(670f) Regulating the Timing and Sequence of Pro- and Anti-Inflammatory Cytokine Deliveries from Magnetically Responsive Biomaterials for Use in Wound Healing Applications

In the United States, approximately 6.5 million patients are diagnosed with chronic wounds. The treatment of chronic wounds costs more than $25 billion annually and this is expected to grow due to the increasing cost of healthcare, an aging population, and a rise in the occurrence of diabetes and obesity worldwide [1]. The process of wound healing includes four major steps: hemostasis (blood clothing), inflammation, proliferation, and tissue maturation [2]. Even a slight perturbation in this process can disrupt proper healing, leading to chronic wounds. Chronic wounds are often a result of arrest in the inflammation phase of healing [2]. Although inflammation critically initiates repair and helps clear infections, a prolonged inflammatory reaction can cause considerable harm to the injury site. After an appropriate duration of inflammation, this inflammatory response can be shifted to a more pro-healing response through the delivery of cytokines like interleukin 4 (IL-4) and interleukin 10 (IL-10). These anti-inflammatory cytokines alter the phenotype of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2), suggesting a potentially powerful drug delivery strategy if these cytokines can be delivered in a delayed manner. We hypothesize that the transition of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) can be controlled through sequenced delivery of interferon gamma (IFN-γ), followed by IL-4 and/or IL-10. The goal of this research was to develop a wound-healing hydrogel system that initially delivers IFN-γ, followed by magnetically triggered delivery of IL-4 and/or IL-10.

Our biomaterial system was composed of two-compartments: (1) a porous gelatin outer compartment designed to recruit macrophages and establish an initial pro-inflammatory (M1) phenotype, and (2) a magnetically responsive alginate inner compartment which was designed to deliver IL-4 and/or IL-10 when magnetically triggered to shift the response to anti-inflammatory by promoting (M2) phenotype. This magnetically responsive inner compartment was made by casting 7 wt% Fe₃O₄in 1 wt% alginate with 2.5 mM adipic acid dihydrazide (AAD) crosslinking in the presence of a magnet to generate biphasic gels. These biphasic gels were then swollen in water, frozen at -20°C, and lyophilized to generate porous, magnetically compressible biphasic gel structures. Gelatin outer compartments were loaded with 1000 ng of IFN-γ and designed to diffusively release IFN-γ early on. Magnetically deformable inner compartment gels were loaded within 100-1000 ng of IL-4 or IL-10 and were designed to deliver these anti-inflammatory proteins to macrophages that were recruited to the outer compartment at a time dictated by the application of the magnetic stimuli. A pulsatile 5 kGauss graded magnetic field was used in these experiments and applied at 1.5 Hz. Validation of these protein delivery profiles over time were quantified for IL-4, IL-10, and IFN-γ using ELISA.

We have demonstrated this systemâ??s ability to initially release a pro-inflammatory cytokine (IFN-γ). We have found that more than 95% of the loaded IFN-γ was released from the outer compartment in the first 6 hours. We have also shown the ability to retain anti-inflammatory cytokines (IL-4 and IL-10) for several days and magnetically trigger their release from the inner compartment. Furthermore, we were able to magnetically control the timing and rate of these deliveries. For example, we showed that when we load 1000 ng of IL-4 in the inner compartment, we were able to retain IL-4 in the gel for 4 days with low release rates prior to magnetic stimulation (29.6 ± 3.3 ng/day for 4 days) and magnetically trigger enhanced release rates on day 4 (345.8 ± 103.29 ng/day over 4 hours). Using gels loaded with 500 ng of IL-10, we again demonstrated excellent retention for 3 days prior to magnetic stimulation (0.17 ± 0.04 ng/day for 3 days) and magnetically enhanced release rates on day 3 (6.99 ± 4.24 ng/day over 3 hours). The timing of IL-10 delivery could be stimulated on day 5 instead of day 3. In this case, IL-10 was retained at a low release rate prior to magnetic stimulation (0.11 ± 0.04 ng/day for 5 days) and magnetically released at enhanced rates on day 5 (3.67 ± 0.776 ng/day for 4 hours). Finally, the rate of IL-4 release could be controlled by using different magnetic stimulation regiments. For example, the release rate of IL-4 when magnetically stimulated at 1.5 Hz for 1 hour every 12 hours for over days was only 20.23 ±7.33 ng/day whereas continuous magnetic stimulation at 1.5 Hz for 4 hours resulted in a much higher release rate (345.85 ±103.29 ng/day). We believe that this biomaterial system will be highly useful for understanding how the timing and sequence of cytokine deliveries impact wound healing outcome.

Keywords: Wound healing, Macrophages, Cytokine delivery, Biomaterial, Magnetic stimulation.

REFERENCES

1. Sen, Chandan K. et al. â??Human Skin Wounds: A Major and Snowballing Threat to Public Health and the Economy.â? Wound Repair and Regenerationâ?¯ 17.6 (2009): 763â??771.

1. Frykberg, Robert G., and Jaminelli Banks. â??Challenges in the Treatment of Chronic Wounds.â? Advances in Wound Care 4.9 (2015): 560â??582.