(C-99) Synthesis of Lipid-Microgels to Deliver Omega-3 Fatty Acid For Ischemic Stroke Repair

Introduction:: Lipids are a major constituent in neural tissue. These lipids perform both structural and bioactive roles, with the delivery of lipid reparative cues like polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA) having a prominent role in anti-inflammatory and neuroregenerative pathways1. We aim to develop granular hydrogels to utilize these regenerative properties following ischemic stroke. We hypothesize that conjugating short lipids to the surface of hydrogel microparticles (HMPs) will enable delivery of DHA via physical lipid-lipid interactions. Previously, we have demonstrated that HMPs in annealed scaffolds are effective in stroke repair2. We first aim to understand how lipid conjugation impacts the availability of microgel norbornenes that can be used in annellation chemistry. We additionally hypothesize that chemical annealing performed between norbornene-modified hyaluronic acid (HA) and tetrazine-modified HA will be effective in crosslinking lipid-microgels to form a scaffold due to excess surface norbornenes on the HA chain, while providing a modest increase to physical stiffness via lipid physical interactions between neighboring beads. We expect that the hydrophobicity of lipid-microgels will enable delivery of DHA and direct cell migration.

Materials and Methods::

Microgels were produced by oil-in-water emulsion on microfluidic devices, with size distribution quantified by fluorescent microscopy. Conjugation of short lipid decanethiol to hydrogel surface was achieved by thiol-norbornene click chemistry in a partial aqueous partial organic solution. Chemical annellation was achieved by mixing the norbornene-modified HMPs with polymer solutions of tetrazine-modified hyaluronic acid. Physical interactions and degree of chemical annellation in scaffolds were measured via oscillatory rheology. To determine cell viability, D1-MSCs were encapsulated in both MAP scaffolds consisting of either plain microgels or lipid-microgels in a 3D scaffold assay. Cells were cultured for 1 day and imaged via fluorescent microscopy under Bright-field. Viability was determined by Calcein AM and EthD-III.

Results, Conclusions, and Discussions:: As the percentage of the lipid-coated microgels increased relative to the amount of unconjugated microgels, the storage modulus (G’) remains similar (Figure 1A), suggesting the same elasticity and stiffness of the microgels when utilizing similar chemical annellation ratios. This result indicates that the norbornene groups we used to crosslink the microgels to form scaffolds are still accessible on the surface of the HMPs, enabling annealing or other further chemistries. The result is expected because decanethiol is a short lipid, suggesting it likely would not impede all potential chemistry of the HMPs as long as there are free norbornene and tetrazine in the system for them to anneal.

Rheology was then performed on bead scaffolds composed of either 0%, 40%, or 100% lipid-modified HMPs mixed with annellation reagent tetrazine-modified HA (Figure 1B). The drop in G’ was expected because the less free norbornene each HMP has, the less norbornene-tetrazine chemistry will happen to anneal the HA-chain beads (HMPs) to increase scaffold stiffness. The result is expected because 100% lipid occupied all free norbornene on the HA-chain surface to block its annealing with tetrazine groups.

Preliminary Bright-field images indicate that D1-MSCs proliferated and spread when encapsulated in lipid-modified microgels scaffolds (Figure 2). Unfortunately, these images lacked the resolution required to quantify cell morphology. We aim to use confocal microscopy in the future to more accurately quantify cell number and viability. 

These findings enable a variety of scaffold formation strategies, allowing customization in how microgel scaffold stiffness is modulated. In the future, we aim to further quantify cell survival and migration in lipid-microgel scaffolds to determine whether local hydrophobicity impacts cell adhesion. Further cell seeding studies are planned to be performed in neural cells (astrocytes) to study the stroke repair function of lipid-microgels.

In conclusion, we generated lipid-coated microgels that increased scaffold stiffness while retaining surface moieties to be used for annellation chemistries. These findings enable tunable customization of scaffold mechanical properties, which we aim to use to study how changing granular microgel viscoelasticity determines cell fate following injury.

Acknowledgements (Optional): : We would like to thank Nhi Phan and Yunxin Ouyang for mentorship. We would like to thank Duke Summer Neuroscience Program for funding.

References (Optional): : 1Jiang, X., Pu, H., Hu, X., Wei, Z., Hong, D., Zhang, W., Gao, Y., Chen, J., & Shi, Y. (2016). A Post-stroke Therapeutic Regimen with Omega-3 Polyunsaturated Fatty Acids that Promotes White Matter Integrity and Beneficial Microglial Responses after Cerebral Ischemia. Translational stroke research, 7(6), 548–561. https://doi.org/10.1007/s12975-016-0502-6
2Sideris, E. et al. Particle Hydrogels Decrease Cerebral Atrophy and Attenuate Astrocyte and Microglia/Macrophage Reactivity after Stroke. Adv Ther (Weinh) 2200048 (2022) doi:10.1002/ADTP .202200048.