X40 - Covalent Cross-linking Enhances Stability and Retains Functionality of Immune Polyelectrolyte Multilayers

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Marian Ackun-Farmmer, PhD

Assistant Professor
Georgia Institute of Technology
Hyattsville, Maryland, United States

Last Author(s)

Christopher Jewell

Professor
Robert E. Fischell Institute for Biomedical Devices/University of Maryland; Veterans Affairs MD Healthcare System/Department of Veterans Affairs, United States

Primary Investigator(s)

Christopher Jewell

Professor
Robert E. Fischell Institute for Biomedical Devices/University of Maryland; Veterans Affairs MD Healthcare System/Department of Veterans Affairs, United States

Introduction: : Multiple sclerosis (MS) is an autoimmune disease characterized by immune cell attack of the protective myelin sheath surrounding neurons in the central nervous system. Towards the goal of treating MS, many therapeutic strategies have emerged; however, these therapeutic options are non-curative and cannot distinguish between healthy and diseased immune cells. One strategy for treating MS is exploiting the inherent interactions between innate and adaptive immune cells in the body. Toll-like receptors (TLRs), which traditionally recognize pathogen-associated molecular patterns (PAMPs), are upregulated in preclinical MS models and MS patients. To achieve immune tolerance without broad immunosuppression, we previously developed self-assembled carriers built entirely from immune cues – termed immune polyelectrolyte multilayers (iPEMs) to co-deliver self-antigen - myelin oligodendrocyte glycoprotein (MOG) and TLR9 antagonist - GpG. GpG competes with TLR9 ligand binding to downregulate innate cell activation and has been shown to reduce inflammation and improve disease severity in a preclinical MS model when co-delivered with MOG in iPEMs. Towards the goal of developing next-generation combinatorial tolerance therapies based on trigger-responsive release of cargo, we pursued pilot studies to cross-link iPEMs with a covalent cross-linker and load Rapamycin (Rapa), a small molecule drug well-documented to expand regulatory T cell (TREGS). Specifically, we hypothesized that covalent cross-links could be used to stabilize MOG and GpG cargo without impeding the biological properties of each component, and Rapa release would promote robust tolerogenic conditioning of immune cells.

Materials and

Methods: : Immune polyelectrolyte multilayers were synthesized via electrostatic assembly of arginine modified MOG peptide (MEVGWYRSPFSRVVLHLYRNGKRRR) and GpG (5’-T*G*A*C*T*G*T*G*A*A*G*G*T*T*A*G*A*G*A*T*G*A*-3’) on calcium carbonate (CaCO3) sacrificial templates. After three bilayer depositions, varying glutaraldehyde (GA) cross-linking solutions were added to iPEMs to achieve covalent cross-links. Subsequently, the CaCO3 sacrificial core was removed using EDTA, followed by multiple washes with ddH2O via centrifugation (Fig. 1A), and cross-linking density was assessed with Fluoraldehyde™ o-Phthaldialdehyde. Since iPEMs are composed entirely of bioactive immune cues that must maintain their function upon cross-linking, we next tested the functional capabilities of iPEMs. TLR9 reporter cells were treated with soluble CpG (TLR9 agonist) and iPEMs containing GpG (TLR9 antagonist), and secreted embryonic alkaline phosphatase (SEAP) secretion was measured 16 hours later by spectrophotometry at 650 nm using a Tecan Spark Multimode Microplate Reader. In parallel, splenic DCs treated with CpG and iPEMs for 24 hours were co-cultured with eFluor450-labeled 2D2 T cells that recognize MOG for 72 hours, and proliferation was measured using flow cytometry. In a subset of studies, drug-loading was accomplished by adding Rapa to iPEMs on cores, and GA was added before core removal and subsequent washes. Rapa loading was quantified using HPLC.

Results, Conclusions, and Discussions:: We found that cross-linking density could be modulated by adjusting GA concentration and incubation times, without impacting the functional capabilities of iPEMs. In reporter assays, TLR9 signaling was comparably restrained by cross-linked and non-cross-linked iPEMs across all doses, indicating that cross-linking does not interfere GpG-mediated restraint of CpG-driven inflammation (Fig. 1B). In co-culture studies, we found that > 80% of 2D2 T cells proliferated in response to soluble and iPEM containing MOG (Fig. 1C). Notably, across a range of iPEM doses, up to 5 divisions were observed at the lowest iPEM dose tested, and up to 4 divisions were observed for the highest dose, indicating that MOG in cross-linked iPEMs remains available for presentation to antigen-specific T cells (Fig. 1C). Finally, we evaluated Rapa loading efficiency. We observed comparable loading between non-cross-linked iPEMs and those cross-linked with 2%, 5%, and 10% GA concentrations. However, iPEMs cross-linked at 20% GA exhibited reduced loading efficiency, suggesting a limitation of drug loading at high cross-linking densities (Fig. 1D). Altogether, these studies demonstrate that chemical cross-links can be effectively incorporated without compromising iPEM functional activity. This work lays the groundwork for continued research in the Ackun-Farmmer lab, which is dedicated to exploiting advanced cross-linking strategies, including trigger-responsive cross-links, to precisely control immune cue kinetics and facilitate the encapsulation of additional immunomodulatory signals.

Acknowledgements and/or References (Optional): :