Uptake and activity of cytokine-nanogel conjugates for macrophage immunotherapy

Introduction: Macrophages are innate immune cells which can adopt inflammatory and regenerative phenotypes in response to environmental cues. Classically activated inflammatory macrophages can cause chronic tissue inflammation, implicated in a range of inflammatory diseases. Cytokine signaling and macrophage cell surfaces can regulate phenotype, inducing or reducing the macrophages’ expression of inflammatory or tolerogenic molecules. However, targeted cytokine delivery is challenging, as cytokines have a short half-life and can lead to significant side effects following off-target accumulation. If delivered to the inflamed site, anti-inflammatory cytokines such as IL-4 and IL-10 could shift macrophages from an inflammatory state to a regenerative state, thus reducing inflammation in the case of inflammatory diseases. Here, we hypothesized that anti-inflammatory cytokine conjugation to nanogels would allow targeted delivery while retaining the cytokine’s activity. To test this hypothesis, we evaluated the uptake and spatial distribution of nanogels in immortalized murine macrophages as well as evaluated the biological activity of cytokine-nanogel conjugates to macrophages, relative to unconjugated recombinant cytokine. Our results span the assessment of synthetic nanogels (poly(acrylamide-co-methacrylic acid) conjugated at up to 10 wt% with interferon gamma (IFN-γ), interleukin 4 (IL-4) and interleukin 10 (IL-10). Taken together, we establish a platform cytokine-nanogel technology for future application in macrophage immunotherapy.

Materials and

Methods: Nanogels were synthesized as described previously (Clegg et al. 2019; Zhong et al. 2018). The conjugation of IFN-γ, IL-4 or IL-10 to the P(AAm-co-MAA) nanogels was performed using a two-step EDC/NHS reaction at 2wt%.
To assess the uptake of the nanogels, Raw 264.7 macrophages and L929 fibroblasts were treated for 24 hours with 100ug/mL fluorescent unconjugated nanogels. The cells were washed, fixed, and stained with Hoechst and Alexa Fluor 647 Wheat Germ Agglutinin and APC-Cy-7 anti-Mouse CD11b and imaged using the EVOS M7000 Microscope.
To assess the impact of cytokine conjugation on the intracellular distribution of nanogels, Raw264.7 macrophages were treated for 24 hours with 1ug/mL IFN-γ-conjugated to 20ug/mL fluorescent nanogels. Washed and fixed cell samples were stained with Hoechst, anti-Murine IFNg primary antibody, anti-Rabbit Alexa Fluor 488 secondary antibody and Alexa Fluor 647 Wheat Germ Agglutinin. Z-stacks were obtained using the Leica SP8 Confocal Microscope. The number and intracellular distribution of cytokine conjugated nanogels was compared with unconjugated nanogels, 1ng/mL free IFN-γ and an untreated control. Image analysis was performed on ImageJ.
To assess the biological activity of the conjugated cytokines, Raw 264.7 macrophages were co-stimulated for 24 hours with 1ng/mL IFN-γ and 1.5ug/mL IL-4 or IL-10 conjugated nanogels. This treatment was compared to co-stimulation with 1ng/mL IFN-γ and 40ng/mL of freely soluble recombinant IL-4/IL-10, individual cytokine, or blank nanogel controls. Macrophage phenotype was characterized by examining pro-inflammatory (MHC II, CD86) and anti-inflammatory (CD206, CD163) membrane markers using spectral flow cytometry (CyTek).
Statistical analysis was performed using GraphPad Prism.

Results, Conclusions, and Discussions: Results and discussion:
Raw 264.7 macrophages showed a 6-fold higher uptake of 100ug/mL unconjugated nanogels compared to fibroblasts, showing a targeted uptake by macrophages. Confocal imaging showed that Raw 264.7 macrophages had a 2-fold higher uptake of the 20ug/mL conjugated nanogels compared to unconjugated nanogel-treated. Nanogels were distributed in the cytoplasm, where they formed several circular agglomerations, indicating potential accumulation in lysosomes or other intracellular compartments.
Co-stimulation of Raw 264.7 macrophages with IFN-γ and IL-4 and IL-10 conjugates showed a 2-fold reduction in CD86 marker for the IL-4 and IL-10 conjugated nanogels compared to unconjugated nanogel-treated cells. Only IL-10 conjugates showed a reduction in MHCII marker expression. CD163 expression showed a 2-fold increase for IL-4 conjugates and a 3-fold increase for IL-10 conjugates. This data shows a decrease in some pro-inflammatory markers’ expression and an increase in anti-inflammatory marker expression in Raw 264.7 macrophages after co-stimulation with IFN-γ and IL-4 and IL-10 conjugates. These results establish that conjugated nanogels retain activity of the conjugated payload, with the potential to induce a regenerative phenotype in murine macrophages in spite of an inflammatory environment.

Conclusions:
Our study demonstrated the uptake into the cytoplasm of cytokine conjugated nanogels. The nanoparticles showed targeted delivery to Raw 264.7 macrophages compared to L929 fibroblasts, indicating cellular specificity. Overall, our study contributes to the development of cytokine-conjugated nanogels for macrophage immunotherapy.

Acknowledgements (Optional): Funding is gratefully acknowledged from the OUHSC-IBEST Collaborative Research Program, American Cancer Society IRG2023-1, Harold Hamm foundation, and the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number 1R35GM150970.