Development of a platform technology to impart immunomodulatory activity to complex biomaterials

Victoria Anne Nash

doi:10.17918/00010569

Tissue regeneration is a complex series of events, driven by highly plastic immune cells: macrophages. Typically, "pro-inflammatory" macrophages act early to support angiogenesis, while later acting "pro-reparative" macrophages support newly sprouted vasculature, assisting in tissue repair. Approaches used in biomaterials engineering to temporally influence macrophage phenotype are surface coating or encapsulation of cytokines, however these are not amenable to a variety of biomaterials. Affinity interactions, such as heparin or albumin have been leveraged for drug delivery and rely on weak interactions, like hydrogen bonding, to retain and deliver the drug. However, these systems require specific biomaterial formulations for drug delivery. While these systems work for small molecules and some amino acids, they are limited for cytokine delivery because weak interactions are not stable enough for effective delivery. Biotin-avidin affinity becomes a favorable option because biotin can be directly conjugated to proteins, biomaterials, and even cells while still retaining high specificity and strength in its binding to avidin and avidin variants. However, biotin-avidin affinity is reduced when biotin is conjugated to larger molecules like proteins, but this reduction in binding affinity has been rarely exploited for controlled release applications. Historically, avidin was first used as a model adjuvant, then explored as a protein carrier for adjuvants in vaccines, but modern uses of the affinity pair, biotin-avidin, range from analytical assays to targeted radioimmunotherapy. Biotin-avidin interactions are rarely used for drug release due to biotin's low dissociation rate from avidin. However, release can be triggered by introducing free biotin to the system, promoting the release of biotinylated molecules from avidin. Yet it is not known how bioconjugation parameters can lead to spontaneous release between biotin and avidin. Or even how biotin or avidin influence macrophage phenotype. Therefore, the goal of this thesis is to determine how bioconjugation parameters can control biotin-avidin interactions to release an immunomodulatory cytokine from a model biomaterial to influence macrophage phenotype. Here, we found that using the biotin-avidin system can indeed impart immunomodulatory properties to biomaterials. First, we used a biotinylated scaffold bound with either streptavidin or CaptAvidin and studied the release of a biotinylated cytokine, interleukin 4 (IL-4), from the modified material. We found that adsorption of the base material, a porous gelatin scaffold (Gelfoam), may have obscured biotin-avidin mediated release, but even with minimal release of biotinylated IL-4 from the scaffold, we could still promote a reparative macrophage phenotype. With that result, we investigated the effect soluble biotin, avidin, streptavidin, and CaptAvidin have on unactivated macrophages using Bulk RNA Sequencing. We found that biotin effected macrophages only at a dose 1000x higher than physiologically relevant. Additionally, we found that CaptAvidin significantly influenced macrophages, while streptavidin only slightly upregulated an inflammatory macrophage phenotype. Interestingly, avidin had almost no effect on macrophage phenotype. We then focused on bioconjugation parameters needed to achieve spontaneous release of biotinylated IL-4 from a modified material. While we were unable to measure how much biotinylated IL-4 initially bound to biotin-avidin modified scaffolds, we found that desthiobiotinylated porous gelatin scaffolds, bound with streptavidin, released the largest mass of biotinylated IL-4. We confirmed that release was driven by biotin-avidin interactions by studying release when free biotin was introduced into the system and saw that increasing the dose of biotinylated IL-4 resulted in an increase in the mass of biotinylated IL-4 released from both biotinylated or desthiobiotinylated scaffold bound with streptavidin and biotinylated IL-4. Finally, we studied the effect biotin-streptavidin mediated presentation of biotinylated IL-4 had on unactivated macrophages using quantitative real-time PCR. Biotinylated or desthiobiotinylated scaffold bound with streptavidin and biotinylated IL-4 significantly increased expression of IL-4 driven genes compared to scaffolds with adsorbed IL-4. Encouragingly, we did not see an increase in expression of key markers of an inflammatory phenotype. Overall, the work in this thesis explored how biotin-avidin interactions can be used to impart immunomodulatory activity to a model biomaterial. We defined the spontaneous release profiles of several different combinations of biotinylated or desthiobiotinylated scaffolds bound with avidin, streptavidin, or CaptAvidin, to release biotinylated IL-4. We saw that the dose of biotin and selection of avidin variant play a role in macrophage polarization. And finally, we saw that biotin-streptavidin mediated presentation of biotinylated IL-4 promoted a stronger reparative phenotype compared to simply adsorbing the biotinylated IL-4 to the scaffold. Future studies should focus on fully characterizing the resulting macrophage phenotype before testing this system in vivo. Additionally, this platform technology should be explored with other biomaterials, such as hydrogels, and engineered tissues to investigate whether biotin-avidin interactions can be used to impart immunomodulatory activity to a wide array of implanted biomaterials.

Development of a platform technology to impart immunomodulatory activity to complex biomaterials

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Development of a platform technology to impart immunomodulatory activity to complex biomaterials

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