Immune Polyelectrolyte Multilayers Retain Functional Capabilities Post-Covalent Cross-linking

Introduction:: Multiple sclerosis (MS) is an autoimmune disease in which autoreactive lymphocytes (e.g., T, B, and natural killer cells) attack the protective myelin sheath surrounding neurons in the central nervous system. Current MS treatments are non-curative and cannot distinguish between healthy and diseased immune cells. Antigen-presenting cells (APCs) – cells that present antigen and costimulatory signals to drive the differentiation of naïve T cells - are altered in MS and are potential targets for use in new therapies. For example, toll-like receptors (TLRs), which traditionally recognize pathogen-associated molecular patterns (PAMPs), are upregulated in preclinical MS models and MS patients. Thus, an emerging therapeutic strategy is to deliver distinct immune signals, such as TLR antagonists and self-antigens, to achieve immune tolerance without broad immunosuppression. To this end, 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. We showed that MOG/GpG iPEMs reduced inflammation and improved disease severity in a preclinical MS model while modestly expanding a key regulatory T cell population-regulatory T cells (T_REGS). Herein, we pursued the co-delivery of Rapamycin (Rapa), a small molecule drug well-documented to expand T_REGS, with self-antigen to target innate and adaptive immune cells involved in MS, towards the goal of promoting durable immune tolerance. Specifically, we hypothesized that Rapa loading could be achieved in iPEMs, and covalent cross-links could be used to stabilize MOG and GpG cargo without disrupting cargo loading and impeding Rapa loading.

Materials and Methods::
Immune polyelectrolyte multilayers (iPEMs) 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 assessment via the 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 using cell-culture techniques. To this end, 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 prior to 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. Specifically, we show that TLR9 signaling is similarly restrained between cross-linked and non-cross-linked iPEMs at distinct iPEM doses, indicating that cross-linking does not hinder GpG-mediated restraint of CpG-driven inflammation (Fig. 1B). Next, we tested if MOG maintained its ability to engage its cognate receptor on primary myelin-specific T cells. In MOG, CpG + MOG, and GpG + MOG soluble controls, > 80% of T cells proliferated as indicated by successive dilution of the fluorescence dye during each round of MOG-induced T cell division (Fig. 1C). Notably, when co-cultures were treated with cross-linked iPEMs using 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 tested, indicating that MOG in cross-linked iPEMs is available for presentation through MHC molecules to the antigen-specific T cells (Fig. 1C). Finally, we tested if cross-linking limits Rapa loading in iPEMs. We observed comparable loading between non-cross-linked iPEMs and 2%, 5%, and 10% GA iPEMs (Fig. 1D). Notably, Rapa loading in 20% GA iPEMs resulted in loading efficiency of ~10%, suggesting a potential limitation in highly cross-linked iPEMs for drug loading (Fig. 1D). Altogether, this is the first time we have shown that incorporating chemical cross-links into precisely controlled self-assembled immune cues - self-antigen and toll-like receptor antagonist – can be accomplished without hindering the function of the assemblies. Most importantly, we show that small molecule Rapa is similarly loaded into cross-linked and non-cross-linked iPEMs, which allows for simultaneous downstream targeting of innate and adaptive immune cells to drive robust immune tolerance in the context of MS. Ongoing efforts include testing Rapa-loaded iPEM efficacy in preclinical MS models and testing if Rapa loaded iPEMs promote memory-like T cells.




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