Peptide-based lipid nanodiscs suppress eosinophil
recruitment and chemotaxis

Alankrita Rani, Denys Balandin, Rohith Ravi, Julia Teppan, Ivan Vidakovic, Melanie Kienzl, Thomas Bärnthaler, Michael Holzer, Lamija Zijadic, Akos Heinemann, et al.

► To cite this version:

Alankrita Rani, Denys Balandin, Rohith Ravi, Julia Teppan, Ivan Vidakovic, et al. Peptide-based lipid nanodiscs suppress eosinophil recruitment and chemotaxis. Journal of Controlled Release, 2025, 385, pp.114060. 10.1016/j.jconrel.2025.114060. hal-05412244

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# Peptide-based lipid nanodiscs suppress eosinophil recruitment and chemotaxis

Alankrita Rani<sup>a</sup>, Denys Balandin<sup>b</sup>, Rohith Ravi<sup>c</sup>, Julia Teppan<sup>a</sup>, Ivan Vidakovic<sup>b</sup>, Melanie Kienzl<sup>a</sup>, Thomas Bärnthaler<sup>a</sup>, Michael Holzer<sup>a</sup>, Lamija Zijadic<sup>a</sup>, Akos Heinemann<sup>a</sup>, Katharina Jandl<sup>a</sup>, Eva Böhm<sup>a</sup>, Karin Kornmueller<sup>b,d</sup>, Mounir Tarek<sup>c</sup>, Ruth Prassl<sup>b</sup>, Gunther Marsche<sup>a,d,*</sup>

## ARTICLE INFO

### Keywords:

- HDL
- Apolipoprotein
- Mimetic peptide
- Eosinophils
- Inflammation
- Asthma
- Migration

## ABSTRACT

Apolipoprotein A-I (ApoA-I) mimetic peptides hold promise for treating inflammatory lung diseases, yet their impact on eosinophils, key mediators of asthma and allergic airway inflammation, remains underexplored. We prepared self-assembling synthetic high-density lipoprotein (sHDL) nanoparticles using the bihelical ApoA-I mimetic peptide 4F-P-4F and phospholipids via microfluidics, yielding stable, nanodisc-shaped structures, as confirmed by *in vitro* and *in silico* analyses. These sHDL nanoparticles potently inhibited eotaxin-induced eosinophil migration *in vitro*, an effect reversed by blocking HDL receptors SR-BI and ABCA1. Mechanistically, sHDL promoted cholesterol efflux, disrupted CCR3 internalization, and modulated ERK1/2, STAT1, and STAT3 signaling. In an IL-5 transgenic mouse model, sHDL significantly reduced eosinophil infiltration in bronchoalveolar lavage. These results show that sHDL nanoparticles suppress eosinophil migration through targeted receptor interactions and signaling modulation, providing a mechanistic basis for further drug development.

## 1. Introduction

High-density lipoproteins (HDL) are heterogeneous nanoparticles composed of a variety of lipids, enzymes, and proteins, acting as important regulators of metabolic and immunological health. Reduced HDL levels, altered composition, or impaired function are linked to inflammatory and infectious diseases [1,2]. Alterations in HDL quality and cargo characteristics have prognostic and therapeutic relevance in cardiometabolic disorders [3,4], infection [2,5], allergy [6], cancer [7], and auto-immune diseases [8]. Several protective functions of HDL are linked to its interaction with immune cells. By altering the cholesterol content of membrane microdomains (lipid rafts), HDL modulates the function of innate immune cells such as monocytes, macrophages [9], polymorphonuclear leukocytes [10], and lymphocytes [11,12]. Lipid rafts organize signaling proteins, and their disruption by HDL suppresses immune cell activation, cytokine production, and inflammation [13,14]. Besides its well-established role in atherosclerosis, immunomodulatory

effects of HDL have been implicated in normal lung development, acute lung injury, asthma, chronic obstructive pulmonary disease, lung cancer, pulmonary fibrosis, pulmonary hypertension, and viral lung infections [15].

HDL-associated apolipoprotein A-I (ApoA-I) drives reverse cholesterol transport and attenuates immune responses [10,16]. Apart from coronary artery disease, ApoA-I has indispensable roles in pulmonary health, such as lung development and protection against infection [17–19]. It has also been shown that APOA-I gene-deletion exacerbates immune cell infiltration and airway hyper-responsiveness [20].

Given its potent anti-inflammatory effects and favorable structural characteristics, ApoA-I has become the primary scaffold for developing reconstituted HDL (rHDL) therapeutics [3,21]. These rHDL nanoparticles are designed to enhance key functions of native HDL, including increased cholesterol efflux capacity and improved binding to bacterial lipopolysaccharides. Currently, rHDL is undergoing clinical evaluation for the treatment of cardiovascular disease [9,22] and sepsis [23].

<footnote><sup>a</sup> Division of Pharmacology, Otto Loewi Research Center, Medical University of Graz, Neue Stiftingtalstrasse 6, 8010 Graz, Austria</footnote>

<footnote><sup>b</sup> Division of Medical Physics and Biophysics, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstrasse 6, 8010 Graz, Austria</footnote>

<footnote><sup>c</sup> University of Lorraine, Laboratory of Theoretical Physics and Chemistry (LPCT), French National Centre for Scientific Research (CNRS), F-54000 Nancy, France</footnote>

<footnote><sup>d</sup> BioTechMed-Graz, 8010 Graz, Austria</footnote>

<footnote>* Corresponding author at: Medical University of Graz, Neue Stiftingtalstraße 6, 8010 Graz, Austria.
E-mail address: gunther.marsche@medunigraz.at (G. Marsche).</footnote>
However, challenges such as protein purification constraints and high production costs limit their application [3,9,21]. To address these challenges, functional mimetics of ApoA-I, known as ApoA-I mimetic peptides, have been developed against inflammation and coronary artery disease [3,9,21,24,25]. These amphipathic helices, which vary in length from 18 to 37 amino acids, mimic the cholesterol efflux function of HDL, exhibiting diverse protective effects against inflammation and coronary artery disease. [9]. Besides their protective role in cardiovascular disease, ApoA-I mimetic peptides have been shown to mitigate lung disease in animal models of pulmonary inflammation [26-28]. Lipidation is proposed to overcome limitations of mimetic peptides, such as aggregation and peptidase susceptibility, thereby increasing their therapeutic potential [29,30]. When ApoA-I mimetic peptides are combined with phospholipids, they form uniform, stable synthetic HDL (sHDL) nanodiscs with improved half-life and stability [3,29]. Critically, these sHDL mimetics often exhibit greater efficiency in their therapeutic actions compared to native HDL, due to their optimized structure and consistent composition [3,29].

Free peptides tend to aggregate and behave unpredictably in biological environments due to their amphipathic properties. With their more consistent and stable formulation, sHDL nanodiscs are particularly well-suited for advanced biomedical investigation [3,9,29,30]. sHDL nanodiscs show promise in treating cardiometabolic diseases [31], however, their effects on allergic inflammation and immune modulation mechanisms remain underexplored.

Eosinophils are vital in combating parasites and allergens [32], yet their overactivation drives lung tissue damage, manifesting as airway hyperresponsiveness, mucus hypersecretion, and tissue remodeling - in conditions like eosinophilic asthma, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, and eosinophilic granulomatosis with polyangiitis [32-34]. These eosinophilic diseases impose a significant health burden [35,36], underscoring the need for targeted therapies.

Migration of eosinophil to tissues is a hallmark of several hyper-
osinophilic diseases and have been associated with worse clinical
outcomes in asthma [37,38], ulcerative colitis [39] and chronic rhino-
sinusitis [40]. Blocking eosinophil chemotaxis, and thus its migration to
the tissues, is an important target for anti-allergic drugs currently used
to treat hypereosinophilic diseases [41].

This study investigates the effect of sHDL nanodiscs on eosinophil migration. We used bihelical, proline-linked dimeric 4F peptide (4F-P-4F) for its structural attributes [42-44] and lipidated it with dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to enhance bioavailability and stability. DMPC exhibits optimal fluidity at physiological temperatures (Tm ~ 23 °C) and has superior anti-inflammatory effects, making it an ideal choice for enhancing the stability of the dimeric 4F peptide [31,45,46]. Moreover, DMPC has been used as an effective stabilizer for vaccine development without any significant disadvantage over natural phospholipid sources [47]. We employed *in vitro* and *in silico* techniques to confirm the structure and stability of the nanodiscs, and assessed their effects using both *in vitro* and *in vivo* models of eosinophil migration.

## 2. Materials & methods

2.1. Synthetic HDL synthesis

4F-P-4F peptide (DWFKAFYDKVAEKFKEAFPDWFKAFYDKVAEKFK EAF) was synthesized by GenScript Biotech (Rijswijk, Netherlands). sHDL nanodiscs were prepared using a microfluidic approach. The system consisted of a herringbone profiled micromixer (Little Things Factory GmbH, Elsoff, Germany) and two programmable syringe pumps NE-1002× (NewEra Pump Systems, Inc., Farmingdale, USA). Before assembly, the microfluidic chip was washed with ethanol, followed by water. The peptide-to-lipid molar ratio was set to 1:20. Reagents were mixed immediately before loading into the syringe setup. The borosilicate microfluidic chip, consisting of three channels, two inlets, and one

outlet, was used. Peptide and DMPC solutions were injected at flow rates
of 5 mL/min and 1 mL/min, respectively. The final sample was
concentrated using 10 K MWCO (Sigma, Vienna, Austria) ultracentrif-
ugal filters to remove free peptides. This concentration step was
repeated three times to remove residual ethanol, and the sample was
then reconstituted in PBS. Phospholipid content was determined using
the Phospholipids FS kit (DiaSys Diagnostic Systems GmbH, Holzheim,
Germany) according to the manufacturer's instructions. Protein con-
centration was determined by measuring absorbance at 280 nm using a
NanoPhotometer P-330 (Implen, Munich, Germany).

## 2.2. Isolation of human HDL

Native HDL was isolated from healthy human donors *via* density gradient ultracentrifugation, a method previously described [48] and approved by the Ethics Committee of the Medical University of Graz (17-291 ex 05/06). To do this, plasma density was adjusted to 1.24 g/ mL with potassium bromide. A two-step density gradient was then created in centrifuge tubes (16 × 76 mm, Beckman Instruments) by layering the density-adjusted plasma (1.24 g/mL) beneath a NaCl-density solution (1.063 g/mL). Tubes were sealed and centrifuged at 90,000 rpm for 4 h in a 90Ti fixed-angle rotor (Beckman Instruments, Krefeld, Germany). The HDL-containing band was collected post-centrifugation and desalted using PD10 columns (GE Healthcare, Vienna, Austria).

2.3. *Dynamic light scattering*

Particle size distribution of the sHDL particles was determined using Dynamic Light Scattering with a Litesizer 300 (Anton Paar GmbH, Graz, Austria). The instrument employs a 40 mW laser operating at a wavelength of 658 nm. sHDL samples were diluted to a final protein concentration of 0.2 mg/mL in 10 mM HEPES buffer (pH 7.4) and loaded into disposable semi-micro cuvettes (Eppendorf, Hamburg, Germany). Scattered light intensity measurements were performed in triplicate at a 90° angle. Data analysis was performed using the Kalliope software (Anton Paar GmbH, Graz, Austria). Results are presented as mean values, with homogeneity of the size distribution characterized by the polydispersity index and reported as D10, D50, and D90 values.

2.4. *Transmission electron microscopy*

To enhance sample contrast for transmission electron microscopy (TEM), samples were subjected to negative staining. CF400-Cu-50 carbon film grids with 400 mesh copper were glow-discharged using a Pelco easiGlow (Ted Pella, USA) to promote even sample distribution. 5 µL of the sample were pipetted onto the grid and incubated for 1 min. The excess sample was gently removed using filter paper. Subsequently, 5 µL of centrifuged 1 % uranyl acetate solution was applied to the grid, incubated for 1 min, and then removed. This staining step was repeated, followed by air-drying. TEM imaging was performed using a Zeiss EM900 transmission electron microscope (Carl Zeiss AG, Jena, Germany) operating at an acceleration voltage of 80 kV. Digital images were analyzed with Image SP software (Version 1.2.7.96, SysProg TRS).

2.5. Circular dichroism

Circular dichroism (CD) spectra were measured using a Jasco J-1500 spectropolarimeter (Jasco Inc., Easton, USA). Measurements were performed from 190 to 260 nm with a scan speed of 50 nm/min, a digital integration time of 2 s, and a bandwidth of 2 nm. Samples were prepared at a final concentration of 0.2 mg/mL for the CD measurements. Measurements were conducted in a 1 mm path-length quartz cuvette (Hellma GmbH & Co. KG, Müllheim, Germany) at 37 °C. For each sample, 10 consecutive measurements were acquired.

To evaluate the secondary structure content derived from molecular
dynamics (MD) simulations and facilitate direct comparison with experimentally acquired CD spectra, far-UV CD spectra were computed from the simulation trajectories. For nanodisc systems, structural snapshots of the peptides around the lipid bilayer were extracted at 10 ns intervals from the last 100 ns of the MD simulations. In the case of peptides only simulation, snapshots were extracted every 5 ns from the last 100 ns. The resulting PDB structures were submitted to the PDBMD2CD web server [49], which utilizes an empirically derived method to predict CD spectra from atomic coordinates.

The deconvolution was performed using the CDSSTR algorithm with the DichroWeb platform [50,51] with reference set SP175, allowing quantitative estimates of secondary structural elements. The average values were plotted as delta epsilon units. Additionally, the Mean Residue Ellipticity values (MRE [θ] in deg. cm² dmol⁻¹ residue⁻¹) at 222 nm were extracted from the CD spectra and the fractional helicity was calculated and comapred, based on the following equation [52]:

$$fH = \frac{[\theta]_{222nm} - 3000}{-36000 - 3000}$$

## 2.6. Molecular dynamics simulations

The ApoA-I mimetic sHDL nanodiscs atomistic MD simulations were carried out using the following protocol: First, the peptides monomers were generated with PEP-FOLD [53], (Supplementary Fig. 1) and then their N-/C-termini were modified by acetylation and amidation using CHARMM-GUI [54]. Based on previous Nuclear Magnetic Resonance (NMR) studies of ApoA-I mimetic peptides [21,55-57], the 4F-P-4F peptides were arranged as dimers in an anti-parallel configuration with most charged residues pointing outward forming therefore an amphipathic construct (Supplementary Fig. 2). The dimers (number depending on the size of the nanodiscs, Supplementary Table 1) were positioned in a circular ring (with 0.5 nm spacing), around an equilibrated DMPC lipid bilayer disk like patch, with their hydrophobic face surrounding the hydrophobic domain of the bilayer patch. The peptide-lipid particles were then solvated in the solvent box at 0.15 M NaCl concentration. All simulations were carried out using the CHARMM36m force field [58] and the open-source software suite GROMACS [59]. After standard minimization protocols, we performed short (a few nanoseconds) constant-temperature, constant-volume (NVT) MD simulations, during which restraints on the peptide backbones and lipid head groups were gradually released. For each system studied, MD simulations employing a 4 fs timestep using Hydrogen Mass Repartitioning [60], were then conducted at constant temperature (303K) and constant pressure (1 atm), using a Parrinello-Rahman barostat [61] and Nosé-Hoover thermostat [62] to probe the structure, dynamics, and stability of the sHDL nanodiscs. Consistent with state-of-the-art MD simulations, the Particle Mesh Ewald [63] was used to account for long-range electrostatics under 3d periodic boundary conditions. We utilized the same protocol to simulate the peptides in water. Further, the specifications of each nanodisc system built are mentioned in Supplementary Table 1.

## 2.7. Isolation and preparation of human eosinophils

Human polymorphonuclear leukocytes (PMNLs), comprising neutrophils and eosinophils, were isolated from peripheral blood obtained from healthy donors. Blood collection was performed according to a protocol approved by the Ethics Committee of the Medical University of Graz (EK17-291 ex 05/06), and all donors provided written informed consent. Whole blood anticoagulated with 3.8 % sodium citrate was centrifuged to separate platelet-rich plasma. Erythrocytes were sedimented using 6 % dextran for 30 min at room temperature. The leukocyte-rich upper phase was layered onto a histopaque gradient (Sigma-Aldrich, Darmstadt, Germany) and centrifuged at 400 xg for 20 min. After removing peripheral blood mononuclear cells (PBMCs), saline, and Histopaque, PMNLs were washed with PBS containing 0.1 %

bovine serum albumin, 10 mM HEPES, and 10 mM glucose (pH 7.4), without Ca²⁺ and Mg²⁺. The remaining red blood cells were lysed with 0.2 % saline. Eosinophils were isolated from the PMNL fraction using a human eosinophil isolation kit (Miltenyi Biotec, Gladbach, Germany) based on negative magnetic selection. Cell purity was assessed by morphological analysis with Kimura staining, typically exceeding 95 %. All functional assays with eosinophils were performed in assay buffer (PBS with Ca²⁺ and Mg²⁺ containing 0.1 % bovine serum albumin, 10 mM HEPES, and 10 mM glucose, pH 7.4), unless otherwise specified.

## 2.8. Eosinophil chemotaxis assay

Purified human eosinophils were pre-treated with vehicle, the 4F-P-4F peptide, or synthetic high-density lipoprotein at specified concentrations for 30 min at 37 °C. The incubation medium was either assay buffer or RPMI media supplemented with 10 % fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, USA). Cells were then allowed to migrate toward 3 nM eotaxin-1 (ImmunoTools, Friesoythe, Germany) in an HTS Transwell 96-well plate with a 5-µm pore size polycarbonate membrane (Corning, Berlin, Germany), seeding 70,000 cells per well, for 1 h at 37 °C in 5 % CO₂. For blocking antibody experiments, eosinophils were pre-incubated with anti-SR-BI and anti-ABCA1 antibodies (Novusbio, Bio-Techne, Oxon, England) and the assay was performed in the presence of the blocking antibodies (30 min pre-incubation without further washing) as described previously [64]. All the cells were collected from the lower compartment, fixed with 100 µL fixative solution, and analyzed by flow cytometry on a BD FACS Canto II. Cell counts were determined using FlowJo 10.8.1.

## 2.9. C-C chemokine receptor type 3 (CCR3) staining

Purified human eosinophils were pre-treated with assay buffer containing vehicle or 25 µg/mL sHDL for 30 min at 37 °C and then stimulated with 10 nM eotaxin-1 for 30 min. In additional control experiments, eosinophils were treated with 8 µM phenylarsine oxide (PAO), an inhibitor of CCR3-internalization [65,66], 4F-P-4F (25 µg/mL) and isolated human HDL (25 µg protein/mL). Cells were subsequently placed on ice, washed, and stained in PBS + 2 %FBS with PE-anti-CCR3 antibody (BioLegend 310,705, San Diego, California, USA) for 30 min at 4 °C in the dark. Cells were then washed with PBS and fixed until acquisition. The surface expression of CCR3 was quantified as the geometric mean of fluorescence in the PE (B585-42 A) channel on a BD FACS Canto II and analyzed using FlowJo 10.8.1.

## 2.10. Cholesterol efflux from human eosinophils

Approximately 500,000 eosinophils per treatment condition were labeled with [³H]-cholesterol (0.5 µCi/mL) for 3 h at 37 °C in a 5 % CO₂ incubator. The labeling medium consisted of RPMI media supplemented with 1 % penicillin/streptomycin and 2 µg/ml of the acyl-CoA cholesterol acyltransferase inhibitor (Sigma, Darmstadt, Germany). Cells were washed three times with assay buffer (PBS with Ca²⁺ and Mg²⁺, 10 mM HEPES, 10 mM glucose, 0.1 % bovine serum albumin, pH 7.4) by centrifugation at 500 xg for 5 min. Subsequently, cells were treated with HDL, sHDL, or 4F-P-4F for 1 h at 37 °C in a 5 % CO₂ incubator. The treatment medium consisted of RPMI media supplemented with 1 % penicillin/streptomycin and 2 µg/mL ACAT inhibitor. After the efflux period, cells were pelleted, supernatants collected, and cell pellets were lysed with 0.1 % SDS in 0.3 N NaOH to assess cell-associated [³H]-cholesterol. For blocking antibody experiments, eosinophils were preincubated with anti-SR-BI and anti-ABCA1 antibodies (Novusbio, Bio-Techne, Oxon, England) and the assay was performed in the presence of the blocking antibodies (30 min pre-incubation without further washing) as described previously [64].

All assays were performed in triplicate, with each sample analyzed in duplicate. Vehicle-treated (PBS) samples served as controls. Cholesterol
efflux capacity was quantified as the ratio of radioactivity in the medium to the total radioactivity (medium plus cells). Specific cholesterol efflux capacity values were then derived by subtracting the background efflux observed in the vehicle control (typically <1 %) from all experimental sample values.

## 2.11. Phosphokinase array

Human proteome profiler array kit (R&D Systems, Minneapolis, USA) was employed to assess the effect of sHDL treatment on the phosphorylation states of multiple kinases in eosinophils. Pooled purified human eosinophils from three individual donors were treated in assay buffer containing vehicle or sHDL nanodiscs, followed by eotaxin-1 stimulation (10 nM), for 5 min at 37 °C. Subsequent assay steps were performed according to the manufacturer's instructions, with overnight incubation of the array buffer membranes with the lysates at 4 °C. Membranes were processed and visualized on a Bio-Rad ChemiDoc™ MP Imaging System, and the signal intensity of duplicate spots was quantified using ImageLab v5.2.

## 2.12. Eosinophil viability assay

Apoptosis-necrosis assay was conducted to assess eosinophil viability using Annexin V and Propidium Iodide (PI) staining. Approximately 50,000 cells per condition were incubated in assay buffer with the vehicle, a positive control (20 % DMSO), or the indicated concentration of sHDL nanodiscs at 37 °C, 5 % CO₂ for 4 h. Following this, the cells were stained with an anti-Annexin V-APC antibody (BioLegend, San Diego, USA) and PI (Sigma, Vienna, Austria). The staining procedure involved diluting the antibodies at 1:50 for Annexin V and 1:20 for PI. Annexin V staining was performed for 30 min at 4 °C, while PI staining was carried out for 1 min at room temperature. Gating strategy: Early apoptosis (Annexin V+ and PI-), Late apoptosis (Annexin V+ and PI+), Necrosis (Annexin V- and PI+), and live cells (Annexin V- and PI-). Cells were acquired without fixation, and live/dead cells were analyzed with FlowJo 10.8.1, where double negative cells for both PI and Annexin V signal were considered viable.

## 2.13. In vivo migration assay

In vivo, eosinophil migration was induced by intranasally administering 4 µg recombinant human eotaxin-2 (R&D Systems, Bio-Techne, Dublin, Ireland) to 7–10-week-old male and female interleukin-5 transgenic (IL-5 Tg) mice (BALB/c background), as previously described and validated [66–68]. The model is specifically designed to study the role of eosinophils in severe asthma presenting with significant pulmonary eosinophilia upon eotaxin stimulation [66,68]. Mice were intravenously administered vehicle or 75 mg/kg sHDL nanodiscs, 30 min before the eotaxin challenge. Broncho-alveolar lavage was collected 4 h after the experiment began and stained for immune cell composition analyses. Gating strategies for the analysis of immune cells included lymphocytes (CD11c-/CD11b-), eosinophils (Siglec F+/CD11b+/CD11c-), alveolar macrophages (Siglec F+/CD11c+), and neutrophils (Siglec F-/CD11b+/Ly6G+), as described previously [67]. The experimental procedure used in this study was approved by the Austrian Federal Ministry of Science, Research, and Economy (protocol numbers: 2023–0402.980, BMWFW-66.010-0041WF/II/3b/14). The experimental procedure conforms to the European Community's Council Directive (2022–0626.093).

## 2.14. Statistical analyses

All data are shown as mean ± SEM for separate experiments. Experiments were repeated with different human donors and mice as indicated in individual experiments. Statistical analyses were performed with GraphPad Prism Version 10.2.2. Comparisons of groups were

performed using statistical tests specified individually for each experiment, and significances were accepted at *p* < 0.05; values are indicated for each experiment.

# 3. Results

## 3.1. Characterization of sHDL nanodiscs

We synthesized sHDL formulations using a microfluidic approach (Fig. 1A). Based on Dynamic Light Scattering (Fig. 1B) the formulation size was determined as 13.2 nm. The polydispersity index (PDI) of the sHDL formulation was 0.216, indicating a relatively uniform size distribution. Transmission electron microscopy (TEM) revealed that sHDL nanodiscs exhibited typical discoidal morphology (Fig. 1C, Supplementary Fig. 1A vii).

We analyzed the helicity of the 4F-P-4F peptide and its lipidated form (sHDL), comparing both the molecular dynamics (MD) simulated and experimental circular dichroism (CD) spectra for their secondary structure composition using DichroWeb [50]. Fractional helicity (fH) was calculated using the equation described in Methods section 2.5 and compared with values derived from DichroWeb. All simulated spectra clearly show helical signatures with minima at 208 and 222 nm, confirming α-helical characteristics (Fig. 1D). However, subtle differences were observed, as outlined in Supplementary Table 2. The deconvolution of the CD spectra using the DichroWeb platform confirms that 4F-P-4F retains a predominantly α-helical structure (~70 %), confirming that the secondary structure of the peptide in the nanodisc form is preserved (Fig. 1D, Supplementary Fig. 2A, Supplementary Table 2A-D).

To model the synthetic high-density lipoprotein nanodiscs, we adjusted the peptide-to-lipid ratio. This allowed us to achieve diameters within the range observed by Transmission Electron Microscopy and Dynamic Light Scattering, which closely matched previous reports on apolipoprotein A-I mimetic peptide nanodisc assemblies [69–71]. We performed MD simulations to examine critical interactions that underpin nanodisc stability, which are difficult to resolve using experimental approaches alone.

Previous studies have indicated that peptides effectively stabilizing these discoidal lipid bilayers, mimicking pre-beta HDL-like particles, are essential for sHDL functionality [72]. To test this hypothesis on 4F-P-4F based sHDL, we constructed peptide-lipid (4F-P-4F:DMPC) complexes informed by prior solid-state NMR data on structurally related apolipoprotein mimetic peptides, given the absence of solid state-NMR data regarding the orientation of 4F-P-4F [56,73,74]. MD simulations of the sHDL nanodiscs yielded average diameters of 11.7 nm, 18.0 nm, and 25.6 nm, respectively, following equilibrium runs. These models remained stable throughout the simulations, owing to salt-bridge interactions between the peptide's polar amino acids and the DMPC lipid head groups. Specifically, lysine-phosphate interactions outnumbered choline-glutamic acid interactions across all systems (Supplementary Table 3, Supplementary Fig. 2B,C). The hydrophobic faces of the 4F-P-4F dimers consistently oriented toward the DMPC hydrophobic tails, with their hydrophilic surfaces exposed to the aqueous phase. Salt-bridge interactions with the lipid head groups anchored the peptides in a belt-like arrangement around the membrane normal, stabilizing the discoidal bilayer structure (Fig. 2 A-C, Supplementary Fig. 2B,C).

MD simulations revealed that polar amino acids at the interface between the hydrophobic peptide residues and lipid headgroups play a pivotal role in stabilizing the lipid bilayer structure. These interactions enable peptides to effectively cover the lipid hydrophobic tails, maintaining structural integrity throughout the simulation across nanodiscs of varying sizes.

Combined atomistic simulations and high-resolution experimental characterizations (Figs. 1, 2) demonstrate the structural stability, quality and robust functional potential of the synthesized sHDL formulations.
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Synthesis and Characterization of sHDL Nanodiscs. (A) Schematic representation of sHDL nanodisc synthesis using microfluidics. (B) Number based particle size distribution of the sHDL formulation, determined by dynamic light scattering (DLS). (C) Representative transmission electron microscopy (TEM) image depicting the discoidal morphology of sHDL nanodiscs. (D) Circular Dichroism (CD) spectra were analyzed to compare the structural characteristics of the sHDL formulation and the non-lipidated 4F-P-4F peptide. This analysis incorporated both experimental values and data from Molecular Dynamics (MD) simulations, utilizing the DichroWeb platform for interpretation.</figcaption></figure>

## 3.2. sHDL nanodiscs suppress chemotaxis in vitro and efflux cholesterol from human eosinophil

Eosinophil accumulation in tissues, driven by chemoattractants such as eotaxin-1 and prostaglandin D₂, significantly amplifies the inflammatory response [75]. We investigated the effects of the 4F-P-4F peptide and sHDL nanodiscs on eosinophil migration induced by eotaxin-1 (3 nM) and prostaglandin D₂ (30 nM). sHDL nanodiscs concentration-dependently inhibited eosinophil migration toward both eotaxin-1 (Fig. 3A, B) and prostaglandin D₂ (Supplementary Fig. 3A), whereas non-lipidated 4F-P-4F peptide showed no effect (Fig. 3B). The lipidation status of sHDL nanodiscs proved critical for their efficacy, with a 1:20 peptide-to-lipid ratio identified as the most potent formulation for suppressing eosinophil migration (Supplementary Fig. 3B).

Eosinophils express the HDL receptors scavenger receptor class B type 1 (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) (Supplementary Fig. 3C), [76]. ABCA1 facilitates unidirectional cholesterol efflux to lipid-poor apolipoproteins, whereas SR-BI mediates cholesterol efflux to mature HDL particles, both critical steps in reverse cholesterol transport [77,78]. The ability of sHDL nanodiscs to suppress eosinophil chemotaxis was abolished when eosinophils were preincubated with blocking antibodies targeting SR-BI and ABCA1 (Fig. 3D). This result

indicates that these receptors are required for sHDL's inhibitory effect on eosinophil migration. Notably, eosinophil shape change, a marker of cell activation, was not impacted by these experimental conditions (Supplementary Fig. 3D), [79]. Importantly, isolated human HDL had no significant impact on eosinophil migration (Supplementary Fig. 3E). Similarly, we found no significant inhibitory effects with the free peptide, even in the presence of serum (Supplementary Fig. 3F). In eosinophils, sHDL nanodiscs induced a substantial reduction in cholesterol levels, contrasting sharply with the significantly lower cholesterol efflux observed with the 4F-P-4F peptide (Fig. 4A). Next, we performed cholesterol efflux experiments in the presence and absence of blocking antibodies against ABCA1 and SR-BI. Intriguingly, blocking these receptors did not significantly decrease cholesterol efflux promoted by either native HDL or sHDL (Fig. 4B). These results indicate that while ABCA1 and SR-BI are crucial for inhibiting the chemotactic response, the cholesterol efflux mediated by sHDL operates independently of these receptors

## 3.3. sHDL treatment modulates eotaxin-induced CCR3 internalization and intracellular signaling

Eotaxin-1 primarily drives eosinophil chemotaxis, adhesion, and
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Molecular Dynamics Simulation of sHDL Nanodiscs. A. Representation of a configuration of the 11.7 nm diameter 4F-P-4F DMPC nanodisc sampled during the MD simulation highlighting the salt bridge interactions between the peptides and the lipid head groups. (i) showcases a lysine-phosphate salt bridge manifested by interactions between the nitrogen of the lysine amino group (blue) and the phosphorus of the lipid phosphate group (orange) and (ii) showcases a glutamic acid-choline salt bridge, manifested by an interaction between the carbon of the glutamate carboxyl group (white) and the nitrogen of the lipid choline group (yellow). B. and C. Alternative views emphasizing the hydrophobic (white) and hydrophilic (pink) residues of the 4F-P-4F dimers to highlight the amphipathic arrangement of the peptide side chains and their orientation with respect to the lipid core and the solvent. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

activation *via* the CCR3 receptor, contributing to inflammation in allergic diseases [80]. CCR3 internalization is a dynamic regulatory process that shapes the magnitude, duration, and directionality of eosinophil chemotaxis [81]. It acts as a critical checkpoint in eosinophil trafficking during immune responses and represents a potential target for the management of eosinophil-driven diseases. Cholesterol-rich microdomains, commonly known as lipid rafts, are critical for organizing intracellular signaling cascades within the cell membrane [82]. Cholesterol extraction by sHDL may influence CCR3 internalization by modifying lipid raft structures within the cell membrane through cholesterol efflux. Strikingly, sHDL treatment substantially reduced eotaxin-1-induced CCR3 internalization (Fig. 5A, B), indicating an interference with receptor trafficking dynamics. We also compared these effects with phenylarsine oxide (PAO), a well established CCR3-internalization inhibitor [65,66]. Rescue of internalization induced by sHDL was comparable to treatment with 8 µM PAO (see Supplementary Fig. 4), suggesting that this effect could explain, at least in part, the inhibition of CCR3-mediated eosinophil chemotaxis. Notably, neither isolated native HDL nor the non-lipidated 4F-P-4F peptide showed significant changes in CCR3 internalization. (Supplementary Fig. 4).

To investigate the downstream impact of sHDL on intracellular signaling, potentially altered by changes in CCR3 internalization and cholesterol efflux, we employed a human proteome profiler array. This analysis revealed that sHDL treatment significantly altered the phosphorylation status of key signaling molecules. Specifically, sHDL downregulated phosphorylation of STAT3 at Tyr705 and Ser727, STAT1 at Tyr701, and ERK1/2 at Thr202/Tyr204 and Thr185/Tyr187

compared to the vehicle treatment. (Fig. 5C, D). Given the roles of STAT3 and STAT1 in mediating inflammatory responses [83] and ERK1/2 in regulating eosinophil migration, cell proliferation, and survival [83,84], these reductions highlight the anti-inflammatory and anti-proliferative properties of sHDL. In contrast, phosphorylation of Akt1/2/3 was markedly increased following sHDL treatment (Supplementary Fig. 5A), pointing to a potential role in promoting cell survival and modulating metabolic pathways [85]. Several other changes in intracellular kinases were also observed, as depicted in Supplementary Fig. 5. Importantly, sHDL nanodiscs did not affect eosinophil viability, even at concentrations and incubation times exceeding those used in the chemotaxis assays (Fig. 5E, Supplementary Fig. 5C).

### 3.4. sHDL nanodiscs inhibit eosinophil migration in vivo

To assess the translational relevance of our *in vitro* findings, we performed migration studies using an established eotaxin-mediated eosinophil migration model in IL-5 transgenic mice, as previously described [67,68,79] and outlined in Fig. 6A. In three individual experiments, mice received a single intravenous injection of sHDL nanodiscs (75 mg/kg) or vehicle, followed by an intranasal challenge with eotaxin-2 (4 µg). The sHDL dose of 75 mg/kg was chosen based on established protocols for sHDL studies [29]. After 4 h, eosinophils in the BAL were identified using the gating strategy shown in Fig. 6B. Treatment with sHDL nanodiscs significantly reduced both eosinophil and neutrophil counts in BAL (Fig. 6C, D). Although eotaxins primarily recruit eosinophils, their activation can indirectly promote neutrophil
<figure><img src="image_6.png" /><figcaption>Fig. 3. sHDL Nanodiscs Inhibit Human Eosinophil Chemotaxis in a Concentration-Dependent Manner *In Vitro*. Human eosinophils pretreated with sHDL nanodiscs were allowed to migrate toward eotaxin-1 for 1 h at 37 °C and 5 % CO₂. A. Representative scatter plots illustrating gated eosinophils at the end of the assay. B. Quantification of eotaxin-1-mediated chemotaxis. C. Quantification of eotaxin-1-mediated chemotaxis following eosinophil preincubation with blocking antibodies (2.5 µg/mL for 30 min) before sHDL treatment (25 µg/mL). Data are expressed as percentages normalized to the vehicle control, presented as mean ± SEM from 5 to 9 independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison test B. and Turkey's multiple comparison test C. with *p*-values as reported and *p* < 0.05 considered significant.</figcaption></figure>

infiltration as previously reported [79,86]. A non-significant trend toward reduced total immune cell infiltration was observed with sHDL treatment (Fig. 6E). However, no significant changes occurred in the counts of pan lymphocytes or alveolar macrophages (Fig. 6F, G).

## 4. Discussion

Tissue eosinophilia is a critical driver of disease exacerbation, with

eosinophil migration to inflamed sites sustaining persistent inflammatory responses. Our data demonstrate a marked suppression of human eosinophil chemotactic migration by 4F-P-4F-based sHDL nanodiscs, without affecting cell viability. An optimal peptide-to-lipid molar ratio of 1:20 was identified for this inhibitory effect. This anti-inflammatory activity was corroborated *in vivo* using IL-5 transgenic mice, a model of chronic eosinophilia, where sHDL nanodisc treatment significantly reduced eosinophil and neutrophil counts in bronchoalveolar lavage
A.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

**Fig. 4.** sHDL Treatment Modulates Cholesterol Efflux Independent of ABCA1 and SR-BI Receptors. A. [³H]-cholesterol efflux from isolated human eosinophils quantified as the percentage of [³H]-cholesterol released following treatment with 25 µg/mL sHDL or 4F-P-4F. Vehicle (<1 % efflux) was subtracted for blank correction from each sample. Data are shown as mean ± SEM from three independent experiments. Statistical significance was determined using Student's t-test. B. Comparison of cholesterol efflux in the presence of blocking antibodies against ABCA1 and SR-BI (2.5 µg/mL for 30 min) before sHDL or native HDL treatment (25 µg/mL). Data are shown as mean ± SEM from 3 independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison test, with p-values as reported and *p* < 0.05 considered significant.

fluid. Conversely, the non-lipidated 4F-P-4F peptide showed no signifi-
cant effect on eosinophil migration, highlighting the critical role of
lipidation in sHDL functionality. These results indicate that lipidation
with DMPC is essential for enhancing the pharmacological properties of
ApoA-I mimetic peptides and eliciting anti-inflammatory effects
[3,29,46]. Mechanistically, blocking antibodies targeting SR-BI and
ABCA1 completely reversed the inhibitory effects of sHDL nanodiscs on
eosinophil migration, underscoring the critical role of these HDL re-
ceptors in their function. ABCA1 facilitates cholesterol efflux to lipid-
poor apolipoproteins, whereas SR-BI mediates cholesterol efflux to
mature HDL particles [77,78]. Interstingly, sHDL as well as isolated HDL
mediated cholesterol efflux from eosinophils could not be significantly
blocked by anti-SR-BI and anti ABCA1 antibodies, suggesting that other
pathways like passive diffusion or ABCG1 mediated mechanisms are
more important in eosinophils. While eosinophil ABCG1 mRNA data
exists [87], the transporter's likely intracellular localization poses a
significant experimental challenge. Since blocking antibodies primarily
target surface proteins, they wouldn't effectively address intracellular
ABCG1. Thus, we didn't investigate ABCG1's role in the proposed
mechanism within this study.

Previous research has demonstrated that SR-BI mitigates neutrophil
infiltration and pulmonary inflammation, with its absence worsening
asthma-like responses in experimental models [88]. Likewise, ABCA1
also modulates inflammatory processes, hinting at a wider impact on

pulmonary inflammatory disorders [89]. The ABCA1 signaling axis
operates across a broad range of cell types, including macrophages, fi-
broblasts, and engineered kidney-derived cell lines, underscoring
ABCA1's role as a versatile signaling receptor beyond just cholesterol
efflux [90,91]. Direct signal initiation by SR-BI leads to the activation of
diverse kinase cascades [92].

G-protein-coupled receptors frequently reside within lipid rafts, where changes in raft architecture can affect receptor conformation, positioning, and engagement with downstream signaling molecules [93]. Disrupting lipid rafts, especially through cholesterol depletion, may hinder receptor internalization and the activation of inflammatory signaling cascades [94]. Reconstituted HDL is recognized for its ability to reduce cholesterol levels in lipid rafts, thereby modifying receptor localization and structure [14]. Cholesterol content of the cell membrane influences membrane fluidity and hence the structure of lipid rafts and associated proteins [95]. Interestingly, our experiments revealed that native HDL did not rescue eotaxin-induced CCR3 internalization. This lack of effect may be due to the lower cholesterol efflux capacity of native HDL compared to sHDL, as shown in Fig. 4B. It is also important to consider the variability in the functional effects of isolated native HDL from different healthy donors on eosinophils, which can be inhibitory or stimulatory. Previous research has demonstrated that the inhibitory effect of isolated HDL on eosinophil shape change and chemotaxis is closely linked to its specific lipid and apolipoprotein composition,
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** sHDL Treatment Modulates CCR3 Internalization and Intracellular Phosphorylations. A. Representative histogram overlay of CCR3 surface expression. Cells were treated with vehicle or 25 µg/mL sHDL for 30 min, followed by 30-min stimulation with 10 nM eotaxin-1. An unstimulated control (no eotaxin) displays baseline CCR3 levels. B. Quantification of panel A, showing the geometric mean of CCR3 signal normalized to respective unstimulated samples, with data presented as mean ± SEM from six independent experiments. C. Phosphokinase array membrane images of pooled eosinophils from three donors, highlighting specific phosphorylation site-changes. D. Quantification of intensity values from (C) expressed as % of vehicle intensity. E. Apoptosis-necrosis assay performed with increasing concentrations of sHDL nanodiscs over a 4-h treatment at 37 °C, with 20 % DMSO used as a positive control for cell death induction. Data are shown as mean ± SEM from six independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison test, with p-values as reported and *p* < 0.05 considered significant.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** sHDL Nanodiscs Suppress Eosinophil Migration *In Vivo*. A. Schematic overview of the migration assay using IL-5tg mice. B. Gating strategy for analyzing immune cell populations following BAL collection, with eosinophils identified as Siglec-F⁺CD11c⁻ within the myeloid cell population. Quantification of immune cell infiltration for C. eosinophils, (D) neutrophils, E. total infiltrating immune cells, F. pan-lymphocytes, and G. alveolar macrophages. Data represent values from three independent experiments (n = 10–11 for treatment groups) normalized to the vehicle mean and presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison test, with p-values as reported, p < 0.05 considered significant; ns: not significant.</figcaption></figure>
particularly its apolipoprotein A-II, apolipoprotein C-III, lysophosphatidylcholine, and triglyceride content [96]. This compositional heterogeneity likely underlies the absence of a consistent effect observed with the native HDL preparations.

It has been shown that eotaxin induces ERK phosphorylation via CCR3 [84]. We demonstrate that sHDL pretreatment reduced eotaxin-1-induced phosphorylation of ERK1/2, STAT1, and STAT3 in eosinophils, critical signaling molecules in inflammation. The ERK1/2 pathway regulates proliferation, differentiation, and survival, while the JAK-STAT pathway drives cytokine-mediated inflammatory responses, both of which are implicated in eosinophilic inflammation and allergic diseases [83,84]. Conversely, sHDL increased phosphorylation of Akt1/2/3, potentially activating pro-survival or anti-apoptotic pathways, which may contribute to maintaining eosinophil integrity during inflammation [85]. Although the exact mechanisms warrant further investigation, the interplay of SR-BI and ABCA1 highlights sHDL as a promising therapeutic option for such conditions.

Eosinophils, while implicated in lung inflammation and tissue damage due to overactivation, play a vital role in maintaining tissue homeostasis and defending against parasitic and certain bacterial infections [97]. Emerging evidence highlights their beneficial contributions, including potential anti-tumorigenic properties [98]. These findings underscore the dual nature of eosinophils, which can both exacerbate and mitigate immune responses depending on the context. The use of therapies such as anti-IL-5, anti-IL-5Rα, anti-IL-4, and anti-IL-13 antibodies to eliminate eosinophils has several side effects [97,99,100]. As their importance in overall health becomes increasingly clear, the complete depletion of eosinophils may carry risks, such as secondary complications [101–103]. Consequently, targeting eosinophil migration with sHDL nanodiscs may offer a more nuanced approach to modulate allergic eosinophil recruitment effectively [33]. The consistent size, structural stability, and customizable composition of sHDLs underscore their promise as adaptable, multifunctional delivery vehicles. For instance, enrichment with miltefosine [79] or lysophosphatidylcholines [66] may further increase the anti-inflammatory potential of sHDL nanodiscs.

*In silico* characterizations are crucial for understanding the structure-function relationship of sHDL nanodiscs and for screening their immunomodulatory functions. Both experimental and simulated helicity data confirm the structural stability of the peptides in their lipidated form. However, comparing the CD spectra of 4F-P-4F and sHDL revealed that the behavior of these mimetic peptides cannot be generalized; unlike the 18A mimetic peptide, we didn't observe an increase in helicity upon lipidation [21]. Developing accurate simulation models as our molecular dynamics simulation model allows for screening of various designs (e.g., modifying peptides, lipids and ligands) early in the development process reducing the need for extensive initial *in vitro* testing.

We acknowledge several limitations in this study. First, the relatively short duration of our *in vivo* experiments limited our ability to assess structural or functional changes in the lung associated with chronic inflammation. Second, treatment with sHDL did not result in significant changes in IL-6 or TNF-α levels in bronchoalveolar lavage fluid (Supplementary Fig. 6), suggesting a limited impact on key pro-inflammatory cytokines within the timeframe examined. Additionally, our analysis did not encompass the broader kinetics of immune cell trafficking within the lung, which may have provided a more comprehensive understanding of the immune dynamics in this model. As a result of these constraints, our findings may not fully capture the long-term pathological features typically observed in chronic asthma.

As clinical trials investigating HDL-based infusion therapies continue to expand, understanding the effects of sHDL on circulating immune cells becomes increasingly important. The sHDL field is rapidly progressing from mimicking native HDL for cholesterol efflux to serving as a versatile drug delivery platform. Recent studies have emphasized the chemical conjugation of therapeutics, enabling precise biodistribution and reduced immunotoxicity [3,104]. The well-documented ability of

HDL mimetics to reduce cytokine production and oxidative stress and modulate G-protein coupled receptors in human immune cells [10,28,46] further supports their potential in managing chronic inflammatory disorders.

In summary, this study demonstrates that 4F-P-4F-based sHDL nanodiscs exert potent inhibitory effects on human eosinophil migration, mediated in part through interactions with the SR-BI and ABCA1 receptors. These findings highlight sHDL nanodiscs as a promising therapeutic platform for targeting eosinophil-driven inflammation in conditions such as asthma and eosinophilic esophagitis, where aberrant eosinophil migration contributes to disease progression.

### CRediT authorship contribution statement

**Alankrita Rani:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Denys Balandin:** Writing – review & editing, Methodology, Formal analysis. **Rohith Ravi:** Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis.

**Julia Teppan:** Writing – review & editing, Methodology, Investigation.

**Ivan Vidakovic:** Writing – review & editing, Visualization, Methodology, Investigation. **Melanie Kienzl:** Writing – review & editing, Methodology. **Thomas Bärnthaler:** Writing – review & editing, Methodology. **Michael Holzer:** Writing – review & editing, Methodology. **Lamija Zijadic:** Writing – review & editing, Investigation. **Akos Heinemann:** Writing – review & editing, Resources. **Katharina Jandl:** Writing – review & editing, Funding acquisition. **Eva Böhm:** Writing – review & editing, Resources, Methodology. **Karin Kornmueller:** Writing – review & editing, Resources. **Mounir Tarek:** Writing – review & editing, Supervision, Resources, Funding acquisition. **Ruth Prassl:** Writing – review & editing, Supervision, Resources, Funding acquisition.

**Gunther Marsche:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Conceptualization.

### Funding

This research was funded by the Austrian Science Fund (FWF) 10.55776/DOC129 (doc. funds RESPImmun), ApoA-I Mimetic Peptide Lipid Assemblies: I 5703-B and the French National Research Agency project ANR-21-CE11-0038 – AAMLA. For open access purposes, the authors have applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission.

### Declaration of competing interest

The authors declare no conflict of interest.

### Acknowledgments

We thank Iris Red, Kathi Rohrer, Sabine Meissl, Ilse Lanz, and Juliana Schwanzer for their excellent technical assistance. Figures were created using GraphPad Prism (v10.2.2), *BioRender.com*, IGOR Pro (WaveMetrics) and UCSF ChimeraX, developed by the Resource for Bio-computing, Visualization, and Informatics at UCSF.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jconrel.2025.114060.

### Data availability

Data will be made available on request.
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