Optimizing microporous annealed particle scaffold therapies for aged models of stroke

Introduction: : Ischemic stroke remains one of the leading causes of death and disability in the United States. While the implementation of therapeutic strategies that remove stroke-causing clots have contributed to reducing stroke mortality, there is no FDA approved treatment that can regenerate already damaged tissue. Aged populations are at an especially high risk - approximately 65-70% of all strokes each year occur in those 65 years of age or older with significantly worse mortality rates and functional outcomes (1). Promisingly, the delivery of bioactive factors, including vascular endothelial growth factor (VEGF), a protein that influences both the circulatory and nervous systems, and extracellular vesicles (EVs), lipid-bilayer nanoparticles ubiquitously released from cells in the body that carry a range of bioactive factors, have been shown to be a potent therapeutic for stroke, enhancing both tissue regeneration and functional recovery in vivo. However, a key limitation to the successful translation of these therapies is their rapid clearance and poor localization in vivo. MAP scaffolds, comprised of crosslinked hydrogel microparticles (HMPs), can be easily injected into the stroke cavity, allow for a higher degree of cell infiltration than standard hydrogels, and can be easily functionalized with additional bioactive components to improve their therapeutic efficacy. We have already observed enhanced tissue regeneration and functional recovery in young mouse models treated with MAP scaffolds functionalized with either VEGF or EVs (2,3). Here, we explore whether these therapeutic strategies can similarly improve tissue regeneration and functional recovery in aged mice post-stroke.

Materials and

Methods: : Hyaluronic acid-norbornene (HA-NB) HMPs were fabricated using a microfluidic flow focusing device (4). VEGF was covalently bound onto the surface of heparin-norbornene nanoparticles (CLUVENA) prior to incorporation into the scaffold (2). For in vivo experiments, CLUVENA was first mixed with HMPs, and then HMPs were crosslinked with a 4-arm-PEG-tetrazine molecule at a ratio of 1:7 (tetrazine/HA-NB monomer) to create MAP scaffolds. Photothrombotic stroke was induced in the motor cortex at 1.5 mm lateral from bregma utilizing the photosensitive dye rose Bengal (10 μL/g) in either young male (8-12 week) or aged female (20-24 month) C57BL/6 mice (2). 5 days post-stroke, mice were treated with MAP + CLUVENA (M+C) or saline via stereotactic injection into the infarct site. 35 days post-stroke, mice were injected with tomato lectin to label blood vessels, and then brains were harvested for histological analysis. Coronal sections of brain tissue were evaluated for angiogenesis (tomato lectin) as well as glial scar thickness and infarct size (glial acidic fibrillary protein (GFAP)). Behavioral assessments were also performed over the course of 4 weeks (5). Rat cortical astrocytes were isolated from postnatal day 1 rat pups and treated with C1q (400 ng/mL) and IL4 (20 ng/mL) to induce a pro-reparative phenotype (3) as well as Ac4ManNAz (50 uM) to metabolically label EVs with azidosugar (6). They were stored at -80°C in 1x PBS until further use. A nanoparticle tracking video microscope (ZetaView x20 Series, Particle Metrix) was used to determine the average median size and concentration of isolated EVs.

Results, Conclusions, and Discussions:: 35 days post-stroke, aged mice were found to have a significantly larger stroke volume than young mice (Figure 1A), supporting the hypothesis that photothrombotic stroke would be more severe in aged mouse populations. Interestingly, a functional assessment of aged mice showed that M+C treated mice had similar functional scores as stroked mice over the course of 4 weeks (Figure 1B), indicating that M+C treatment led to no significant behavioral improvements in aged populations. We next assessed the underlying histology of both young and aged M+C treated mice. We found that while M+C treatment reduced glial scar thickness in both young and aged populations in comparison to no treatment groups, the glial scar in M+C treated aged mice was noticeably thicker in comparison to treated young mice (Figure 1C). We also found that while some vessel infiltration was observed in M+C treated aged mice, there were minimal differences compared to the stroke only condition (Figure 1D). Because we found that aged mice had significantly larger stroke volumes than young mice, we hypothesized that the ability of M+C treatment to enhance tissue regeneration and functional recovery in aged populations post-stroke would be more difficult. Our histological analysis supported this, with M+C treated aged mice having increased glial scar thickness and lower levels of vessel infiltration in comparison to young mice. We are now exploring whether EVs, which can deliver a multitude of different bioactive components, will be more effective than the delivery of only VEGF in enhancing stroke recovery in aged populations. EVs harvested from C1q/IL4 treated cortical rat astrocytes (C1q/IL4-EVs) (3) were found to have an average median size of 149.3 nm (n = 3 technical replicates) (Figure 1E). Future studies will utilize fluorescently labelled EVs metabolically labelled with azidosugar to covalently bind to MAP scaffolds to evaluate both EV biodistrubtion and therapeutic efficacy in a photothrombotic stroke model (Figure 1F-G). The azidosugar-labelled EVs will be fluorescently tagged with DBCO-fluorophores via azide-alkene click chemistry. DBCO-modified HMPs will then be used to bind EVs via their remaining free azidosugars.

Acknowledgements and/or References (Optional):: Figure 1F-G were created in biorender.com

References

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