WO2018187184A1 - Injectable therapeutic angiogenic material for brain repair - Google Patents

Abstract

An injectable therapeutic angiogenic material includes an in situ gelling hyaluronic acid-based hydrogel containing a first plurality of clustered VEGF heparin nanoparticles in which VEGF is immobilized to the heparin nanoparticles and a second plurality of naked heparin nanoparticles that do not contain VEGF immobilized to the heparin nanoparticles. In one aspect of the invention, the injectable therapeutic angiogenic material is used to repair ischemic tissue in a subject (e.g., mammal). To treat the subject or patient, the stroke cavity in the brain tissue is located and the therapeutic angiogenic material is injected into the stroke cavity along with a crosslinker.

Description

INJECTABLE THERAPEUTIC ANGIOGENIC MATERIAL FOR BRAIN REPAIR

Related Application

[0001] This Application claims priority to U.S. Provisional Patent Application No.

62/481,587 filed on April 4, 2017, which is hereby incorporated by reference in its entirety. Priority is claimed pursuant to 35 U.S.C. § 119 and any other applicable statute.

Statement Regarding Federally Sponsored

Research and Development

[0002] This invention was made with Government support under Grant No. NS079691, awarded by the National Institutes of Health. The Government has certain rights in the invention.

Technical Field

[0003] The technical field generally relates to therapeutic angiogenic materials that are used to repair ischemic tissues through the formation of vascular networks. In particular, the field of the invention relates to an injectable hydrogel -based therapeutic angiogenic material that incorporates heparin nanoparticles with highly clustered Vascular Endothelial Growth Factor (VEGF) in combination with "naked" heparin nanoparticles.

Background

[0004] The administration of hydrogels in the damaged brain has been shown to be insufficient to promote significant infiltration of cells and complex networks such as vessels and axons. Attempts have been made to promote vessel development using VEGF.

However, the administration of VEGF in the brain has been shown to be associated with severe side effects such as hemorrhage, edema, intracranial brain pressure, an increased post- stroke inflammation and an increase infarct volume, as well as the formation of leaky and unorganized vessels.

[0005] The lack of a successful medical therapy that promotes long-term recovery after stroke represents a substantial clinical burden, establishing a need for a medical treatment outside the confines of conventional therapies. In the developing body, angiogenesis pioneers a vascular network that leads to the growth and maturation of the nervous system. Angiogenesis after stroke is associated with better outcomes in stroke patients and promotes the formation of new neurons through an endogenous neural stem cell response (post-stroke neurogenesis). However, therapeutic manipulation of angiogenesis is problematic, for example systemic or brain administration of vascular endothelial growth factor (VEGF), as noted above, is associated with an increase in blood brain barrier (BBB) opening, brain edema and, worsening neurological deficits.

[0006] Stroke inherently causes tissue damage and results in the formation of a cavity. In this cavity, there is no extracellular matrix that supports cell infiltration into the lesion or to physically support a growing tissue. Indeed, there is a glial barrier and physical barrier to cell entry that is formed. Stroke also stimulates a massive local inflammatory response that may also impede recovery. Over time the cavity reorganizes into a fibrotic scar devoid of neural tissue. Yet, the stroke cavity represents a potential transplant location because it can accept a large volume injection without damaging normal brain. In addition, the delivery of therapies to the stroke cavity targets the area adjacent to stroke: the peri-infarct area. This is the site of the most robust neuronal and vascular plasticity after stroke and of functional recovery. In total, a successful strategy for brain repair after stroke would deliver a molecule that stimulates angiogenesis and neural regeneration, reduces local inflammation, removes the barrier to cellular infiltration in the stroke site, and introduces a scaffold that can serve as a physical support onto which a new neuronal network can grow. Recent advances in biopolymer hydrogels have developed materials with extracellular matrix motifs that directly support survival and cell infiltration. The problem remains, however of the adverse effects of incorporating VEGF into the brain.

Summary

[0007] In one embodiment, a therapeutic angiogenic hydrogel material is delivered to a stroke cavity and includes VEGF in a clustered conformation with heparin nanoparticles within the hydrogel matrix using to the stroke cavity forms axonal networks along newly generated blood vessels. In one embodiment, the therapeutic angiogenic hydrogel material is a in situ gelling hyaluronic acid hydrogel that contains a first plurality of clustered VEGF heparin nanoparticles in which VEGF is immobilized to the heparin nanoparticles and a second plurality of naked heparin nanoparticles that do not contain VEGF immobilized to the heparin nanoparticles. The hydrogel is cross-linked in one embodiment with a biodegradable crosslinker such as a matrix metalloproteinase (MMP) labile peptide as the crosslinker, resulting in a hydrogel that is both hyaluronidase degradable and MMP degradable. The hydrogel material may also be modified with a cell adhesion peptide such as RGD derived from fibronectin to allow for integrin-medicated cell attachment to the scaffold. [0008] The therapeutic angiogenic hydrogel material forms axonal networks along newly generated blood vessels. This regenerated tissue produces functional recovery through the established axonal networks. Thus this biomaterials approach generates a vascularized network of regenerated functional neuronal connections within previously dead tissue. The use of the clustered VEGF immobilized heparin nanoparticles in combination with the naked heparin nanoparticles bypasses the difficulties associated with VEGF.

[0009] The VEGF -containing hydrogel material may be injected directly within the stroke cavity. The therapeutic angiogenic hydrogel material induces the formation of a novel vascular and neuronal structure that leads to behavioral improvement. The therapeutic angiogenic hydrogel material results in the formation of a robust, mature and highly developed vascular bed within the stroke cavity that helps develop and partem the nervous system with axons associating closely with vessels. The formation of vascular bed also enhances the proliferation and migration of immature neurons. The axonal projections into this network are causally associated with recovered motor function. Thus, the local delivery of the engineered VEGF containing hydrogel created a neuronal structure through the sequential growth of vascular, neuronal and axonal elements. The axonal and vascular ingrowth and formation of this new neural structure within the stroke cavity produces a functionally active and essential new brain tissue that leads to the recovery of motor control.

[0010] In one aspect of the invention, an injectable therapeutic angiogenic material is provided that includes an in situ gelling hydrogel material that incorporates heparin nanoparticles therein. In one preferred embodiment of the invention, the hydrogel is a hyaluronic acid-based hydrogel (e.g., hyaluronic acid functionalized with acrylamide groups) that incorporates a first plurality of clustered VEGF heparin nanoparticles in which VEGF is immobilized to the heparin nanoparticles, and a second plurality of heparin nanoparticles that are "naked" and do not contain VEGF immobilized to the heparin nanoparticles. In one preferred embodiment, the number of the second plurality of naked heparin nanoparticles is much greater than the number of first plurality of clustered VEGF heparin nanoparticles. A biodegradable crosslinker (e.g., a matrix metalloproteinase (MMP) labile peptide) is used with the injectable therapeutic angiogenic material to form the crosslinked hydrogel in situ within the stroke cavity of a mammalian subject. The injectable therapeutic angiogenic material includes, in some embodiments, a cell adhesion peptide.

[0011] This mixture containing both clustered VEGF heparin nanoparticles as well as naked heparin nanoparticles induces the formation of a robust vascular bed within the stroke cavity and enhances the proliferation and migration of neurons. Importantly, the addition of VEGF in the injectable therapeutic angiogenic material described herein does not suffer from the known adverse side effects such as hemorrhage, edema, intracranial brain pressure, an increased post-stroke inflammation and an increase infarct volume, as well as the formation of leaky and unorganized vessels.

[0012] In one embodiment, the VEGF concentration per weight of heparin for the clustered VEGF heparin nanoparticles is within the range of 1 mg VEGF/mg heparin to 20 mg VEGF/mg heparin. In another aspect of the invention, the VEGF concentration per weight of heparin for the clustered VEGF heparin nanoparticles is at least 10 mg VEGF per mg of heparin. In another aspect of the invention, the VEGF concentration per weight of heparin for the clustered VEGF heparin nanoparticles is within the range of 1 mg to 20 mg.

[0013] In another aspect of the invention, during clinical use, the patient or subject (e.g., human or other mammalian subject) will typically be first given a scan such as a magnetic resonance imaging (MRI) scan to localize the location and volume of the stroke site. The first three days (e.g., at about five days) after stroke are associated with a massive inflammatory response where cellular debris resulting from cell death in the damaged site are cleared by specialized inflammatory cells (microphages/microglia) leaving behind an empty cavity. The specific localization of both the infarct (stroke cavity) and the peri-infarct areas are determined with three-dimensional intra-cerebral coordinates (x, y and z). To access the stroke cavity, a hole or access passageway is drilled in the subject's skull (e.g., craniotomy) adjacent to the site of the stroke. Most strokes occur in the cerebral cortex or outer layer of brain tissue which can be then be readily accessed after the formation of the craniotomy. A delivery device, which may be a syringe or the like that contains the injectable therapeutic material described herein, is then inserted into the craniotomy and the therapeutic material is then delivered to the stroke cavity. The injectable therapeutic angiogenic material will then gel within the stroke cavity and provides the therapeutic benefits. Notably, the injectable therapeutic angiogenic material may provide therapeutic benefits even though administered days after the stroke onset.

Brief Description of the Drawings

[0014] FIG. 1 illustrates a cross-sectional view of a mammalian brain showing a stroke cavity. An injectable therapeutic angiogenic material is being delivered to the stroke cavity via a delivery device. [0015] FIG. 2 illustrates a schematic representation of a stroke cavity that contains a crosslinked hydrogel that forms the therapeutic angiogenic material according to one embodiment.

[0016] FIG. 3 illustrates a sequence of operations or flowchart that outlines a method of making the injectable therapeutic angiogenic material that is delivered to the stroke cavity.

[0017] FIG. 4 illustrates schematically a stroked mouse brain that has been treated with an injectable therapeutic angiogenic material that includes highly clustered VEGF heparin nanoparticles (hcV).

[0018] FIG. 5A schematically illustrates the HA-AC polymer, MMP degradable peptide, and cell adhesion peptide that was used to make the injectable therapeutic angiogenic material according to one embodiment.

[0019] FIG. 5B schematically illustrates three different cluster configurations of clustered VEGF heparin nanoparticles. Different clustering densities of VEGF were created by mixing the same amount of VEGF with different amounts of heparin nanoparticles, leading to a low (lcV), medium (mcV) and high cluster density (hcV) of the growth factor onto the heparin nanoparticle's surface. Note that medium (mcV) cluster densities were only used for in vitro studies.

[0020] FIG. 5C schematically illustrates the therapeutic angiogenic hydrogel in the crosslinked or gelled state.

[0021] FIG. 6 illustrates the various experimentally tested gel conditions in the murine model. This includes the empty gel (containing only HA hydrogel), gel + soluble VEGF (Vs); gel + heparin nanoparticles (nH); gel + low cluster density VEGF heparin nanoparticles (lcV); gel + high cluster density VEGF heparin nanoparticles (hcV) and naked nanoparticles; and gel + high cluster density VEGF heparin nanoparticles (hcV) without naked

nanoparticles.

[0022] FIG. 7 A is a graph showing the loading of VEGF ^g VEGF / mg heparin) for the various clustering configurations (high (h), medium (m), low (1)) as tested by Dot Plot and ELISA.

[0023] FIG. 7B is a graph of the relative fluorescence as a function of VEGF (ng/mL) for the different clustering configurations.

[0024] FIG. 7C illustrates VEGF activity after binding to heparin particles, by quantifying VEGFR-2 phosphorylation in presence of increasing concentration of soluble unbound VEGF and heparin nanoparticle-bound VEGF. The Western blot assay shows the presence of a band for each corresponding protein, each band thickness being correlated to the amount of protein measured. The results show that VEGFR-2 phosphorylation is proportional to the total amount of VEGF added to endothelial cell culture.

[0025] FIG. 7D illustrates the effect of high, medium and low clusters of VEGF on the activation of VEGF-R2 intracellular signaling cascade, with the quantification of

phosphorylated sites Y1214 and Yl 175 of the receptor, as well as the proteins p38 and p42/44. The Western blot assay shows the presence of a band for each corresponding protein, each band thickness being correlated to the amount of protein measured.

[0026] FIG. 7E illustrates the quantification of the Western blot shown in FIG. 7D, each plot corresponding to the different levels of phosphorylated proteins (Y1214, Yl 175, p38 and p42/44) in the different conditions (high, medium and low clusters, soluble VEGF, buffer and nanoparticles). Each column represents the quantitative value corresponding to each band obtained in Western blot.

[0027] FIG. 8A illustrates a graph of measured stroke volume (mm³) at day ten after gel injection. No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with ^g heparin nanoparticles (nH), gel + lcV = HA hydrogel with 1 μg nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test with * and *** indicating P < 0.05 and P < 0.001, respectively.

[0028] FIG. 8B illustrates a graph of cortex volume ratio (cortex volume) for the tested gel conditions referred to in FIG. 8A.

[0029] FIG. 8C illustrates a graph of Ipsi/Contra volume ratio (hemisphere volume) for the tested gel conditions referred to in FIG. 8A.

[0030] FIG. 9 A illustrates fluorescent images of microglial cells (Iba-1, upper row) and astrocytes (GFAP, lower row) at the stroke site (*) at day 10 after gel transplantation. Scale bar: 100 μιτι.

[0031] FIG. 9B illustrates a graph of infarct positive area for Iba-1 for the different test gel conditions. No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with ^g heparin nanoparticles (nH), gel + lcV = HA hydrogel with ^g nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with * and ** indicating P < 0.05 and P < 0.01, respectively.

[0032] FIG. 9C illustrates a graph of peri-infarct positive area for Iba-1 for the different test gel conditions.

[0033] FIG. 9D illustrates a graph of GFAP -labeled astrocytic scar thickness (μιη) for the different test gel conditions.

[0034] FIG. 9E illustrates images of brain sections showing Evans blue extravasation in the stroke site after intravenous injection of the dye. The arrow in one panel indicates the presence of Evans blue showing evidence of leakage.

[0035] FIG. 9F illustrates a graph showing blood brain barrier leakage quantified by spectrophotometry of the amount of Evans blue measured per grams of brain tissue (Abs/g tissue).

[0036] FIG. 10 is a graph showing the tail vein bleeding time (seconds) for mice injected intravenously with saline (PBS), 2μg of heparin or heparin nanoparticles (nH). Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **** indicating P < 0.0001.

[0037] FIG. 11A illustrates fluorescent images of vessels (Glut-1) with Dapi in and around the stroke site (*) at day 10 after gel transplantation. Scale bar: 100 μιτι.

[0038] FIG. 1 IB illustrates fluorescent images a marker of proliferation (BrdU) in and around the stroke site (*) at day 10 after gel transplantation. Scale bar: 100 μιτι.

[0039] FIG. l lC illustrates fluorescent images of pericyte/smooth muscle cells (PDGFR- β) in and around the stroke site (*) at day 10 after gel transplantation. Scale bar: 100 μιτι.

[0040] FIG. 1 ID illustrates a graph showing the quantification of the vascular density (% Glut-1 area) in the infarct and peri-infarct areas. No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with ^g heparin nanoparticles (nH), gel + lcV = HA hydrogel with ^g nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with *, ** and **** indicating p < 0.05, p < 0.01 and p < 0.0001, respectively.

[0041] FIG. 1 IE illustrates a graph showing the quantification of angiogenesis (Glut- 1/BrdU cells) in the infarct and peri-infarct areas. [0042] FIG. 1 IF illustrates a graph showing the pericyte vascular coverage (% PDGFR-β area) in the infarct and peri-infarct areas.

[0043] FIG. 12A illustrates fluorescent images of vessels (Glut-1) in and around the stroke site (*) 16 weeks after gel transplantation for the empty gel, Gel + Vs, Gel + lcV, and Gel + hcV. Scale bar: 100 μηι.

[0044] FIG. 12B illustrates a graph showing the quantification of the vascular density (% Glut-1 area) in and around the stroke site (*) in the infarct and peri-infarct areas. In all plots (for FIGS. 12B-12D), the dotted line and number indicates the value for the given quantification of the contralateral side. Empty gel = gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = ^g nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with *, ** and **** indicating p < 0.05, p < 0.01 and p < 0.0001, respectively.

[0045] FIG. 12C illustrates graphs showing the quantification of the vessel morphology: vessel tortuosity = total vessel length/shortest distance and Number of ramifications = the number of branches/vessel.

[0046] FIG. 12D illustrates graphs showing the vessel diameter and maximum infiltration distance of the vessels into the stroke site.

[0047] FIG. 13A illustrates fluorescent images of neuroblasts (Dcx) and the proliferation marker BrdU in and around the stroke site (*) at 2 weeks after gel transplantation. Scale bar: 100 μιη.

[0048] FIG. 13B illustrates fluorescent images of axonal neurofilaments (NF200) in and around the stroke site (*) at 2 weeks after gel transplantation. Scale bar: 100 μιτι.

[0049] FIG. 13C illustrates graphs showing the quantification of neuroblasts (Dcx) and proliferating neuroblasts (Dcx/BrdU) in the ipsilateral ventricle at 2 weeks after stroke (10 days after gel transplantation).

[0050] FIG. 13D illustrates graphs showing the number of neuroblasts migrating and their migration distance and number at 2 weeks after stroke (10 days after gel transplantation).

[0051] FIG. 13E illustrates graphs showing the axonal area (NF200) in and around the stroke site (infarct and peri-infarct) at 2 weeks after stroke (10 days after gel transplantation).

[0052] FIG. 13F illustrates graphs showing infiltration distance and penetration angle in the stroke site at 2 weeks after stroke (10 days after gel transplantation). [0053] FIG. 13G illustrates fluorescent images of axonal neurofilaments (NF200) in and around the stroke site (*) at 16 weeks after gel transplantation. Scale bar: 100 μιη.

[0054] FIG. 13H illustrates a graph showing the quantification of axonal area (NF200) in the infarct site 16 weeks after gel injection.

[0055] FIG. 131 illustrates a graph showing the quantification of axonal area (NF200) in the peri -infarct area 16 weeks after gel injection.

[0056] FIG. 13J illustrates a graph of the infiltration distance 16 weeks after gel injection.

[0057] FIG. 13K illustrates a graph of axonal penetration angle 16 weeks after gel injection. In all of FIGS. 13C-13F and 13H-13K), the dotted line and number indicates the value for the give quantification of the contralateral side. Empty gel = gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = ^g nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH, Endo = a daily i.p (intraperitoneal) injection of endostatin day 5 to 15. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with *, ** and **** indicating p < 0.05, p < 0.01 and p < 0.0001, respectively.

[0058] FIG. 14A illustrates fluorescent images of vessels (Glut-1), Angiopoietin-2 and Dapi in the peri-infarct area in the different conditions 2 weeks after stroke. Scale bar: 50 μιτι.

[0059] FIG. 14B illustrates a graph showing the quantification of the positive area for Angiopoetin-2 in the peri-infarct area.

[0060] FIG. 14C illustrates a graph showing the quantification of the positive area around vessels (10 μιτι distance from vessels). For FIGS. 14A-14C, No gel = stroke only condition, empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + nH = HA hydrogel with 1 μg unloaded nH, gel + lcV = HA hydrogel with ^g nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH, Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with * and ** indicating P < 0.05 and P < 0.01, respectively.

[0061] FIG. 15A illustrates a graph showing the quantification of the stroke volume (mm³) 16 weeks after gel transplantation.

[0062] FIG. 15B illustrates fluorescent images of astrocytes (GFAP) and microglial cells (Ibal) (and merged images) in and around the stroke site (*) in the different conditions. Scale bar: 100 μιη. [0063] FIG. 15C illustrates graphs showing the quantification of the infarct and peri- infarct positive area for Iba-1.

[0064] FIG. 15D illustrates graphs showing the quantification of the infarct and peri- infarct positive area for GFAP. The average value obtained for the contralateral side of the same mice was added as a positive control (average shown on the left upper comer and the dotted line). Empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + lcV = HA hydrogel with ^g nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH, gel + hcV + Endo = Gel + hcV + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **** indicating P < 0.0001, respectively. P values between gel + hcV and gel + hcV + endostatin were determined by t test, with $ indicating P < 0.05.

[0065] FIG. 16 illustrates graphs quantifying ipsilateral neuroblast proliferation 16 weeks after stroke. The upper graph shows Dcx/Brdu cell number for the different tested gels while the lower graph shows Dcx cell number for the different tested gels. Empty gel = HA hydrogel, gel + Vs = HA hydrogel loaded with 200ng of soluble VEGF, gel + lcV = HA hydrogel with ^g nH loaded with 200 ng VEGF, gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot, gel + hcV + Endo = gel + hcv + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test.

[0066] FIG. 17 illustrates graphs (migrating Dcx cell number and Dcx migration distance) quantifying neuroblast migration 16 weeks after stroke for the different tested gels.

[0067] FIG. 18A illustrates fluorescent images of vessels (Glut-1) and axonal

neurofilaments (NF200) in and around the stroke site (*) 16 weeks after gel transplantation. Empty gel = gel = HA hydrogel, Vs = 200 ng of soluble VEGF, lcV = ^g nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH, Endo = a daily i.p injection of endostatin days 5 to 15. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with ** and **** indicating p < 0.01 and p < 0.0001, respectively. Data represent the average. $$$ indicates p < 0.001 vs Gel+hcV. Scale bar: 100 μιη (For FIGS. 18D-18F as well). [0068] FIG. 18B illustrates a graph illustrating the quantitative assessment of the proximity between the two networks with the quantification of NF200 positive signal on vessels.

[0069] FIG. 18C illustrates a graph illustrating the NF200 positive area a distance of 50 μιτι from vessels.

[0070] FIG. 18D illustrates fluorescent images of the peri-infarct astrocytic scar (GFAP) and BDA-traced neurons in the ipsilateral hemisphere of gel + hcV injected mice 16 weeks after gel transplantation.

[0071] FIG. 18E illustrates fluorescent images of astrocytes (GFAP) co-stained with vessels (Glut-1) and pericytes/smooth muscle cells (PDGFR-β) in the stroke site of hcV- treated mice, 16 weeks after gel transplantation.

[0072] FIG. 18F illustrates fluorescent images of astrocytes (GFAP) co-stained with DAPI and astrocytes (Aqua-4) in the stroke site of hcV -treated mice, 16 weeks after gel transplantation.

[0073] FIG. 19A illustrates a graph of Glut-1 Area percentage quantifying vascular density in the infarct and peri-infarct areas growth 16 weeks after stroke. Gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH, Gel + hcV + Endo = Gel + hcV + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with ** and **** indicating P < 0.01 and P < 0.0001, respectively.

[0074] FIG. 19B illustrates two graphs (vascular tortuosity and Nb ramification/vessel) quantifying morphology changes of newly formed vessels in the infarct and peri-infarct areas growth 16 weeks after stroke. Gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH, Gel + hcV + Endo = Gel + hcV + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **** indicating P < 0.0001, respectively.

[0075] FIG. 19C illustrates two graphs (vessel diameter and max vessel infiltration distance) quantifying and describing the newly formed vessels in the infarct and peri-infarct areas 16 weeks after stroke. Gel + hcV = HA hydrogel with 0.01 μg nH loaded with VEGF and 0.99 μg unloaded nH, Gel + hcV + Endo = Gel + hcV + i.p administration of endostatin from day 5 to 15 after stroke. Data is presented using a min to max box plot. Each dot in the plots represent one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **** indicating P < 0.0001, respectively.

[0076] FIG. 20A schematically illustrates the experiment timeline for the behavioral tests. Mice were injected 5 days after stroke with one of the following treatments: empty gel, gel + Vs, gel + IcV, and gel + hcV. Mice were subjected to different behavioral tests (illustrated by vertical boxes on the timeline) at week 0, 1, 4, 8, 12 and 16 after stroke.

[0077] FIG. 20B illustrates the results of four different behavioral tests performed on the mice (cylinder, grid, pasta - time/piece, and pasta - adjustment). The cylinder test (panel Bl) was used to measure the dexterity of their contralateral forelimb, the Grid test (panel B2) was looked at the contralateral hindlimb, and the Pasta test (panels B3 and B4) were used for the contralateral forepaw, normally sensitive to post-stroke lateralized impairments. Empty gel = HA hydrogel, Vs = 200 ng of soluble VEGF, IcV = 2μg nH loaded with 200 ng VEGF, hcV = 0.01 μg nH loaded with 200 ng VEGF and 0.99 μg unloaded nH. Data represent the average ± SEM (n = 12 mice) and p values were determined by One-way ANOVA, Tukey's post-hoc test, * indicating P < 0.05.

[0078] FIG. 20C illustrates the results of the same tests (C1-C4) as those in FIG. 20B with a supplemental set of gel+hcV animals that were administered with endostatin, a VEGF- independent angiogenic inhibitor for 10 days after the gel injection. This was done in order to determine the role of gel+hcV-induced vascularization on behavioral recovery.

[0079] FIG. 20D illustrates the results of the same tests (D1-D4) as those in FIG. 20B with a supplemental set of gel+hcV animals received a brain injection of an AAV5 viral construct expressing hM4 DREADD receptors (designer receptors exclusively activated by a designer drug) directly in the stroke area on week 13. This was done in order to determine the role of gel+hcV-induced axonal growth on recovery. Transfected neurons are silenced after i.p administration of the DREADD ligand, clozapine-N-oxide (CNO) on week 16.

[0080] FIG. 21A illustrates fluorescent images of vessels (Glut-1), astrocytic scar (GFAP), microglia (Iba-1) and axonal neurofilaments (NF200) in and around the stroke site (*) of gel + (hcV - nH) and LcV + nH) conditions, 2 weeks post-stroke. Scale bar: 100 μιτι.

[0081] FIG. 21B illustrates graphs showing the percentage Glut-1 Area for the infarct and peri -infarct areas at 2 weeks after gel transplantation. hcV - nH = 0.01 μg nH loaded with 200 ng VEGF, IcV = ^g nH loaded with 200 ng VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **, *** and **** indicating p < 0.01, p < 0.001 and p < 0.0001, respectively. Data represent the average. [0082] FIG. 21C illustrates graphs showing the percentage Iba-1 area for the infarct and peri-infarct areas at 2 weeks after gel transplantation.

[0083] FIG. 21D illustrates two graphs showing the percentage NF200 area for the infarct and peri-infarct areas at 2 weeks after gel transplantation.

[0084] FIG. 21E illustrates two graphs showing the astrocytic scar and axonal infiltration distance at 2 weeks after gel transplantation.

[0085] FIG. 22A illustrates fluorescent images of vessels (Glut-1), astrocytic scar (GFAP), microglia (Iba-1) and axonal neurofilaments (NF200) in and around the stroke site (*) of gel + (hcV - nH) and LcV + nH) conditions, 16 weeks post-stroke. Scale bar: 100 μιη.

[0086] FIG. 22B illustrates graphs showing the percentage Glut-1 Area for the infarct and peri-infarct areas at 16 weeks after gel transplantation. hcV - nH = 0.01 μg nH loaded with 200 ng VEGF, lcV = ^g nH loaded with 200 ng VEGF and 0.99 μg unloaded nH. Data is presented using a min to max box plot. Each dot in the plots represents one animal and p values were determined by One-way ANOVA with a Tukey's post-hoc test, with **, *** and **** indicating p < 0.01, p < 0.001 and p < 0.0001, respectively. Data represent the average.

[0087] FIG. 22C illustrates graphs showing the percentage Iba-1 area for the infarct and peri-infarct areas at 16 weeks after gel transplantation.

[0088] FIG. 22D illustrates graphs showing the percentage NF200 area for the infarct and peri-infarct areas at 16 weeks after gel transplantation.

[0089] FIG. 22E illustrates two graphs showing the astrocytic scar and axonal infiltration distance at 16 weeks after gel transplantation.

[0090] FIG. 23 illustrates graphs (for 3 and 10 days post gel injection) that measures the levels of the cytokines TNF-a for the no stroke, stroke, and gel + nH conditions.

Detailed Description of the Illustrated Embodiments

[0091] FIG. 1 illustrates a cross-sectional view of a mammalian brain 10 that includes stroke cavity 12 formed therein. As explained herein, the delivery site is a stroke cavity 12 such as that illustrated in FIG. 1 that naturally forms after stroke. After initial cell death that follows a stroke, the clearance of debris in the lesion leaves a compartmentalized cavity 12 that can accept a large volume of the injectable therapeutic angiogenic material 20 described herein without further damaging the surrounding healthy parenchyma. This stroke cavity 12 is situated directly adjacent to the peri-infarct tissue area 14, the region of the brain that undergoes the most substantial repair and recovery, meaning that any therapeutic delivered to the cavity 12 will have direct access to the tissue target for repair. In addition to being deliverable to the stroke cavity 12, the injectable therapeutic angiogenic material 20 may also be transplanted in the peri-infarct area 14, or the brain surface 16.

[0092] FIG. 1 further illustrates a delivery device 22 that is used to deliver the therapeutic angiogenic material 20 to the stroke cavity 12. As seen in FIG. 1, the delivery device 22 is in the form of a syringe that includes a needle 24 and barrel 26 that holds the injectable therapeutic angiogenic material 20. A depressor 28 is used to eject the injectable therapeutic angiogenic material 20 from the end of the needle 24 and into the stroke cavity 12. During clinical use, the patient or subject (e.g., human or other mammalian subject) will typically be first given a scan such as a magnetic resonance imaging (MRI) scan to localize the location and volume of the stroke cavity 12. The first three days (e.g., at about five days) after stroke are associated with a massive inflammatory response where cellular debris resulting from cell death in the damaged site are cleared by specialized inflammatory cells

(microphages/microglia) leaving behind an empty cavity. The therapeutic angiogenic material 20 is preferably s injected within fifteen (15) days of stroke onset, after day three (3) post-stroke to avoid the severe post-stroke inflammation and edema in the damaged brain.

[0093] The specific localization of both the infarct (stroke cavity 12) and the peri-infarct areas are determined with three-dimensional intra-cerebral coordinates (x, y and z). While a syringe is illustrated as the delivery device 22 the therapeutic angiogenic material 20 may also be delivered using a catheter-based device or the like to deliver the injectable therapeutic angiogenic material 20 from a location outside the subject's brain to the stroke cavity 12.

[0094] To access the stroke cavity, a hole or access passageway is drilled in the subject's skull (e.g., craniotomy) adjacent to the site of the stroke. Most strokes occur in the cerebral cortex or outer layer of brain tissue which can be then be readily accessed after the formation of the craniotomy. The delivery device 22, which may be a syringe or the like as described above that contains the injectable therapeutic angiogenic material 20, is then inserted into the craniotomy and the therapeutic material 20 is then delivered to the stroke cavity 12. The injectable therapeutic angiogenic material 20 then crosslinks or gels within the stroke cavity 12 and provides the therapeutic benefits. Notably, the injectable therapeutic angiogenic material 20 may provide therapeutic benefits even though administered days after the stroke onset. The delivery device 22 may be manually or automatically controlled to dispense the injectable therapeutic angiogenic material 20 into the stroke cavity 12. For example, the delivery device 22 may be mounted on a robotic arm or the like that can be used to precisely place the tip of the needle 24 within the stroke cavity 12 using surgical robotic techniques known to those skilled in the art. [0095] FIG. 2 illustrates the injectable therapeutic angiogenic material 20 that has gelled in situ within the stroke cavity 12. In one embodiment, the injectable therapeutic angiogenic material 20 is formed from a hyaluronic acid-based hydrogel that forms an amorphous non- fibrous hydrogel composed of hyaluronic acid, which has been shown to promote neural differentiation, to serve as the backbone into which a VEGF delivery system would serve as the initial physical support for infiltrating cells, angiogenesis and axonogenesis. As explained herein, the hyaluronic acid is functionalized with acrylamide functionality (HA- AC) because its kinetics are slower than those of acrylates or vinyl sulfones, which allowed for enough time for injection and ensure that the entire stroke cavity 12 was full of gel before complete crosslinking. The injectable therapeutic angiogenic material 20 precursor remains liquid for a period after mixing, such that it can be injected into the brain 10 through a minimally invasive needle 24; and will gel within the stroke cavity 12, conforming to the boundaries of this damaged brain tissue. The mechanical properties of this injectable therapeutic angiogenic material 20 are similar to those of normal brain.

[0096] As seen in FIG. 2, the injectable therapeutic angiogenic material 20 crosslinks or gels via the crosslinker 30. In one preferred embodiment, the crosslinker 30 is a

biodegradable crosslinker 30. For example, the crosslinker 30 may include a matrix metalloproteinase (MMP) labile or degradable peptide. An example, of such an MMP labile peptide includes (Ac-GCREGPQGIWGQERCG-NH2, MMP-degradable [SEQ ID NO: 1]. In stroke, local production of hyaluronidases and matrix metalloproteases modify the tissue environment and can be coopted to alter the duration of effect of an injectable material. Thus, in the injectable therapeutic angiogenic material 20 that uses a degradable peptide, the resulting hydrogel is both hyaluronidase degradable and MMP degradable, and is designed with a stiffness corresponding to the brain to reduce the local inflammatory response. In other embodiments, the crosslinker 30 may be non-degradable. An example of a non- degradable crosslinker includes Ac-GCREGDQGIAGFERCG-NH2 [SEQ ID NO: 2].

[0097] FIG. 2 also illustrates the injectable therapeutic angiogenic material 20 that includes a first plurality of clustered VEGF heparin nanoparticles 32 in which VEGF is immobilized (via a covalent bond) to the heparin nanoparticles and a second plurality of naked heparin nanoparticles 34 that do not contain VEGF immobilized to the heparin nanoparticles. The term "nanoparticles" refers to small nanometer-sized particles of heparin and in particular heparin particles that have a diameter or width within the range of about 200 nm to less than 1 μιη. The nanoparticles of heparin are formed using an inverse emulsion polymerization process that generates spherically-shaped nanoparticles of heparin. The diameter of the unbound or naked nanoparticles 34 is generally within the range of about 90 nm to about 200 nm when contained in water or aqueous-based fluid. The size or diameters of clustered VEGF heparin nanoparticles 32 are larger than the unbound or naked nanoparticles 34 and are typically several hundred nanometers in diameter, e.g., within a range of about 500 nm to about 800 nm.

[0098] The injectable therapeutic angiogenic material 20 is populated with both clustered VEGF heparin nanoparticles 32 and naked heparin nanoparticles 34. In one preferred embodiment, the injectable therapeutic angiogenic material 20 contains more naked heparin nanoparticles 34 than clustered VEGF heparin nanoparticles 32. In particular, it is preferable that there are significantly more naked heparin nanoparticles 34 than clustered VEGF heparin nanoparticles 32 (i.e., number of naked heparin nanoparticles 34 » clustered VEGF heparin nanoparticles 32). Because there are more naked heparin nanoparticles 34 than clustered VEGF heparin nanoparticles 32, the weight percentage of all heparin nanoparticles contained in the injectable therapeutic angiogenic material 20 is dominated by the naked heparin nanoparticles 34. For example, in one preferred embodiment, the weight percentage of the second plurality of naked heparin nanoparticles 34 (as compared to the total weight of all heparin nanoparticles) in the hyaluronic acid-based hydrogel is about 99%. Thus, in one preferred embodiment, the beneficial results are achieved with about 1% clustered VEGF heparin nanoparticles 32 and the remaining 99% of heparin nanoparticles being naked heparin nanoparticles 34.

[0099] As explained herein, the heparin nanoparticles used in the first plurality of clustered VEGF heparin nanoparticles 32 and the second plurality of naked heparin nanoparticles 34 were generated through inverse emulsion polymerization of methacrylated heparin. This resulted in heparin nanoparticles having diameters within the range between about 90 nm and about 200 nm (when in a naked or non-immobilized state and contained in water). The heparin nanoparticles are designed such that they retained their ability to bind growth factors, including VEGF, but not the native heparin ability to reduce blood coagulation (see FIG. 10), such that heparin the nanoparticles could sequester and retain endogenously expressed heparin binding signals after stroke. For the first plurality of clustered VEGF heparin nanoparticles 32, VEGF molecules are covalently crosslinked to the heparin nanoparticle using a photosensitive crosslinker such as p-azidobenzyl hydrazide (ABH).

[00100] The packing density of VEGF may be controlled by the concentration of heparin nanoparticles and VEGF. Generally, a high packing density may be accomplished by using a higher concentration of VEGF with a lower number of heparin nanoparticles. As described herein, the clustered VEGF heparin nanoparticles 32 should have a high density of VEGF formed thereon (described in experiments herein as high cluster VEGF or hcV). In one embodiment, the VEGF concentration per weight of heparin for the first plurality of clustered VEGF heparin nanoparticles 32 is at least 10 mg VEGF/mg heparin. In another embodiment, the VEGF concentration per weight of heparin for the first plurality of clustered VEGF heparin nanoparticles 32 is within the range of 17 mg to 20 mg VEGF/mg heparin.

[00101] It was found that through engineering a VEGF -containing therapeutic angiogenic material 20 and injecting it directly within the stroke cavity 12, this induced the formation of a novel vascular and neuronal structure that leads to behavioral improvement. The formation of a robust, mature and highly developed vascular bed within the stroke cavity 12 helps develop and pattern the nervous system with axons associating closely with vessels. The formation of vascular bed also enhances the proliferation and migration of immature neurons. The axonal projections into this network are causally associated with recovered motor function. Thus, the local delivery of the engineered VEGF containing hydrogel created a neuronal structure through the sequential growth of vascular, neuronal and axonal elements. The axonal and vascular in-growth and formation of this new neural structure within the stroke cavity 12 produces a functionally active and essential new brain tissue that leads to the recovery of motor control.

[00102] The injectable therapeutic angiogenic material 20 may also include cell adhesion peptides. For example, the hyaluronic acid-based hydrogel may be functionalized with a cell adhesion peptide. As example of an adhesion peptide includes fibronectin-derived RGD adhesion peptide Ac-GCGYGRGDSPG-NH2 [SEQ ID NO: 3] (RGD, Genscript, Piscataway, NJ). As explained herein, during formation of the injectable therapeutic angiogenic material 20 the RGD peptide may be clustered in the hyaluronic acid-based hydrogel. This may be accomplished by crosslinking of a smaller sub-volume (e.g., around 15%) of the hyaluronic acid precursor (HA- AC) material followed by the addition of RGD-free hyaluronic acid precursor material (e.g., around 85%).

[00103] FIG. 3 illustrates a flowchart of operations used to generate the injectable therapeutic angiogenic material 20. As seen in FIG. 3 in operation 200, the precursor solution of HA-AC is formed. For example, this precursor solution may be made by dissolving lyophilized HA-AC in 0.3 M HEPES buffer for 15 minutes at 37°C. Next, as seen in operation 210, the cell adhesion peptide (e.g., RGD peptide) is added to this solution. As described above, it is preferably to create clusters of RGD peptide within the precursor solution of HA-AC. This may be accomplished by adding the RGD peptide to a smaller sub- volume (e.g., around 15%) of the hyaluronic acid precursor (HA-AC) material to obtain a degree of clustering of around 1.17, and reacted at room temperature for around 10-20 minutes to allow for crosslinking (waiting operation 220). This may be followed by the addition of the remaining RGD-free hyaluronic acid precursor material (e.g., around 85%). In operation 230, the mixture containing the first plurality of clustered VEGF heparin nanoparticles 32 and the second plurality of naked heparin nanoparticles 34 are added to this solution. As seen in operation 240, prior to delivery to the stroke cavity 12 the crosslinker 30 is added. This may include a small aliquot of the MMP-degradable (or non-degradable) crosslinker 30 dissolved in an appropriate buffer solution (e.g., 0.3 M HEPES). Next, in operation 250, the mixture is well mixed and loaded into the delivery device 22. As seen in operation 260, the injectable therapeutic angiogenic material 20 is then delivered to the stroke cavity 12 with the delivery device 22.

[00104] In some embodiments, the injectable therapeutic angiogenic material 20 may be provided as part of a kit. For example, the kit may include a hyaluronic acid-based hydrogel precursor solution containing a first plurality of clustered VEGF heparin nanoparticles 32 in which VEGF is immobilized to the heparin nanoparticles and a second plurality of naked heparin nanoparticles 34 that do not contain VEGF immobilized to the heparin nanoparticles. The kit may also contain a biodegradable crosslinker 30 for crosslinking the hyaluronic acid- based hydrogel precursor solution into a crosslinked hydrogel. The crosslinker 30 may be provided in a separate vial or container which can be added just prior to delivery. The kit may also include a cell adhesive peptide such as RGD. The adhesive peptide may also be provided in a separate vial or container that is added to the hyaluronic acid-based hydrogel precursor solution as explained herein. The kit may also include in some embodiments, the delivery device 22. Alternatively, the operating room may use an existing delivery device 22 which is loaded with solutions as part of the kit.

[00105] Experimental

[00106] An in situ gelling injectable therapeutic angiogenic material was selected as the platform for these studies. The injectable therapeutic angiogenic material is a hyaluronic acid hydrogel based on thiol-acrylamide Michael-type addition as described herein with a MMP labile peptide used as the crosslinker which resulted in a hydrogel that is both hyaluronidase degradable and MMP degradable, designed with a stiffness corresponding to the brain to reduce the local inflammatory response. FIG. 4 illustrates one preferred embodiment of injectable therapeutic angiogenic material that was used in mouse brain studies. The injectable therapeutic angiogenic material illustrated in FIG. 4 is a hyaluronic acid hydrogel that includes clustered VEGF heparin nanoparticles in a "high cluster" or (hcV) configuration and naked heparin nanoparticles. This particular version of the injectable therapeutic angiogenic material resulted in increased angiogenesis, axonogenesis, and behavioral recovery. Likewise, this version of the injectable therapeutic angiogenic material resulted in and decreased physical barrier properties and inflammation.

[00107] FIG. 5A schematically illustrates the HA-AC polymer, MMP degradable peptide, and cell adhesion peptide that was used to make the injectable therapeutic angiogenic material. FIG. 5B illustrates three different cluster configurations of clustered VEGF heparin nanoparticles. Different clustering densities of VEGF were created by mixing the same amount of VEGF with different amounts of heparin nanoparticles, leading to a low (IcV), medium (mcV) and high cluster density (hcV) of the growth factor onto the heparin nanoparticle's surface. The low (IcV) and high (hcV) VEGF clustering densities were tested in vivo as explained herein. FIG. 5C schematically illustrates the therapeutic angiogenic hydrogel in the gelled state.

[00108] Various experimental groups were evaluated to test the efficacy of the hydrogel material. As seen in FIG. 6, this included the empty hydrogel containing the raw hydrogel without any VEGF or heparin nanoparticles; the hydrogel containing soluble VEGF (Vs) without any heparin nanoparticles; the hydrogel plus heparin nanoparticles (nH) without any VEGF; the hydrogel plus heparin nanoparticles with a clustering of VEGF in a low cluster density (IcV); the hydrogel plus heparin nanoparticles with a clustering of VEGF in a high cluster density (hcV) which also includes naked heparin nanoparticles; the hydrogel plus heparin nanoparticles with a clustering of VEGF in a high cluster density (IcV) with the naked heparin nanoparticles removed (hcV - nH). Not illustrated in FIG. 6 is the configuration of the hydrogel plus heparin nanoparticles with a clustering of VEGF in a medium cluster density (mcV).

[00109] The composition of each injected treatment condition was as follows:

• No gel = stroke only condition

• Empty gel = 6 HA hydrogel

• Gel + Vs = 6 HA HA hydrogel + 200ng Vs

• Gel + nH = 6 HA HA hydrogel + 1 μg nH

• Gel + IcV = 6 HA μΐ, HA hydrogel + 1 μg nH bound to 200 ng VEGF

• Gel + hcV = 6 HA μί HA hydrogel + 0.01 μg nH bound to 200 ng VEGF + 0.99 μg naked nH. [00110] It is important to note that the injected high and low cluster treatments

(respectively hcV and lcV) were designed to contain equal amounts of heparin and VEGF. Therefore, the VEGF distribution in the hcV treatment is reduced to a low amount of heparin in the hcV, leaving a high amount of naked nanoparticles. As explained herein, it was experimentally shown that the injection of heparin nanoparticles only (nH) reduced inflammation in the stroke brain at both short and long term. In order to understand the contribution of these naked nanoparticles in the pro-repair effect of the hcV, an additional hcV group where the naked nanoparticles were removed (hcV-nH) was tested. The results showed that the hcV- nH treatment did improve vessel formation in the stroke site; however, this angiogenesis was not followed by the formation of axons, and was not associated with a reduced glial inflammation.

[00111] Characterization of the heparin nanoparticles was undertaken using transmission electron microscopy (TEM) and dynamic light scattering (DLS) (95.9 nm, and a PDI of 0.268). The size of heparin nanoparticles was measured after each step of the formation (surfactants, hexane then water) showing an increase in diameter after attachment with VEGF in the 3 cluster conditions (lcV, mcV, hcV). Table 1 below illustrates size details of the various 3 cluster conditions as well as naked heparin nanoparticles.

Table 1

[00112] Two methods were used to determine the VEGF content in the heparin

nanoparticles, a direct method that measures the amount of VEGF on the nanoparticle surface using dot blots and an indirect method that measures the amount of VEGF not bound to nH during synthesis using ELISA. The two methods showed the same VEGF concentration per weight of heparin for each of the clustering densities: 10 mg, 1 mg and 100 μg VEGF/mg heparin for high, medium and low cluster, respectively as seen in FIG. 7A. To ensure that the process of VEGF clustering did not impact the activity of VEGF, the ability of VEGF clusters to enhance endothelial cell (EC) proliferation (results seen in FIG. 7B) and to induce VEGF- receptor-2 (VEGFR-2) phosphorylation on the Yl 175 and Yl 114 sites and the MAP-Kinase protein p38 and p-42/44 were tested (results in FIGS. 7C-7E). For the data illustrated in FIG. 7B, endothelial cells were cultured in 2D and were exposed to bound VEGF in high, medium and low cluster presentation, in order to observe their behavioral response to VEGF. As expected (normal behavior with soluble VEGF), the cells proliferated as indicated by the measured fluorescence (the fluorescence measured is proportional to the total number of cells present in culture). Data represent the average ± SEM (n = 6) and P values were determined by One-way Anova, Tukey's post-hoc test, with *** indicating P < 0.001.

[00113] To test the effects of these hydrogels on tissue repair in stroke, a mouse model with distal and permanent occlusion of the middle cerebral artery (MCAo) was utilized. Injection into the stoke cavity and in situ gelation 5 days after stroke did not significantly affect the cortex volume, size or shape compared to the contralateral side at 2-weeks post-stroke as seen in FIGS. 8A (stroke volume), 8B(cortex volume), 8C(hemisphere volume), indicating that the injection volume (6μί) and crosslinked hydrogel could be accommodated by the stroke cavity. The same experiment was performed at 16 weeks and showed that only the gel+nH group was associated with an increased infarct volume (FIG. 15 A).

[00114] FIG. 9A illustrates fluorescent images of microglial cells (Iba-1, upper row) and astrocytes (GFAP, lower row) at the stroke site (*) at day 10 after hydrogel transplantation. The dashed line represents the boundary of the stroke cavity. FIG. 9B illustrates a graph of the quantification of the infarct positive area for Iba-1 for the different tested hydrogels. FIG. 9C illustrates a graph of the quantification of the peri-infarct positive area for Iba-1 for the different tested hydrogels. FIG. 9D illustrates the measured GFAP-labeled astrocytic scar thickness. FIG. 9E illustrates images of brain sections showing Evans blue extravasation in the stroke site (white arrow) after intravenous injection of the dye. FIG. 9F illustrates a graph showing the quantification of blood brain barrier leakage by spectrophotometry of the amount of Evans blue measured per g of brain tissue. Injection of the gel + hcV resulted in statistically decreased astrocyte activation as seen in FIG. 9D compared to a sham condition. However, this decrease in astrocyte scar thickness was not accompanied with an increase in brain repair as measured by angiogenesis, axonogenesis, and behavioral improvement, indicating that additional factors might be required. The same experiment was performed at 16 weeks, with a staining of microglia and astrocytes (images illustrated in FIG. 15B) and showed that gel + hcV group was associated with a reduction of microglial density and astrocytic scar (FIG. 15C and 15D respectively) in the peri-infarct area compared with the other groups injected with VEGF (gel + Vs and gel + lcV). [00115] Heparin nanoparticles alter the inflammatory and glial environment after stroke

[00116] Heparin is an extracellular matrix component that naturally binds growth factors, cytokines and other molecules and in turn regulates their bioavailability and signaling.

Heparin nanoparticles were designed so that they retained their ability to bind growth factors, including VEGF, but did not retain the native heparin ability to reduce blood coagulation as seen in FIG. 10, such that the nanoparticles could sequester and retain endogenously expressed heparin binding signals after stroke. The dominant post-stroke inflammatory response of the brain is defined by the activation and recruitment to the damaged site of macrophages and microglia. The initial extent of stroke damage correlates with intensity of microglia/macrophage activation in peri-infarct tissues. HA gel + nH injection into the stroke cavity in distal MCAo significantly decreases microglial/macrophage levels (measured by IBA-1) as compared to empty gel or sham controls (FIG. 9C).

[00117] The decrease in markers of inflammation was accompanied by a significantly increased vascularization within the stroke cavity and the peri-infarct area as seen in FIGS. 1 lA-1 IF, 12A, 12B, 12C, 12D and an increase in immature neuron (DCX positive) cell number (FIG. 13C). Recent evidence indicates that astrocyte activation in stroke and CNS lesions occurs in direct response to initial damage and restricts this damage, providing a barometer of the evolving ischemic injury. As seen in FIG. 9D, the presence of nH significantly reduces the region of reactive astrocytes around the stroke cavity. These gels do not modify the size of the stroke lesion itself (FIG. 8C), suggesting that nH gel delivery to the stroke cavity reduces measures of long term damage in the tissue bordering the infarct.

However, there was no difference of in-growth of axons within or around the stroke cavity in this condition.

[00118] VEGF clusters differentially activate endothelial cells

[00119] The HA- AC based hydrogel was further engineered to promote angiogenesis. VEGF, bound to the extracellular matrix, signals through the clustering of VEGF receptors leading to sustained VEGF receptor-2 activation and altered downstream signaling compared to non-matrix bound VEGF. Increasing ligand avidity through ligand clustering can enhance cellular activation, producing a more controlled tissue morphogenesis than with soluble factors. Clustered VEGF nanoparticles were generated as a means to control VEGF -induced angiogenesis through the immobilization of VEGF to nH. Different clustering densities of VEGF were synthesized: low (lcV), medium (mcV) and high cluster (hcV). All VEGF loaded nH nanoparticles promote the same level of endothelial cell proliferation as seen in FIG. 7B; however, VEGF receptor-2 (VEGFR-2) activation was significantly different between the different VEGF loading densities. Yl 175 and Y1214 in VEGFR-2 are major phosphorylation sites and we have previously shown differential activation upon presentation with bound or soluble VEGF. These sites are also associated with the activation of the downstream signaling MAP kinases Erkl/2 (p42/44) and p38. Phosphorylation at Yl 175 and Y1214 by clustered VEGF displayed statistically higher phosphorylation for hcV than mcV or lcV (FIGS. 7C, 7D, 7E, p < 0.05) in response to a 5-min exposure to nH bound VEGF. There was no statistically significant difference between hcV and soluble VEGF (Vs) phosphorylation at Yl 175 or Y1214. Interestingly, although no differences in downstream signal activation were observed for the different clustered VEGF presentations, there were differences when compared to Vs-induced activation: soluble VEGF preferentially activated p42/44, while clustered VEGF preferentially activated p38. The differences in downstream signal are reminiscent of what has been observed for collagen bound and heparin bound VEGF versus soluble VEGF, indicating that VEGF bound to nH respond similarly to VEGF bound to bulk matrices.

[00120] hcV results in long-term revascularization of the stroke cavity

[00121] The brain tissue response to the angiogenic biomaterial (HA gel + hcV) was assessed after injection into the stroke cavity in distal MCAo 5-days post stroke.

Angiogenesis was measured at 2- and 16-weeks post stroke, representing a vessel maturation period and chronic period in tissue and behavioral recovery in stroke , respectively. A 5 -day time point for hydrogel injection was chosen in order to activate the endogenous post-stroke angiogenesis known to peak at day 7 after the stroke onset, by increasing the local presentation of VEGF in the damaged brain at a time of high VEGF production. Further, this sub-acute time point after stroke is when most stroke patients are in rehabilitation and would be available for such a therapy. All injections contained 6μ1 of gel. If heparin was part of the condition, a total of 1 μg heparin was delivered. If VEGF is part of the condition, a total of 200ng VEGF was delivered.

[00122] At 2-weeks after stroke mice injected with HA gel containing the high clustered VEGF condition (HA gel + hcV) significantly increased endothelial cell (Glut-1) and pericyte/smooth muscle cell (PDGFR-β) density in and around the stroke cavity (FIG. 11 A- 1 ID) compared to the control conditions of HA gel containing the low VEGF cluster (lcV), nanoparticles only (nH), soluble VEGF (Vs) or HA gel only. Interestingly, the heparin nanoparticle-only group also outperformed every other group but was significantly lower than HA gel + hcV. In this stimulated vascular network in the infarct and peri-infarct tissue, HA + hcV induced a significant increase of proliferating endothelial cells (FIG. 11C). VEGF delivery to the brain has previously been associated with immature vessels, lacking pericyte coverage. Thus, co-localization of endothelial and pericyte makers were tested in the condition with the most significant increase in endothelial cell proliferation and vascular density. HA gel + hcV stimulated vascular structures with pericyte coverage (FIG. 1 ID). Angiopoietin-2 has a distinct role in post-stroke angiogenesis and in coupling of angiogenesis to other elements of tissue repair. The quantification of Angiopoietin-2 (FIGS. 14A-14C) showed a significant increase in the peri-infarct area compared with the No gel and gel + Vs conditions. Angiopoietin-2 was significantly increased in the close vicinity of vessels (10 μιτι) in the gel + hcV condition compared with any other group, except of gel + nH.

[00123] Therapeutic angiogenic materials often have a short-term angiogenic response that subsides and vessels regress as the angiogenic stimulus (e.g., VEGF) is depleted due to a reducing tissue demand for new vessels at distant time points from the ischemic stimulus and lack of vessel stability. Endothelial cell density was assessed 16-weeks post stroke in the infarct and peri-infarct regions (FIG. 12A). HA gel + hcV resulted in a significantly increased vascular area inside the stroke cavity; no significant difference between groups was observed in the peri-infarct area (FIG. 12B). Inside the infarct, the increased angiogenesis led to a vascular area greater than the contralateral side, indicating a vascular network in the stroke cavity that is more substantial than the original network in this cortical area. In stroke, the normal process of angiogenesis leads to a tortuous and dilated vasculature. The vascular tortuosity, branching points, vessel diameter, and infiltration distance within the infarct cavity of the induced vessels was assessed compared to normal vessels in the contralateral side at the 16-week time point. HA gel + hcV showed a lower degree and variability of vessel tortuosity and was closer to the tortuosity of normal vessels (FIG. 12C). There was no significant difference in the number of vascular ramifications between groups. Finally, the stroke cavity with HA gel + hcV displayed a significantly reduced vascular diameter compared with HA gel + soluble VEGF, with a value close to the contralateral normal control side (7. 28 μιτι) and a significantly greater vascular infiltration distance into the lesion site compared with any other group (FIG. 12D). In stroke, the cavity is characterized by a reduced blood vessel density and the presence of a fibrotic scar. Transplantation of HA gel + hcV hydrogel induces robust angiogenesis into the stroke cavity, with the long-term production of mature vessels with normal vascular morphology.

[00124] Interestingly, while no effect on blood-brain barrier (BBB) permeability was observed at the 2-week time point (FIG. 9F), a significantly increased infarct size was found in the Vs condition (FIG. 8A), consistent with previous reports that soluble VEGF administration in stroke may worsen stroke outcome. This increased stroke volume observed in the Vs condition 2 weeks after stroke is maintained at 16 weeks post-stroke (FIG. 15 A). The stroke volume observed in gel + hcV group is significantly lower than gel + Vs, showing long-term beneficial effect of the hcV transplantation compared to its soluble form. In addition, gel+hcV+endostatin group does not show a reduction of stroke volume at 16 weeks, confirming the necessity to induce functional vessel growth after stroke in order to promote tissue growth and stroke volume reduction.

[00125] Revascularization of the stroke cavity leads to neurogenesis and axonogenesis

[00126] The stroke cavity lacks both vascular and neuronal structures and connections. Experiments were conducted to test the hypothesis that by promoting a dense and well- formed vascular bed inside the stroke cavity, the development of an accompanying neural structure would follow. Neurogenesis after stroke occurs to a limited degree as newly bom neurons (neuroblasts) migrate from their origin in the sub-ventricular zone to areas of damage. Neurogenesis in the present experiments was assessed by staining neuroblasts (Dcx) 10-day s or 16 weeks after gel transplantation in distal MCAo stroke. The number of proliferative neuroblasts (Dcx/BrdU) was significantly increased in the hcV compared with the No gel control group (FIG. 13A and 13C). The total number of Dcx cells in the SVZ was also significantly increased in both the HA gel + hcV and HA gel + nH conditions compared with any other group. Similarly, these two conditions display a significantly increased number of Dcx cells along the corpus callosum, the migratory path towards the lesion, compared with any other group, with no significant difference in the migrating distance (FIG. 13D). These results may be due to the increased angiogenic signal in HA gel + hcV and HA gel + nH conditions (FIG. 1 ID), or due to reductions in tissue barriers at this site. The addition of VEGF did not change the inflammatory response to HA gel + nH, which continued to have significantly reduced the microglial/macrophage positive area and astrocytic scar thickness compared with mice injected with low cluster VEGF (Gel + lcV), soluble VEGF (Gel + Vs), Empty gel and No gel control 2 weeks after stroke (FIGS. 9D), FIG. 15D. This early increase in newly born, immature neurons in the peri-infarct cortex was not sustained: at 16 weeks there were no significant differences between groups in the number of Dcx cells (FIGS. 16 and 17). This data suggests that the presence of naked heparin nanoparticles found in both gel + nH and gel + hcV conditions induces migration of immature neuroblasts into the damaged tissue adjacent to the stroke cavity at initial stages during tissue recovery after stroke. [00127] Next it was determined if angiogenesis could promote the infiltration of axons into the stroke lesion site as it does in the normal body during development. Three separate assessments were performed to assess axonogenesis at 2- and 16-weeks utilizing a marker for axons, neurofilaments (NF200) (images shown in FIG. 13B for the 2 week assessment and FIG. 13G for the 16 week assessment) and quantify its density in the infarct and peri -infarct areas, neurofilament infiltration distance, and neurofilament infiltration angle (a measure of the orientation of the axonal network). At both 2 weeks (FIGS. 13B, 13E, 13F) and 16 weeks after stroke (FIGS. 13H-13K), only the hcV had significantly more neurofilament area compared with the other conditions. These axons had a significantly deeper axonal infiltration distance into the infarct, and a penetrating angle that is similar to the angle measured between the NF200 axons and the cortex in the contralateral side (FIG. 13K) which suggests that the newly formed axonal network in the hcV condition is a structured in a similar linear partem to the normal cortical axonal network. Interestingly at 16 weeks the NF200 positive area in the high cluster VEGF condition is higher than the contralateral value (dotted line) in the homologous cortex that is not affected by stroke (FIGS. 13H, 131) indicating that similar to the newly formed vasculature in the hcV -treated brains, the axonal ingrowth into the stroke cavity establishes a network of connections that is greater than the underlying brain structure of cortex.

[00128] To further study the connection of the vascular network with the newly formed axonal network, a co-staining operation was performed to identify vessels (Glut-1) and neurofilament networks (NF200) at 16-weeks. A very close association between the vessels and neurofilaments was found in the hcV brains (FIG. 18A, 18B). In the hcV, there is a statistically higher percent of neurofilaments that are either in direct contact with (FIG. 18C) or within 50μπι of a vessel (FIG. 18D), further confirming the role of vessels in forming a reparative neurovascular niche that leads to axonogenesis within the stroke cavity.

[00129] To prove that the observed axonogenesis was due to the revascularization of the stroke cavity and not a direct effect of VEGF on sprouting neurons, angiogenesis was blocked through a VEGF independent mechanism using endostatin during days 5 to 15 after stroke and analyzed the neurofilament network at 16 weeks. This approach blocks angiogenesis in this stroke-hydrogel-VEGF approach. A significant reduction of the vascular area in both the infarct and peri-infarct areas was found (FIG. 19A), growth (number of ramication) (FIG. 19B) and infiltration in the wound (FIG. 19C) of endostatin-injected HA + hcV mice at 16 weeks post-stroke. The blockade of angiogenesis in HA gel + hcV mice is associated with a statistical reduction of the axonal network area in and around the stroke (FIGS. 18A, 18B), particularly in the vicinity of vessels (FIG. 18C), indicating that angiogenesis is causally linked to the axonogenesis observed in the hcV condition. These results show that inducing angiogenesis into the infarct can generate an elaborate neuronal architecture within this normally fibrotic cavity. In order to identify the neuronal population that sends axonal projections to the stroke site of gel + hcV -treated mice, BDA (biotinylated dextran amine), a marker for bidirectional axonal tracing in the gel injection coordinates was delivered on Week 16 after gel treatment (FIG. 18D). BDA uptake was visualized fluorescently using a streptavidin-fluorochrome detection. Brain sections were co-stained with the astrocytic marker GFAP in order to visualize the site of lesion. It was found that neurons sending axonal projections in the injected cavity of hcV mice are located in the in the ipsilateral peri-infarct cortex, with a clear detection of retrogradely traced cell bodies up to 800μηι distant from the site of injection.

[00130] Last, experiments were performed to determine whether the newly formed vasculature in the gel + hcV brains present characteristics of functional maturity with both coverage with pericytes/smooth muscle cells (PDGFR-β; FIG. 18E) and astrocytic endfeet (Aquaporin-4, FIG. 18F. Fluorescent staining shows a strong vascular coverage with both pericytes and end-feet astrocytes, associated with GFAP-expressing astrocyte cell bodies in the vicinity of vessels, further indicating that in this condition, normal brain architecture is achieved.

[00131] From a therapeutic angiogenic material to a functional axonal network

[00132] To determine whether the induced vascular and neuronal tissue in the stroke cavity was functional, behavioral recovery was measured after hydrogel transplantation.

Photothrombotic stroke in the forelimb motor cortex causes limb use deficits in mice for at least 16 weeks after the infarct. Mice received this stroke and were transplanted 5 days later with HA gel, HA gel + Vs, HA gel + IcV and HA gel + hcV (FIGS. 20A-20D). Limb control was measured with 3 different behavioral tests every 4 weeks for 16 weeks, in order to assess the dexterity of the contralateral forelimb (Cylinder test/asymmetry score, FIG. 20B panel Bl), hindlimb (Grid test/footfault, FIG. 20B panel B2) and forepaw (Pasta test/paw adjustment and time in sec, FIG. 20B, panels B3, B4) in measures that reflect human motor control patterns after stroke. Stroke causes a deficit in limb motor control in exploratory rearing, gait and dexterous forepaw use that is maximal at 4 and 8 weeks after the infarct with some recovery but still a persistent deficit at 12 and 16 weeks. HA gel + hcV-injected animals displayed a significantly increased use of their contralateral forelimb in exploratory rearing in the cylinder task beginning 12 weeks after stroke (FIG. 20B, panel Bl). HA gel + hcV injected animals showed a significant decrease in the number of contralateral footfaults on the grid walk task at the same point (FIG. 20B, panel B2). The pasta handling test showed the HA gel + hcV condition produced a significantly reduced time to manipulate and eat a piece of pasta on weeks 8 to 16 (FIG. 20B, panel B3), while showing a significantly increased use of contralateral digits (adjustments) in handling the pieces of food (FIG. 20B, panel B4). No other gel or VEGF condition was associated with this degree of enhanced functional recovery. These results indicate a dramatic decrease in limb use deficits for high cluster VEGF condition, suggesting that the axonal network observed in HA + hcV delivery is functionally active.

[00133] To prove that the observed functional recovery was linked to angiogenesis due to the newly formed axonal network in the stroke cavity, two separate tests were performed. First, endostatin was used to block angiogenesis as previously done (FIGS. 19A-19C).

Because blocking angiogenesis significantly reduced neurofilament density and infiltration distance (FIGS. 13G-13J), blocking angiogenesis should also prevent the functional improvement observed if the newly formed axonal network or vascular network are responsible for the observed improvement. Treatment with endostatin impaired the recovery observed in the high cluster VEGF condition, showing a significantly decelerated recovery compared with the HA gel + hcV animals; and this was present in all the behavioral tasks performed (FIGS. 20C, panels C1-C4), demonstrating that VEGF induced angiogenesis is critical in the observed functional recovery. To test if the enhanced functional recovery in the HA + hcV condition was due to the new axonal connections observed in the infarct (FIGS. 20D, panels D1-D4), the neurons that extend these axons into the infarct were retrogradely transfected (FIG. 18A, 18D) by injecting an AAV construct that expresses a neuronal silencing receptor, hM4D DREADD directly into the infarct site. Neurons that form a new connection into the damaged tissue and the HA gel + hcV gel will be inhibited when CNO, the ligand for hM4D, is administered. This technique has been used to silence neuronal activity in a specific area of rodent brain. At week 13, a time period of enhanced motor recovery in HA gel + hcV, neurons that project into the hcV gel were silenced with CNO (See FIG. 20A). Initially, HA gel + hcV treated stroke animals showed enhanced functional recovery, replicating the earlier results of this gel. However, when these animals were treated with CNO, the improved motor functions were lost and the animal deficit was worsened (FIG. 20D, panels D1-D4), demonstrating that the newly formed axonal network is responsible for the observed functional improvement. In total, these studies indicate HA gel + hcV promotes development of a neurovascular brain tissue within a normally fibrotic scar and promotes functional recovery through angiogenesis and stimulated axonal ingrowth into a network of connections from adjacent brain.

[00134] Naked heparin nanoparticles are essential for the pro-repair properties of hcV

[00135] The injected high and low cluster treatments (respectively hcV and IcV) were designed to contain equal amounts of heparin and VEGF. Though it was shown that heparin nanoparticles have a reduced blood thinning ability compared to polymeric heparin (FIG. 10), the introduction of naked heparin nanoparticles to the hcV group complicates clinical translation. Thus, a goal of the experiments was to determine if the delivery of high cluster VEGF alone would perform similarly as high cluster VEGF + heparin nanoparticles. Mice were injected with gels containing only heparin nanoparticles coated with VEGF at high cluster density and the unloaded naked nanoparticles were then removed (hcV-nH). The results showed that similarly to the hcV treatment, the hcV-nH treatment enhanced post- stroke angiogenesis at 2 weeks (FIG. 21A, 21B). This pro-angiogenic effect was maintained in the peri-infarct 16 weeks after stroke (FIGS. 22A, 22B). However, the increase in angiogenesis was accompanied with an increase in inflammation (FIG. 21 A, 21 C), characteristic of VEGF delivery in brain and other organ systems. Both microglia area and glial scar thickness were significantly increased in the hcV-nH condition compared with hcV, showing an inflammation worse than in the empty gel control group (FIG. 21E). Further, no axonal growth was observed in this group (FIG. 21D). The pro-inflammatory effect of the hcV - nH was also observed at 16 weeks, where the microglia area and the scar thickness was maintained significantly higher than in hcV (FIGS. 22C, 22E).

[00136] The results obtained with the hcV-nH conditions show for the first time that axonal formation that follows therapeutic angiogenesis requires immune-modulation of injury- induced inflammation. The absence of post-stroke immune-modulation results in vasculature that is not followed by tissue repair. Similarly, experiments were conducted to test whether the addition of naked heparin nanoparticles to IcV, a VEGF cluster group that did not show pro-repair effect in the brain, would increase its beneficial effects. This experimental condition, IcV + nH, was tested for the same repair characteristics at 2 weeks (FIG. 21A-21E) and 16 weeks (FIGS. 22A-22E). The results show that the IcV + nH treatment did not promote vascular (FIG. 21B) or axonal growth (FIG. 21D), did not reduce the microglial inflammation (FIG. 21C) or the scar thickness (FIG. 21E) 2 weeks after stroke. The same results were observed at 16 weeks post-stroke (FIGS. 22A-22E). The results obtained with IcV + nH show that the combination of VEGF and naked nanoparticles is not enough to promote brain repair, as the therapeutic effect of heparin nanoparticles is only observed with high VEGF clusters.

[00137] To investigate the effect of heparin nanoparticles on inflammatory and antiinflammatory cytokine levels after stroke, a multiplex ELISA was performed. Mice were stroked and injected with gel + nH five days post stroke. Three and ten days after gel transplantation, the infarct core with the injected gel were harvested and quantified for their levels of cytokines (TNF-a, IL-6, IFN-γ, IL-1 a, IL-1 β, IL-2, IL-4 and IL-10). It was found that gel + nH significantly decreased brain concentration of TNF-a 3 days after the gel injection (P =0.0076). At this time point, gel + nH does not show any significant difference with the no stroke condition as seen in FIG. 23. The other cytokines tested showed no significant difference between groups, however. The technical challenges of dissecting the injected gel without the surrounding peri-infarct tissue may have hindered the quality of the harvested samples. In addition, the presence of the hydrogel itself may have increased the background in the injected brains, reducing the sensitivity of cytokines detection. A perfected and customized protocol may be able detect significant differences of the other inflammatory cytokines. Nevertheless, the data is consistent with previous reports.

[00138] The stroke cavity is a fibrotic region devoid of neurons and with a sparse, disordered vasculature. The cavity represents the tissue lost after stroke and is associated with functional disability in stroke patients. The experimental data described herein shows that engineering a VEGF-containing hydrogel biomaterial and injecting it directly within the stroke cavity induces the formation of a novel vascular and neuronal structure that leads to behavioral improvement. The HA gel + hcV induce the formation of a robust, mature and highly developed vascular bed within the stroke cavity and patterned axonal ingrowth along these vessels. This vascular bed is mature as compared to the normal vascular structure in cortex in its morphology and pericyte coverage. This is unexpected in a traditional delivery of VEGF in the brain and indicates that the clustered nanoparticle presentation of VEGF in the hydrogel promotes elements of normal vascular development. This distinctive vascular development in hydrogel-presented nanoparticle-clustered VEGF is supported by the differential VEGFR2 phosphorylation and downstream p38 signaling of clustered VEGF.

[00139] This hydrogel also modifies the adjacent peri-infarct tissue, by reducing microglia/macrophage activation, the size of the reactive astrocyte border and by promoting in-migration of immature neurons from the sub-ventricular zone. HA + hcV hydrogels promote vascular in-growth into the infarct cavity and an accompanying axonal network. These newly formed axons inside the stroke cavity are tightly physically associated with the new vascular network, and selective blockade of vascular in-growth blocks the development of this axonal network inside the cavity. These findings support a process of coordinated vascular and axonal growth in a developing neural tissue inside a normally fibrotic brain scar. In the developing body, and particularly in skin, development of the vascular and neuronal networks are tightly coordinated through shared molecular signaling systems, such as Slits, semaphorins, netrins and VEGF. The co-delivery of nanoparticle-clustered VEGF and heparin nanoparticles was sufficient to induce this ingrowth and co-patterning of vascular and neuronal systems, but is likely to have done so by initiating a process that may be locally sustained by these other signaling systems.

[00140] Materials and Methods

[00141] Heparin nanoparticle synthesis and VEGF binding

[00142] Heparin was first modified with p-azidobenzyl hydrazide (ABH) through 1-ethyl- 3-(3-dimethylaminopropyl)carbodiirnide (EDC) mediated conjugation in a 1 :3 molar ratio of ABH to available carboxylic acids at pH 5.5 in a lOOmM solution of 2-(N-morpholino) ethanesulfonic acid (MES) buffer. The remaining carboxylic acid groups on heparin were then conjugated with N-(3-Aminopropyl) methacrylamide in 27 molar excess through EDC coupling chemistry overnight at room temperature in MES buffer. The solution was then dialyzed against distilled (DI) water and lyophilized for two days. The final product was dissolved in a 100 mg/ml solution of sodium acetate at pH 4, then combined with Tween-80 and Span-80 (8% HLB) and sonicated to form nanoparticles. The radical polymerization was initiated by mixing heparin in a ten-fold volume of hexane combined with Ν,Ν,Ν',Ν'- tetramethyl-ethane-l,2-diamine (TEMED) and ammonium persulfate (APS). The resultant nanoparticles were purified using liquid-liquid extraction in hexane and bubbling nitrogen gas was used to evaporate off the excess of hexane. The nanoparticles were then dialyzed in 100 kD MWCO dialysis units for 12 hours and stored at +4C. The amount of heparin in the solution was determined by lyophilizing a small aliquot of the solution. A total concentration of 20μg/ml VEGF was mixed with different concentrations of heparin nanoparticles ranging from 0.1 to 0.001 mg/mL to form different packing densities of VEGF onto the nanoparticle's surface, incubated overnight and exposed to a 365 nm wavelength UV light for 10 minutes to lock VEGF covalently to the surface. The VEGF nanoparticles were then washed from excess with 0.05% Tween-20 in PBS, then with PBS, using a 100 kD MWCO dialysis units. The washes were collected and an Elisa and Dot blot were performed to estimate the amount of VEGF bound to nanoparticles by subtracting the washes to the total amount of VEGF mixed. [00143] Heparin nanoparticle characterization

[00144] Dynamic Light Scattering (DLS) was used to characterize the Z-average (diameter) and polydispersity index (PDI) of heparin nanoparticles after each preparation step. Samples were loaded into a filtered DI water quartz cuvette and analyzed by a Malvern Zetasizer where ten runs of three measurements each were performed. Transmission Electron

Microscopy (TEM) with a T12 Quick CryoEM was performed in order to confirm the morphology and size distribution of nanoparticles: a drop of sample solution (1 mg/mL) was placed onto a 300 mesh copper grid coated with carbon. The nanoparticles were then negatively stained by 2 wt % photungstic acid (PTA) solution.

[00145] Enzyme linked immunosorbent assay (ELISA) and Dot Blot

[00146] The amount of VEGF immobilized onto nanoparticles in the different clustering conditions was measured using a standard ELISA technique: a high binding 96-well plate was coated overnight at room temperature with 1 μg/ml VEGF capture antibody (R&D Systems, Minneapolis, MN), non-specific binding sites were blocked with blocking buffer (1% BSA in PBS, pH 7.4) for 1 h at room temperature and samples collected from the washes were added to the wells and incubated for 2 h at room temperature. Washes with a solution of 0.05% Tween-20 in PBS at pH 7.4 were performed before adding a biotinylated detection antibody (1 μg/ml in blocking buffer, R&D Systems, Minneapolis, MN) for 2 h at room temperature. Finally, streptavidin-HRP (200 μg/ml in blocking buffer, R&D Systems, Minneapolis, MN) was added and incubated for 20 min at room temperature, then exposed to TMB substrate (Cell Signaling Technology, Boston, MA) for 20 min at room temperature. The resulting absorbance at 645 nm (applying a correction at 570 nm) was measured using a plate reader (BioTek PowerWave XS, Winooski, VT). A Dot blot was also realized in order to confirm the ELISA results: a 2μ1 drop of the sample was deposited on a nitrocellulose membrane. The membrane then was soaked in blocking buffer (1% BSA in PBS, pH 7.4) for 1 h at room temperature before addition of the biotinylated detection antibody, followed by streptavidin-HRP. The samples were then visualized by chemifluorescence (ECL detection reagents, GE Healthcare) using a Molecular Imager Chemi Doc XRS+ scanner (Bio Rad). Images of the stained membrane were analyzed using Image Lab software.

[00147] VEGFR-2 phosphorylation assay

[00148] Human Umbilical Vein Endothelial Cells (HUVECs) were grown to confluency in a 6 well plate, and submitted to serum depravation for 5 h before cell lysis with 0.1 mM sodium vanadate for 5 min. The cells were then treated with 5 ng/ml of either soluble or bound VEGF at 37 °C for 5 min. 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% Nonidet, 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 (DC assay, Bio-Rad) were mixed with NuPAGE LDS sample buffer (4x) and NuPAGE Reducing Agent (lOx) (Novex, life technology) with the ratio of 2.5: 1, boiled for 10 min at 70 °C, separated by SDS-PAGE (4-12% resolving, 1 h at 200 V), and transferred to nitrocellulose membranes (2 h at 350 mA). 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 (pVEGFR-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 Molecular Imager Chemi Doc XRS+ scanner (Bio Rad). The images were analyzed with Image Lab software.

[00149] RNA Isolation and Real Time q-PCR

[00150] HUVECs were grown in complete EGM media (Lonza, Switzerland) in a 24 well plate at 70% confluency. Cells were submitted to serum depravation for 5 h., before exposing them to fetal bovine serum basal EGM-2 media at different time points (2, 4, 6 h). Cells were trypsinized and the cell pellet was collected. Lysis buffer from the RNAqueous micro total RNA isolation kit (Ambion, Life Technologies) was immediately added to cell pellet. Total RNA was isolated from the cells following the manufacturer's protocol. RNA concentration was evaluated by UV absorbance (λ = 260 nm). Reverse transcription was carried out by loading 0.25 μg RNA per reaction of the iScript Advanced cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR (qPCR) was carried out using 10 ng cDNA per reaction of the Maxima SYBR® Green/ROX qPCR master mix (Thermo Scientific, Pittsburgh, PA, USA) following the manufacturer's recommended protocol for three-step cycling using the StepOnePlus real-time PCR system (Applied Biosystems, Life

Technologies). Each 20-μ1 reaction contained 5μ1 of cDNA, 12.5 μΐ SYBR Green master mixes (life technology), 250 nM forward and reverse primers, and nuclease free water. Threshold cycles (CT) were evaluated by the bundled software and expression fold change was calculated using the delta-delta CT method assuming 100% efficiency. GAPDH was used as the housekeeping gene.

[00151] Proliferation assay

[00152] Proliferation rate of HUVEC cells exposed to different clusters of VEGF was measured by the CyQUANT® Cell Proliferation Assay Kit (Invitrogen). Briefly, cells were grown in complete EGM-2 media in a 96 well-plate for 2-4 hrs for cell attachment and exposed to VEGF nanoparticle of different cluster density, in basal EBM media with 2% fetal bovine serum (Lonza, Basal, Switzerland) and compared to a negative control condition containing no VEGF. After 48 h, cells were lysed with Cy quant lysis buffer and the relative fluorescence was measured at 485 nm excitation and 528 nm emissions. The data are expressed as relative fluorescence to the negative control where only heparin nanoparticles were added in the media.

[00153] Tail vein bleeding assay

[00154] Animal procedures were performed in accordance with the US National Institutes of Health Animal Protection Guidelines and approved by the Chancellor's Animal Research Committee as well as the UCLA Office of Environment Health and Safety. Briefly, under isoflurane anesthesia (2-2.5% in a 70% N₂O/30% O2 mixture), young adult C57BL/6 male mice (8-12 weeks) obtained from Jackson Laboratories were placed on a warming pad and injected intravenously with heparin, heparin nanoparticles or saline (4 U/kg, 50 μΐ) 30 minutes before testing. A transverse incision was made with a scalpel over a lateral tail vein at a position where the diameter of the tail is 2.5 mm. The depth of the incision is designated to macerate the tail vein. The tail is then hung over the edge of a table and immersed in normal saline at 37°C in a hand-held conical tube. The time (in sec) from the incision to the cessation of bleeding is recorded as the tail vein bleeding time.

[00155] Hyaluronic acid modification and hydrogel gelation

[00156] Hyaluronic acid (60,000 Da, Genzyme, Cambridge, MA) was functionalized with an acrylamide groups using a two-step synthesis as previously described in Lei, S. et al, The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels, Biomaterials 32, 39-47 (2011) and P. Moshayedi et al, Systematic optimization of an engineered hydrogel allows for selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain, Biomaterials 105, 145- 155 (2016), which are incorporated herein by reference.

[00157] After dissolving the HA (2.0 g, 5.28 mmol) in water, it was reacted with adipic dihydrazide (ADH, 18.0 g, 105.5 mmol) in the presence of l-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC, 4.0 g, 20 mmol) overnight at a pH of 4.75. The hydrazide-modified hyaluronic acid (HA-ADH) was purified with decreasing amounts of NaCl (100, 75, 50, 25 mmol) for 4 hours each via dialysis (8,000 MWCO). The solution was then purified via dialysis (8000 MWCO) in deionized water for 2 days. After 2 days purifying against deionized water, the HA-ADH was lyophilized. The HA-ADH was re- suspended in 4-(2-hydroxyethyl)-l-piperazine ethane-sulfonic acid (HEPES) buffer (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH 7.4) and reacted with N-acryloxysuccinimide (NHS-AC), 1.33 g, 4.4 mmol) overnight. After purification via dialysis as described earlier, the HA-acrylamide (HA-AC) was lyophilized. The product was analyzed with 1H NMR (D₂0) and the degree of acrylamide modification (14.9%) determined by dividing the multiplet peak at δ = 6.2 (cis and trans acrylate hydrogens) by the singlet peak at δ = 1.6 (singlet peak of acetyl methyl protons in HA).

[00158] This hydrogel was chosen because of its biocompatibility with human tissue, as it is constituted of naturally occurring brain extracellular matrix constituents, the acrylamide functionality was used because its kinetics are slower than those of acrylates or vinyl sulfones, which allowed for enough time for injection and ensure that the entire stroke cavity was full of gel before complete crosslinking. The gel precursor remains liquid for a period after mixing, such that it can be injected into the brain through a minimally invasive needle; and will gel within the stroke cavity, conforming to the boundaries of this damaged brain tissue. The mechanical properties of this hydrogel are similar to those of normal brain. Finally, HA has been shown to promote angiogenesis in a mouse model of skin wound healing.

[00159] Gelation

[00160] The hydrogel was made by dissolving the lyophilized HA-AC in 0.3 M HEPES buffer for 15 minutes at 37°C. Studies with stroke mice contained 500 μΜ of the adhesion peptide Ac-GCGYGRGDSPG-NH2 [SEQ ID NO: 3] (RGD, Genscript, Piscataway, NJ). It has been previously found that clustered bioactive signals such as the adhesion peptide RGD results in significant differences in cell behavior when encapsulated inside three-dimensional HA. The highest degree of cell spreading, integrin expression and proliferation of encapsulated mouse mesenchymal stem cells was obtained with a ratio of 1.17 mole of RGD- reacting HA for 1 mole of RGD. The RGD peptide was dissolved in 0.3 M HEPES and added to 16% of the total HA-AC required to obtain a degree of clustering of 1.17, and reacted for 20 minutes at room temperature before being added to the rest of non-RGD reacting HA-AC. A total of 200 ng of soluble (Gel + Vs) or heparin nanoparticle-bound VEGF 165 (Gel + hcV, Gel + lcV) was added to the gel precursor solution. To crosslink the gels, an aliquot of the desired crosslinker (Ac-GCREGPQGIWGQERCG-NH2 [SEQ ID NO: 1], MMP-degradable or Ac-GCREGDQGIAGFERCG-NH2 [SEQ ID NO: 2], MMP- nondegradable) was dissolved in 0.3 M HEPES and added to the gel precursor solution. For viability and animal injections, the precursor was loaded into the Hamilton syringe directly after mixing in the desired crosslinking peptide.

[00161] Animal experiment design

[00162] Animal procedures were performed in accordance with the US National Institutes of Health Animal Protection Guidelines and approved by the Chancellor's Animal Research Committee as well as the UCLA Office of Environment Health and Safety. Two different stroke models were used in this study: A permanent and distal Middle cerebral artery occlusion (MCAo) and photo-thrombotic (PT). The MCAo model is one of the models that most closely simulate human ischemic stroke and its peri-infarct penumbra as approximately 70% of human infarcts originate from the MCA. The MCAo model is considered to be suitable for reproducing cell death, inflammation, and blood-brain barrier (BBB) damage after stroke and has therefore been used in the majority of studies that address post-stroke repair mechanisms such as neurogenesis and angiogenesis. This model was chosen for the 2 week time point in order to determine the effect of the gel + hcV treatment on

vascularization. However, because the permanent and distal MCAo model doesn't induce a long-term neurological deficit, the PT model was used to assess the neurological deficit for 16 weeks.

[00163] Middle Cerebral Artery Occlusion (MCAO) stroke model

[00164] Focal and permanent cortical stroke was induced by a middle cerebral artery occlusion (MCAo) on young adult C57BL/6 male mice (8-12 weeks) obtained from Jackson

Laboratories. Briefly, under isoflurane anesthesia (2-2.5% in a 70% N₂O/30% O2 mixture), a small craniotomy was performed over the left parietal cortex. One anterior branch of the distal middle cerebral artery was then exposed, electrocoagulated and cut. Bilateral jugular veins were clamped for 15 min. Body temperature was maintained at 36.9 ± 0.4 °C with a heating pad throughout the operation. In this model, ischemic cellular damage is localized to somatosensory and motor cortex and was chosen because of the high re-vascularization process after stroke in this region.

[00165] Photothrombotic (PT) stroke model

[00166] Mice were anesthetized and maintained with 2% isoflurane in Ν2Ο 2 (2: 1).

Stroke was produced with a light-sensitive dye in a photothrombotic approach. Briefly, mice were positioned in a stereotaxic instrument and administered Rose Bengal (10 mg/ml, i.p.) and the closed skull at the stereotaxic coordinate 1.5 mm medial/lateral was illuminated with a white light for 18 minutes.

[00167] Hydrogel and VEGF intracranial transplantation

[00168] Five days following stroke surgery, 6 of RGD - functionalized HA hydrogel containing the different forms of VEGF was loaded into a 25 Hamilton syringe (Hamilton, Reno, NV) connected to a syringe pump. The solution was then injected in liquid form directly into the stroke cavity using a 30-gauge needle at stereotaxic coordinates 0.26 mm anterior/posterior (AP), 3 mm medial/lateral (72), and 1 mm dorsal/ventral (DV) for the MC AO-strokes mice and at 1.5 mm medial/lateral for PT-stroked mice at an infusion rate of 1 μΐνηϋη. The control group was injected with an empty RGD-functionalized gel (Empty). The needle was withdrawn from the mouse brain immediately after the injection was complete. This time point for VEGF delivery was chosen because it falls within the time frames of post-stroke ipsilateral VEGF up-regulation and the peak of peri-infarct

microvascular density. Ten days following the hydrogel transplantation, animals were given the DNA synthesis marker 5 -bromo-2'-deoxy uridine (BrdU, Sigma, St Louis, MO; 100 mg/kg in 0.9% NaCl) intraperitoneally 4 and 2 hours before euthanasia to assess cell proliferation. In all experiments, the researchers were blind to the treatment given to each animal.

[00169] Mouse tissue processing and immunohistochemistry

[00170] At 2 weeks post-stroke (10 days after transplantation), mice were transcardially perfused with 0.1 M PBS followed by 40 mL of 4% (wt/vol) paraformaldehyde (PFA). After isolation, the brain was post-fixed in 4% PFA overnight, cryoprotected in 30% sucrose in phosphate buffer for 24 hours and frozen. Tangential cortical sections of 30 μιη-thick were sliced using a cryostat and directly mounted on gelatin-subbed glass slides. Brain sections were then washed in PBS and permeabilized and blocked in 0.3% Triton and 10% Normal Donkey Serum before being immunohistochemically stained. Primary antibodies were as follows: Rabbit anti-Glucose Transporter 1 (Glut-1-) (1 :200; Abeam, Cambridge, MA) for vascular Endothelial Cells; goat anti- Platelet-derived Growth Factor Receptor β (1 :400; PDGF-R , R&D Systems, Minneapolis, MN) for pericytes; goat anti-doublecortin (DCX) (CI 8, 1 :500; Santa Cruz Biotechnology, Santa Cruz, CA) for sub-ventricular neural progenitor cells; rat anti-BrdU (1 :300; Abeam, Cambridge, MA); rat anti-GFAP (1 :400; Zymed, San Francisco, CA) for astrocytes; rabbit anti-microglial-specific ionized calcium binding adaptor molecule 1 (Iba-1) (1 :250; Wako Pure Chemical Industries, Japan) for microglial cells; rabbit anti-Neurofilament 200 (NF200) (1 : 100; Sigma- Aldrich, St Louis, MO), Angiopoietin-2 (1 :250; Abeam, Cambridge, MA). Primary antibodies were incubated overnight at +4°C followed by fluorescently labeled secondary antibody (Molecular Probes, Cergy-Pontoise, France, 1 :400) for 1 h at room temperature. Cell nuclei were then counterstained with the nuclear marker 4', 6-diamidino-2-phenylindole (DAPI, 1 :500, Invitrogen) for 10 minutes at room temperature. After 3x 10 minute washes in PBS, the slides were dehydrated in ascending ethanol baths, and dewaxed in xylene and coverslipped over fluorescent mounting medium (Dako). Sections stained for BrdU were pretreated with 2N HC1 for 30 min and neutralized with sodium borate buffer, pH 8.4, before incubation in primary antibody.

[00171] Microscopy and Morphoanalvsis

[00172] Analyses were performed on microscope images of three coronal brain levels at +0.80 mm, -0.80 mm and -1.20 mm according to Bregma, which consistently contained the cortical infarct area. Each image represents a maximum intensity projection of 10 to 12 Z- stacks, 1 um apart, captured at a 20x magnification with a Nikon C2 confocal microscope using the NIS Element software. The different surfaces for positively stained signals were quantified in 4 to 8 randomly chosen regions of interest (ROI of 0.3 mm²). In each ROI, the positive area was measured using pixel threshold on 8-bit converted images (ImageJ vl.43, Bethesda, Maryland, USA) and expressed as the area fraction of positive signal per ROI. Values were then averaged across all ROI and sections, and expressed as the average positive area per animal. The thickness of scar was measured on the ischemic boundary zone within the ipsilateral hemisphere on three sections stained for GFAP. The NF200 infiltration within the ischemic core represents the average of the length of axonal sprouts penetrating in the infarct area. The Dcx migration was measured on the ipsilateral hemisphere and represents the length of migration of Dcx positive neuroblasts along the Corpus Callosum.

[00173] Assessment of Infarct, hemispheres and cortex volume

[00174] Quantification of infarct, ipsilateral and contralateral hemispheres and cortex was performed using a upright Leica DMLB microscope, equipped with hardware and software from Microbrightfield (Williston, VT, USA). For each animal, every 10th coronal sections were stained for NeuN and Dapi and digitized using a computer-assisted analysis system, Stereo Investigator (Microbrightfield). The volumes were calculated by integrating the appropriate areas with the section interval thickness (250 μιτι). All measurements were averaged to obtain a single value per animal for every region of interest.

[00175] Evaluation of Blood-Brain Barrier (BBB) permeability [00176] BBB permeability was evaluated by assessing the extravasation of intravenously injected Evans blue dye in mouse brain. Briefly, the animals were anesthetized as previously described before injection of 2% Evans Blue dye/PBS (Sigma-Aldrich, St Louis, MO) into the left jugular vein (4 ml/kg). Brains were rapidly removed and each hemisphere placed separately in 1 ml of formamide and left to soak for 48h at room temperature. The amount of extracted Evans Blue from the tissue was quantified by spectrophotometry. The absorbance of the supernatant solution was measured at 625 nm and a ratio ipsilateral/contralateral was obtained. Results were expressed as the relative absorbance (unit/g dry weight) and as a percentage of the PBS group.

[00177] Endostatin treatment

[00178] Recombinant mouse endostatin (100 μg/ml; Alpha Diagnostic, San Antonio, TX), a VEGF-independent angiogenesis inhibitor, was given as a single daily subcutaneous injection during days 5-15 after stroke to hcV -treated mice. hcV/Endostatin mice were submitted to behavioral tests and their brain used for immunohistology at the 16 week time point.

[00179] AAV5 brain injection

[00180] An additional set of hcV -injected mice were injected at week 13 after stroke with a viral construct AAV5 construct expressing hM4 DREADD receptors (designer receptors exclusively activated by a designer drug), capable of silencing transfected neurons after attachment to an i.p administered drug clozapine-N-oxide (CNO) on week 16. A total volume of 1 of AAV5 was injected in the stroke site at an infusion rate of 0.1 μΙ7ιηίη. The AAV5 vector as a tool to study the direct association between brain activity in the infarcted zone and the behavioral outcome. Indeed, the CNO/DREAAD system was shown to inhibit the action potential (AP) of transfected cells through the activation of Inwardly Rectifying K+ channels, provoking a massive entrance of K+ ions and a subsequent hyperpolarization of the cell. Thus, the administration of CNO inhibits the neurotransmitter transport within the injected site, here the infarcted area, 30 minutes after injection and remains active for a period of 2 hours without compromising cells integrity. A behavioral test was then performed within the AP silent window, to assess whether hcV -induced axons in the stroke site participate to the behavioral outcome.

[00181] BP A brain injection

[00182] A total volume of 1 μΐ. of BDA (biotinylated dextran amine), an efficient and powerful marker for bidirectional axonal tracing, was injected in the stroke site at an infusion rate of 0.1 μΙ7ιηιη, in gel + hcV-treated mice, 16 weeks after stroke. Mice were sacrificed five days later and BDA staining was visualized fluorescently by immunohistology using one-step streptavidin-fluorochrome detection.

[00183] Cytokine analysis

[00184] Animals were stroked and injected with gel + nH five days later. The contralateral brain at 10-day s (no stroke) and stroke only were used as positive and negative controls, respectively. At three and ten days after gel transplantation, animals were anesthetized and their blood flushed out with 20 mL cold PBS via transcardial perfusion. Brains were harvested and the infarct core with the injected gel were dissected and homogenized. A total of 1.5 mg/mL of sample was then diluted 1 : 1 with PBS+ 0.5% fetal bovine serum and quantified for their levels of cytokines using a multiplex Elisa analysis through the Bio-Plex kit (Bio-rad Laboratories Inc.) for the following cytokines: TNF-a, IL-6, IFN-γ, IL-1 a, IL-1 β, IL-2, IL-4 and IL-10.

[00185] Behavioral deficit assessment

[00186] Mice were videotaped during walking and exploratory behavior in the cylinder, grid-walking, and pasta-handling tasks, two weeks before surgery to establish baseline performance levels. For all of the studies, animals were tested every four weeks after stroke at approximately the same time each day at the end of their dark cycle. Behavioral tests were scored by observers, who were masked to the treatment group in the study.

[00187] Cylinder test / spontaneous forelimb task

[00188] The spontaneous forelimb task encourages the use of forelimbs for vertical wall exploration/press in a cylinder as previously described. When placed in a cylinder, the animal rears to a standing position, while supporting its weight with either one or both of its forelimbs on the side of the cylinder wall. Mice were placed inside a Plexiglas cylinder (15 cm in height with a diameter of 10 cm) and videotaped for 5 min. Videotaped footage of animals in the cylinder was evaluated quantitatively in order to determine forelimb preference during vertical exploratory movements. While the video footage was played frame by frame, the time (seconds) during each rear that each animal spent on either the right forelimb, the left forelimb, or on both forelimbs were calculated. The percentage of time spent on each limb was calculated and these data were used to derive an asymmetry index ((contralateral use - ipsilateral use) / (contralateral + ipsilateral + bilateral)). The 'contact time' method of examining the behavior was chosen over the 'contact placement' method.

[00189] Grid-walking test

[00190] The grid-walking apparatus was manufactured using a 12-mm square wire mesh with a grid area of 32/20/50 cm (length/width/height). A mirror was placed beneath the apparatus to allow video footage in order to assess the animals' stepping errors (foot faults). Each mouse was placed individually on top of the elevated wire grid and allowed to freely walk for a period of 5 min. Video footage was analyzed offline by raters blind to the treatment groups. The total number of contralateral foot faults, along with the total number of steps was counted, and a ratio was calculated (number of foot faults / total number of steps).

[00191] Pasta

[00192] Manual dexterity is central to daily activities and it is commonly disrupted with any brain damage. In some capacities homologous to humans, rodents use their forepaws and digits to skillfully manipulate the food. Mice were submitted to food restriction one day prior to the test, then placed in a cylinder and were given a total number of four pieces of uncooked vermicelli (7 cm length, 1.5 mm diameter; De Cecco Capellini), once piece at a time and videotaped while eating. A total of 3 criteria were quantified: 1) Adjustment: The primary quantitative variable recorded was the number of adjustments made with each forepaw per each pasta piece. An adjustment was defined as any visible release-regrasp of the pasta piece or reformation of the paw hold on the pasta piece using extension-flexion and/or abduction- adduction of the digits. Only adjustments made after eating had commenced were counted. 2) The time to eat: beginning when the pasta piece was grasped and placed in the mouth and ending when the piece was released by the paws and disappeared into the mouth, was also recorded (reported in seconds). 3) Atypical pasta handling behaviors: paws together when long, guide and grasp switch, failure to contact, drop, paws apart when short, mouth pulling, hunched posture, guide around grasp, angling with head tilt.

[00193] Statistics

[00194] Tests were analyzed blindly to experimental condition. Animals were randomly assigned to control and treatment groups. Power analysis tool (Statistical Solutions LLC, Cottage Grove, WI) was employed to calculate sample size with the expected variance and changes in predicted proliferation and differentiation rates based on preliminary data. In each figure, data represent the average ± SEM (n = 6-8) and P values were determined by Oneway Anova, Tukey's post-hoc test. A value of PO.05 was considered significant (Prism 5.03, graph Pad, San Diego, USA). For non-parametric variables (behavioral scores), multiple comparisons were performed using Kruskal-Wallis test with post-hoc Dunn test (n = 12). In all the figures, *, **, ***, and **** indicate P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 respectively, and represent statistical differences with all the other groups. When there is a statistical difference between two groups only, it is indicated with a line between the two groups. P values between gel + hcV and gel + hcV + endostatin were determined by t test, with $, $$, and $$$ indicating P < 0.05, P < 0.01, and P < 0.001, respectively.

[00195] While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited, except to the following claims, and their equivalents.

Claims

What is claimed is:

1. An injectable therapeutic angiogenic material comprising:

an in situ gelling hyaluronic acid-based hydrogel containing a first plurality of clustered VEGF heparin nanoparticles in which VEGF is immobilized to the heparin nanoparticles and a second plurality of naked heparin nanoparticles that do not contain VEGF immobilized to the heparin nanoparticles.

2. The injectable therapeutic angiogenic material of claim 1, wherein the VEGF is covalently bound to the heparin nanoparticles in the first plurality of clustered VEGF heparin nanoparticles.

3. The injectable therapeutic angiogenic material of claim 1, wherein the heparin nanoparticles, in a naked or non-immobilized state in water, have a size range between about 90 nm and about 200 nm.

4. The injectable therapeutic angiogenic material of claim 1, wherein the injectable therapeutic angiogenic material comprises a larger number of the second plurality of naked heparin nanoparticles than the first plurality of clustered VEGF heparin

nanoparticles.

5. The injectable therapeutic angiogenic material of claim 4, wherein the number of the second plurality of naked heparin nanoparticles is » than the number of first plurality of clustered VEGF heparin nanoparticles.

6. The injectable therapeutic angiogenic material of claim 1, wherein the weight percentage of the second plurality of naked heparin nanoparticles is » than the weight percentage of the first plurality of clustered VEGF heparin nanoparticles.

7. The injectable therapeutic angiogenic material of claim 1, wherein the weight percentage of the second plurality of naked heparin nanoparticles in the hyaluronic acid- based hydrogel is about 99%.

8. The injectable therapeutic angiogenic material of claim 1, wherein the first plurality of clustered VEGF heparin nanoparticles and the second plurality of naked heparin nanoparticles are well-mixed in the injectable therapeutic angiogenic material.

9. The injectable therapeutic angiogenic material of claim 1, wherein the in situ gelling hyaluronic acid-based hydrogel comprises a matrix metalloproteinase (MMP) labile peptide as a crosslinker.

10. The injectable therapeutic angiogenic material of claim 1, wherein the hydrogel comprises a cell adhesion peptide.

11. The inj ectable therapeutic angiogenic material of claim 1 , wherein the VEGF concentration per weight of heparin for the first plurality of clustered VEGF heparin nanoparticles is at least 10 mg VEGF/mg heparin.

12. The injectable therapeutic angiogenic material of claim 1, wherein the VEGF concentration per weight of heparin for the first plurality of clustered VEGF heparin nanoparticles is within the range of 17 mg to 20 mg VEGF/mg heparin.

13. The injectable therapeutic angiogenic material of claim 1, wherein the in situ gelling hyaluronic acid-based hydrogel further comprises a biodegradable crosslinker.

14. The injectable therapeutic angiogenic material of claim 1, wherein the hyaluronic acid-based hydrogel comprises hyaluronic acid functionalized with acrylamide groups.

15. A method of repairing ischemic tissue in a mammalian subject comprising: locating a stroke cavity in the brain tissue of the mammalian subject; and injecting the therapeutic angiogenic material of any of claims 1-14 into the stroke cavity.

16. The method of claim 15, wherein the therapeutic angiogenic material is injected with a syringe.

17. The method of claim 15, wherein the angiogenic material is injected within 15 days of stroke onset.

18. A kit comprising:

a hyaluronic acid-based hydrogel precursor solution containing a first plurality of clustered VEGF heparin nanoparticles in which VEGF is immobilized to the heparin nanoparticles and a second plurality of naked heparin nanoparticles that do not contain VEGF immobilized to the heparin nanoparticles; and

a biodegradable crosslinker for crosslinking the hyaluronic acid-based hydrogel precursor solution into a crosslinked hydrogel.

19. The kit of claim 18, further comprising a delivery device.

20. The kit of claim 19, wherein the delivery device comprises a syringe.

21. The kit of claim 18, wherein the hyaluronic acid-based hydrogel precursor solution comprises a cell adhesion peptide.

22. The kit of claim 18, wherein the biodegradable crosslinker comprises a matrix metalloproteinase (MMP) labile peptide.

23. A method of using the kit of claim 18 comprising mixing the biodegradable crosslinker and the hyaluronic acid-based hydrogel precursor solution and delivering the mixed solution to a stroke cavity with the delivery device.