E13 - Payload-Free Macrophage Modulation by Synthetic Nanogels: Correlation of Particle Core Chemistry with Immunological Outcome

Introduction: : Inflammation is a key contributor to major fatal diseases, including cancer, autoimmune disorders, and chronic infections. Among immune cells, macrophages play a crucial role in maintaining immune homeostasis and controlling inflammatory responses. Their ability to polarize into distinct phenotypes—pro-inflammatory (M1) and anti-inflammatory (M2)—is essential for processes such as tumor suppression, tissue regeneration, and immune regulation. However, dysregulated macrophage activity leads to disease progression, making macrophage polarization a critical target for therapeutic intervention. Targeted therapies use drug delivery systems, such as nanoparticles, designed to carry agents that modulate macrophage function. Recent research has highlighted an unexpected finding: beyond their intended function, blank drug delivery systems themselves can influence macrophage behavior. These carrier particles, even without active pharmaceutical agents, have been observed to affect macrophage polarization, gene expression, and functional states. Understanding this intrinsic immunomodulatory effect is critical for designing more effective and predictable delivery systems. Nanogels, a next-generation drug-delivery platform, have gained significant attention due to their biocompatibility, tunable properties, and responsiveness to stimuli. These three-dimensional nanostructures, formed by crosslinking hydrophilic polymers in aqueous environments, can be precisely engineered by selecting specific functional monomers, polymerization techniques, and crosslinking strategies. In this study, we investigate the potential of stimulus-responsive nanogels synthesized via activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) to modulate macrophage polarization. By characterizing their physicochemical properties and evaluating their impact on macrophage gene and protein expression, we aim to gain deeper insights into how nanogel chemistry influences intrinsic immunomodulatory effects and explore their potential in immunotherapy.

Materials and

Methods: : To establish a pH-responsive nanogel platform for immunomodulation, 20 formulations were synthesized. Each featured a cationic and hydrophobic core, crosslinked with a disulfide linker, and decorated with a poly(ethylene glycol) (PEG) macromer. Cores included a cationic monomer (DMAEMA or DEAEMA) and one of five hydrophobic co-monomers (MMA, BzMA, CHMA, tBMA, or BMA). Nanogels were synthesized at 75:25 and 25:75 cationic:hydrophobic ratios via ARGET ATRP miniemulsion polymerization using bis(2-methacryloyl)oxyethyl disulfide as a crosslinker and PEG(2000) methacrylate for surface modification. Nanogels were characterized by FT-IR, DLS, and zeta potential to confirm composition and pH-responsiveness. Structural validation and crosslinking efficiency were assessed by 1D and 2D NMR (TOCSY, COSY, HSQC, HMBC) in D₂O with TCEP (to degrade disulfide bonds) and TMSP (internal standard). In vitro testing was conducted using RAW264.7 macrophages. Cytotoxicity and viability were assessed via LDH and MTS assays (24 h at 50 and 200 µg/mL). Flow cytometry evaluated polarization markers—M1 (CD86, MHCII, iNOS) and M2 (CD163, CD206, Arg-1)—after 24 h at 200 µg/mL. For inflammatory conditions, macrophages were co-treated with IFN-γ (2 ng/mL), and compared to IFN-γ alone or IFN-γ + IL-4 (80 ng/mL). Cytokine secretion (IL-6, TNF-α) was measured by ELISA. Gene expression profiling used NanoString nCounter with a custom 163-gene panel for macrophage polarization. RNA was isolated (ZYMO Quick-RNA™), quantified (NanoDrop, Qubit), and stored in DNA/RNA Shield™ at −80 °C. Samples (≥20 ng/µL) were hybridized at 65 °C overnight. Data were acquired with the nCounter Digital Analyzer and analyzed using nSolver v4.0. Statistics were performed using GraphPad Prism.

Results, Conclusions, and Discussions:: FT-IR analysis confirmed successful polymerization through the disappearance of the =C–H stretch (~1635 cm⁻¹) and C=C stretch (~695 cm⁻¹) in nanogel spectra. Dynamic light scattering revealed particle sizes below 200 nm and pH-responsive surface charge behavior across all formulations. Zeta potential decreased with increasing pH, reflecting reduced cationic charge. Cytotoxicity and viability assays showed minimal toxicity at both 50 and 200 µg/mL, with RAW264.7 macrophage viability exceeding 80%, confirming nanogels' biocompatibility. For structural analysis, 2D NMR analyzed crosslinking and monomer incorporation. Spectra revealed two distinct peaks for TCEP and its oxidized form (TCEP-oxide), confirming disulfide-based crosslinking, as this pattern was absent in pure TCEP. Quantification showed combined TCEP/TCEP-oxide levels of 8.5 ± 0.4 mM, consistent with feed ratios and supported by calibration (8.7 mM). Peak integration enabled accurate assessment of crosslinker and monomer incorporation. Flow cytometry revealed that nanogels containing 75% cationic monomer—especially DEAEMA-BzMA, DEAEMA-BMA, and DMAEMA-MMA—induced significant pro-inflammatory macrophage polarization. CD86 increased five-fold (p < 0.0001), and iNOS increased 73-fold (p < 0.005), relative to untreated macrophages. IL-6 secretion also rose 33-fold in response to 75% DEAEMA nanogels, while 25% cationic formulations suppressed IL-6 secretion. Transcriptomic profiling further supported this trend. Heatmap and hierarchical clustering analyses revealed that macrophages treated with 75% DEAEMA nanogels exhibited gene expression profiles closely aligned with those stimulated by LPS + IFN-γ, indicating M1-like polarization. Upregulation of pro-inflammatory chemokines (CCL5, CXCL11) and cytokines (TNF) suggested activation of NF-κB and STAT1 signaling pathways. Marked increases in transcription factors IRF1 and REL, along with genes involved in phagocytosis (CLEC4E), apoptosis (BID), oxidative stress responses (TXNRD1, SRXN1), and metabolic adaptation (C3), further underscored broad inflammatory activation and metabolic programming. These findings demonstrate that DEAEMA nanogel treated macrophages exhibit a classical inflammatory phenotype (Figure 1), compared to alternative phenotypes in other nanogel-treated macrophages. Our findings demonstrate successful synthesis of pH-responsive nanogels and highlight how specific monomer combinations influence their payload-free immunomodulatory effects on macrophages. This work supports the development of tailored platforms for immune therapy with translational potential. Future studies will expand formulations and evaluate efficacy in primary bone marrow-derived macrophages.

Acknowledgements and/or References (Optional):: Funding is gratefully acknowledged from the Harold Hamm foundation and the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number 1R35GM150970. We also thank the ImmunoEngineering Core at OU Norman and the Genomics Core at OUHSC for their support.