FKN-aptamer functionalized hydrogels for local enrichment of M2 macrophages after traumatic brain injury

摘要

Introduction: Traumatic brain injury (TBI) has progressively become the single leading cause of disability and death in the United States amongst those aged between 1 and 44 years. Death, however, is rarely due to the primary insult and instead due to an onslaught of secondary reactions after TBI. Of these, the inflammatory reaction post-injury is highly significant and consists of a breach in the blood-brain-barrier, which allows systemic monocytes into the site of injury. There is also an overburdening pro-inflammatory (M1)compared to anti-inflammatory (M2) macrophage polarization response resulting in death and scarring of potentially viable brain tissue. Altering this balance towards M2 macrophages may be possible by enriching Fractalkine (FKN), an M2 recruiting chemokine, to quell the inflammation and allow for regeneration. We are developing a scaffold to enrich endogenously synthesized FKN via FKN-binding DNA aptamers coupled to a hydrogel backbone. Endogenous enrichment may equal or supersede conventional delivery as it obviates the need for exogenous delivery of proteins. Cytokines typically possess short half-lives which poses another limitation to exogenous delivery and may be circumvented by the scaffold as endogenous cells are continuously producing cytokines. Furthermore, the scaffold could potentially act as a 'depot' to enrich FKN and prevent premature dissipation of FKN at the site of injury. Materials and Methods: DNA aptamers specific to Fractalkine protein and scrambled aptamers were modified with 5' Acrydite ends and then conjugated independently to polyethylene glycol diacrylate (PEGDA) in a 1:50 ratio via free radical initiation. The gels were washed in deionized water three times over two days. Presence of conjugated aptamer was ascertained via 6-FAM labelled complementary sequence. Functionalized hydrogels were placed in a 48-well-plate incubation bath of Fractalkine protein dissolved in phosphate buffered saline (PBS) of different concentrations for 24 hours. The gels were then moved to fresh wells with PBS and complementary sequence was added to release any Fractalkine taken up by the gels over 24 hours. The baths were sampled and Fractalkine amount was quantified via enzyme linked immunosorbent assay (ELISA). Results and Discussion: Hydrogels formed within seconds of addition of catalyst. Fluorescence microscopy confirmed acrydite modified aptamers conjugated to PEGDA while absence of acrydite did not result in conjugation. Furthermore, smaller hydrogel segments with higher surface area resulted in enhanced aptamer activity vis-a-vis FKN enrichment capacity. ELISA for Fractalkine showed that FKN aptamer-functionalized hydrogels took up FKN from the incubation bath in a concentration dependent manner while scrambled aptamer-functionalized gels did not take up any FKN. This confirms the specificity of FKN towards the FKN aptamer-functionalized hydrogels. Conclusion: We have demonstrated that FKN aptamer-functionalized hydrogels take up FKN from a solution. Future work will demonstrate enrichment from an in vitro bath model of the post TBI injury with multiple cytokines, M2 macrophage attraction towards the material, and in vivo testing of aptamer-functionalized microgels in a rat controlled cortical impact model over the next couple of months.