Structural and functional characterization of a
histidylated liposome for mRNA delivery

Albert Ngalle Loth, Manon Maroquenne, Ayoub Medjmedj, Franck Coste, Thomas Bizien, Chantal Pichon, Delphine Logeart-Avramoglou, Federico Perche

► To cite this version:

Albert Ngalle Loth, Manon Maroquenne, Ayoub Medjmedj, Franck Coste, Thomas Bizien, et al. Structural and functional characterization of a histidylated liposome for mRNA delivery. Journal of Controlled Release, 2025, 379, pp.164-176. 10.1016/j.jconrel.2025.01.010. hal-04984847

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# Structural and functional characterization of a histidylated liposome for mRNA delivery

Albert Ngalle Loth¹, Manon Maroquenne², Ayoub Medjmedj¹, Franck Coste¹, Thomas Bizien⁵, Chantal Pichon³,⁴, Delphine Logeart-Avramoglou²*, Federico Perche¹*

Affiliations :

¹Centre de Biophysique Moléculaire, CBM, CNRS UPR4301, Orléans, France

²Université Paris Cité, CNRS, INSERM, ENVA, B3OA, 75010, Paris, France

³Inserm UMS 55 ART ARNm and LI2RSO, Université d'Orléans, F-45100 Orléans

⁴Institut Universitaire de France, 1 rue Descartes, F-75035 Paris, France

⁵Université Paris-Saclay, Synchrotron Soleil, 91190, Saint-Aubin, France

* Corresponding Authors:

Federico Perche, federico.perche@cnrs-orleans.fr +33 2 38 25 56 29

Delphine Logeart-Avramoglou, delphine.logeart@cnrs.fr, +33 1 57 27 85 63

## Graphical Abstract

<figure><img src="image_2.png" /></figure>

## Abstract

The development of lipid-based mRNA delivery systems has significantly facilitated recent advances in mRNA-based therapeutics. Liposomes, as the pioneering class of mRNA vectors, continue to lead in clinical trials. We previously developed a histidylated liposome that demonstrated efficient
nucleic acid delivery. In this study, the liposome preparation process was optimized by freeze-drying followed by extrusion to homogenize size distribution and improve storage stability. A comprehensive characterization of these LYX liposomes was performed, including evaluation in cellular and murine animal models.

LYX liposomes can be stored for up to one year at 4°C, maintaining a stable size (150 ± 10 nm) and polydispersity index (0.10 ± 0.02), while preserving their transfection efficacy. They exhibit high encapsulation efficacy (~95%) and protect mRNA from RNase degradation. Lamellar organization was confirmed by Small Angle X-ray Scattering and CryoTEM, and intracellular trafficking was examined using confocal microscopy. LYX-mRNA lipoplexes can transfect both cell lines and primary cells, albeit with a lower transfection efficacy compared to the commercial Lipofectamine MessengerMAX™ vector. Our data suggest that this could be attributed to slower cell uptake and reduced endosomal escape of LYX. LYX liposomes effectively delivered mRNA encoding therapeutic BMP2 and BMP9 molecules, producing significant amounts of functional proteins that successfully induced BMP signaling. In addition, in vivo studies demonstrated the potential of LYX lipoplexes when incorporated into hydrogels and implanted subcutaneously in mice. These findings provided evidence that LYX liposomes are a promising platform for mRNA delivery, offering versatility for multiple applications.

**Keywords** : mRNA therapy, histidylated liposome, lipoplexes morphology, delivery, endosomal escape, intracellular trafficking

# 1. Introduction

Messenger RNA (mRNA)-based technology harnesses the cellular protein synthesis machinery to produce any protein or peptide of interest in transfected cells. mRNA therapeutics offer numerous advantages over conventional vaccine and drug delivery approaches, including rapid and scalable manufacturing, as well as the ability to address diverse applications through simple changes in mRNA sequence [1, 2]. Unlike DNA gene delivery, which requires translocation to the nucleus before its expression can begin, mRNA expression occurs in cytosol, thereby avoiding the risk of opportunistic insertional mutagenesis [3]. Consequently, mRNA is increasingly being utilized in various therapeutic areas, including vaccination [4, 5], cancer immunotherapy [5-7] and regenerative medicine [7, 8].

One limitation of this approach is that exogenous mRNA, synthesized by *in vitro* transcription (IVT), is recognized as viral RNA by cytoplasmic and endosomal nucleic acid sensors [9, 10].

The clinical success of mRNA therapies has been partly achieved by the reduction of the immunogenicity of synthetic mRNA through the use of chemically modified mRNA (cmRNA). These cmRNA contain naturally modified nucleoside analogs like pseudouridine, N6-methyladenosine, or 5-methylcytidine, which prevent IVT mRNA-induced TLR activation, thereby increasing mRNA expression without adverse effects [4, 6, 10].

The development of suitable delivery systems has been another key factor for the efficacy of mRNA therapy, as naked mRNAs are hardly taken up by cells due to their high molecular weight and negative charge [2]. A wide range of mRNA delivery systems have been developed to protect the synthetized transcripts from degradation and to promote their cellular uptake into the targeted cells. In most cases, these systems are made with cationic carriers that compact the mRNA via electrostatic interaction, complexation or encapsulation, leading to the formation of nanoparticles that the cell can take up by endocytosis [11, 12]. Lipid-based systems including liposomes or lipid nanoparticles (LNPs) are the most used mRNA delivery carriers.

LNPs are currently the most advanced mRNA formulation and are composed of ionizable lipids and helper lipids combined with mRNA by microfluidic mixing, resulting in self-assembled 80-200 nm spherical nanoparticles [13]. For instance, the LNPs used in the approved mRNA COVID19 vaccines were formulated by mixing 4 components: ionizable lipid, phospholipid, cholesterol and polyethylene glycol-lipid (PEG-lipid). Each of these components must be produced in compliance with Good Manufacturing Practice (GMP) standards [14].
However, the use of other lipid-based delivery systems, in particular liposomes, one of the earliest mRNA formulations for cell transfection and for mRNA vaccination [15], remains a promising strategy. This approach is supported by numerous preclinical studies and ongoing clinical vaccine development projects (reviewed in [13, 16]). Liposomes provide a simple and versatile method for formulating mRNA by mixing the mRNA directly with liposomes to form lipoplexes, typically within minutes [17]. Moreover, the liposomes used in clinical trials are composed of only two lipids in contrast to the four components used in LNs. This reduced number of components not only decreases the costs associated with GMP-compliant lipid production, but is also expected to simplify regulatory approval requirements and lower manufacturing expenses [17, 18].

We have previously reported the formulation of a histidylated liposome (Lip100) that demonstrated efficient nucleic acid delivery [19-22]. Lip100 is made of equimolar quantities of 2 lipids: the cationic lipid O,O-dioleyl-N-(3N-(N-methylimidazolium iodide)propylene) phosphoramidate (KLN25) and the fusiogenic ionizable lipid O,O-dioleyl-N-histamine phosphoramidate (MM27) (Figure 1A). KLN25 possesses an N-methylimidazolium polar head conferring a permanent positive charge for nucleic acids condensation. The imidazole group of MM27, is protonated in endosomes facilitating mRNA release in the cytosol (endosome escape) [23]. However, Lip100 liposomes were of variable size (polydispersity index (PDI) of 0.34) [24] resulting in polydisperse mRNA lipoplexes (PDI 0.3) [25]. In fact, high polydispersity may present several drawbacks such as inconsistent mRNA encapsulation and delivery, variable cellular uptake and stability issues [26, 27].

In this study, we have optimized the preparation process of Lip100 histidylated liposomes by incorporating lyophilization and extrusion steps. We show that the improved protocol produces monodisperse liposomes, here designed as LYX. We reported a detailed characterization of physicochemical properties (size, morphology and structure by cryoTEM and SAXS analyses) of LYX liposomes and lipoplexes along with an analysis of intracellular trafficking. We demonstrated that the optimized LYX formulation efficiently delivers mRNA in cell lines, primary cells and 3D cultures. This study is the first to evaluate histidylated liposomes for mRNA delivery in vivo and the first to compare the transfection potency of histidylated liposomes with the commercialized product Lipofectamine MessengerMAX™.

## 2. Materials and methods

### 2.1. Lipid synthesis

Unless otherwise stated, all reagents were purchased from Merck (Saint-Quentin-Fallavier, France). O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] Phosphoramidate (KLN25) and O,O-dioleyl-N-histamine phosphoramidate (MM27) were synthetized by WuXi AppTec Co., Ltd. (Chengdu, China) as described previously [21, 22].

### 2.2. EGFP-Rab plasmids and mRNA synthesis

The EGFP-rab7 WT plasmid was provided by R. Pagano (Addgene plasmid #12605) [28], while the EGFP-Rab11a-7 plasmid was provided by M. Davidson (Addgene plasmid #56444) [29]. EGFP-Rab27A plasmid was provided by W. Gahl (Addgene plasmid # 8923) [30], and pFX-EGFP-Rab5 plasmid was provided by Y. Ohba (Addgene plasmid # 174454) [31].

Plasmids containing a T7 promoter, the 5'UTR of β-globin, and the 3'UTR of mtRNR1 and AES were used to produce the mRNA [32] encoding for either the enhanced green fluorescent protein (EGFP) [33] or the human Bone Morphogenetic Protein 2 (hBMP2) [34]. The sequence of hBMP2 was cloned from pVAX1-BMP2, provided by G. Feichtinger (Addgene plasmid # 137909) [34]. The RNA molecules containing an anti-reverse cap analog (ARCA) and a poly(A) tail were synthesized by IVT using the High Yield T7 RNA Synthesis Kit (Jena Bioscience) with 35% 5-iodo-uridine and 7.5 % of 5-iodocytidine [3]. The mRNA quality was assessed by capillary electrophoresis using an Agilent 2100 Bioanalyzer and RNA 600 LabChipKit® (Agilent Technologies) following the manufacturer's instructions [35]. Cap1-capped and 5-methoxyuridine modified firefly luciferase mRNA (Fluc-cmRNA) was purchased from Oz Biosciences, Marseille, France.
## 2.3. Liposome and lipoplex formulation

The liposomes were prepared as previously described by Perche et al. [36], then freeze-dried and extruded. Briefly, the lipids KLN25 and MM27 (Figure 1A) were mixed in ethanol at a 1:1 molar ratio. Ethanol was then evaporated using a rotary evaporator until a lipid film was formed. The film was then hydrated overnight in HEPES ((2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) buffer 10 mM pH 7.4, yielding liposomes with a lipid concentration of 10.8 mM. The liposomes were sonicated at 25°C and 37 kHz using an ultrasonic bath (Bioblock Scientific, Illkirch, France) for 5 min and dialyzed against 10 mM pH 7.4 HEPES buffer using dialysis cassettes (G2 Slide-A-Lyzer™, Thermo Scientific™) with a molecular weight cut-off of 10 kDa at 25°C for 4 h. Liposomes were then freeze-dried and stored at -20°C until use. The liposomes (hereafter referred to as LYX) were rehydrated and downsized by stepwise extrusion through 0.4 and 0.2 µm pore size membranes using an Avanti Polar Lipids mini extruder (Figure 1B).

The LYX-mRNA lipoplexes were prepared at a 2:1 N/P ratio (ratio of amino groups of the lipid to phosphate groups of the mRNA). 1 µg mRNA and 1.2 µl LYX liposomes (10.8 mM) were incubated separately in 250 µl Opti-MEM™ for 5 min at room temperature. The LYX suspension was then added to the mRNA solution and incubated for 15 min. Complexes with Lipofectamine Messenger MAX™ (LFM, Thermo Fisher) were prepared following manufacturer's instructions.

## 2.4. Particle characterization

Particle size was measured using dynamic light scattering (DLS) on an SZ-100 nanoparticle analyzer (Horiba Scientific) in 10 mM HEPES buffer at pH 7.4. LYX liposomes stability was assessed by DLS on different batches stored for 2, 4, 7 or 12 months at 4°C. Then, their performances were controlled by flow cytometry using cap1-capped and 5-methoxyuridine modified enhanced green fluorescent protein mRNA (EGFP-mRNA).

Then, the stability of LYX-mRNA complexes was assessed in 7% fetal bovine serum - HEPES at 37°C by measuring the lipoplex size every 30 min using dynamic light scattering. The encapsulation efficacy was determined using Quant-iT™ RiboGreen (Thermo Fisher) after dissociation of lipoplexes with 2% Triton-X100, as described in [37].

The RNase protection assay was performed as described in [36]. Briefly, 1 µg of free or complexed mRNA was incubated with an RNase A/T1 mix (Thermo Scientific) for 1 h at 37°C. Following RNase inhibition with Protector RNase Inhibitor (Roche), the remaining mRNA was mixed with RNA loading dye (Thermo Fisher) and ran on a 1% agarose gel containing Quant-iT™ RiboGreen (Thermo Fisher). Gels were imaged using a GelDocXR+ imager (Biorad).

The structure of LYX-mRNA lipoplexes was analyzed using small angle X-ray scattering (SAXS) as a function of decreasing pH (from 7.5 to 4.5) in order to evaluate their morphology in the acidic environment found in endosomes after cell internalization. SAXS data were collected at the SWING beamline of the SOLEIL synchrotron (Saclay, France) using an incident beam of 12 keV and recorded on an EIGERX 4M detector at a sample-to-detector distance of 0.5 m. We used the Foxtrot software (version 3.5.10) for data reduction and first analysis. The LYX-mRNA lipoplex morphology was further analyzed by cryo (TEM) as previously described [23, 36] using a JEM-1400Plus electron microscope (Jeol, Japan) operating at 120 kV.

## 2.5. Cell culture

DC2.4 murine immune cells were a gift from Pr Kenneth L. Rock [38]. RAW-264 murine immune cells, HEK293 human kidney cells and HeLa human ovarian carcinoma cells were purchased from the American Type Culture Center (ATCC). C2C12-BRE/Luc cells containing a BMP-sensitive reporter gene were obtained after stable transfection with the BMP-responsive element (BRE) fused to the luciferase gene [39]. C2C12-BRE/luc, RAW-264 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma), containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 1% antibiotics (Sigma). DC2.4 cells were cultured in RPMI 1640 medium, containing 10% FBS and 1% antibiotics. HeLa cells were cultured in Minimum Eagle's medium containing 10% FBS, 1% GlutaMAX™ (Gibco), 1% MEM non-essential amino acids ('Gibco™') and 1% antibiotics.
Rat mesenchymal stem cells (rMSCs) derived from the bone marrow of 8-week-old male Wistar rats were collected as previously described [40]. Briefly, after euthanasia, the rat femurs and tibias were harvested aseptically, and bone marrow was flushed from the bone shafts with alpha-MEM supplemented with 10% FBS and 1% antibiotics and seeded onto 0.1% gelatin-coated tissue culture dishes. Once the cells had adhered, approximately 5-7 days, non-adherent cells were eliminated by changing the culture medium, and the adherent cells were used as rMSCs. Human MSCs (hMSCs) were harvested from bone marrow from discarded bone tissue during routine bone surgery from 3 donors at the Lariboisière Hospital, Paris, France. The tissues were harvested with the respective donor's consent in accordance with the regulations of the Lariboisière Hospital. hMSCs were isolated from the bone marrow using a procedure as previously reported [23, 41].

## 2.6. In vitro transfection and protein production

Lipofectamine Messenger Max™ (LFM, Thermo Fisher), a commercial transfection reagent, was used as a control. The transfection efficacy of lipoplexes was assessed both in a 2D culture (well plate) and a 3D environment (fibrin-based hydrogel). For 2D culture, cells at 70-80% confluence were transfected with 0.5 µg of mRNA per well complexed with either LYX or LFM. Transfection efficacy was assessed by flow cytometry 24 h post-transfection using EGFP chemically modified mRNA (EGFP-cmRNA).

Toxicity-mediated cell death was assessed by adding propidium iodide (PI) to the samples prior to analysis (10 µg/mL final concentration) [42]. Cell-associated fluorescence intensity was measured using a FORTESSA X20 flow cytometer with $\lambda_{ex}$ = 488 nm and $\lambda_{em}$ = 530 ± 30 nm for EGFP detection, and $\lambda_{ex}$ = 561 nm; $\lambda_{em}$ = 586 ± 15 nm for PI detection (Becton Dickinson). The fluorescence intensity was expressed as a mean fluorescence intensity (MFI) of 2,500 events for primary cells and 10,000 events for cell lines.

For 3D culture, 100,000 rMSCs were mixed with lipoplexes containing 2 µg of Fluc-cmRNA (Oz Biosciences, Marseille) and embedded in a 130 µL fibrin gel (18 mg/mL fibrinogen and 25 U/mL thrombin) [43, 44]. The gels were then incubated in culture medium at 37°C and imaged 24 h and 48 h post-transfection in the presence of 150 µg/mL D-luciferin (Interchim) using a bioluminescence imaging system (IVIS Lumina II; Caliper Life Sciences).

The efficacy of the vectors in delivering mRNA to produce therapeutic proteins was tested in HEK293 and rMSCs using cmRNA (35% of iodo-uridine and of 7.5% iodo-cytidine) encoding either hBMP2 or hBMP9. Transfections were performed at 30 pg cmRNA/cell. At defined time points, the levels of secreted hBMP2 and hBMP9 in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) kit (Quantikine, R&D Systems, MN, USA) according to the manufacturer's instructions. The functionality of the produced hBMP2 and hBMP9 was evaluated by transfecting C2C12-BRE/luc cells with increasing amounts (5, 10, and 20 pg/cell) of cmRNA. Twenty-four hours post-transfection, the cells were lysed, and luciferase activity was measured using the Luciferase assay system (Promega) and expressed as relative light units (RLU) per milligram of protein. The results were normalized to the luminescence levels of non-transfected control cells.

## 2.7. Confocal microscopy

C2C12-BRE/Luc cells (25,000) were seeded on glass coverslips in 24-well plates. The next day, the cells were transfected with pDNA encoding either EGFP-Rab5 (small GTPase associated with early endosomes), EGFP-Rab7 (small GTPase associated with late endosomes), EGFP-Rab11 (small GTPase associated with recycling endosomes) or EGFP-Rab27 (small GTPase associated with exosomes) with 25K branched polyethyleneimine at 0.5 µg mRNA per well. After 24 h of incubation at 37°C, cells were incubated with LYX- or LFM-mRNA lipoplexes containing 0.5 µg of Cy3-labelled mRNA for 1 h or 4 h. Cells were then fixed at 1 h, 4 h, or 24 h post-transfection and imaged to assess the subcellular distribution of labeled lipoplexes using a LSM 980 Airyscan 2 confocal microscope (Carl Zeiss, Oberkochen, Germany) [37]. Weighted Pearson's colocalization coefficients between Cy3-labelled mRNA and GFP-labelled organelles were determined using Zen Blue software version 3.2 (Carl Zeiss, Oberkochen, Germany).
## 2.8. In vivo transfection efficacy

The animals were handled following the European Directive 2010/63/EU regarding the protection of animals used for scientific purposes. The protocols of in vivo experiments were approved by the local Ethics Committee on Animal Research and authorized by the French Ministry of Agriculture (APAFIS#35857). The in vivo transfection efficacy of lipoplexes combined in a fibrin gel was assessed through subcutaneous implantation. Lipoplexes containing 5 µg of Fluc-cmRNA were embedded in 130 µl fibrin gel (18 mg/mL fibrinogen and 25 U/mL thrombin). The loaded gels were then implanted subcutaneously in 8-week-old male SWISS mice as previously described [43, 44]. Mice were then imaged in a bioluminescence imaging system (IVIS Lumina II®; Caliper Life Sciences) at 24 h, 48 h, and 72 h post-implantation after local injection of 75 µl of 20 mg/mL D-luciferin substrate at each implantation site.

## 2.9. Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.0.1 (GraphPad software). All numerical data are expressed as mean ± standard deviation (SD). The t-test (for normally distributed variables) or Mann-Whitney test was used to compare the means of two data groups. The one-way analysis of variance (ANOVA) was used to compare the means of more than two data groups. Experiments were performed in triplicate in three independent experiments. For all analyses, differences at p < 0.05 (*) were considered statistically significant.
3. Results

3.1. Liposome and lipoplex formulation and characterization

<figure><img src="image_3.png" /><figcaption>**Figure 1:** Liposome and lipoplex formulation and characterization. (A) Chemical formula of the two lipids used in the liposome. (B) Main steps of the liposome synthesis. (C) Diagram of lipoplex formulation with corresponding cryo-TEM images of liposome and lipoplex (scale bar = 100 nm). (D) RNase protection assay: 1-Free mRNA; 2- Free mRNA after RNase treatment; 3- LYX-mRNA lipoplexes; 4-LYX-mRNA lipoplexes after RNase treatment followed by RNase inactivation and complex dissociation; 5-LYX-mRNA lipoplexes after RNase treatment.</figcaption></figure>

The LYX liposomes containing equal molar amounts of KLN25 and MM27 (Figure 1A) were formulated and prepared as shown in Figure 1B. CryoTEM observation of LYX liposomes confirmed the presence of unilamellar vesicles (Figure 1C), whereas Lip100 exhibited both unilamellar and multilamellar vesicles (Supplementary Figure S1). When complexed with mRNA, the obtained LYX lipoplexes exhibit a lamellar organization (Figure 1C). Analysis of the lipoplexes by gel electrophoresis (Figure 1D) confirmed mRNA complexation (lane 3) and its protection from RNase degradation. While naked RNA was degraded by RNase (lane 2), LYX lipoplexes protected it from degradation (lanes 4 and 5).
<figure><img src="image_4.png" /><figcaption>**Figure 2: Liposome and lipoplex stability.** (A) DLS analysis performed after rehydration and extrusion of lyophilized liposomes stored at -20°C for 0, 2 or 6 months. (B) Stability of freshly prepared LYX lipoplexes as a function of time, in presence of serum. (C) DLS analysis of size, and (D) polydispersity of Lip100 and LYX liposomes from multiple batches, stored at 4°C for up to 12 months. (E) Percentages of encapsulation and cell transfection (left axis) and MFI per cell (right axis) in DC2.4 cells after 24 h, using different batches of LYX liposomes stored at 4°C for up to 12 months (statistics analysis were performed in comparison with the earliest time point).</figcaption></figure>

Once lyophilized, Lip100 liposomes could be stored at -20°C for at least 6 months without significant structural modification (Figure 2A). When needed, the freeze-dried liposomes were rehydrated and extruded resulting in LYX liposomes. To assess the compatibility of LYX lipoplexes with physiological fluids, their size was monitored by DLS in presence of serum at 37°C and no aggregation was observed after 9h (Figure 2B). LYX liposomes can be stored in solution at 4°C for up to 12 months without any structural changes, maintaining a stable size of 150 ± 10 nm and a polydispersity index of 0.1 ± 0.02 whereas Lip100 liposomes showed aggregation over time and a higher polydispersity (~ 0.3) (Figure 2C and 2D). Furthermore, the transfection efficacy of lipoplexes formulated with LYX liposomes stored at 4°C in DC2.4 cells remained stable (73-79 %) for up to 12 months (Figure 2E). However, their ability to encapsulate mRNA decreased (from 95 % to 70 % between the 2^nd^ and 12^th^ month of storage) with a concomitant decrease in the mean fluorescence intensity (MFI) of transfected cell (Figure 2E). Once lyophilized, Lip100 liposomes could be stored at -20°C for at least 15 months without significant loss of transfection efficacy (Supplementary Figure S2)."
## 3.2. pH-dependent morphology of lipoplexes

<figure><img src="image_5.png" /></figure>

To investigate the morphology of LYX lipoplexes during the endosomal acidification, small angle X-ray scattering (SAXS) and cryo-TEM analyses were performed at decreasing pH levels to mimic the acidic microenvironment encountered during the endosomal trafficking. The SAXS profile of LYX liposomes without mRNA displayed a decreasing curve without any distinct peaks, indicating the presence of unilamellar vesicles (Figure 3A). In contrast, the SAXS profile of mRNA-containing lipoplexes displayed a single peak at $q = 0.1155 \AA^{-1}$ (Figure 3A), indicating an ordered structure and, therefore, a lamellar organization. The distance (d) between the repeating lipid layers, calculated from the position of the peak (q value) and using the equation $d = 2\pi/q$ [45], was estimated to be
5.44 nm. Interestingly, when comparing the SAXS profiles at decreasing pH, a single peak was observed from 7.5 to 6.5, while at pH below 6.0, a second peak appeared at higher *q* value (Figure 3B). The position of the first peak remained constant at a *q*-value of 0.1155 Å⁻¹ across all pH levels tested (except at the most acidic pH 4.5), corresponding to a spacing distance of 5.44 nm (Figure 3C). The position of the second peak slightly increased with acidification, from 0.1226 Å⁻¹ (*d* = 5.12 nm) at pH 6.0 to 0.1243 Å⁻¹ (*d* = 5.05 nm) at pH 4.5. The appearance of a second peak suggests the presence of a secondary lamellar structure [46], which emerged only at acidic pH. Cryo-TEM images were taken at pH 6.5, 5.5 and 4.5 to examine the morphology of the LYX-mRNA lipoplexes (Figures 3D, 3E and 3F). Under acidic conditions, the inner structure of the lipoplexes do not seems to be changed (Figures 3H, 3J and 3L), with the same lamellar organization as that observed at neutral pH (Figure 1C). However, whereas the rim of LYX-mRNA lipoplexes appeared thin at pH 6.5 (Figure 3G), an amorphous and blurred peripheral layer appeared at pH 5.5 and 4.5 (Figures 3J and 3K). The size and PDI of the LYX liposomes and lipoplexes were also measured across decreasing pH levels (from pH 7.5 to 4.5). No significant changes were observed in liposome size and PDI under acidic condition. However, a slight decrease in the size of lipoplexes was observed (from 203 nm at pH 7.5 to 188 nm at pH ≥ 6.5) with the PDI remaining stable (Supplementary Figure S3).
### 3.3. In cellulo transfection efficacy of LYX lipoplexes

<figure><img src="image_6.png" /><figcaption>**Figure 4: LYX and LFM transfection efficacy on various murine and human cell types. (A)** Transfection rates, (B) mean fluorescence intensity per cell, and (C) toxicity percentage (n=3) 24h post-transfection using 10 pg of EGFP cmRNA per cell. Values are represented as mean ± SD. Mann-Whitney statistical test was used (*) p < 0.05.</figcaption></figure>

The transfection efficacy of LYX was evaluated in comparison with Lipofectamine MessengerMAX™ (LFM) in several cell types using 10 pg/cell of EGFP cmRNA. Expression was assessed 24 h post-transfection in a panel of murine and human cell lines, including murine C2C12 myoblasts, DC2.4 dendritic cells, RAW 264.7 macrophages, human HeLa cancer cells, HEK293
immortalized human kidney cells and, primary bone marrow-derived mesenchymal stem cells from rats (rMSC) and humans (hMSC). Overall, compared to LFM, LYX induced a lower transfection rate (ranging from 75% to 17%) compared to LFM (87% to 29%) across all cell types tested (Figure 4A). A notable exception was the similarly low transfection percentage of RAW macrophages by LFM (25%) and LYX (30%) (Figure 4A). Interestingly, the cellular mean fluorescence intensities (MFI) induced by LYX were either greater or similar to those induced by LFM, except for HeLa and HEK293 human cell lines (Figure 4B). In addition, LFM induced less cytotoxicity (5-10% range in all tested cells) compared to LYX (10-20%) (Figure 4C). Note that LYX could also deliver unmodified mRNA albeit with lower efficiency than modified mRNA (Supplementary Figure 4). These data provided evidence that LYX is a potent and versatile mRNA delivery system for human and murine cell lines. An evaluation of liposome-mediated cytotoxicity using the MTT assay revealed dose- and cell type dependent cytotoxicity, with LYX exhibiting overall higher cytotoxicity compared to LFM (Supplementary Figure 5).
### 3.4. LYX induce the production of functional therapeutic proteins

Next, we assessed the efficacy of LYX in delivering mRNA to produce therapeutic proteins such as osteoinductive BMP2 and BMP9 [47].

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

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

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

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

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

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

**Figure 5: Production of bioactive hBMP2 and hBMP9.** (A) hBMP2 and (B) hBMP9 levels secreted by HEK293 and rMSC cells transfected with LFM or LYX lipoplexes containing cmRNA encoding hBMP-2 or hBMP-9. (C) Schematic representation of the BMP signaling activation pathway in C2C12-BRE/LUC cells, leading to firefly luciferase expression. (D) Quantification of the luciferase activity of C2C12-BRE/Luc cells treated with increasing doses of recombinant hBMP-2 or recombinant hBMP-9. (E) Quantification of the luciferase activity of C2C12-BRE/Luc cells transfected with increasing doses
of LFM or LYX lipoplexes containing cmhBMP-2 or (F) cmhBMP-9 mRNA (n=3). Values are represented
as mean + SD. Two-way ANOVA statistical test was used (*) p < 0.05.

ELISA quantification confirmed the synthesis of correctly folded BMP proteins after mRNA
transfection with LFM and LYX lipoplexes in both human HEK293 cells and rMSC. Although overall
BMP protein production was higher with LFM than with LYX, it still depended on both the RNA
molecule and the cell type. While rMSCs produced little to no BMP2 regardless of the vector, they
produced significant amounts of BMP9 with LFM but none with LYX. Additionally, HEK293 cells, which
are more transfection-permeable than primary rMSCs, produced 6-fold more BMP2 with LFM than
with LYX, but produced comparable amounts of BMP9 with both vectors (Figures 5A and 5B).

To confirm lipoplex bioactivity, C2C12-BRE/LUC cells (Figure 5C), were transfected with increasing
doses of cmhBMP-2 and cmhBMP-9 mRNA [39]. The luciferase activity in C2C12-BRE/LUC cells
increased proportionally with higher doses of recombinant proteins and mRNA, except for rhBMP9
(Figure 5D). Notably, transfection with 20 pg/cell induced luciferase activity equivalent to
approximately 100 ng/mL of BMP2 with LFM, and 30-40 ng/mL of BMP2 with LYX (Figures 5D and
5E).
### 3.5. Comparison of LYX and LFM transfection mechanisms

To further understand the differences in transfection efficiency between LYX and LFM (Figures 4 and 5), we analyzed and compared the cellular uptake and intracellular trafficking of these two delivery systems. C2C12-BRE/LUC cells were transfected with LYX or LFM complexed with Cy3-labeled mRNA and observed by confocal microscopy (Figure 6).

<figure><img src="image_13.png" /><figcaption>**Figure 6:** Cellular uptake and intracellular trafficking analyses. (A) Percentage of Cy3-positive C2C12-BRE/LUC cells 4h or 24h post-transfection with LFM or LYX transfection reagent. Values are represented as mean ± SD. Two-way ANOVA statistical test was used (*) p < 0.05. (B) Diagram illustrating endosomal trafficking following lipoplex uptake. Confocal microscopy images of cells labeled with Rab5-EGFP fusion protein, 4 hours post-transfection with LFM-Cy5 mRNA (C) or LYX-Cy5 mRNA labelled complexes (D) (scale bar = 20 µm or 5µm for zoomed images). (E) Coefficient of colocalization of Cy3 mRNA-containing LYX or LFM lipoplexes with EGFP-labelled Rab5, (F) Rab7, (G) Rab11 and (H) Rab27 proteins in C2C12-BRE/LUC cells. Values are represented as mean ± SD. Student's statistical test was used (*) p < 0.05.</figcaption></figure>
A transfection control using free mRNA showed minimal cell internalization with 1.0 % and 4.2 % of the labelled mRNA taken up by cells at both 4h and 24h post-transfection, respectively. The proportion of cells with internalized labelled complexes was lower when mRNA was complexed with LYX than when complexed with LFM (77% versus 96%) 4h post-transfection. However, this difference was reduced (93% for LYX versus 99% for LFM) 24h post-transfection (Figure 6A). These results indicated different internalization kinetics for the two types of complexes into the cells, with LYX lipoplexes being internalized more slowly than LFM complexes.

In addition, the subcellular trafficking of internalized complexes was analyzed 1h and 4h post-transfection using confocal microscopy. Although Rab7 is also present in lysosomes, endosomal complexes were pre-labelled with EGFP-Rab5, EGFP-Rab7, EGFP-Rab11 and EGFP-Rab27 fusion proteins to detect early endosomes, late endosomes, recycling endosomes, and secretory vesicles, respectively (Figure 6B) [48-50]. Pearson's colocalization coefficients and representative confocal image are presented in Figures 6C, 6D, 6E and 6F.

Results showed that 1h post-transfection, the number of LYX lipoplexes in early Rab5 endosomes was lower than that of LFM complexes. However, from 4 hours post-transfection, the number of LYX lipoplexes increased over time and became higher than that of LFM (Figure 6C), confirming the effective uptake of LYX lipoplexes, but with slower kinetics than with LFM lipoplexes. Both LYX and LFM complexes were observed to be routed towards Rab7 endosomes, recycling Rab11 endosomes, and Rab27 exocytosis compartments (Figures 6D, 6E and 6F). These data suggested that LYX lipoplexes escape endosome, for mRNA release, more slowly than LFM complexes and, were more susceptible to exocytosis.
3.6. Transfection efficacy of lipoplexes in hydrogels in vitro and in vivo

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

Lipoplex and cells
in fibrin hydrogels

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

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

**Figure 7:** *In cellulo* and *in vivo* LYX lipoplexes delivery embedded in fibrin hydrogel using Fluc-cmRNA. (A) Bioluminescence measured after rMSC transfection using LYX or LFM hydrogels using Fluc-cmRNA. (B) Bioluminescence kinetic after subcutaneous implantation of LYX-mRNA or LFM-mRNA embedded in fibrin hydrogels. Values are represented as mean ± SD. Two-way ANOVA statistical test was used (*) p < 0.05.*

To assess the suitability of lipoplexes for therapeutic applications, they were incorporated into a
fibrin hydrogel, which is recognized for its biocompatibility, ease of preparation and use, ability to
mimic the natural extracellular matrix, and most importantly, good degradability in vivo [51, 52].

The transfection efficacy was first assessed in an *in vitro* 3D model comprising either LFM or LYX complexes prepared with Fluc-mRNA mixed with rMSCs into fibrin gels. The bioluminescence signal of gels containing each of the two types of lipoplexes decreased with the incubation time and reached background levels after 72h. It is interesting to note that, albeit not significant, LYX-containing gels showed a trend towards a higher bioluminescence signal than LFM gels (Figure 7A).

Finally, the LYX lipoplexes-loaded fibrin hydrogels were implanted subcutaneously in mice to evaluate the *in vivo* capability of local mRNA production *in vivo*. The kinetic of FLuc expression was similar to *in vitro* with the 3D models, with a decrease in bioluminescence signal from 24h to 48h after implantation (Figure 7B). Noteworthy, 24 hours after implantation, gels complexed with LYX induced significantly higher bioluminescence than those complexed with LFM.
## 4. Discussion

Liposomes are a promising delivery system for mRNA therapy supported by numerous preclinical studies and ongoing clinical vaccine development. Liposome dispersity and stability represents a critical challenge for mRNA delivery. In fact, variable size results in polydisperse mRNA lipoplexes solution, which can lead to inconsistent mRNA encapsulation and delivery, variable cellular uptake and stability issues.

### LYX is an optimized version of Lip100 liposomes for mRNA delivery

In this study, we report an optimized version of "Lip100" liposomes, designed LYX for enhanced mRNA delivery [20, 21]. Both liposomes have the same lipid composition with equal amounts of the KLN25 cationic lipid, which enables mRNA complexation, and the MM27 ionizable lipid, which ensures endosome escape [23]. However, LYX liposomes possess an improved preparation process with a lyophilization and an extrusion step. The implementation of a lyophilization step in the preparation of LYX liposomes enabled the production of large volume batches (5 to 10 mL) which can be stored at -20°C for at least 6 months after lyophilization, without altering their structures or performances. Compared to the preparation of Lip100 liposomes, which exhibit a PDI of ~ 0.3, the introduction of an extrusion step in the LYX formulation resulted in monodisperse liposomes with a size of 150 nm and a reduced PDI of ~ 0.1, which is considered optimal for mRNA delivery. Liposome size and size distribution are considered as critical quality attributes for achieving optimal cell transfection. A size range of 50 to 200 nm is generally considered optimal for most drug delivery applications, while the PDI of 0.1 to 0.3 reflects a consistent and uniform size distribution, which is essential for ensuring reliable performance and predictable behavior [26].

After reconstitution and extrusion, LYX liposomes can be stored at 4°C for 12 months without any changes in size or homogeneity, which was not the case with Lip100 (Figures 2C and 2D). Consequently, LYX transfection efficacy (percentage of transfected cells) remained high and stable during liposomes storage (from 79% to 73%). However, their ability to encapsulate mRNA decreased (from 95% to 70% between the 2^nd^ and 12^th^ month of storage) with a concomitant decrease in the mean fluorescence intensity (MFI) of transfected cell (Figure 2E). A decrease in encapsulation efficiency suggests that fewer mRNA copies per lipoplex were encapsulated and delivered to cells, explaining the loss of MFI.

Additionally, the formulation process allows a high formulation reproducibility that is essential to obtain reliable results. Moreover, the LYX lipoplexes do not aggregate, even in the presence of serum (Figure 2B), and protect mRNA from RNAse degradation (Figure 1D). All these features suggest a strong potential of LYX as a vector for *in vivo* mRNA delivery [6, 53].

### Morphological reorganization of LYX lipoplexes according to pH acidification

Once lipoplexes enters the cells, endosomal trafficking induces vesicle acidification ([54], illustration Figure 6B). Our study reports a detailed analysis of lipoplex morphology reorganization under acidic condition, using SAXS and cryo-TEM in tandem to characterize and track structural changes.

On the one hand, SAXS and cryo-TEM analysis performed at neutral pH showed that, in absence of mRNA, LYX liposomes exhibited a spherical morphology (Figure 1C) with an unilamellar structure (Figure 3A)[55-57]. In contrast, LYX lipoplexes complexed with mRNA exhibited a single peak on SAXS curves (Figure 3A) and a multilamellar structure on cryo-TEM images, confirming complexation between cationic lipids and anionic mRNA molecules (Figure 1C) [59, 60, 62]. Both techniques indicated a 5-6 nm interlamellar spacing between the lipid bilayers, in agreement with values observed in others reports [46, 55, 58] [58-60].
On the other hand, SAXS analysis performed under acidic conditions to mimic the microenvironment encountered during endosomal trafficking showed the appearance of a second peak at pH 6.0 and below (Figures 3B and 3C). This suggests a structural transition of LYX lipoplexes in a pH range corresponding to endosomal trafficking [46], a phenomenon already reported with mRNA LNPs [61, 62]. The appearance of the second peak at a pH value below MM27 ionizable lipid pKa (~6.2) suggests that the first peak corresponds to mRNA interaction with the cationic KLN25 lipids, while the second peak reflects mRNA interaction with protonated MM27 lipid (Figures 3B and 3C). MM27, which contains an imidazole moiety, becomes protonated at low pH [19, 63-66], supporting our hypothesis. In the cryo-TEM images, as the pH decreases, the edges of the lipoplexes change from clear to blurred (Figures 3G, 3I and 3K), with a hollow structure clearly visible at pH 4.5 (Figure 3K), similar to the findings reported by [67].

Based on these results, we hypothesize that endosome acidification triggers a chain reaction that enhances mRNA release into the cytosol. First, acidification progressively increases the proportion of cationic MM27, which facilitates electrostatic interaction with mRNA such as KLN25 (Figure 3C). Second, the stepwise protonation of lipids leads to an excess of positive charges, leading in an imbalanced distribution of lipids on the lipoplexe surface and resulting in a hollow structure (Figures 3I and 3K). We propose that this hollow structure, composed of cationic lipids, favors electrostatic interactions with the negatively charged endosome membrane, ultimately leading to endosome destabilization and mRNA release.

## LYX liposomes are effective vectors for mRNA delivery both in vitro and in vivo

We demonstrated, in the present study, the ability of LYX liposomes to deliver mRNA in a variety of cellular models and in vivo when loaded in hydrogels.

LYX successfully delivered EGFP reporter gene mRNA in a wide range of cell lines and primary cells (Figure 4). However, the transfection efficiency of LYX was lower than that of the commercial gold standard Lipofectamine MessengerMAX™ (LFM), up to 60% fewer transfected cells in C2C12 cells. LFM has been established as a commercial solution for mRNA delivery in hard-to transfect cells such as human induced pluripotent stem cells and peripheral blood mononuclear cells [68, 69]. A key finding was the high transfection of human MSC with both LFM (82% transfected cells) and LYX (70% transfected cells), which is particularly desirable for therapeutic mRNA applications. However, a limitation of LYX was its observed cytotoxicity, with up to 25% cell mortality in cell lines and primary cells compared to a maximum of 10% for LFM (Figure 4C). Although not assessed in the present study, the higher cytotoxicity with LYX can be attributed to its weak biodegradability, a known issue reported with the first generation lipids used in LNPs [70].

The potential of LYX in delivering mRNA of therapeutic protein production was confirmed using mRNA encoding the osteoinductive BMP2 and BMP9 growth factors for bone regeneration. LYX successfully produced BMP2 and BMP9 proteins in rat MSC and HEK293 cells (Figure 5A and 5B) though at lower levels compared to LFM, consistent with cell transfection analysis (Figure 4 and 6). The proteins produced by LYX lipoplexes are functional, as evidenced by a dose-dependent induction of BMP signaling in C2C12-BRE/Luc cells (Figure 6E), similar to the induction observed with recombinant human BMP2 and BMP9 proteins. Also, our results have shown that HEK293 cells produced more BMPs, overall, than primary rat MSCs. These observations are consistent with the frequent use of HEK293 cells in bioproduction due to their high transfection rate and ability to rapidly produce protein in a stable and abundant manner [71, 72].

A comprehensive analysis of the cellular uptake and intracellular trafficking was conducted to better understand the differences in transfection efficacy between LYX and LFM (Figures 4, 5 and 6). The results revealed two major differences in the cellular processing of LYX mRNA complexes compared to LFM mRNA complexes. First, LYX complexes were internalized more slowly into cells (Fig. 6A) and exhibited lower escape from Rab5 endosomes (Fig. 6C). A recent study reported that endosomal escape of mRNA LNP occurs during the first 2h after transfection mainly from Rab5 endosomes [73]. This is in agreement with our findings and suggests improving Rab5 endosomal escape as a promising approach to increase the efficiency of mRNA delivery. Second, LYX complexes were more susceptible
to exocytosis by translocation into the Rab11 recycling and Rab27 secretory compartments. We noted a lack of correlation between intracellular delivery of mRNA quantitated using Cy3-labeled mRNA (Fig. 6A) and expression degree of mRNA (Fig. 4). The lack of correlation between uptake and expression degree was also evidenced by Jiang et al [74]. They quantified a higher correlation between Fluc reporter mRNA expression degree with endosomal escape than between Fluc expression degree and uptake.

Altogether, this suggests that LYX are less likely than LFM complexes to escape the endosome for mRNA release, resulting in a lower yield of mRNA translation [75]. Among them, incorporation of biodegradable groups in the vector formulation can facilitate their degradation, thus promoting particle disassembly after internalization [76]. In parallel, biomimetic strategies are being explored, combining nonviral delivery vectors with viral fusogenic peptides or bacterial toxins, potentially leading to more efficient mRNA delivery systems [77].

Given the significant potential of mRNA for directing the synthesis of therapeutic proteins for tissue repair and regeneration, we evaluated the efficacy of LYX liposomes compared to LFM for mRNA delivery within an implantable hydrogel matrix commonly used in tissue engineering approaches [51]. Lipoplexes were incorporated into a fibrin hydrogel, which offers numerous advantages as a delivery platform for bioactive molecules, particles, or cells, and supports tissue regeneration.

Interestingly, while the transfection potential of mRNA with LYX lipoplexes in rat MSCs in vitro was 2-fold lower than that of LFM under 2D conditions (cell culture in a monolayer) (Figure 4A), it was similar in a 3D fibrin gel environment for 48h of incubation (Figure 7A). Advantageously, when the gels loaded with lipoplexes (without cells) were implanted subcutaneously in mice, LYX-loaded hydrogels induced higher luciferase expression during the first 24 hours compared to LFM-loaded hydrogels (Figure 7B). Overall, our results demonstrated that LYX liposomes are more efficient at mRNA delivery in a 3D environment compared to a 2D environment, especially in comparison to LFM.

## 5. Conclusion

Our study focused on characterizing and evaluating the therapeutic potential of LYX, an optimized version of the Lip100 liposomes, designed for nucleic acid delivery, previously reported. LYX are stable, monodisperse, and maintain their performances upon storage. Detailed analysis of LYX lipoplex morphology led us to hypothesize a pH-based mechanism for mRNA release into the cytosol triggered by acidification. Finally, analyses of cell transfection both in vitro and in vivo highlighted the limitations and advantages of the LYX liposomes, demonstrating its versatility as a platform for mRNA delivery.

## 6. Acknowledgments

The authors thank the P@CYFIC division of the MO2VING facility (CBM, Orléans, France), more particularly David Gosset. We also thank Julien Burlaud Gaillard and the Plateforme IBiSA de Microscopie Electronique de l'Université de Tours for cryo-TEM studies. Then, we thank Céline Landon (CBM, Orléans) for lending us the extrusion apparatus.

Funding: This work was supported by the Agence Nationale de la Recherche (CREABONE Grant Number ANR-21-CE18-0037-01). SAXS studies were supported by Synchrotron SOLEIL (proposal # 20230060).

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