# Deciphering the role of polyethylene glycol-lipid anchors in siRNA-LNP efficacy for P2y2 inhibition in bone marrow-derived macrophages

Sezen Gül, Juliette Vergnaud, Qinglin Wang, Mélanie Hery, Jhon Mino, Jihana Achour, Séverine Domenichini, Claudine Delomenie, Jean-Luc Perfettini, François Fay, et al.

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Sezen Gül, Juliette Vergnaud, Qinglin Wang, Mélanie Hery, Jhon Mino, et al. Deciphering the role of polyethylene glycol-lipid anchors in siRNA-LNP efficacy for P2y2 inhibition in bone marrow-derived macrophages. International Journal of Pharmaceutics, 2025, 684, pp.126186. 10.1016/j.ijpharm.2025.126186. hal-05322517

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# Deciphering the role of polyethylene glycol-lipid anchors in siRNA-LNP efficacy for P2y2 inhibition in bone marrow-derived macrophages

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Sezen Gül<sup>a</sup>, Juliette Vergnaud<sup>a, b</sup>, Qinglin Wang<sup>a, b</sup>, Mélanie Hery<sup>a</sup>, Jhon Mino<sup>a</sup>, Jihana Achour<sup>a</sup>, Séverine Domenichini<sup>b</sup>, Claudine Delomenie<sup>c</sup>, Jean-Luc Perfettini<sup>d</sup>, François Fay<sup>a, e, *</sup>, Elias Fattal<sup>a, *</sup>

## ARTICLE INFO

Keywords:

Lipid nanoparticles
siRNA

Pegylated lipids
Cytoplasmic release
P2Y2 inhibition

## ABSTRACT

Macrophages play a vital role in controlling tissue homeostasis, yet their dysregulation can drive the patho-
genesis of various diseases. In this context, P2Y2, a purinergic receptor that binds ATP, could be involved in
several biological mechanisms in inflammatory, infectious, or cancer diseases. Lipid nanoparticles (LNPs)
varying by the difference of lipid moiety of the polyethyleneglycol-lipid (PEG-lipid) conjugate entering their
composition were prepared by microfluidic mixing technology and loaded with a siRNA targeting the P2y2
mRNA. Three PEG-lipids with varying saturated alkyl chain lengths — DMG-PEG (C14), DPPE-PEG (C16), and
DSPE-PEG (C18) — were evaluated for formulation optimization. The results showed that all siRNA-LNPs were
efficiently taken up by unpolarized (M0) and M2-polarized bone marrow-derived macrophages (BMDMs).
Despite comparable cellular uptake across formulations, their performance in gene silencing differed signifi-
cantly. While (DMG-PEG)-LNPs demonstrated the highest efficacy in both cellular models, (DSPE-PEG)-LNPs
remained ineffective in knocking down the target gene. Our findings further revealed that gene silencing is more
efficient in M2 BMDMs than in M0 BMDMs, emphasizing the influence of macrophage polarization. Additionally,
the PEG-lipid type notably influenced the intracellular distribution patterns of lipids and siRNA, with DPPE-PEG
and DSPE-PEG formulations leading to higher siRNA compartmentalization. In contrast, the DMG-PEG formu-
lation enabled more effective siRNA delivery to the cytoplasm. This research provides valuable insights into the
impact of different PEG-lipid anchors on the performance of siRNA-LNPs targeting macrophages in vitro, paving
the way for developing new macrophage-based immunotherapies.

## 1. Introduction

Purinergic signaling is essential for many biological processes in humans (Ai et al., 2023). Among P2Y purinergic receptors, the P2Y2 receptor binds ATP and mediates several aspects of immune cell function (Idzko et al., 2014). Activation of P2Y2 receptors on macrophages during inflammation contributes to the expression of inflammatory mediators, including *cyclo-oxygenase 2* and inducible nitric oxide synthase (Eun et al., 2014). *P2y2* gene deletion was recently shown to protect mice from bacterial endotoxin and sepsis-associated liver injury and mortality. In this publication, P2Y2 antagonist pre-treatment of

macrophages attenuated LPS/TLR4-mediated cytokine and chemokine (MCP-1, IL-6, IL-10) release in Raw264.7 macrophages (Arunachalam et al., 2023).

A functional interaction between the NOD-like receptor (NLR) family member NLRP3 and P2Y2 was recently discovered (Paoletti et al., 2019). NLRP3 is an intracellular pattern recognition receptor that recognizes pathogen-associated and damage-associated molecular patterns involved in the so-called NLRP3 inflammasome (Swanson et al., 2019). It was paradoxically demonstrated that the activation of the P2Y2 receptor represses the biological activity of NLRP3 and regulates susceptibility to HIV-1 infection. Indeed, NLRP3 represses F-actin remodeling,

<footnote><sup>a</sup> Institut Galien Paris-Saclay, CNRS UMR 8612, Université Paris-Saclay, 91400 Orsay, France</footnote>

<footnote><sup>b</sup> UMS-IPSIT Plateforme MIPSIT, Université Paris-Saclay, CNRS, Inserm, Ingénierie et Plateformes au Service de l'Innovation Thérapeutique, 91400 Orsay, France</footnote>

<footnote><sup>c</sup> UMS-IPSIT Plateforme ACTAGEN, Université Paris-Saclay, CNRS, Inserm, Ingénierie et Plateformes au Service de l'Innovation Thérapeutique, 91400 Orsay, France</footnote>

<footnote><sup>d</sup> Université Paris-Saclay, Gustave Roussy, Inserm UMR1030, Laboratory of Molecular Radiotherapy and Therapeutic Innovation, Villejuif F-94805, France</footnote>

<footnote><sup>e</sup> Institut Universitaire de France (IUF), France</footnote>

<footnote>* Corresponding authors at: Institut Galien Paris-Saclay, CNRS UMR 8612, Université Paris-Saclay, 91400 Orsay, France.
E-mail addresses: francois.fay@universite-paris-saclay.fr (F. Fay), elias.fattal@universite-paris-saclay.fr (E. Fattal).</footnote>
reducing the virus’s entry into host cells such as macrophages (Swanson et al., 2019). Additionally, in an oncology setting, Mostefa-Kara et al. identified P2Y2 as a negative regulator of pro-inflammatory macrophage activation, with its loss promoting pro-inflammatory responses in macrophages after irradiation (Mostefa-Kara et al., 2024). Thus, the inhibition of P2Y2-dependent signaling pathways could be proposed to reprogram macrophages from an alternatively activated anti-inflammatory M2 profile to a classical activated pro-inflammatory one, as a combination with radiotherapy. For all these reasons, we propose to consider nanoparticle-based systems for delivering P2y2 expression inhibitors in treating inflammatory, infectious, or cancer diseases.

To knock down P2y2, we considered targeted delivery of small interfering RNA (siRNA). SiRNA are short double-stranded RNA molecules that silence genes by degrading target messenger RNA (mRNA) and stopping protein production (Dong et al., 2019; Fattal and Fay, 2021; Saw and Song, 2020; Tatiparti et al., 2017). They can effectively modulate the immune response of macrophages by silencing selected genes. However, delivering free siRNA by injection faces degradation, quick elimination, and limited cellular uptake (Singh et al., 2018). Lipid nanoparticles (LNPs) have emerged as highly effective vehicles for overcoming the challenges associated with siRNA delivery. These nanocarriers protect siRNA from nuclease degradation, decrease uptake by the mononuclear phagocyte system (MPS), reduce renal clearance, and facilitate transmembrane transport and endosomal escape (Kulkarni et al., 2021). Today, a few preclinical studies use siRNA-loaded LNPs to reprogram macrophages in various diseases, including cancer (Shobaki et al., 2020; Walters et al., 2021; Yong et al., 2022; Zhou et al., 2024), autoimmune and inflammatory diseases (Courties et al., 2014; Koga et al., 2015; Zhao et al., 2024; Zhou et al., 2022), as well as infectious diseases (Tang et al., 2024). However, further research is needed to uncover the full therapeutic potential of this approach.

LNPs typically comprise four main constituents: an ionizable cationic lipid, a helper phospholipid, cholesterol, and a polyethylene glycol (PEG)-lipid conjugate (Kulkarni et al., 2021; Tieu et al., 2021). PEG-lipids' influence on LNP behavior is complex, giving rise to a term commonly called the 'PEG dilemma' (Sarode et al., 2022). PEG-lipids reduce protein adsorption to the LNP surface by providing a hydrophilic steric barrier, consequently decreasing the uptake by MPS and increasing the *in vivo* circulation time. However, the hydrophilic PEG corona might reduce cellular uptake efficiency or endosomal escape processes. Both the PEG chain and the anchoring lipid significantly affect the performance of LNPs (Berger et al., 2023). An intermediate length with a molecular weight of 2000 g/mol is widely utilized for the PEG chain to balance prolonged circulation time and effective intracellular delivery (Berger et al., 2023; Chen et al., 2019; Ge et al., 2021). Regarding the lipid anchor, PEGs with short lipid chains can rapidly dissociate from the lipid membrane of LNPs in serum, leading to a shorter *in vivo* half-life. Previous studies have demonstrated that the DMG-PEG2000 (C14) quickly diffuses from the LNPs in blood circulation, allowing for ApoE binding to the surface, resulting in optimal hepatocyte targeting (Akinc et al., 2019; Albertsen et al., 2022). Conversely, PEGs with longer lipid chains [(e.g., DSPE-PEG (C18)] enable longer blood circulation time, which could enhance accumulation in tumors or inflammatory sites (Berger et al., 2023; Paoletti et al., 2024). Therefore, to optimize the transfection efficiency of siRNA-LNPs, the type of PEG-lipid should be selected depending on the specific therapeutic target and the administration route (Albertsen et al., 2022).

In this study, we utilized microfluidic mixing technology to encapsulate siRNA targeting the P2y2 gene (siP2Y2) in LNPs composed of diverse PEG-lipid anchors to modulate macrophage function *in vitro*. Three PEG-lipids with saturated alkyl chains, namely DMG-PEG (C14), DPPE-PEG (C16), and DSPE-PEG (C18), were assessed to optimize the LNP formulations in terms of siRNA delivery efficiency and gene silencing. Murine BMDMs were employed as the cellular model. While the primary focus was on M2-polarized BMDMs, unpolarized BMDMs

were also examined to evaluate the potential differences in gene silencing efficiency between these two models. Following siRNA-LNP treatment, a highly efficient and comparable cellular uptake was observed amongst different LNP variants in both macrophage subsets. Our results demonstrated that the choice of PEG-lipid significantly impacts gene silencing efficiency in macrophages, and this efficiency is dependent on their polarization state.

Additionally, we provided insights into the influence of distinct PEG-lipid anchors on the intracellular fate of siRNA-LNP systems. While DPPE-PEG and DSPE-PEG formulations exhibited higher intracellular compartmentalization of siRNA, DMG-PEG formulation enabled more effective siRNA delivery to the cytoplasm. These findings highlight the critical importance of PEG-lipid optimization in siRNA-LNP design, providing a basis for therapeutic strategies targeting macrophage reprogramming.

## 2. Material and methods

### 2.1. Materials

The ionizable cationic lipid (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).1,2-distearoyl-*sn*-glycero-3-phosphocholine (DSPC) and N-(carbonyl-methoxy) polyethylene glycol-2000)-1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG2000) were obtained from Lipoid GmbH (Ludwigshafen, Germany). 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dipalmitoyl-*sn*-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DPPE-PEG2000) and 1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N-(Cyanine 5.5) (DSPE-Cy5.5) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol was provided by Sigma-Aldrich (St. Louis, MO, USA). siRNAs used in this study were obtained from Kaneka Eurogentec S.A. (Seraing, Belgium), and the sequences of their sense and anti-sense strands are listed in Supplementary Table S1. Glacial acetic acid and absolute anhydrous ethanol were purchased from Carlo Erba (Milan, Italy). Nuclease-free water was obtained from Sigma-Aldrich (St. Louis, MO, USA). QuantiFluor® RNA System was purchased from Promega Corporation (Madison, WI, USA). Lipofectamine™ RNAiMAX transfection reagent was provided by Thermo Fisher Scientific (Waltham, MA, USA). Recombinant murine macrophage colony-stimulating factor (M-CSF), interleukin 4 (IL-4), and interleukin 13 (IL-13) were acquired from PeproTech EC Ltd. (London, UK). Lipopolysaccharide (LPS from Escherichia coli O55:B5) was purchased from InvivoGen (Toulouse, France).

### 2.2. Preparation of siRNA-LNPs

siRNA-LNPs were prepared via a microfluidic method, employing a staggered herringbone micromixer (Cat\#10000019, microfluidic Chip-Shop, Jena, Germany). The ionizable cationic lipid DLin-MC3-DMA, DSPC, cholesterol, and lipid-PEG2000 (DSPE-PEG2000, DPPE-PEG2000, or DMG-PEG2000) were dissolved in ethanol at the molar ratios of 46.5:9.3:41.9:2.3, resulting in a total lipid concentration of 14.33 mM. The siRNA solution, siP2Y2 or scrambled siRNA (siScr) was prepared in 25 mM sodium acetate buffer at pH 4.0. The lipid and siRNA solutions were combined in the microfluidic mixer at a total flow rate of 6 mL/min, with an aqueous-to-organic volumetric flow rate ratio of 3:1, using two syringe pumps (NE-300 InfusionOne, New Era Pump Systems Inc., Farmingdale, NY, USA). The resulting siRNA-LNPs had a nitrogen-to-phosphate (N/P) ratio of 11.9 considered high. Still, values superior to 9 were described as optimal by us (Wang et al., 2025) and confirmed by others (Mo et al., 2025). Subsequently, three rounds of ultrafiltration (3600 g, 30 min, 4–6 °C) were performed using Amicon Ultra Centrifugal Filters (100 kDa MWCO, Merck Millipore, Burlington, MA, USA) to
remove the residual ethanol solvent and adjust the pH to 7.4 with
phosphate-buffered saline (PBS). Finally, the formulations were steril-
ized using a 0.22-µm PVDF filter (Merck Millipore, Burlington, MA,
USA) and stored at 4 °C in PBS under a nitrogen or argon atmosphere.

## 2.3. Characterization of siRNA-LNPs

The hydrodynamic diameter, polydispersity index (PDI), and zeta potential (ζ-potential) of the LNPs were determined by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern Panalytical, Worcestershire, UK) at 25 °C. For the measurements, LNP solutions were diluted 1:20 with PBS. The size and polydispersity were determined in PBS (pH 7.4), while the zeta potential was measured in NaCl solution (1 mM, pH 4 or 7). All measurements were conducted in triplicate using independent samples.

The siRNA encapsulation efficiency (EE%) was determined using the QuantiFluor® RNA System. Briefly, 200 µL of QuantiFluor® RNA dye working solution was added to 10 µL of diluted LNPs (1:10, 1:20, and 1:40 diluted), which had been lysed with 2% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Following a 10-minute stirring process on a plate shaker (450 rpm, room temperature, dark), siRNA was quantified by measuring the fluorescence (λ<sub>ex</sub> = 492 nm, λ<sub>em</sub> = 540 nm) using an Envision Xcite reader (Perkin Elmer, Waltham, MA, USA). The RNA concentration was calculated based on a linear standard curve (0.31–20 ng/µL, R<sup>2</sup> ≥ 0.99) established for each siRNA tested. The EE% of siRNA was calculated using the following formula: $EE\% = (Amount of siRNA encapsulated (ng)/Total amount of siRNA injected (ng)) \times 100$. Additionally, agarose gel electrophoresis was conducted to confirm the siRNA encapsulation within LNPs qualitatively. In brief, lysed or unlysed LNP samples were mixed with gel loading buffer (Sigma-Aldrich, St. Louis, MO, USA) and loaded onto a 3 % agarose gel prepared in Tris/Borate/EDTA (TBE) buffer (Invitrogen™, Waltham, MA, USA) buffer containing 25 µg/µL ethidium bromide (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gel was run for 60 min at 120 V. Nucleic acid bands were visualized using a ChemiDoc Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA).

LNPs' stability in biological solutions was analyzed by incubating
LNPs samples in 10 % fetal bovine serum (FBS) at 37 °C for various
periods ranging from 10 min to 24 h. After incubation, LNPs were lysed
with 2 % (v/v) Triton X-100, and the siRNA quantities were determined
by agarose gel electrophoresis and ImageJ quantification.

2.4. Bone marrow-derived macrophage (BMDM) isolation

Male C57BL/6 mice (Envigo), aged 8 to 12 weeks, were euthanized, and the bone marrow was collected from femurs and tibias. Following the lysis of red blood cells with RBC lysis buffer (BioLegend, San Diego, CA, USA), bone marrow cells were cultured at a density of 2x10⁶ cells per T25 flask in 5 mL of DMEM high glucose medium (Gibco, Waltham, MA, USA) supplemented with 10 % heat-inactivated FBS (Gibco, Waltham, MA, USA), 1 % penicillin/streptomycin/amphotericin B (Gibco, Waltham, MA, USA), and 25 ng/mL recombinant mouse M-CSF. Cultures were maintained at 37 °C in a 5 % CO₂ atmosphere for 7 days, adding 2.5 mL of fresh differentiation medium on day 4. After the differentiation of monocytes into BMDMs, the cells were either given media alone to obtain an unpolarized M0 state or treated for 24 h with 20 ng/ mL IL-4 and 20 ng/mL IL-13 for M2 polarization.

2.5. Evaluation of siRNA-LNP uptake by flow cytometry

Fluorescently labeled nanoparticles were prepared to investigate the
cellular uptake of M0 and M2 BMDMs, by incorporating DSPE-cyanine
5.5 (Cy5.5) into the lipid formulation at a molar ratio of 0.2 % and
loading them with siP2Y2-6-carboxyfluorescein (6-FAM). BMDMs were
treated with LNPs at 1 µM siP2Y2-6-FAM for 4 h in a humidified incu-
bator (37 °C and 5 % CO₂). Untreated cells and cells treated with free

siP2Y2-6-FAM were used as negative controls. Following the treatment,
cells were washed twice with 2.5 mL of warm PBS and harvested by
scraping in 1 mL of ice-cold PBS. Subsequently, the cell suspension was
analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences,
Franklin Lakes, NJ, USA) to measure the fluorescence intensity of Cy5.5
(λ<sub>ex</sub> = 678 nm, λ<sub>em</sub> = 707 nm) and 6-FAM (λ<sub>ex</sub> = 495 nm, λ<sub>em</sub> = 517 nm).
A minimum of 10,000 gated events, based on forward scatter and side
scatter, were recorded for each sample. Post-acquisition analysis was
performed using FlowJo software (BD Biosciences, Franklin Lakes, NJ,
USA). For data analysis, the mean fluorescence intensity values (6-FAM
or Cy5.5) obtained for LNP-treated samples were initially normalized to
the fluorescence of each LNP formulation. This was done to account for
potential variations in fluorescence across the three LNP variants. To
this end, LNPs were lysed with 2 % (v/v) Triton X-100 (Sigma-Aldrich,
St. Louis, MO, USA) and diluted 1:100 with PBS. Two hundred µl of the
diluted LNP solution was added to each well of a black flat-bottom 96-
well plate (#3915, Corning, NY, USA) in quadruplicate. Then each
formulation's 6-FAM and Cy5.5 fluorescence was measured at the pre-
viously stated excitation and emission wavelengths using SAFAS Xenius
XC spectrofluorometer (SAFAS, Monaco). Subsequently, these values
were further normalized to the mean fluorescence intensity of untreated
negative control cells determined by the flow cytometer and multiplied
by 10⁵ for illustrative purposes.

2.6. Assessment of target gene silencing by RT-qPCR

Unpolarized or M2-polarized BMDMs were treated with three LNP formulations encapsulating 50 or 100 nM siRNA. The cells were incubated for either 24 or 48 h at 37 °C. For the 48-hour incubations, the medium was replaced after 24 h, and the cells were subsequently cultured in a regular growth medium for an additional 24 h. The experimental design included two types of negative controls: an untreated sample and a sample treated with LNPs encapsulating siScr. A positive control was also included, consisting of a sample transfected with the commercial transfection reagent LipofectamineTM RNAiMAX. After the specified incubations, total RNA was extracted using the NucleoSpin® RNA Kit (Macherey-Nagel, Dueren, Germany), following the manufacturer's instructions. Specifically, 350 µL of extraction buffer was added directly to the T25 flask, the cells were dislodged using a cell scraper, and the final elution was performed with 40 µL of nuclease-free water. Genomic DNA contamination was eliminated with the RNase-Free DNase Kit (Macherey-Nagel, Dueren, Germany). The RNA concentration was measured with NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cDNA synthesis, 0.5 to 1 µg of total RNA was reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Quantitative real-time PCR (RT-qPCR) reactions were then performed on the cDNA using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA) in the CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The qPCR conditions were as follows: an initial denaturation at 95 °C for 30 s, followed by 45 cycles of 95 °C for 5 s and 60 °C for 20 s. A melting curve analysis was performed with 0.5 °C increments every 5 s between 65 °C and 95 °C. The mRNA expression level of the P2y2 gene was assessed using CFX Maestro software (Bio-Rad Laboratories Inc., Hercules, CA, USA). Relative gene expression was analyzed by the ΔΔ Ct method and normalized to the internal reference genes *β*-actin and *Gapdh*.

Furthermore, the gene expression kinetics in M2-polarized BMDMs
were studied over 72 h at various time points (0, 2, 4, 8, 24, 48, and 72
h). The cells were treated with various LNPs encapsulating 50 nM siRNA
to confirm the downregulation of the target gene *P2y2*. RT-qPCR was
performed, and the data were analyzed as described above. PCR primers
used in this study were obtained from Eurofins Genomics (Ebersberg,
Germany), and the sequences of the forward and reverse primers are
listed in Supplementary Table S2.
## 2.7. Investigation of siRNA-LNPs' intracellular fate by confocal microscopy

A confocal microscopy experiment was carried out to evaluate the intracellular distribution of siRNA delivered by LNPs composed of three lipid-PEGs. On day 7, BMDMs were harvested by gentle scraping in ice-cold PBS, centrifuged at 200 g for 5 min, and resuspended in differentiation medium (DMEM high glucose supplemented with 10 % heat-inactivated FBS, 1 % penicillin/streptomycin/amphotericin B, and 25 ng/mL recombinant mouse M-CSF). The cells were seeded in 8-well high ibidi µ-slides (ibiTreat, Cat#80806, ibidi GmbH, Gräfelfing, Germany) at a density of 75,000 cells/well in 300 µL of differentiation medium and allowed to adhere overnight. Cells were then polarized to the M2 phenotype by incubating with 20 ng/mL IL-4 and 20 ng/mL IL-13 for 24 h. Following polarization, M2 BMDMs were treated with three Cy5.5-LNPs encapsulating 1 µM 6-FAM-siP2Y2 for 4, 8, 24, and 48 h. After the specified incubation times, cells were washed three times with PBS and fixed with 4 % paraformaldehyde (#P087.5, Carl Roth, Karlsruhe, Germany) for 10 min at room temperature. Subsequently, cells were rewashed three times with PBS, and VECTASHIELD® antifade mounting medium with DAPI (Cat#H-1200, Vector Laboratories, Newark, CA, USA) was applied to cover the surface of the wells. Images were acquired using an inverted TCS SP8-gated STED confocal microscope (Leica Microsystems, Wetzlar, Germany) controlled by Leica Application Suite X (LAS X) v3.5.5 software. The microscope was equipped with an HC PL APO 63x/1.40NA oil-immersion objective, a 405 nm diode laser for DAPI staining (cyan channel), and a WLL laser set at 498 nm for 6-FAM (green channel) and 670 nm for Cy5.5 (red channel). Fluorescence emissions were collected using a sequential mode with a variable beam splitter configured to specific ranges: 415–480 nm for the cyan channel, 507–580 nm for the green channel, and 676–800 nm for the red channel. A photomultiplier tube (PMT) was used for the blue channel, while an internal hybrid detector (HyD) with gating was employed for the green and red channels. The detection offset was adjusted to minimize zero-value pixels, and detector gains were set to optimize the dynamic range (12-bit images) while minimizing pixel saturation. The detector settings were calibrated based on the strongest signal condition and kept consistent across all acquisitions within the same analysis (e.g., all formulations simultaneously). The pinhole was set to 1.0 Airy unit, resulting in an optical section thickness of 0.89 µm per slice. For each well, a minimum of three fields were imaged using a 2 × 2 tile z-stack series, with identical z-step parameters across all samples. Image processing, including merging and maximum intensity projection, was performed using LAS X software (Leica Microsystems, Wetzlar, Germany).

## 2.8. Statistical analysis

All data were analyzed with GraphPad Prism version 8.0.1 (GraphPad Software Inc., San Diego, CA, USA) and expressed as mean ± standard deviation (SD) of independent repeats. The statistical comparison between the two groups was evaluated by a two-tailed unpaired student's t-test, and the difference between multiple groups was tested using either one-way or two-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The specific statistical analysis methods employed are described in the figure legends. Values were considered statistically significant for *p* < 0.05. The statistical significance was represented as follows: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

# 3. Results and discussion

## 3.1. Preparation and characterization of siRNA-loaded LNPs

In this study, as a proof of concept, we tested an LNP formulation (Fig. 1a) designed with a lipid composition analogous to the clinically

approved Onpattro® (Patisiran) formulation (Hu et al., 2020). Three lipid-PEGs with different alkyl chains were utilized to evaluate the performance of LNPs for siRNA delivery, including one neutral diglyceride PEG lipid, DMG-PEG, and two anionic phosphoglyceride PEG lipids, DPPE-PEG and DSPE-PEG (Fig. 1b). Saturated alkyl chains characterize all these lipid-PEGs, although the length of the hydrocarbon chains differs. While DMG-PEG has a lipid chain of C14, DPPE-PEG and DSPE-PEG possess a C16 and C18 chain, respectively. Previous studies have shown that increasing the length of the lipid anchor results in enhanced stability and blood circulation times but reduced efficiency of cellular uptake and endosomal escape (Berger et al., 2023; Ge et al., 2021). While PEGs with short lipid chains, such as C14, improve transfection efficiency thanks to their ability to rapidly detach from the LNP in the presence of a lipid sink, PEGs with longer lipid chains, such as C16 or C18, could interfere with gene silencing (Berger et al., 2023; Hattori et al., 2020). However, these longer PEG anchors could allow prolonged circulation time and enhanced tumor accumulation (Berger et al., 2023). Therefore, to optimize the transfection efficiency of siRNA-LNPs in macrophages, we examined the effects of three different PEG anchors on cellular uptake, intracellular distribution, and in vitro gene silencing. The molecular weight of PEG was fixed at 2000 Da, as intermediate PEG lengths have been demonstrated to provide an optimal balance between stability and effective gene delivery (Ge et al., 2021). The molar percentage of lipid-PEG was kept constant at 2.3 %.

The LNPs were obtained via microfluidic mixing of DLin-MC3-DMA, DSPC, cholesterol, and lipid-PEG2000 at a molar ratio of 46.5:9.3:41.9:2.3, respectively. LNPs encapsulating either siP2Y2 or siScr with an N/P ratio of 11.9 were purified by centrifugal filtration and characterized by DLS and QuantiFluor® RNA System, as described in Section 2.3. All LNP variants exhibited similar average sizes ranging from 76 to 92 nm, and homogeneous size distribution with a PDI of less than 0.2 (Fig. 1c). The surface charge was slightly negative at pH 7 (−3.4 to −15.9 mV) and positive at pH 4 (+14.4 to + 22.7 mV) (Fig. 1d), attributable to the pKa value of 6.44 for the ionizable cationic lipid DLin-MC3-DMA (Jayaraman et al., 2012). The positive surface potential at pH 4 is critical for the endosomal escape of siRNA into the cytoplasm (Han et al., 2021). When LNPs composed of the same lipid-PEG anchor were evaluated independently, the zeta potential values of siP2Y2 and siScr-loaded formulations were similar. Moreover, a comparative analysis of zeta potentials at pH 4 across all LNP variants revealed no significant differences. As expected, zeta potentials at pH 7 were less negative for LNPs coated with neutral DMG-PEG (−4.9 ± 1.2 mV). When compared to LNP(DMG-PEG), LNPs coated with anionic DPPE-PEG or DSPE-PEG exhibited approximately a 2.7-fold decrease in zeta potential at pH 7 (−13.2 ± 2.1 mV and −12.7 ± 1.2 mV, respectively, \*\* *p* < 0.01). The different lipid-PEG anchors did not significantly impact the siRNA encapsulation efficiency, which was consistently high at over 80 % across all LNP formulations (Fig. 1e). These encapsulation efficiency values are consistent with the existing literature (Schober et al., 2024). Additionally, agarose gel electrophoresis confirmed the siRNA entrapment in LNPs and stability in 10 % FBS (Supplementary Figs. S1 and S2). Finally, stability assay demonstrated that when the LNPs were kept at 4 °C in PBS, the average size, PDI, and zeta potential values remained stable over 30 days (Supplementary Fig. S3).

## 3.2. Cellular uptake of siRNA-loaded LNPs in BMDMs

To evaluate the intracellular uptake of both the LNP vehicle and the siRNA cargo, primary murine BMDMs were treated with LNPs composed of a fluorescently labeled lipid (DSPE-Cy5.5) and a fluorescently labeled siRNA (siRNA-6-FAM). While Cy5.5 is chosen to tag LNPs due to its near-infrared fluorescence, high photostability, bright signal, and compatibility with LNP formulations, 6-FAM is selected to label siRNA thanks to its stable fluorescence, brightness, minimal interference with siRNA function, ease of conjugation, cost-effectiveness, and compatibility with standard detection systems. Importantly, control measurements did not
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Design and physicochemical characterization of siRNA-LNPsyc. (a) Schematic representation of the structure of siRNA-LNPs. (b) Chemical structures of the lipid-PEGs used in this study: DMG(C14:0)-PEG, DPPE(C16:0)-PEG, and DSPE(C18:0)-PEG. (c) Hydrodynamic size (bars), polydispersity index (blue circles), and (d) surface charge at pH 7 and pH 4 of the LNP variants loaded with either siP2Y2 (dashed bars) or siScr (empty bars), as determined by DLS. (e) Encapsulation efficiency of siP2Y2 and siScr in LNP variants, as measured by the QuantiFluor® RNA System. Green, purple, and red colors represent the LNP(DMG-PEG), LNP (DPPE-PEG), and LNP(DSPE-PEG), respectively. Data are expressed as mean ± S.D. (n = 3). Only significant differences are indicated on the graphs, as determined by two-way ANOVA and Tukey's post hoc test (\*\*p ≤ 0.01). The statistical significance was determined relative to the LNP(DMG-PEG) counterpart, which served as the standard.</figcaption></figure>

reveal any alteration of the LNPs' physical characteristics after using those two markers (Supplementary Table S3). The experiment involved treating both unpolarized and M2-polarized BMDMs with three Cy5.5-LNP formulations (consisting of either DMG-PEG, DPPE-PEG, or DSPE-PEG) encapsulating 1 µM of siP2Y2-6-FAM. At 4 h post-transfection, nearly 100% of M2 BMDMs had taken up the LNP and the encapsulated siRNA (Fig. 2a). In M2 BMDMs, Cy5.5 signal mean fluorescence intensity (MFI) levels were comparable between formulations, with LNP (DMG-PEG) displaying the highest signal, followed by LNP(DSPE-PEG) and LNP(DPPE-PEG) (Fig. 2b). Moreover, the MFI observed for 6-FAM's signal was slightly higher in LNP(DMG-PEG) compared to LNP (DSPE-PEG), with both formulations exhibiting a greater fluorescence than LNP(DPPE-PEG). The observed differences suggest that the type of lipid-PEG anchor could affect the interaction of nanocarriers with M2 BMDMs. Since the fluorescence of each formulation has been tested and the normalizations have been performed accordingly, the observed differences are unlikely to be caused by variations in fluorescence across the three LNP variants. The higher fluorescence of Cy5.5 and 6-FAM in LNP(DMG-PEG) treated cells could indicate an enhanced cellular uptake in the case of the DMG-PEG conjugate. This can be attributed to its shorter hydrophobic tail that facilitates the detachment of the PEG moiety from the LNP surface, promoting endocytosis (Kulkarni et al., 2021). In contrast, longer PEG-lipid anchors result in a more rigid structure, decreasing the dissociation of PEG and thereby causing reduced interaction between the LNPs and the cellular membrane. Incorporating DSPE-PEG into LNPs has been known to facilitate cellular uptake (Berger et al., 2023). Similar to M2 BMDMs, M0 BMDMs had efficiently internalized both the LNP and the siRNA following 4 h of treatment (Supplementary Fig. S4a). In M0 BMDMs, the mean

fluorescence intensities of Cy5.5 or 6-FAM did not show significant differences between different treatment conditions after 4 h, indicating similar uptake among the three different formulations (Supplementary Fig. S4b).

The intracellular delivery of siRNA-6-FAM into M2 BMDMs by Cy5.5-LNPs was further validated by confocal microscopy. The images are displayed in Fig. 2c and Supplementary Fig. S5. While 6-FAM fluorescence was visualized as small green spots around the nuclei of all LNP-treated cells, Cy5.5 signal was observed to be spread through the cytoplasm. These signals indicate effective cellular internalization of siP2Y2-LNPs by M2-polarized BMDMs after 4 h of treatment. 6-FAM fluorescence intensity was comparable across all three formulations. Interestingly, the Cy5.5 fluorescence was more saturated in cells treated with LNP(DSPE-PEG) than the other two formulations, possibly due to a higher concentration of intact LNPs within cellular compartments, such as endosomes or lysosomes. The enhanced stability of the LNP(DSPE-PEG) lipid bilayer might slow the release of siRNA from the LNPs, accumulating Cy5.5-labeled nanocarriers inside the cells without releasing their cargo.

### 3.3. P2Y2 gene silencing in BMDMs

The efficiency of siRNA-mediated P2Y2 gene silencing was evaluated in unpolarized and M2-polarized BMDMs using three different LNP formulations: LNPs consisting of either DMG-PEG, DPPE-PEG, or DSPE-PEG. The experiments included two types of negative controls: an untreated sample and a sample treated with LNPs encapsulating siScr. A positive control, a sample transfected with the commercial transfection reagent Lipofectamine RNAiMAX, were included in the study. Two
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Cellular uptake of (6-FAM-siP2Y2)-LNPs-Cy5.5 in M2 BMDMs. (a) Flow cytometry histograms of siRNA-LNP uptake in M2 BMDMs, indicated by Cy5.5-LNP and 6-FAM-siRNA signals following 4 h treatment. Black and blue colors represent untreated negative control and free siP2Y2-6-FAM-treated cells, while green, purple, and red colors correspond to the BMDMs treated with LNP(DMG-PEG), LNP(DPPE-PEG), and LNP(DSPE-PEG) containing 1 µM siP2Y2-6-FAM, respectively. (b) Mean fluorescence intensity (MFI) of Cy5.5 and 6-FAM after 4 h treatment measured by flow cytometry. Values were initially normalized to the fluorescence of each LNP formulation and then further normalized to the untreated M2 negative control. Data are represented as mean ± S.D. (n = 3). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 (ordinary one-way ANOVA followed by Tukey's post hoc test). (c) Representative confocal images of M2 BMDMs treated with fluorescently tagged siRNA-LNPs composed of different lipid-PEGs 4 h post-transfection. Negative control image corresponds to the cells treated with unstained LNP(DMG-PEG) for 16 h, confirming the absence of background fluorescence. Micrographs show the merged images of 6-FAM-siRNA (green), Cy5.5-LNP (red) and DAPI (cyan; nucleus) signals, taken with a 60 × oil immersion objective at 2 × magnification. Regions of interest (86 × 111 µm) were cropped from the original images. Scale bar = 10 µm.</figcaption></figure>

siRNA concentrations, 50 and 100 nM, and two incubation times, 24 and 48 h, were tested. After the siRNA-LNP treatment, P2y2 gene expression was assessed using RT-qPCR. The analysis of M2 BMDMs revealed that LNP(DSPE-PEG) remained ineffective in silencing the target gene, whereas 50 nM siP2Y2-loaded LNPs composed of DMG-PEG and DPPE-PEG significantly reduced the P2y2 mRNA levels relative to corresponding scramble controls (Fig. 3a). LNP(DMG-PEG) exhibited the highest silencing effects (~90 %) among the three variants after 24 or 48 h of treatment (Supplementary Fig. S6a), performing even more effectively than Lipofectamine RNAiMAX (Fig. 3a; \*\*\*p < 0.001 at 24 h, \*\*p < 0.01 at 48 h). LNP(DPPE-PEG) also achieved approximately a 60–70 % decrease in P2y2 mRNA levels after 24 and 48 h. The potency of gene silencing was not enhanced by increasing the siRNA dose to 100 nM (Supplementary Fig. S7a), and therefore, 50 nM siRNA concentration was selected for further experiments to limit possible off-target effects. Additionally, lower concentrations of siRNAs, 10 and 25 nM, were tested only with the DMG-PEG-containing formulation, as it was the most efficient. These concentrations were also effective with nearly 80–85 % P2y2 knockdown after 24 h, and 85–90 % P2y2 knockdown after 48 h of treatment (Supplementary Fig. S8).

In M0 BMDMs, Lipofectamine RNAiMAX-siP2Y2 (50 nM) complexes induced a ~40–50 % reduction in the target gene P2y2 compared to the scramble control following a 24 or 48-hour incubation period (Fig. 3b). While LNP(DPPE-PEG) and LNP(DSPE-PEG) failed to knock down the P2y2 gene in M0 BMDMs, LNP(DMG-PEG) loaded with 50 nM siP2Y2 successfully silenced the target gene after 24 or 48 h of treatment. The

efficacy of gene silencing following (DMG-PEG)-LNP treatment was comparable to that of the Lipofectamine RNAiMAX transfection reagent (p > 0.05). Similar results were observed with 100 nM siRNA treatment in unpolarized BMDMs (Supplementary Fig. S7), except that LNP(DPPE-PEG) demonstrated a modest silencing effect at the 100 nM concentration after 48 h.

It was observed that treatment of M2 BMDMs with LNP(DMG-PEG) and LNP(DPPE-PEG) resulted in a more pronounced P2y2 silencing compared to M0 BMDMs (Fig. 3a vs 3b) at both 24 and 48 h time points. The silencing efficiency of LNP(DMG-PEG) was notably higher in M2 BMDMs compared to M0 BMDMs (~90 % vs. ~40–60 %, respectively). Moreover, while LNP(DPPE-PEG) did not exhibit statistically significant gene silencing in M0 BMDMs compared to the scramble control, it effectively knocked down the P2y2 gene in M2 BMDMs (~70 %). These observations might be attributed to the enhanced endocytic activity of M2 BMDMs (Kapellos et al., 2016; Li et al., 2012; Schulz et al., 2019).

These data indicate that LNP(DMG-PEG) was the most effective formulation for siRNA-mediated P2y2 gene knockdown *in vitro* at 24 and 48 h. It is crucial to comprehend the underlying mechanisms that govern the differences in the gene-silencing performance of siRNA-LNPs composed of various PEG moieties. The question arises as to why LNP (DMG-PEG) and LNP(DPPE-PEG) exhibit potent gene silencing in M2 BMDMs while LNP(DSPE-PEG) fails to knock down the target gene efficiently. Two possible explanations have been proposed to interpret this observation. The first hypothesis was that LNPs containing shorter PEG anchors (DMG-PEG or DPPE-PEG) are internalized to a greater
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Influence of lipid-PEG species on gene silencing in M2 and M0 BMDMs following transfection with siP2Y2-LNPs. P2y2 mRNA levels were measured by RT-qPCR after 24 or 48 h in (a) M2 or (b) M0 BMDMs treated with siP2Y2 (50 nM)-loaded LNP formulations containing various lipid-PEGs. The experimental design included a negative control sample transfected with LNPs encapsulating scramble siRNA and a positive control sample treated with the commercial transfection reagent Lipofectamine RNAiMAX. Expression levels were normalized to an untreated control group of the corresponding macrophage subset (M2 or M0). Data are presented as mean ± S.D. (n = 3) of three independent experiments, except for (DPPE-PEG)-LNP treatments on M0 BMDMs (two measurements at both 24 and 48 h) and (DSPE-PEG)-LNP treatments on M0 BMDMs (two measurements at 24 h and one measurement at 48 h). Dashed bars: siP2Y2, Empty bars: Scramble siRNA. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 (two-tailed unpaired student's t-test against corresponding scramble control, when applicable).</figcaption></figure>

extent than those with longer PEG anchors (DSPE-PEG). However, after a 4-hour treatment period, a comparable uptake of siRNA and LNPs was observed for the DMG-PEG and DSPE-PEG formulations (Fig. 2a), suggesting that the inefficacy of LNP(DSPE-PEG) in silencing the target gene was not caused by inefficient cellular uptake. The second hypothesis was that LNPs with shorter PEG anchors, DMG-PEG or DPPE-PEG, would disrupt the endosomal membrane and release siRNAs more efficiently following their uptake than those LNP with a longer PEG anchor, DSPE-PEG. Therefore, to determine if the inefficient siRNA release could be responsible for the non-potency of LNP(DSPE-PEG), we analyzed the intracellular fate of siRNA-LNPs in M2 BMDMs at 8, 24, and 48 h using confocal microscopy (Section 3.4). Finally, we monitored the kinetics of siRNA-mediated P2y2 silencing for three different formulations using RT-qPCR (Section 3.5). In this experiment, we extended the observation period to 72 h to evaluate the persistence of silencing with LNP(DMG-PEG) and LNP(DPPE-PEG) while also investigating whether LNP (DSPE-PEG) has a delayed but potentially significant gene silencing effect.

### 3.4. Intracellular fate of siRNA-loaded LNPs in M2 BMDMs

A confocal microscopy experiment was performed to investigate the fate of siRNAs delivered by LNPs containing various lipid-PEGs. To observe the intracellular distribution of siRNA, M2 BMDMs were incubated with 1 µM of 6-FAM-tagged siRNAs encapsulated in Cy5.5-LNPs for 8, 24, and 48 h. Representative confocal micrographs displaying the merged images of three channels (6-FAM-siRNA, Cy5.5-LNP, and DAPI) are given in Fig. 4. The images for individual channels are provided in Supplementary Fig. S9. Additionally, confocal images with a broader field of view showing the intracellular distribution of siRNA-6-FAM at the 8 and 24-hour time points are presented in Supplementary Fig. S10. At the 8-hour time point, M2 BMDMs treated with LNP(DMG-PEG), LNP(DPPE-PEG), and LNP(DSPE-PEG) exhibited comparable levels of intracellular 6-FAM fluorescence, characterized by a diffuse pattern with some localized concentrated regions. However, at 24 and 48 h, notable differences in the siRNA distribution patterns became apparent. Treatments with DPPE-PEG and DSPE-PEG formulations resulted in significantly more concentrated green spots, suggesting
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Intracellular fate of (6-FAM-siP2Y2)-LNPs-Cy5.5 in M2 BMDMs. Representative confocal images of M2 BMDMs treated with fluorescently tagged siRNA-LNPs containing different lipid-PEGs for 8, 24, or 48 h. Micrographs display merged images of three channels, corresponding to 6-FAM-siRNA (green), Cy5.5-LNP (red), and DAPI (cyan; nuclei), captured using a 60 × oil immersion objective at 2 × magnification. The scale bar represents 10 µm.</figcaption></figure>

intracellular compartmentalization of 6-FAM-labeled siRNA, compared to the DMG-PEG formulation. LNP(DMG-PEG), the most effective formulation for gene silencing, showed a predominantly diffuse pattern with only a few concentrated spots at these later time points, indicating more extensive siRNA delivery to the cytoplasm. Moreover, at 8 and 24 h post-treatment, the DSPE-Cy5.5 signal was higher and more saturated in cells treated with LNP(DPPE-PEG) and LNP(DSPE-PEG) compared to those treated with LNP(DMG-PEG). At 48 h, cells treated with LNP(DSPE-PEG) exhibited the highest DSPE-Cy5.5 signal, with intermediate levels observed for LNP(DPPE-PEG) and the lowest levels for LNP(DMG-PEG). This trend could be due to differences in the stability and desorption rate of the PEG-lipid molecules. DSPE-PEG likely facilitates a slower and sustained lipid exchange with endosomal membranes, resulting in an enriched intracellular Cy5.5 signal. Conversely, the less stable structure of DMG-PEG could allow for enhanced lipid exchange dynamics, leading to reduced cellular accumulation.

### 3.5. Kinetics of siRNA-mediated P2Y2 gene silencing in M2 BMDMs

Three formulations were further utilized to investigate the kinetics of P2y2 gene silencing throughout 72 h following the treatment with 50 nM siRNA in M2 BMDMs. In the case of LNP(DMG-PEG), P2y2 mRNA levels decreased by 49 % and 80 % for 4 and 8 h, respectively, relative to the corresponding scramble controls (Figs. 5a & 5d). Subsequent time points, including 24, 48, and 72 h, exhibited a consistent silencing efficiency of approximately 90 %. In contrast, LNP(DPPE-PEG) and LNP(DSPE-PEG) demonstrated a progressively delayed gene silencing response, with the knockdown effects becoming observable after 24 and 48 h, respectively. LNPs containing DPPE-PEG achieved a 70 % decrease in P2y2 mRNA levels after 24 h, reaching approximately 85 % after 48 and 72 h (Figs. 5b & 5d). While a pronounced increase in silencing efficiency was observed between 2 and 8 h for LNP(DMG-PEG), this effect was monitored between 8 and 24 h for LNP(DPPE-PEG), which achieved

comparable silencing efficiency to LNP(DMG-PEG) by 48 h. This delayed knockdown profile of LNP(DPPE-PEG) may be advantageous for applications requiring sustained release. In addition to showing the slowest onset of silencing, LNP(DSPE-PEG) exhibited the least efficient P2y2 knockdown, with an efficiency of ~30 % at 48 h and reaching only ~40–45 % at 72 h (Figs. 5c & 5d). Further experiments are needed to evaluate whether its knockdown efficiency continues to improve at later time points. Notably, earlier time points following siRNA-LNP treatment (up to 8 h) exhibit higher variability in target gene silencing, as evidenced by the larger error bars (Fig. 5d). This variability is probably due to the heterogeneity of cellular states during the initial stages of siRNA-LNP uptake and processing. In contrast, the error bars are markedly reduced at later time points (beyond 24 h), suggesting a shift towards a more homogeneous cellular population. This is potentially due to the stabilization of the intracellular distribution of siRNA, and its consistent engagement with the RNAi machinery.

The observed kinetics of target gene silencing (Fig. 5) correlate strongly with the intracellular distribution patterns revealed by confocal microscopy (Fig. 4). LNP(DMG-PEG), which demonstrated a primarily diffuse intracellular siRNA distribution with minimal compartmentalization at all time points, achieved a rapid and effective P2y2 knockdown, with efficiency reaching ~80 % by 8 h, increasing to ~90 % by 24 h and then remaining stable over 72 h. This suggests that the efficient cytoplasmic delivery of siRNA enabled by LNP(DMG-PEG) formulation contributes to rapid RNA interference. In contrast, the concentrated fluorescent spots indicated that LNP(DPPE-PEG) showed siRNA compartmentalization to some extent at 24 and 48 h. This might correspond to a delayed but effective gene silencing profile, with ~70 % knockdown at 24 h and ~85 % over 48–72 h. The gradual siRNA release provided by LNP(DPPE-PEG) was sufficient to achieve comparable silencing to LNP(DMG-PEG) at later time points. LNP(DSPE-PEG), which exhibited the highest degree of siRNA compartmentalization, was the least effective formulation, with a slow onset of silencing and only ~30
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Impact of PEG-lipid anchors on siRNA-mediated P2y2 gene silencing kinetics in M2 BMDMs. P2y2 knockdown kinetics in M2-polarized BMDMs transfected with (a) LNP(DMG-PEG), (b) LNP(DPPE-PEG), or (c) LNP(DSPE-PEG) containing either 50 nM siP2Y2 (blue) or scramble siRNA (red) over 72 h. Expression levels were normalized to an untreated control group at the corresponding time point. Data are presented as mean ± S.D. (n = 3). (d) Comparison of P2y2 silencing, quantified by RT-qPCR, in M2 BMDMs using LNP formulations containing DMG-PEG, DPPE-PEG, or DSPE-PEG over 72 h. Silencing (%) was calculated as the relative normalized expression of P2y2 in siP2Y2-treated samples divided by the relative normalized expression in siScr-treated controls, multiplied by 100. Data are presented as mean ± S.D. (n = 3).</figcaption></figure>

% knockdown at 48 h. It is important to note that increased cellular internalization of LNPs does not directly correlate with greater siRNA-mediated gene knockdown efficiency (Yu et al., 2014). These findings highlight the critical impact of PEG-lipid anchors on intracellular trafficking of siRNA-LNPs, which subsequently affects the efficacy of siRNA-mediated gene silencing.

## 4. Conclusion

In this study, we successfully synthesized siRNA-loaded LNPs incorporating diverse PEG-lipid anchors using a microfluidic method. To optimize siRNA delivery to M2-polarized macrophages, three PEG-lipids with saturated alkyl chains — DMG-PEG (C14), DPPE-PEG (C16), and DSPE-PEG (C18) — were evaluated. Notably, various PEG-lipid anchors did not significantly influence the physicochemical properties of siRNA-LNPs, as evidenced by comparable particle size, PDI, zeta potential, and encapsulation efficiency across all formulations. Flow cytometry experiments revealed efficient and comparable cellular uptake of these siRNA-LNPs by both unpolarized and M2-polarized BMDMs. Even though the cellular uptake was similar among the different formulations, the silencing efficiency of siRNA-LNPs was strongly affected by the PEG-lipid used, with (DMG-PEG)-LNPs outperforming the other formulations in M0 and M2 macrophages. While (DPPE-PEG)-LNPs did not show a significant gene knockdown in M0 BMDMs, they markedly reduced the target gene mRNA levels in M2 BMDMs. Furthermore, (DSPE-PEG)-LNPs remained ineffective in silencing the target gene in

both cellular models. Notable differences were observed in the target gene silencing efficiency of the same formulation across distinct macrophage polarization states, with a more pronounced knockdown detected in M2 BMDMs relative to M0 BMDMs. Furthermore, confocal microscopy revealed that the intracellular distribution patterns of lipid and siRNA were influenced by the type of PEG-lipid employed. The results indicated that the siRNA is compartmentalized within cells to a greater extent after treatment with DPPE-PEG and DSPE-PEG formulations, while treatment with DMG-PEG results in enhanced siRNA delivery. The results of the present study highlight the importance of PEG-lipid optimization in siRNA-LNP design, with DMG-PEG emerging as a superior anchor for achieving robust gene silencing in M2-polarized macrophages in vitro. The findings provide a foundation for developing more effective therapeutic strategies for immune modulation of macrophages in the context of cancer, inflammatory diseases, and beyond.

## 5. Funding declaration

This work benefited from the support of the 2021–084 project from the French Institut National du Cancer.

### CRediT authorship contribution statement

Sezen Gül: Writing – original draft, Validation, Software, Methodology, Investigation, Data curation. **Juliette Vergnaud:** Writing –
original draft, Validation, Supervision, Methodology, Investigation, Data curation. **Qinglin Wang:** Methodology, Investigation. **Mélanie Hery:** Methodology, Investigation. **Jhon Mino:** Investigation. **Jihana Achour:** Investigation. **Séverine Domenichini:** Methodology, Investigation. **Claudine Delomenie:** Methodology, Investigation. **Jean-Luc Perfettini:** Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization. **François Fay:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Elias Fattal:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

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

The authors acknowledge all core facilities from IPSIT for access to their equipment/infrastructure and advice. In particular, they would like to thank Emy Ponsardin at ACTAGen-IPSIT, Delphine Courilleau at CIBLOT-IPSIT, Marie-Laure Aknin at CYM- IPSIT, Valerie Domergue at AnimEx- IPSIT, and Catherine Cailleau at IGPS-UMR CNRS 8612. They would also like to thank the Region Ile-de-France for its financial support to the IPSIT platforms. Fig. 1a was made using Biorender Fay, F. (2025).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2025.126186.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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