Production, characterization and biodistribution of
therapeutic high-density lipoprotein-like nanoparticles
reconstituted with or without histidine-tagged
recombinant ApoA1

Sarah Rosanaly, Marie Laurine Apalama, Matthieu Bringart, Pierre Giraud, Benoit Allard, Bryan Veeren, Olivier Meilhac, Joël Couprie, Philippe Rondeau

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To cite this version:

Sarah Rosanaly, Marie Laurine Apalama, Matthieu Bringart, Pierre Giraud, Benoit Allard, et al. Production, characterization and biodistribution of therapeutic high-density lipoprotein-like nanoparticles reconstituted with or without histidine-tagged recombinant ApoA1. Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids, 2025, 1870 (3), pp.159606. 10.1016/j.bbalip.2025.159606. hal-05009726

HAL Id: hal-05009726

https://hal.science/hal-05009726v1

Submitted on 3 Feb 2026

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# Production, characterization and biodistribution of therapeutic high-density lipoprotein-like nanoparticles reconstituted with or without histidine-tagged recombinant ApoA1

Sarah Rosanaly<sup>a,1</sup>, Marie Laurine Apalama<sup>a,1</sup>, Matthieu Bringart<sup>a</sup>, Pierre Giraud<sup>a</sup>, Benoit Allard<sup>a</sup>, Bryan Veeren<sup>a</sup>, Olivier Meilhac<sup>a,b,*</sup>, Joël Couprie<sup>a</sup>, Philippe Rondeau<sup>a</sup>

## ARTICLE INFO

**Keywords:**

rHDL

Apolipoprotein A-1

High-density lipoprotein

Nanoparticles

Vectorization

Inflammation

## ABSTRACT

High-density lipoproteins (HDLs) are known for their cardiovascular protection due to apolipoprotein A-1 (ApoA1), their primary protein. ApoA1 promotes cholesterol reverse transport and exhibits antioxidant and anti-inflammatory properties. Although increasing HDL levels has not consistently reduced cardiovascular mortality in clinical trials, reconstituted HDL (rHDL) nanoparticles containing ApoA1 show potential in treating acute inflammation, such as in ischemic stroke, sepsis, and even COVID-19. ApoA1 is commonly produced in bacteria due to its simplicity and potential therapeutic optimisation. Addition of a histidine tag to recombinant ApoA1 may improve purification, stability and therapeutic efficacy, although its functional impact remains a subject of debate. In this study, ApoA1 with a poly-histidine tag (His-rApoA1) was produced in a clear coli system for simplified purification, followed by an evaluation of the tag's effects on rHDL nanoparticle properties. rHDL and His-rHDL nanoparticles were prepared using the sodium cholate dialysis method, combining recombinant rApoA1 or His-rApoA1 with phosphatidylcholine at a 1:75 M ratio. Nuclear magnetic resonance confirmed that both forms of rApoA1 structurally resembled plasma ApoA1, whether lipid-free or in nanoparticle form. Dynamic light scattering and electron microscopy revealed nanoparticle sizes around 7 nm with native HDL-like morphology. Testing on endothelial cells (EA.hy926) showed rapid uptake of rHDL and His-rHDL while preserving cell viability. Additionally, both nanoparticles reduced interleukin-6 and ICAM-1 expression in cells, demonstrating their anti-inflammatory and protective effects, unaffected by the poly-histidine tag. Intravenous injection in mice shows homogeneous distribution of His-rHDL in the liver, lungs, and spleen, with no cytotoxicity, indicating potential use for treating inflammatory diseases.

## 1. Introduction

High-density lipoproteins (HDLs) are natural nanoparticles which have long been associated with protection from cardiovascular events, including ischemic heart disease [1,2]. HDLs are composed of lipids and proteins, the main one being apolipoprotein A-1 (ApoA1). They are known to reverse transport cholesterol from peripheral tissues back to the liver to promote its elimination by the biliary route [3]. Clinical studies showing an inverse association between HDL levels and cardiovascular disease are based primarily on the levels of cholesterol contained in these particles (HDL-C). However, ApoA1, which

constitutes >60 % of the protein mass of HDLs, also has vasculoprotective functions, conferring antioxidant, anti-inflammatory, anti-apoptotic and potentially anti-infectious properties to HDL, which it carries alone or in combination with other proteins that compose HDL particles [4]. Numerous clinical investigations have focused on increasing circulating HDL levels, either by pharmacological means with drugs acting on endogenous HDL metabolism or by intravenous supplementation with reconstituted HDL composed of ApoA1 and phospholipids (rHDL) [5,6]. These approaches, although leading to an increase in HDL-C levels in target subjects, did not reduce cardiovascular mortality. A large-scale study conducted to determine the efficacy of

<footnote><sup>a</sup> Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI), Saint-Pierre de La Réunion, France</footnote>

<footnote><sup>b</sup> Centre hospitalier universitaire de La Réunion, Saint-Pierre, France</footnote>

<footnote>* Corresponding author at: DéTROI - Université de la Réunion, 77 Avenue du docteur Jean Marie Dambreville, 97410 Saint Pierre, La Réunion, France. E-mail address: olivier.meilhac@inserm.fr (O. Meilhac).</footnote>

<footnote n="1">¹ These two authors contributed equally to this work and should be considered co-first authors.</footnote>
rHDL injections in over 18,000 coronary patients has been published recently, with negative results. In fact, intravenous injection of a novel formulation of rHDLs (CSL112) 4 times a week for secondary prevention had no effect on the recurrence of cardiovascular events at 90 days [7]. CSL112 was obtained from human pooled plasma-purified ApoA1 combined with phosphatidylcholine using the sodium cholate dialysis method containing 0.2 % alpha-tocopherol to achieve a 55-fold molar excess over ApoA1 [8]. Beyond the disappointing effects of increasing HDL levels (by any means) to prevent cardiovascular disease, many preliminary studies show beneficial effects of ApoA1 and rHDLs in acute situations of moderate or severe inflammation. For example, we have shown that HDL injections in the acute phase of ischemic stroke could decrease the size of cerebral infarction or limit hemorrhagic complications [9,10]. In 3 models of sepsis, injection of rHDLs (CSL111) reduced mortality and inflammation in mice [11–13]. Similarly, rHDLs have been injected into humans in various pilot studies, to limit metabolic disorders [14–16] and more recently in COVID-19 patients [17,18], showing improvements in inflammatory profiles. Several pharmaceutical companies produce synthetic rHDLs, which are composed of ApoA1 and phospholipids of variable composition such as CSL Behring for CSL111 and CSL112 and Abionyx for CER 001. ApoA1 can come from blood bank plasma pools or can be produced by genetic engineering. The production of ApoA1 through recombinant methods, followed by the subsequent reconstitution of HDL nanoparticles, enhances their properties by modifying their lipid composition or the ApoA1 sequence. This approach also allows ApoA1 to be modified in order to improve its effects (production of gain-of-function variants) or to follow it after administration by adding a label [19,20].

Different strategies exist for the production of ApoA1, on the one hand in eukaryotic cells such as the Chinese hamster ovary (CHO) cell systems [21,22] or in the baculovirus-insect cell system [23], or on the other hand in prokaryotic cells with mainly E. coli bacteria for human ApoA1 expression [24,25]. Since ApoA1 does not undergo post-translational modification such as glycosylation, the eukaryotic system is not required for the production of the mature protein. Therefore, expression of the protein in a bacterial system is more relevant as transfection is easier than in eukaryotic cells and the expression yield is higher [26]. In addition, it is important to use a contaminant-free protocol for the production of ApoA1 to avoid its degradation by the proteases of the bacterial strain [26]. For these compelling reasons, the development of the custom production of ApoA1 in a recombinant bacterial system is therefore the preferred approach in this study.

Histidine tag is one of most commonly used tags for protein expression and purification in particular in recombinant protein production. The polyhistidine-tag, best known as His-tag, which consists of an amino acid motif with at least six histidine (His) incorporated in expression plasmids ensure improved production levels of the protein by limiting and facilitating purification stages. This peptide tag indeed offers numerous other advantages such as enhancing the folding, solubility, and stability of the produced protein, which appear crucial for ensuring its therapeutic efficacy and circulation lifespan [27]. This histidine tag appears useful for tracking the protein after in vivo injection and could also contribute to its functional aspects as a therapeutic vector, such as its endosomolytic activity, which facilitates its intracellular delivery [28]. Due to its relatively small size (~2.5 kDa), His-tag is not supposed to significantly interfere with the function and structure of a majority of proteins and its deleterious impact is up to now not clearly established and remains controversial [29]. We therefore decided to verify this hypothesis for ApoA1 when it is incorporated into a nanoparticle.

To determine this impact, we reconstituted ApoA1 nanoparticles using recombinant ApoA1 produced in bacteria lacking active endotoxins, on which the tag was either cleaved or not. We then studied their structural and functional characteristics as well as their biodistribution after intravenous administration in mice. The development of production techniques of these nanoparticles will allow us to modulate their

composition by varying the ApoA1 and the phospholipids, then in a second time to enrich them with protective molecules according to the target pathologies to improve their therapeutic properties.

## 2. Materials and methods

### 2.1. Materials

MA145 ClearColi® BL21(DE3) bacteria were obtained from Lucigen and the kanamycin from EUROMEDEX. LB broth medium, Isopropyl β-D-1-thiogalactopyranoside (IPTG), and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Sigma-Aldrich. Antibodies against ApoA1 and against Histidine-Tag were respectively obtained from Calbiochem and from Abcam. Histidine-tagged protein purification 5 mL columns (HisTrap HP) and Gel Filtration Superdex® 200 Increase 10/300 GL were obtained from GE Cytiva. Sodium Cholate was purchased from Bio Basic. SOC medium and 1,1'-dioctadecyl-3,3',3'',3'''-tetramethylindocarbocyanine perchlorate (DilC₁₈) dye were purchased from Thermofisher.

### 2.2. Plasmid constructs

A pET28a plasmid encoding the sequence of a mature human ApoA1 (243 amino acids) was designed by Gene Cust France and the nucleic acid sequence was optimized for an overexpression in bacteria (codon optimized ApoA1 sequence). A short sequence encoding for the TEV cleavage site (ENLYFQG) was added upstream the ApoA1 gene sequence to allow cleavage of the N-terminal 6× His-tag sequence encoded by the pET28a vector (Supplementary Fig. S1).

### 2.3. Production of recombinant ApoA1

A schematic overview of the different steps starting from the expression, purification and cleavage of ApoA1 to the reconstitution of nanoparticles is shown in Fig. S2 (Supplementary data).

#### 2.3.1. Escherichia coli bacterial competent cell

Non competent *Clear coli* bacteria were cultured in 250 mL LB broth medium [30]. The bacterial growth was monitored by spectrophotometry at 600 nm until the OD600 reached 0.55. Cells were then harvested by centrifugation at RT (26 °C), for 10 min at 5000g. The supernatant was discarded, and the pellet was resuspended in 80 mL of Inoue buffer (PIPES 10 mM pH 6.7, MnCl₂ 55 mM, CaCl₂ 15 mM and KCl 250 mM). The sample was centrifuged again at RT, for 10 min at 3900g, and the pellet was resuspended in 20 mL of Inoue buffer. 1.5 mL of sterile DMSO was added and the sample was left on ice for 4 h.

#### 2.3.2. Transformation and induction

The ApoA1 pET28a plasmid vector was used to chemically transform competent *Clear coli* cells by heat shock as described below. About 2 µL (100 ng) of plasmids were added to 50 µL of competent bacteria suspension and the mixture was consecutively incubated on ice for 30 min then at 42 °C for 30 s. The solution was then left on ice for 2 min, 250 µL of SOC medium pre-heated to 42 °C were added and the mixture was incubated at 37 °C for 1 h. Transformed *Clear coli* cells were cultured on selective LB-broth agar medium plates containing 30 µg/mL kanamycin and incubated overnight at 37 °C to select transformed clones. Bacterial transformed colonies were selected and inoculated in 10 mL of liquid LB media containing 30 µg/mL kanamycin. This bacterial inoculum was then used for pre-culture in 200 mL of LB medium supplemented with 200 µL kanamycin (30 µg/mL) at 37 °C with 200 rpm shaking. After overnight incubation, the preculture was diluted into 1 L of LB medium (supplemented with 30 µg/mL kanamycin) at an initial absorbance of 0.4 at 600 nm (OD600). The culture was then incubated at 37 °C with 200 rpm agitation and the bacterial cells were grown until the OD600 reached 0.6 to 0.8. Induction of T7 RNA polymerase expression of
ApoA1 was initiated by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Bacteria cells were harvested after an additional 4 h incubation at 37 °C (200 rpm shaking) by centrifugation for 20 min at 4000 g and 4 °C.

### 2.3.3. Purification and cleavage

The pellet was resuspended in 20 mL of lysis buffer (0.1 mg/mL lysozyme in phosphate buffer Saline (PBS), pH 7.4) per liter of bacterial culture. The mixture was incubated at 4 °C for 30 min followed by sonication (5 min, 4 s ON and 10 s OFF, 60 % power) to disrupt the cells. The cell lysates were centrifuged for 20 min at 12000 g at 4 °C and the supernatant containing recombinant ApoA1 was collected.

Recombinant ApoA1 was purified by affinity chromatography on an AKTA PURE 25 system (GE Healthcare) using a 5 mL HisTrap HP column. Loading buffer and elution buffer consisted of PBS containing 20 mM imidazole at pH 7.4, and 500 mM of Imidazole pH 7.4, respectively. Then, the supernatant was applied to the HisTrap affinity column and a chromatographic method with a four steps gradient was used. Fractions were collected and analyzed by SDS-PAGE and fractions containing ApoA1 protein were pooled and concentrated by ultrafiltration on Amicon® Ultra-4 10 K filters (MWCO = 10 kDa) up to 2 mg/mL. The 6xHis-Tag grafted to ApoA1 was removed by using a homemade recombinant TEV protease that also included a 6xHis tag at the N-terminus. TEV protease was added to His-tag-ApoA1 at a ratio of 1:100 (w/w), and the mixture was incubated at 30 °C for different times (2 h, 4 h and 20 h). The cleavage efficiency was checked by SDS-PAGE and the sample was applied once again to the HisTrap column to elute cleaved and to retain the noncleaved ApoA1 and the TEV as described previously.

## 2.4. SDS PAGE and Western blot analysis

The overexpression and purification of ApoA1, as well as the efficiency of cleavage of the histidine Tag were checked on a SDS-12 % PAGE and by Western Blot on lysed cell pellet and supernatant after centrifugation. Ten µg of proteins (cleaved and noncleaved) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane for immunoblot analysis or stained with Coomassie blue. For the immunoblot analysis, the membranes were first blocked overnight at 4 °C with 5 % (w/v) non-fat dry milk in TRIS-buffer containing 0.1 % (v/v) Tween 20 (TBS-Tween), before an incubation with the primary rabbit-anti-human ApoA1 (Calbiochem, 1/500 dilution) or the primary mouse-anti-His-Tag (Abcam, 1/1000 dilution) for 2 h at RT. Nitrocellulose membranes were then incubated with the corresponding HRP conjugated secondary antibodies for 1 h. Between each step of incubation, membranes were washed three times with PBS/0.1 % Tween 20 buffer. Protein detection was performed using the enhanced chemiluminescence reagent (ECL, GE Healthcare) and Amersham Imager 600 (GE Healthcare).

## 2.5. NMR analysis of purified ApoA1

Plasma ApoA1 (pApoA1), purified noncleaved and cleaved recombinant ApoA1, plasma HDLs and rHDLs were analyzed on a 600 MHz BRUKER Avance III NMR spectrometer equipped with a ¹H/¹⁹F, ¹³C and ¹⁵N cryoprobe to check the structure of the protein. For that, 50 µL of deuterium oxide (D₂O) and 2 µL of TMS (used as reference) were added to 500 µL of protein concentrated at 600 µM. 1D ¹H, 2D TOCSY and 2D NOESY spectra were recorded at 37 °C. Spectra were recorded with a sweep width of 10 ppm and the carrier was placed at 4.7 ppm and analyzed with Topspin 3.6.

## 2.6. Mass spectrometry for ApoA1 characterization

Purified cleaved and noncleaved recombinant ApoA1 was analyzed in positive mode using a heated electrospray ionization (HESI)-Orbitrap mass spectrometer (Q-Exactive™ Plus, Thermo Scientific, Bremen,

Germany). In brief, the desalted samples (200 fmol) were then directly infused into the mass spectrometer at a flow rate of 8 µL/min via a syringe pump connected to the HESI probe. The electrospray conditions were as follows: a sheath gas flow rate of 8 units, an auxiliary gas flow rate of 12 units, a spray voltage of 3 kV, a capillary temperature of 320 °C and an S-lens RF level of 80 %. Nitrogen was used as the drying gas.

For the mass spectrometer conditions, mass spectra were recorded in full scan mode from *m/z* 800 to 2500 at a resolving power of 140,000 FWHM at *m/z* 400. The automatic gain control was set at 1e6 with an injection time of 200 ms. The analysis time per sample was 1 to 2 min. The spectra were acquired in intact protein mode using the Tune 2.11 QF2 Build 3007 software and processed with the Freestyle software (Thermo Fisher Scientific Inc.).

## 2.7. rHDL and His-rHDL nanoparticles preparation

The reconstitution of nanoparticles was performed by the cholate sodium dialysis method and POPC (2-Oleoyl-1-palmitoyl-*sn*-glycero-3-phosphocholine; Sigma) was used as a lipid source [31]. A molar ratio of 1:75 for ApoA1:POPC was prepared. Briefly, POPC (29.4 mg/mL) was dissolved in chloroform: methanol (2:1, v/v) and dried for 2 h with nitrogen gas. After resuspending the lipids in 500 µL of TEN buffer (Tris 10 mM, NaCl 150 mM, EDTA 1 mM), 387 µL of sodium cholate (30 mg/mL) was added to the POPC. Then, 10 freeze/thaw cycles were performed in order to promote multilamellar vesicles formation. Cleaved or uncleaved ApoA1 was added to the mixture followed by an incubation with shaking overnight at 37 °C. The sample was dialyzed against the TEN buffer for 3 days (5 bath changes every 12 h) at 4 °C in order to eliminate the excess of sodium cholate. The resulting rHDLs were kept at 4 °C until use. For the aerosolization experiment, the nanoparticles were prepared by addition of DilC₁₈ fluorescent dye (DilC₁₈ [3]; 20 mg/mL; Invitrogen) to POPC dissolved in chloroform:methanol (1:100, mol/mol). To eliminate free DilC₁₈ labeled nanoparticles were isolated by ultracentrifugation as follows: nanoparticles density was adjusted to d = 1.22 with KBr and overlaid with KBr solution (d = 1.21). Ultracentrifugation was performed at 250000 g for 20 h at 10 °C with a 50.4Ti rotor using a Beckman Coulter Optima L-80 XP Ultracentrifuge. The DilC18 labeled nanoparticles fraction was recovered and sterile-filtered.

## 2.8. Dynamic light scattering analysis

The particle size and distribution of rHDLs were recorded on a Dynamic Light Scattering (DLS) Zetasizer Nano (Malvern Instruments). The analyses were conducted in aqueous solution after filtration of rHDL samples (in TRIS 10 mM pH 8, NaCl 150 mM and EDTA 1 mM buffer) through a 0.22 µm pore size PTFE syringe filter to remove small polymer aggregates. Each analysis was performed at 25 °C in triplicate with an angle detection of 175°.

## 2.9. rHDL characterization by TEM microscopy

Transmission electron microscopy (TEM) analyses were performed at the Institute of Mineralogy, Physics of Materials and Cosmochemistry (IMPMC, University of Jussieu, Paris). The morphology and the size of nanoparticles were determined from negative staining images recorded on UltraScan 1000, 2 k × 2 k CCD camera (Gatan, USA), working with a LaB6 JEOL JEM2100 (JEOL, Japan) Transmission electron microscope operating at 200 kV. The negative staining was achieved with 2 % uranyl acetate solution. Commercial 300 mesh copper grids covered with a carbon membrane were used (Delta Microscopies France). The grid was glow discharged (Edwards Auto 306 United Kingdom) for 30 s to obtain a hydrophilic carbon surface for a better deposition. Then the solution of the different samples was deposited on the carbon grids. The negative staining was then obtained with a 2 % solution of Uranyl Acetate. After waiting for the samples deposited on the carbon grids to dry completely
in the open air, they were mounted on a single tilt holder and introduced into the microscope for observation on a JEOL Japan JEM 2100 Transmission Electron Microscope operating at 200 kV with a cryopole piece. The images were recorded on a Gatan USA camera UltraScan US1000 2 k by 2 k CCD camera.

## 2.10. Cell culture and stimulation

Endothelial cell lines (EA.hy926 and bEnd.3 cell lines) were obtained from American Type Culture Collection (ATCC, USA, CRL-2922™). Cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Pan Biotech, Germany) supplemented with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 250 µg/mL amphotericin B. For EA.hy926 cells, the culture medium was also supplemented with HAT (100 µmol/L hypoxanthine, 0.4 µmol/L aminopterin and 16 µmol/L thymidine). Cells were maintained at 37 °C in a 5 % CO₂ humidified atmosphere. After reaching confluence, cells were serum-deprived for 3 h before stimulation by replacing the cell culture media by DMEM supplemented with 5 % delipidated FBS.

## 2.11. Determination of rHDL uptake in cells

rHDL and His-rHDL uptake by EA.hy926 and bEnd.3 cells was determined by immunofluorescence. Cells were plated on a Millicell 8 well-glass slide (PEZGS0816 Millipore) and incubated with rHDL or His-rHDL solutions (0.4 mg/mL). Immunofluorescence detection of rHDL (ApoA1) was done as described below. Cells were washed three times with PBS, fixed for 15 min in 4 % paraformaldehyde solution and washed again with PBS. Cells were permeabilized with 0.5 % Triton X-100 (in PBS) twice for 5 min, blocked with 1 % BSA solution (in PBS) for 30 min and incubated with primary rabbit anti-human ApoA1 (Calbiochem, 1/500 dilution). After overnight incubation at room temperature, cells were washed three times with PBS and incubated with secondary goat anti-rabbit Alexa 488 (Jackson, 1/2000 dilution) for 2 h at room temperature. The slides were rinsed 3 times with PBS and cell nuclei were stained with DAPI (Sigma, 0.1 µg/mL) for 20 min at room temperature. Slides were washed with PBS before mounting in fluorescence medium (Fluoromount, Thermofisher) and image captured using a NanoZoomer S60 digital slide scanner (Hamamatsu) or a confocal microscope (Nikon Eclipse Ti2).

## 2.12. Neutral red test for cytotoxicity of nanoparticles

Neutral red colorimetric test was used for the quantitative assessment of cell viability following exposure to rHDLs. This test is based on the ability of viable cells to incorporate neutral red into lysosomes [32]. EA.hy926 cells were seeded in a 96-well plate (5.10⁴ cells/well). After reaching confluency and 3 h serum starvation, cells were stimulated with rHDLs or His-rHDLs (0.4 mg/mL) for 24 h. Then, the medium was removed and the cells were washed 3 times with PBS. DMEM supplemented with 80 µg/mL neutral red was added to each well and cells were incubated for 2 h at 37 °C. After removing the medium, cells were washed 3 times with PBS and 200 µL of a destaining solution of water: ethanol 96 %:acetic acid (50:50:1 v/v) were added in each well to solubilize the dye and the absorbance was read at 540 nm.

## 2.13. RNA extraction and RT-qPCR

EA.hy926 cells were seeded in a 6-well plate (3.10⁵ cells/well). After reaching the confluency and 3 h serum deprivation step, cells were stimulated with rHDLs or His-rHDLs (0.4 mg/mL) for 6 h in the absence or presence of TNFα (5 ng/mL). Total RNA was isolated with TRIzol™ reagent (Invitrogen). About 2 µg of total RNA were reverse-transcribed (RT) using Random hexamer primers and NxGen® M-MuLV (Lucigen). Quantitative polymerase chain reaction (qPCR) was performed using the

BrightGreen™ master mix (ABM) and results analyzed using CFX96 system software (Biorad). The relative expression of pro-inflammatory cytokine IL-6 (Forward: GGT ACA TCC TCG ACG GCA TCT and Reverse: GTG CCT CTT TGC TGC TTT CAC) and adhesion protein ICAM-1 (Forward: GGC CTC AGT CAG TGT GA and Reverse: AAC CCC ATT CAG CGT CA) was normalized to the expression of ribosomal 18S RNA (Forward: ATT AAG GGT GTG GGC CGA AG and Reverse: TGG CTA GGA CCT GGC TGT AT).

## 2.14. Quantification of IL-6 secretion by ELISA

EA.hy926 cells were seeded in a 12-well plate (1.10⁵ cells/well). After reaching the confluency and 3 h serum starvation, cells were stimulated with rHDLs or His-r HDLs (0.4 mg/mL) for 24 h in the absence or presence of TNFα (5 ng/mL). Cell culture media were collected and analyzed using human IL-6 ELISA Ready-SET-Go® assays (Thermofisher). Absorbance was measured using a microplate reader (FLUOstar Optima, BMG Labtech) at 450-570 nm.

## 2.15. Biodistribution of His-rHDL in mice after intravenous administration

Nine months old C57BL/6 mice were fed *ad libitum* with standard laboratory chow and water. All animal experiments were approved by the local ethics committee (CYROI APAFIS#29999-2020062212483247v4). All mice were anesthetized using intraperitoneal (i.p.) ketamine (100 mg/kg) and xylazine (10 mg/kg). Retro-orbital intravenous administration started after anesthesia. Mice received either 50 µL of PBS solution or 50 µL carbocyanine-labeled (DilC₁₈)-rHDL (20 mg/kg of body weight). The mice were warmed and observed until awake. After 24 h, mice were sacrificed by exsanguination after ketamine and xylazine anesthesia. Six female mice (25.7-41.6 g) were studied. 4 % paraformaldehyde (PFA) was used for washing and infusing the vascular compartment; engulfed tissues (brain, liver, spleen, heart, lung and kidney) were fixed overnight in 4 % PFA, kept in 30 % sucrose solution overnight, and then frozen in OCT at -80 °C. Frozen sections (10 µm) were obtained using a cryostat. For direct detection of DilC18-labeled His-rHDL, OCT was eliminated by 5 rinses with PBS and incubated with DAPI (1/500, 0.1 µg/mL), before microscopic observation. Images were observed on the NanoZoomer S60 digital slide scanner (Hamamatsu).

## 2.16. Statistical analysis

All the results are expressed as the mean +/− SEM from three to four experiments. Statistical analysis was achieved using Prism software (Prism software, GraphPad, USA). Significant differences (*p* < 0.05) between the means were determined by *t*-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

# 3. Results

## 3.1. Overexpression of ApoA1

### 3.1.1. ApoA1 overexpression monitoring

After ApoA1 overexpression in *Clear coli* induced by IPTG, bacteria were pelleted, sonicated, and centrifuged to collect the soluble material. The presence of ApoA1 in the bacterial suspension at different stages of induction and purification were examined by SDS-PAGE (Fig. 1A). The fractions corresponding to bacterial samples after induction with IPTG (AI) and the supernatant (S) after centrifugation were found to contain the largest amount of ApoA1 as evidenced by a more intense band around 25-28 kDa (see red boxes), and confirmed by plasma ApoA1 migration (right side). The absence of an equivalent band in the pellet (P) attests that ApoA1 is mainly expressed as a soluble protein. Given that our recombinant protein expressed in bacteria contains a poly-
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Overexpression of recombinant His-ApoA1. A) Coomassie blue staining after SDS-12 % PAGE Different samples were analyzed on the gel, Bl: protein sample before IPTG induction; AI: after IPTG induction; S: supernatant; P: pellet and pApoA1: plasma ApoA1 used as control. The red boxes indicate the bands corresponding to the His-ApoA1 protein at 25–28 kDa. B) Western Blot analysis of plasma ApoA1 (pApoA1) and recombinant His-ApoA1 (rApoA1) by using a rabbit anti-human ApoA1 antibody. C) Typical native MS spectrum of purified His-ApoA1 showing its Gaussian distribution charge states (21+ to 38+) and the average mass 31,073.9069 Da (inset) obtained after deconvolution. Purified His-ApoA1 was analyzed with a Q-Exactive™ Plus mass spectrometer in intact protein mode. D) Identical 1D ¹H NMR spectra show evidence for no significant structural difference between the recombinant His-ApoA1 (bottom blue spectrum) and the plasma ApoA1 (top red spectrum). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Cleavage of the histidine Tag does not affect the configuration of rApoA1. A) Coomassie blue staining of the different forms of rApoA1 protein following SDS-12 % PAGE: uncleaved form (BC), the partial cleaved forms (2 h and 4 h) and the totally cleaved form (20h). B) Western Blot analysis of rApoA1 samples before and after cleavage (BC and AC). The detection of the histidine Tag was performed by using a mouse anti His-Tag antibody. C) Typical native MS spectrum of cleaved rApoA1 showing its Gaussian distribution charge states (19+ to 35+) and the average mass 28,117.5668 Da (inset) obtained after deconvolution. D) Identical 1D ¹H NMR spectra for the totally cleaved form rApoA1 (red) and uncleaved form of rApoA1 (blue) show evidence for structural similarities. HE1 and HD2 circled in black correspond to the signal of the histidine Tag proton. Top left: molecular structure of histidine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
histidine tag (His-tag), it was purified by HisTrap affinity chromatography. The two purification methods tested showed that a four-step gradient in elution strength in imidazole provided a better separation and purification of the His-tagged ApoA1 (His-ApoA1) than a gradual increase (Supplementary Fig. S3). After this purification step, Western blot analysis using a rabbit anti-ApoA1 antibody confirmed that the 25–28 kDa protein expressed in the soluble fraction was indeed ApoA1 (Fig. 1B).

Mass spectrometry analysis of the entire protein on the deconvolution of the obtained total ion chromatogram (Fig. 1C) indicate that the average molecular mass of the obtained protein is 31,073.9069 Da, which corresponds to the theoretical mass of the His-tagged ApoA1 (31,092 Da) with a loss of a water molecule. ¹H NMR was used to assess the folding state of the recombinant protein and to compare it with ApoA1 purified from plasma. The signal dispersion in both spectra highlights the presence of many peaks in the NH region (green circles) and the methyl region (pink circles) that attests the partial structuring of lipid free plasma and recombinant ApoA1 in solution. The similarity between the two NMR spectra shows that His-ApoA1 has a similar structure to plasma ApoA1 (Fig. 1D). All these results show that the protein expressed *via* the pET28a plasmid in the *Clear coli* strain corresponds to a purified His-ApoA1 with similar structure compared to plasma ApoA1.

### 3.1.2. Histidine Tag cleavage by TEV protease

In order to obtain a recombinant protein with the native ApoA1 sequence, the His-ApoA1 tag was cleaved with the TEV protease during

different incubation times with the ratio of 1:100 (TEV: His-ApoA1 (w/w)) at 30 °C. Analysis on the Coomassie blue-stained gel of the corresponding samples at 2 and 4 h of incubation with TEV showed two distinct bands at 28 kDa and 31 kDa corresponding to the cleaved and uncleaved forms of the protein, respectively (Fig. 2A). 20 h of incubation with TEV protease were found to be sufficient for complete cleavage as evidenced by the single band at 28 kDa for the corresponding sample. Western blot analysis using an anti-His-Tag antibody showed the drastic decrease in intensity of the band relative to the protein tag following the incubation of His-ApoA1 in the presence of the TEV protease (Fig. 2B).

The cleaved protein was characterized by mass spectrometry and the resulting multiple charge state spectrum was deconvoluted. This revealed that the molecular mass of the cleaved ApoA1 was 28,117.5668 Da, which precisely matched the theoretical mass of unlabeled ApoA1 (28,135 Da) with a loss of a water molecule (obtained *via* the nucleic acid sequence of the serial cloner software and the ExPASy translation tool) (Fig. 2C). ¹H NMR analyses were performed on His-ApoA1 (uncleaved form) and ApoA1 (cleaved form) to assess the impact of histidine tag removal on the structure of recombinant ApoA1. The similarity in spectra between cleaved and uncleaved ApoA1, in particular in the amide, aromatic and methyl regions demonstrate that the cleavage occurred correctly without altering the structure of the protein (Fig. 2D). The disappearance of the chemical shifts of the histidine label proton signals in cleaved ApoA1 in the HE1 (about 7.8 ppm) and HD2 (about 7 ppm) region (dark insets) confirms the efficiency of cleavage. All these results show that His-ApoA1 could be efficiently cleaved without altering the structure of the protein.

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** rHDL and His-rHDL nanoparticles display typical morphological structure of plasma HDLs. Particle size distribution in intensity and in number of particles determined by DLS analysis for rHDL (A and C) and for His-rHDL (B and D) nanoparticles. Representative negative staining transmission electron microscopic (TEM) images of rHDL and His-rHDL nanoparticles (E and F, respectively). Images show that nanoparticles exhibit a discoidal shape of about 8–10 nm in diameter. Some nanodiscs are also stacked in the form of rouleau (see arrows). Scale bar = 200 nm.</figcaption></figure>
## 3.2. Production and characterization of rHDL nanoparticles

ApoA1 nanoparticles were reconstituted by mixing rApoA1, His-ApoA1 or lipid free plasma ApoA1 with POPC (at a molar ratio of 1:75) according to the sodium cholate method. The size of nanoparticles was determined by Differential Light Scattering (DLS) and their morphology visualized through (TEM) (Fig. 3).

The intensity size of rHDLs and His-rHDLs were distributed between 7 and 120 nm (Fig. 3A and B) with a low polydispersity (value below 0.2). Because a small fraction of large particles can generate a disproportionate signal, the intensity profile emphasizes the larger particles and, therefore, does not reflect the proportions of different particle populations. In contrast, the number distributions provide an estimate of the actual particle counts, showing here that the majority of particles for both nanoparticle types presented an apparent size around 7–8 nm (Fig. 3C and D). The particle sizes were confirmed by TEM, which showed the discoidal shape of the nanoparticles stacked in the form of “rouleau” (Fig. 3E and F). NMR also demonstrated that rApoA1 conformation in rHDLs was similar to that of plasma ApoA1 in nanoparticles (Supplementary Fig. S4).

## 3.3. rHDL nanoparticles are taken-up by endothelial cells

The intracellular uptake of rHDL and His-rHDL nanoparticles by endothelial cells (EA.hy926) was investigated by using anti-ApoA1 antibody for their immunodetection. Fig. 4A and B shows that both rHDL nanoparticles are taken up by endothelial cells after 6 h incubation.

Indeed, ApoA1 was detected in endothelial cells and confocal microscopy analysis further confirmed the internalization of rHDLs (here His-rHDLs) by EA.hy926 cells (Supplementary Fig. S5). We also found that rHDL nanoparticle uptake by endothelial cells occurred already after 2 and 4 h of incubation (Supplementary Fig. S6). It is important to note that endothelial cells viability was not affected by either rHDL or His-HDL nanoparticles after 24 h incubation (Fig. 4C and D). We have also demonstrated that at concentrations up to 1 mg/mL, His-rHDL nanoparticles exhibit no significant cytotoxicity on endothelial cells (Supplementary Fig. S7). The capacity of our rHDL nanoparticles to be taken up was confirmed using another type of endothelial cells: bEnd3 a model of microvascular endothelial cells (Supplementary Fig. S8).

## 3.4. rHDL nanoparticles reduce IL-6 and ICAM-1 expression by endothelial cells stimulated by TNFα

We tested the capacity of our rHDL particles to limit TNFα-induced endothelial cell activation under our experimental conditions. EA.hy926 cells were exposed to TNFα (5 ng/mL) in the presence or absence of rHDLs or His-rHDLs (0.4 mg/mL). After 6 h of exposure, TNFα significantly increased the expression of IL-6 (Fig. 5A-D). While His-rHDL significantly decreased IL-6 expression at the mRNA and protein levels, the decrease of IL-6 mRNA with rHDL did not reach statistical significance (p = 0.063). As a result, TNFα induced an increase of IL-6 secretion after 24 h exposure and this increase was significantly reduced in the presence of either rHDLs or His-rHDLs (Fig. 5C and D). Similarly, TNFα-induced endothelial activation as evidenced by the marked increase in the expression of ICAM1 (intercellular adhesion

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** rHDL and His-rHDL nanoparticles are internalized by EA.hy926 endothelial cells. Immunostaining of endothelial EA.hy926 cells after 6 h of incubation without (CTRL) or with 0.4 mg/mL of A) rHDL or B) His-rHDL nanoparticles. Rabbit anti-human ApoA1 antibody was used for detection of rHDL or His-rHDL (green) and cell nuclei were stained with DAPI (blue). Magnification x20. Scale bar = 30 µm. Cell viability after 24 h of incubation with C) rHDLs or D) His-rHDLs, expressed as a percentage of neutral red value compared with CTRL (DMEM). Data are expressed as the mean ± SEM of three independent experiments.</figcaption></figure>

<figure><img src="image_9.png" /></figure>
A

<figure><img src="image_10.png" /></figure>

C

<figure><img src="image_11.png" /></figure>

E

<figure><img src="image_12.png" /></figure>

B

<figure><img src="image_13.png" /></figure>

D

<figure><img src="image_14.png" /></figure>

F

<figure><img src="image_15.png" /></figure>

**Fig. 5.** rHDL and His-rHDL nanoparticles prevent TNFα-induced IL-6 production and ICAM-1 expression by EA.hy926 endothelial cells. Relative gene expression of IL-6 by EA.hy926 cells after 6 h of incubation with A) rHDLs or B) His-rHDLs (0.4 mg/mL) in absence or presence of TNFα (5 ng/mL). IL-6 secretion by EA.hy926 cells quantified by ELISA. Data are expressed as percentage of IL-6 secretion compared to the condition in the presence of TNFα. Cells were previously co-incubated for 24 h with C) rHDLs or D) His-rHDLs (0.4 mg/mL) in absence or presence of TNFα (5 ng/mL). Relative gene expression of ICAM-1 and by EA.hy926 cells after incubation for 6 h with E) rHDL or F) His-rHDL (0.4 mg/mL) in the absence or presence of TNFα (5 ng/mL). Data are the mean ± SEM of three independent experiments. \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001.

molecule 1), which was inhibited by both rHDL and His-rHDL treatment (Fig. 5E and F).

### 3.5. rHDL accumulate in multiple mouse organs after intravenous administration

To further explore how rHDLs may be taken up *in vivo*, we evaluated the biodisponibility of our particles in mice.

24 h after intravenous administration of 50 µL of His-rHDL (12.5 µg/ µL), we observed a maximal accumulation of the nanoparticles in the liver, which is the principal site of HDL metabolism (Fig. 6). Nanoparticles were also clearly detectable in the spleen and in the lung with a homogeneous distribution in these organs. A fraction of nanoparticles

also appeared to reach the brain.

## 4. Discussion

In this study, we have produced, in a *Clear coli* strain, recombinant human apolipoprotein A1 with a final yield of around 30 mg of protein per liter of culture medium, comparable to the other procedures used for the production of ApoA1 [23]. Such a high yield is only possible with the presence of an affinity tag incorporated upstream the recombinant protein by genetic fusion. The addition of a His tag improves the purification yield because of its high affinity for nickel or cobalt chromatography resins, which streamline and increase the efficiency of the purification steps [33].
<figure><img src="image_16.png" /><figcaption>**Fig. 6.** DilC18 labeled rHDLs are mainly detectable in mouse liver, lung and spleen 24 h after injection. DAPI (blue) was used for nucleus staining. Representative illustration of DilC18 labeled rHDLs (red) after injection of 50 µL of PBS solution or His-rHDL (625 µg ApoA1) (from 3 experiments with *n* = 6 mice). Scale bar =5 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
Cleavage of the label following the purification step can have several drawbacks, particularly the impact of cleavage conditions on protein structuration. We have observed that using more extreme cleavage conditions to increase cleavage efficiency (longer time or higher temperature) can denature recombinant ApoA1, which tends to form aggregates. Furthermore, following this cleavage step, removal of the cleaved tag and TEV protease requires additional purification steps which clearly reduce the overall yield [34].

In addition to avoiding this potentially disruptive cleavage step for protein structuration and yield, the relevance of keeping the histidine tag has several advantages: first, it makes it easier to purify the protein by affinity chromatography using a nickel column. Moreover, it could also enable the purification of ApoA1-containing nanoparticles (Histagged ApoA1) following an enrichment of the nanoparticles with a therapeutic drug. In addition, ApoA1 nanoparticles with the poly histidine chain might be a potential carrier for delivery of miRNA mimics [35]. Finally, it can facilitate *in vivo* tracking after injection for preclinical studies (anti-His antibodies are very specific). Up to now, there is not much extensive and detailed experimental data describing the immunogenicity of the 6xHis tag to support or exclude the use of Histagged drugs in clinical practice. However, several malaria vaccines that include histidine-rich tags have shown their safety in various phase II clinical trials conducted on African children [36] as well as in phase I studies carried out in Kenyan adults [37]. These studies suggest that the addition of a histidine-rich motif does not affect the safety of the therapeutic agent.

In our study, the main drawback of preserving this label would be to affect biochemical and biological properties of the apolipoprotein and hence the nanoparticles. However, we showed by NMR that following the cleavage of the histidine tag by TEV protease, the structure of the protein is unmodified. Moreover, the tag does not appear to have a notable impact on the nanoparticle reconstitution.

Several protocols have previously been used for reconstituting nanoparticles from phospholipids and ApoA1, including sonication, thermal cycling, sodium cholate, and microfluidics [38]. Here, using a method that combined use of sodium cholate with freeze/thaw thermal cycling, we mainly reconstitute nanoparticles with a diameter of 7–8 nm after incubation with ApoA1 and POPC at a 1:75 M ratio of protein to phospholipid. DLS, TEM and NMR results indicate that the method used allows ApoA1 to be complexed correctly with POPC and that the use of ApoA1 with a poly-histidine extension in the N-terminal position does not seem to affect the size of the nanoparticles with the used method.

As the functions of rHDL nanoparticles are closely related to their structural integrity [6] as well as that of ApoA1, some biological properties have been tested. Our study focused on the anti-inflammatory activity and endothelial cell uptake of ApoA1 nanoparticles, which are the key properties required for their use in limiting the complications of sepsis. Our data revealed the ability of rHDL and His-rHDL to be rapidly internalized by endothelial cells. The internalization of HDL by endothelial cells has already been shown to be mediated by scavenger receptor, class B, type I (SR-BI) [4], and, as described by Rohrer L et al., transcytosis appears to be one of the main mechanisms in which the ATP-binding cassette transporter G1 (ABCG1) is also involved [39]. ATP-binding cassette transporter A1 (ABCA1) could also modulate the transcytosis of apoA1 through endothelial cells [40]. HDL uptake can also occur *via* endocytosis involving tubular endosomal vesicles and multivesicular bodies in human endothelial cells [41]. Whatever the mechanism involved, the ApoA1 nanoparticles we have produced, with or without His tag, are taken up by endothelial cells.

Similarly, the occurrence of the Tag on the apolipoprotein does not affect the anti-inflammatory and endothelial-protective properties of rHDL. We tested the ability of rHDL and His-rHDL to limit inflammation and endothelial activation induced by TNFα on immortalized human vascular endothelial cells (EA.hy926). Indeed, we found that both types of ApoA1 nanoparticles equivalently inhibited IL-6 and ICAM-1 expression in response to TNFα. As for the decrease in IL-6 mRNA

levels and its secretion in TNFα-stimulated endothelial cells, the levels of protection appear similar to those found in another study using reconstituted or plasma HDLs [42]. In addition, inhibition of ICAM-1 expression by rHDLs and His-HDLs indicates a significant protective role of nanoparticles towards endothelial activation. This protection is particularly significant as, in another study, the levels of protection were equivalent solely when at least 5 % sphingomyelin were included in the rHDL lipid composition [43].

To investigate the fate of our His-HDL nanoparticles *in vivo*, a bio-distribution study was carried out in C57BL/6 mice by intravenous injection. Twenty-four hours after administration, the mice were found to be healthy, with the nanoparticles localized mainly in the liver, lung and spleen and to a lesser extent in the brain. This suggests that our rHDL may cross the blood-brain barrier, as previously reported *in vitro* and *in vivo* [10,44]. The presence of the N-terminal extension containing a poly-histidine label did not prevent homogeneous biodistribution in these tissues. This is consistent with some studies showing that the histidine tail does not significantly affect protein biodistribution in NMRI mice [45,46]. The specific distribution of His-HDL, which we only observe in some organs, is in agreement with *in vivo* PET imaging of discoidal HDL nanoparticles in murine, rabbit and porcine models, where nanoparticles accumulate mainly in the liver, spleen and lungs [47]. It is not entirely surprising that POPC-related DilC18 labelling is not observed in the kidney, since it is ApoA1 that accumulates mainly in this organ, whereas in the liver, spleen and lungs, it is phospholipids that accumulate the most [48]. In addition, we quantified IL-6 levels in the plasma of PBS- and His-HDL-treated mice and found no difference in basal expression (data not shown), ruling out any pro-inflammatory effect of His-HDL treatment.

Taken together, these results demonstrate that the introduction of a poly-histidine tag into recombinant human ApoA1 produced in the *Clear* *coli* strain do not prevent the reconstitution of HDL-like nanoparticles and do not alter their protective functions. This opens the way to make use of the tag to enrich rHDLs with miRNAs or therapeutic drugs targeting the liver, lung or other organs.

### CRediT authorship contribution statement

**Sarah Rosanaly:** Writing – review & editing, Writing – original draft, Methodology, Investigation. **Marie Laurine Apalama:** Investigation, Formal analysis. **Matthieu Bringart:** Methodology, Investigation. **Pierre Giraud:** Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization. **Benoit Allard:** Writing – review & editing, Writing – original draft, Investigation, Conceptualization. **Bryan Veeren:** Methodology. **Olivier Meilhac:** Writing – review & editing, Writing – original draft, Validation, Supervision, Conceptualization. **Joël Couprie:** Writing – review & editing, Supervision. **Philippe Rondeau:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

### Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used ChatGPT in order to make adjustments and improve readability of the text. After using this tool, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

### Funding

This work was supported by, the Université de La Réunion and by the European Regional Development Funds RE0001897 (EU, Région Réunion, French State national counterpart). SR and MLA were recipients of a fellowship grant from the Ministère de l'Éducation Nationale, de l'Enseignement Supérieur et de la Recherche, La Réunion
University.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Special thanks for J.M. Guigner, IMPMC Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, Sorbonne Université for performing Transmission Electron Microscopy.

Appendix A. Supplementary data

**Supplementary Materials: Fig. S1:** Schematic of the codon-optimized pet28a plasmid designed by GeneCust for bacterial transformation and overexpression of ApoA1. **Fig. S2:** Protocol design of recombinant ApoA1 production and rHDL reconstitution. **Fig. S3:** Two methods of purification of uncleaved rApoA1 by affinity chromatography. **Fig. S4:** rApoA1 conformation is similar to that of plasma ApoA1 conformation in nanoparticles as evidenced by NMR. **Fig. S5:** His-rHDL nanoparticles are taken up by EA.hy 926 endothelial cells. **Fig. S6:** Kinetic of rHDL nanoparticle uptake by EA.hy 926 cells. **Fig. S7:** Cytotoxicity assays on endothelial EA.hy926 cells with increasing concentration of His-rHDL nanoparticles for 24 h. **Fig. S8:** Kinetic of rHDL nanoparticle uptake by bEnd.3 cells. Supplementary data to this article can be found online at doi:https://doi.org/10.1016/j.bbalip.2025.159606.

Data availability

No data was used for the research described in the article.

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