Novel Voriconazole-Loaded Hyalurosomes Optimized for Enhanced Skin Penetration and Antifungal Activity against Candida albicans

Novel Voriconazole-Loaded Hyalurosomes Optimized for Enhanced Skin Penetration and Antifungal Activity against Candida albicans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel Voriconazole-Loaded Hyalurosomes Optimized for Enhanced Skin Penetration and Antifungal Activity against Candida albicans Amparo Nácher, José-Esteban Peris, Raquel Taléns-Visconti, Octavio Díez-Sales, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7270257/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract Cutaneous candidiasis, mainly caused by Candida albicans , is a growing global health concern and is listed by WHO as a high-priority fungal threat. Suboptimal penetration of conventional vehicles limits the efficacy of current topical antifungals, increasing the risk of severe and invasive infections. Therefore, there is an innovative research field in advanced topical delivery systems to improve drug deposition, retention and antifungal efficacy. The main objective of this work was to develop nanocarriers based on hyalurosomes for the delivery of voriconazole (VCZ) and evaluate their potential to enhance the drug’s cutaneous penetration and antifungal activity. Four VCZ-loaded hyalurosomal formulations were prepared (H1-H4) by modulating the proportions of phospholipid and polyols. Although changes in some physicochemical properties were observed, all the VCZ-loaded nanosystems were nanosized ( 72 %), excellent biocompatibility with human keratinocytes and potent antifungal activity against C. albicans . VCZ release from formulation H1 (1 % phospholipid, 10 % ethanol) followed a Fickian mechanism, while H2–H4 (4-10 % phospholipid, 2.5-10 % ethanol) exhibited anomalous diffusion involving both diffusion and matrix relaxation or erosion. Additionally, H1 and H2 (1-4 % of phospholipid, 10 % ethanol) achieved significantly enhanced drug penetration into deeper skin layers and superior in vivo antifungal efficacy compared to VCZ dispersion. The results highlight the potential of hyalurosomes as a next-generation topical antifungal delivery system, effective against both superficial and invasive candidiasis, with formulations H1 and H2 emerging as the most promising candidates for the treatment of the more invasive forms. voriconazole hyalurosomes candida albicans topical administration antifungal activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Skin fungal infections, or dermatomycoses, rank among the most prevalent infectious diseases, with estimates suggesting that more than a quarter of the world’s population will experience a dermatomycosis at some stage in their lifetime [ 1 ]. Recent trends indicate a rising incidence and severity of these conditions, primarily driven by risk factors such as the widespread use of broad-spectrum antibiotics, antineoplastic agents and immunosuppressive drugs [ 2 ]. This includes paediatric patients with leukaemia, who are at increased risk of developing chronic disseminated candidiasis [ 3 ]. Importantly, these infections are not limited to immunocompromised patients as fully healthy individuals, including athletes and gym users, are also frequently affected, underscoring the substantial clinical burden and propensity for recurrence that dermatomycoses generate [ 2 ], thus posing a significant clinical challenge. In this context, candidiasis is a common fungal infection mostly caused by yeasts of the Candida species , mainly Candida albicans . In fact, it was classified as one of the four highest priority fungi for global public health in the first-ever fungal priority pathogens list published by WHO in 2022 [ 4 ]. Candidiasis can affect various parts of the body, ranging from superficial mucocutaneous infections to invasive systemic disease, which may involve multiple organ systems and become life-threatening, particularly in vulnerable patients [ 5 , 6 ]. In this sense, in April 2025, WHO also published its first-ever reports on fungal tests and treatments, showing the urgent need for innovative research and development [ 7 ]. The most widely used antifungal agents are included in the class of azoles, especially imidazoles. Voriconazole (VCZ) a second-generation triazole agent approved in 2002 [ 8 ], specifically inhibits the enzyme 14α-demethylase, essential for the synthesis of ergosterol, a component of the fungal cell membrane. By blocking ergosterol production, VCZ destabilizes the cell membrane and leads to fungal cell death [ 9 ]. However, VCZ, as many azoles, is poorly water soluble, which limits its bioavailability and antifungal effect. Formulation and drug delivery strategies could improve water-based hydration and dissolution properties, thus increasing its pharmacokinetics and a sustained release will prolong the retention of a high concentration of the azole localized at the infection site, therefore enhancing its bioavailability and therapeutic efficacy [ 10 ]. For cutaneous candidiasis, topical dosage forms are preferred, mainly due to site-specific drug delivery. The problem with conventional marketed formulations, based on gels, powders, creams, solutions or foams of imidazoles, is that high and repeated doses have to be administered, which may result in local or even systemic toxicity reducing patient adherence [ 11 ]. Moreover, their efficacy is limited in treating more invasive fungal infections [ 12 ]. Additionally, the increasing prevalence of drug-resistant strains has further compromised the efficacy of currently available formulations [ 9 ]. Consequently, the risks of more invasive and deadly infections among the general population have also increased. Hence, the need to develop new topical delivery systems for new-generation, broad-spectrum antifungals, such as VCZ, to overcome biopharmaceutical challenges associated with conventional drug delivery systems like poor retention and low bioavailability. In this context, nanocarrier systems are gaining relevance due to their ability to enhance the diffusion of active ingredients across the skin barrier [ 10 – 12 ]. Among them, we demonstrated that nanostructured lipid carriers (NLCs) loaded with VCZ showed biocompatibility with human keratinocytes and improved the penetration and distribution of VCZ into deeper skin layers, offering superior antifungal activity [ 13 ]. Therefore, the vehicle composition of a topical delivery system may significantly affect drug release and skin penetration, thereby affecting biological activity. In this sense, liposomes have been successfully used to improve the topical efficacy of some drugs, proteins and natural ingredients by modifying their local penetration capacity into the skin and their biodistribution [ 14 – 16 ]. Hyalurosomes are a special type of liposomes, enriched with hyaluronic acid (HA), which enhance the penetration of drugs into the deeper skin layers. In addition, HA acts as a gelling agent and skin regenerator, prolonging the action of the treatment and favoring skin recovery [ 17 , 18 ]. It also intervenes by improving the epithelial barrier and the innate immune response, which could protect the skin from microbial infections. In fact, HA has been shown to possess antimicrobial activity against Candida [ 19 ]. The aim of this study is the design and characterization of novel nanosystems for voriconazole delivery enriched with HA, thus offering a novel strategy for the topical management of candidiasis. If successful, our approach could not only increase the efficacy of VCZ but also address current challenges such as poor retention, low bioavailability and the emergence of drug resistance, ultimately reducing systemic complications that could lead to hospitalizations and associated deaths. Materials and Methods Reactants The following compounds were purchased from Sigma-Aldrich (Madrid, Spain): dimethyl sulfoxide (DMSO), RPMI 1940 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium salt. Polysorbate 80 was obtained from Guinama (Valencia, Spain) and ethanol was purchased from PanReac Applichem (Barcelona, Spain). VCZ and hyaluronic acid (HA) (supplied as sodium hyaluronate, molecular weight 200–400 KDa) were provided by Glentham Life Sciences (Corshman, UK). Sabouraud dextrose agar (SDA) and acetonitrile (AcN) were obtained from VWR chemicals (Barcelona, ​​Spain). 4-Morpholinepropanesulfonic acid (MOPS) came from Alfa Aesar (Kandel, Germany) and Eugon LT 100 from BIOKAR Diagnostics (Beauvais, France). Glucose and cyclophosphamide monohydrate were obtained from Thermo scientific (Madrid, Spain). Phospholipon® 90 G was provided by Lipoid GmbH (Ludwigshafen, Germany) and glycerin from Acofarma (Madrid, Spain). Cell medium, fetal bovine serum (FBS), penicillin, streptomycin, fungizone and all the other reagents for cell studies, unless otherwise specified, were purchased from Gibco (Paisley, UK). Analytical Method VCZ was quantified by means of a high-performance liquid chromatography assay (HPLC) with ultraviolet (UV) detection at 255 nm, as described previously by our research group [ 13 ]. A Waters “Nova-Pack” C 18 analytical column (4 µm, 3.9 mm × 150 mm) was used, and the mobile phase consisted of a mixture of acetonitrile, water and 0.6% trietylamine, pH 6 (35/65, v/v). The injection volume was 25 µL, and the flow rate was 1 mL/min. The HPLC equipment consisted of a quaternary pump SpectraSYSTEM P4000, an autosampler SpectraSYSTEM AS3000 and a spectrophotometric detector SpectraSYSTEM UV 6000LP. Data were processed through “Chromquest Chromatography Workstation Software Version 1.63”. Preparation of hyalurosomes Four VCZ-loaded hyalurosome formulations (H1–H4) were developed and selected based on their promising physicochemical characteristics. The main differences among them were the phospholipid concentration and the composition of the hydrating solvent mixtures (Table 1 ). Table 1 Composition of VCZ-loaded hyalurosomes. Formulation Component (%) H1 H2 H3 H4 Sodium hyaluronate 0.1 0.1 0.1 0.1 Phospholipon® 90G 1 4 10 4 Polysorbate 80 0.5 0.5 0.5 0.5 Glycerin - - - 7.5 Ethanol 10 10 10 2.5 Water 90 90 90 90 Voriconazole 0.05 0.05 0.05 0.05 The components of each formulation were mixed under magnetic stirring at 200 rpm and then hydrated overnight with 10 mL of either ethanol/water (10:90, v/v) for H1, H2, and H3, or glycerol/ethanol/water (7.5:2.5:90, v/v/v) for H4. Thereafter, formulations were sonicated with 3 cycles of 5 minutes (5 s on and 2 s off, 60% and 45% amplitude) with an ultrasonic disintegrator (CY-500, Optic Ivymen system, Barcelona, Spain) to homogenize the preparation. Finally, formulations were sterilized by filtration (CA syringe filter; cellulose acetate; 0.22 µm). The final concentration of VCZ in hyalurosomes was 0.5 mg/ml. Empty hyalurosomes were also prepared and used as controls for physicochemical characterization and antimicrobial testing. Characterization of hyalurosomes Transmission electron microscopy (TEM) was used to confirm vesicle formation and evaluate the morphology. The samples were stained with 2% phosphotungstic acid aqueous solution and examined under a JEM-1010 (Jeol Europe, Paris, France) transmission electron microscope equipped with a digital camera AMT RX80 and the AmtV602 software, version 602.579 at an accelerating voltage of 80 kV. Photon correlation spectroscopy was used to analyze the mean diameter (MD) and polydispersity index (PI) using a Zetasizer nano (Malvern Instruments, Worcestershire, UK). The same equipment was also used to measure the zeta potential (ZP) by means of the M3-PALS (Phase Analysis Light Scattering) technique, which measures particle electrophoretic mobility. The MD, PI and ZP were monitored over 3 months of storage at 4 ± 1°C to evaluate the stability of the formulations. The entrapment efficiency (EE) was calculated as the percentage of the concentration of VCZ after dialysis versus that initially used [ 20 ]. VCZ-loaded hyalurosomes (1 mL) were loaded into the dialysis tube (Spectra/Por® membranes, 12–14 kDa MW cut-off, 3 nm pore size; Spectrum Laboratories Inc., DG Breda, the Netherlands) and kept at room temperature (25 ± 1°C) in 100 mL of distilled water under continuous stirring for 10 h. After reaching the dialysis equilibrium, a sample of 0.5 mL of the exterior aqueous medium was taken and added to 0.5 mL of AcN. The mixture was injected into the HPLC to determine the VCZ concentration in the external aqueous medium, which is assumed to be equal to the free (unencapsulated) VCZ concentration in the hyalurosomes. Additionally, the total VCZ concentration inside the dialysis tube was quantified by HPLC after disrupting the vesicles with AcN (1/10). In vitro Release Study The release studies of the hyalurosomes were conducted using the dialysis membrane method. 1 mL of VCZ-loaded hyalurosomes was loaded into a Spectra/Por® 2 standard regenerated cellulose dialysis tube with an MWCO of 12–14 kDa and clipped by standard closures. The dialysis tube was immersed into 100 mL of distilled water with a magnetic stirrer stirring at 300 rpm. At 0, 1, 2, 3, 4, 6, 8 and 10 hours, 0.5 mL of the medium were removed and replaced with an equal volume of distilled water. VCZ released amounts were determined by HPLC. To determine the release kinetics of VCZ from hyalurosomes, different commonly used mathematical models including zero order, first order, Higuchi, Korsmeyer–Peppas and Peppas–Sahlin were used. The values of the kinetic parameters were obtained by using Sigmaplot 10.0® (Systat Software, Inc., San Jose, CA, USA). The equations that represent each drug release model are summarized in Table 2 . Table 2 Equations of the models used for fitting drug release data. Model Equation Zero order \(\:{Q}_{t}={{Q}_{0}+K}_{0}\bullet\:t\) First order \(\:{Q}_{t}={Q}_{0}·{e}^{-{K}_{1}·t}\) Higuchi \(\:{Q}_{t}/{Q}_{\infty\:}={K}_{H}\bullet\:\surd\:t\) Korsmeyer-Peppas \(\:{Q}_{t}/{Q}_{\infty\:}={K}_{K-P}\bullet\:{t}^{n}\) Peppas-Sahlin \(\:{Q}_{t}/{Q}_{\infty\:}={K}_{P-S\left(1\right)}\bullet\:{t}^{n}+\:{K}_{P-S\left(2\right)}\bullet\:{t}^{2n}\) Q t : amount of drug released over time t; \(\:{Q}_{0}\) : initial amount of drug in the formulation, \(\:{Q}_{t}/{Q}_{\infty\:}\) : fraction of drug released; K 0 , K 1 , K H : release rate constants for zero-order, first-order and Higuchi release kinetics, respectively; K K−P and K P−S(1) : diffusion constants, K P−S(2) : relaxation constant; n: exponent that characterizes the diffusion process. In vitro Cytotoxicity of Formulations Immortalized human keratinocytes (HaCaT, ATCC, Manassas, VA, USA) were cultured as monolayers in 75 cm² flasks using Dulbecco’s Modified Eagle’s Medium (DMEM) with low glucose (1 g/L), sodium pyruvate and GlutaMAX, supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (10,000 U/mL penicillin, 10,000 µg/mL streptomycin) and 0.1% fungizone. Cells were maintained under standard incubation conditions of 37°C, 5% CO₂, and saturated humidity. For experimental procedures, cells were seeded into 96-well plates at a density of 7.5 × 10 3 cells per well and allowed to adhere for 24 hours. Subsequently, cells were exposed to VCZ, either in aqueous dispersion or encapsulated within hyalurosomes. The formulations were diluted in cell culture medium to obtain final concentrations of 0.5, 5, 50, and 500 ng/mL. These concentrations were selected to mimic potential in vivo dilution scenarios, with 500 ng/mL considered a likely maximum concentration of VCZ capable of reaching deeper skin layers [ 21 ]. After 48 hours of incubation with the treatments, the culture medium was replaced with an MTT solution (0.5 mg/mL in phosphate-buffered saline, PBS). Following a 3-hour incubation period, the MTT solution was removed and the resulting formazan crystals were dissolved using DMSO. Absorbance was then measured at 570 nm using a Multiskan EX microplate reader (Thermo Scientific, Waltham, MA, USA). All experiments were performed in six independent runs, each conducted in triplicate. Cell viability results are expressed as percentages relative to untreated control cells (set at 100%). In vitro Skin Permeation The transdermal permeation of VCZ, either in aqueous dispersion or encapsulated in hyalurosomes, was investigated using vertical Franz diffusion cells (Vidrafoc, Barcelona, Spain), featuring an effective diffusion area of 0.785 cm² and a receptor chamber volume of approximately 6 mL. Donor compartments were filled with 500 µL of VCZ-loaded formulations at a concentration of 0.5 mg/mL, while the receptor compartments were filled with 0.9% sodium chloride solution. Dermatomed porcine ear skin, with a uniform thickness of 600 µm, was positioned between the donor and receptor chambers. The skin samples were obtained from pig ears supplied by the Faculty of Medicine at the University of Valencia (Valencia, Spain), collected post-mortem from animals previously used in unrelated research protocols. The assembled diffusion cells were maintained in a thermostatic water bath set at 37 ± 1°C, ensuring that the skin surface remained at a physiological temperature of 32 ± 1°C, with continuous magnetic stirring throughout the experiment. After a 10-hour permeation period, the amount of VCZ present in the donor as well as receptor compartments was quantified using an HPLC. Prior to skin extraction, the surface was washed with 0.5 mL of AcN/H₂O (50:50) to remove any formulation remaining on the surface that had not been absorbed. Subsequently, cryostat sectioning of the treated skin samples was performed (Leica CM1950) to obtain slices being 10, 40, 100 and 450 µm thickness, simulating stratum corneum, epidermis, superficial dermis and deep dermis, respectively. The drug content within the skin layers was then extracted with AcN and analyzed by HPLC. Microbial Strains The antifungal activity of VCZ both in aqueous dispersion and formulated as hyalurosomes was tested against Candida albicans (CECT 1394). Cultures were kept for 24 h at 36 ± 1°C. After 24 h of incubation, the fungal suspensions were diluted with PBS in order to obtain an adequate density expressed as colony forming units per milliliter (CFU/ml). In vitro Antifungal Activity The procedure followed to carry out this type of test was described previously by our research group [ 14 ]. Briefly, vials with 500 µl of a 1/5 diluted VCZ-based formulations and 500 µl of inoculum containing 1–5 x 10 5 CFU/mL were used. Thus, the concentration of VCZ in the diluted formulations was 100 µg/mL and the final concentration in the assay vials was 50 µg/mL. The assays also included a positive control composed of an aqueous dispersion of VCZ (50 µg/mL), in which maximal C. albicans inhibition was expected, and a negative control containing 500 µl of water. All vials were incubated at 36 ± 1 ºC, taking samples from each vial at 0, 6, 24 and 48 hours. To collect them, 50 µL of each were diluted in 5 ml of PBS and serial decimal dilutions were subsequently prepared and seeded in Petri dishes with SDA. Following incubation at 36 ± 1 ºC for 48 hours, CFU were enumerated. The total C. albicans burden in each sample was calculated based on plates exhibiting 30 to 300 colonies, with appropriate adjustments for dilution factors and plated volumes. In vivo Antifungal Activity Protocols for the in vivo studies using mice, were approved by the Animal Care Committee of the Faculty of Pharmacy at the University of Valencia (Spain) [reference: 2024-VSC-PEA-0081]. Male 5–6 weeks old ICR (CD-1) mice, weighing 30–35 g, (Envigo, Barcelona, Spain), were obtained from the animal facility of the Faculty of Pharmacy at the University of Valencia and were kept in a clean room at a temperature of 23 ± 1°C, a relative humidity of 60% and a light/dark cycle of 12 h. Mice were fed a standard laboratory diet and had access to water ad libitum. The methodology employed for these in vivo studies was based on a procedure previously published by our research team [ 22 ]. Briefly, to induce immunosuppression prior to fungal infection, mice received intraperitoneal injections of cyclophosphamide at a dose of 100 mg/kg/day for three consecutive days. On the final day of immunosuppressive treatment, a working culture of C. albicans , grown for 24 hours at 35°C on SDA, was used to prepare a yeast suspension containing 10⁷ CFU/mL in a mixture of RPMI 1640 and yeast extract-peptone-dextrose (YPD) medium (50/50, v/v). On the same day, the dorsal area of each mouse was shaved using an electric clipper. A 100 µL aliquot of the C. albicans suspension was then applied to the shaved area using a custom-designed cylindrical plastic chamber (4.5 mm i.d. × 6 mm height), which was affixed to the skin with cyanoacrylate adhesive to maintain localized contact of the suspension with the skin surface under aerobic conditions. 24 hours post-inoculation, the infected skin areas were treated with 100 µL of one of the following formulations: normal saline (control), a VCZ aqueous dispersion or VCZ-loaded hyalurosomes. Additionally, in order to compare the influence of the nanocarrier system in the antifungal activity of VCZ, the most promising VCZ-loaded NLCs previously developed by our research group in a previous work (formulations C and D) were evaluated [ 13 ]. All animals were sacrificed after 24 hours and the corresponding skin regions were excised. The surface of each skin sample was scraped and transferred to 1 mL of Eugon LT100 broth in microcentrifuge tubes. Samples were vortexed for 30 seconds and centrifuged at 2,000 × g for 5 minutes. The supernatant was discarded and the pellet was resuspended in 1 mL of fresh Eugon LT100 broth. This wash step was repeated once. The resulting pellet was finally resuspended in 1 mL of Eugon LT100 broth and serial tenfold dilutions were prepared using the same medium. An adequate volume of sample was seeded in Petri dishes with SDA, plates were incubated at 36 ± 1°C for 48 hours and the colonies observed were counted. Statistical Analysis Data are presented as mean ± standard deviation (SD). The Student’s t test was used for two-group comparison. One-way analysis of variance (ANOVA) was used for comparisons of more than two groups; when statistically significant differences were found, Tukey’s test was applied to determine which groups were statistically different. P values of < 0.05 were considered statistically significant. All calculations were performed with IBM SPSS Statistics 26 (SPSS Inc., Chicago, IL). Results Characterization of hyalurosomes VCZ-loaded formulations were mainly multilamellar, as detected by TEM analyses (Fig. 1 C and 1 D). The hyalurosomes were small in size, spherical shape and slightly aggregated. Empty formulations were also prepared in order to assess the effect of VCZ on hyalurosomes assembly (Fig. 1 A and 1 B). The physicochemical properties of hyalurosomes were evaluated measuring the mean diameter (MD), polydispersity index (PI), zeta potential (ZP) and entrapment efficiency (EE) (Table 2 ). Nanovesicles with sizes ranging from 64 to 160 nm and PI values between 0.19 and 0.42 were obtained by varying the concentrations of phospholipid and co-solvents. Formulation H1, prepared with the lowest phospholipid concentration (1% Phospholipon 90G) and an ethanol/water mixture (10/90, v/v), produced particles with the highest MD (142 nm), and the lowest PI (0.19) and EE (72%). Furthermore, it was observed that increasing the phospholipid concentration led to a reduction in vesicle size and a slight increase in PI. EE exceeded 94% when phospholipid content was above 4%. Except for formulation H1 which was slightly negative (-12.5 mV), all formulated hyalurosomes exhibited a neutral surface charge, ranging between − 10 and + 10 mV [ 23 ]. After 1 month of storage at 4 ºC, except in formulation H3, particle size remained stable with a variation of less than 4 nm and PI below 0.4 (Fig. 2 ). Similarly, the ZP remained unchanged, in the neutral range (Fig. 3 ). The only formulation that remained stable after 3 months of storage was H2. In vitro Release Studies The percentages of VCZ released over time from the tested formulations are shown in Fig. 3 . Between 85 and 93% of VCZ was released from hyalurosomes within 10 hours. Among the formulations, H3, which contained the highest phospholipid concentration (10%), exhibited the slowest release, with only 40% of VCZ released after 3 hours. In contrast, formulation H1, containing the lowest phospholipid concentration (1%), released approximately twice that amount in the same period. Formulations H2 and H4, both containing 4% of phospholipid, demonstrated intermediate release rates, suggesting that the different solvents used did not significantly influence the release of the active compound. To describe the release kinetics, zero-order, first-order, Higuchi, Kosmeyer–Peppas and Peppas–Shalin models were tested and compared (Table 4 ). Among the applied models, the Peppas-Shalin model exhibited the best fit with the experimental data, giving rise to the highest correlation coefficient values (0.9963–0.9997). As can be observed in Table 4 , the n values obtained were equal or higher than 0.5 in the case of formulations H2, H3 and H4. Therefore, in this formulations, the non-Fick or anomalous diffusion (0.5 < n < 1) process was dominant [ 24 ]. Moreover, as can be seen from the results, the relaxation rate constants (K P−S(2) ) have much lower values than the diffusion rate constants (K P−S(1) ), suggesting that the matrix nature has a relative importance compared to the Fick diffusion. In contrast, formulation H1gave rise to an n value of 0.22, suggesting a Fickian release. Table 4 Parameters obtained from those adjusted using the equations from release kinetics for the models used. Mean ± Standard Error (SE), n = 4. Formulation Model Parameter H1 H2 H3 H4 Zero order r 0.7122 0.8991 0.9596 0.8905 K 0 (%·h − 1 ) 6.29 ± 2.53 7.67 ± 1.52 7.73 ± 0.92 7.99 ± 1.67 First order r 0.9129 0.9643 0.9939 0.9935 K 1 (h − 1 ) 0.23 ± 0.04 0.19 ± 0.02 0.13 ± 0.01 0.20 ± 0.01 Higuchi r 0.8893 0.9820 0.9888 0.9802 K H (%·h − 1/2 ) 26.68 ± 5.60 28.47 ± 2.24 27.05 ± 1.67 29.89 ± 2.46 Korsmeyer-Peppas r 0.9974 0.9863 0.9912 0.9866 n 0.16 ± 0.02 0.42 ± 0.05 0.59 ± 0.05 0.40 ± 0.05 K K−P (h − n ) 66.59 ± 1.70 34.69 ± 3.20 21.52 ± 2.10 37.91 ± 3.37 Peppas-Sahlin r 0.9997 0.9965 0.9963 0.9976 n 0.22 ± 0.01 0.67 ± 0.08 0.86 ± 0.11 0.65 ± 0.06 K P−S(1) (h − n ) 64.12 ± 0.81 29.37 ± 2.05 17.94 ± 1.74 32.28 ± 1.82 K P−S(2) (h − 2n ) ~ 0 ~ 0 ~ 0 ~ 0 Biocompatibility of Formulations The viability of human keratinocytes (Fig. 5 ) incubated for 48 hours with VCZ, either in dispersion or encapsulated in hyalurosomes, was generally higher than 87% at all tested concentrations (0.5, 5, 50, and 500 ng/mL), relative to untreated cells, considered 100% viable. These findings confirm the high biocompatibility of the formulations, in line with established international standards [ 25 ]. Moreover, the results indicate that variations in the vehicle used to prepare the hyalurosomes did not affect their biocompatibility. In vitro Skin Permeation Percentages of VCZ present in the remaining formulation in the washing liquid, in the donor compartment, in the skin and accumulated in the receptor compartment at the end of the transdermal absorption study are summarized in Table 5 . No statistically significant differences between formulations and dispersion were obtained when the percentage of VCZ in the washing liquid was compared. However, the penetration of formulation H1 and dispersion through the skin to the receptor compartment were significantly higher than those of formulations H2, H3 and H4, with higher drug percentages detected after the 10-hour penetration period. When the VCZ load on the skin was compared, statistically significant differences were obtained when formulations H1 and H3 were compared, showing a higher VCZ accumulation in the case of formulation H1. Table 5 VCZ percentage present in the remaining formulation in the washing liquid (% Wash), in the donor compartment (% Donor), in the skin (% Skin) and accumulated in the receptor compartment at the end of the transdermal absorption study (% Receptor). Each value represents the mean value ± SD of four replicates (n = 4). Formulation Dispersion H1 H2 H3 H4 % Wash 2.02 ± 0.71 1.22 ± 0.33 2.21 ± 0.34 1.56 ± 0.43 2.42 ± 0.34 % Donor 87.1 ± 7.1 a 79.9 ± 3.7 a 93.6 ± 4.6 b 94.8 ± 2.1 b 92.3 ± 4.1 b % Skin 0.32 ± 0.13 a,b 0.51 ± 0.27 a 0.12 ± 0.07 a,b 0.10 ± 0.05 b 0.13 ± 0.05ª ,b % Receptor 10.73 ± 7.37 a 18.41 ± 3.05 a 4.08 ± 2.31 b 3.56 ± 2.11 b 7.28 ± 2.14 b The same superscript letter indicates values that are not statistically different (p > 0.05). The study of the drug distribution across skin layers revealed differences between the dispersion and formulations H1 and H2 (Fig. 6 ). In these formulations, VCZ was distributed more homogeneously, reaching the deepest layers of the dermis. This highlights a greater drug load in both the epidermis and dermis, which may represent a more effective reservoir for the treatment of more invasive fungal skin diseases. Antifungal Activity on C. albicans Growth The growth curves of C. albicans in the presence of different VCZ formulations are shown in Fig. 7 A. The final concentration of all formulations in the incubation medium was 50 µg/ml. In this assay, a positive control composed of an aqueous dispersion of VCZ was also tested and it was checked that it provoked the maximum inhibition of the growth. By contrast, negative control marked the absence of inhibition. As can be observed, the growth inhibition achieved by the formulations and the dispersion was similar, with a reduction of approximately 1 log after 24 hours of incubation. Moreover, empty formulations were tested to confirm the absence of antifungal activity from the vehicle; the results are shown in Fig. 7 B. In vivo antifungal activity The antifungal activity of VCZ-loaded hyalurosomes was evaluated in vivo using mice carrying a dense layer of C. albicans on their dorsal skins. According to the guidelines of the ethical committee and aiming at reducing the number of used animals, only the three most promising formulations were tested, being these formulations H1, H2 and H4. The same criterion was used in the case of VCZ-loaded NLCs, only formulations C and D were tested. Saline was used in the control group (no inhibition of C. albicans growth) and the dispersion was employed for comparative purposes. Both the VCZ dispersion and the VCZ-loaded formulations reduced the count of the colonies of C. albicans (CFU) compared to the control group (Fig. 8 ). However, the reduction was significantly greater (p < 0.05) when VCZ was administered as a dispersion or formulated in hyalurosomes than when it was loaded into NLCs. Discussion The development of lipid-based delivery systems loaded with VCZ has garnered significant attention in recent years, primarily to address the drug’s poor aqueous solubility, systemic side effects, and the need for targeted, sustained antifungal action. Among the most prominent VCZ-loaded nanosystems developed for topical, ocular or transungual administration are Solid Lipid Nanoparticles (SLNs) [ 26 , 27 ], NLCs [ 13 , 28 – 32 ] and different types of liposomes [ 33 – 39 ]. In general, they have shown an encouraging performance with regard to particle size, entrapment efficiency and in vitro release. Specifically, VCZ-loaded liposomes seem to offer the most promising features to promote deeper skin penetration, a critical factor for enhancing drug delivery to target sites within the dermis and epidermis [ 34 , 38 , 39 ]. However, the antifungal activity against C. albicans in cutaneous infections of any of these nanovesicles has been tested. In this sense, an important contribution of the present study is the in vitro and in vivo evaluation of the antifungal activity of new VCZ-loaded hyalurosomes against C. albicans . Four different liposome-based formulations were tested in this study (Table 1 ). In all cases, 0.1% HA was incorporated offering unique advantages for topical therapy, including enhanced skin hydration, improved drug penetration and the potential for synergistic wound healing and antifungal effects [ 17 – 19 ]. Despite these benefits, the use of HA in VCZ-loaded liposomes has so far been limited to ocular applications. In this sense, VCZ-loaded cubosomes [ 37 ] and ultradeformable elastosomes [ 40 ] have been developed. The concentrations of HA used in those systems that gave rise to the best results were in the range of 0.2–0.4%. However, a concentration of 0.1% was used in the four formulations developed in this work as a direct relationship between HA % and MD has been described [ 40 , 41 ]. Additionally, polyols such as glycerol and/or ethanol were incorporated into the formulations, representing 10%. These components enhance the solubility of VCZ and contribute to improved skin penetration by increasing the flexibility and elasticity of the liposomal bilayer. As a result, the vesicles become more deformable and capable of penetrating deeper into the skin layers [ 42 ]. Furthermore, the presence of these polyols is associated with a reduction in particle size, which further facilitates dermal delivery [ 43 ]. Another component present in the designed formulations that increase the flexibility of liposomes by destabilizing their lipid bilayer is the non-ionic surfactant polysorbate 80 [ 44 ], that also provides steric stabilization of colloidal systems despite a low ZP [ 45 , 46 ]. The main difference between formulations lies in the proportion of phospholipon® 90G involved, which varies from 1 to 10%. Phospholipon® 90G is a blend of purified phospholipids, derived mainly from soy lecithin. Its main component is phosphatidylcholine (≥ 94%) and may contain small proportions of other phospholipids such as phosphatidylethanolamine and phosphatidylinositol, in addition to a very low amount of triglycerides and free fatty acids. The concentration of this component can influence several characteristics of the resulting hyalurosomes, including MD, EE and drug release behavior. In this sense, an increase in Phospholipon® 90G content led to a reduction in vesicle size (Table 3 ), a trend previously reported by Ahad et al. in the development of eprosartan mesylate-loaded transfersomes [ 47 ]. Table 3 Mean diameter (MD), polydispersity index (PI), zeta potential (ZP) and entrapment efficiency (EE) of empty and VCZ loaded hyalurosomes. Each value represents the mean value ± standard deviation (SD) of three replicates (n = 3). MD (nm) PI ZP (mV) EE (%) Empty Formulation H1 160.1 ± 3.0 a 0.21 − 9.70 ± 0.60 - Formulation H1 142.2 ± 1.0 b 0.19 − 12.50 ± 0.36 71.83 ± 6.74 a Empty Formulation H2 78.9 ± 1.1 c 0.25 − 5.06 ± 0.31 - Formulation H2 101.3 ± 2.2 d 0.30 − 7.32 ± 0.20 94.54 ± 2.41 b Empty Formulation H3 64.1 ± 0.5 e 0.42 − 3.86 ± 0.12 - Formulation H3 90.6 ± 1.7 f 0.37 1.60 ± 0.30 94.16 ± 0.64 b Empty Formulation H4 91.6 ± 0.9 f 0.39 1.81 ± 0.19 - Formulation H4 88.2 ± 0.5 f 0.39 0.88 ± 0.62 96.94 ± 1.36 b The same superscript letter indicates values that are not statistically different (p > 0.05). Similarly, EE was found to be dependent on the phospholipid concentration, with the lowest value (72%) observed at the lowest tested concentration (1% of Phospholipon® 90G) (Table 3 ). This enhancement of EE with increasing phospholipid content is likely related to the hydrophobic nature of VCZ, which favors its incorporation into the lipid bilayer through hydrophobic interactions, thereby improving drug retention within the vesicles [ 39 ]. Despite the differences observed in size and EE between formulations, no relevant practical impact would be expected, since all obtained hyalurosomes had MD lower than 140 nm, aligning with the optimal size range for enhanced deposition in the epidermis and dermis (reported to be below 300 nm [ 48 , 49 ] or around 100–150 nm [ 32 , 50 , 51 ], depending on the source), and high EE values (above 72%). Additionally, the slight size variations observed after at least 3 months of storage at 4°C did not compromise the particle size suitability for topical skin administration (MD < 180 nm), considering the above reported optimal size. Regarding VCZ release kinetics from hyalurosomes, an increase in the n-index was observed with increasing Phospholipon® 90G content (Table 4 ). This trend suggests a shift in the dominant release mechanism from Fickian diffusion, associated with a less compact and more permeable bilayer structure, to mechanisms in which matrix relaxation and/or restructuring of the lipid bilayer play a more significant role. At lower phospholipid content, hyalurosomes display a greater structural disorder, the bilayer is less compact and more permeable, facilitating drug diffusion. In fact, formulation H1 (1% of phospholipon® 90G) exhibited an n-value of 0.22, indicative of a Fickian release mechanism, governed by diffusion and dependent on concentration gradient. Conversely, as the phospholipid content increases, the vesicle structure becomes more compact and stable, reducing diffusion and promoting alternative release mechanisms. This behavior was evident in formulations H2, H3, and H4. In formulations H2 and H4 (4% Phospholipon® 90G), n = 0.6, indicating anomalous (non-Fickian) diffusion, in which both diffusion and matrix relaxation contribute to drug release. Formulation H3 (10% Phospholipon® 90G) presented an n-value of 0.86, suggesting a release mechanism closer to relaxation- and/or erosion-controlled transport (anomalous or overlapping Case II), with drug release predominantly driven by structural reorganization of the lipid bilayer, swelling of hyaluronic acid, or slow disintegration of the vesicle. Therefore, in the proposed formulations, to achieve prolonged release profiles (K P−S(1) = 17.94 h − n ), a higher proportion of phospholipid is recommended and, if desired to promote a faster release (K P−S(1) = 64.12 h − n ), a lower proportion of Phospholipon® 90G is preferable. Consistent with the release rate, in vitro skin permeation studies revealed a significantly higher concentration of VCZ in the receptor compartment for the formulation with the fastest release (formulation H1), compared to the other formulations developed (Table 5 ). This effect could be attributed to the rapid saturation of the skin, allowing faster drug diffusion through the tissue. Moreover, formulation H1 demonstrated increased VCZ deposition in the deeper layers of the skin compared to the dispersion (Fig. 6 ), probably due to the smallest concentration of Phospholipon® 90G. The inverse correlation observed in our study aligns with findings by Montenegro et al. [ 52 ], who reported that higher concentrations of this phospholipid reduce liposome flexibility and, consequently, their ability to penetrate through the stratum corneum and epidermis. However, although formulations H2 and H4 share the same phospholipid concentration, the higher ethanol content in formulation H2 (10%) enhanced VCZ penetration into deeper skin layers by disrupting stratum corneum lipids and increasing vesicle deformability [ 53 ]. In contrast, formulation H4, with less ethanol (2.5%) and more glycerin (7.5%), promoted retention in the stratum corneum, as glycerin improves hydration but lacks the lipid-disruptive properties needed for deeper permeation [ 54 ]. Despite the differences between formulations, all of them were biocompatible with human keratinocytes and demonstrated antifungal activity against C. albicans comparable to that of VCZ dispersion. However, the stability of formulation H3 (containing the highest phospholipid concentration) was the most compromised, showing loss of size homogeneity after 1 month (PI > 0.6), and significantly lower skin retention compared to formulation H1 (0.1% vs. 0.51%, respectively). Consequently, formulation H3 was excluded from in vivo testing. In addition, in vivo assays confirmed the promising properties of formulations H1, H2 and H4, giving rise to a significant reduction in C. albicans growth compared to the control group (untreated). However, although VCZ-loaded NLCs previously designed by our research group exhibited favorable characteristics [ 13 ], they failed to significantly reduce C. albicans colony-forming units (CFU) when administered in mice. These findings highlight the superior performance of hyalurosomes over NLCs for topical antifungal therapy. The main differences between VCZ-loaded hyalurosomes and NLCs involve higher EE (mean values: 89% vs. 77%, respectively) and slower release rate (complete release at 10 vs. 6 hours, respectively), and higher flexibility, which taken together favored higher penetration depth (mean value of VCZ retained in the dermis of the formulations that penetrated deeper into the skin: 41% vs. 24%, respectively) and enhanced accumulation in the receptor compartment (mean values: 8.3% vs. 1.7%, respectively). These results are consistent with those reported by Santos et al. [ 38 ], who evidenced the importance of the transfollicular route for VCZ topical delivery from liposomes and attributed the faster release of VCZ from NLCs to the drug’s lower affinity for the lipid components of NLCs compared to the phospholipids in liposomes. Additionally, the slightly hydrophilic nature of VCZ favors its retention within the internal aqueous core of liposomes [ 33 ]. Conclusions This study demonstrates that hyalurosomes represent a promising next-generation nanosystem for the topical delivery of VCZ, significantly enhancing antifungal activity and promoting deeper drug deposition within the skin. These results highlight the considerable potential of hyalurosomes as an advanced delivery system for the topical treatment of superficial mycoses and, notably, for more invasive forms of cutaneous candidiasis. Furthermore, by facilitating targeted local therapy and potentially reducing the risk of systemic complications, hyalurosomes address a critical need for safer and more effective antifungal interventions. Such innovation directly aligns with the World Health Organization's global health priorities, emphasizing the urgent demand for novel solutions in the management of fungal infections by C. albicans . Declarations Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of University Valencia (2024-VSC-PEA-0081). Consent for publication Not applicable. Availability of data and materials All data and materials generated or analyzed during this study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests in this work. Funding This work was supported by Generalitat Valenciana through project CIGE/2022/112. Authors’ contributions Conceptualization: A.N., J.E.P. and I.U.; investigation: A.N., J.E.P., R.T.V. and I.U.; data curation: A.N, O.D.S., M.L.M., M.M. and I.U.; methodology and supervision: I.U. and J.E.; writing - original draft: A.N, J.E.P., O.D.S., R.T.V., M.L.M., M.M. and I.U.; writing - review & editing. I.U. and J.E. All authors have read and agreed to the published version of the manuscript. 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Cite Share Download PDF Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Major Revisions Needed 20 Sep, 2025 Reviewers agreed at journal 28 Aug, 2025 Reviewers invited by journal 27 Aug, 2025 Editor assigned by journal 06 Aug, 2025 First submitted to journal 01 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7270257","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506613961,"identity":"105133d6-41f9-4bcd-9ba6-e85484f51cd1","order_by":0,"name":"Amparo Nácher","email":"","orcid":"","institution":"Universidad de Valencia Facultad de Farmacia: Universitat de Valencia Facultat de Farmacia","correspondingAuthor":false,"prefix":"","firstName":"Amparo","middleName":"","lastName":"Nácher","suffix":""},{"id":506613962,"identity":"168a6c88-2990-4a66-b63f-0fcfb9699c68","order_by":1,"name":"José-Esteban Peris","email":"","orcid":"","institution":"Universidad de Valencia Facultad de Farmacia: Universitat de Valencia Facultat de Farmacia","correspondingAuthor":false,"prefix":"","firstName":"José-Esteban","middleName":"","lastName":"Peris","suffix":""},{"id":506613963,"identity":"08686894-c714-4fa7-9930-06dd05178c18","order_by":2,"name":"Raquel Taléns-Visconti","email":"","orcid":"","institution":"Universidad de Valencia Facultad de Farmacia: Universitat de Valencia Facultat de Farmacia","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Taléns-Visconti","suffix":""},{"id":506613964,"identity":"73ac433c-fa5b-4223-bef5-121c1b861266","order_by":3,"name":"Octavio Díez-Sales","email":"","orcid":"","institution":"Universidad de Valencia Facultad de Farmacia: Universitat de Valencia Facultat de Farmacia","correspondingAuthor":false,"prefix":"","firstName":"Octavio","middleName":"","lastName":"Díez-Sales","suffix":""},{"id":506613965,"identity":"114adda4-c613-4f1d-b8bd-58dc212dc678","order_by":4,"name":"Maria Letizia Manca","email":"","orcid":"","institution":"University of Cagliari: Universita degli Studi Di Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Letizia","lastName":"Manca","suffix":""},{"id":506613966,"identity":"7f21034f-4e00-4972-88c9-b756cf450057","order_by":5,"name":"Maria Manconi","email":"","orcid":"","institution":"University of Cagliari: Universita degli Studi Di Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Manconi","suffix":""},{"id":506613967,"identity":"9ef06e9e-e7d6-42c0-80f6-3d523c21a0ec","order_by":6,"name":"Iris Usach","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACNgbGBghLgvkAyVrYEki1T4LHgDiFfNKHmz8wttXJ88/u+SbNw2BjT9hhfIltEoxthw1n3Dm7DaglLbGBoBYexjYGxrYDjBskcrdJzmA4nEDYFh5GsMPsN0jkPANq+U+Ew3gYG4AOY04EamGT+MBwABaA+B0mkXDucPKMG2nGFh8Mkgn7Rb6H/fGHD2V1tv0zkh/eSKiwI+wwMEiAs4iMmlEwCkbBKBgFBAAAbnEzBEtzDPIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5786-5668","institution":"Universidad de Valencia Facultad de Farmacia: Universitat de Valencia Facultat de Farmacia","correspondingAuthor":true,"prefix":"","firstName":"Iris","middleName":"","lastName":"Usach","suffix":""}],"badges":[],"createdAt":"2025-08-01 10:24:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7270257/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7270257/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-025-02007-3","type":"published","date":"2025-11-10T15:58:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90611335,"identity":"9fe916af-9fee-4791-9e69-1cea5dca8200","added_by":"auto","created_at":"2025-09-04 17:06:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1520166,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy (TEM) images of empty formulation H3 (A and B) and VCZ-loaded formulation H3 (C and D).\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/0aecdbeffe4134948ca61558.jpg"},{"id":90612085,"identity":"d8b7a03a-589e-455f-be82-cbc1cdff0b62","added_by":"auto","created_at":"2025-09-04 17:14:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":197593,"visible":true,"origin":"","legend":"\u003cp\u003eMean diameter (MD) and polydispersity index (PI) of VCZ loaded hyalurosomes stored for 3 months at 4 °C. Data are reported as mean values ± standard deviations (SD) (n = 3). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/9ee212eab2d898ee62a9e354.jpg"},{"id":90611333,"identity":"6fe379b6-7308-484a-87c5-2d08c35acf68","added_by":"auto","created_at":"2025-09-04 17:06:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94472,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential (ZP) of VCZ loaded hyalurosomes stored for 3 months at 4 °C. Data are reported as mean values ± SD (n = 3).\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/1e8d4d8f5991da338b45a8bf.jpg"},{"id":90611336,"identity":"20ca4d67-9d26-4a5f-8892-c84f7b6eeac6","added_by":"auto","created_at":"2025-09-04 17:06:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":883721,"visible":true,"origin":"","legend":"\u003cp\u003eRelease profiles of VCZ from the different formulations over 10 hours (mean + S.D., n=4). The curves show the predicted values using the Peppas-Sahlin equation.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/b5c496cd58fe216c7a7dcaa3.jpg"},{"id":90611339,"identity":"42b8a695-26b9-4a23-96cd-ccf6aea2916c","added_by":"auto","created_at":"2025-09-04 17:06:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1017461,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of HaCaT cells treated for 48 h with VCZ in dispersion or loaded in hyalurosomes diluted to reach 500, 50, 5 or 0.5 ng/ml of VCZ. Data are reported as mean values (n=17) ± SD of cell viability expressed as the percentage of untreated cells (100 % of viability).\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/e5504ba7b31509178c1dfcb4.jpg"},{"id":90612086,"identity":"d1cb8750-124b-40f7-8b1e-ac1dc80de96e","added_by":"auto","created_at":"2025-09-04 17:14:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":930253,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of VCZ per gram of tissue accumulated into skin layers after the administration of formulations and VCZ dispersion, following a 10-hour penetration period. Data are reported as mean values (n=4) + SD. Symbols indicate that the % VCZ/g of skin treated with formulations is statistically different from that of skin treated with the dispersion. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/dd4272ac3e3c7b40958b58b4.jpg"},{"id":90611341,"identity":"66d870ba-9f04-42fd-8181-eafbd15bd919","added_by":"auto","created_at":"2025-09-04 17:06:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":132803,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of \u003cem\u003eC. albicans \u003c/em\u003eobtained in the presence of different formulations with (A) or without VCZ (B). The curves corresponding to the positive (VCZ 50 µg/mL) and negative (without VCZ) controls are also shown for comparative purposes. Except in the negative control, the VCZ concentration in the incubation medium was 50 µg/mL. Mean + SD, n=6.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/b41f4f356fd5038cdaa80793.jpg"},{"id":90612088,"identity":"f18a8246-897f-4b2a-b01f-e08643f92944","added_by":"auto","created_at":"2025-09-04 17:14:19","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":967669,"visible":true,"origin":"","legend":"\u003cp\u003eTotal count of \u003cem\u003eC. albicans\u003c/em\u003e(CFU) in the skin samples of mice 24 h after the administration of formulations H1, H2, H4 and VCZ dispersion. Moreover, results obtained in the case of administration of NLCs have been included (formulations C and D). Symbols indicate statistically different values from control group (saline). Mean + SD, n=5. **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/66d9ae478ab93bb324182a8f.jpg"},{"id":96105116,"identity":"829bcdea-1411-4b39-b804-a0caf4535f72","added_by":"auto","created_at":"2025-11-17 16:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6824428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7270257/v1/27f0c364-7a24-437e-a1be-3b93fb4cd046.pdf"}],"financialInterests":"","formattedTitle":"Novel Voriconazole-Loaded Hyalurosomes Optimized for Enhanced Skin Penetration and Antifungal Activity against Candida albicans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin fungal infections, or dermatomycoses, rank among the most prevalent infectious diseases, with estimates suggesting that more than a quarter of the world\u0026rsquo;s population will experience a dermatomycosis at some stage in their lifetime [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent trends indicate a rising incidence and severity of these conditions, primarily driven by risk factors such as the widespread use of broad-spectrum antibiotics, antineoplastic agents and immunosuppressive drugs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This includes paediatric patients with leukaemia, who are at increased risk of developing chronic disseminated candidiasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Importantly, these infections are not limited to immunocompromised patients as fully healthy individuals, including athletes and gym users, are also frequently affected, underscoring the substantial clinical burden and propensity for recurrence that dermatomycoses generate [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], thus posing a significant clinical challenge. In this context, candidiasis is a common fungal infection mostly caused by yeasts of the \u003cem\u003eCandida species\u003c/em\u003e, mainly \u003cem\u003eCandida albicans\u003c/em\u003e. In fact, it was classified as one of the four highest priority fungi for global public health in the first-ever fungal priority pathogens list published by WHO in 2022 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Candidiasis can affect various parts of the body, ranging from superficial mucocutaneous infections to invasive systemic disease, which may involve multiple organ systems and become life-threatening, particularly in vulnerable patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this sense, in April 2025, WHO also published its first-ever reports on fungal tests and treatments, showing the urgent need for innovative research and development [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe most widely used antifungal agents are included in the class of azoles, especially imidazoles. Voriconazole (VCZ) a second-generation triazole agent approved in 2002 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], specifically inhibits the enzyme 14α-demethylase, essential for the synthesis of ergosterol, a component of the fungal cell membrane. By blocking ergosterol production, VCZ destabilizes the cell membrane and leads to fungal cell death [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, VCZ, as many azoles, is poorly water soluble, which limits its bioavailability and antifungal effect. Formulation and drug delivery strategies could improve water-based hydration and dissolution properties, thus increasing its pharmacokinetics and a sustained release will prolong the retention of a high concentration of the azole localized at the infection site, therefore enhancing its bioavailability and therapeutic efficacy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor cutaneous candidiasis, topical dosage forms are preferred, mainly due to site-specific drug delivery. The problem with conventional marketed formulations, based on gels, powders, creams, solutions or foams of imidazoles, is that high and repeated doses have to be administered, which may result in local or even systemic toxicity reducing patient adherence [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, their efficacy is limited in treating more invasive fungal infections [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, the increasing prevalence of drug-resistant strains has further compromised the efficacy of currently available formulations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, the risks of more invasive and deadly infections among the general population have also increased. Hence, the need to develop new topical delivery systems for new-generation, broad-spectrum antifungals, such as VCZ, to overcome biopharmaceutical challenges associated with conventional drug delivery systems like poor retention and low bioavailability.\u003c/p\u003e\u003cp\u003eIn this context, nanocarrier systems are gaining relevance due to their ability to enhance the diffusion of active ingredients across the skin barrier [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among them, we demonstrated that nanostructured lipid carriers (NLCs) loaded with VCZ showed biocompatibility with human keratinocytes and improved the penetration and distribution of VCZ into deeper skin layers, offering superior antifungal activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, the vehicle composition of a topical delivery system may significantly affect drug release and skin penetration, thereby affecting biological activity. In this sense, liposomes have been successfully used to improve the topical efficacy of some drugs, proteins and natural ingredients by modifying their local penetration capacity into the skin and their biodistribution [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Hyalurosomes are a special type of liposomes, enriched with hyaluronic acid (HA), which enhance the penetration of drugs into the deeper skin layers. In addition, HA acts as a gelling agent and skin regenerator, prolonging the action of the treatment and favoring skin recovery [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It also intervenes by improving the epithelial barrier and the innate immune response, which could protect the skin from microbial infections. In fact, HA has been shown to possess antimicrobial activity against \u003cem\u003eCandida\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aim of this study is the design and characterization of novel nanosystems for voriconazole delivery enriched with HA, thus offering a novel strategy for the topical management of candidiasis. If successful, our approach could not only increase the efficacy of VCZ but also address current challenges such as poor retention, low bioavailability and the emergence of drug resistance, ultimately reducing systemic complications that could lead to hospitalizations and associated deaths.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eReactants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe following compounds were purchased from Sigma-Aldrich (Madrid, Spain): dimethyl sulfoxide (DMSO), RPMI 1940 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium salt. Polysorbate 80 was obtained from Guinama (Valencia, Spain) and ethanol was purchased from PanReac Applichem (Barcelona, Spain). VCZ and hyaluronic acid (HA) (supplied as sodium hyaluronate, molecular weight 200\u0026ndash;400 KDa) were provided by Glentham Life Sciences (Corshman, UK). Sabouraud dextrose agar (SDA) and acetonitrile (AcN) were obtained from VWR chemicals (Barcelona, ​​Spain). 4-Morpholinepropanesulfonic acid (MOPS) came from Alfa Aesar (Kandel, Germany) and Eugon LT 100 from BIOKAR Diagnostics (Beauvais, France). Glucose and cyclophosphamide monohydrate were obtained from Thermo scientific (Madrid, Spain). Phospholipon\u0026reg; 90 G was provided by Lipoid GmbH (Ludwigshafen, Germany) and glycerin from Acofarma (Madrid, Spain). Cell medium, fetal bovine serum (FBS), penicillin, streptomycin, fungizone and all the other reagents for cell studies, unless otherwise specified, were purchased from Gibco (Paisley, UK).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalytical Method\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVCZ was quantified by means of a high-performance liquid chromatography assay (HPLC) with ultraviolet (UV) detection at 255 nm, as described previously by our research group [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A Waters \u0026ldquo;Nova-Pack\u0026rdquo; C\u003csub\u003e18\u003c/sub\u003e analytical column (4 \u0026micro;m, 3.9 mm \u0026times; 150 mm) was used, and the mobile phase consisted of a mixture of acetonitrile, water and 0.6% trietylamine, pH 6 (35/65, v/v). The injection volume was 25 \u0026micro;L, and the flow rate was 1 mL/min. The HPLC equipment consisted of a quaternary pump SpectraSYSTEM P4000, an autosampler SpectraSYSTEM AS3000 and a spectrophotometric detector SpectraSYSTEM UV 6000LP. Data were processed through \u0026ldquo;Chromquest Chromatography Workstation Software Version 1.63\u0026rdquo;.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of hyalurosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFour VCZ-loaded hyalurosome formulations (H1\u0026ndash;H4) were developed and selected based on their promising physicochemical characteristics. The main differences among them were the phospholipid concentration and the composition of the hydrating solvent mixtures (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComposition of VCZ-loaded hyalurosomes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eFormulation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComponent (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eH2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eH4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSodium hyaluronate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePhospholipon\u0026reg; 90G\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePolysorbate 80\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGlycerin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEthanol\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWater\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVoriconazole\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe components of each formulation were mixed under magnetic stirring at 200 rpm and then hydrated overnight with 10 mL of either ethanol/water (10:90, v/v) for H1, H2, and H3, or glycerol/ethanol/water (7.5:2.5:90, v/v/v) for H4. Thereafter, formulations were sonicated with 3 cycles of 5 minutes (5 s on and 2 s off, 60% and 45% amplitude) with an ultrasonic disintegrator (CY-500, Optic Ivymen system, Barcelona, Spain) to homogenize the preparation. Finally, formulations were sterilized by filtration (CA syringe filter; cellulose acetate; 0.22 \u0026micro;m). The final concentration of VCZ in hyalurosomes was 0.5 mg/ml. Empty hyalurosomes were also prepared and used as controls for physicochemical characterization and antimicrobial testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of hyalurosomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTransmission electron microscopy (TEM) was used to confirm vesicle formation and evaluate the morphology. The samples were stained with 2% phosphotungstic acid aqueous solution and examined under a JEM-1010 (Jeol Europe, Paris, France) transmission electron microscope equipped with a digital camera AMT RX80 and the AmtV602 software, version 602.579 at an accelerating voltage of 80 kV.\u003c/p\u003e\u003cp\u003ePhoton correlation spectroscopy was used to analyze the mean diameter (MD) and polydispersity index (PI) using a Zetasizer nano (Malvern Instruments, Worcestershire, UK). The same equipment was also used to measure the zeta potential (ZP) by means of the M3-PALS (Phase Analysis Light Scattering) technique, which measures particle electrophoretic mobility.\u003c/p\u003e\u003cp\u003eThe MD, PI and ZP were monitored over 3 months of storage at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C to evaluate the stability of the formulations.\u003c/p\u003e\u003cp\u003eThe entrapment efficiency (EE) was calculated as the percentage of the concentration of VCZ after dialysis versus that initially used [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. VCZ-loaded hyalurosomes (1 mL) were loaded into the dialysis tube (Spectra/Por\u0026reg; membranes, 12\u0026ndash;14 kDa MW cut-off, 3 nm pore size; Spectrum Laboratories Inc., DG Breda, the Netherlands) and kept at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) in 100 mL of distilled water under continuous stirring for 10 h.\u003c/p\u003e\u003cp\u003eAfter reaching the dialysis equilibrium, a sample of 0.5 mL of the exterior aqueous medium was taken and added to 0.5 mL of AcN. The mixture was injected into the HPLC to determine the VCZ concentration in the external aqueous medium, which is assumed to be equal to the free (unencapsulated) VCZ concentration in the hyalurosomes. Additionally, the total VCZ concentration inside the dialysis tube was quantified by HPLC after disrupting the vesicles with AcN (1/10).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro Release Study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe release studies of the hyalurosomes were conducted using the dialysis membrane method. 1 mL of VCZ-loaded hyalurosomes was loaded into a Spectra/Por\u0026reg; 2 standard regenerated cellulose dialysis tube with an MWCO of 12\u0026ndash;14 kDa and clipped by standard closures. The dialysis tube was immersed into 100 mL of distilled water with a magnetic stirrer stirring at 300 rpm. At 0, 1, 2, 3, 4, 6, 8 and 10 hours, 0.5 mL of the medium were removed and replaced with an equal volume of distilled water. VCZ released amounts were determined by HPLC.\u003c/p\u003e\u003cp\u003eTo determine the release kinetics of VCZ from hyalurosomes, different commonly used mathematical models including zero order, first order, Higuchi, Korsmeyer\u0026ndash;Peppas and Peppas\u0026ndash;Sahlin were used. The values of the kinetic parameters were obtained by using Sigmaplot 10.0\u0026reg; (Systat Software, Inc., San Jose, CA, USA). The equations that represent each drug release model are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEquations of the models used for fitting drug release data.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEquation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZero order\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{t}={{Q}_{0}+K}_{0}\\bullet\\:t\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFirst order\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{t}={Q}_{0}\u0026middot;{e}^{-{K}_{1}\u0026middot;t}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHiguchi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{t}/{Q}_{\\infty\\:}={K}_{H}\\bullet\\:\\surd\\:t\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKorsmeyer-Peppas\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{t}/{Q}_{\\infty\\:}={K}_{K-P}\\bullet\\:{t}^{n}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeppas-Sahlin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{t}/{Q}_{\\infty\\:}={K}_{P-S\\left(1\\right)}\\bullet\\:{t}^{n}+\\:{K}_{P-S\\left(2\\right)}\\bullet\\:{t}^{2n}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eQ\u003csub\u003et\u003c/sub\u003e: amount of drug released over time t; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{0}\\)\u003c/span\u003e\u003c/span\u003e: initial amount of drug in the formulation, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Q}_{t}/{Q}_{\\infty\\:}\\)\u003c/span\u003e\u003c/span\u003e: fraction of drug released; K\u003csub\u003e0\u003c/sub\u003e, K\u003csub\u003e1\u003c/sub\u003e, K\u003csub\u003eH\u003c/sub\u003e: release rate constants for zero-order, first-order and Higuchi release kinetics, respectively; K\u003csub\u003eK\u0026minus;P\u003c/sub\u003e and K\u003csub\u003eP\u0026minus;S(1)\u003c/sub\u003e: diffusion constants, K\u003csub\u003eP\u0026minus;S(2)\u003c/sub\u003e: relaxation constant; n: exponent that characterizes the diffusion process.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro Cytotoxicity of Formulations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmortalized human keratinocytes (HaCaT, ATCC, Manassas, VA, USA) were cultured as monolayers in 75 cm\u0026sup2; flasks using Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) with low glucose (1 g/L), sodium pyruvate and GlutaMAX, supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (10,000 U/mL penicillin, 10,000 \u0026micro;g/mL streptomycin) and 0.1% fungizone. Cells were maintained under standard incubation conditions of 37\u0026deg;C, 5% CO₂, and saturated humidity.\u003c/p\u003e\u003cp\u003eFor experimental procedures, cells were seeded into 96-well plates at a density of 7.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and allowed to adhere for 24 hours. Subsequently, cells were exposed to VCZ, either in aqueous dispersion or encapsulated within hyalurosomes. The formulations were diluted in cell culture medium to obtain final concentrations of 0.5, 5, 50, and 500 ng/mL. These concentrations were selected to mimic potential \u003cem\u003ein vivo\u003c/em\u003e dilution scenarios, with 500 ng/mL considered a likely maximum concentration of VCZ capable of reaching deeper skin layers [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After 48 hours of incubation with the treatments, the culture medium was replaced with an MTT solution (0.5 mg/mL in phosphate-buffered saline, PBS). Following a 3-hour incubation period, the MTT solution was removed and the resulting formazan crystals were dissolved using DMSO. Absorbance was then measured at 570 nm using a Multiskan EX microplate reader (Thermo Scientific, Waltham, MA, USA). All experiments were performed in six independent runs, each conducted in triplicate. Cell viability results are expressed as percentages relative to untreated control cells (set at 100%).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro Skin Permeation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe transdermal permeation of VCZ, either in aqueous dispersion or encapsulated in hyalurosomes, was investigated using vertical Franz diffusion cells (Vidrafoc, Barcelona, Spain), featuring an effective diffusion area of 0.785 cm\u0026sup2; and a receptor chamber volume of approximately 6 mL. Donor compartments were filled with 500 \u0026micro;L of VCZ-loaded formulations at a concentration of 0.5 mg/mL, while the receptor compartments were filled with 0.9% sodium chloride solution. Dermatomed porcine ear skin, with a uniform thickness of 600 \u0026micro;m, was positioned between the donor and receptor chambers. The skin samples were obtained from pig ears supplied by the Faculty of Medicine at the University of Valencia (Valencia, Spain), collected post-mortem from animals previously used in unrelated research protocols. The assembled diffusion cells were maintained in a thermostatic water bath set at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, ensuring that the skin surface remained at a physiological temperature of 32\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, with continuous magnetic stirring throughout the experiment. After a 10-hour permeation period, the amount of VCZ present in the donor as well as receptor compartments was quantified using an HPLC. Prior to skin extraction, the surface was washed with 0.5 mL of AcN/H₂O (50:50) to remove any formulation remaining on the surface that had not been absorbed. Subsequently, cryostat sectioning of the treated skin samples was performed (Leica CM1950) to obtain slices being 10, 40, 100 and 450 \u0026micro;m thickness, simulating stratum corneum, epidermis, superficial dermis and deep dermis, respectively. The drug content within the skin layers was then extracted with AcN and analyzed by HPLC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobial Strains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antifungal activity of VCZ both in aqueous dispersion and formulated as hyalurosomes was tested against \u003cem\u003eCandida albicans\u003c/em\u003e (CECT 1394). Cultures were kept for 24 h at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. After 24 h of incubation, the fungal suspensions were diluted with PBS in order to obtain an adequate density expressed as colony forming units per milliliter (CFU/ml).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro Antifungal Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe procedure followed to carry out this type of test was described previously by our research group [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, vials with 500 \u0026micro;l of a 1/5 diluted VCZ-based formulations and 500 \u0026micro;l of inoculum containing 1\u0026ndash;5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/mL were used. Thus, the concentration of VCZ in the diluted formulations was 100 \u0026micro;g/mL and the final concentration in the assay vials was 50 \u0026micro;g/mL. The assays also included a positive control composed of an aqueous dispersion of VCZ (50 \u0026micro;g/mL), in which maximal \u003cem\u003eC. albicans\u003c/em\u003e inhibition was expected, and a negative control containing 500 \u0026micro;l of water. All vials were incubated at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C, taking samples from each vial at 0, 6, 24 and 48 hours. To collect them, 50 \u0026micro;L of each were diluted in 5 ml of PBS and serial decimal dilutions were subsequently prepared and seeded in Petri dishes with SDA. Following incubation at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C for 48 hours, CFU were enumerated. The total \u003cem\u003eC. albicans\u003c/em\u003e burden in each sample was calculated based on plates exhibiting 30 to 300 colonies, with appropriate adjustments for dilution factors and plated volumes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo Antifungal Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProtocols for the \u003cem\u003ein vivo\u003c/em\u003e studies using mice, were approved by the Animal Care Committee of the Faculty of Pharmacy at the University of Valencia (Spain) [reference: 2024-VSC-PEA-0081]. Male 5\u0026ndash;6 weeks old ICR (CD-1) mice, weighing 30\u0026ndash;35 g, (Envigo, Barcelona, Spain), were obtained from the animal facility of the Faculty of Pharmacy at the University of Valencia and were kept in a clean room at a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, a relative humidity of 60% and a light/dark cycle of 12 h. Mice were fed a standard laboratory diet and had access to water ad libitum.\u003c/p\u003e\u003cp\u003eThe methodology employed for these \u003cem\u003ein vivo\u003c/em\u003e studies was based on a procedure previously published by our research team [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, to induce immunosuppression prior to fungal infection, mice received intraperitoneal injections of cyclophosphamide at a dose of 100 mg/kg/day for three consecutive days. On the final day of immunosuppressive treatment, a working culture of \u003cem\u003eC. albicans\u003c/em\u003e, grown for 24 hours at 35\u0026deg;C on SDA, was used to prepare a yeast suspension containing 10⁷ CFU/mL in a mixture of RPMI 1640 and yeast extract-peptone-dextrose (YPD) medium (50/50, v/v). On the same day, the dorsal area of each mouse was shaved using an electric clipper. A 100 \u0026micro;L aliquot of the \u003cem\u003eC. albicans\u003c/em\u003e suspension was then applied to the shaved area using a custom-designed cylindrical plastic chamber (4.5 mm i.d. \u0026times; 6 mm height), which was affixed to the skin with cyanoacrylate adhesive to maintain localized contact of the suspension with the skin surface under aerobic conditions. 24 hours post-inoculation, the infected skin areas were treated with 100 \u0026micro;L of one of the following formulations: normal saline (control), a VCZ aqueous dispersion or VCZ-loaded hyalurosomes. Additionally, in order to compare the influence of the nanocarrier system in the antifungal activity of VCZ, the most promising VCZ-loaded NLCs previously developed by our research group in a previous work (formulations C and D) were evaluated [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. All animals were sacrificed after 24 hours and the corresponding skin regions were excised. The surface of each skin sample was scraped and transferred to 1 mL of Eugon LT100 broth in microcentrifuge tubes. Samples were vortexed for 30 seconds and centrifuged at 2,000 \u0026times; g for 5 minutes. The supernatant was discarded and the pellet was resuspended in 1 mL of fresh Eugon LT100 broth. This wash step was repeated once. The resulting pellet was finally resuspended in 1 mL of Eugon LT100 broth and serial tenfold dilutions were prepared using the same medium. An adequate volume of sample was seeded in Petri dishes with SDA, plates were incubated at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for 48 hours and the colonies observed were counted.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The Student\u0026rsquo;s t test was used for two-group comparison. One-way analysis of variance (ANOVA) was used for comparisons of more than two groups; when statistically significant differences were found, Tukey\u0026rsquo;s test was applied to determine which groups were statistically different. P values of \u0026lt;\u0026thinsp;0.05 were considered statistically significant. All calculations were performed with IBM SPSS Statistics 26 (SPSS Inc., Chicago, IL).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of hyalurosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVCZ-loaded formulations were mainly multilamellar, as detected by TEM analyses (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The hyalurosomes were small in size, spherical shape and slightly aggregated. Empty formulations were also prepared in order to assess the effect of VCZ on hyalurosomes assembly (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eThe physicochemical properties of hyalurosomes were evaluated measuring the mean diameter (MD), polydispersity index (PI), zeta potential (ZP) and entrapment efficiency (EE) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Nanovesicles with sizes ranging from 64 to 160 nm and PI values between 0.19 and 0.42 were obtained by varying the concentrations of phospholipid and co-solvents. Formulation H1, prepared with the lowest phospholipid concentration (1% Phospholipon 90G) and an ethanol/water mixture (10/90, v/v), produced particles with the highest MD (142 nm), and the lowest PI (0.19) and EE (72%). Furthermore, it was observed that increasing the phospholipid concentration led to a reduction in vesicle size and a slight increase in PI. EE exceeded 94% when phospholipid content was above 4%. Except for formulation H1 which was slightly negative (-12.5 mV), all formulated hyalurosomes exhibited a neutral surface charge, ranging between \u0026minus;\u0026thinsp;10 and +\u0026thinsp;10 mV [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eAfter 1 month of storage at 4 \u0026ordm;C, except in formulation H3, particle size remained stable with a variation of less than 4 nm and PI below 0.4 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, the ZP remained unchanged, in the neutral range (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The only formulation that remained stable after 3 months of storage was H2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro Release Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentages of VCZ released over time from the tested formulations are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Between 85 and 93% of VCZ was released from hyalurosomes within 10 hours. Among the formulations, H3, which contained the highest phospholipid concentration (10%), exhibited the slowest release, with only 40% of VCZ released after 3 hours. In contrast, formulation H1, containing the lowest phospholipid concentration (1%), released approximately twice that amount in the same period. Formulations H2 and H4, both containing 4% of phospholipid, demonstrated intermediate release rates, suggesting that the different solvents used did not significantly influence the release of the active compound.\u003c/p\u003e\n\u003cp\u003eTo describe the release kinetics, zero-order, first-order, Higuchi, Kosmeyer\u0026ndash;Peppas and Peppas\u0026ndash;Shalin models were tested and compared (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the applied models, the Peppas-Shalin model exhibited the best fit with the experimental data, giving rise to the highest correlation coefficient values (0.9963\u0026ndash;0.9997). As can be observed in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the \u003cem\u003en\u003c/em\u003e values obtained were equal or higher than 0.5 in the case of formulations H2, H3 and H4. Therefore, in this formulations, the non-Fick or anomalous diffusion (0.5\u0026thinsp;\u0026lt;\u0026thinsp;n\u0026thinsp;\u0026lt;\u0026thinsp;1) process was dominant [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, as can be seen from the results, the relaxation rate constants (K\u003csub\u003eP\u0026minus;S(2)\u003c/sub\u003e) have much lower values than the diffusion rate constants (K\u003csub\u003eP\u0026minus;S(1)\u003c/sub\u003e), suggesting that the matrix nature has a relative importance compared to the Fick diffusion. In contrast, formulation H1gave rise to an \u003cem\u003en\u003c/em\u003e value of 0.22, suggesting a Fickian release.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eParameters obtained from those adjusted using the equations from release kinetics for the models used. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error (SE), n\u0026thinsp;=\u0026thinsp;4.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZero order\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8905\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003e0\u003c/sub\u003e (%\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFirst order\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9939\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9935\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003e1\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHiguchi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9888\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9802\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003eH\u003c/sub\u003e (%\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1/2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.68\u0026thinsp;\u0026plusmn;\u0026thinsp;5.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKorsmeyer-Peppas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9863\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9912\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9866\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003eK\u0026minus;P\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;n\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeppas-Sahlin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9976\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003eP\u0026minus;S(1)\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;n\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003eP\u0026minus;S(2)\u003c/sub\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;2n\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e~\u0026thinsp;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e~\u0026thinsp;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e~\u0026thinsp;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e~\u0026thinsp;0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocompatibility of Formulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe viability of human keratinocytes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) incubated for 48 hours with VCZ, either in dispersion or encapsulated in hyalurosomes, was generally higher than 87% at all tested concentrations (0.5, 5, 50, and 500 ng/mL), relative to untreated cells, considered 100% viable. These findings confirm the high biocompatibility of the formulations, in line with established international standards [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, the results indicate that variations in the vehicle used to prepare the hyalurosomes did not affect their biocompatibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro Skin Permeation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePercentages of VCZ present in the remaining formulation in the washing liquid, in the donor compartment, in the skin and accumulated in the receptor compartment at the end of the transdermal absorption study are summarized in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. No statistically significant differences between formulations and dispersion were obtained when the percentage of VCZ in the washing liquid was compared. However, the penetration of formulation H1 and dispersion through the skin to the receptor compartment were significantly higher than those of formulations H2, H3 and H4, with higher drug percentages detected after the 10-hour penetration period. When the VCZ load on the skin was compared, statistically significant differences were obtained when formulations H1 and H3 were compared, showing a higher VCZ accumulation in the case of formulation H1.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eVCZ percentage present in the remaining formulation in the washing liquid (% Wash), in the donor compartment (% Donor), in the skin (% Skin) and accumulated in the receptor compartment at the end of the transdermal absorption study (% Receptor). Each value represents the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of four replicates (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDispersion\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Wash\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Donor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e87.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Skin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u0026ordf;\u003csup\u003e,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Receptor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.73\u0026thinsp;\u0026plusmn;\u0026thinsp;7.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eThe same superscript letter indicates values that are not statistically different (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe study of the drug distribution across skin layers revealed differences between the dispersion and formulations H1 and H2 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). In these formulations, VCZ was distributed more homogeneously, reaching the deepest layers of the dermis. This highlights a greater drug load in both the epidermis and dermis, which may represent a more effective reservoir for the treatment of more invasive fungal skin diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntifungal Activity on C. albicans Growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth curves of \u003cem\u003eC. albicans\u003c/em\u003e in the presence of different VCZ formulations are shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA. The final concentration of all formulations in the incubation medium was 50 \u0026micro;g/ml. In this assay, a positive control composed of an aqueous dispersion of VCZ was also tested and it was checked that it provoked the maximum inhibition of the growth. By contrast, negative control marked the absence of inhibition. As can be observed, the growth inhibition achieved by the formulations and the dispersion was similar, with a reduction of approximately 1 log after 24 hours of incubation. Moreover, empty formulations were tested to confirm the absence of antifungal activity from the vehicle; the results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo antifungal activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antifungal activity of VCZ-loaded hyalurosomes was evaluated \u003cem\u003ein vivo\u003c/em\u003e using mice carrying a dense layer of \u003cem\u003eC. albicans\u003c/em\u003e on their dorsal skins. According to the guidelines of the ethical committee and aiming at reducing the number of used animals, only the three most promising formulations were tested, being these formulations H1, H2 and H4. The same criterion was used in the case of VCZ-loaded NLCs, only formulations C and D were tested. Saline was used in the control group (no inhibition of \u003cem\u003eC. albicans\u003c/em\u003e growth) and the dispersion was employed for comparative purposes.\u003c/p\u003e\n\u003cp\u003eBoth the VCZ dispersion and the VCZ-loaded formulations reduced the count of the colonies of \u003cem\u003eC. albicans\u003c/em\u003e (CFU) compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). However, the reduction was significantly greater (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when VCZ was administered as a dispersion or formulated in hyalurosomes than when it was loaded into NLCs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of lipid-based delivery systems loaded with VCZ has garnered significant attention in recent years, primarily to address the drug\u0026rsquo;s poor aqueous solubility, systemic side effects, and the need for targeted, sustained antifungal action. Among the most prominent VCZ-loaded nanosystems developed for topical, ocular or transungual administration are Solid Lipid Nanoparticles (SLNs) [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], NLCs [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] and different types of liposomes [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. In general, they have shown an encouraging performance with regard to particle size, entrapment efficiency and \u003cem\u003ein vitro\u003c/em\u003e release. Specifically, VCZ-loaded liposomes seem to offer the most promising features to promote deeper skin penetration, a critical factor for enhancing drug delivery to target sites within the dermis and epidermis [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e in cutaneous infections of any of these nanovesicles has been tested. In this sense, an important contribution of the present study is the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evaluation of the antifungal activity of new VCZ-loaded hyalurosomes against \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFour different liposome-based formulations were tested in this study (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In all cases, 0.1% HA was incorporated offering unique advantages for topical therapy, including enhanced skin hydration, improved drug penetration and the potential for synergistic wound healing and antifungal effects [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Despite these benefits, the use of HA in VCZ-loaded liposomes has so far been limited to ocular applications. In this sense, VCZ-loaded cubosomes [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e] and ultradeformable elastosomes [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] have been developed. The concentrations of HA used in those systems that gave rise to the best results were in the range of 0.2\u0026ndash;0.4%. However, a concentration of 0.1% was used in the four formulations developed in this work as a direct relationship between HA % and MD has been described [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, polyols such as glycerol and/or ethanol were incorporated into the formulations, representing 10%. These components enhance the solubility of VCZ and contribute to improved skin penetration by increasing the flexibility and elasticity of the liposomal bilayer. As a result, the vesicles become more deformable and capable of penetrating deeper into the skin layers [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Furthermore, the presence of these polyols is associated with a reduction in particle size, which further facilitates dermal delivery [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Another component present in the designed formulations that increase the flexibility of liposomes by destabilizing their lipid bilayer is the non-ionic surfactant polysorbate 80 [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], that also provides steric stabilization of colloidal systems despite a low ZP [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe main difference between formulations lies in the proportion of phospholipon\u0026reg; 90G involved, which varies from 1 to 10%. Phospholipon\u0026reg; 90G is a blend of purified phospholipids, derived mainly from soy lecithin. Its main component is phosphatidylcholine (\u0026ge;\u0026thinsp;94%) and may contain small proportions of other phospholipids such as phosphatidylethanolamine and phosphatidylinositol, in addition to a very low amount of triglycerides and free fatty acids. The concentration of this component can influence several characteristics of the resulting hyalurosomes, including MD, EE and drug release behavior. In this sense, an increase in Phospholipon\u0026reg; 90G content led to a reduction in vesicle size (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), a trend previously reported by Ahad et al. in the development of eprosartan mesylate-loaded transfersomes [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean diameter (MD), polydispersity index (PI), zeta potential (ZP) and entrapment efficiency (EE) of empty and VCZ loaded hyalurosomes. Each value represents the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of three replicates (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMD (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZP (mV)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEE (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEmpty Formulation H1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e160.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulation H1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e142.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;12.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.74\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEmpty Formulation H2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulation H2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;7.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEmpty Formulation H3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;3.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulation H3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEmpty Formulation H4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulation H4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eThe same superscript letter indicates values that are not statistically different (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly, EE was found to be dependent on the phospholipid concentration, with the lowest value (72%) observed at the lowest tested concentration (1% of Phospholipon\u0026reg; 90G) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This enhancement of EE with increasing phospholipid content is likely related to the hydrophobic nature of VCZ, which favors its incorporation into the lipid bilayer through hydrophobic interactions, thereby improving drug retention within the vesicles [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDespite the differences observed in size and EE between formulations, no relevant practical impact would be expected, since all obtained hyalurosomes had MD lower than 140 nm, aligning with the optimal size range for enhanced deposition in the epidermis and dermis (reported to be below 300 nm [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e] or around 100\u0026ndash;150 nm [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e], depending on the source), and high EE values (above 72%). Additionally, the slight size variations observed after at least 3 months of storage at 4\u0026deg;C did not compromise the particle size suitability for topical skin administration (MD\u0026thinsp;\u0026lt;\u0026thinsp;180 nm), considering the above reported optimal size.\u003c/p\u003e\n\u003cp\u003eRegarding VCZ release kinetics from hyalurosomes, an increase in the n-index was observed with increasing Phospholipon\u0026reg; 90G content (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). This trend suggests a shift in the dominant release mechanism from Fickian diffusion, associated with a less compact and more permeable bilayer structure, to mechanisms in which matrix relaxation and/or restructuring of the lipid bilayer play a more significant role.\u003c/p\u003e\n\u003cp\u003eAt lower phospholipid content, hyalurosomes display a greater structural disorder, the bilayer is less compact and more permeable, facilitating drug diffusion. In fact, formulation H1 (1% of phospholipon\u0026reg; 90G) exhibited an n-value of 0.22, indicative of a Fickian release mechanism, governed by diffusion and dependent on concentration gradient. Conversely, as the phospholipid content increases, the vesicle structure becomes more compact and stable, reducing diffusion and promoting alternative release mechanisms. This behavior was evident in formulations H2, H3, and H4. In formulations H2 and H4 (4% Phospholipon\u0026reg; 90G), n\u0026thinsp;=\u0026thinsp;0.6, indicating anomalous (non-Fickian) diffusion, in which both diffusion and matrix relaxation contribute to drug release. Formulation H3 (10% Phospholipon\u0026reg; 90G) presented an n-value of 0.86, suggesting a release mechanism closer to relaxation- and/or erosion-controlled transport (anomalous or overlapping Case II), with drug release predominantly driven by structural reorganization of the lipid bilayer, swelling of hyaluronic acid, or slow disintegration of the vesicle.\u003c/p\u003e\n\u003cp\u003eTherefore, in the proposed formulations, to achieve prolonged release profiles (K\u003csub\u003eP\u0026minus;S(1)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.94 h\u003csup\u003e\u0026minus;\u0026thinsp;n\u003c/sup\u003e), a higher proportion of phospholipid is recommended and, if desired to promote a faster release (K \u003csub\u003eP\u0026minus;S(1)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;64.12 h\u003csup\u003e\u0026minus;\u0026thinsp;n\u003c/sup\u003e), a lower proportion of Phospholipon\u0026reg; 90G is preferable. Consistent with the release rate, \u003cem\u003ein vitro\u003c/em\u003e skin permeation studies revealed a significantly higher concentration of VCZ in the receptor compartment for the formulation with the fastest release (formulation H1), compared to the other formulations developed (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). This effect could be attributed to the rapid saturation of the skin, allowing faster drug diffusion through the tissue. Moreover, formulation H1 demonstrated increased VCZ deposition in the deeper layers of the skin compared to the dispersion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), probably due to the smallest concentration of Phospholipon\u0026reg; 90G. The inverse correlation observed in our study aligns with findings by Montenegro et al. [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e], who reported that higher concentrations of this phospholipid reduce liposome flexibility and, consequently, their ability to penetrate through the stratum corneum and epidermis. However, although formulations H2 and H4 share the same phospholipid concentration, the higher ethanol content in formulation H2 (10%) enhanced VCZ penetration into deeper skin layers by disrupting stratum corneum lipids and increasing vesicle deformability [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. In contrast, formulation H4, with less ethanol (2.5%) and more glycerin (7.5%), promoted retention in the stratum corneum, as glycerin improves hydration but lacks the lipid-disruptive properties needed for deeper permeation [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDespite the differences between formulations, all of them were biocompatible with human keratinocytes and demonstrated antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e comparable to that of VCZ dispersion. However, the stability of formulation H3 (containing the highest phospholipid concentration) was the most compromised, showing loss of size homogeneity after 1 month (PI\u0026thinsp;\u0026gt;\u0026thinsp;0.6), and significantly lower skin retention compared to formulation H1 (0.1% vs. 0.51%, respectively). Consequently, formulation H3 was excluded from \u003cem\u003ein vivo\u003c/em\u003e testing.\u003c/p\u003e\n\u003cp\u003eIn addition, \u003cem\u003ein vivo\u003c/em\u003e assays confirmed the promising properties of formulations H1, H2 and H4, giving rise to a significant reduction in \u003cem\u003eC. albicans\u003c/em\u003e growth compared to the control group (untreated). However, although VCZ-loaded NLCs previously designed by our research group exhibited favorable characteristics [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e], they failed to significantly reduce \u003cem\u003eC. albicans\u003c/em\u003e colony-forming units (CFU) when administered in mice. These findings highlight the superior performance of hyalurosomes over NLCs for topical antifungal therapy. The main differences between VCZ-loaded hyalurosomes and NLCs involve higher EE (mean values: 89% vs. 77%, respectively) and slower release rate (complete release at 10 vs. 6 hours, respectively), and higher flexibility, which taken together favored higher penetration depth (mean value of VCZ retained in the dermis of the formulations that penetrated deeper into the skin: 41% vs. 24%, respectively) and enhanced accumulation in the receptor compartment (mean values: 8.3% vs. 1.7%, respectively). These results are consistent with those reported by Santos et al. [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e], who evidenced the importance of the transfollicular route for VCZ topical delivery from liposomes and attributed the faster release of VCZ from NLCs to the drug\u0026rsquo;s lower affinity for the lipid components of NLCs compared to the phospholipids in liposomes. Additionally, the slightly hydrophilic nature of VCZ favors its retention within the internal aqueous core of liposomes [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that hyalurosomes represent a promising next-generation nanosystem for the topical delivery of VCZ, significantly enhancing antifungal activity and promoting deeper drug deposition within the skin. These results highlight the considerable potential of hyalurosomes as an advanced delivery system for the topical treatment of superficial mycoses and, notably, for more invasive forms of cutaneous candidiasis. Furthermore, by facilitating targeted local therapy and potentially reducing the risk of systemic complications, hyalurosomes address a critical need for safer and more effective antifungal interventions. Such innovation directly aligns with the World Health Organization's global health priorities, emphasizing the urgent demand for novel solutions in the management of fungal infections by \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of University Valencia (2024-VSC-PEA-0081).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials generated or analyzed during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Generalitat Valenciana through project CIGE/2022/112.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: A.N., J.E.P. and I.U.; investigation: A.N., J.E.P., R.T.V. and I.U.; data curation: A.N, O.D.S., M.L.M., M.M. and I.U.; methodology and supervision: I.U. and J.E.; writing - original draft: A.N, J.E.P., O.D.S., R.T.V., M.L.M., M.M. and I.U.; writing - review \u0026amp; editing. I.U. and J.E. All authors have read and agreed to the published version of the manuscript. Amparo N\u0026aacute;cher (A.N), Jos\u0026eacute;-Esteban Peris (J.E.P.), Raquel Tal\u0026eacute;ns-Visconti (R.T.V.), Octavio D\u0026iacute;ez-Sales (O.D.S.), Maria Letizia Manca (M.L.M.), Maria Manconi (M.M.) and Iris Usach (I.U.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors acknowledge the valuable assistance and help provided by Inmaculada Noguera and Inmaculada Ballester for carrying out the \u003cem\u003ein vivo\u003c/em\u003e experiments in a successful way. In addition, the authors thank Lipoid GmbH (Ludwigshafen, Germany) for providing the gift sample of phospholipid for the research work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability Statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are included in the manuscript. Additional data are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMehrmal S, Uppal P, Giesey RL, Delost GR. Identifying the prevalence and disability-adjusted life years of the most common dermatoses worldwide. J Am Acad Dermatol 2020;82(1):258-259.\u003c/li\u003e\n\u003cli\u003eZiental D, Anaya-Plaza E, Talarska-Kulczyk P, Kubicka A, Żurawski J, Dlugaszewska J, et al. Quaternized phthalocyanines as a tool against melanoma and a broad spectrum of bacteria and fungi. 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Proc Natl Acad Sci U S A 2003;100(12):7360-7365.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"voriconazole, hyalurosomes, candida albicans, topical administration, antifungal activity","lastPublishedDoi":"10.21203/rs.3.rs-7270257/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7270257/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCutaneous candidiasis, mainly caused by \u003cem\u003eCandida albicans\u003c/em\u003e, is a growing global health concern and is listed by WHO as a high-priority fungal threat. Suboptimal penetration of conventional vehicles limits the efficacy of current topical antifungals, increasing the risk of severe and invasive infections. Therefore, there is an innovative research field in advanced topical delivery systems to improve drug deposition, retention and antifungal efficacy. The main objective of this work was to develop nanocarriers based on hyalurosomes for the delivery of voriconazole (VCZ) and evaluate their potential to enhance the drug’s cutaneous penetration and antifungal activity. Four VCZ-loaded hyalurosomal formulations were prepared (H1-H4) by modulating the proportions of phospholipid and polyols. Although changes in some physicochemical properties were observed, all the VCZ-loaded nanosystems were nanosized (\u0026lt; 140 nm), spherical, multilamellar and exhibited high entrapments efficiencies (\u0026gt; 72 %), excellent biocompatibility with human keratinocytes and potent antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e. VCZ release from formulation H1 (1 % phospholipid, 10 % ethanol) followed a Fickian mechanism, while H2–H4 (4-10 % phospholipid, 2.5-10 % ethanol) exhibited anomalous diffusion involving both diffusion and matrix relaxation or erosion. Additionally, H1 and H2 (1-4 % of phospholipid, 10 % ethanol) achieved significantly enhanced drug penetration into deeper skin layers and superior \u003cem\u003ein vivo\u003c/em\u003e antifungal efficacy compared to VCZ dispersion. The results highlight the potential of hyalurosomes as a next-generation topical antifungal delivery system, effective against both superficial and invasive candidiasis, with formulations H1 and H2 emerging as the most promising candidates for the treatment of the more invasive forms.\u003c/p\u003e","manuscriptTitle":"Novel Voriconazole-Loaded Hyalurosomes Optimized for Enhanced Skin Penetration and Antifungal Activity against Candida albicans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 17:06:14","doi":"10.21203/rs.3.rs-7270257/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-09-20T10:18:39+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-28T17:58:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-28T01:24:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T07:15:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2025-08-01T06:23:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ed16290-6845-4611-a702-7087428a220c","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:03:14+00:00","versionOfRecord":{"articleIdentity":"rs-7270257","link":"https://doi.org/10.1007/s13346-025-02007-3","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2025-11-10 15:58:24","publishedOnDateReadable":"November 10th, 2025"},"versionCreatedAt":"2025-09-04 17:06:14","video":"","vorDoi":"10.1007/s13346-025-02007-3","vorDoiUrl":"https://doi.org/10.1007/s13346-025-02007-3","workflowStages":[]},"version":"v1","identity":"rs-7270257","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7270257","identity":"rs-7270257","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}