X30 - PEG Surface Modification as a Strategy to Attenuate Cationic Nanogel-Induced Acute Toxicity and Inflammatory Activation of Macrophages

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Kevin Phuc Nguyen-Do

Undergraduate Researcher
University of Oklahoma
Oklahoma City, Oklahoma, United States

Co-Author(s)

Mojtaba Ghanbari Mehrabani

Graduate Research Assistant
The University of Oklahoma
Norman, Oklahoma, United States

Primary Investigator(s)

John R. Clegg

Assistant Professor
The University of Oklahoma
Norman, Oklahoma, United States

Introduction: :

Introduction: Nanogels are an emerging class of nanoscale hydrogels, formed by chemically cross-linking hydrophobic or amphiphilic monomers in an aqueous environment. Nanogels (NGs) have potential for targeted drug delivery due to their cytocompatibility, pH responsiveness, immune modulation, and properties that allow for delivery to immune cells such as macrophages. Macrophages are regulators of inflammation and can polarize into pro-inflammatory (“M1-Like”) or anti-inflammatory (“M2-Like”) phenotypes. The modulation between phenotypic states is valuable for cancer treatment and enabling controlled regulation of adoptive cell therapeutics. Due to their tunable features, the design of the nanogels can be engineered by selecting different monomers and grafted polymers. This design versatility enables control over physiochemical aspects such as surface charge, size, degradability, and drug loading capacity, which are critical for delivering specific cell types or locations. Furthermore, their ability to respond to external stimuli, such as pH, allows for site-specific release of therapeutic drugs, minimizing side effects. Our recent studies suggest that nanogels can influence macrophage polarization based on their chemical composition. Our current study focuses on grafting hydrophilic polymer onto a nanogel core composition at varying molecular weights and millimolar ratios, with the goal of evaluating how these variations in composition influence physiochemical properties of nanogels. We hypothesize that increasing the grafting millimolar ratio and molecular weight will enhance nanogel colloidal stability and reduce the surface charge in a predictable manner. This study sets a foundation for future research on immune cells like macrophages, where grafted polymers and millimolar ratios may affect polarization.

Materials and

Methods: : Materials and

Methods: A previously established nanogel core composed of one cationic monomer, 2-(Diethylamino) ethyl methacrylate (DEAEMA), and one hydrophobic monomer, Methyl Methacrylate (MMA) in a 75:25 cationic:hydrophobic molar ratio. This formulation was synthesized and tested within our lab. Onto this core, Poly(Ethylene Methyl Ether Methacrylate (PEG-MA) (mw = 500, 2000, 10000) and Poly(Ethylene Methyl Ether Acrylate (PEG-A) (MW = 2000), were grafted onto it at 3 different millimolar ratios. Nanogels were synthesized through a mini-emulsion technique via ARGET ATRP (Activators Regenerated by Electron Transfer Atom Transfer Radical Polymerization) , with the reaction initiated by Ethylene bis(2-bromoisobutyrate) (EBIB) and Copper (II) Bromide, with bis(2-methacryloyl)oxyethyl disulfide (DSDMA) as a crosslinking agent and Tris(2-pyridylmethyl) amine (TPMA) as a ligand. Following synthesis, nanogels underwent washing, dialysis, and lyophilization. Nanogels were characterized by Dynamic Light Scattering (DLS) at 2mg/ml in 5 mM sodium phosphate and phosphate buffer solution to assess particle size, zeta potential, and colloidal stability under various pH conditions. FT-IR and ¹H NMR spectroscopy were used to confirm chemical composition and grafting millimolar ratio. In vitro cell studies will be conducted utilizing RAW264.7 macrophages to assess the cell cytotoxicity and viability via MTS and LDH assays at nanogel treatment concentrations of 50 ug/mL and 200 ug/mL following incubation for 24 hours. Flow cytometry will be conducted to evaluate macrophage phenotype after 24 hours (200 ug/mL), measuring for inflammation/classically activated markers (CD86, MHC II, iNOS) and anti-inflammation/alternately activated markers (CD163, CD206, Arg-1).

Results, Conclusions, and Discussions:: Results and

Discussion: FT-IR analysis confirmed monomer incorporation and removal of unreacted monomers by the disappearance of double bond peaks associated with acrylic monomers. FT-IR spectra (Figure 1A) showed a disappearance of double bond peaks; =C–H stretch at approximately 1635 cm^-1 (2mg/mL D2O) (Figure 1B) confirmed polymerization, where the peaks at 5 and 6ppm related to vinyl protons disappeared. Additionally, increasing the grafting millimolar ratio resulted in higher signal intensity at ~3.5 ppm, corresponding to -O-CH₂- protons of the grafted polymer utilized. DLS analysis revealed that the particle size remained less than 400 nm (Figure 2A). Colloidal stability was assessed by titrating from pH 3 to 9 in 0.5-unit step increments. At low grafting millimolar ratios, nanogels aggregated between pH 5–7. Increasing grafting improved stability across this range. Molecular weight significantly impacted stability; formulations grafted with PEG-MA and PEG-A (MW=500, 2000) aggregated at lower ratios, whereas PEG-MA (MW=10k) remained stable at all ratios throughout the titration (Figure 2A). This finding demonstrates that increasing molecular weight and grafting millimolar ratio of PEG macromers enhances colloidal stability and surface shielding. Surface Charge measurements during titration (Figure 2B) showed that surface charge decreased with increasing grafted polymer ratio, further supporting the hypothesis. Higher molecular weights of grafted polymer reduced surface charge by a significant amount, which highlights differences between grafted polymers.

Conclusions: We have established a method to synthesize tunable nanogels through a controlled mini-emulsion technique, as well as being able to graft different hydrophilic polymers onto this core. The study highlights how polymer type, molecular weight, and grafting millimolar ratio affect physiochemical properties, such as size, surface charge, and colloidal stability. These insights form a foundation for future toxicity and intrinsic activity studies.

Acknowledgements and/or References (Optional): :