Female 9–10 week old C57BL/6J mice (JAX #000664) and SJL/J mice (JAX #000686) were purchased from the Jackson Laboratory. Mice were housed in an animal facility at Stanford University on a 12-hour light/dark cycle. All procedures were approved by the Stanford University Institutional Animal Care and Use Committee and performed in accordance with approved university protocols.

EAE was induced in 11–12 week old female mice using Hooke kits. For C57BL/6J mice, the MOG_35-55/CFA Emulsion PTX kit (EK-2110) was used. Mice were injected subcutaneously with 100 μL of MOG_35-55/CFA Emulsion in two sites in the back, followed by intraperitoneal injections of 100 ng of pertussis toxin (PTX) four and 24 hours later. For SJL/J mice, the [Ser¹⁴⁰]-PLP_139-151/CFA Emulsion PTX kit (EK-2120) was used. Mice were injected subcutaneously with 50 μL of [Ser¹⁴⁰]-PLP_139-151/CFA Emulsion in four sites in the back, followed by the same PTX regimen.

Liposomes were produced by the ADDReB laboratory of the Stanford Cardiovascular Institute. Briefly, 333 mg of distearoylphosphatidylcholine (DSPC), 67 mg of DSPE-PEG-NH2-3400 (Laysan Bio), and 167 mg of Cholesterol dissolved in chloroform were evaporated and rehydrated with 10 mL of water using a 20-minute path sonicator and probe sonicator for 5 minutes. For the nonPEGylated liposomes, DSPE (18:0/18:0 PE, Echelon Biosciences) was used instead of DSPE-PEG-NH2-3400. To evaluate the giant nanoparticle formation, we used dynamic light scattering (DLS), available in ADDReB (90 Plus Particle Size Analyzer, Brookhaven Instruments Corporation).

The samples for SEM were made by drop-casting 10 μL of liposomal solutions on cleaned Si wafers for 10 minutes. The wafers were then gently washed with deionized water twice. The samples were sputtered in a Denton Desk IV sputterer with 10 nm of gold in Argon plasma and imaged in a Quanta 200 SEM (FEI-ThermoFisher) with Everhart-Thornley secondary electrons detector at 30 kV accelerating voltage and 3000X magnification (Supplementary Figure S1).

To assess the ability of PEG to treat EAE, we treated mice daily with 100 μL of 30% PEG 1500 (w/v) in 75 mM Hepes (pH 8.0) solution (Roche) intraperitoneally starting either day 3 or 13 post-EAE induction. To assess the ability of PEGylated liposomes to treat EAE, we treated mice daily with 150 μL of PEGylated liposomes of varying sizes intraperitoneally starting either day 3 or 16 post-EAE induction. Both treatments were continued until the end of the experiment.

To assess the ability of PEGylated liposomes to prevent relapse in SJL/J mice, we treated mice daily with 150 μL of PEGylated liposomes intraperitoneally starting day 13 post-EAE induction. Treatment was continued until day 25 post-EAE induction.

Classical EAE disease manifestations were scored as follows: 0, normal; 0.5, limp tip of tail; 1.0, limp tail; 1.5, hindlimb inhibition; 2.0, weakness of hindlimbs; 2.5, dragging of hindlimbs; 3.0, complete paralysis of hindlimbs; 3.5, complete hindlimb paralysis and inability to right itself; 4.0, complete hindlimb paralysis and partial forelimb paralysis; 4.5, complete hindlimb and partial forelimb paralysis and no movement around cage; 5.0, moribund. After euthanasia, mice were given a score of 5.0 for the remainder of the experiment. For the spontaneous relapsing-remitting EAE model, relapse was defined as a sustained increase of at least 0.5 in clinical score from the lowest score during remission.

Mice were euthanized with CO₂, and femurs were isolated and crushed in complete Dulbecco’s minimum essential medium supplemented with 10 mM L-glutamine, 100 U/mL penicillin and streptomycin, and 10% fetal bovine serum (cDMEM) using a mortar and pestle. The single-cell suspension was passed through a 70 μm filter, resuspended in ACK Lysing Buffer (Gibco) for 5 minutes, and centrifuged. Cells were then resuspended at a concentration of 2 x 10⁶ cells/mL in cDMEM with 50 ng/mL GM-CSF (PeproTech) for 7 days (37°C and 5% CO₂). Fresh media with 50 ng/mL GM-CSF was added 2 and 5 days later. By day 7, the bone marrow cells were fully differentiated and used for downstream applications.

BMDMs were plated (1 x 10⁵ cells/well) in a 96-well plate, pre-treated with varying levels of PEG or L-PEGylated liposomes for 2 hours, and stimulated with 500 ng/mL LPS (Invitrogen) overnight (37°C and 5% CO₂). After 18 hours, supernatant was collected and incubated with anti-IL-1β (BD Biosciences), anti-IL-6 (BD Biosciences) and anti-TNF-α (BD Biosciences) capture and detection antibodies. Samples were then analyzed using a Fortessa Flow Cytometer. Data were analyzed using FlowJo software.

Mice were euthanized with CO₂ and perfused with 20 mL PBS and 10 mL 4% paraformaldehyde (PFA, Electron Microscopy Sciences) each. Spinal cords were isolated, fixed in 4% PFA for 24 hours, cryopreserved in 30% sucrose (Sigma-Aldrich) for 4–5 days, and frozen in OCT (Fisher Scientific). 14-micron thick sections were processed and stored at -20°C until the day of staining. For immunofluorescence, sections were permeabilized in 0.3% Triton-X100 (Sigma-Aldrich) for 15 minutes at room temperature (RT), blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich), 5% normal goat serum (ThermoFisher), and 5% normal donkey serum (Abcam) for 1 hour at RT, stained with 1:100 rat anti-mouse CD11b (BioLegend, clone M1/70) and rabbit anti-mouse CD4 (clone RM1013, Abcam) overnight at 4°C, incubated with 1:500 goat anti-rat IgG-Cy5 (Abcam) or 1:500 donkey anti-rabbit IgG-TRITC (Abcam) for 1 hour at RT, and then mounted with Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories). For myelin staining, sections were washed in distilled water, dehydrated in 70% ethanol (Sigma-Aldrich), incubated in 0.5% Sudan Black B (Sigma-Aldrich) for 30 minutes, washed in ethanol (70%, 50%, 25%), rehydrated in distilled water, and mounted with 70% glycerol (Sigma-Aldrich). For H&E staining, we used the H&E Stain Kit (Abcam) and mounted with Limonene Mounting Medium (Abcam). Images were then acquired using a Keyence BZ-X1000 microscope (4X or 20X objective lens).

To evaluate potential toxicity, we treated mice daily with 150 μL of L-PEGylated liposomes intraperitoneally for 7 days. Mice were weighed on days 0 and 7. On day 7, the mice were euthanized with CO_2, and the kidneys, heart, brain, and liver were weighed. The livers were isolated and processed for sectioning as described earlier. We used the H&E Stain Kit (Abcam) and mounted with Limonene Mounting Medium (Abcam). Images were then acquired using a Keyence BZ-X1000 microscope (4X objective lens).

Mice were euthanized with CO₂ and perfused with 20 mL PBS each. The spinal cords were then isolated and homogenized in cDMEM using a pre-chilled Dounce homogenizer. The cell suspension was passed through a 70 μm filter and resuspended in 30% Percoll in cDMEM. After centrifugation, cells were labeled with Live/Dead™ Fixable Blue Dead Cell stain (Invitrogen) and Fc block (BioXcell, clone 2.4G2). The cells were then labeled with monoclonal antibodies against cell-specific markers. For intracellular staining of IL-1β, cells were activated in vitro with 500 ng/mL lipopolysaccharide (LPS, Invitrogen) in the presence of 5 μg/mL brefeldin A (BioLegend) for 4 hours, permeabilized in Cytofix/Cytoperm (BD Biosciences), and then stained with an anti-IL-1β monoclonal antibody. Stained cells were fixed with 1% PFA and then analyzed using a Fortessa Flow Cytometer. 123 count eBeads™ Counting Beads (Invitrogen) were used to calculate absolute counts. Data were analyzed using FlowJo software. Antibody information is provided in Supplementary Table S1. Gating strategy for flow cytometry can be found in Supplementary Figure S2.

To assess the effect of treatment on macrophage activation, BMDMs were plated (5 x 10⁴ cells/well), incubated overnight, pre-treated with L-PEGylated liposomes for 4 hours, and then stimulated with 100 ng/mL LPS (Invitrogen) overnight (37°C and 5% CO₂). After 18 hours, the cells were labeled with Live/Dead™ Fixable Blue Dead Cell stain (Invitrogen) and Fc block (BioXcell, clone 2.4G2). The cells were then labeled with monoclonal antibodies against cell-specific markers. For intracellular staining, the cells were permeabilized in Cytofix/Cytoperm (BD Biosciences) and then stained with anti-iNOS and anti-Arg-1 monoclonal antibodies. Stained cells were fixed with 1% PFA and then analyzed using a Fortessa Flow Cytometer. Antibody information is provided in Supplementary Table S1.

To assess the effect of treatment on macrophage size, BMDMs were plated (1 x 10⁶ cells/well), incubated overnight, pre-treated with L-PEGylated liposomes for 4 hours, and then stimulated with 100 ng/mL LPS (Invitrogen) overnight (37°C and 5% CO₂). After 18 hours, the cells were fixed with 4% PFA for 15 minutes, blocked with 1% BSA, 1% goat serum, and 0.1% Fc block for 1 hour, stained with 1:100 rat-anti-mouse CD11b (BioLegend, clone M1/70) for 1 hour, stained with 1:500 goat anti-rat IgG-Cy5 (Abcam) for 1 hour, and then mounted with Vectashield Plus Antifade Mounting Medium with DAPI (Vector Laboratories). Images were then acquired using a Keyence BZ-X1000 microscope (20X objective lens).

All statistical analyses were carried out using GraphPad Prism 10 software. All analyses with 2 groups were tested using unpaired 2-tailed t tests. All analyses with 3 or more independent groups were tested with an ordinary one-way ANOVA with Tukey’s correction for multiple comparisons. All analyses with two independent variables were tested with a two-way ANOVA with Šidák multiple comparisons. A p value less than 0.05 (*) was considered significant; **p < 0.01, ***p < 0.001, and ****p < 0.0001.

We first investigated the effects of systemic PEG prior to EAE development. We induced EAE, treated mice with PEG daily starting day 3 post-induction, and monitored their neurological disease manifestations and weight over time (Figure 1A). Mice treated prophylactically with PEG exhibited no change in clinical score (Figure 1B) or weight change (Figure 1C) compared to untreated mice. Next, we evaluated the effects of systemic PEG on EAE progression after disease manifestations had already developed. We induced EAE, treated mice with PEG daily starting day 13 post-induction, and tracked their neurological disease manifestations and weight over time (Figure 1D). Similarly, therapeutic PEG treatment resulted in no change in clinical score (Figure 1E) or weight change (Figure 1F). These results demonstrate that systemic PEG treatment, whether initiated before or after disease onset, fails to suppress EAE progression.

PEG is known to affect many different cell types, including but not limited to enterocytes (32), epithelial cells (29, 33), and neurons (34). PEGylated nanoparticles also suppress the production of pro-inflammatory cytokines and chemokines by macrophages (35). However, these effects were observed under non-inflammatory conditions, and it remains unclear whether they were attributable to PEG itself or to the lipid components of the nanoparticles. Following EAE induction, monocyte-derived macrophages infiltrated the spinal cord and expressed higher levels of pro-inflammatory cytokine interleukin (IL)-1β (Supplementary Figure S3). Given the lack of efficacy of PEG in dampening EAE (Figure 1), we hypothesized that PEG may not have reached the macrophages in sufficient quantities.

To directly investigate the effects of PEG on macrophages, we pre-treated bone marrow-derived macrophages (BMDMs) with varying concentrations of PEG for four hours and then stimulated them with lipopolysaccharide (LPS). After 18 hours of stimulation, we analyzed the supernatant for pro-inflammatory cytokines using flow cytometry-based cytokine bead array (Figure 1G). PEG pre-treatment led to a decrease in IL-1β, but not IL-6, secretion by the BMDMs (Figure 1H, Supplementary Figure S4). These results demonstrate that PEG can modulate macrophage IL-1β production.

PEGylated liposomes have been widely used for drug delivery, extending the half-life and controlling the release of encapsulated drugs (36, 37). They can deliver drugs to the desired target through passive and/or active targeting, thus avoiding off-target side effects and improving the therapeutic benefits (38). Many factors, such as size, charge, and composition, determine the efficacy and targeting of liposomes (39–41). Previous studies have shown that liposome size can shape immune responses, with larger particles preferentially targeting macrophages (42, 43). Therefore, we examined how PEGylated liposome size influences targeting of macrophages and other immune cells in the spinal cord during EAE. We fluorescently labeled 150, 550, and 700 nm PEGylated liposomes with fluorescein isothiocyanate (FITC) and began treatment two days after disease induction (Figure 2A). Although 150 nm PEGylated liposomes primarily targeted monocytes and macrophages, most of these cells were not FITC-positive (Figure 2B). In contrast, larger PEGylated liposomes (500 and 700 nm) more effectively and selectively targeted CNS-infiltrating monocytes and macrophages, increasing FITC positivity by approximately two-fold compared to 150 nm PEGylated liposomes (Figures 2C, D). Notably, the PEGylated liposomes failed to target CNS-resident microglia, suggesting that PEGylated liposomes primarily target monocytes in the periphery before their infiltration into the spinal cord during EAE.

We also tracked the disease severity of mice treated prophylactically with PEGylated liposomes of different sizes. Treatment with the larger (~700 nm) PEGylated liposomes effectively prevented EAE development, as treated mice exhibited no neurological manifestations (Figure 2E), minimal demyelination (Figures 2F, G), and reduced immune infiltration (Figures 2H, I) at day 15 post-induction compared to control mice. There were no differences in body weight (Supplementary Figure S5A) or the weights of the kidneys, heart, or brain (Supplementary Figures S5B–I) between control and liposome-treated mice. Liposome-treated mice displayed a slight, but not statistically significant, increase in liver weight (Supplementary Figures S5J, K), but the livers showed no signs of hepatotoxicity (Supplementary Figure S5L). Based on these results, we utilized the large macrophage-targeted PEGylated (L-PEGylated) liposomes in subsequent experiments.

When given prophylactically, the L-PEGylated liposomes were superior to PEG alone in preventing EAE development (Figures 1 and 2). Because MS patients only initiate treatment after diagnosis, when the disease is already established (44), we next tested whether L-PEGylated liposomes exhibited therapeutic potential when administered to EAE mice after disease onset, compared to similarly sized non-PEGylated liposomes or PBS (Figure 3A). Only mice treated with L-PEGylated liposomes exhibited a decrease in neurological manifestations (Figure 3B), a reversal of weight loss (Figure 3C), and reduced demyelination (Figures 3D, E) and immune infiltration (Figures 3F, G). We further evaluated their efficacy in the relapsing-remitting EAE model in SJL/J mice. Following EAE induction, mice were treated daily with L-PEGylated liposomes from days 13 to 25 post-induction. Mice treated with the L-PEGylated liposomes remained relapse-free throughout treatment and only started relapsing 8 days after cessation of treatment (Supplementary Figure S6).

The development and progression of EAE are driven by increased infiltration of immune cells, particularly monocytes/macrophages and CD4+ T cells, into the spinal cord (45, 46). To investigate the effect of L-PEGylated liposomes on immune cell infiltration, we isolated the spinal cords from mice treated with these liposomes following disease onset. Only mice treated with L-PEGylated liposomes exhibited a significant decrease in the absolute number of infiltrating immune cells (Figure 3H), including macrophages (Figure 3I; Supplementary Figures S7A, B). Despite preferentially targeting monocytes and macrophages (Figure 2D), L-PEGylated liposomes also significantly reduced the absolute number of CD4+ T cells (Figure 3J; Supplementary Figures S7C, D). These results demonstrate that macrophage-targeted PEGylated liposomes administered following the onset of active disease reduce immune cell infiltration and reverse disease manifestations.

Macrophages are key orchestrators of the inflammatory response of other immune cells, like T cells, through their potent antigen presentation capabilities and cytokine secretion (9–11). Because pro-inflammatory cytokines such as IL-1β play central roles in promoting neuroinflammation and demyelination during EAE (47, 48), we first examined cytokine expression in spinal cord-infiltrating macrophages following treatment. Macrophages isolated from mice treated with L-PEGylated liposomes displayed a significant reduction in intracellular IL-1β following ex vivo stimulation (Figure 4A). In contrast, MHC-II expression on these cells remained unchanged (Figure 4B), suggesting that antigen presentation capacity was largely unaffected. These findings indicated that the therapeutic efficacy of L-PEGylated liposomes may lie in its modulation of cytokine production, rather than changes in antigen presentation, in macrophages.

To determine whether this reflected a direct effect on macrophages, we next assessed the impact of L-PEGylated liposomes on BMDMs in vitro. Pretreatment of BMDMs with L-PEGylated liposomes followed by overnight LPS stimulation significantly reduced IL-1β secretion (Figure 4C), whereas IL-6 and TNF-α production were unchanged (Supplementary Figure S8). We then evaluated whether these effects were accompanied by alterations in macrophage activation phenotype. The liposome-treated BMDMs expressed similar levels of MHC-II (Figure 4D) and exhibited modest decreases in canonical M1 markers, including CD86, CD40, and iNOS (Figures 4E–G), as well as M2 markers, including CD206 and Arg-1 (Supplementary Figure S9), compared to untreated BMDMs. Cell size, which can influence macrophage polarization (49), was also unchanged (Supplementary Figure S10). Together, these data demonstrate that macrophage-targeted PEGylated liposomes do not broadly reprogram macrophage activation but instead selectively suppress IL-1β production in macrophages, which may contribute to their therapeutic efficacy.