# Robust micelles formulation to improve systemic corticosteroid therapy in sepsis in multiple healthcare systems

Younes Louaguenouni, Qinglin Wang, Thomas Baticle, Catherine Cailleau, Elodie Lamy, Stanislas Grassin-Delyle, Julie Mougin, David Chapron, Juliette Vergnaud, E. Fattal, et al.

► To cite this version:

Younes Louaguenouni, Qinglin Wang, Thomas Baticle, Catherine Cailleau, Elodie Lamy, et al. Robust micelles formulation to improve systemic corticosteroid therapy in sepsis in multiple healthcare systems. Journal of Controlled Release, 2025, 381, pp.113635. 10.1016/j.jconrel.2025.113635. hal-05029253

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# Robust micelles formulation to improve systemic corticosteroid therapy in sepsis in multiple healthcare systems

Younes Louaguenouni<sup>a</sup>, Qinglin Wang<sup>a</sup>, Thomas Baticle<sup>a</sup>, Catherine Cailleau<sup>a</sup>, Elodie Lamy<sup>b</sup>, Julie Mougin<sup>a</sup>, David Chapron<sup>a</sup>, Stanislas Grassin-Delyle<sup>b</sup>, Juliette Vergnaud<sup>a</sup>, Nicolas Tsapis<sup>a</sup>, Elias Fattal<sup>a,*</sup>, François Fay<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Sepsis

Micelles

Dexamethasone

Inflammation

Nanomedicine

## ABSTRACT

Sepsis is a life-threatening condition resulting from an imbalanced immune response to an infection that causes over 10 million deaths annually, particularly in low and middle-income countries. Current clinical management of sepsis relies on infection control, homeostasis restoration, and systemic corticosteroid therapy. Unfortunately, while beneficial, corticosteroid regimens, including dexamethasone, can lead to adverse effects such as neurological and metabolic complications, limiting their use. In this work, we decided to develop a scalable production method using only approved and cost-effective materials. We also conceived our formulation to be freeze-drying friendly to allow its use within various healthcare systems. Following those concepts, we designed DSPE-PEG (2000)-based micelles to encapsulate dexamethasone, and improve its *in vivo* efficacy by extending blood circulation time and targeting innate blood immune cells. First, the physicochemical properties, stability, *in vitro* release kinetics, and efficacy of dexamethasone-loaded micelles were comprehensively measured to demonstrate the platform's robustness. The therapeutic *in vivo* efficacy of dexamethasone-loaded micelles and their ability to increase animal survival was exhibited in two murine sepsis models, an endotoxemia model, and the cecal ligation and puncture model. Various biodistribution and *ex vivo* fluorescence imaging assays revealed that using micelles led to an improved blood circulation time and a preferential accumulation within immune cells that could explain the enhanced efficacy of dexamethasone-loaded micelles compared to the soluble form of the drug used clinically. Altogether, our results indicate that this robust micellar delivery system can potentially improve the anti-inflammatory therapy of dexamethasone, offering a safer and more effective alternative to conventional corticosteroid regimens in sepsis.

## 1. Introduction

Sepsis is characterized by a profound dysregulated immune response of the immune system to an infection that can lead to hypotension, organ failure, and, in 25% of cases, death [1,2]. Based on an estimation by the World Health Organization, every year, almost 50 million people are affected by sepsis, leading to more than 10 million deaths, mostly in low- and middle-income countries, making this disease a global health priority. With the world's population over 60 years likely to double in the next three decades, the societal impact of sepsis is bound to increase as the disease is more frequent and associated with higher mortality in elderly patients [3].

The mechanisms underlying immune dysregulation during sepsis are

heterogeneous and complex. Still, one of the typical events is the activation of circulating monocytes by foreign pathogens, leading to a massive secretion of proinflammatory cytokines and an excessive systemic immune reaction. At the clinical level, the management of patients is based solely on controlling the infection and restoring cardiovascular and respiratory homeostasis [4]. Unfortunately, despite its severity, no approved immunomodulatory treatment for sepsis currently exists, except systemic corticosteroid therapy [5].

Corticosteroids, including dexamethasone, have been shown to reduce mortality in deadly inflammatory conditions, such as sepsis, acute respiratory distress syndrome, and, more recently COVID-19 [6]. However, while beneficial, corticosteroid regimens may lead to adverse effects such as neurological and metabolic complications [7], preventing

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

<footnote><sup>b</sup> Département de Biotechnologie de la Santé, Université Paris-Saclay, UVSQ, INSERM U1173, Infection et inflammation, 78180 Montigny le Bretonneux, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: elias.fattal@universite-paris-saclay.fr (E. Fattal), francois.fay@universite-paris-saclay.fr (F. Fay).</footnote>
the use of high-dose by systemic route [8,9]. Hence, following on our previous works about nanoparticle-based dexamethasone delivery in rheumatoid arthritis and acute lung injury [10,11], our group has focused on developing a new platform to enhance dexamethasone's pharmacokinetic profile and improve treatment outcomes for sepsis patients.

In this project, our delivery strategy has been centered on three pivotal ideas: 1) to extend dexamethasone blood circulation time and to reduce fluctuations in drug concentration and high blood concentration 2) to target blood innate immune cells and improve dexamethasone anti-inflammatory efficacy due to the crucial role of those cells in innate immunity, 3) to employ already approved, cost-effective materials to produce a robust and storable dosage form that could be administered in a large array of healthcare systems. Based on those concepts, we focused on the development of micelles composed of distearoylphosphatidylethanolamine-Poly(ethylene glycol) (DSPE-PEG) as they have emerged as versatile and effective drug carriers with high loading capacity and extended circulation time [12].

The first step of this study was to develop a new dexamethasone-loaded DSPE-PEG micelles (DXM-micelles) formulation and to comprehensively investigate its physicochemical properties, including stability, in vitro release kinetics, and in vitro immunomodulatory ability. The in vivo therapeutic efficacy of DXM-micelles was then assessed in two different sepsis murine models and demonstrated improved effects compared to saline treatment and free dexamethasone. The tissular and cellular distribution of the DXM-micelles were subsequently characterized using fluorescent imaging, mass spectrometry, and flow cytometry to comprehend the in vivo benefits of dexamethasone micelles encapsulation. Our results demonstrate that DXM-micelles are an elegant and robust dosage form that can significantly improve dexamethasone's efficacy in vivo.

## 2. Methods

### 2.1. Materials

Dexamethasone (DXM) was provided from Interchim (France). Dexamethasone disodium phosphate (DSP) was purchased from Fagron (The Netherlands). [N-(carbony-methpypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt] (DSPE-PEG (2000)) was purchased from Lipoid Gmbh (Germany). High-quality ultra-pure water was obtained using a Milli-Q Reference system from Merck-Millipore. Solvents were of HPLC analytical grade and were obtained from Carlo Erba (Italy). All experiments were performed in amber vials due to the photosensitivity of dexamethasone and DSPE-PEG (2000). Phosphate-buffered saline (PBS) tablets were purchased from Sigma-Aldrich.

### 2.2. DXM-micelles preparation and characterization

DXM-micelles were prepared using a thin-film rehydration method as described by Sahib et al. [13] DSPE-PEG(2000) and DXM were dissolved in a methanol:chloroform mixture (7:3 v/v). The mixture was then placed under ultrasonication for 2 min and vortexed for 1 min. The organic solvent was then evaporated under reduced pressure using a rotary evaporator (Büchi, Switzerland) to obtain a dried lipid film. The film was rehydrated with 4 mL of PBS and then gently stirred for 40 min at 40 °C to allow micelles formation. The final concentration of DSPE-PEG and DXM were 25 mg/mL and 0.5 mg/mL, respectively. The unencapsulated DXM was removed by filtration using a 0.22 µm Medical Millex-GV Syringe Filter Unit. Micelles were stored in amber vials at 4 °C protected from light.

After each preparation, DXM-micelles size ($d_H$), polydispersity index (PDI), and surface charge were evaluated using a Malvern NanoZS at 173° angled laser (measurements were performed 3 times on each sample). Size ($d_H$) and polydispersity index (PDI) were recorded on non-

diluted samples, while ζ potential measurements were carried out in a 1/10 dilution of 1 mM NaCl. The stability of DXM-micelles was monitored for up to 18 days on samples stored at 4 °C.

The quantity of encapsulated DXM was determined by high-performance liquid chromatography (HPLC, Ultimate 3000, Thermo Scientific, France). Before each HPLC measurement, the micelles were diluted in acetonitrile to solubilize the micelles and release the encapsulated DXM. The analysis was performed at 238 nm wavelength using an Interchrom Reverse Phase Nucleosil 5 (150 × 4.0 mm) column. The column temperature was maintained at 30 °C. The mobile phase was composed of 50 % acetonitrile and 50 % water. The flow rate was set at 1 mL/min and the injection volume at 20 µL. Each run lasted 10 min with a retention time of 3.6 min. The calibration curve of DXM was linear in the range 31.625–1000 µg/mL ($R^2 = 0.9997$, $y = 0.66 \times + 6.13$). The absorbance peaks of the different solutions were recorded at a wavelength of 238 nm, and the DXM concentrations were quantitatively determined using a pre-established standard curve.

Fluorescent micelles were prepared following the same procedure as the DXM-micelles described above. For FRET analysis, 3,3-dioctadecylox-acarbocyanine perchlorate (DiO) and 1,1-diocta-decyl-3,3-tetramethylindocarbocyanine perchlorate (DiL), were individually loaded or co-loaded within the DSPE-PEG(2000) micelles at a 1 % w/w ratio. In the case of co-loading, DiO and DiL were incorporated at 1 % and 2 % w/w ratio, respectively, to have a final DiL/DiO ratio = 2, as described by Sanchez-Gaytan et al [14] For in vivo imaging and flow cytometry studies, Cy7-DXM-micelles and DiO-DXM-micelles were prepared by incorporating DSPE-PEG-Cy7 and DiO to the lipidic mixture at 1.5 % w/w ratio and 0.5 % w/w ratio respectively.

### 2.3. In vitro drug release profile of DXM from micelles

The kinetics of drug release were examined using dialysis cassettes from (Slide-A-Lyzer™ Dialysis Cassette, Thermo Scientific, France) with a cut-off of 3.5 kDa. Two mL of the micelle's suspension were deposited inside a dialysis cassette, which was then placed into a beaker containing 300 mL of release media (1:1 v/v human serum:PBS) and maintained under gentle stirring at 37 °C. 50 µL samples were drawn from the cassette at different time intervals. The samples were then diluted two times by adding an equivalent volume of acetonitrile to break the micelles and release the encapsulated DXM. All samples were then stored at -20 °C until analysis by HPLC was performed as described above. For comparative purposes, a control solution of DSP in PBS was prepared to achieve a concentration of 0.5 mg/mL. This DSP solution was subjected to the same procedure employed for DXM micelles. Notably, the retention times for DSP were recorded as 1.1 min, while its calibration curve exhibited linearity across the range of 31.625 to 1000 µg/mL ($R^2 = 0.9998$, $y = 0.1019 \times + 0.66$).

### 2.4. Freeze drying and stability

Aliquots of 800 µL DXM-micelle solution were transferred into vials without adding any cryoprotectant agent. The vials were plunged into liquid nitrogen for 5 min and lyophilized during a 24-h cycle below 0.01 mbar. The lyophilized powders were then stored at 4 °C for over 14 months. The particle size, PDI, ζ potential, and encapsulation efficiency were measured after the micelles reconstitution by adding 800 µL of PBS.

### 2.5. Transmission Electron microscopy

Transmission electron microscopy (TEM) was performed at I2BC (CNRS, Gif-sur-Yvette, France). Micelle samples were diluted in PBS just prior to deposition. Five µL of micelles suspension at 5 mg/mL and 25 mg/mL of DSPE-PEG were deposited for 1 min on 400 mesh formvar copper grids. Excess liquid was then removed, and the grids were allowed to air dry for another 2 min. The grids were negatively stained
by adding uranyl acetate at 2 % w/w for 30 s. The excess solution was removed, and grids were left to dry before observation. Samples were analyzed using a JEOL1400 microscope (JEOL, Japan) operating at 80 kV. Pictures were acquired using a Rio9 camera (Gatan, USA).

## 2.6. Differential scanning calorimetry

Differential scanning calorimetry (DSC) thermograms were obtained by using a microcal VP-DSC (Malvern Panalytical, France) apparatus. DSC analyses were performed from 0 to 70 °C at a 1.5 °C/min rate. Several heat/cool cycles were performed. A Hamilton syringe was filled with at least 580 µL of the suspension and then inserted on the top of the calorimetric cell through the filling tunnel. The mentioned transition temperatures are the average of the peak temperatures. Enthalpy values were obtained by integrating the area under the peak. Thermograms were analyzed using the Origin 7 software (Origin Lab, USA).

## 2.7. Structural stability of micelles in serum using Förster resonance energy transfer

Förster resonance energy transfer analyses were carried out on a spectrofluorometer from Horiba (Fluoromax series). Micelles were incubated within a 1:1 v/v human serum:PBS at 37 °C at a 40-dilution fold. Emission spectra were recorded with a 458 nm excitation and an emission scan from 480 to 700 nm with 1.5 nm bandwidth. Monitoring studies were performed following the same conditions with a FRET signal detected at 580 nm.

## 2.8. Cell culture

Murine macrophage cell line RAW 264.7 cells (ATCC TIB-71) obtained from ATCC (USA) were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 % fetal bovine serum, 50 U/mL penicillin and 50 U/mL streptomycin at 37 °C with 5 % CO₂. Cells were split twice weekly at a 1/10 ratio using a scraper and used between passages 3 to 14 after thawing.

## 2.9. In vitro cytotoxicity and anti-inflammatory effect

RAW 264.7 cells were seeded in 96-well plates (Pierce® 96-Well Plates-Corner Notch) at a density of $9 \times 10^3$ cells/well in 100 µL of DMEM and were left overnight in the incubator. The medium was then replaced by fresh medium alone (for negative control) or fresh medium containing LPS (Escherichia coli strain O55:B5, Sigma-Aldrich, USA) at 25 ng/mL and IFN-γ (PeproTech, France) at 50 ng/mL and left for 18 h to induce inflammation. Afterward, DXM-micelles were added to fresh medium at different concentrations (250, 100, 50, 25, 10, and 1 µg/mL) to a final volume of 200 µL per well. Cells were then incubated at different time intervals (4, 8, and 24 h). Cell supernatants were then collected and frozen at -20 °C until analysis, while cell viability was assessed in fresh medium.

TNFα and IL-6 concentrations were measured through ELISA (uncoated mouse TNFα and IL-6 ELISA Kits, ThermoFisher, USA) according to the manufacturer protocols. Cell viability was assessed then by adding fresh medium containing tetrazolium 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) at a final concentration of 500 µg/mL followed by an incubating period of 1 h. After the crystals were formed, the medium was discarded and replaced with 200 µL dimethyl sulfoxide (DMSO) to dissolve the crystals. The absorbance of the different wells was measured at 570 nm using a 96-well plate reader and compared to a non-treated cell sample.

## 2.10. In vivo studies

### 2.10.1. Mice

C57BL/6J OlaHsd mice were purchased from Envigo for efficacy,

biodistribution, and immune cell targeting studies. Mice were kept in controlled conditions (12 h/12 h light-dark cycle; temperature, 21-24 °C; relative humidity, 40-70 %) and had access to food and water *ad libitum*. Experiments were approved by the Animal Care Committee of the University Paris-Saclay, following principles of laboratory animal care and European legislation 2010/63/E under the protocol APAFIS#40336-2,023,011,220,041,066 v3.

### 2.10.2. Evaluation of in vivo efficacy of DXM-micelles on endotoxemia mouse models

Endotoxemia was induced in mice using LPS from *Escherichia coli* (strain O55:B5, Sigma-Aldrich, USA). A single intraperitoneal (IP) injection of LPS at a 22 mg/kg dose was administered to induce endotoxemia. The selection of the LPS dose was based on pilot experiments. DXM-micelles or DSP were administered retro-orbitally *via* intravenous (IV) injection. Two IV injections of 2.5 mg/kg and 1 mg/kg DXM-micelles or DSP were performed at 1 h and 6 h after LPS administration to evaluate the therapeutic efficacy and sustained effect. Control mice received an equal volume of sterile saline. At the designated timepoint ($T = 8$ h), blood samples were collected from each mouse *via* cardiac puncture. The levels of proinflammatory cytokines TNFα and IL-6 in the plasma were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. In addition, plasmatic levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, and creatinine were measured by Cerbavet Laboratory (France). These biochemical markers are indicative of liver and renal function and can assess the potential protective effect of DXM-micelles on these organs.

### 2.10.3. Cecal and ligation puncture (CLP) model

The procedure was adapted from Toscano et al. [15] Mice were anesthetized with Ketamine/Xylazine mixture (100 µg/10 µg per g bodyweight). The cecum was exposed and then ligated below the ileo-cecal junction while passing the ligation silk below the mesenteric artery to avoid inducing ischemia. The cecum was finally punctured with an 18-gauge needle and gently pressed to squeeze out a small amount of feces. The incision was sutured, and the animals were resuscitated with a subcutaneous injection of 1 mL saline. During the first 48 h, animals received a subcutaneous injection of buprenorphine (0.05 mg/kg) twice per day.

### 2.10.4. Efficacy assessment on CLP mice

The therapeutic efficacy of DXM-micelles was evaluated in CLP model. 6- and 24-h post-surgery, DXM-micelles (0.1 mg/kg) or an equivalent dose of DSP were intravenously injected via the retro-orbital sinus. CLP mice injected with an equivalent volume of saline were used as controls. For 5 days, all mice were monitored twice a day using murine sepsis score (MSS) adapted from Shrum et al. [16], until endpoint (defined as a total score of 16) and consequent euthanasia. MSS involves observing six parameters: appearance, posture, activity, response to stimulus, eyes, rectal temperature, and body weight loss. (Table S2).

In another set of experiments, DSP or DXM-micelle-treated animals underwent a submandibular blood collection at $t = 30$ h. Plasmatic cytokine levels were measured using the MSD® V-Plex® proinflammatory panel 1 (mouse) kit, which can simultaneously measure IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, and TNFα levels.

### 2.10.5. Biodistribution of Cy7-DXM-micelles and blood half-life

Biodistribution measurements were carried out on CLP mice by intravenously administrating Cy7-DXM-micelles 6 h after surgery. This timepoint was chosen following the efficacy studies. *Ex vivo* biodistribution imaging studies were then performed at different time intervals (30 min, 60 min, 3 h, and 24 h). At each timepoint, selected mice were euthanized with an intraperitoneal (IP) administration of ethanol® (150 mg pentobarbital per kg of body weight). An intracardiac perfusion of saline was then carried out to remove blood residues within
organs. Subsequently, the spleen, kidneys, liver, heart, and lungs were collected and imaged. All fluorescence images were acquired after excitation at 754 nm, and the signal was recovered using a 780 nm emission filter with the IVIS Lumina LT Series III (PerkinElmer, USA). Images were processed using the Living Image software (PerkinElmer, USA). Regions of interest (ROIs) were measured in each well, and data were fitted using a nonlinear one-phase decay regression in GraphPad Prism. Organs collected from non-treated CLP mice (control) were used to determine the baseline level of fluorescence (i.e., autofluorescence), which was subtracted from the recorded signal.

To estimate the blood circulation time of the Cy7-DXM-micelles, blood was drawn from CLP animals at predetermined timepoints after injection: 5, 10, 20, 30, 40, 50, 60, 90, 120, 180, and 1440 min. In order to rationalize the number of mice used, each mouse underwent three samplings (two submandibular samplings and one final cardiac puncture). The blood samples were then placed on a black 96-well plate, and the fluorescence images were acquired and analyzed similarly as above.

### 2.10.6. *Dexamethasone in vivo plasma concentration and tissue distribution*

The study was conducted on CLP mice. Animals were randomly divided into two experimental groups: one group received dexamethasone in its micellar form, and the other group received dexamethasone in its free soluble form, DSP. At predetermined timepoints (15 min, 30 min, 2 h, and 6 h) following IV dexamethasone administration, animals were euthanized using euthasol® (150 mg per kg of body weight) via IP administration. Blood samples were collected by intracardiac puncture. Plasma was recovered and stored at -80 °C. Organs, including the spleen, kidneys, heart, lungs, and liver, were carefully excised from each animal and immediately transferred to a liquid nitrogen container before storage at -80 °C.

### 2.10.7. Quantification of dexamethasone using mass spectrometry

#### 2.10.7.1. Sample preparation.
For plasma samples, 50 µL of each sample was transferred to 1.5 mL tubes, then 10 µL of dexamethasone-d4 (1000 ng/mL in methanol) and 500 µL of methanol were added. Samples were mixed for 5 min and centrifuged at 10000 rpm for 10 min at +4 °C. 400 µL of the supernatant were transferred to a 1.5 mL tube and evaporated to dryness under vacuum at 45 °C for 1 h in a Concentrator Plus (Eppendorf, France). One hundred microliters (100 µL) of a mixture of water/methanol (95/5, v/v) were added to the dry residues. Each sample was mixed and transferred to a glass vial for LC-MS/MS analysis. Before analysis, samples were centrifuged at 3000 rpm for 15 min at +4 °C.

For organs, 20 mg of each organ were weighed in 2.0 mL tubes, then 10 µL of dexamethasone-d4 (4000 ng/mL in methanol) and 250 µL of methanol were added. Samples were crushed for 10 min at 50 Hz in a TissueLyser (Qiagen, France), centrifuged at 15000 rpm for 10 min at +4 °C and 100 µL of the supernatant was transferred to a 1.5 mL tube. One hundred microliters (100 µL) of methanol were then added. Samples were finally mixed and transferred to a glass vial for LC-MS/MS analysis. Before analysis, samples were centrifuged at 3000 rpm for 15 min at +4 °C.

#### 2.10.7.2. LC-MS analysis.
Chromatography was performed by injecting 10 µL of sample onto a 1.9 µm Hypersil gold column (100 × 2.1 mm, ThermoFisher Scientific, France) maintained at 40 °C. The mobile phase consisted of a gradient of methanol and 20 mM of ammonium carbonate in water adjusted to pH 9 (by the addition of ammonium hydroxide) at a flow rate of 500 µL/min. Compounds were ionized in negative mode using a heated electrospray ionization source and detected using Multiple Reaction Monitoring analysis on a TSQ Quantiva mass spectrometer (ThermoFisher Scientific). Dexamethasone was detected using the 391.3 → 361.3 (m/z) transition and quantified using a calibration curve

covering 5–1000 ng/mL for plasma and 0.1–50 ng/mg for organs. The quantification was achieved using dexamethasone-d4 as an internal standard.

#### 2.10.8. Myeloid cells in vivo uptake of DiO-DXM-micelles

We used a protocol similar to the one previously reported [17]. At different timepoints following DiO-DXM-micelles intravenous administration in CLP mice model, blood was collected via an intracardiac puncture in heparinized tubes, and red blood cells were removed from the blood sample by lysis buffer (BioLegend, USA) following manufacturer protocol. Finally, the cells were stained with antibodies for flow cytometry. DiO was detected on the 530/30 channel. The panel consisted of Viability™ Fixable Dyes 405, CD11C-APC (clone N418), CD11b-PE(clone M1/70), Ly6C-PerCP-Cy5.5 (clone HK1.4), CD90-PE-Cy7 (clone 53–2.1), CD45-PE-Cy7 (clone RA3-6B2), CD49b-PE-Cy7 (clone DX5), NK1.1-PE-Cy7 (clone PK136), TER 119-PE-Cy7 (clone TER-119), Ly6G-PE-Cy7 (clone 1AB-Ly6G). All samples were measured on an LSRII FORTESSA flow cytometer (Becton Dickinson, USA). Results were analyzed with FlowJo software (Becton Dickinson, USA) with the gating strategy being detailed in fig. S7.

### 2.11. Statistics

All analyses were done with Graphpad Prism 9.0 statistical program (USA). ***, * * P < 0.001, **, * * P < 0.01, * P < 0.05, ns, no significant difference.

## 3. Results and discussion

### 3.1. DXM-micelles formulation and in vitro characterization

Using a thin-film hydration procedure, we were able to produce a monodisperse population of PEGylated micelles loaded with DXM with an average size of 13 nm (Fig. 1a), a polydispersity index (PDI) of 0.08, and a slightly negative (−3 mV) zeta potential value. Interestingly, subsequent stability analysis demonstrated that the storage of the DXM-micelles at 4 °C for 18 days did not lead to any modifications in size or polydispersity (Fig. 1b). Those features, key to enable sustained circulation in blood [18], were further confirmed by transmission electron microscopy (TEM) imaging (Fig. 1c). HPLC measurements of the DXM in our micelles revealed an 80 % DXM encapsulation rate, which was confirmed by LC/MS analyses. This high efficiency was expected thanks to the hydrophobicity of the micelle core conferred by the C18 lipidic chains. The *in vitro* release behavior of the DXM-micelles was subsequently evaluated by dialysis against a mixture of 1:1 human serum:PBS at 37 °C using *Dexamethasone* Sodium Phosphate (DSP, the water-soluble free form of the drug) as control. Fig. 1d shows that almost 80 % of DSP *passed through* the dialysis bag within 15 min, but DXM-micelles demonstrated a sustained drug release over 6 h. To better comprehend the interaction between DXM and DSPE-PEG molecules, a DSC analysis was performed and revealed a reversible transition observed around 12 °C with a transition enthalpy of 11.04 J/g (Fig. 1e) for micelles made solely of DSPE-PEG. Interestingly, the incorporation of DXM in the micelle formulation led to a slight shift and shouldering of the transition peak to the right alongside an increase in the measured enthalpy estimated at 13.03 J/g, demonstrating the presence of physical interaction between DXM and the DSPE lipidic chains. While only a few studies have reported on the transition of DSPE-PEG in micelles’ monolayer, our results are in agreement with previous results obtained by Kastantin et al. [19] In their work, no transition was observed between 30 and 90 °C. In contrast, a reversible transition was reported at 12 °C along with a significant transition enthalpy of 39 ± 4.2 kJ/mol (≈ 21 J/g) attributed to the melting of DSPE lipid chains in the center of the DSPE-PEG(2000) micelles. Altogether, our data are indicative of the DXM’s localization within the micelles’ core and explain the slow release profile observed [12, 20].
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Physical characterization of the DXM-micelles. a, Size, polydispersity index (PDI) and surface zeta potential of freshly prepared DXM-DSPE-PEG(2000) micelles (n = 3). b, Size and PDI monitoring up to 18 days on samples stored at 4 °C, graphs are presented as mean ± SD (n = 3). c, TEM images after negative staining of DXM-DSPE-PEG(2000) micelles at 5 and 25 mg/mL of DSPE-PEG(2000) respectively. d, *In vitro* release study of dexamethasone from the DSPE-PEG(2000) micelles within human serum media (50 % PBS). e, DSC thermograms of fresh unloaded DSPE-PEG(2000) micelles (blue), fresh DXM-DSPE-PEG(2000) micelles (green), and reconstituted lyophilized DXM-DSPE-PEG(2000) micelles following a 14 month storage period at 4 °C (black). f, Representative pictures of DXM-DSPE-PEG(2000) micelles freshly prepared solution (Fre), after lyophilization (Lyo) and the reconstituted solution obtained from the lyophilized powder (Rec). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

As long-term stability has been a critical characteristic that drives the translation of novel nanomedicine from bench to bedside [21], we decided to investigate if our micellar platform could be preserved through lyophilization. As successful reconstitution of DSPE-PEG micelle formulations without a cryoprotectant has been previously reported [13,22,23], we chose to lyophilize our micelles without cryoprotectant before storing them at 4 °C as proof of concept. After 14 months, the lyophilized formulations were reconstituted in PBS by gently vortexing for 30 s. Upon rehydration, all samples produced clear and colorless solutions with no visual precipitates (Fig. 1f). Subsequent DLS and HPLC analysis revealed that lyophilization and reconstitution processes did not significantly alter the particle sizes, the PDI and zeta potential of the micelles, nor did they lead to major DXM release (Table S1). DSC analysis of reconstituted lyophilized samples also revealed a similar pattern to the fresh sample (Fig. 1e), demonstrating our micelles' excellent stability after freeze-drying and long-term storage.

The stability of DSPE-PEG micelles was further investigated through a Förster resonance energy transfer (FRET)-based approach by loading in the micelles two hydrophobic fluorescent dyes to form a FRET pair using respectively 3,3-dioctadecylox-acarbocyanine perchlorate (DiO) and 1,1-diocta-decyl-3,3-tetramethylindocarbocyanine perchlorate

(DiL) as electron donor and acceptor (Fig. 2a). First, data obtained using DiO-only-micelles and DiL-only-micelles (fig.S1) demonstrated that on their own, DiO and DiL do not emit significant signals at 568 nm when excited at 458 nm. Subsequently, the fluorescent spectra with an excitation wavelength at 458 nm of the micelles containing the two fluorescent probes (FRET-micelles) was recorded and revealed the presence of a FRET signal within DiO/DiL micelles, unlike a mixture of the probes solubilized in ethanol (Fig. 2b). This confirms that in our system, FRET signal only occurs in intact micelles in which DiO and DiL are kept nearby each other. We then conducted measurements of the FRET signal from DiO/DiL-micelles incubated in a 50:50 v/v human serum:PBS solution over 20 h to assess their long-term stability. As presented in Fig. 2c, the FRET signal remained consistently strong throughout the experiment, demonstrating the stability of our micelle platform. Importantly, in a control experiment (blue dashed line), adding ethanol at t = 5 h led to a significant drop in the FRET signal, validating that the recorded FRET signal only originated from unaltered DiO/DiL micelles. Altogether, these data demonstrated that our DSPE-PEG micelles can maintain their structural integrity in blood and be effectively used to deliver a hydrophobic payload without significant micelle dissociation.

The anti-inflammatory activity of DXM-micelles was first assessed *in vitro* on LPS/IFNγ-activated RAW 267.4 murine macrophages using
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** *In vitro* characterization of the DXM-micelles. a, Schematic representation of the particles and the FRET process. b, Emission spectra of FRET-Micelles in PBS and a physical mixture of DiO-DiL in ethanol with an excitation wavelength at 458 nm. c, Kinetic monitoring spectra of FRET-Micelles in a solution 50:50 v/v human serum:PBS with an excitation wavelength at 458 nm and an emission signal recovered at 570 nm (black). The addition of ethanol at 1/3 volume ratio (at *t* = 5 h) is illustrated with the discontinuous blue line. d, Inhibitory activity of DXM-DSPE-PEG(2000) micelles on LPS-induced TNFα and IL-6 production by RAW 264.7 cells. Data in bar graphs are presented as mean ± SD (n = 4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

TNFα and IL-6 secretions as a read-out. Results displayed in Fig. 2d revealed that a 4–8-h incubation of DXM-micelles led to a significant dose-dependent reduction in TNFα secretion. At the same time, no cytotoxic effects were observed for the entire DXM concentration range, i.e. up to 250 µg/mL (fig.S2). Importantly, this *in-vitro* anti-inflammatory effect was preserved after lyophilization and storage at 4 °C for 14 months (fig.S3), demonstrating the robustness of our formulation and its potential use in a large spectrum of healthcare systems.

## 3.2. DXM-micelles contribute to improved outcomes in sepsis models

The anti-inflammatory effect of DXM-micelles *in vivo* was then evaluated in an LPS-induced non-lethal endotoxemia mice model, using DSP as control. In this model, endotoxemia was first induced in mice through a single intraperitoneal injection of 22 mg/kg of LPS from *Escherichia coli* followed by IV injection of saline, DSP, or DXM-micelles after 1 and 6-h at a respective dose of 2.5 mg/kg and 1 mg/kg (Fig. 3a).

This regimen was chosen to tackle the rapid inflammatory reaction that arises after the LPS injection in this model [24–26] and to mimic the continuous/frequent administration of dexamethasone used in the clinic [5]. Analysis of the plasmatic concentrations of IL-6 and TNFα 8-h post-LPS injections (Fig. 3b) revealed that both DXM-micelles and DSP regimens led to a significant decrease in proinflammatory cytokines compared to the group that received only saline, indicative of strong anti-inflammatory effects. Notably, the plasmatic concentration of liver enzymes alanine transaminase (ALAT) and alkaline phosphatase (ALP), showed only a slight increase of the latest for DXM-micelles compared to the control group (Fig. 3c). Additionally, DXM-micelles and DSP treatment groups exhibited a similar and moderate increase in creatinine and urea plasmatic concentrations compared to the control group. Altogether, the absence of significant differences between the two treatment groups suggests that while demonstrating potent anti-inflammatory efficacy, DXM-micelles did not cause additional renal impairment compared to DSP.
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** *In vivo* therapeutic efficacy of DXM-micelles in LPS endotoxemia model. a, Schematic overview of the induced LPS endotoxemia model followed by two retro-orbital IV injections DXM-micelles or equivalent in DSP, after 1 h (at 2,5 mg/kg) and 6 h (at 1 mg/kg) following the LPS injection. b, Plasmatic levels of TNFα and IL-6 measured by ELISA 8 h after LPS administration. c, ALAT and ALP values, creatinine, and urea concentration in mouse plasma 8 h after exposure to different treatments. Statistical significance was assessed via one-way ANOVA with Tukey's *post hoc* correction \*P < 0.05, \*\*P < 0.01 (n = 5 mice per treatment group, n = 3 for control).</figcaption></figure>

Next, the DXM-micelles were tested on a sepsis cecal ligation and puncture (CLP) model, considered the gold standard murine model in experimental research on the inflammatory phase of polymicrobial sepsis [27] (Fig. 4). This model consists in surgically ligating the animal's cecum, perforating it before replacing it within the abdominal cavity to induce peritonitis, evolving into sepsis. In this slow-progressing model, small doses (0.1 mg/kg) of DXM-micelles or DSP were intravenously injected 6- and 24-h post-surgery (Fig. 4a). At the same time, control animals were administered with a corresponding volume of saline. The survival curves in Fig. 4b revealed that while free DSP led to only a small, non-significant, reduction of the mortality compared to the control group, DXM-micelles regimen significantly improved the survival of animals compared to both DSP- or saline-treated mice. Five days after surgery, 80 % of the animals treated with DXM-micelles survived, compared to only 30 % in the DSP group. In parallel, the evaluation of murine sepsis scores (Fig. 4c, table S2) revealed a significant improvement in the DXM-micelles-treated animals' clinical score group

compared to the saline-treated groups. The reduction in this score for the DXM-micelles group (especially during the first two days following the surgery) denotes a decrease in the severity of sepsis-associated manifestations indicative of better control of the inflammation and its effects. Similarly, measurement of plasmatic proinflammatory cytokines levels at 30 h (Fig. 4d, fig.S4) shows that DXM-micelles-treated animals exhibited lower plasma concentrations for an array of proinflammatory cytokines, including TNFα and IL-1β. Our data indicate that DXM-micelles are more effective than free dexamethasone in reducing the hyper-proinflammatory response associated with sepsis, thereby contributing to improved clinical outcomes and survival.

### 3.3. Mechanisms behind DXM-micelles improved efficiency

To elucidate the mechanisms behind dexamethasone's improved efficiency when administered as micelles, blood circulation and biodistribution analysis of DXM-micelles were evaluated in vivo using DXM-micelles incorporating 1.5 % w/w of near-infrared fluorescent, Cy7. Following our efficacy studies, Cy7-DXM-micelles were administered 6 h after CLP surgery. Blood samples were collected at different timepoints, and the Cy7-DXM-micelle content was analyzed by fluorescent imaging. Using a nonlinear one-phase decay regression model, the blood circulation half-time was calculated to be around 90 min (Fig. 5a.i, fig.S5), consistent with previous results (120 min in murine tumor model) reported by Lukyanov et al. [28] Next, analysis of Cy7-DXM-micelles accumulation in different organs (Fig. 5.a.ii, fig.S6) revealed the presence of Cy7-DXM-micelles in the liver 30 min after administration that persisted for a minimum of 24 h. Kidneys and lungs exhibited a low signal level, while signals from the spleen and heart were almost at baseline. Altogether, these results indicate that similar to most PEGylated nanocarriers [29], Cy7-DXM-micelles predominantly accumulated in the liver, with limited uptake occurring in other organs during the studied timepoints.

As following micelles distribution using fluorescence imaging analysis has limitations and could not be used to comprehend the difference observed between treatments entirely, a follow-up experimental protocol was carried out using mass spectrometry to quantify DXM in plasma and various organs after administration of the DSP or DXM-micelles in CLP models. Interestingly, the results reveal a significant difference in plasma concentrations (Fig. 5b.i) as DSP rapidly declined within two hours, indicating a quick clearance from the bloodstream. DXM-micelles showed a significantly prolonged plasmatic level. Similar to the fluorescent biodistribution data, the DXM biodistribution results (Fig. 5b.ii) revealed accumulation of DXM in the liver following administration of both DXM-micelles and free soluble DSP. After 2 h, the amount of dexamethasone administered as DXM-micelles still present in the liver was substantially higher than when injected in the free DSP form. Altogether, these observations suggest that micelles prevent the rapid elimination of dexamethasone, providing a sustained source of the drug between injections that could partly explain the better anti-inflammatory efficacy of the DXM micelles and the improved survival of the animals.

To further understand the different mechanisms behind the enhanced effect of DXM-micelles, we subsequently assessed the interactions between DXM-micelles and circulatory innate immune cells in vivo. DiO-labeled DXM-micelles were administered to CLP mice using the same regimen as the therapeutic protocol (Fig. 5c.i) before the animals were sacrificed at different timepoints. Flow cytometry was then performed on isolated peripheral blood leukocytes to discriminate the various cell populations and measure their interaction with the DiO-labeled DXM-micelles (gating strategy illustrated in fig. S7). Results displayed in Fig. 5c show that the ratios of DiO-positive cells are low 1-h after the first injection and negligible at the 24-h mark. However, when focusing on blood samples collected 1 h after the second injection of DiO-DXM-micelles, a surprisingly significantly higher number of DiO-positive cells were detected. Interestingly, when comparing the
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** *In vivo* therapeutic efficacy of DXM-micelles in cecal ligation and puncture model. a, Schematic of the procedure, sepsis is induced by a CLP procedure which is followed by two retro-orbital IV injections of 0.1 mg/kg of DXM-micelles or equivalent in DSP, after 6 and 24 h following the surgery. At *T* = 30 h, mice underwent a submandibular blood collection for plasma cytokine quantification. b, Survival followed for 96 h after CLP procedure (n = 10 mice per group). A Log-rank (Mantel-Cox) test was used to compare treatments, \*P < 0.05, \*\*P < 0.01. c, Clinical score of animals. A score of 16 was attributed for each dead animal. (n = 10 mice per group). A two-way ANOVA interaction test using Geisser-Greenhouse correction was used to compare treatments with the saline group, \*P < 0.05. d, Heatmap of the plasma proinflammatory cytokine levels for the different treatments. The concentrations of various cytokines are expressed as fold changes relative to the mean concentration obtained with the saline group. The concentrations increase as the colour changes from white to red (n = 7 mice per group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

different cell populations that have interacted with DiO-micelles, Ly6C<sup>low</sup> non-classical Mo represents the population with the highest positive ratio in the first injection, while micelles injected at 24 h interacted more with dendritic cells DC (Fig. 5c.iv). These differences in DiO-positive cell levels in the blood and the absence of positive cells after 24 h, could be explained by the rapid turn-over of the WBC during the early phase of sepsis inflammation as they are recruited to the site of infection [30]. These observations are supported by recent publications by our laboratory and others [31–33] demonstrating that long-circulating PEGylated liposomes are taken up by circulatory innate immune cells in the blood before their chemotactic recruitment, which drives the accumulation of their content into inflamed tissues. Accordingly, our *in vivo* results could indicate that in addition to better plasma pharmacokinetics, DSPE-PEG micelles lead to circulatory innate immune cells uptake and dexamethasone gradual release within the cellular environment. This sustained release profile may extend the exposure of immune cells to dexamethasone and could partly explain the better and prolonged suppression of proinflammatory cytokine production and immune cell activation. In contrast, DSP may be subject to rapid clearance, limiting its duration of action. Additionally, as previously demonstrated with other long-circulating nanocarriers [34], the use of DXM-micelles could also reduce the systemic exposure of non-targeted organs and tissues to dexamethasone and reduce the incidence of adverse effects commonly associated with dexamethasone therapy, thus improving the safety profile of DXM-micelles compared to

DSP for the patients.

## 4. Conclusion

In conclusion, we explored the development of a new nanomedicine-based strategy to reduce the proinflammatory reaction in the early stages of sepsis to improve its clinical outcomes while using cost-effective materials. In this project, we used a conventional anti-inflammatory drug (i.e. dexamethasone) loaded within PEGylated micelles to create a robust delivery platform. Our micellar platform demonstrated its ability to increase survival rate in a murine sepsis model compared to DSP. *In vivo*, the enhanced cellular uptake, sustained release, and immunomodulatory effects provided by DXM-micelles collectively contribute to the superior outcomes observed compared to DSP. The interaction between DSPE-PEG micelles and the circulatory myeloid immune cells unveils new prospects for treating other systemic inflammatory disorders. Finally, as our micelles showed a promising ability to sustain lyophilization and extended storage at 4 °C while preserving their efficacy, we hope that one day they can be administered in a variety of healthcare systems, particularly in low-income countries.

## CRediT authorship contribution statement

Younes Louaguenouni: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Qinglin Wang: Writing
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Biodistribution studies in CLP model. Biodistribution of DXM-micelles a, i) Cy7-DXM-micelles blood half-life as fitted with a one phase decay function (n = 3), ii) Biodistribution analysis via fluorescence imaging of Cy7-DXM post intravenous administration. Data in bar graphs are presented as mean ± SD (n = 3 mice). b, i) Plasma concentrations and ii) biodistribution analysis of DXM after IV injection of DXM-micelles or DSP Data in bar graphs are presented as mean ± SD, A two-way ANOVA interaction test using Geisser-Greenhouse correction was used to compare the kinetics curve in plasma, \*P < 0.05, (n = 4 mice per group). c, DiO-DXM-micelles uptake in WBC, i) schematic of the procedure, Percentage of positive DiO viable cells for each immune cell population and median fluorescence positive cells. ii) 1 h after the first injection. iii) 24 h after the first injection and iv) 1 h after the second injection. Data are presented as mean ± SEM (n = 3 mice per group).</figcaption></figure>

– review & editing, Investigation, Data curation. **Thomas Baticle:** Investigation. **Catherine Cailleau:** Writing – review & editing, Methodology, Data curation. **Elodie Lamy:** Writing – review & editing, Validation, Methodology, Data curation. **Julie Mougin:** Writing –

review & editing, Methodology, Data curation. **David Chapron:** Writing – review & editing, Methodology, Data curation. **Stanislas Grassin-Delyle:** Writing – review & editing, Supervision, Methodology. **Juliette Vergnaud:** Writing – review & editing, Validation, Methodology.
**Nicolas Tsapis:** Writing – review & editing, Validation, Methodology.
**Elias Fattal:** Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.
**François Fay:** Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.

## Acknowledgments

YL and TB received a scholarship from the doctoral school: ED Innovation Thérapeutique – Du Fondamental à l'Appliqué [ITFA n. 569]. QW received a scholarship from the Chinese Scholarship Council. This work benefited from the support of the project NANOSEPSIS (see: https://anr.fr/Projet-ANR-20-CE18-0019) of the French National Research Agency (ANR). This work benefited from the support of IPSIT-Printemps des plateformes. The authors acknowledge Claudine Delomenie and ACTAGen, Marie-Laure Aknin at CYM, and Valerie Domergue and AnimEx as well as all core facilities from IPSIT, for access to their equipment/infrastructures and advice. We also thank the Region Ile-de-France for financial support of the IPSIT platforms. SGD, NT, EF, and FF are members of the FHU SEPSIS (Saclay and Paris Seine Nord Endeavour to PerSonalize Interventions for Sepsis) and the IHU Prometheus Comprehensive Sepsis Center.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jconrel.2025.113635.

## Data availability

Data will be made available on request.

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