

[0005] In various embodiments, 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. [0006] In various embodiments, 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.

[0007] In various embodiments, the composition includes greater than 10, 15, 20 μg heparin-binding factor per mg heparin. In some embodiments, the composition includes less than 45, 35, or 30 μg heparin-binding factor per mg heparin.

[0008] In various embodiments, 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.

[0009] In certain embodiments, at least some nanoclusters in the composition are encapsulated in a biodegradable shell. In specific embodiments, the biodegradable shell is susceptible to degradation by a protease, such as a matrix metalloproteinase. In particular embodiments, the biodegradable shell includes a polyethylene glycol, a carbohydrate, a peptide, or a combination thereof.

[0010] In some embodiments, 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.

[0011] In particular embodiments, the nanoclusters are present in a gel, such as a fibrin gel. In some embodiments, 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.

[0012] 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.

[0013] 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.

[0014] In specific embodiments, where the heparin-binding factor is an angiogenic growth factor, 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. In illustrative embodiments, the composition is administered to a damaged or diseased site and or at the site of an implant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : VEGF modified particle synthesis. (A) 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. The heparin-binding domain of VEGF has many available amines on the lysine groups that interact with the sulfate groups on heparin. (B) For the heparin nanoparticle, 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. Once the modified polymer is purified, it is combined with surfactants into a hexane solution for sonication. During the inverse emulsion sonication process, 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

concentration with different number of particles. (D) With VEGF bound, 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.

[0016] 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. (B) DLS characterization of heparin nanoparticles shows a smaller average diameter than the polystyrene particles, but high polydispersity (n = 3, *p < 0.05, **p < 0.01).

[0017] 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. (C) VEGF is stably bound to heparin nanoparticles with different binding densities. Each successive wash has less VEGF until leveling (n = 3).

[0018] Figure 4: Particle activity characterization. (A) Micrographs of cell migration experiment indicate movement of front from time 0 (top pictures) to 18 hours later (bottom pictures) in every condition except the negative control (Scale bar = 100 μιη). (B)

Quantification of the migration study shows particle bound VEGF is active. No statistical significance is observed between Vc, hNP, and Vs (n = 3).

[0019] Figure 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.

[0020] Figure 6: cdc42 activation assay. Heparin nanoparticles with high density loading have a statistically significant increase in cdc42 activation over soluble VEGF. The polystyrene particles do no have a significant enhancement over the soluble condition, but do over the negative control, Vn. (n = 2, *p < 0.05).

[0021] Figure 7: Tube formation assay with polystyrene particles. (A) Phase micrographs of cytodex beads with endothelial cell sprouts after 9 days in culture. The arrow heads indicate branching points of tubes that sprout from the cytodex bead (Scale bar = 50 μιη). (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)

Quantification of the number of sprouts emanating from the cytodex beads is not statistically different between conditions. (E) Tube thickness also did not change significantly between the conditions, (n = 10).

[0022] Figure 8: Tube formation assay with heparin nanoparticles. (A) Phase micrographs of cytodex beads with endothelial cell sprouts after 9 days in culture. The arrow heads indicate branching points of tubes that sprout from the cytodex bead (Scale bar = 50 μιη). (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. (E) hNP high led to a significant increase in tube thickness over Vs (**p < 0.01), as did Vs-hNP (over Vs, **p < 0.01). (n = 10).

[0023] Figure 9: CAM micrographs of fibrin implant and surrounding tissue show

VEGF leads to induction of blood vessels surrounding implant. Since the low density conditions required more polystyrene particles, the fibrin gels became more opaque. The negative control, fibrin only (Vn), does not show the vascular induction of the other conditions with VEGF. Characteristic radial vessels originating from the implants are observed in every condition except the negative control (First and third column scale bar = 100 μιη, second and fourth column scale bar = 60 μιη).

[0024] Figure 10: CAM fluorescent micrographs of fibrin implant show that covalently bound VEGF leads to infiltration and branching of blood vessels within implant. Fluorescent micrographs were taken both of the implant itself and just outside the implant to show vessel morphology at the interface of the fibrin gel with the CAM (the star denotes the inside of the gel). Electrostatically bound VEGF releases and leads to radial vessel formation similar to the soluble VEGF condition (Scale bar = 25 μιη).

[0025] Figure 11 : FfUVEC migration induced by VEGF-heparin coated polystyrene particles. Polystyrene particles of 10 μιη and 3 μιη diameters were coated with heparin and either electrostatically or covalently conjugated to VEGF. A wound was created in vitro by dragging a pipet tip over a confluent monolayer of HUVECs. Pictures were taken immediately after the wound was created and 18 hours later. The percent wound closure was determined by measuring the distance between cell fronts on either side of the wound at 18 hours and comparing to the distance at 0 hours. All of the conditions tested led to a significant increase in wound closure (Vc is covalently bound VEGF, Ve is electrostatically bound VEGF, scale bar = 100 μπι, *p<0.05).

[0026] Figure 12: VEGF-heparin coated particles internalized by HUVECs.

Polystyrene particles of 10 μιη diameters were coated with heparin and either

electrostatically or covalently conjugated to VEGF. The particles were exposed to HUVECs for either 20 min or 1 hour, then subjected to an acid wash to remove particles that were not internalized. VEGF coated 10 μιη diameter polystyrene particles lead to specific

internalization into HUVECs after 1 hour of exposure. After 20 min, background internalization is observed for VEGF coated polystyrene particles. By coating the particles with VEGF, internalization significantly increased (Vc is covalently bound VEGF, Ve is electrostatically bound VEGF, Vn is no VEGF, *** p < 0.001).

[0027] 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. 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.

[0028] Figure 14: VEGF-hNP dose response curve. VEGF alone and VEGF conjugated to heparin nanoparticles (VEGF-hNP) 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.

[0029] Figure 15: Cellular growth curves of HUVECs treated with 0.1 ng/ml and

1 ng/ml VEGF and VEGF-hNP in minimal media. The data shows that growth factors conjugated to heparin nanoparticles increase the cellular growth rate over growth factors alone.

[0030] 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.

DETAILED DESCRIPTION

Introduction

[0031] In certain embodiments, 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. In particular embodiments, the present invention further relates to reducing the effective dose of heparin-binding factors (e.g., growth factors) by conjugating them to heparin nanoparticles. In some embodiments, the invention relates to enhancing the cell signaling activity of heparin-binding factors (e.g., growth factors) by conjugating them to heparin nanoparticles. In certain embodiments, 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.

[0032] 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. Those of skill in the art will appreciate that the considerations discussed below with respect to VEGF apply to other angiogenic growth factors, as well as to growth factors more generally. In particular, 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. For ease of discussion, 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.) 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.

[0033] In particular embodiments, 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. In particular, 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. In illustrative embodiments, 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.

[0034] In certain embodiments, 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

regeneration. 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. When the vasculature is in place, 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.

[0035] Advantages of the compositions discribed herein include that the

presentation of 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. In addition, 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.

[0036] Experimental findings related to embodiments of the invention are described in Examples 1-5 below. These can be summarized as follows:

(1) 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.

(2) In vitro clustered covalently bound VEGF and clustered electrostaic bound VEGF were able to activate VEGFR-2 in HUVECs similar to soluble VEGF.

(3) In vitro clustered covalently bound VEGF and clustered electrostatic bound VEGF induced HUVEC vessel branching to a higher extent than soluble VEGF in vitro.

(4) 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.

(5) Clustered covalently bound VEGF and clustered electrostatic bound VEGF facilitated particle internalization in to HUVECs to a greater extent than particles without bound VEGF.

(6) Heparin nanoparticles fabricated by different chemistries result in particles with similar dimensions and size distribution.

(7) VEGF bound to heparin nanoparticles lowers the effective dose of the growth factor and results in greater cell growth rate.

(8) VEGF bound to heparin nanoparticles can be encapsulated in a protective polymer shell that can be degraded by treatment with an enzyme.

Definitions

[0037] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0038] As used herein, a "nanoparticle" is any particle that has an average characteristic dimension, such as diameter, that is less than 1000 nm. [0039] As used herein, 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.

[0040] As used herein, the term "heparin-binding factor" refers to any moiety that has binding affinity for heparin. For example, the term includes, but is not limited to, factors that bind heparin with a Kd of less than 1 μΜ. 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.

[0041] As used herein, the term "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). Other examples of 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. The term 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).

[0042] The term "extracellular matrix protein" ("ECM protein") includes collagen, fibronectin, laminin, vitronectin, fibrin, and the like.

[0043] The term "fragment" is used herein with reference to a polypeptide to describe a portion of a larger molecule. Thus, 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%>).

[0044] The term "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).

[0045] As used herein, the term "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

polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass; and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a preexisting natural polymer.

[0046] The publications and other materials used herein to illustrate the background of the invention, and in particular cases, to provide additional details regarding the use of the disclosed compositions and methods, are incorporated herein by reference in their entireties. Compositions Comprising Growth Factor-Heparin Nanoclusters

[0047] 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. For some applications, it is advantageous to include one or more soluble heparin-binding factor(s) (e.g., growth factor(s)), together with the nanoclusters, in the compositions. In some embodiments, useful, e.g., in tissue engineering the compositions include a gel, e.g., a hydrogel, including encapsulated and/or non-encapsulated nanoclusters with optional soluble heparin-binding factor(s).

Nanoclusters

[0048] In certain embodiments, 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. 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, β-D-glucuronic acid, 2-acetamido-2-deoxy- a D-glucose, and a -L-iduronic acid as major saccharide units. These are joined through l->4 glycosidic linkages. The presence and frequency of these saccharide units vary with the tissue source from which heparin is extracted. In addition to unfractionated pharmaceutical heparin, there are partially depolymerized forms called low molecular weight (LMW) heparins and a synthetic heparin pentasaccharide that are currently in clinical use. 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.

[0049] Any growth factor that can bind, or be engineered to bind, heparin can be used in the nanoclusters described herein. Angiogenic growth factors are of particular interest, although the invention is not limited to such. 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). Example 1 illustrates the preparation and function of VEGF-heparin nanoclusters. Example 3 demonstrates different chemistries that can form heparin nanoparticles with similar physical dimensions and size distributions.

[0050] The growth factor is covalently bound to the nanocluster at an average density of greater than 200 molecules per nanocluster. In various embodiments, the average density in greater than 400, 600, 800, or 900 molecules per nanocluster. In various embodiments, the average density can have an upper limit of 1800, 1600, 1400, 1200, or 1100 molecules per nanocluster. Thus, 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 μg growth factor per mg heparin. This relative concentration can, variously, be less than 45, 35, or 30 μg 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 μg growth factor per mg heparin, 10-20 μg growth factor per mg heparin, or 30-35 μg growth factor per mg heparin.

[0051] Nanoclusters can be produced in any desired size, which can vary, depending on the specific application. Generally, a nanocluster of heparin in combination with a growth factor has an average characteristic dimension, such as diameter, that is less than 1000 nm. In various illustrative embodiments, the average characteristic dimension can be less than 500, 300, 250, 220, 200, 180, 160, 140, 120, 100, 80, or 60 nm. In various illustrative embodiments, 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.

[0052] In particular embodiments, the nanoclusters comprise nanoparticles including heparin and a growth factor. The growth factor can be covalently bound to the nanocluster via the heparin molecules. For example, the nanoparticles can include heparin immobilized on a support (e.g., a bead) made of a different material. [0053] 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. A variety of 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.

[0054] 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. Alternatively, heparin can be added to the support after nanoparticle formation. In either case, 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.

[0055] In alterative embodiments, nanoparticles are formed from heparin. Any method that produces appropriately sized heparin nanoparticles that can bind growth factor can be employed. In certain embodiments, 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. Next, 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. Once the reaction is complete, 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. During the heparin- ABH-cystamine reaction with the acid labile polymer 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.

[0056] 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. In particular embodiments, 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.

Encapsulated Nanoclusters

[0057] In certain embodiments, compositions including growth factor-heparin nanoclusters can have some or all of the nanoclusters encapsulated in a biodegradable shell. For example, 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. In tissue engineering applications, for example, such a composition can take the form of a biocompatible, biodegradable hydrogel including the non-encapsulated and encapsulated nanoclusters. 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.

[0058] In particular embodiments, 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. In tissue engineering applications, for example, 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). In various embodiments, the biodegradable shell includes one or more of poly(ethylene glycol), carbohydrate, peptide polyacrylamide, methacrylamide, or a combination thereof. See Example 5.

[0059] In an illustrative embodiment, 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. To accomplish this, a MMP-2 sensitive peptide diacrylate can be incubated with the particles, along with acrylamide, ammomium persulate and Ν,Ν,Ν',Ν'-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.

Nanoclusters and Soluble Growth Factors and/or Clustered, Non-Covalently Bound Growth Factors

For certain applications, 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. In tissue engineering applications, where the object is promote blood vessel growth into an implant, 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. 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.

Gels Including Nanoclusters

[0060] In certain embodiments, the nanocluster-containing composition is a gel. For in vivo applications, 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.

[0061] In various embodiments, the nanocluster-containing gel can also include:

(1) one or more soluble growth factor(s) and/or (2) clustered, non-covalently bound growth factors, as discussed above for implants. 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). In particular embodiments, the nanocluster-containing gel serves as the implant, which can be injected, or otherwise implanted into the body. Applications

[0062] In various embodiments, 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. See Example 4. 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. In specific, illustrative embodiments, 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.

[0063] 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. In certain embodiments, 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.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Vascular endothelial growth factor (VEGF) presentation within fibrin matrices dictates endothelial cell branching

Abstract

[0064] Vascular endothelial growth factor (VEGF) has been extensively

investigated to promote vascularization at damaged or diseased sites and in tissue implants. Here we are interested in determining if the manner in which VEGF is presented from a scaffold to endothelial cells influences the architecture of the blood vessels formed. We bound VEGF to nanoparticles and placed these nanoparticles inside 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. In addition, we used a CAM assay to determine the role of VEGF presentation on angiogenesis in vivo. We tested 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

electrostatically exhibited no significant difference with covalently bound VEGF in the tube formation assay, this method failed to sequester host vessel infiltration into the fibrin implant on the CAM. Together our data suggest that the mode of VEGF presentation to endothelial cells influences the vessel architecture and vascularization of implants in vivo.

Introduction

[0065] Tissue regeneration involves the growth of specific tissue types for replacement of damaged tissue that the body is incapable of regenerating[l]. In order to assimilate the new tissue implant into the body and to support survival of the growing cells within the implant, a vascular supply is typically required[2]. Infiltration of blood vessels into the implant, however, is not enough to guarantee adequate blood supply, nutrient delivery, and waste removal for the cells inhabiting the implant. To maintain the integrity of the new tissue, perfusion of the implant by branched capillaries is needed to provide a feasible infrastructure upon which the new tissue can mature [3]. [0066] 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].

[0067] VEGF has also been covalently bound to the polymer backbone of a biomaterial without an engineered release mechanism. However, 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].

[0068] In this present report, we are interested in understanding the role of VEGF presentation on vessel branching. To study the role of growth factor presentation on branching, we established a system in which VEGF is bound with increasing affinity for the matrix, and in increasing heterogeneity with respect to its distribution in the gel. Polystyrene particles 260 nm in diameter were coated with heparin which was modified with a photoactive crosslinker. VEGF is covalently bound to the particle in a bind-and-lock approach[18]. To vary the affinity of VEGF for the matrix from covalent to electrostatic, the photoactive crosslinker is omitted. To modify the distribution of the growth factor in the gel, VEGF is bound in low density and high density forms, where the low density form has less VEGF molecules bound per particle. By maintaining a constant growth factor concentration between the conditions, 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. Finally, the particle-fibrin gels are introduced to the CAM of a chicken embryo and assayed for angiogenic potential. In addition to the polystyrene particles, 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.

Materials and Methods

Materials

[0069] Heparin sodium salt from porcine intestinal mucosa was purchased from

Alfa Aesar (Ward Hill, MA). Vascular Endothelial Growth Factor (VEGF) was kindly provided by the National Cancer Institute. Human umbilical vein endothelial cells

(HUVEC) were purchased from Lonza (Walkersville, MD). 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.

Cell culture

[0070] HUVECs were cultured in EGM-2 complete medium (Lonza, Walkersville,

MD) at 37°C and 5% C0 ₂ . 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 polystyrene coated nanoparticle preparation

[0071] 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,

Rockford, IL) for 2 hours at room temperature. The solution was then diluted to 1 mg/ml heparin and adjusted to pH 9-9.5 before incubating with amine functionalized polystyrene particles 260 nm in diameter for 2 hours at room temperature. Then, the particles were incubated with 50 mM sodium cyanoborohydride for 5 minutes at room temperature. The amount of polystyrene particles in the solution was determined by the following relation using information provided by the manufacturer (from Spherotech Technical Notes): πΡΌ ³ where N is the number of particles, Wis the weight of the polymer (g), P is the density of the polymer (g/cm ³ ), and D is the diameter (μιη). Overnight incubation of VEGF at 4°C in 1% BSA-PBS at 100 μg/ml was 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.

Heparin nanoparticle synthesis

[0072] Heparin was first modified with p-azidobenzyl hydrazide (ABH, Pierce,

Rockford, IL) through EDC mediated conjugation in a 1 :3 molar ratio of ABH to available carboxylic acids. The pH was monitored during the course of the reaction and the conjugation took place at pH 4.75 in PBS. After 2 hours of reaction at room temperature, the carboxylic acid groups on heparin were reactivated with EDC, but this time reacted with adipic dihydrazide (ADH) in 27 molar excess in order to saturate reaction binding sites and prevent unwanted crosslinking. Again, the reaction pH was monitored at pH 4.75 in PBS. This reaction was allowed to proceed overnight at room temperature, at which point the reaction solution was dialyzed against DI water. The dialysis units were then placed in PBS at pH 7.4 for buffer exchange. Heparin with ABH and ADH was then reacted with NHS- acrylate to convert the amine groups to acrylates. The reaction proceeded overnight at room temperature, and then dialyzed again. After dialysis, the solution was lyophilized for two days. 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. 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 μg/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.

Dynamic light scattering (DLS)

[0073] Heparin-polystyrene coated nanoparticles were analyzed in a Malvern

Zetasizer to determine particle diameter after each preparation step. Heparin nanoparticles were analyzed after formation, purification, and dialysis. Samples were loaded into a filtered DI water cleaned quartz cuvette. Ten runs each comprised three measurements, and data was reported as Z-average with polydispersity index (PDI).

Enzyme linked immunosorbant assay (ELISA)

[0074] Following dialysis, nanoparticles were collected and quantified for amount

VEGF bound using the human VEGF Duoset from R&D Systems (Minneapolis, Minnesota) following manufacturer's instructions. Briefly, the plate was coated with "capture" antibody overnight at room temperature, then non-specific binding sites were blocked via incubation with 1% BSA in PBS. The samples were added for 2 hours at room temperature with gentile agitation. The plates were washed several times, and "detection" antibody probed the 96- well plate for presence of VEGF. Streptavidin-HRP diluted 1 :200 in 1% BSA-PBS was then incubated with the wells for 20 minutes. TMB substrate (Cell Signaling, Boston, MA) was added to the wells for 20 minutes at room temperature, and then read at 645 nm with 570 nm correction.

Cell migration

[0075] 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.

VEGFR-2 phosphorylation assay

[0076] 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. After aspirating all remnants of liquid from the wells, 100 μΐ of 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 μg/ml of aprotinin) was added to the surface and scraped. 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 (BCA assay, Bio-Rad) were diluted in 5X loading buffer (1 M Tris-HCL, pH 6.8, 20% SDS, 50% glycerol) supplemented with 5% (v/v) β-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.

Cdc42 GLISA

[0077] Cells were treated for 5 minutes following the same method mentioned earlier. After treatment, the cells were lysed and quantified with ProteinRed. The samples were assayed with the CytoSkeleton (Denver, CO) cdc42 GLISA kit following manufacturer's instructions. Samples were loaded at 0.5 mg/ml, and absorbance read at 490 nm.

Tube formation assay and branching quantification

[0078] 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 μg/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. 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).

Chorioallantoic membrane assay (CAM)

[0079] 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 ½ 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 μg/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.

Statistical analysis

[0080] Data are presented as mean ± standard deviation. To identify significant trends in data, statistical comparisons were performed by one-way ANOVA with post-test using the Tukey method. Data were considered significantly different if p < 0.05.

Results

Heparin-coated polystyrene and heparin nanoparticle synthesis

[0081] To understand the role of VEGF presentation on branching morphogenesis in biomaterials, polystyrene nanoparticles 260 nm in diameter were coated with oxidized heparin as outlined in Figure 1. Heparin underwent periodate-mediated oxidation and conjugation to p-azidobenzyl hydrazide (ABH). The reaction was done in excess of aldehyde groups to ensure remaining binding sites for conjugation to amine-functionalized polystyrene particles. After reducing the Schiff base with sodium cyanoborohydride, the particles were washed several times, and then incubated with VEGF. Upon exposure to UV light, the VEGF became covalently bound to the polystyrene particle (Figure 1)[18].

[0082] 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. 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). 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).

[0083] 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 nanoparticle loading characterization

[0084] 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. As a control, 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. 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.

[0085] The heparin nanoparticles were washed and the washes were analyzed for

VEGF content. With each successive wash, the amount of VEGF decreased until remaining steady (Figure 3C). After these washes, 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 μg VEGF/mg heparin while the low binding heparin nanoparticle had 10 μg VEGF/mg heparin (Figure 3C). Again, 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

phosphorylation

[0086] To test the activity of VEGF modified nanoparticles, in vitro wound closure,

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).

[0087] VEGF modified nanoparticles were able to phosphorylate VEGFR-2 at two different tyrosine residues, Yl 175 and Y1214 (Figure 5). Interestingly, 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.

[0088] We next tested the levels of cdc42 when the cells were presented with the particle-bound VEGF. We observed a statistically significant increase in cdc42 activation for VEGF modified heparin nanoparticles at both high (hNP _h i _gh ) and low (hNPi _0W ) surface concentrations compared to the heparin nanoparticles without VEGF (Vn) attached (p < at least 0.05, Figure 6). In addition, hNP _h i _gh achieved higher activation of cdc42 than soluble VEGF (Vs) (p < 0.05). Although there was activation of cdc42 with the polystyrene modified particles at high and low density and for both covalent (PS-Vchi _g h/iow) and electrostatically (PS-Ve _h i _gh /iow) immobilized VEGF, there was no statistical significance between any of the polystyrene modified VEGF particles and Vs (p > 0.05). VEGF presentation inside fibrin hydrogels modulates HUVEC branching

[0089] To determine the effect of VEGF presentation on endothelial tube formation, 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]. During 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).

[0090] The number of branching points for PS-Vc i _gh and PS-Ve i _gh was statistically higher than for that observed for Vs (p < at least 0.01, Figure 7B). However, lowering the amount of VEGF displayed per particle, PS-Vci _0W and PS-Vei _0W , resulted in no significant increase in branching over Vs (p > 0.05), indicating that the presentation of the VEGF inside the fibrin hydrogel affected the architecture of the vessels formed. Comparing between PS-Vc _h i _gh vs. PS-Vci _0W and PS-Ve _h i _gh vs. 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).

[0091] 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). However, 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. Comparing between PS-Vc _h i _gh vs. PS-Vci _0W and PS-Ve _h i _gh vs. PS- Vei _ow there was a statistically significant difference for PS-Vc (p < 0.001) but not for the PS-Ve (p > 0.05), further indicating that the presentation of VEGF affected the extent of the network. No difference was observed between PS-Vchigh and PS-Vehigh (p > 0.05).

[0092] Next, the number of sprouts from the cytodex bead and tube thickness were quantified. No differences were observed for any of the particles tested compared to Vs (Figure 7D,E), indicating that the induction of angiogenesis was not dependent on VEGF presentation nor was the thickness of the vessel formed.

[0093] Since no major differences between covalent and electrostatically bound

VEGF were observed, when we sought to translate the PS particles to a more biocompatible particle, only covalently bound VEGF was used. VEGF modified heparin nanoparticles were synthesized and similar analysis as those done with PS nanoparticles were performed (Figure 8). Similarly to the VEGF modified PS nanoparticles, 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). These findings further pointed to the role of VEGF presentation on endothelial cell branching. Comparing between hNP _h i _gh and hNPi _0W showed statistically higher branching for hNP _h i _gh (p < 0.01, Figure 8B). To determine if the preloading of VEGF to the nanoparticles was necessary or the presence of the heparin nanoparticles alone was sufficient to recruit soluble VEGF into nanodomains, unloaded heparin nanoparticles were introduced to the hydrogel. A statistically higher number of branching points were observed for the hydrogels that contained unloaded nanoparticles along with soluble VEGF (VS-hNP) than those with only Vs (p < 0.05). This data indicated that the presence of the nanoparticles alone was sufficient to recruit VEGF into clusters that changed the growing vessel architecture.

[0094] Similar to that observed with the VEGF modified polystyrene nanoparticles, total network length for hNP _h i _gh and hNPi _0W was statistically higher than that observed for Vs (p < 0.001 , Figure 8C), further suggesting the presentation of VEGF affected the extent of the vessel network. However, as was the case for the VEGF coated PS nanoparticles, the binding density on hNP did not result in a statistical difference for hNP _h i _gh and hNPi _0W . 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).

[0095] Similar to PS, 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

[0096] 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.

[0097] To further study the extent of vessel induction and infiltration into the implant, the embryo was perfused with FITC-Dextran. In PS-Vchigh and hNPhigh,

microvessels inside the fibrin implant were observed, indicating that not only were vessels inside the implant formed, but that the vessels were integrated with the host vasculature (Figure 10). The PS-Ve gel did not facilitate infiltration of blood vessels into the fibrin gel. However, there were radial blood vessels originating from the gel, indicating that the VEGF was released from the particles and diffused out of the gel. The Vs fibrin gel also did not show blood vessel formation in the fibrin gel, but did show characteristic radial vessel formation outside of the gel (Figure 10).

Discussion

[0098] Vascular endothelial growth factor (VEGF) has been extensively

investigated to promote vascularization at damaged or diseased sites and in tissue implants for tissue engineering applications [21]. In this current report, we studied if the manner in which VEGF is presented from a scaffold to endothelial cells influences the architecture of the blood vessels formed. We bound VEGF to nanoparticles and placed these nanoparticles inside 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]. We tested 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. We found that VEGF covalently bound to nanoparticles at high density led to increases in HUVEC tube branching, thickness, and total vessel network length compared to soluble VEGF. Our data indicates that the presentation of VEGF within matrices determines the architecture of the resulting blood vessels. [0099] VEGF modified nanoparticles were synthesized using either a polystyrene core (PS) or a heparin core. For the PS nanoparticles the surface of the nanoparticles contained amines, which were used to immobilize heparin. To immobilize VEGF covalently or electrostatically to the PS-heparin particle surface we used either heparin modified with a photoreactive group or unmodified heparin as we have previously described[18]. For the nanoparticles that contained a heparin core, 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.

[0100] 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.

[0101] To determine if the process of immobilization affects VEGF activity,

HUVEC migration and VEGFR-2 phosphorylation induced by VEGF modified

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. At the molecular level, 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). Interestingly, 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]. In addition phosphorylation at Y1214 leads to the activation of cdc42 [27], which is involved in branching. To determine if VEGF modified nanoparticles result in activation of cdc42, 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].

[0102] The trends observed in the in vitro tube formation studies highlight the importance of pre-loading of VEGF for hNP and high density binding. Interestingly, 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. However, 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. By binding VEGF to nanoparticles in the high binding density format, growth factor reservoirs are created, mimicking the physiological environment[33-35]. Growth factors in the

extracellular matrix (ECM) 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].

[0103] 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]. [0104] 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. This serves as a motivation for development of the heparin nanoparticles, since polystyrene is not a biocompatible material[37]. However, in the course of the material characterization, the heparin nanoparticles have been found to be consistently polydisperse. Despite this, no significant negative impact of this polydispersity is observed for high PDI heparin nanoparticles compared to low PDI polystyrene nanoparticles, suggesting that this polydispersity may be tolerable. This evidence suggests that cluster size, specifically, may not affect the morphological behavior of endothelial tubes, but nanoscale clustering in general does increase vascular network size and degree of branching.

Conclusion

[0105] 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. When administered to the CAM of an embryonic chicken, the clustered, covalently bound VEGF nanoparticles successfully guided host vasculature into the implant, leading to a perfused capillary network within the fibrin gel.

References

[0106] 1. Lanza RP, Langer RS, Vacanti J. Principles of tissue engineering. 3rd ed. Amsterdam ; Boston: Elsevier Academic Press, 2007. [0107] 2. Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev Mar 17.

[0108] 3. Kannan RY, Salacinski HJ, Sales K, Butler P, Seifalian AM. The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review. Biomaterials 2005 May;26(14): 1857-1875.

[0109] 4. Gu F, Amsden B, Neufeld R. Sustained delivery of vascular endothelial growth factor with alginate beads. J Control Release 2004 May 18;96(3):463- 472.

[0110] 5. Huang M, Vitharana SN, Peek LJ, Coop T, Berkland C.

Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor. Biomacromolecules 2007 May;8(5): 1607-1614.

[0111] 6. Edelman ER, Mathiowitz E, Langer R, Klagsbrun M. Controlled and modulated release of basic fibroblast growth factor. Biomaterials 1991 Sep;12(7):619-626.

[0112] 7. Ehrbar M, Djonov VG, Schnell C, Tschanz SA, Martiny-Baron G,

Schenk U, et al. Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ Res 2004 Apr 30;94(8): 1124-1132.

[0113] 8. Zisch AH, Lutolf MP, Ehrbar M, Raeber GP, Rizzi SC, Davies N, et al. Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth. FASEB J 2003;17(15):2260-2262.

[0114] 9. Joung YK, Bae JW, Park KD. Controlled release of heparin-binding growth factors using heparin-containing particulate systems for tissue regeneration. Expert Opin Drug Deliv 2008 Nov;5(l 1): 1173-1184.

[0115] 10. Lee J, Lee KY. Local and sustained vascular endothelial growth factor delivery for angiogenesis using an injectable system. Pharm Res 2009

Jul;26(7): 1739-1744.

[0116] 11. Zieris A, Prokoph S, Levental KR, Welzel PB, Grimmer M,

Freudenberg U, et al. FGF-2 and VEGF functionalization of starPEG-heparin hydrogels to modulate biomolecular and physical cues of angiogenesis. Biomaterials Nov;31(31):7985- 7994. [0117] 12. King TW, Patrick CW, Jr. Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic

acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single

emulsion/solvent extraction technique. J Biomed Mater Res 2000 Sep 5;51(3):383-390.

[0118] 13. Chung YI, Kim SK, Lee YK, Park SJ, Cho KO, Yuk SH, et al.

Efficient revascularization by VEGF administration via heparin- functionalized nanoparticle- fibrin complex. J Control Release May 10;143(3):282-289.

[0119] 14. Fairbrother WJ, Champe MA, Christinger HW, Keyt BA, Starovasnik

MA. Solution structure of the heparin-binding domain of vascular endothelial growth factor. Structure 1998 May 15;6(5):637-648.

[0120] 15. Lee S, Jilani SM, Nikolova GV, Carpizo D, Iruela-Arispe ML.

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.

[0121] 16. Moon JJ, Saik JE, Poche RA, Leslie-Barbick JE, Lee SH, Smith AA, et al. Biomimetic hydrogels with pro-angiogenic properties. Biomaterials

May;31(14):3840-3847.

[0122] 17. Phelps EA, Landazuri N, Thule PM, Taylor WR, Garcia AJ.

Bioartificial matrices for therapeutic vascularization. Proc Natl Acad Sci U S A Feb 23;107(8):3323-3328.

[0123] 18. Anderson SM, Chen TT, Iruela-Arispe ML, Segura T. The phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently immobilized VEGF. Biomaterials 2009

Sep;30(27):4618-4628.

[0124] 19. Nakatsu MN, Davis J, Hughes CC. Optimized fibrin gel bead assay for the study of angiogenesis. J Vis Exp 2007(3): 186.

[0125] 20. Nakatsu MN, Sainson RC, Perez-del-Pulgar S, Aoto JN, Aitkenhead

M, Taylor KL, et al. VEGF(121) and VEGF(165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model. Lab Invest 2003 Dec;83(12): 1873-1885. [0126] 21. Phelps EA, Garcia AJ. Engineering more than a cell: vascularization strategies in tissue engineering. Curr Opin Biotechnol Jul 15.

[0127] 22. Missirlis D, Tirelli N, Hubbell JA. Amphiphilic hydrogel nanoparticles. Preparation, characterization, and preliminary assessment as new colloidal drug carriers. Langmuir 2005 Mar 15;21(6):2605-2613.

[0128] 23. Chen TT, Luque A, Lee S, Anderson SM, Segura T, Iruela-Arispe

ML. Anchorage of VEGF to the extracellular matrix conveys differential signaling responses to endothelial cells. J Cell Biol Feb 22;188(4):595-609.

[0129] 24. Takahashi T, Yamaguchi S, Chida K, Shibuya M. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. Embo J 2001 Jun

l;20(l l):2768-2778.

[0130] 25. Dayanir V, Meyer RD, Lashkari K, Rahimi N. Identification of

Tyrosine Residues in Vascular Endothelial Growth Factor Receptor-2/FLK-l Involved in Activation of Phosphatidylinositol 3-Kinase and Cell Proliferation. J Biol Chem 2001 May 18, 2001;276(21): 17686-17692.

[0131] 26. Fujio Y, Walsh K. Akt Mediates Cytoprotection of Endothelial Cells by Vascular Endothelial Growth Factor in an Anchorage-dependent Manner. J Biol Chem 1999 June 4, 1999;274(23): 16349-16354.

[0132] 27. Lamalice L, Houle F, Jourdan G, Huot J. Phosphorylation of tyrosine

1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of

SAPK2/p38. Oncogene 2004 Jan 15;23(2):434-445.

[0133] 28. Rousseau S, Houle F, Landry J, Huot J. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 1997 Oct;15(18):2169-2177.

[0134] 29. Mori M, Murata Y, Kotani T, Kusakari S, Ohnishi H, Saito Y, et al.

Promotion of cell spreading and migration by vascular endothelial-protein tyrosine phosphatase (VE-PTP) in cooperation with integrins. J Cell Physiol Jul;224(l): 195-204. [0135] 30. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A,

Abramsson A, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003 Jun 23;161(6):1163-1177.

[0136] 31. Krueger J, Liu D, Scholz K, Zimmer A, Shi Y, Klein C, et al. Fltl acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo. Development May;138(10):2111-2120.

[0137] 32. Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S,

Fujisawa H, et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 2002 Oct 15;16(20):2684-2698.

[0138] 33. Laise P, Di Patti F, Fanelli D, Masselli M, Arcangeli A. Deterministic and stochastic aspects of VEGF-A production and the cooperative behavior of tumoral cell colony. J Theor Biol Mar 7;272(l):55-63.

[0139] 34. Hubbell JA. Matrix-bound growth factors in tissue repair. Swiss Med

Wkly 2007 Mar 2; 137 Suppl 155:72S-76S.

[0140] 35. Xie Y, Upton Z, Richards S, Rizzi SC, Leavesley DI. Hyaluronic acid: Evaluation as a potential delivery vehicle for vitronectin: growth factor complexes in wound healing applications. J Control Release Mar 30.

[0141] 36. Lei Y, Huang S, Sharif-Kashani P, Chen Y, Kavehpour P, Segura T.

Incorporation of active DNA/cationic polymer polyplexes into hydrogel scaffolds.

Biomaterials Dec;31 (34) : 9106-9116.

[0142] 37. Rayavarapu RG, Petersen W, Hartsuiker L, Chin P, Janssen H, van

Leeuwen FW, et al. In vitro toxicity studies of polymer-coated gold nanorods.

Nanotechnology Apr 9;21(14): 145101.

Example 2

Internalization of VEGF-Heparin Coated Particles

Results

[0143] To investigate the internalization of VEGF-heparin coated particles, a 10 μιη particle with a polystyrene core was used as a proxy to visualize internalization into human umbilical vein endothelial cells (HUVECs). First, 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.

[0144] In a separate experiment, 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 μιη diameter polystyrene particles are internalized by HUVECs after one hour of exposure.

Particle synthesis

[0145] 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,

Rockford, IL) for 2 hours at room temperature. The solution was then diluted to 1 mg/ml heparin and adjusted to pH 9-9.5 before incubating with amine functionalized polystyrene particles 260 nm in diameter for 2 hours at room temperature. Then, the particles were incubated with 50 mM sodium cyanoborohydride for 5 minutes at room temperature. The amount of polystyrene particles in the solution was determined by the following relation using information provided by the manufacturer (from Spherotech Technical Notes):

6W 12

N = r x lO

πΡϋ ³

[0146] where N is the number of particles, W is the weight of the polymer (g), P is the density of the polymer (g/cm ³ ), and D is the diameter (μιη). Overnight incubation of VEGF at 4°C in 1% BSA-PBS at 100 μ^πιΐ was 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. Cell migration

[0147] 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.

Particle Internalization

[0148] Confluent cell monolayers were exposed to 10 and 3 μιη 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.

Micrographs were acquired by the Zeiss Observer microscope and the particles internalized were counted using ImageJ.

References

[0149] 1. Zisch AH, Schenk U, Schense JC, Sakiyama-Elbert SE, Hubbell JA.

Covalently conjugated VEGF-fibrin matrices for endothelialization. J Control Release. 2001;72(1-3): 101-13.

[0150] 2. Yotsumoto F, Sanui A, Fukami T, Shirota K, Horiuchi S, Tsujioka H, et al. Efficacy of ligand-based targeting for the EGF system in cancer. Anticancer Res. 2009 Nov;29(l l):4879-85.

[0151] 3. Wong A. Modified epidermal growth factor receptor (EGFR)-bearing liposomes (MRBLs) are sensitive to EGF in solution. PLoS One. 2009;4(10):e7391.

[0152] 4. Baumann MD, Kang CE, Stanwick JC, Wang Y, Kim H, Lapitsky Y, et al. An injectable drug delivery platform for sustained combination therapy. J Control Release. 2009 Sep 15;138(3):205-13. [0153] 5. Chung YI, Kim SK, Lee YK, Park SJ, Cho KO, Yuk SH, et al. Efficient revascularization by VEGF administration via heparin- functionalized nanoparticle-fibrin complex. J Control Release May 10;143(3):282-289.

[0154] 6. Zisch AH, Lutolf MP, Ehrbar M, Raeber GP, Rizzi SC, Davies N, et al. Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth. FASEB J. 2003;17(15):2260-2.

[0155] 7. Moon JJ, Saik JE, Poche RA, Leslie-Barbick JE, Lee SH, Smith AA, et al. Biomimetic hydrogels with pro-angiogenic properties. Biomaterials.

May;31(14):3840-7.

[0156] 8. Ehrbar M, Zeisberger SM, Raeber GP, Hubbell JA, Schnell C, Zisch

AH. The role of actively released fibrin-conjugated VEGF for VEGF receptor 2 gene activation and the enhancement of angiogenesis. Biomaterials. 2008 Apr;29(l 1): 1720-9.

Example 3

Heparin nanoparticles synthesized from different chemistries

Results

[0157] Heparin nanoparticles (hNP) 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).

Michael addition method

[0158] Heparin was first modified with p-azidobenzyl hydrazide (ABH, Pierce,

Rockford, IL) through EDC mediated conjugation in a 1 :3 molar ratio of ABH to available carboxylic acids. The pH was monitored during the course of the reaction and the conjugation took place at pH 4.75 in PBS. Successful incorporation of ABH into the heparin polymer was confirmed by UV absorbance readings as outlined in Chapter 4. After 2 hours of reaction at room temperature, the carboxylic acid groups on heparin were reactivated with EDC, but this time reacted with adipic dihydrazide (ADH) in 27 molar excess in order to saturate reaction binding sites and prevent unwanted crosslinking. Again, the reaction pH was monitored at pH 4.75 in PBS. Incorporation of amines into the polymer was assayed by TNBSA following manufacturer's instructions (Pierce, Rockford, IL). This reaction was allowed to proceed overnight at room temperature, at which point the reaction solution was dialyzed against DI water. The dialysis units were then placed in PBS at pH 7.4 for buffer exchange. Heparin with ABH and ADH was then reacted with NHS-acrylate to convert the amine groups to acrylates. The reaction proceeded overnight at room temperature, and then dialyzed again. The disappearance of amines was quantified by the TNBSA assay. After dialysis, the solution was lyophilized for two days. 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. 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 μg/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.

Acrylate method

[0159] Heparin was first modified with p-azidobenzyl hydrazide (ABH, Pierce,

Rockford, IL) through EDC mediated conjugation in a 1 :3 molar ratio of ABH to available carboxylic acids. The pH was monitored during the course of the reaction and the conjugation took place at pH 4.75 in PBS. After 2 hours of reaction at room temperature, the carboxylic acid groups on heparin were reactivated with EDC, but this time reacted with adipic dihydrazide (ADH) in 27 molar excess in order to saturate reaction binding sites and prevent unwanted crosslinking. Again, the reaction pH was monitored at pH 4.75 in PBS. This reaction was allowed to proceed overnight at room temperature, at which point the reaction solution was dialyzed against DI water. The dialysis units were then placed in PBS at pH 7.4 for buffer exchange. Heparin with ABH and ADH was then reacted with NHS- acrylate to convert the amine groups to acrylates. The reaction proceeded overnight at room temperature, and then dialyzed again. After dialysis, the solution was lyophilized for two days. 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. 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 μg/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.

Methacrylate method

[0160] Heparin was first modified with p-azidobenzyl hydrazide (ABH, Pierce,

Rockford, IL) through EDC mediated conjugation in a 1 :3 molar ratio of ABH to available carboxylic acids. The pH was monitored during the course of the reaction and the conjugation took place at pH 4.75 in PBS. After 2 hours of reaction at room temperature, the carboxylic acid groups on heparin were reactivated with EDC, but this time reacted with aminopropyl methacrylamide (APMA) in 27 molar excess in order to saturate reaction binding sites. Again, the reaction pH was monitored at pH 4.75 in PBS. This reaction was allowed to proceed overnight at room temperature, at which point the reaction solution was dialyzed against DI water. After dialysis, the solution was lyophilized for two days. 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. 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 μ§/ηι1, 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.

Example 4

VEGF-hNP dose response

Results

[0161] VEGF was conjugated to heparin nanoparticles at a ratio of 10 μg VEGF to

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.

[0162] At day 2, 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).

Particle synthesis

[0163] Heparin was first modified with p-azidobenzyl hydrazide (ABH, Pierce,

Rockford, IL) through EDC mediated conjugation in a 1 :3 molar ratio of ABH to available carboxylic acids. The pH was monitored during the course of the reaction and the conjugation took place at pH 4.75 in PBS. After 2 hours of reaction at room temperature, the carboxylic acid groups on heparin were reactivated with EDC, but this time reacted with adipic dihydrazide (ADH) in 27 molar excess in order to saturate reaction binding sites and prevent unwanted crosslinking. Again, the reaction pH was monitored at pH 4.75 in PBS. This reaction was allowed to proceed overnight at room temperature, at which point the reaction solution was dialyzed against DI water. The dialysis units were then placed in PBS at pH 7.4 for buffer exchange. Heparin with ABH and ADH was then reacted with NHS- acrylate to convert the amine groups to acrylates. The reaction proceeded overnight at room temperature, and then dialyzed again. After dialysis, the solution was lyophilized for two days. 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. 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 μg/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.

Particle exposure to cells

[0164] Human umbilical vein endothelial cells (HUVECs) 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.

Example 5

Protease degradable shell formation around VEGF-hNP

Results

[0165] 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. In this example, 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.

Shell formation

[0166] 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. With initiation of radical polymerization from addition of 1% APS and 10% TEMED, 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.