Therapeutic Activity of Internalized Synthetic Nanogels on Murine Macrophages

Introduction: Nanogels represent a next-generation drug-delivery system, distinguished by their cytocompatibility and stimuli responsiveness. These three-dimensional nanostructures are formed by chemically crosslinking hydrophilic polymers in an aqueous environment. Due to their tunable features, the design of these nanocarriers can be precisely engineered by selecting specific functional monomers, methods of polymerization, and crosslinking agents. This enables them ideal candidates for advanced treatment approaches in different biological processes. Here, we evaluated the impact of nanogel composition on their intrinsic activity to immortalized murine macrophages. Modulation of macrophages from pro- to anti-inflammatory phenotypes is useful in context of cancer treatment, reduction of inflammatory disease severity, or exerting engineered control over the persistence of adoptive cell therapeutics. This study explores the hypothesis that the polymer chemistry of internalized nanogels will drive distinct macrophage responses. Here, we developed and characterized stimulus-responsive nanogels synthesized using ARGET-ATRP (activators regenerated by electron transfer atom transfer radical polymerization) as an enhanced controlled polymerization technique.

Materials and

Methods: To establish a pH-responsive nanogel platform for immunomodulatory purposes, a small library of 20 nanogel formulations was synthesized. These formulations incorporated one of two cationic (DMAEMA and DEAEMA) and one of five hydrophobic (MMA, BzMA, CHMA, tBMA, and BMA) methacrylic monomers, at ratios of 75:25 and 25:75 cationic:hydrophobic co-monomer. Nanogels were prepared using the miniemulsion technique, with bis(2-methacryloyl)oxyethyl disulfide as a crosslinking agent and poly(ethylene glycol MW=2,000) methacrylate macromer for surface modification. Fourier Transform Infrared Spectroscopy (FT-IR) was utilized to validate the incorporation of feed monomers. Dynamic Light Scattering (DLS) was used to evaluate hydrodynamic particle diameter and zeta potential as a function of environmental pH. In vitro cytotoxicity and cell viability were assessed using LDH and MTS assays, respectively, following a 24-hour incubation with Raw264.7 cells at two concentrations (50 and 200 µg/ml). Flow Cytometry analysis of nanogel-treated on Raw264.7 macrophages (200 g/mL, 24h) for markers of a classical/inflamed (CD86, MHCII, iNOS) or alternative (CD163, CD206, Arg-1) activation was used to determine nanogel-induced changes to murine macrophage phenotype. Additionally, we evaluated the immunomodulatory function of these synthesized formulations in inflammatory conditions, where macrophages were co-stimulated with IFN-γ at 2ng/ml. Marker expression was compared to control macrophages treated with IFN-γ alone, or IFN-γ in combination with anti-inflammatory cytokine IL4 (80ng/ml), by spectral flow cytometry. Secretion levels of IL6 and TNFa cytokines in supernatants from the exact test conditions were assayed using an appropriate mouse ELISA kit. All statistical analyses were performed using GraphPad Prism.

Results, Conclusions, and Discussions: FT-IR analysis confirmed successful monomer incorporation and complete extraction of unreacted monomers by the disappearance of double bond peaks associated with acrylic monomers. Specifically, comparison of polymer and monomer FTIR spectra revealed two distinct disappearance peaks: =C–H stretch at approximately 1635 cm^-1 and C=C stretch at around 695 cm^-1. Most nanogels had a particle size below 200 nm, exhibiting pH-dependent behavior. However, samples containing DEAEMA aggregated, as evidenced by an increase in particle diameter and polydispersity, at pH 9 and pH 4 for the 75:25 and 25:75 cationic:hydrophobic monomer ratios in the formulations, respectively. Furthermore, all formulations displayed a positive surface charge, which was reduced, approaching neutrality, at pH 9. Remarkably, cytotoxicity assays demonstrated no toxicity for all formulations at both 50 and 200 µg/ml concentrations after 24h treatment, with approximately 80% macrophage viability (Figure 1). This highlights the biocompatibility and dose-independent toxicity of the synthesized nanogels. Flow cytometry analysis revealed a significant difference in the expression of inflammatory CD86 and iNOS markers for nanogels formulated with 75% cationic monomers. At this ratio, specifically treatment with DEAEMA-BzMA, DEAEMA-BMA, and DMAEMA-MMA nanogels increased CD86 expression by an average of 5-fold (p < 0.0001), and iNOS expression was significantly higher (73-fold on average) with formulations containing DEAEMA and DMAEMA-BMA (p < 0.005), when compared with the untreated macrophages. Secreted IL-6 levels showed agreement with the flow cytometry analysis. Macrophages treated with 75% DEAEMA cationic monomer demonstrated, on average, a 33-fold increase in IL-6 secretion, while treatment with 25% cationic monomer resulted in a decrease compared to the untreated group (Figure 2).
In conclusion, we have laid the groundwork for a tailored drug delivery platform for macrophage immunomodulation. Our findings demonstrate the successful synthesis of pH-responsive nanogels and underscore the influence of specific monomer combinations on the immunomodulatory properties of nanogels to macrophages. This study can contribute to the development of a platform for immune therapy with improved targeting responsiveness, which may have translational application for advanced smart drug delivery systems.

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.