SDF-1 Bound Heparin Nanoparticles Recruit Progenitor Cells for Their Differentiation and Promotion of Angiogenesis after Stroke

Abstract

Angiogenesis after stroke is correlated with enhanced tissue repair and functional outcomes. The existing body of research in biomaterials for stroke focuses on hydrogels for the delivery of stem cells, growth factors, or small molecules or drugs. Despite the ability of hydrogels to enhance all these delivery methods, no material has significantly regrown vasculature within the translatable timeline of days to weeks after stroke. Here, two novel biomaterial formulations of granular hydrogels are developed for tissue regeneration after stroke: highly porous microgels (i.e., Cryo microgels) and microgels bound with heparin-norbornene nanoparticles with covalently bound SDF-1α. The combination of these materials results in perfused vessels throughout the stroke core in only 10 days, in addition to increased neural progenitor cell recruitment, maintenance, and increased neuronal differentiation.

Keywords: cryogels; hydrogel; microgel/microparticle; microporous annealed particle; neural stem cells; stroke; stromal cell‐derived factor 1.

Figure 1.. Formation of cryo microgels and material characterization.

A) Schematic overview of cryogel formation by lyophilizing HA-NB microgels in Tris HCl, NaCl, and CaCl2 buffer. B) Confocal images and Nikon volume renderings of microgels compared to C) cryogels with void fraction shown in red. D) IMARIS surface renderings of MAP scaffolds (green) and void fraction (red) compared to E) Cryo scaffolds. F) Quantification of percent void space from IMARIS renderings. G) Frequency sweep of MAP and Cryo Scaffolds. H) Storage modulus of MAP scaffold verses Cryo scaffold. Significance was reported at p < 0.05 (*), <0.01 (**), <0.005 (***), and <0.001 (****).

Figure 2:. Effects of porosity on Glut1+ vessel infiltration 10 days after stroke.

A) Depicts in vivo experimental overview of photothrombotic stroke on Day 0, gel injections on Day 5, and sacrificing on Day 15. B) Depicts imaging of ROI brain sections and orientation of 20x representative images in C. C) For each image, the fourth section of the slide was imaged with a 5×5 tile grid at 20x magnification and oriented with the stroke in the top of the image and peri infarct towards the bottom of the image. Representative images stained for vessels (Glut1+) in white, astrocytes (GFAP+) in red, MAP in green, and nuclei (DAPI+) in blue. Representative images from D) stroke only, E) MAP, and F) Cryo scaffold conditions are shown stained for astrocytes (GFAP+), microglia/macrophages (Iba1+), vessels (Glut1+), and neurons (NF200+). Dotted line indicates the region between peri-infarct and infarct (with an asterisk). G) Analysis of astrocyte (GFAP) infiltration and scar thickness. The percent positive area of H) microglia/macrophages (Iba1), I) vessels (Glut1), and J) neurons (NF200) was measured across conditions in the stroke infarct area as well as the peri infarct area next to the stroke. Scale bars represent 500 microns (N=5). Significance was reported at p < 0.05 (*), <0.01 (**), <0.005 (***), and <0.001 (****).

Figure 3:. Synthesis of Heparin Nanoparticles for Delivery of SDF-1.

A) Reaction schematic for synthesizing heparin-norbornene nanoparticles (nH) from heparin modified with norbornene and covalent binding of SDF-1 soluble growth factor to nH. B) SEM image of gold coated nH. C) DLS data of nH, PDI = 0.23. D) ELISA data of measured SDF-1 bound to nH and ratio of SDF-1 to nHs. E) Release assay of SDF-1 nH in MAP scaffolds. Blue line indicates the amount of SDF-1 loaded in the scaffold.

Figure 4:. SDF-1 Effects on NPCs in vitro.

A) RT-PCR measuring CXCR4 expression after NPC exposure to concentrations of 100 ng, 200 ng, and 400 ng of soluble SDF-1. B) Representative live/dead immunofluorescent images of NPCs spreading in MAP Scaffolds at D4 stained with CalceinAM. Scale bars represent 100 μm (N=3). C) Quantification of NPC spreading after 2 hours and 1 day of exposure to nH soluble SDF-1, bound and unbound SDF-1 nH at 200 ng SDF-1. Significance was reported at p < 0.05 (*), <0.01 (**), <0.005 (***), and <0.001 (****).

Figure 5:. SDF-1 nanoparticles recruit SOX2+ and Tuj1+ cells inside SDF-1 Cryo Scaffolds.

A) Depicts in vivo experimental timeline for stroked mice injected with MAP and schematic explaining imaging of fixed and sectioned brains. Large image scans and 20x magnified images for mice treated with Cryo scaffolds and Cryo scaffold with SDF-1 nH, stained for nuclei marker DAPI (blue), labeled Cryo scaffolds (green), and either B) embryonic stem cell marker SOX2 (white) or C) neuron marker Tuj1 (white). D) Quantification of colocalization of SOX2+ cells and DAPI. E) Quantification of Tuj1+ percent area. Quantification was performed on large image scans (5×5, 20x). Cryo scaffolds (green), Tuj1+ cells (white), SOX2+ cells (White), Nestin+ cells (red), DAPI (blue). Scale bars represent 500 microns (N=5). Significance was reported at p < 0.05 (*), <0.01 (**), <0.005 (***), and <0.001 (****).

Figure 6:. Perfused vasculature characteristics in SDF-1 treated mice 15 days after stroke.

A) A 5×5 tiled large image scan of stroke infarct with SDF-1 nH Cryo scaffold (green), TL vessels (white), nuclei (blue), and Nestin+ cells (red). B) TL vessel channel only showing the ROI outlined in yellow. Insets show higher magnification of i) the vasculature depicting how it was traced for analysis of co-localization of ii) Nestin+ cells. 5×5 20x large image scans followed by a single 20x representative images of C) Cryo scaffold vs D) Cryo scaffold SDF-1 nH. Sections of all 5 biological replicates from Cryo scaffold SDF-1 nH are shown. Scale bars represent 500 microns (N=5). Quantification of vessel architecture and Nestin+ cells with vasculature include E) perfused vessel area in the infarct, F) perfused vessel area in the peri-infarct, G) Nestin+ area in the infarct, H) SOX2+ cells, I) furthest perfused vessel infiltration, J) total perfused vessel length, K) furthest perfused vessel infiltration normalized to infarct length, L) # branches, M) maximum branch length, and N) average branch length. Significance was reported at p < 0.05 (*), <0.01 (**), <0.005 (***), and <0.001 (****).

Figure 7:. Inflammatory response following the delivery of SDF-1 nanoparticles.

Representative large scan and magnified images of A) Cryo scaffold and B) Cryo scaffold SDF-1 nH treated mice stained for astrocyte (GFAP+, white) or microglial/macrophages (Iba1+, white), DAPI (blue), and Cryo scaffold (green). Scale bars represent 500 microns in large image scans and 100 microns in other images (N=5). Quantification of C) astrocyte scar thickness, D) infiltration, and E) microglia/macrophage percent positive area.