(251f) Apoptotic Bio-Inspired Materials for Targeting and Engineering Macrophage Function

Introduction. A major initiative factor of various pathological conditions such as type 1 diabetes, obesity, and other autoimmune diseases is chronic low-grade inflammation. The immune cells responsible for such chronic inflammation are macrophages^1–3. Currently, there exists several small molecule drugs that bind cognate receptors on macrophages and promote an anti-inflammatory response in the presence of inflammatory stimuli^1,4,5. However, most of these small molecule drugs suffer from off-target and off-site side effects^6,7. Therefore, there is a need for targeted therapies. Macrophage recognize apoptotic cells through the “eat me” signal, phosphatidylserine (PS)⁸. Interestingly, the recognition of PS on apoptotic cells leads to the internalization of the expressing apoptotic cell and subsequent initiation of an anti-inflammatory and regenerative gene program by macrophages^9,10. This study aims to 1) Synthesize micron-sized polymer drug delivery carriers that present PS on their surface like that of an apoptotic cell and 2) Test the ability of these carriers to target macrophages and potentially modulate macrophage inflammatory function.

Methods. PS-presenting polymer microparticles were synthesized using the single oil-in-water emulsion/solvent extraction method, with poly(lactide-co-glycolide) (PLG) as our polymer of interest¹¹. An annexin V binding assay was used to investigate the presence of functional PS on particle surface. The PS-presenting PLG microparticles (PS:PLG) were loaded with coumarin 6 (C6), to track particle uptake by bone marrow-derived macrophages (BMDMs) using flow cytometry and live cell imaging. BMDMs were co-treated with an inflammatory stimuli, lipopolysaccharide (LPS) and PS:PLG particles to monitor macrophage inflammatory response.

Results and Discussion. We show that PS:PLG particles of different sizes can be synthesized using the single oil-in-water emulsion/solvent extraction method (Figure 1). Thus, indicating that particle size can be modified to fit the right application. For example, the utility of larger PS:PLG particles where extended drug release rates are desired¹¹. An annexin V binding assay demonstrated that PS:PLG particles bound fluorescently labeled Annexin V at increasing levels with more addition of PS to the emulsion during particle synthesis, while particles lacking PS did not (PLG particles) (Figure 2).

PS functions as an “eat me” signal to macrophages that promotes binding and uptake of apoptotic bodies. Confocal microscopy showed increased PS:PLG-C6 particle interaction with BMDMs compared to PLG particles having no surface PS (Figure 3A). Congruent to this visual data, flow cytometry shows a higher percentage of macrophages positive for PS:PLG-C6 particles compared to PLG-C6 particles (Figure 3B). Hence indicating that these PS:PLG particles could be employed in future drug formulations to improve the delivery of small molecule drug payloads to macrophages.

In addition, a co-treatment of macrophages with PS:PLG particles and LPS, significantly promoted an anti-inflammatory response characterized by a decrease in TNF-α and an increase in IL-10 (Figure 4). This suggests that the PS:PLG particles could have physiological effects as a drug carrier by promoting the resolution of inflammation without any drug payload.

Conclusion and Future Work. In summary, we report the development of macrophage-targeted drug delivery carriers that could be applied in therapeutic interventions to actively get drugs or bioactive agents to macrophages for better clinical outcomes and at the same time potentially act as therapeutics to treat low-grade inflammation. These macrophage-targeted carriers could also be applied to repurpose and improve the therapeutic efficacy of some FDA approved drug release formulations made from PLG. Future studies aim to test these PS:PLG particles in vivo. This information will further inform development of optimized particle formulations best suited to be delivered in vivo.

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