# Increased anti-psoriatic effect of anti-inflammatory dendrimers using fluid catanionic vesicle-based topical formulations

Ranime Jebbawi, Abdelouahd Oukhrib, Emily Clement, Helene Labie, Stephanie Cassel, Séverine Fruchon, Anne-Marie Caminade, Michel Simon, Rémy Poupot, Cédric-Olivier Turrin, et al.

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## To cite this version:

Ranime Jebbawi, Abdelouahd Oukhrib, Emily Clement, Helene Labie, Stephanie Cassel, et al. In-creased anti-psoriatic effect of anti-inflammatory dendrimers using fluid catanionic vesicle-based topical formulations. Advanced Healthcare Materials, 2025, 10.1002/adhm.202503282. hal-05385228

HAL Id: hal-05385228

https://hal.science/hal-05385228v1

Submitted on 19 Feb 2026

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RESEARCH ARTICLE

OPEN ACCESS

# Increased Anti-Psoriatic Effect of Anti-Inflammatory Dendrimers Using Fluid Catanionic Vesicle-Based Topical Formulations

Ranime Jebbawi<fnref n="1" />,² | Séverine Fruchon² | Muriel Blanzat<fnref n="1" /> ID

Abdelouahd Oukhrib<fnref n="1" />,³ | Anne-Marie Caminade³ |

Emily Clement²,⁴ | Michel Simon² |

Hélène Labie<fnref n="1" /> | Rémy Poupot² |

Stéphanie Cassel<fnref n="1" /> | Cédric-Olivier Turrin³,⁴ ID

**Correspondence:** Cédric-Olivier Turrin (cedric-olivier.turrin@lcc-toulouse.fr) | Muriel Blanzat (muriel.blanzat@cnrs.fr)

**Received:** 2 July 2025 | **Revised:** 25 November 2025 | **Accepted:** 26 November 2025

**Keywords:** catanionic vesicles | dendrimer | drug delivery | formulation | psoriasis | skin

## ABSTRACT

Psoriasis is a chronic inflammatory skin disease requiring effective anti-inflammatory treatments. The **IMD-006** dendrimer exhibits promising immunomodulatory effects, but optimized formulations are needed to improve skin penetration. This study describes fluid vesicle formulations to enhance **IMD-006** delivery and evaluates their anti-psoriatic efficacy in a murine model, offering potential for innovative topical therapies. Due to **IMD-006**'s hydrolytic instability at pH 7, more stable analogues, **IMD-036** and **IMD-046**, are designed with phosphoramidate and triazine branching points, improving hydrolytic resistance. Fluorescent derivatives are also synthesized using copper-free click chemistry for biological studies. These dendrimers are actively internalized by monocytes, promoting an anti-inflammatory phenotype. Encapsulation in highly fluid **TriCat** catanionic vesicles significantly enhances cellular uptake and skin penetration, particularly for **IMD-006** and **IMD-036**, facilitating deeper tissue diffusion without systemic exposure. A xanthan-based hydrogel incorporating **TriCat** vesicles loaded with these dendrimers is developed for topical application, improving stability, sustained drug release, and skin permeability. In a psoriatic mouse model, **TriCat/IMD-006** and **TriCat/IMD-036** formulations significantly reduced disease severity (*p* < 0.0001 and *p* < 0.05 respectively), producing clinical and histopathological outcomes equivalent to corticosteroid treatment. These findings highlight the potential of dendrimer-based formulations as effective topical treatments for psoriasis, offering an alternative to conventional corticosteroids.

## 1 | Introduction

Psoriasis is an immune-mediated chronic inflammatory disease that primarily affects the skin, with a gruelling variety of symptoms that dramatically impact the quality of life of patients. It is characterized by keratinocyte hyperproliferation and impaired differentiation, as well as immune cell activation

that drives the development of skin erythematous plaques. Its prevalence is 2–3% in Western populations, with over a third of patients presenting moderate to severe psoriasis that may require systemic treatments, and 5–10% presenting related joint inflammation requiring even stronger treatments. Psoriasis is treated in a gradual manner to reduce inflammation during a flare-up [1]. First, local administration of steroidal drugs and

<footnote n="1">¹Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Toulouse, France | ²Toulouse Institute for Infectious and Inflammatory Diseases (INFINITy), Université de Toulouse, INSERM UMR1291 – CNRS UMR5051, Toulouse, France | ³Laboratoire De Chimie de Coordination (LCC), Université de Toulouse, CNRS UPR 8241, Toulouse, France | ⁴IMD-Pharma, Toulouse, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.</footnote>

<footnote>© 2025 The Author(s). *Advanced Healthcare Materials* published by Wiley-VCH GmbH</footnote>
vitamin D are commonly prescribed for mild/moderate psoriasis [2], whereas moderate/severe forms require phototherapy and/or systemic drugs, mostly immunosuppressant synthetic molecules with potentially severe side effects (methotrexate, cyclosporine) or manufactured proteins (soluble receptors, cytokine-signalling inhibitors or monoclonal antibodies (mAb)) [3]. The latter class of biological compounds (biologics) is a market of billions of euros dominated by injectable products, mostly T-cell blockers and drugs that inhibit the production/action of pro-inflammatory cytokines. Despite the fact that psoriasis is not always considered a true auto-immune disease [4], clinicopathological correlations show similarities with other auto-immune diseases, in particular the implication of pro-inflammatory T cells, monocytes and dendritic cells as well as pro-inflammatory relays between the innate and the adaptive immune systems leading to the production, *inter alia*, of pro-inflammatory cytokines like tumor necrosis factor (TNF), interleukin (IL)1β, IL22 and IL17. As a matter of fact, many systemic immunosuppressant drugs (biologic immunomodulators) used for the treatment of other chronic inflammatory diseases have recently been authorized for the treatment of psoriasis, for instance the TNF-antagonist etanercept, the anti-IL12/23 ustekinumab and the anti-TNF adalimumab. However, biologics are highly expensive [5] and they can have severe (sometimes lethal) side effects. Indeed regulatory agencies have withdrawn or restricted several of them such as efalizumab [6] (withdrawn in 2009 due to the risk of progressive multifocal leukoencephalopathy), alemtuzumab [7] (still available but with severe usage restrictions due to serious adverse effects), and natalizumab [8] (temporarily withdrawn in 2005 and reintroduced under strict monitoring due to cases of progressive multifocal leukoencephalopathy). Therefore, there is an unmet need to develop new classes of anti-inflammatory molecules for the treatment of chronic diseases.

Polyphosphorhydrazone (PPH) dendrimers with a cyclotriphosphazene core bearing poly(phosphorhydrazone) branches, and ending with aza-bisphosphonate (ABP) groups have immunomodulatory effects on human immune cells in vitro [9–12]. Among them, the **IMD-006** dendrimer inhibits the proliferation of pro-inflammatory CD4+ T lymphocytes [13] and shifts human monocytes toward an anti-inflammatory activation [14]. The **IMD-006** dendrimer has shown a strong anti-inflammatory efficacy in vivo in animal models of acute and chronic inflammatory diseases (uveitis [15], Rheumatoid Arthritis [16] and Multiple Sclerosis [17]). In addition, in a preliminary study performed in vivo, topical application of **IMD-006** in the Imiquimod (IMQ)-induced psoriatic mouse model significantly reduced the severity of skin lesions, including histopathological abnormalities [18]. A recent study performed in vitro and *ex vivo* has demonstrated the anti-proliferative and pro-differentiating effects of **IMD-006** dendrimer on human keratinocytes (KC) [19]. In 2D cultures of keratinocyte, **IMD-006** was internalized and distributed to mitochondria, inducing mitochondrial reorganization and degradation, a key step in keratinocyte differentiation. The pro-differentiating and anti-proliferative effects of **IMD-006** validate its great potential for the treatment of psoriasis. However, since the epidermal barrier in IMQ-treated mice is severely altered, potentially allowing the passage of molecules more easily than in human skin, the **IMD-006** dendrimer will necessitate structural tuning and suitable formulations to increase its skin permeation and related therapeutic efficacy.

In this context, various supra-assemblies have been explored for their ability to enhance transdermal delivery [20], including liposomes [21] or surfactants [22]. Among these approaches, formulation strategies using liposomes or vesicles appear to be most promising [23, 24]. In particular, liposomes with highly deformable or highly fluid bilayers have been specifically designed to overcome the *stratum corneum* barrier and improve drug penetration [25]. Highly deformable liposomes [26], known as transfersomes, are obtained by incorporating edge activators into their bilayer, reducing membrane elastic energy and making them up to five orders of magnitude more deformable than conventional liposomes. Alternatively, highly fluid vesicles, such as ethosomes, are obtained by adding ethanol to the liposomal bilayer, where it interacts with lipid polar heads. Upon contact with the skin, ethosomes disrupt the *stratum corneum*’s organization and temporarily alter its integrity, thereby enhancing permeability [27, 28]. To date, most of the studies with highly deformable or highly fluid bilayers are based on modified liposomes incorporating detergents or permeation enhancers. As an alternative to liposomes, catanionic vesicles [29–35], comprising oppositely charged amphiphiles, were synthesised without additives and exhibit bilayer physicochemical properties highly favorable to membrane fluidity. As we have previously described [36, 37], catanionic vesicles are made of **TriCat** glycolipids capable of spontaneously fusing with the lipid membranes of cells, thus greatly facilitating the cellular uptake of drugs [38, 39]. Their spontaneous fusion behavior is attributed to electrostatic interactions at closely apposed interfaces, driven by the local electric field generated by the charged vesicle surface. Their merging is further enhanced since the lipid assembly is already fluid, initiating fusion with the lipid bilayer of the plasma membrane [40]. It is this remarkable ability of catanionic vesicle to fuse with lipid bilayers that we have taken advantage of, in order to boost the dendrimers’ diffusion through the different skin layers.

This study aims to explore the synthesis of **IMD-006** analogues, develop suitable fluid vesicle formulations, and investigate their ability to deeply penetrate the skin. Moreover, the anti-psoriatic efficacy of these formulations is validated in a murine psoriasis model, offering insights into their potential for clinical application. Through this work, we seek to provide an innovative solution for the topical treatment of psoriasis [41–45], leveraging the combined benefits of optimized compounds and advanced drug delivery systems.

## 2 | Results and Discussion

### 2.1 | Synthesis of Dendrimers and Their NIR-Fluorescent Analogues

The **IMD-006** dendrimer was prepared as already described in the literature [9]. This compound has two sites prone to hydrolysis (Figure 1). Previous work showed that the saline form of dendrimer **IMD-006** undergoes a spontaneous hydrolysis at pH 7.2 in aqueous solutions [17] that is characterized by a progressive loss of the branches resulting from the breaking of hydrazone bonds (C=N). Quantification by ¹H NMR revealed that 20% of these bonds are hydrolysed after 24 h in solution (pH 7.2, 37°C) with a zero order kinetics [46] (see ESI S1.9.). At pH 4, another hydrolysis profile is observed and characterized by the progressive decrease
<figure><img src="image_4.png" /><figcaption>FIGURE 1 | pH-dependent degradation pathways of dendrimer IMD-006.</figcaption></figure>

of the signal corresponding to the branching point centred on 64 ppm and the appearance of a new singlet at 49 ppm (see ESI S1.12.). This chemical shift is characteristic of a HOP(S)(OAr)₂ moiety resulting from the hydrolysis of the P-N bond.

Remarkably, the absence of an aldehydic proton in the 9.8–10.0 ppm region on ¹H NMR spectra and the absence of signal in the 68–69 ppm region on ³¹P NMR spectra at pH 4 demonstrates there is no hydrolysis of the CH=N that is observed at neutral pH. Conversely, under less drastic conditions, typically at 4<PH <6 (pH of the skin external surface), the ³¹P NMR monitoring of the hydrolytic process reveals the appearance of one singlet at 49 ppm showing the breaking of the P-N bond as well as one singlet at 68.5 ppm showing the breaking of the CH=N bond. Taking into account the relative sensitivity of the PPH linkage to hydrolytic degradation, we considered the synthesis of dendrimers with a similar structure to **IMD-006**, but with a lower sensitivity to hydrolysis. In this regards the PPH linkage was replaced by alternative branching points that were reported to be more stable. Two new compounds were then prepared, namely **IMD-036** and **IMD-046**, which in place of the PPH divergent point have a phosphoramidate and a triazine group respectively (Figure 2), all other structural components, including core and terminal functions, remaining identical.

The synthesis of **IMD-036** (ESI S1.3.) was designed according to a semi-convergent strategy (Scheme 1), starting from 4-hydroxybenzaldehyde protected with a tetrahydropyranyl (THP) group. After an aminoreductive step conducted with methy-lamine, the secondary amine is reacted on a large excess of thiophosphoryl chloride with triethylamine as a base to gener-ate dendron 3, equipped with a dichlorothiophosphoramidate divergent point that resonates on ³¹P NMR spectra as a typical singlet at 63.2 ppm. This dendron is then reacted with the aminobis(methylenephosphonate) derivative [9] of tyramine 4 to yield the THP-protected dendron 5. The nucleophilic substitu-tion of the chlorine atoms induces a significant deshielding of the thiophosphoramidate group, which is found to resonate at 68.5 ppm on ³¹P NMR spectra. This finding is in contrast with what is generally observed on PPH dendrimers, for which the substitu-tion of the chlorine atoms of the dichlorothiophosphorhydrazone

by phenols is generally accompanied by a slight shielding (1 to 3 ppm) of the phosphorus atom.

The THP-protecting group is easily removed by means of pyridinium *para*-toluenesulfonate to generate the free phe-nol which is in turn reacted with hexachlorocyclotriphos-phazene in the presence of cesium carbonate to produce the dimethylphosphonate-terminated dendrimer **6**. The relative steric hindrance of the phenol function required a smooth heating of the reaction mixture at 45°C. The dendrimer **6** is finally converted into the corresponding phosphonic acid-terminated dendrimer **IMD-036A** by means of a routine procedure involving a persilylation of the phosphonates with bromotrimethylsilane followed by methanolysis. As already observed with other bis(methylenephosphonate) terminated dendrimers [47], the acidic form is scarcely soluble in water, giving poorly resolved sig-nals in NMR. It is then suitably converted into the corresponding sodium salt **IMD-036** that is in turn highly soluble in aqueous media and mixtures of water with polar solvents. This compound is unambiguously characterized by multinucleus NMR (see ESI S1.3.) in D₂O/CD₃CN mixtures that provide a suitable solvation for all structural compartments. The ³¹P NMR spectrum shows typical singlets at 6.9, 9.6 and 68.8 ppm corresponding to the phosphonate, cyclotrisciphosphazene and thiophosphoramidate groups respectively. The proton spectrum is well-defined with typical doublet (²J<sub>HP</sub> = 10.7 Hz) centred on 2.76 ppm for the NMe groups and a typical doublet (²J<sub>HP</sub> = 11.9 Hz) centred on 3.47 ppm for the methylene groups located on the aminophosphonate tweezers. The shelf stability of aqueous solutions of **IMD-036** was monitored by ¹H and ³¹P NMR at room temperature. At pH 7, no trace of hydrolysis could be detected after 8 months of storage on ¹H and ³¹P NMR spectra, indicating the excellent resistance of **IMD-036** toward hydrolytic degradation at neutral pH (see ESI S1.10.). At pH 4, the onset of the hydrolysis of the P-N bond was detected after a period of 3 months (see ESI S1), as indicated by the appearance of a typical singlet at 47 ppm accounting for the HO(S)P(OAr)₂ sub-product whereas no trace of hydrolysis could be detected after the same period of time at pH 4.5 (see ESI S1.12.).

Following a full convergent strategy, another dendron with a triazine cycle as a divergent point instead of the thiophos-
<figure><img src="image_5.png" /><figcaption>FIGURE 2 | Comparison of dendrimers IMD-006, IMD-036 and IMD-046.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>SCHEME 1 | Synthesis of dendrimer IMD-036.</figcaption></figure>

phoramidate was prepared (Scheme 2; ESI S1.4.). The triazine scaffold has been extensively studied for its biological properties such as anti-inflammatory and antimicrobial activities [48]. In addition, the nucleophilic substitution of the chlorine atoms of trichlorotriazine can be finely controlled with a large variety of nucleophilics [49], making it a suitable building block to prepare dendrimers [50]. The controlled substitution of two chlorine atoms of trichlorotriazine required the sequential addition of one equivalent of the aminobis(methylenephosphonate) derivative of tyramine [9] 4 at 0°C and the further addition of a second equivalent at 0°C to afford the key intermediate 8 which requires a purification by column chromatography

to remove traces of mono- and tri-substituted triazines. The third chlorine atoms being much less reactive, it required heating at 60°C to be substituted by the amine function the bifunctional linker 4-hydroxy-N-methylbenzylamine obtained by the reductive amination of 4-hydroxybenzaldehyde with methylamine.

The resulting unprotected dendron **9** could then be grafted on hexachlorocyclotriphosphazene in the presence of cesium carbonate at 40°C to generate the dimethylphosphonate-terminated dendrimer **10**, which was converted into the dendrimer **IMD-046** following the same procedure as for **IMD-036**.
<figure><img src="image_7.png" /><figcaption>SCHEME 2 | Synthesis of dendrimer IMD-046.</figcaption></figure>

¹³C NMR was found to be more accurate to monitor the stability of this compound. At pH 7 the dendrimer presented a good stability, evidenced by the absence of new signals on the ¹³C spectrum over a period of 45 days. At pH 4, a new signal at 115 ppm appeared after 10 days of storage at room temperature. In the absence of modification of the ³¹P NMR signature, in particular for the singlet at 8 ppm for the cyclophosphazene core, this new signal at 115 ppm on ¹³C NMR spectra was attributed to the loss of phenol moieties on the triazine cycle (see ESI S1.11).

In order to evaluate the encapsulation efficiency of the dendrimers in the TriCat vesicles and to monitor and document their biological properties like cell internalization or skin penetration, fluorescent analogues have been synthesized. As an alternative to the stochastic modification and statistic grafting of a fluorescent probe on the outer shell of dendrimers [51], and taking into account that the reactivity of the chlorine atoms of hexachlorocyclotriphrosphazene can be finely controlled [52], we have chosen a rational approach consisting of replacing one unique branch by a fluorescent probe equipped with a phenol function that can be easily grafted on the cyclophosphazene core. This approach has already proved its efficiency, as fluorescent analogues having 5 branches terminated with aminobis(methylene phosphonate) termini possess the same overall biological properties as their non-fluorescent counterparts equipped with 6 branches terminated with aminobis(methylene phosphonate) termini [9, 11, 46]. In order to develop versatile dendrimeric platforms, we have designed a synthetic approach based on the preparation of dendrimeric analogues equipped with an azadibenzocyclooctyne (ADIBO) moiety [53] that could further be reacted with fluorescent compounds having an azide function in copper-free click reactions. A phenol with an ADIBO moiety and an azido-fluorescent probe were then synthesized separately.

An NIR probe with large Stoke shift, suitable for in vivo and ex vivo imaging and avoiding autofluorescence issues, was then prepared according to a published procedure [54] that was optimized and adapted to yield NIR probes as a chlorine salt instead of the commercially available harmful hexafluorophosphate salt. Briefly, the NIR dye was prepared from lepidine and 4-diethylaminosalicylaldehyde as starting scaffolds according to a semi convergent approach (Scheme 3). The azido-modified lepidine withdrawing group was obtained in a three-step procedure from lepidine while 4-diethylaminosalicylaldehyde was alkylated

with 2-bromo-1,1-diethoxyethane to yield intermediate **13** that could undergo a Bronsted acid catalysed intramolecular cyclization to afford the 6-diethylaminobenzo[b]furan-2-carbaldehyde donating group **14**. Both counterparts **12** and **14** were then crotonized in the presence of a catalytic amount of piperidine to produce the NIR probe **15** (ESI S1.5.).

Strain-promoted azide–alkyne cycloaddition (SPAAC) click chemistry is widely used to obtain relevant products in chemical biology and medicinal chemistry [55]. Using Cu-free click chemistry has several advantages: it allows a simple work-out without any additives which simplifies the purification of the final product. In addition, the presence of phosphonate functions in the surface of dendrons requires the absence of metal-based catalysts in order to avoid metal-phosphonate complexation, which would further make purification impossible, as well as contamination by copper (I), which could influence the biological activity of final products. The route to such species (Scheme 4) is based on the controlled substitution of the chlorine atoms of hexachlorocyclotriphrosphazene by a phenol equipped with an ADIBO moiety [56] and one of the dendrons employed to generate **IMD-036** or **IMD-046**, and the subsequent click reaction with a NIR fluorescent probe equipped with an azide function (see ESI S1.6. and S1.7.). The choice of ADIBO was motivated by the fact that its well-understood chemistry is compatible with aqueous reactions. In addition, ADIBO moieties are reported to be suitable for in vivo and in vitro experiments and to react in fair rates and to accept a broad variety of bulky substrates [57]. This strategy is complementary to the fully divergent approach employed to generate **IMD-006-NIR** which was prepared according to a published procedure [58]. The ADIBO pattern was elicited for its high reactivity toward azides in copper-free click cycloadditions under mild conditions [59]. An ABIBO-phenol derivative was then readily prepared from commercially available activated alkyne ADIBO-amine (dibenzocyclooctyne-amine) and 4-hydroxybenzenepropanoic acid using standard peptide coupling conditions (Scheme 4).

The SPAAC reactions were all performed in acetonitrile at room temperature with a dendrimer-alkyne concentration set to approximately 0.01 M [60]. The progress of the reaction was monitored by ¹³C NMR which showed the total disappearance of both sp carbons of the octyne pattern in 48 h, indicating the formation of triazole. Finally, the phosphonate groups were converted to the corresponding sodium salts according to the pro-
<figure><img src="image_8.png" /><figcaption>**SCHEME 3** | NIR probe preparation.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**SCHEME 4** | Synthesis of ADIBO-phenol and dendron-based approach to generation 1 fluorescent dendrimers IMD-036-NIR and IMD-046-NIR (only one regioisomer of the triazole is represented).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**FIGURE 3** | In vitro internalization of **IMD-0X6-NIR** (X = 0; 3; 4) by primary human monocytes from 3 independent donors. Data are presented as the mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple-comparison test; \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001, ns = non-significant.</figcaption></figure>

procedure described hereabove with bromotrimethylsilane to afford dendrimers **IMD-036-NIR** and **IMD-046-NIR**. The nature of the dendrimeric scaffold was found to have a limited influence on the fluorescence properties of the dye, as all fluorescent analogues showed a typical fluorescence pattern in water that was suitable for confocal microscopy (λex in the 615–650 nm range, λem in the 790–805 nm range, ESI S1.8.) while their solubility and stability were found to be very similar to the ones of their non-fluorescent counterparts.

## 2.2 | Biological Activity of Dendrimers on Monocytes

We have previously shown that **IMD-006** is internalized by monocytes, leading to their activation toward an anti-inflammatory phenotype [14]. To evaluate cellular uptake of our new dendrimers, human primary monocytes from 3 healthy donors were incubated with increasing concentrations (2, 20, and 100 µM) of the fluorescent analogues of dendrimers **IMD-006**, **IMD-036** and **IMD-046** (IMD-006-NIR, IMD-036-NIR and IMD-046-NIR) for 30 mins at 37°C or 4°C (temperature at which active internalization processes such as endocytosis and phagocytosis are blocked). At 37°C, mean fluorescence intensity of cells rose with increasing concentrations of the dendrimers and at comparable rates for all 3 molecules (Figure 3), demonstrating that **IMD-036-NIR** and **IMD-046-NIR** are uptaken similarly to **IMD-006-NIR** by cells. Moreover, at 4°C, cellular fluorescence is greatly reduced after incubation with all 3 molecules, indicating that the uptake of dendrimers by monocytes is an active process.

After 3 days of incubation at 37°C with 0.2, 2 or 20 µM of **IMD-006**, **IMD-036** and **IMD-046**, human primary monocytes exhibited morphological changes in a dose-dependent manner, notably an increase in size and granularity (as attested by the increase in FSC and SCC observed in Figure 4A). Additionally, we have assessed the surface marker HLA-DR on monocytes and we show that these dendrimers significantly downregulate the expression of HLA-DR at 20 µM (Figure 4B). These results show that **IMD-036** and **IMD-046** are able to activate monocytes to an anti-inflammatory phenotype in a similar manner to **IMD-006**, as we have already shown [9].

## 2.3 | Preparation and Characterization of Fluid, Dendrimer-Charged, Catanionic Vesicles

Previously described catanionic vesicles [36, 37, 39] named **TriCat**, synthesized quantitatively and without counter ions by an in situ acid-base reaction between N-dodecylamino-1-deoxylactitol and α-bis(hydroxyl) dodecylphosphinic acid, were chosen to encapsulate **IMD-0X6** (X = 0; 3; 4) dendrimers. The size (mean diameter at 100–200 nm) and shape (spherical) of these vesicles, previously determined with light scattering (DLS), nanoparticle tracking analysis (NTA), small angle neutron scattering (SANS) and freeze-fracture scanning electron microscopy (cryo-SEM) [61], are quite consistent with the prerequisite for drug delivery systems. Our study on these vectors also showed that they have the remarkable property of spontaneously fusing with the lipid membranes of cells, thus greatly facilitating the cellular uptake of drugs [38, 39].

The first encapsulation attempts were performed by dispersing the catanionic surfactant in an aqueous solution of highly water soluble **IMD-0X6** dendrimer. Despite being in the correct size range, only the encapsulation efficiency (EE) is only around 25% of the dendrimer was encapsulated and the separation of the non-encapsulated drug was complicated.

The method for preparing the formulation was therefore adapted, using the non-soluble dendrimer precursor, in its protonated phosphonic acid form (IMD-0X6A, X = 0; 3; 4), which increases its insertion in the hydrophobic part of the vesicles' bilayer (Figure 5). This procedure allowed us to obtain an average encapsulation efficiency of 70% to 80% depending on the dendrimer encapsulated [62]. The non-encapsulated active dendrimer was then easily separated after membrane filtration.

The **TriCat/IMD-006** nano-aggregates were first characterized by multi-angle dynamic light scattering (MADLS). This technique was used to obtain accurate hydrodynamic radii, avoiding errors that could be obtained at a single angle while working with large or polydisperse distributions of nanoparticles. The fluctuations in scattering intensity were then analyzed using a technique called autocorrelation (Figure 6A), which shows very similar profiles for the three **TriCat/IMD-0X6** systems. The correlograms obtained
<figure><img src="image_11.png" /><figcaption>**FIGURE 4** | In vitro anti-inflammatory properties of **IMD-0X6** (X = 0; 3; 4) on primary human monocytes at 0.2, 2, or 20 µM by flow cytometry. (A) Analysis of morphological changes, size (FSC, Forward Scatter) and granularity (SSC, Side Scatter) of monocytes (ellipse) incubated for 3 days in the presence of H₂O (control), **IMD-0X6** (X = 0; 3; 4). Results from one representative donor. (B) Analysis of HLA-DR expression on the surface of monocytes. Data are represented as the mean ± SEM from three independent donors (except for the 0.2 µM dose of **IMD-006**, n = 2 donors). Statistical analyses of the HLA-DR expression were carried out by a One Way ANOVA test followed by a Tukey post-test to compare the groups with each other.
\*p<0.05, \*\*p<0.01, \*\*\*p<0.001, ns = non-significant.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**FIGURE 5** | Chemical structures of dendrimer **IMD-006A** and **TriCat** surfactant's precursors (Lhyd12 and the phosphinic acid); Schematic representation of a **TriCat/IMD-006** vesicle.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>FIGURE 6 | (A) Correlograms and (B) hydrodynamic radius intensity-averaged distribution of **TriCat/IMD-006** (blue), **TriCat/IMD-036** (orange), **TriCat/IMD-046** (green) obtained with MADLS; (C) Particles average hydrodynamic diameter distribution for **TriCat/IMD-006** obtained with NTA and (D) cryo scanning electron micrograph of **TriCat/IMD-006** (scale bar represents 200 nm).</figcaption></figure>

<table><caption>TABLE 1 | Compilation of physico-chemical data for TriCat and TriCat/IMD-0X6 vesicles.</caption><thead><tr><th></th><th>Rh (nm)<sup>a)</sup></th><th>Dh (nm)<sup>b)</sup></th><th>Rg (nm)<sup>c)</sup></th><th>Rg/Rh</th><th>Tm (°C)</th></tr></thead><tbody><tr><td>TriCat</td><td>50</td><td>127</td><td>54</td><td>1.08</td><td>36</td></tr><tr><td>TriCat/IMD-006</td><td>57</td><td>112</td><td>58</td><td>1.02</td><td>33</td></tr><tr><td>TriCat/IMD-036</td><td>53</td><td>106</td><td>55</td><td>1.04</td><td>33</td></tr><tr><td>TriCat/IMD-046</td><td>65</td><td>106</td><td>66</td><td>1.01</td><td>34</td></tr></tbody></table>

were then processed with the Multi Storms software with the NNLS method to obtain a Rh value for each angle. The average Rh value (Table 1) is then obtained after a linear regression. As observed in the size distributions for the three systems (Figure 6B), the encapsulation of the **IMD-0X6** dendrimer in **TriCat**, results in the formation of a relatively monodisperse distribution of nanocarriers, with a mean hydrodynamic radius (Rh) measured at 57 nm for the **TriCat/IMD-006** system. To complete this analysis, another technique was used to confirm the DLS data obtained. Nanoparticle Tracking Analysis (NTA) is a

convenient method for polydisperse distributions as each particle is analysed, thus giving rise to an accurate distribution of the concentration of nanoparticles for each size. With this technique, an average hydrodynamic diameter (Dh) of about 113 nm was obtained, with a major population at 81 nm (Figure 6C). Finally, the spherical shape of the vesicles and the size distribution of 100–150 nm diameter vesicles were also confirmed by freeze-fracture scanning electron microscopy (Figure 6D). All these results highlight there is no change in the behavior of the vesicles' structure after dendrimer incorporation.

<footnote>a)measured from H₂O samples at 25°C by MADLS;</footnote>

<footnote>b)measured from H₂O samples at 25°C by NTA;</footnote>

<footnote>c)measured from H₂O samples at 25°C by MASLS.</footnote>
Concerning the other dendrimer families, **IMD-036** and **IMD-046** (Table 1) and their fluorescent analogues **IMD-036-NIR** and **IMD-046-NIR**, similar results were obtained in terms of hydrodynamic diameters, shape and encapsulation efficiency (see ESI S2.1. and S2.2.). All the dendrimer-charged vesicular systems are in the 100–150 nm range. A kinetic study of the stability of **TriCat/IMD-0X6** vesicles was performed using DLS measurements. No significant variations in mean diameter size were observed, indicating a good stability of the systems for at least 1 month when stored at 4°C in solution.

The radii of gyration ($R_g$) were determined from multi angle static light scattering (MASLS) experiments (see ESI S2.2.), using the Guinier plot. For all **TriCat/IMD-0X6** systems, the Rg were measured between 55 to 66 nm, which is consistent with the data obtained with other physico-chemical techniques. Moreover, the ratio Rg/Rh was found to be in the 1.0-1.1 range. These values are in agreement with the theoretical hollow sphere shape ratio, confirming thus the presence of vesicles [63].

In order to gain more information on the membrane fluidity of catanionic vesicles and to better control their skin diffusion properties, the thermotropic behavior of a **TriCat** vesicles suspension was measured by Differential Scanning Calorimetry (DSC) [64]. The main transition peak, corresponding to the transition from the gel phase to the liquid phase of the fatty chains [61], was observed for pure catanionic **TriCat** vesicles at 36°C. This temperature, which is close to that of skin (32–35°C) [65], is the main determinant of the fluidity or deformability of the catanionic vesicles when applied to the skin. DSC is also sensitive to variations in polar head group spacing or to the organisation of the lipophilic part of the bilayer induced by the penetration of external molecules [66]. Thus, any variations in thermodynamic features of the **TriCat** bilayer transition in the presence of the **IMD-0X6** dendrimer can be related to interactions between **TriCat** and the active ingredient inserted in the bilayer. These could have a non-negligible impact on the vesicles' fluidity and thus on their diffusion through the epidermis. After encapsulation of the **IMD-006** dendrimer, the **TriCat/IMD-006** formulation has a phase transition temperature measured at 33°C (see ESI S2.3. and Table 1), which is more than 2°C below the one of pure **TriCat** catanionic vesicles. Similar results were obtained with **IMD-036** and **IMD-046** formulations in **TriCat** vesicles (see ESI S2.3. and Table 1). These results confirm the interaction between the dendrimers and the hydrophobic part of the **TriCat** vesicles' bilayer. Below 33°C, the membrane bilayer of the vesicle is rigid and stable, which is beneficial for storage of the dendrimer formulation at room temperature. Above this temperature, the vesicles' membrane becomes fluid and we expect that the bio-addressing properties of the vesicles are thus improved, facilitating their diffusion in tissues and the release of active ingredients.

## 2.4 | Promotion of Skin Penetration on Pig Ear Skin Explants

To assess whether encapsulation in catanionic vesicles enhances dendrimer skin penetration, *ex vivo* tests were performed on pig ear skin using Franz cells (Figure 7). Porcine skin is widely regarded as the closest analog to human skin in terms of both

morphology and biochemistry, with a similar thickness and lipid composition of the stratum corneum. Porcine skin exhibits trans-epidermal water loss values close to human skin, suggesting a similar barrier function. For many compounds, the permeability through porcine skin correlates strongly with human data. Therefore, porcine skin allows accurate prediction of human skin absorption while being accessible, ethically acceptable, and experimentally practical [67–69]. Topical application of the fluorescent analogues **IMD-0X6-NIR**, either in water or formulated in **TriCat** vesicles, was performed at skin temperature (35°C), which is above the transition temperature of **TriCat/IMD-0X6** as previously determined. After 24 h, the contents of receptor chambers were analysed by fluorescence spectroscopy to evaluate trans-cutaneous penetration. Moreover, to evaluate dendrimer retention and localisation in the skin, both the total fluorescence of skin samples after homogenization and the presence of fluorescent dendrimers in skin cryosections were assessed.

First of all, it could be established that after 24 h, no fluorescence was detected in the receptor chambers indicating that neither **IMD-0X6-NIR** solution, nor **TriCat12/IMD-0X6-NIR** promote the transdermal penetration of the dendrimers limiting thus the occurrence of systemic exposure.

Figure 8 shows the cumulative amounts of **IMD-0X6-NIR** retained in the skin after 24 h of treatment with pure **IMD-0X6-NIR** or with **TriCat/IMD-0X6-NIR**. For all three dendrimers, when the pig ear skin is treated with pure dendrimer water solutions, the skin retention amount was around 2 to 3 nmol (9 ± 1 µg of **IMD-006-NIR**, 13 ± 2 µg of **IMD-036-NIR** and 8 ± 1 µg of **IMD-046-NIR**). However, when the skin is treated with the **TriCat/IMD-0X6-NIR** formulations, the amount of dendrimer retained in the skin is significantly increased to, 30 ± 2 µg and 20 ± 2 µg for **IMD-006-NIR** and **IMD-036-NIR** respectively, which is almost twice, or even three times in the case of **IMD-006-NIR**, the quantity measured without vesicles. This clearly shows the effectiveness of catanionic vesicles in improving the retention of dendrimers in the skin. However, it is noteworthy that in the case of dendrimer **IMD-046-NIR**, both the pure dendrimer solution and the vesicular solution show lower quantities of dendrimer diffused in the skin and a statistically non-significant difference between both.

<figure><img src="image_14.png" /><figcaption>**FIGURE 7** | Schematic representation of the cutaneous penetration studies using Franz cells.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>FIGURE 8 | Cumulative amounts of (A) IMD-006-NIR, (B) IMD-036-NIR and (C) IMD-046-NIR retained in the skin, determined by in vitro dermal penetration studies on pig ear skin using Franz cells after 24 h, from water solutions of pure IMD-0X6 or TriCat/IMD-0X6. Statistical analyses were carried out by using a Mann-Whitney test. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.</figcaption></figure>

After showing that the vesicular formulations significantly increase the skin retention of both **IMD-006** and **IMD-036** dendrimers, we aimed to qualitatively assess the depth of skin penetration of the dendrimer by confocal microscopy on cryosections of skin samples. As previously, we treated pig ear skin samples with pure **IMD-0X6-NIR** or **TriCat/IMD-0X6-NIR**. Then, after 24 h of treatment, samples were disassembled from Franz cells, and cryo-sections of 10 µm were prepared for visualization with a confocal microscope.

Figure 9 shows that pig ear skins, treated with pure **IMD-006-NIR** dendrimer solutions, exhibit red fluorescence distributed only within the uppermost layer of the skin, the stratum corneum. However, when **IMD-006-NIR** dendrimer is formulated in **TriCat** vesicles, a broader distribution of red fluorescence is observed in the stratum corneum and also deeper into the viable epidermis. Furthermore, the red fluorescence of the dendrimer also appears in some areas of the dermis. **IMD-006-NIR** dendrimer deeply penetrates into the skin only when it is formulated in **TriCat** vesicles, validating thus the benefit of using **TriCat** vesicles for deep dermal delivery. In the case of dendrimer **IMD-036-NIR** (see ESI S3.1.), the same behavior was observed with a clear and significant increase of the fluorescence in the viable epidermis when delivered with **TriCat** vesicles. However, in the case of the dendrimer **IMD-046-NIR** (see ESI S3.1.), both the skin samples treated with the non-formulated and the formulated dendrimer exhibit a faint red fluorescence in the epidermis. This observation suggests that **IMD-046** dendrimer might be less performant than the two other dendrimers for topical administration. As previously observed in the literature with transfersomes or ethosomes [25], the deformability and/or the fluidity of vesicular vectors can strongly improve dermal or transdermal drug delivery. In the present case, the fluidity of the bilayer does not require the addition of detergent edge activators or irritant permeation enhancers like ethanol. In fact, the inherent structure of the catanionic vesicles provides fluidity to the vesicles' bilayer at physiological temperature.

## 2.5 | Incorporation of TriCat/IMD-0X6 Vesicles in a 1% Xanthan Formulation

The challenge was to incorporate the highly fluid vesicles charged with the dendrimers in pharmaceutical formulations adapted to topical cutaneous administration. As the vesicles are dispersed in water, the use of a hydrogel, a hydrophilic galenic form, is thus preferred. At first, a screening of different hydrogels commonly used in dermatology or cosmetology [70, 71], such as chitosan, hydroxyethyl cellulose or xanthan gum, allowed us to identify xanthan gum as the most appropriate hydrogel in terms of ease to proceed, stability and drug release profile. To incorporate the vesicles in the gel, a post-loading protocol was chosen. A twice-concentrated solution of **TriCat/IMD-0X6** was prepared with the classical protocol described above and was then added to xanthan 2% w/v water solution. The presence of the vesicles within the xanthan hydrogel was confirmed using freeze-fracture scanning electron microscopy, where one can observe spherical vesicles of about 100 nm incorporated in the polymeric networks for **TriCat/IMD-046/xanthan** formulation (Figure 10) as well as for **TriCat/IMD-006/xanthan** and **TriCat/IMD-036/xanthan** formulations (see ESI S2.4.). The stability of the formulations was then evaluated using Turbiscan. This technique consists of measuring the transmitted light on the whole height of a sample over time, as a modification of the transmitted light during the time of experiment can traduce a flocculation or a sedimentation process. In presence of the **TriCat/IMD-006/xanthan** formulation, a very low and non-significant variation of ΔTransmission (less than 5%) was observed over one month (see ESI S2.5.), indicating a homogeneous repartition of **TriCat/IMD-006** aggregates throughout the gel and confirming thus a good stability of the vesicles in the xanthan gel formulation when stored at room temperature.

In vitro release studies were then performed to determine the **IMD-0X6-NIR** release profile from **TriCat12/IMD-0X6-NIR/Xanthan** formulations. A 0.45 µm nylon membrane was
<figure><img src="image_16.png" /><figcaption>**FIGURE 9** | Observation of ex vivo skin penetration depth on porcine skin. Confocal fluorescence microscopy images (left) and bright field (BF, middle) images were superimposed to observe localisation of **IMD-006-NIR** (red) in 10 µm thick porcine skin sections obtained after diffusion of (A) PBS (control), (B) pure **IMD-006-NIR** and (C) **TriCat/IMD-006-NIR** formulation for 24 h at 35°C. Scale bar represents 50 µm.</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>**FIGURE 10** | Cryo-scanning electron micrograph obtained after freeze fracture of **TriCat/IMD-046/Xanthan**. Scale bar represents 300 nm.</figcaption></figure>

used to retain the xanthan gel, while the dendrimer and the vesicles can cross the membrane. The receptor media was regularly sampled and replaced in order to quantify the **IMD-0X6-NIR** released by spectrofluorimetry. As an example, the cumulative percentage (Figure 11) of **IMD-036-NIR** release from the **IMD-036-NIR** solution (blue curve), the **TriCat/IMD-036-NIR** (red curve) and the **TriCat/IMD-036-NIR/xanthan** (grey curve) through the Nylon membrane was plotted over a period of 96 h. In this experiment, the solutions of pure **IMD-036-NIR** and **TriCat/IMD-036-NIR** exhibit a very fast release profile with respectively 76% and 74% of **IMD-036-NIR** quantified in the receptor media after the first 8 h, while only 37% of the dendrimer was released from the **TriCat/IMD-036-NIR/xanthan** formulation. However, 74% of **IMD-036-NIR** was released from the **TriCat/IMD-036-NIR/xanthan** formulation after 96 h. Similar results were obtained with the 2 other dendrimers (see ESI S2.6.). These results confirm the slow, sustained but complete release of the dendrimer when formulated in the vesicle/gel formulation.

Finally, in vitro tests were performed on pig ear skin using Franz cells (Figure 12) to evaluate the influence of **TriCat/IMD-0X6** vesicle incorporation in xanthan gel on their skin diffusion properties. When **TriCat/IMD-006-NIR/Xanthan** formulation was applied on pig ear skins, red fluorescence was observed in the stratum corneum as well as in the deep viable epidermis and in some areas of the dermis (Figure 12), as previously observed with the TriCat/IMD-006-NIR vesicular solution (Figure 9). Equivalent deep skin penetration was also obtained with **TriCat/IMD-036-NIR/Xanthan** and **TriCat/IMD-046-NIR/Xanthan** formulations (see ESI S3.1.). These experiments confirm that incorporating the vesicles into the gel does not hinder the enhanced skin diffusion provided by the **TriCat**
<figure><img src="image_18.png" /><figcaption>**FIGURE 11** | Cumulative percentage release of IMD-036-NIR from IMD-036-NIR solution (blue curve), TriCat/IMD-036-NIR (red curve) and TriCat/IMD-036-NIR/Xanthan (grey curve) through a Nylon membrane to a receptor medium of H₂O at 37°C. Error bars represent SD.</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**FIGURE 12** | Observation of ex vivo skin penetration depth on porcine skin. Confocal fluorescence microscopy images, IMD-006-NIR, bright field (BF) and merge images of skin sections 10 µm thick obtained after 24 h diffusion at 35°C of TriCat/IMD-006-NIR/Xanthan formulation in pig ear skin. Nuclei are stained in blue with 4',6-diamidino-2-phenylindole. Scale bar represents 50 µm.</figcaption></figure>

catanionic vesicles, despite the sustained drug release previously observed with the gel. This establishes that the combined vesicle-in-gel formulation is suitable for topical administration of **IMD-0X6**.

## 2.6 | Anti-Psoriatic Effect on Psoriatic Mouse Model Induced by Imiqussimod

The anti-psoriatic effect of the formulations was evaluated in an IMQ-induced psoriatic mouse model, which was previously used to assess the **IMD-006** dendrimer efficacy [18]. Mice were treated by applying water (vehicle), pure dendrimer solutions (at 13 mg/kg/day or 50 mg/kg/day, an effective reference dose whose therapeutic effect was already observed in a previous study [18]), **TriCat/IMD-0X6** or **TriCat/IMD-006-NIR/Xanthan** formulations (at 13 mg/kg/day of dendrimer) to the shaved back skin of mice. Dermoval, a potent corticosteroid-based cream commonly prescribed to topically treat psoriasis, was used as a benchmark. Mice treated daily with xanthan and untreated mice that did

not receive cutaneous application of IMQ served as healthy controls. Clinical assessment of disease severity was based on daily evaluations of erythema, scaling and thickening of the skin. The sum of the score for each criterion was used to calculate an overall clinical score for the severity of the disease (Figure 13A). Skin biopsies were collected on D7 for histological analysis of the skin after hematoxylin/eosin (HE) staining. The evaluation of acanthosis, spongiosis, hyperkeratosis, parakeratosis, immune infiltrates and Munroe's abscesses allowed us to determine a total histological score (Figure 13B).

All mice treated with IMQ alone present skin inflammation which gradually evolves from D1 to reach a maximum on D6-D7 (total clinical score of 9.6 ± 0.3) (Figure 13A). As expected, topical application of **IMD-006** in water at a dose of 50 mg/kg reduced skin inflammation induced by IMQ from D5 until reaching a final clinical score of 6.3 ± 0.5 at D7, proving a significant therapeutic efficacy in comparison to the non-treated group (p < 0.001). The histological score at D7 also decreased from of 16.4 ± 0.3 (non-treated group) to 11.1 ± 0.5 (p < 0.0001) with
<figure><img src="image_20.png" /><figcaption>**FIGURE 13** | Clinical and histological scores for the **IMD-006** formulations. (A) Evaluation of the total clinical score (erythema + scales + thickness) left over 7 days, represented on a scale from 0 to 12, and right total clinical score at D7. Data are presented as the mean ± SEM (B) Evaluation of the total histological score at D7 on a scale from 0 to 20 (sum of scores of all histological parameters). Error bars represent SEM. Statistical analyses of the total clinical and histological scores on D7 were carried out using a Kruskal-Wallis test followed by a Dunn's Tukey post-test to compare the groups with each other. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001. n = 33 for IMQ, n = 23 for IMQ + **IMD-006** (50 mg/kg), n = 15 for IMQ + **IMD-006** (13 mg/kg), n = 10 for IMQ + **TriCat/IMD-006** (13 mg/kg), n = 5 for IMQ + Dermoval.</figcaption></figure>

IMD-006 treatment. When applied at a lower dose of 13 mg/kg, the **IMD-006** dendrimer demonstrated an earlier effect, around D4-D5, with a final clinical score of 6.5 ± 0.5 at D7, showing approximately the same clinical efficacy as the dose of 50 mg/kg in comparison to the non-treated group. The histological score at D7 decreased down to 14.5 ± 0.7 with the dose at 13 mg/kg, this dose being significantly less efficient to decrease the histological score in comparison with the 50 mg/kg dose (*p* < 0.01). Taken together, these data show that both doses of **IMD-006**, 13 and 50 mg/kg, are effective. At 13 mg/kg, the therapeutic efficacy was significantly improved with the **TriCat/IMD-006** solution. Indeed, the severity of lesions significantly decreased and an improvement of the clinical score from D6 was observed with a final clinical score of 5.7 ± 0.7 on D7, lower than that observed in the non-treated group (*p* < 0.0001). The histological score also traduced a significant improvement of the efficacy, with a score of 10.4 ± 0.8 at D7 at 13 mg/kg dose of **TriCat/IMD-006**, significantly lower than the score of 16.4 ± 0.3 observed at D7 for the non-treated group (*p* < 0.0001), and more interestingly, significantly lower than the score of 14.5 ± 0.7 observed at D7 for the group treated with **IMD-006** alone (*p* < 0.001).

When mice were treated with the **TriCat/IMD-006/Xanthan** formulation, a delay in the therapeutic response was observed as shown by the total clinical score at D4 higher than that of the other groups. This observation was in accordance with the fact that drug-release kinetics is significantly lowered in the gel formulation in comparison with vesicular formulation (Figure 11).

After D5, the total clinical score decreased more rapidly than in other groups, reaching a clinical score of 5.4 ± 0.5 (*p* < 0.001 in comparison with non-treated group) on D7. The histological score of the group treated with the **TriCat/IMD-006/Xanthan** formulation at 13 mg/kg on D7 was 11.3 ± 0.5, significantly lower than the non-treated group (16.4 ± 0.3, *p* < 0.0001) and the group treated with **IMD-006** alone (14 ± 0.7, *p* < 0.05).

Taken together these data indicate that the preformulation of **IMD-006** in **TriCat** vesicles does increase the therapeutic efficacy, **TriCat/IMD-006** at 13 mg/kg being for instance as efficient as **IMD-006** alone at 50 mg/kg. This is likely due to enhanced skin penetration of the dendrimer, as demonstrated on porcine skin. Conversely, the formulation in the Xanthan gel did not affect the overall therapeutic efficacy at D7 nor the skin penetration of the dendrimer: both the **TriCat/IMD-006** and **TriCat/IMD-006/Xanthan** formulations exhibited comparable efficacy to Dermoval as exemplified by the images of animals shown in Figure 14, and by the clinical and histological scores of treated groups (Figure 13).

Mice treated with **IMD-036** at 50 mg/kg showed a clinical score of 10.9 ± 0.3 and a histological score of 17.5 ± 0.4 (see ESI S3.2.). These scores are slightly higher, but non-statistically different from, the clinical score (9.6 ± 0.4) and histological (15.6 ± 0.5) scores observed in the non-treated group. At 13 mg/kg both clinical and histological scores were decreased to values of 6.8 ± 0.6 and 13.0 ± 0.8 respectively but no statistically significant
<figure><img src="image_21.png" /><figcaption>**FIGURE 14** | Photos of the back skin of representative mice from the study presented in Figure 13. Animals were treated daily with xanthan + water (healthy group), IMQ + water, IMQ + **IMD-006** at 50 mg/kg, IMQ + **IMD-006** at 13 mg/kg, IMQ + **TriCat/IMD-006** at 13 mg/kg, IMQ + **TriCat/IMD-006/Xanthan** at 13 mg/kg and Dermoval 0.05% at D7. n = 33 for IMQ, n = 23 for IMQ + **IMD-006** (50 mg/kg), n = 15 for IMQ + **IMD-006** (13 mg/kg), n = 15 for IMQ + **TriCat/IMD-006** (13 mg/kg), n = 10 for IMQ + **TriCat/IMD-006/Xanthan** (13 mg/kg) and n = 5 for IMQ + Dermoval.</figcaption></figure>

difference was found when compared to untreated animals. Only the **TriCat** formulation at 13 mg/kg of **IMD-036** induced significant decreases in the clinical score (6.3 ± 0.4, *p* < 0.05) and in the histological score (11.9 ± 0.7, *p* < 0.05) in comparison with the non-treated group. The **TriCat/IMD-036** formulation at 13 mg/kg in xanthan failed to maintain statistically meaningful therapeutic efficacy, with clinical (7.1 ± 0.7) and histological (13.5 ± 0.7) scores comparable to those of non-formulated **IMD-036** at 13 mg/kg. As is the case for **IMD-006**, a slower drug release kinetics from the xanthan gel and a concomitant slower skin diffusion may be responsible for the absence of therapeutic enhancement of **IMD-036** in the gel, taking into account its lower intrinsic activity in comparison with **IMD-006**. The surprising absence of efficacy at 50 mg/kg with slightly higher clinical and histological scores than in the non-treated group was putatively attributed to a possible cytotoxic or irritant effect of **IMD-036** at this concentration. Indeed, the odd clinical and histological scores of the 50 mg/kg group are statistically different from all other treated groups (see ESI S3.2.).

Taking into account the strong structural similarities between **IMD-006** and **IMD-036**, the potential irritant or cytotoxic profile of the latter was difficult to explain. Indeed, both compounds are not expected to have significantly different bioactive profiles. This puzzling observation illustrates a paradox which is often observed

in medicinal chemistry applied to small molecules, when tiny structural modification can induce significant modifications in the therapeutic and/or toxicologic profiles. This paradox is somehow understandable in target-driven strategies, when the design of a drug candidate is driven by the strength of drug-target interactions. In the more adventurous field of phenotype-driven drug discovery, as it is the case for this series of dendrimers for which the biological target(s) have not been formally identified yet, it is much harder to explain or understand such paradox, precisely when the analogues were conceived to show comparable biological properties. This mild cytotoxicity observed at higher concentrations may relate to its increased stability and cellular retention, which could enhance off-target interactions. However, this increased stability may also confer stronger and more sustained biological activity, suggesting that lower doses or less frequent administration may achieve comparable therapeutic effects, potentially minimizing adverse outcomes. Despite its puzzling aspect, this observation should be kept in mind during all the next steps of preclinical development, as it may later give insights to identify possible lead-optimization options or risk-mitigation strategies.

When mice were treated with **IMD-046**, no significant therapeutic effect of the dendrimer could be observed whether pure or formulated in **TriCat** vesicles, with histological scores of
14.5 ± 0.9 and 14.7 ± 0.5, respectively, compared to 15.9 ± 0.7 in the untreated group (see ESI S3.2). Although conceived as a close analogue of **IMD-006**, sharing the same core and the same outer-shell, **IMD-046** has a triazine divergent point that is significantly different from the thiophosphonate divergent points of both dendrimers **IMD-006** and **IMD-036**. Taking into account that i) the nature of the divergent point strongly affects the spatial distribution of the terminal amino-bismethylene tweezers, ii) the latter that should be viewed as “first-in-line” entities when considering interaction with biological entities, and taking into account the dramatic influence of the spatial distribution of such amino-bismethylene tweezer previously observed for a series of amino-bismethylene-terminated dendrimers both on the in vitro immuno-modulating properties [12] and on the in vivo therapeutic activities in a mouse model of rheumatoid arthritis [11], we can hypothesize that the spatial arrangement of the phosphonate moieties probably also affect these molecules’ anti-psoriatic therapeutic efficacy.

### 3 | Conclusions

Psoriasis remains a challenging inflammatory disorder with significant impacts on patients' quality of life. Although current treatments, particularly biologic drugs, have shown effectiveness in managing the disease, they come with high costs and potential severe side effects. This highlights the need for new therapeutic strategies that offer better safety profiles and more targeted actions. The **IMD-006** dendrimer represents a promising alternative for topical treatment, showing strong anti-inflammatory effects and the ability to modulate immune responses. Its potential to influence keratinocyte differentiation could help address the excessive skin proliferation characteristic of psoriasis, offering a novel approach to managing the disease at the epithelial cell level.

The first challenge was to design analogues of **IMD-006** to overcome its long-term hydrolytic instability in aqueous environments. Modifications to the branching points in **IMD-036** and **IMD-046** improved the hydrolytic stability of the dendrimers, especially at superficial skin pH.

Then, to optimize the pharmacokinetic profile of these dendrimers through topical treatments, highly fluid catanionic vesicles were produced to enhance their skin penetration. **TriCat** catanionic vesicles, with an average size of 100 nm, demonstrated a high loading capacity for **IMD-0X6** (X = 0; 3; 4) and thermoresponsive bilayer properties that allow control over membrane fluidity when applied to the skin. Further, to provide a suitable medium for dermal administration, the **TriCat**/**IMD-0X6** vesicles were incorporated into a xanthan hydrogel. This approach resulted in high colloidal stability and a more sustained drug release. The formulations have shown great potential in enhancing the permeation of the **IMD-0X6** dendrimer family. By improving the penetration and delivery of the dendrimers to the deeper layers of the skin, these formulations can increase the bioavailability of the drug, ensuring that it reaches the target areas in a more controlled and efficient manner.

We finally validated the promising anti-psoriatic effects of **IMD-006** and **IMD-036** dendrimer formulations in an IMQ-induced

mouse model of psoriasis. The clinical and histological analyses confirmed that the **TriCat**-based formulations enhanced the dendrimer's therapeutic effect by reducing markers of skin inflammation, compared to the unformulated dendrimers. The results highlight the potential of these catanionic formulations as effective treatments for psoriasis at lower doses, providing strong therapeutic benefits.

In summary, **IMD-006** dendrimer and its more stable analogues **IMD-036** dendrimer, and in a lesser extend **IMD-046** dendrimer, particularly when formulated with fluid **TriCat** catanionic vesicles, demonstrate significant potential as effective alternatives for psoriasis treatment. By enhancing skin penetration and improving drug delivery, these formulations show promising anti-psoriatic effects at lower doses. This could represent a significant step forward in psoriasis therapy, offering patients a treatment option with fewer side effects compared to conventional biologics or corticosteroids. Continued research into their long-term effects, scalability, dose range optimization and ability to address additional therapeutic areas will be crucial in establishing these dendrimer-based treatments as a valuable tool in dermatology.

### 4 | Materials and Methods

#### 4.1 | Syntheses of IMD-0X6 and IMD-0X6-NIR Dendrimers

All the syntheses and characterizations of **IMD-0X6** and **IMD-0X6-NIR** dendrimers are described in the supplementary information document.

#### 4.2 | Isolation of Primary Human Monocytes

Peripheral blood mononucleated cells (PBMCs) are obtained from normal blood donors of the Etablissement Français du Sang (EFS), Toulouse, France. Informed written consent was obtained from all blood donors, and the study protocol was approved by the INSERM-EFS ethical committee. CD14+ monocytes are purified as previously described [10]. All experiments using primary human monocytes were performed with cells from 3 independent donors.

#### 4.3 | In Vitro Internalization Assays in Primary Human Monocytes

Monocytes are seeded at 1 million cells/mL, then cultured at 37°C, 5% CO₂, 100% humidity in RPMI-1640-Glutamax (Gibco; Paisley, UK) containing 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/mL; Gibco), streptomycin (100 µg/mL; Gibco). 20 min after seeding, cells are either maintained at 37°C or placed at 4°C, then **IMD-0X6-NIR** was added at the indicated concentrations for 30 min. After treatment, the cells are washed with PBS then transferred to a cytometry tube in cold PBS and kept on ice until cytometry analysis (BD LSR Fortessa Flow Cytometer; BD Biosciences). Cytometer and sample analysis parameters are set using DIVA software. Forward and side scatter profiles are used to select the population of living cells and exclude cellular debris.
Singlets were then identified to eliminate doublets, and for each sample, the NIR fluorescence is analyzed (APC-Cy7 channel) on at least 10000 events within this gate.

## 4.4 | In Vitro Anti-Inflammatory Properties on Primary Human Monocytes

Monocytes are cultured as described above. After 3 days of incubation with **IMD-0X6** at the indicated concentrations, cells are washed with PBS supplemented with 2% of FBS and labeled with an anti-HLA-DR-VioBlue antibody (clone REA805, Miltenyi Biotec), along with its corresponding isotype control (clone REA293, Miltenyi Biotec). After treatment, the cells are washed with PBS then transferred to a cytometry tube in cold PBS and kept on ice until cytometry analysis (BD LSR Fortessa Flow Cytometer; BD Biosciences). Cytometer and sample analysis parameters are set using DIVA software. Forward and side scatter profiles are used to select the population of living cells and exclude cellular debris. Singlets were then identified to eliminate doublets, and for each sample, the morphological and phenotypic changes of the monocytes are then evaluated on at least 10000 events within this gate. All flow cytometry data are analyzed using Flowlogic software (Miltenyi Biotec).

## 4.5 | Preparation of TriCat/IMD-0X6 and TriCat/IMD-0X6-NIR Vesicles

The **TriCat/IMD-0X6** vesicles are obtained by mixing the cation-ionic surfactant **TriCat** and the acidic dendrimeric precursor **IMD-0X6A** in water. 5.29 mg of **IMD-006A** dendrimer (5.21 mg for **IMD-036A** dendrimer, 5.30 mg for **IMD-046A** dendrimer and 5.31 mg for **IMD-0X6A-NIR** dendrimer), 1.02 mg of N-dodecylamino-1-deoxylactitol (L-Hyd 12) and 0.87 mg of bis-α-(hydroxydodecyl)phosphinic acid are weighted and placed in 2 mL of ultrapure water at room temperature. The mixture is vortexed for 5 min then sonicated using an ultrasonic probe for 15 min at power 3 with 30% active cycles whilst maintaining the temperature at 45°C with a water bath.

Prepared vesicles were stored at -20°C. Stability was evaluated at both 4°C and -20°C, demonstrating consistent vesicle size for up to 2 months. Vesicles were thawed immediately before each experiment to ensure reproducibility.

## 4.6 | Determination of the Encapsulation Efficiency

The encapsulation efficiency of **IMD-0X6-NIR** dendrimer in **TriCat** vesicles was determined by a spectrofluorimetric assay. A calibration curve was first established by preparing standard solutions of IMD-0X6-NIR in methanol at concentrations ranging from $2.5 \times 10^{-6}$ to $1.5 \times 10^{-5}$ mol/L. Fluorescence intensity was measured using a spectrofluorometer ($\lambda$ex = 632 nm), and the calibration curve was constructed by plotting fluorescence intensity versus concentration. The method exhibited excellent linearity within the tested range with a correlation coefficient $R^2 = 0.98$, confirming a direct proportionality between fluorescence intensity and concentration.

The total amount of **IMD-0X6-NIR** was determined by measuring the fluorescence intensity (IF) of a sample at 800 nm after breaking the vesicular structures by adding methanol to the solution (1 mL of methanol per 20 µL of vesicular solution). The unencapsulated **IMD-0X6-NIR** was then separated from the vesicles by filtration at 1.2 µm (Minisart Cellulose Acetate filter, Sartorius). After dilution of the filtrate in methanol (1 mL methanol per 20 µL of filtrate), the amount of encapsulated **IMD-0X6-NIR** was quantified by measuring fluorescence and calculating the difference between total amount of **IMD-0X6-NIR** and unencapsulated **IMD-0X6-NIR**. The encapsulation efficiency is thus determined with the Equation (1):

$$EE (\%) = \frac{IF (IMD - 0 \times 6 - NIR \text{ encapsulated})}{IF (IMD - 0 \times 6 - NIR \text{ total})} \times 100 \quad (1)$$

## 4.7 | Formulation of TriCat/IMD-0X6 Vesicles in Xanthan 1%

A solution of 2 mL of **TriCat/IMD-0X6** vesicles is prepared as described above. The solution is then freeze-dried and resuspended in 1 mL mQ water. Meanwhile, a 2% xanthan gel is prepared by adding 20 mg of xanthan to 1 mL of mQ water whilst stirring at 500 rpm. The mixture is stirred for at least 1 h. Then, both solutions are mixed (1 mL of **Tricat/IMD-0X6** vesicles and 1 mL 2% Xanthan gel) and left under agitation at 500 rpm for at least 4 h.

## 4.8 | Determination of the IMD-0X6-NIR Release Profile from Xanthan Gel

300 µL of the **TriCat/IMD-0X6/xanthan** formulation is placed in a vial covered with a 0.45 µm Nylon membrane (GVS). Then the inverted vial was immersed in 1 mL of ultrapure water to a pH 4. The receptor media was regularly sampled and replaced by a new one in order to quantify the amount of **IMD-0X6-NIR** released by spectrofluorimetry. Before the fluorimetric assay, the receptor media was adjusted to pH 7 (by dilution in PBS).

## 4.9 | Assessment of the Stability of Formulations with Turbiscan

The stability of **TriCat/IMD-0X6/xanthan** formulations was measured using a Turbiscan Lab Expert (Formulaction). The Turbiscan is composed of a pulsed near-infrared light source ($\lambda$ = 880 nm) and two synchronous detectors for transmitted (0° from light source) and back scattered light (135° from light source). With a vertical scan of the samples, the Turbiscan is able to track destabilisation phenomena and thus detect the variations of the intensity of the transmitted light at different heights of the sample as a function of time. 5 mL of the samples were placed in cylindrical glass cells. Measurements are performed according to the protocol of 145 scans/1 h, then 145 scans/24 h at 25°C and the transmitted light was analysed. The variations of the transmitted light ($\Delta$T) were plotted as a function of time. All data treatments were performed on Turbiscan Lab software.
## 4.10 | Multi-Angle Light Scatstering

DLS analyses were carried out at 25°C on a 3D LS Spectrometer (LS Instruments AG, Switzerland) equipped with a Cobolt laser ($\lambda$ = 660 nm, 100 mW). The samples are filtered at 0.2 µm (PTFE). The measurements were carried out in 2D configuration with a 100 mW laser, scanning scattering angles $\theta$ between 15° and 150°, corresponding to the scattering wave vectors $q = [4\pi \cdot n \cdot \sin(\theta/2)]/\lambda$ ranging from $2 \times 10^{-4} \AA^{-1}$ to $2 \times 10^{-3} \AA^{-1}$, with n = 1.330 (refractive index of water). 3 measurements of 90 to 300 s were recorded for each angle (38 angles script). Fluctuations in scattering intensity are then analyzed using a technique called autocorrelation. The deconvolution of the measured intensity autocorrelation function of the sample was realized with a custom-made program named STORMS [72] with the non-negative least squares (NNLS) method to obtain the distribution of diffusion coefficients (D). This program was designed with Matlab, and makes it more accurate to fit DLS correlograms. The apparent equivalent hydrodynamic radius ($R_h$) was then obtained using the Stokes-Einstein equation for each angle. The mean Rh value is then obtained after a linear regression. Hydrodynamic radii values were obtained from three different runs.

SLS measurements were carried out in 2D configuration with a 100 mW laser. Measurements were performed at 25°C for scattering angles ranging from 12° to 150°, 10 acquisitions of 5 s for each angle. The radii of gyration ($R_g$) were determined using the Guinier approximation $I(q) = I(0) \exp(-q^2 R_g^2/3)$ and performing a linear fit to the plot $\ln(I)$ versus $q^2$, called the Guinier plot.

## 4.11 | Nanoparticle Tracking Analysis

Individual particle tracking analysis technology combines the light scattering properties and Brownian motion to determine the size distribution and concentration of particles in solution. The device used is the NanoSight NS300 (Malvern Instruments, Ltd, UK). It is equipped with an sCMOS camera. The camera level was fixed at 13 with a detection threshold at 5. NTA analyses were performed at 25°C on samples diluted 100 times with mQ water filtered at 0.2 µm. The samples were then injected with a syringe pump at a constant flow rate. Measurements of each sample were performed in triplicate, each for 60 s. The concentration of particles is presented as a function of the size of the particles.

## 4.12 | Cryo-SEM

Vesicles were observed through Scanning electron microscopy with cryofracture (cryo-SEM) using an ESEM Quanta FEG250 (FEI), operating at 5 kV and about $10^{-4}$ Pa. The samples were placed on the sample holder then frozen in pasty nitrogen (–210°C). They were fractured in a vacuum chamber at –140°C, then sublimed at –95°C for 20 min. Finally, the samples undergo a platinum coating using an Ion-sputter JFC-1100 (JEOL) for 60 s. The samples thus prepared were transferred to the observation SEM chamber kept at –140°C under a flow of nitrogen gas.

## 4.13 | Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was used to measure the phase transition temperature of bilayers. The measurement consists in studying the transition signal during heating to the maximal temperature to determine the thermal transition temperature (melting temperature, $T_m$). The measurements were carried out on 75 µL of sample in sealed crucibles at medium pressure. A medium pressure crucible filled with the mQ water was used as a reference. The DSC samples were prepared at a concentration of $1 \times 10^{-2}$ M in 200 µL of water. Sonication is done externally in a water bath for 30 min at power 4 and 40% active cycles. The measurements were carried out by the DSC device Mettler Toledo equipped with a MultiSTAR FRS5 sensor. The script developed for this technique includes 4 temperature cycles between 5°C and 60°C with a speed of 2°C/min. Isothermic steps of 10 min at 10°C were also programmed between heating/cooling cycles. Thermograms and calorimetric data were elaborated and calculated by Perkin Elmer "Pyris Series" software.

## 4.14 | In Vitro Skin Permseation Studies

Freshly cut pig ears were provided by the local slaughterhouse of Montauban, France. Their skin was excised with a scalpel and cut into patches of 3.5 × 3.5 cm, which were kept frozen at –80°C until the diffusion experiment. After removing the hairs with scissors, all the subcutaneous fat was cut out. As skin thickness can influence diffusion, particular attention was paid to maintaining a consistent thickness across all samples. Pig ear skin patches were mounted on Franz diffusion cells (PermeGear, Hellertown, PA, USA), with an active diffusion area of 1.77 cm². At least six cells were used for each condition to get acceptable statistics. The lower receptor chamber was filled with 12 mL phosphate buffered saline (PBS pH 7.4), thermostated at 37°C with a water jacket to maintain the skin temperature at $35 \pm 1°C$ (measured with an infrared thermometer). The receptor is kept under constant agitation at 300 rpm. The outer surface of the cell is covered with parafilm to prevent evaporation and volume variations. The entire assembly is placed under a polystyrene cage to maintain the constant temperature at the surface of the skin at $35 \pm 1°C$ (measured with an infrared thermometer). The skin separated the receptor from the donor chamber, in which the formulations $5 \times 10^{-4}$ M of **IMD-0X6-NIR** (790 µg **IMD-006-NIR**; 780 µg **IMD-036-NIR**; 780 µg **IMD-046-NIR**) in 300 µL water or in 300 µL of a $1 \times 10^{-3}$ M **TriCat** solution, were placed. After 24 h, the receptor samples were analysed by spectrofluorimetry at 632 nm and measured at 800 nm, to quantify **IMD-0X6-NIR** in the receptor cell. Then, the skin was gently washed with PBS then dried with a paper towel. The treated zone was cut in small pieces, put in a vial with 3 mL PBS and homogenised with an Ultra-Turrax T25 Basic (Ika) for 5 min at 21500 rpm. The dispersions were thus filtered at 0.45 µm (Minisart, Sartorius) and all the samples were analysed by spectrofluorimetry at 632 nm and measured at 800 nm in order to evaluate the **IMD-0X6-NIR** quantity retained in the skin.

Data were analyzed with Graphpad Prism 9.0 software (GraphPad, San Diego, CA, USA). The data are represented as the mean $\pm$ SEM (standard error mean).
## 4.15 | Confocal Microscopy

After 24 h incubation as described above, the porcine skins are gently rinsed with PBS. The treated area is cut into three equal slices to be representative of the entire skin. The three slices of skin were then placed, using pliers, in a plastic mold (Tissue-Tek Cryomold), the bottom of which is filled with O.C.T (Tissue-Tek), and covered again with OCT up to the edge of the mold. The skin slices embedded in OCT, in the mold, are then frozen in isopentane cooled to -80°C for 1 min. Sections of 10 µm thickness obtained using a cryostat (CM1950, Leica, Wetzlar, Germany) are mounted between slides and coverslips using a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) to counterstain nuclei (ProLong Gold anti-fade reagent with DAPI, Invitrogen) or without DAPI (Prolong Gold antifade reagent, Invitrogen) (one drop per slide). The slides are then observed using a Leica SP8 confocal microscope with an inverted objective (63X), oil immersion. The parameters used during the observation on the LAS X software are: λex = 635 nm, λem = 650 to 800 nm. The obtained images are then processed using ImageJ software.

## 4.16 | Imiquimod-Induced Mouse Model of Psoriasis

Eight-week old female BALB/c mice were purchased from Envigo (France). Housing, care and experimentations were performed in the US 006/CREFRE INSERM/UPS—Service Zootechnie Expéri-mentation Purpan animal facility (Toulouse, France), in accor-dance with French regulations and the European Union Council Directive (2010/63/EU). Mouse experiments were performed by authorized investigators in accordance with ARRIVE guidelines and were approved by the government-accredited animal care and use committee (Comité d'Ethique de l'US 006/CREFRE (Toulouse, France); reference number: 21245-2019062511322839). Mice were housed in groups of 5 in plastic cages in a controlled environment (temperature of 22°C ± 2°C and relative humidity of 50 ± 15%) with 12 h cycles of light and darkness. Food and drinking water were provided ad libitum. Mice were acclimatized for a week before starting the experiment. Upon arrival, mice were randomly assigned to cages prior to acclimatization. Animals were excluded if they showed signs of illness or skin irritation before treatment, or if baseline body weight differed by more than 20% from the group mean. To induce the psoriasis model, first, fur was removed from the back skin of mice by shaving then applying depilatory cream on a surface area of approximately 1.5 cm × 2.5 cm that was removed with a humid cotton pad 2 min later before washing skin gently with warm water. Twenty-four hours later, and then every afternoon for 7 consecutive days, 80 mg of a cream containing 5% IMQ (Aldara, Meda), or a 1% Xanthan-based hydrogel (80 mg) used as a control, was applied to this area and massaged into skin. Every morning, the skin was cleaned with a wet cotton pad then 100 µL of water or each solution/formulation at the indicated dose (50 mg/Kg corresponding to a 1.7 mM (10 mg/mL) solution, 13 mg/Kg corresponding to a 0.5 mM (2.65 mg/mL) solution) was applied and massaged into skin. Each afternoon, before applying IMQ or Xan, an unblinded clinical score was performed based on three criteria: erythema, scales, and thickness. Each criterion was

scored on a scale of 0 to 4: 0 = normal, 1 = slight, 2 = moderate, 3 = severe, and 4 = very severe. The sum of the individual scores gives the total score (scale from 0 to 12). For the histological analysis, the skin samples collected from the back of the mice on D7 are immediately fixed in a 4% paraformaldehyde solution for 24 h and then embedded in paraffin blocks. Haematoxylin & eosine (HE) staining is performed on 5 µm sections of deparaffinized skin. Images were obtained using a Panoramic Slide Scanner 250 (3DHISTECH, Budapest, Hungary), with all slides scanned at 40X magnification. The severity of inflammation at the histological level is assessed, in a non-blinded manner, on the whole skin section based on the following histopathological characteristics of psoriasis: acanthosis, spongiosis, hyperkeratosis, parakeratosis, and immune infiltration. For each of these criteria, a score rang-ing from 0 (normal) to 4 (severe) was assigned, and these scores are summed to give the total histological score (scale from 0 to 20).

## 4.17 | Statistical Analyses

Values are presented as mean ± standard error of the mean (SEM). Normality of each group was evaluated using the Shapiro-Wilk test. For comparisons between two groups, statistical significance was assessed using Student's t-test if data were normally distributed, or the Mann-Whitney U test if normality was not met. For comparisons between more than two groups, one-way ANOVA was performed for normally distributed data, with Tukey's post-hoc test if variances were homogeneous (assessed by Bartlett) or Dunnett post-hoc test if variances were unequal. If the data were not normally distributed, Kruskal-Wallis tests were performed, followed by Dunn's post-hoc test. For two independent variables, two-way ANOVA was applied, followed by Tukey's multiple-comparison test. *P* values below 0.05 (*), < 0.01 (**), < 0.001 (***) , and < 0.0001 (****), were deemed as significant, ns: non-significant. Graphs and statistical analysis were performed using GraphPad Prism software (version 9; GraphPad, San Diego, CA, USA).

### Acknowledgements

This research was funded by the "Agence Nationale de la Recherche" (program ANR-18-CE18-0011). We thank, Bruno Payre of the TRI-Genotoul platform, Université de Toulouse, France for technical support in cryo-SEM and gratefully acknowledge Christophe Déjugnat, Laboratoire Softmat, Toulouse for MASLS data analysis. Flow cytometry and imaging experiments were performed at the Infinity-INSERM UMR1291 core facility connected to Toulouse Réseau Imagerie network, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04). We thank Sophie Allart, Simon Lachambre and Lhorane Lobjois for their technical assistance with imaging. We thank Fatima L'Faqihi, Valérie Duplan-Eche, Anne-Laure Iscache and Hugo Garnier for their technical assistance with flow cytometry. We also thank all the staff from the Zootechnical core facility and the Experimental Histopathology Facility of the INSERM / UPS US006 CREFRE) for animal care and histological analyses.

### Conflicts of Interest

C.O. Turrin is CEO and shareholder of IMD-Pharma, R. Poupot and A.M. Caminade are shareholders of IMD-Pharma.
# Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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### Supporting Information

Additional supporting information can be found online in the Supporting Information section.

**Supporting File:** adhm70597-sup-0001-SuppMat.docx