The skin has evolved a complex network of interconnected barriers, i.e., the physical/mechanical, biochemical, immune and microbiome barriers, which protect from external insults while maintaining internal homeostasis (22,24). The outermost lipid-rich layer of the SC, composed of tightly linked dead corneocytes, and the underlying tight junction (TJ)-mediated paracellular sealing form a major physical barrier against penetrating molecules, particularly highly hydrophilic or lipophilic drugs (22,25,26).

Barrier function also involves biochemical elements, including skin pH, natural moisturizing factors (NMFs) and skin enzymes (22-24,27,28). The 'acid mantle' on the skin's surface not only protects against pathogens with its antimicrobial action but also affects drug partitioning from the vehicle into different skin layers. Notably, pH-responsive delivery systems can utilize local pH changes in lesional skin for targeted drug release (27). The NMFs are essential for maintaining skin moisture and SC integrity. Increased skin hydration can enhance drug solubility and permeability by rearranging the lipid matrix to create aqueous pores that facilitate drug transport across the skin (22-24). In addition, cutaneous bioavailability can be further altered since penetrating substances may be trapped and biotransformed by drug-metabolizing enzymes, mostly found in the viable epidermis (28).

Additionally, the skin harbors various immunocompetent cells strategically located in the epidermis and dermis (24). In the context of drug delivery, initial views of the skin's immune surveillance have demonstrated a dynamic immune-epithelial crosstalk. Activated Langerhans cells (LCs) have been shown to extend their dendrites through TJs to uptake invading molecules without compromising TJ integrity (29,30). This interaction may provide useful insights for translating the immune aspects of the skin barrier into clinical implications for topical drug delivery.

Furthermore, the microbiome barrier, comprising a diverse ecosystem of commensal microbes and their genetic material, forms an additional barrier covering the entire skin surface (24,31). Although the exact role of skin flora in penetration processes remains elusive, emerging evidence suggests that Staphylococcal strains may affect TJ functionality, indicating an interplay between the physical and microbiome barriers (24,25,31). In this regard, unraveling the enzymatic pathways by which gut commensals metabolize pharmaceuticals may partly decipher the skin microbiota-drug interactions offering a tool for optimizing topical dermatotherapy (31).

However, despite its complex barriers, the skin also offers several opportunities for targeted manipulation of its components in the context of topical drug delivery.

As shown in Fig. 2, drug transport across intact skin involves three main pathways: the transcellular route (traversing corneocytes, preferred by hydrophilic or polar compounds), the intercellular route (across the lipid matrix, mostly used by lipophilic molecules) and the transappendageal route (through hair follicles and sweat glands) (22). While the type of pathway depends on the physicochemical properties of the drug, the intercellular path remains the preferred route for entry into the skin. However, the importance of transfollicular routes in skin penetration, especially for TAC, cannot be ignored (22,32).

In addition to skin barriers, it is well known that the physicochemical properties of the permeant agent are equally important in determining its penetration into the skin. Since drug movement across the skin primarily occurs by diffusion via the intercellular lipid matrix, parameters that favor topical delivery include high pharmacological potency, low molecular weight (MW; <500 Da) and moderate lipophilicity (octanol-water partition coefficient-logP-between 1 and 3) (14,22,33). Thus, larger and highly hydrophilic or lipophilic molecules, including topical calcineurin and mTOR inhibitors, can hardly overcome the SC barrier and reach deeper skin compartments due to their size and solubility profiles.

Topical drug absorption is mainly governed by the pathophysiological status of the skin, as specific dermatoses can cause distinct changes in skin barrier elements and permeability (22,30,34). For instance, in conditions such as psoriasis and AD, impaired barrier function due to SC disruptions, increased transepidermal water loss and alterations in lipid composition can enhance drug diffusion in both lesional and nonlesional skin areas (30,35,36). However, the barrier resistance in diseased skin is not always compromised and may allow only modest increases in penetration compared with normal skin. This suggests that an effective barrier function can still be maintained in certain disease stages (22,30,37). Indeed, chronic psoriatic plaques may retain some barrier properties that limit drug absorption, while acute lesions might exhibit enhanced permeability (38,39). This variability underscores the need to exploit disease-specific alterations when designing drug delivery systems tailored to particular skin conditions.

TAC and PIM are grouped, along with cyclosporine A (CsA), in the class of calcineurin inhibitors. The two TCIs interact with the cytosolic protein macrophilin-12 (FK506-binding protein; FKBP12) to form inhibitory complexes that block the calcineurin-mediated dephosphorylation of the nuclear factor of activated T-cells, thereby suppressing T-cell activation and cytokine production (6,10,11). In addition, TCIs can inhibit mast cell and neutrophil activation (6,11,40). Notably, PIM prevents mast cell degranulation and release of pro-inflammatory mediators without affecting LCs (6,41).

Topical preparations of TCIs are currently available in two forms in clinical practice: TAC 0.1% or 0.03% ointment for treating moderate to severe AD and PIM 1% cream for mild to moderate atopic eczema (6,11). Of note, the therapeutic success of TCIs in AD, along with their minimal risk of skin atrophy and systemic absorption, have motivated their off-label use as steroid-sparing agents in multiple inflammatory dermatoses, including non-atopic dermatitis, psoriasis and vitiligo (5,11,42-45).

However, while TCIs have shown efficacy in managing AD flares, their topical application faces a number of limitations (46-48). Specifically, the low and variable skin absorption of the commercial formulation of TAC ointment cannot ensure adequate delivery to its target site in the deeper skin layers, which can ultimately limit therapeutic outcomes (46,47). In addition, its greasy nature and sticky sensation, combined with application-site reactions such as irritation, discomfort and/or pruritus, may compromise patient compliance and satisfaction (6,48,49).

Furthermore, therapeutic outcomes with TCIs appear to vary across different body areas and disease states. While facial, genital and inverse psoriasis can respond well to TCIs, refractory cases of the most common plaque-type psoriasis have already been reported, particularly in difficult-to-treat areas such as the scalp (2,11,12,50-52). Additionally, topically applied TAC is often ineffective in thick psoriatic plaques unless used under occlusion (10,53). Indeed, drug absorption in hyperkeratotic psoriatic skin presents challenges. Although key barrier elements, including TJ functionality and lipid composition, are compromised, epidermal hyperplasia and excessive hyperkeratosis can hamper penetration through the follicular routes. This may explain the relative ineffectiveness of TCIs in treating plaque psoriasis (12,38,39).

Considering the challenges and limitations associated with the topical use of TAC, the skin penetration behavior of TCIs requires special attention. The dermatokinetic profiles of topically applied TAC and PIM are briefly covered in the following section.

This section focuses on the dermatopharmacokinetic profiles of TCIs by summarizing the evidence from experimental and clinical studies exploring their penetration and biodistribution in normal and inflamed (psoriatic and atopic) skin.

TAC (FK506), the prototype of macrolactam immunomodulators, is a natural metabolite produced by the fungus-like bacterium Streptomyces tsukubaensis. Classified as a lipophilic macrolactone (logP=3.2; MW 822 Da), TAC is a crystalline compound that is unstable in alkaline conditions and nearly insoluble in water, but it dissolves in organic solvents such as methanol, ethanol, acetone and chloroform (10,54,55). The newer ascomycin derivative PIM (SDZ ASM 981) is isolated from Streptomyces hygroscopicus var. ascomyceticus. This macrolide lactone has a molecular weight of 810 Da and is more lipophilic than TAC (logP=6.99) (10,56-58) (Fig. 3).

The large size and high lipophilicity of TCIs indicate a greater affinity for the skin compartment with low potential for percutaneous absorption into the bloodstream, offering a more skin-selective pharmacological profile compared with topical corticosteroids (45). However, these properties remain not ideally suited for topical use (6,14,16,58).

In this respect, previous studies have explored the ability of conventional topical TAC formulations to penetrate into, and permeate through, healthy or inflamed skin using preclinical models and human volunteers. Early ex vivo findings demonstrated that TAC penetrates more readily in intact human skin than topical CsA (MW 1202 Da), partly because of its higher potency and 30% smaller molecular size (59-62). However, skin absorption remains highly variable and usually low depending on skin barrier integrity and TAC concentration (10).

Dermally applied TAC has been shown to reside mostly in the SC without entering deeper layers in healthy skin (34,61,63). SC integrity represents a crucial factor limiting TAC transport within the skin. Following a 24-h application on ex vivo human skin, ~20-25% of TAC was found in the SC with no drug penetrating deeper skin layers (34). Similarly, in human cadaver skin topically treated with TAC for 24 h, only 13-23 and 0.5-1.1% of the total dose could reach the epidermis (including the SC) and dermis, respectively (61). In a series of ex vivo studies, topical TAC could hardly penetrate the intact human skin, whereas flux rates were 7-fold higher after the SC was removed (63). Intradermal TAC delivery enhanced as the formulation strength increased, albeit with no evidence of depot effects (61,63). Notably, occlusion could not affect skin absorption of TAC (60).

While barrier dysfunction in diseased skin may theoretically enhance drug penetration, earlier ex vivo studies demonstrated similar amounts of topically applied TAC in the viable epidermis and dermis of both normal and inflamed porcine skin (64). Penetration kinetics of TAC ointment across inflamed skin were further explored in AD patients. In barrier-disrupted human skin, TAC penetrated efficiently into inflamed lesions to control mild to moderate AD relapses. TAC levels beneath the SC increased with prolonged treatment but declined with increasing skin depth. Despite drug depot formation, only a minor proportion (3%) of TAC concentration was sustained 7 days post-treatment, indicating short-term retention of minimal drug amounts in upper skin layers (65).

Preclinical ex vivo and in vivo studies on the penetration behavior of PIM in normal skin yielded comparable results. When applied to human and porcine skin under non- and semi-occlusive conditions, >93% of PIM remained unabsorbed creating a depot on the skin surface, with minimal amounts entering the epidermis. PIM levels in the SC of minipig skin were far higher in vivo compared with the viable epidermis and dermis (2.2 vs. 0.21 and 0.14% of the applied dose, respectively), while dose proportions of 3.1 and 2.9% accumulated ex vivo in the human epidermis (including the SC) and dermis, respectively. Despite drug depot formation in the SC, skin deposition of PIM decreased with increasing treatment duration and was not sustained after repeated dermal application for up to 13 weeks. In fact, only 0.35% of topically applied PIM was retained in the dermis 10 days post-treatment and it was completely eliminated after a wash-out period of 4 weeks (66).

Despite structural and functional similarities, TCIs display distinct differences in skin penetration dynamics. The higher lipophilicity of PIM (logP=6.99) vs. TAC (logP=3.2) results in a greater affinity for the skin tissue during penetration and thus lower systemic exposure, offering a more favorable safety profile (6,56,67). However, preclinical studies on human and animal models showed that both TCIs diffuse similarly across the skin (58,64), corroborating in vitro findings (67). In all models studied, both TAC and PIM could cross the SC to a similar degree, achieving comparable levels in the viable epidermis and dermis under normal and inflamed skin conditions (58,64). Notably, permeation of TAC beyond human and animal skin was consistently higher compared with PIM, regardless of the skin origin (animal or human) and formulation composition. Moreover, absorption of both TCIs beyond inflamed skin was up to 6-fold higher compared with healthy skin. Skin inflammation seemed to enhance only the transdermal permeation of TCIs without affecting their skin deposition (64).

Although damaged or inflamed skin may exhibit increased permeability, potentially leading to systemic exposure, accumulated evidence from adult and pediatric pharmacokinetic studies has consistently shown very low or undetectable serum concentrations of both TCIs following dermal application, far below the levels required for systemic immunosuppression (6,45,49,58,65,68,69). Percutaneous permeation of PIM in AD patients remains minimal, even with prolonged or extensive use (58,68). Additionally, systemic absorption of TAC appears to decrease with treatment duration as the skin heals and barrier integrity is restored, indicating a favorable long-term safety profile (45,65,69).

As reported by skin metabolism studies, no evidence supporting the biotransformation of topical TAC and PIM has been observed in human and porcine skin models. This suggests a low potential for intradermal interactions that could influence the in vivo skin bioavailability of TCIs (60,63,66).

Sirolimus (rapamycin) and its derivatives (rapalogs) belong to a novel class of macrolactam immunomodulators. These agents exert potent antiproliferative and immunosuppressive effects by inhibiting mTOR, a multifunctional serine/threonine kinase of the PI3K family. Similar to TAC, mTORIs form initial complexes with the cytosolic FKBP12 protein but then primarily inhibit mTOR complex 1. As a result, mTORIs induce cell cycle arrest at the G₁ to S transition, while calcineurin inhibitors block the cell cycle at an earlier phase (G₀/G₁) (1,5,10,70).

While the newer macrolides, sirolimus and everolimus, have currently no approval for inflammatory dermatoses, the aberrant activation of the PI3K/Akt/mTOR axis has already been involved in the pathogenesis of psoriasis and AD (8,71-76). The upregulated PI3K/Akt/mTOR pathway in psoriatic epidermis appears to play a key role in disease initiation and progression by mediating Th1/Th2/Th17 imbalance, secretion of pro-inflammatory mediators, abnormal keratinocyte proliferation and differentiation and neovascularization (8,74-76). Although less studied in AD, the PI3K/Akt/mTOR cascade has recently been implicated in regulating epidermal barrier formation by influencing filaggrin processing and lipid synthesis (71-73).

Systemic mTORIs, especially everolimus, have shown potential in managing both psoriasis and AD (77-81). Despite limited evidence to support the beneficial effects of topical mTORIs on inflamed skin, cutaneous mTOR blockade appears promising in immune-mediated skin diseases that require modulation or control of inflammation, epidermal and vascular proliferation and keratinocyte differentiation (4).

Indeed, topically applied sirolimus has already demonstrated efficacy in a small clinical trial on psoriasis patients (82). Although preclinical data from contact dermatitis (CD) animal models were initially not promising (83-85), subsequent studies in skin models of AD and psoriasis indicated the broad anti-inflammatory effects of dermally applied sirolimus both in vitro and in vivo (82,86-92). Traditional topical formulations of sirolimus, investigated in animal and human inflamed skin, have shown potential for addressing several clinical, histological and molecular aspects of cutaneous inflammation (82,86-90). Table I summarizes the key findings from studies exploring the effects of conventional topical sirolimus formulations on inflamed skin.

From a clinical perspective, dermally applied sirolimus, although displaying moderate efficacy in controlling inflammation in irritant CD models, could markedly improve the clinical features of psoriatic and atopic skin lesions while reducing itch and scratching behavior (82,86-88,90).

Laboratory analyses of psoriatic and atopic lesional skin topically treated with sirolimus revealed several biological effects. These include reduced epidermal thickness and neutrophilic microabscesses and decreased dermal neoangiogenesis and inflammatory cell infiltration (comprising T lymphocytes, eosinophils and mast cells). Sirolimus also normalized markers of keratinocyte proliferation and differentiation, such as Ki-67 and keratins. Notably, this compound partially restored skin barrier integrity, as indicated by the increased expression of caspase-14, involucrin and loricrin and further contributed to regulating cellular homeostasis, by modulating cell structure, autophagy and oxidative stress, through the normalization of tropomyosins and related markers (82,86-90).

Furthermore, topical sirolimus could target multiple signaling pathways involved in skin inflammation, including mTOR, ERK 1/2 and NF-κB. It also achieved a broad downregulation of key inflammatory mediators, such as TNF-α, interferon-γ (IFN-γ), interleukins (IL-4, IL-6, IL-13, IL-17A, IL-22 and IL-23) and thymic stromal lymphopoietin (TSLP) (86-90). In addition, topical sirolimus showed potential to impact pruritus onset and severity by reducing nerve growth factor (NGF) levels (86).

Beyond local effects, a trend towards reduced migration of immune cells, including lymphocytes, monocytes, neutrophils, LCs, myeloid and plasmacytoid dendritic cells (DCs), to draining lymph nodes has been observed (88). This suggests that sirolimus may counteract the complex feedback loop sustaining chronic inflammation in the atopic and psoriatic skin microenvironment (93,94). Additionally, topical sirolimus appeared to exert extracutaneous effects, as evidenced by decreased serum levels of IgE, total leukocytes, neutrophils and monocytes (86,88). This may indicate its potential to normalize serum biomarkers involved in the so-called 'atopic or psoriatic march', thereby mitigating systemic inflammation and related comorbidities (94).

These findings indicate that locally applied sirolimus may have diverse biological effects beyond its known antiproliferative and immunosuppressive properties. Further research is warranted to elucidate the underlying mechanisms and the precise impact of topical sirolimus on the inflammatory and structural components of the psoriatic and atopic skin microenvironments.

Sirolimus is a lipophilic macrolactone initially derived from the soil fungus-like bacteria Streptomyces hygroscopicus found in Rapa Nui (Easter Island). In its natural form, sirolimus is a white crystalline solid with a molecular weight of 914 Da, a partition coefficient (logP) of 5.8 and low water solubility (95,96) (Fig. 3). As a result, sirolimus is difficult to formulate into traditional vehicles for dermal application. Currently, topical formulations of sirolimus are not commercially available and are typically prepared from crushed tablets, solution, or powder forms incorporated into ointment, cream, or gel vehicles (4,70,96,97). In addition, the physicochemical properties of sirolimus appear suboptimal for topical use, limiting its capacity to efficiently penetrate the epidermal barrier and reach deeper skin compartments (17).

Thus, understanding the dermatokinetic profile of locally applied sirolimus is important for the development and optimization of topical formulations that ensure maximum skin bioavailability. In this context, an early study by Ormerod et al (82) evaluated the penetration of topical sirolimus in ex vivo barrier-impaired and in vivo normal human skin after a single application of 2.2 and 8% solutions. As the results showed, topical sirolimus could diffuse into human skin with no evidence of systemic absorption. Penetration depth seemed to enhance as the formulation strength increased.

Several topical preparations of sirolimus have also been explored to elucidate factors influencing intradermal penetration, such as formulation composition and skin barrier integrity (98-100). Tanaka et al (98) compared the skin absorption of 0.2% sirolimus formulated in gel and ointment forms using an in vitro three-dimensional (3D) cultured human skin model. Their findings demonstrated that the hydrogel facilitated greater skin accumulation than the lipophilic ointment (1,360 pg/mg vs. 680 pg/mg, respectively) with lower irritation.

In an in vivo murine study, the dermatokinetics of sirolimus were investigated utilizing different formulations (gel, cream and lotion) as delivery vehicles. Overall, superior skin bioavailability was observed with topical application than oral administration of sirolimus. When locally applied, sirolimus displayed dose-dependent skin penetration, showing enhanced absorption and retention when applied as a gel or cream but was less adherent to the skin when incorporated into a lotion (99).

In a series of in vitro (on synthetic membranes) and ex vivo (on human skin) studies, Le Guyader et al (100) also compared the penetration of sirolimus hydrogel, cream and ointment formulations, demonstrating that skin flux and deposition were higher with the hydrogel and enhanced with increased formulation strength. This study emphasized the importance of the formulation type and using sirolimus solubilized (rather than dispersed) at close-to-saturation concentrations to maximize skin bioavailability.

Penetration of topically applied sirolimus was further evaluated in barrier-disrupted ex vivo human skin. As expected for a hydrophobic macromolecule such as sirolimus, absorption into the intact SC was minimal after topical application as a solution or gel. However, in barrier-impaired skin, sirolimus could cross the entire SC and reach deeper epidermal layers. The penetration depth increased as the skin barrier damage enhanced (101).

These findings suggest that the skin absorption and deposition of topical sirolimus depend not only on dosage but also on the administration route and the nature of the formulation/vehicle (hydrophilic vs. lipophilic; hydrogel > cream > ointment). The hydrogel appears to be the preferable vehicle in terms of release and penetration profiles. Notably, the integrity of both the SC and TJ barriers seems to have a greater impact on sirolimus penetration into the skin than the formulation vehicles.

Regarding safety, adverse effects of topical sirolimus appear to be rare and mainly consist of mild application-site reactions that are easily managed without requiring treatment withdrawal. Systemic absorption of locally applied sirolimus is minimal and rarely detectable, independent of the formulation strength (4,82,97).

The use of nanoproducts for encapsulating bioactive compounds offers a novel strategy to address unfavorable physicochemical drug properties, such as lipophilicity or hydrophilicity, high molecular weight, stability and bioavailability. This approach can provide enhanced skin penetration with targeted and controlled drug release, ultimately improving the efficacy and safety profiles of topical therapeutics (20,21,46).

As aforementioned, due to the complexities involved in intradermal delivery of challenging pharmaceutics, such as calcineurin and mTOR inhibitors, more advanced nanocarrier-based prospects are being explored to improve the penetration kinetics of these 'hard-to-formulate' macromolecules within the skin. This section summarized the experimental evidence on various nanovectors investigated as prospective carriers for topical TAC, PIM and sirolimus, focusing on research specifically conducted in psoriasis and AD skin models.

Several types of nanosystems have been explored to overcome the limitations of intradermal penetration and effectively deliver topically applied TAC to its target sites within the viable skin.

Lipid-based nanoformulations have been widely studied for delivering TAC in both normal and inflamed skin. Exploring this concept, Kovačević et al (102) fabricated nanostructured lipid carriers (NLCs) for TAC encapsulation after screening 20 selected lipids. The prepared NLCs, composed of mixed lipid cores, displayed favorable physicochemical properties and entrapment efficiency (EE) of nearly 99%. The use of polyethylene glycol (PEG)-free stabilizers ensured optimal particle stability and prevented irritation effects (102). TAC-loaded solid lipid nanoparticles (SLNs) and NLCs, comprising stearic acid (SA) or beeswax as solid lipids, exhibited satisfactory physicochemical features, with beeswax-based nanoparticles (NPs) providing superior loading capacity (3.3 and 2.9% for NLC- and SLN-Beeswax/TAC, respectively vs. 2.7 and 2.3% for NLC- and SLN-SA/TAC, respectively). No incompatibility between TAC and lipid components was observed (103).

In this context, Wang et al (104) formulated TAC-loaded SLNs, demonstrating that in vitro drug release was fast from free TAC solution (96% at 8 h) but sustained from TAC-SLNs (55 and 86% at 8 h and 72 h, respectively). In ex vivo studies, SLNs achieved enhanced skin penetration and deposition of TAC in healthy skin compared with the commercial ointment.

Kang et al (18) developed thermosensitive SLNs for the targeted release of TAC in response to thermal variations within the skin. The designed TAC-SLNs could cross the SC (32°C) as intact particles and release TAC in the dermis (37°C) without deeper diffusion. Ex vivo studies showed temperature-dependent skin penetration; TAC-loaded SLNs failed to traverse the skin at higher temperatures. While TAC was mainly confined to the SC, SLNs delivered greater drug amounts into deeper skin layers (up to 300-450 μm depth) compared with the marketed ointment (undetectable below 150 μm). The TAC-SLN nanoformulation was more tolerated in vivo, showing only occasional mild erythema, whereas the commercial ointment induced severe erythema.

Chitosan (CS) has been employed in the design of NPs (105-108). Khan et al (105) fabricated TAC-loaded SLNs to investigate the effects of CS coating and gel formulation on skin delivery of TAC. In vitro drug release was faster from uncoated SLNs and sustained from both CS-coated SLNs and SLN-gels. Ex vivo studies showed that both SLNs and SLN-gels achieved similar intradermal penetration, but the SLN-gel resulted in higher retention of TAC in normal skin. Notably, the CS coating did not markedly affect drug retention in the skin (105).

Furthermore, NLCs, considered successors to SLNs, displayed controlled release patterns in vitro (10% of TAC/24 h) and facilitated skin deposition of 61.7% of dermally applied TAC ex vivo (109). Minimal toxicity to murine fibroblasts was observed. Notably, this study also explored a novel combination therapy using NLCs for co-delivering TAC and TNFα small interfering RNA (siRNA) in psoriatic skin. This system exhibited synergistic anti-psoriatic effects in vivo, preventing the onset of erythematosquamous lesions and achieving greater TNFα reduction (7-fold) than TAC-loaded in NLCs and commercial vehicles (2.5- and 2-fold TNFα reduction, respectively) (109). In addition, emulsions containing TAC-loaded NLCs outperformed the marketed ointment in terms of TAC penetration and deposition in ex vivo skin (NLCs: 8.6 mg/cm²; marketed ointment: 5.4 mg/cm²) (110).

CS-coated NLCs prepared for co-loading TAC and clobetasol propionate (CP) provided an EE >90% (98% for TAC; 92.8% for CP). In vitro analysis demonstrated that co-encapsulated NLCs released TAC more slowly than CP. While CS coating favored TAC retention in the SC, TAC failed to diffuse beneath the SC when loaded alone in NLCs, regardless of the coating. Co-encapsulation of CP, especially in CS-coated NLCs, enhanced the capacity of TAC to penetrate deeper skin layers (106).

Vesicular nanocarriers have also gained attention in this field (111-117). An in vivo murine study first reported that a topical liposomal lotion enhanced cutaneous bioavailability, resulting in 9-fold higher TAC skin levels compared with intravenous administration. This formulation also showed superior suppression of skin inflammation than systemic TAC vehicles, either liposomal or traditional, as evidenced both clinically (no erythema or edema) and pathologically (reduced cell infiltration) (111). Similarly, liposomal encapsulation improved the skin flux and deposition of TAC ex vivo with respect to free TAC. Liposomal and conventional TAC vehicles both showed similar anti-inflammatory activity in vivo in CD skin models (112).

In ex vivo comparison studies by Li et al (113,114), both ethosomal and traditional liposomal systems achieved higher epidermal accumulation of TAC than the commercial ointment. While classic liposomes facilitated the highest SC deposition, ethosomes delivered greater amounts of TAC to deeper epidermal layers compared with liposomal and conventional vehicles. The in vivo capacity to reduce AD-like skin inflammation was in the order of ethosomes > classic liposomes > dexamethasone cream > commercial TAC ointment.

Using transfersomes (TFs), topical TAC was effectively delivered into normal, atopic and psoriatic skin. Transfersomal and liposomal carriers both exhibited improved penetration profiles ex vivo and in vivo in terms of TAC release, deposition and retention in the skin compared with commercial formulations. TFs displayed enhanced delivery properties, as evidenced by the penetration depth and amount of TAC in both ex vivo and in vivo viable skin. Notably, TFs were superior to classic liposomes and conventional vehicles in vivo in restoring the clinical and pathological features of atopic and psoriatic skin (115-117).

Liquid crystalline nanoparticles (LCNPs) have also been used for topical TAC delivery, providing controlled drug release and enhanced skin penetration and retention. Ex vivo studies showed that LCNPs increased the skin concentration and retention of TAC by 6- and ~3-fold, respectively, compared with free TAC. When loaded into LCNPs, TAC exhibited greater anti-psoriatic efficacy in vivo, as supported by clinical [Psoriasis Area Severity Index (PASI) score] and pathological (skin thickness and inflammatory infiltration) evaluations. LCNP formulations, with or without oleic acid, were equally effective in repairing psoriatic skin (118,119).

When comparing lipid-based nanoformulations, in vitro TAC release at 24 h was 34, 62, 65 and 69% from LCNP, SLN, NLC and liposomes, respectively. LCNP displayed slow, constant release patterns, while SLN, NLC and liposomes showed an initial burst followed by sustained release. LCNP, SLN and NLC increased TAC skin bioavailability ex vivo by 2.5-, 2- and ~2-fold, respectively, compared with marketed formulations. Penetration depth decreased in the order of SLN > NLC > LCNP > liposomes, with SLN and NLC reaching deeper skin strata than the commercial ointment. The in vivo anti-psoriatic efficacy was as follows: NLC=SLN > LCNP > commercial ointment > liposomes. Unlike conventional vehicles, all nanovectors, especially liposomes, achieved lower transepidermal water loss values without causing skin irritation (120).

Lipid NPs (LN) and modified nanolipid carriers (MNLC) have been exploited to deliver TAC in normal and atopic skin. A series of in vitro and ex vivo studies showed improved profiles in terms of skin release, penetration and deposition of TAC with all studied nanovehicles compared with traditional vehicles. When delivered by LN or MNLC in vivo, TAC achieved higher levels in total skin, especially in the deeper epidermis and dermis, without entering the bloodstream. While nano-based and marketed formulations showed similar occlusive and hydration effects in vitro and in vivo, the former achieved improved skin moisture restoration ex vivo, as well as earlier and superior AD control in vivo. Neither skin irritation nor histological changes occurred in intact or inflamed skin with all tested NPs, even after repeated application (48,121-124). Additionally, when TAC was loaded into natural rhamnolipid-based NPs, no cytotoxicity was observed in human dermal fibroblasts (125).

Microemulsions (MEs) and nanoemulsions (NEs) have also been employed as topical vehicles for TAC (32,126-130). In vitro, MEs released greater amounts of TAC at higher rates than the commercial ointment (127,128). In ex vivo studies, MEs improved the cutaneous bioavailability of TAC, as indicated by the higher drug concentrations in animal and human skin compared with the marketed ointment (126-129). The latter delivered 3-fold lower TAC amounts in the deeper SC than the lecithin-based MEs (128). Although MEs and conventional vehicles carried similar amounts of TAC in the viable epidermis (below the SC), MEs delivered 9-14% of TAC into the dermis, whereas only 6.5% could reach the dermis when applied as a traditional ointment (126). Based on the formulation type, TAC retention in ex vivo skin decreased in the order of conventional solution > ME-based cream > commercial ointment (127). MEs were well tolerated in vitro and in vivo, with no observed toxicity or skin irritation (126-128).

ME systems have already shown therapeutic potential for AD (127,129). As reported by Wang et al (129), TAC-loaded MEs achieved enhanced control of AD in vivo compared with marketed formulations, as demonstrated by clinical and laboratory investigations, corroborating previous findings reported by Lalan et al (127).

Building on the benefits of MEs, a Kalonji oil-based NE system (NE and NE-gel) was developed for the local delivery of TAC to psoriatic plaques. In vitro TAC release followed a slower, sustained pattern from NEs compared with free TAC gel. With respect to the latter, NEs displayed improved ex vivo penetration kinetics in terms of total delivery and retention of TAC in the SC and viable epidermis. In vivo, the Kalonji oil plus TAC-loaded NE gel showed greater anti-psoriatic activity than the commercial ointment, as assessed by clinical and laboratory evaluations. TAC-loaded NEs exhibited stronger antiproliferative effects on epidermal cell lines than the free drug (130).

In a comparative study, Savić et al (32) fabricated NLCs and NEs to explore their potential as topical carriers of TAC. In vitro TAC release from NLCs was superior to that from NEs and marketed formulations. In ex vivo studies, both NLCs and NEs achieved greater TAC deposition in the SC than the commercial ointment, with the highest follicular drug uptake obtained with NLCs followed by NEs and the ointment. The latter resulted in higher transdermal permeation of TAC compared with nanovehicles. Overall, NLCs proved superior to NEs for dermal delivery of TAC.

Natural plant agents combined with TAC have been used as topical antipsoriatic agents. Lipospheres co-loaded with TAC and curcumin (CUR) displayed a slow release of both cargos in vitro and compatibility with healthy skin in vivo. In in vivo studies, the marketed ointment and TAC-CUR lipospheres were equally effective but superior to betamethasone in restoring the clinical features of psoriatic skin, such as scaling and thickness, while the decrease in erythema followed the order of TAC-CUR lipospheres > commercial TAC ointment > betamethasone ointment. Among the studied formulations, only TAC-CUR lipospheres normalized the histological psoriatic changes. Notably, TAC-CUR lipospheres achieved the strongest suppression of TNFα and IL-22, while IL-17 inhibition followed the order of TAC-CUR lipospheres ≈ commercial TAC ointment > TAC lipospheres > betamethasone ointment (47).

Using micellar systems, TAC was successfully delivered into normal and inflamed skin (131,132). While micelles remained largely unabsorbed on the skin surface, they facilitated greater skin deposition of TAC ex vivo compared with marketed formulations, without permeating beyond human and porcine skin. Improved penetration profiles were observed in porcine compared with human skin, indicating the importance of transfollicular pathways for micelle-mediated delivery (131).

Using X-ray microscopy, the penetration of topical TAC incorporated into methoxy-poly(ethylene glycol)-hexyl-substituted polylactide (mPEG-hexPLA) micelles was investigated in an ex vivo psoriasis murine model. Slightly increased SC deposition was observed when TAC was formulated in micelles rather than in the commercial ointment. SC levels of TAC-loaded micelles gradually increased until saturation and then decreased due to deeper absorption, while amounts in the viable epidermis remained stable. This study, although indicating intercellular delivery routes, also suggested that micelles might penetrate corneocytes (132).

Similar attempts have been made by Gabriel et al (133) to design mPEG-hexPLA-based NPs for the in vivo evaluation of TAC penetration and anti-psoriatic effects on intact and psoriasiform murine skin. TAC embedded into mPEG-hexPLA NPs showed enhanced absorption in inflamed compared with normal skin, reaching ~2-fold higher levels in psoriatic lesions than with a commercial ointment. This nanoformulation was superior to CP in lesion clearance and demonstrated comparable antipsoriatic efficacy to the marketed ointment. No toxicity was observed after repeated application on healthy skin.

In a novel approach, Zabihi et al (134) exploited polymer-based biodegradable NPs composed of poly(lactide-co-glycerol) (PLG) to deliver TAC into human skin. In ex vivo experiments, PLG-NPs facilitated 80, 16 and 4% of encapsulated TAC to reach the SC, viable epidermis and dermis, respectively, resulting in 1.74-, 1.44- and 2-fold higher drug concentrations in the SC, epidermis and dermis, respectively, compared with the marketed ointment. The anti-inflammatory efficacy was assessed in vitro on a 3D reconstructed filaggrin-deficient human skin model by measuring pro-inflammatory mediators. While the downregulation of IL-2 was comparable between TAC-PLG-NPs and the marketed product, the NPs were more effective in suppressing TSLP. No evidence of cytotoxicity on primary human keratinocytes and fibroblasts was noted.

Fereig et al (107) developed CS-based NPs for topical application of TAC on psoriatic skin. In vitro drug release from CS-NPs followed sustained biphasic patterns. When delivered by CS-NPs, 82% of dermally applied TAC was accumulated in ex vivo skin, whereas a 34% total deposition was achieved with a conventional ointment. CS-NPs facilitated reduced flux rates into and permeation of TAC beyond the skin compared with a traditional ointment (24 vs. 61% permeated drug, respectively). In in vivo experiments, CS-NPs also displayed enhanced skin deposition of TAC (54.6 vs. 13.8% for the ointment) and achieved faster and superior control of the clinical and pathological psoriatic features compared with the ointment. Notably, only TAC-loaded CS-NPs promoted hair growth in the treated areas.

CS-based NPs were further explored in combination with nicotinamide (NIC) or hyaluronic acid (HA) to transfer TAC into atopic skin (108,135). Under normal ex vivo and AD-like in vivo conditions, the synergistic effect of NIC and CS achieved greater TAC concentrations in total skin compared with the commercial ointment. NIC-CS-NPs showed superior in vivo anti-AD activity compared with the marketed formulation, despite containing equal or lower TAC doses, as confirmed by clinical, pathological and molecular analyses (108). HA coating ensured controlled and sustained in vitro release patterns, while enhancing TAC deposition and retention beneath the SC in ex vivo skin. HA-covered NPs exhibited in vivo greater anti-AD efficacy than both the uncovered CS-NPs and the commercial TAC ointment, based on clinical and laboratory evaluations (135).

NPs prepared by HA and cholesterol (Chol) conjugations in a NIC solution (NIC-HA-Chol NPs) synergistically enabled deeper penetration and enhanced deposition of TAC in ex vivo and in vivo studies on intact skin compared with the commercial ointment. HA-Chol-NPs, with or without NIC, showed improved drug uptake in HaCaT cells (136). Further evaluation showed that the NIC-HA-Chol nanoformulation achieved 2.4- and 2.5-fold higher TAC permeation and retention in ex vivo psoriatic skin, respectively, compared with the marketed product. The in vivo anti-psoriatic efficacy of the studied NPs, assessed via the PASI score and epidermal thickness, was comparable to CP but superior to the marketed ointment. The NIC-HA-Chol NPs exhibited enhanced cellular uptake and strong anti-proliferative effects in murine macrophage and HaCaT cells (136,137).

In this context, the dermatokinetics and therapeutic efficacy of TAC-loaded polymeric core-multishell (CMS) nanocarriers have been examined in vivo using an AD-like murine model. While CMS nanoparticles delivered TAC into all skin layers, they failed to enhance drug deposition compared with the commercial ointment (mean TAC levels in epidermis and dermis: 36 and 77 ng/cm² for CMS nanocarriers vs. 93 and 118 ng/cm² for the ointment, respectively). However, both CMS nanocarriers and the marketed ointment showed similar efficacy in improving the clinical and pathological features of atopic skin (138,139).

Thermoresponsive nanogels offered a new approach for the topical delivery of TAC in ex vivo human skin. Comparative studies showed that the commercial ointment demonstrated superior penetration, particularly in breast vs. abdominal skin and in barrier-impaired vs. intact skin. After barrier disruption, nanogels, although primarily confined to the SC, could enter deeper viable skin. TAC levels increased over time in ointment-treated sites but remained constant in areas treated with nanogels. The increase in IL-6 and IL-8 in damaged skin treated with TAC was attributed to the irritative effects of TAC and/or the vehicles used. Nanogels and the marketed ointment were both equally effective in inhibiting T-cell proliferation (140).

As reported by Limón et al, nanostructured hydrogels could entrap TAC both in interstitial spaces and within gel fibers, offering a reservoir for controlled release and protection from degradation. In vitro tests revealed moderate rates of biphasic drug release. In ex vivo human skin, TAC hydrogel could cross the SC and remain in the epidermis and upper dermis with minimal percutaneous permeation. In in vivo psoriasis models, TAC nano-hydrogel showed superior efficacy and tolerability compared with a free TAC solution. The latter achieved only a 9% reduction in skin thickness and failed to prevent local adverse effects, whereas TAC nano-hydrogel achieved a 50% reduction in psoriasiform hyperplasia without causing desquamation or hair loss (141).

Recently, mesoporous silica nanoparticles (MSNs) have been employed as carriers for topical TAC, demonstrating potential in managing AD. TAC-loaded MSNs displayed improved in vitro release kinetics than a free TAC gel (73 and 55% at 24 h, respectively) without any evidence of cytotoxicity. Although both TAC-MSNs and TAC gel showed low transdermal permeation ex vivo (13 vs. 11%, respectively), a markedly higher amount of TAC was deposited in the skin with MSNs than with TAC gel (75 vs. 36%, respectively). In in vivo AD-like skin, TAC-MSNs were more effective than TAC gel in restoring both clinical and pathological alterations (142).

Hybrid nanosystems, emerging as superior alternatives to conventional nanocarriers, have been used for delivering TAC in psoriatic and atopic skin (143,144). Wan et al (143) developed a polymer-based ME as a topical delivery system of TAC. Ex vivo and in vivo studies on normal and psoriatic skin demonstrated that the studied ME outperformed the commercial ointment in terms of delivery and deposition of TAC across the entire skin. This system displayed in vivo anti-psoriatic activity comparable to CP but superior to the marketed TAC ointment, as evidenced by clinical (PASI score) and pathological (epidermal thickness) outcomes. In addition, this formulation showed enhanced uptake and growth inhibition of HaCaT cells.

Shams et al (144) fabricated polymeric nanofibers incorporating TAC-loaded ME for topical application on atopic skin. This system displayed sustained in vitro release profiles, with 22% of TAC released over 3 days. The tested ME-nanofibers, applied every two days, proved equally effective as the daily use of commercial TAC ointment in improving the AD histological features.

Unlike TAC, only a few studies have investigated the skin penetration and anti-inflammatory effects of topical sirolimus nanoformulations (91,92,145,146). New generations of bioresponsive nanocarriers, capable of exploiting key aspects of the inflamed skin microenvironment, can release their cargos in response to specific stimuli such as pH, temperature, or redox variations within the skin (147). In this regard, redox-sensitive CMS nanocarriers have been explored as topical vehicles for sirolimus in ex vivo models of inflamed human skin co-cultured with activated T-cells in comparison with conventional preparations. All tested formulations delivered sirolimus into barrier-disrupted inflamed skin, suppressing T-cell proliferation and release of IL-2 and IL-17A. The strongest inhibition of IL-2 release was observed with sirolimus incorporated in CMS nanocarriers, while no effect on IL-1α, IL-6 and IL-8 pro-inflammatory cytokines was detected. Only the sirolimus-loaded CMS nanocarriers could downregulate mTOR activity and target skin DCs, preventing their activation and migration (91,92).

Furthermore, aqueous formulations of sirolimus-loaded polymeric micelles have been developed for dermal application. Micellar solution and hydrogel both increased the bioavailability of sirolimus in total skin compared with a conventional ointment. Greater amounts of sirolimus were deposited in the SC, viable epidermis and dermis with micelle-based systems. Sirolimus skin levels increased over time, indicating sustained release kinetics. Transdermal drug permeation was undetectable for all tested formulations (145).

Based on these results, Le Guyader et al (146) investigated the penetration of sirolimus-loaded polymeric micellar preparations applied topically to ex vivo human skin. Although micelle-based and conventional vehicles carried similar amounts of sirolimus to the dermis (700-800 ng/cm²), a 2.5-fold higher drug deposition in the epidermis was observed with sirolimus formulated in micelles rather than a hydroalcoholic gel (1,900 vs. 700 ng/cm², respectively).

In a comparative study, Quartier et al (54) used polymeric micelles for the topical delivery of TAC, PIM and sirolimus. PIM exhibited a lower linear release from the micelles in vitro, while TAC and sirolimus showed higher releases, reaching a plateau before further increase. In ex vivo skin, PIM was deposited in higher amounts, especially in the epidermis, due to its greater lipophilicity and stronger binding with skin components. TAC and sirolimus showed lower but similar deposition levels, with greater accumulation in the dermis. The higher water solubility of sirolimus was associated with increased dermal levels. Transdermal drug permeation was not observed. These findings indicate the need for tailored micelles, even for closely related drugs, as minor variations in drug properties, particularly aqueous solubility and lipophilicity, can affect dermatokinetic profiles.

The wide range of nanomaterials discussed in this section underscores their potential to overcome challenges associated with conventional dosage forms. The key outcomes from preclinical studies utilizing nanoparticulate systems to explore the dermatokinetic profiles, efficacy and safety of topical TAC, PIM and sirolimus in normal and inflamed skin models are summarized in Table II.

However, clinical translation still needs to be improved, as only a few studies have progressed to real-world settings. Notably, a recent clinical trial investigating a topically applied TAC-loaded ME on scalp psoriasis patients reported promising results, paving the way for future clinical applications (12).