Development of biopolymer films loaded with fluconazole and thymol for resistant vaginal candidiasis.

Vulvovaginal candidiasis (VVC) is an opportunistic infection caused by a fungus of the Candida genus, affecting approximately 75 % of women during their lifetime. Fungal resistance cases and adverse effects have been the main challenges of oral therapies. In this study, the topical application of thin films containing fluconazole (FLU) and thymol (THY) was proposed to overcome these problems. Vaginal films based only on chitosan (CH) or combining this biopolymer with pectin (PEC) or hydroxypropylmethylcellulose acetate succinate (HPMCAS) were developed by the solvent casting method. In addition to a higher swelling index, CH/HPMCAS films showed to be more plastic and flexible than systems prepared with CH/PEC or only chitosan. Biopolymers and FLU were found in an amorphous state, contributing to explaining the rapid gel formation after contact with vaginal fluid. High permeability rates of FLU were also found after its immobilization into thin films. The presence of THY in polymer films increased the distribution of FLU in vaginal tissues and resulted in improved anti-Candida activity. A significant activity against the resistant C. glabrata was achieved, reducing the required FLU dose by 50 %. These results suggest that the developed polymer films represent a promising alternative for the treatment of resistant vulvovaginal candidiasis, encouraging further studies in this context.

Development of ophthalmic nanoemulsions of ß-caryophyllene for the treatment of Acanthamoeba keratitis.

Although rare, amoebic keratitis (AK) is a disease caused by Acanthamoeba spp. that can lead to blindness. The drugs currently available for its treatment are very toxic, which has motivated the investigation for more effective and safe therapeutic options. In this study, the in vitro activity of ß-caryophyllene (BCP) was exploited taking into account its action against other protozoans as well as its well-known healing and anti-inflammatory properties (aspects relevant for the AK pathogenesis). On the other hand, high volatilization and oxidation phenomena are found for this compound, which led to its incorporation into nanoemulsions (NEs). Two emulsifying agents were tested, resulting in monodisperse systems with reduced droplet size (<265 nm) and high surface charge (positive and negative for NEs prepared with cetrimonium bromide -CTAB and Phosal® 50+, respectively). NEs prepared with CTAB were shown to be more stable after long-term storage at 4 and 25 °C than those prepared with Phosal®. Pure BCP, at the highest concentration (500 µM), resulted in a level of inhibition of Acanthamoeba trophozoites equivalent to that of reference drug (chlorhexidine). This activity was even greater after oil nanoencapsulation. The reduced droplet size could improve the interaction of the oil with the microorganism, justifying this finding. Changes in surface charge did not impact the activity. Positively charged NEs improved the interaction and retention of BCP in the cornea and thus should be prioritized for further studies.

Revisiting the Effect of Aging on the Transport of Molecules through the Skin.

Both intrinsic and extrinsic aging lead to a series of morphological changes in the skin including the flattening of the dermal-epidermal junction, increased stratum corneum dryness, reduction in sebaceous gland activity and enzyme activity as well as atrophy of blood vessels. In this study, the impact of these changes on the transport of molecules through the skin was revised. The increase in the number of transdermal formulations on the market in recent decades and life expectancy represent the main reasons for an in-depth discussion of this topic. Furthermore, elderly subjects have often been excluded from clinical trials due to polypharmacy, raising concerns in terms of efficacy and safety. In this way, ex vivo and in vivo studies comparing the transport of molecules through the mature and young skin were analyzed in detail. The reduced water content in mature skin had a significant impact on the transport rate of hydrophilic molecules. The lower enzymatic activity in aged skin, in turn, would explain changes in the activation of prodrugs. Interestingly, greater deposition of nanoparticles was also found in mature skin. In vivo models should be prioritized in future experimental studies as they allow to evaluate both absorption and metabolism simultaneously, providing more realistic information.

The rheology of injectable hyaluronic acid hydrogels used as facial fillers: A review.

Injectable hyaluronic acid (HA) hydrogels have been popularized in facial aesthetics as they provide a long-lasting effect, low risk of complications, allergenicity tests are not required before application and can be easily removed by the action of hyaluronidases. On the other hand, the development of these systems requires in-depth studies of chemical mechanisms involved in hydrogel formation. Ideal dermal fillers should temporarily fluidize during extrusion through the needle and quickly recover their original shape after application. Hydrogels with more elastic properties, for example, are difficult to inject while viscous materials are too liquid. A balance between both properties should be achieved. Each region of the face requires products with distinct rheological properties. High G' dermal fillers are preferable for deeper wrinkles whereas the counterpart with lower values of G' is more indicated in superficial wrinkles or lip augmentation. Factors such as molecular weight and concentration of HA, pH, type and concentration of the crosslinking agent, particle size, crosslinking reaction time and crosslinking agent/polysaccharide ratio should be modulated to achieve specific rheological properties. In this review, the effect of each variable is discussed in detail to guide the rational development of new dermal fillers.

Nanoemulsions and nanocapsules loaded with Melaleuca alternifolia essential oil for sepsis treatment.

Sepsis represents a complex clinical syndrome that results from a harmful host response to infection. The infections most associated with sepsis are pneumonia, intra-abdominal infection, and urinary tract infection. Tea tree oil (TTO) has shown high antibacterial activity; however, it exhibits low aqueous solubility and high volatility, which have motivated its nanoencapsulation. In this study, the performance of nanoemulsions (NE) and nanocapsules (NC) loaded with TTO was compared. These systems were prepared by spontaneous emulsification and nanoprecipitation methods, respectively. Poly-ε-caprolactone or Eudragit® RS100 were tested as polymers for NCs whereas Tween® 80 or Pluronic® F68 as surfactants in NE preparation. Pluronic® F68 and Eudragit® RS100 resulted in more homogeneous and stable nanoparticles. In accelerated stability studies at 4 and 25 °C, both colloidal suspensions (NC and NE) were kinetically stable. NCs showed to be more stable to photodegradation and less cytotoxic than NEs. After sepsis induction by the cecal ligation and puncture (CLP) model, both NE and NC reduced neutrophil infiltration into peritoneal lavage (PL) and kidneys. Moreover, the systems increased group thiols in the kidney and lung tissue and reduced bacterial growth in PL. Taken together, both systems showed to be effective against injury induced by sepsis; however, NCs should be prioritized due to advantages in terms of cytotoxicity and physicochemical stability.

A Comparative Analysis of Biological and Synthetic Skin Models for Drug Transport Studies.

Ethical restrictions as well as practical or economic issues related to use of animal and human skin has been the main reason the growth in the number of investigations with alternative models. Reconstructed skin models, for example, have been useful to evaluate the in vitro toxicity of compounds; however, these models usually overestimate the amount of drug permeated due to impaired barrier properties. In this review, the performance of synthetic and biological skin models in transport studies was compared by considering two compounds with different physicochemical properties. The advantages and limitations of each skin model are discussed in detail. Although synthetic and reconstructed skin models have shown to be useful in the formulation optimization step, they present many limitations: (1) impaired barrier properties; (2) lack of follicular transport; (3) no metabolism in synthetic membranes; (4) differences in terms of lipid organization; (5) more affected by formulation constituents. Therefore, animal and human tissues should still be prioritized in drug transport studies until new advances in alternative models are achieved. Investigations of the impact of cell-culture conditions on skin formation, in turn, bring perspectives related to the development of unhealthy/injured skin models (an aspect that still deserves attention).

Effect of Vehicle Composition on the Preparation of Different Types of Dapsone Crystals for Topical Drug Delivery.

Topical formulations composed of API-pure crystals have been increasingly studied, especially in regards to the impact of particle size in penetration efficiency. Less attention, however, has been devoted to the solid-state properties of drugs delivered to the skin. In this study, we address the effect of formulation composition on the crystal form existing in topical products. Dapsone (DAP) gel formulations were prepared by mixing an organic solution containing DAP with an aqueous solution containing polymers and preservatives. The organic solvent was chosen as ethoxydiglycol (DEGEE), polyethylene glycol (PEG), or 1-methyl-2-pirrolidone (MPR) to assess the impact of composition on DAP crystal form. Such solvent variations resulted in different particulate matter. In terms of crystalline nature, the presence of DEGEE in formulations induced the crystallization of DAP hydrate, while PEG cocrystal and a mixture of hydrate and MPR solvate crystallized from the same amounts of PEG and MPR, respectively. Microscopic analysis of the gels showed heterogeneous particles with different characteristics. The behavior of gels after application to the skin was also tested. Interestingly, the different formulations seemed to accumulate in different regions of the skin. This could be the result of the effect of vehicle composition/excipients on the characteristics of the skin, such as hydration. The site-specific accumulation, however, was more pronounced in crystal-loaded gels as opposed to blank formulations. These results indicate that future studies should consider the effect of formulation composition on the API crystal form landscape as part of the strategies used to successfully target drug delivery to the skin.

How to define the experimental conditions of skin permeation assays for drugs presenting biopharmaceutical limitations? The experience with testosterone.

Cutaneous permeation assays are crucial to attest the performance or bioequivalence of topical or transdermal products. Although the official guidelines (e.g., FDA/EMA) play a key role in harmonizing the experimental design, alternative methods are often proposed by the scientific community, which makes it difficult to compare results from different studies. In this review, permeation assays with testosterone (TST) were selected to show this high variability in drug transport rate. The main sources of variation discussed were tissue thickness, animal model, donor and receptor fluid constitution, type of solubilizing agent used in aqueous fluids, drug concentration, degree of supersaturation, skin lipid content, number of experimental times and the physical-chemical stability of the molecule in test fluids. This variation becomes even more critical for molecules that present biopharmaceutical limitations such as TST. In addition, the skin presents specific receptors for this hormone due to its physiological action in this region of the body, which makes the evaluation of the TST transport rate in this tissue even more challenging. The impact of each experimental parameter mentioned above on the flux or permeation coefficient of TST is discussed in detail in the review. Assays used to evaluate tissue integrity are also presented.

Thermosensitive hydrogels for vaginal delivery of secnidazole as an approach to overcome the systemic side-effects of oral preparations.

Secnidazole (SEC) has been suggested as an alternative agent against Trichomonas vaginalis to overcome the adverse effects, antimicrobial resistance problems and poor adherence to the currently available therapy. Once no topical formulation may be found in the market until now, SEC was incorporated in thermosensitive bioadhesive systems to extend the contact time in the mucosa and to avoid a systemic drug disposition. Formulations containing 20% poloxamer 407, 1% poloxamer 188 and 1 or 2.5% chitosan showed suitable sol-gel transition temperature (> 30 °C), presenting a fast gelation time (100-115 s). Rheological, dynamic light scattering and infrared spectroscopy analysis suggested molecular interactions among polymers. Chitosan increased the mucoadhesion strength of the formulations. In addition, hydrogels showed a tendency to decrease the drug transport rate through mucosa when compared to the control. Mucin was also added onto mucosa for a more realistic simulation of permeability/retention. In the presence of this agent, hydrogels containing chitosan reduced the permeability/retention of the drug in approximately 2.0-fold when compared to the control. Therefore, the hydrogels presented suitable characteristics to remain in the vaginal environment, which would result in effective local treatment of trichomoniasis.

Development and characterization of thermopressed polyvinyl alcohol films for buccal delivery of benznidazole.

Given that oral preparations of benznidazole (BZN) have demonstrated limited efficacy in the treatment of Chagas' disease due to pharmacokinetic or toxicological problems, the development of buccal polymeric films was purposed in this study. These systems ensure high patient acceptability and direct access to the systemic circulation, improving drug bioavailability and toxicological profile. Polymer films were prepared through a thermopressing method by mixing BZN and polyvinyl alcohol (PVAL). In some preparations, propylene glycol (PG) and thymol (TM) were also included as plasticizer and chemical absorption enhancer, respectively. Morphology, X-ray diffraction, spectroscopic, thermal, mechanical, and water uptake properties, as well as ex vivo permeability studies, were performed to characterize the film formulations. BZN remained stable and in an amorphous form over 90 days. The addition of PG and TM improved the mechanical properties of the films, making them soft, flexible and tear-resistant. Also, these additives increased the water sorption rate of the films at 50 and 75% relative humidity and the TM increased the film erosion properties and drug permeability (close to 6×) compared to control. It was hypothesized that the permeability improvement of thymol-based films that follow a drug release profile through erosion is also associated with the inhibition of the crystallization of BNZ when the film is in contact with the buccal mucosa. Once the thymol has previously demonstrated a significant in vivo and in vitro trypanocidal action and even improved film characteristics, these systems may be considered promising for Chagas' disease treatment.

Emerging Technologies to Target Drug Delivery to the Skin - the Role of Crystals and Carrier-Based Systems in the Case Study of Dapsone.

Dapsone (DAP) is a long-established molecule that remains a promising therapeutic agent for various diseases mainly because it combines antimicrobial and anti-inflammatory activities. Its oral application, however, is limited by the dose-dependent hematological side effects that may rise from systemic exposure. As an alternative to overcome this limitation, the administration of DAP to the skin has witnessed prominent interest in the past 20 years, particularly when applied to the treatment of dermatological disorders. In this review, all technological strategies proposed to the topical delivery of DAP are presented. Most of the reported studies have been devoted to the clinical use and safety of a gel formulation containing both solubilized and microcrystalline drug, however, the technological characteristics of such preparation are still missing. In parallel, the incorporation of DAP into vesicular and particulate carriers (e.g. nano- and microemulsions, niosomes, invasomes, bilosomes, cubosomes, solid lipid nanoparticles, nanostructured lipid carriers, polymeric nanocapsules and polymer-lipid-polymer hybrid nanoparticles) appears to be an alternative to provide greater drug release control, enhanced drug solubilization and follicular targeting. Indeed, the main application of DAP topical formulations reported in the literature was the treatment of acne vulgaris, a disease located in the hair follicle. Other diseases affecting different regions of the skin (e.g. cutaneous lupus erythematosus and cutaneous leishmaniasis), however, may also benefit from a topical therapeutic regimen containing DAP. Therefore, the investigation of appendageal route in comparison to passive transmembrane diffusion as a function of targeted disease, as well as pharmacokinetic studies, are perspectives highlighted herein. Such studies may drive future efforts towards the rational development of safe and effective technologies to deliver DAP to the skin. Graphical abstract.

Transbuccal Delivery of Isoniazid: Ex Vivo Permeability and Drug-Surfactant Interaction Studies.

The oral administration of isoniazid (INH) may lead to discontinuation of tuberculosis treatment due to drug-related hepatotoxicity events, and thus, the transbuccal delivery of this drug was investigated, for the first time, as an alternative administration route. Ex vivo permeability assays were performed in Franz-type diffusion chambers, applying INH alone and in combination with sodium dodecyl sulfate (SDS) and sodium taurocholate (ST). After confirming the formation of micelle structures by dynamic light scattering analysis, UV-visible spectroscopy and zeta potential analyses were used to investigate drug-micelle interactions. In zeta potential analyses, no electrostatical interactions were identified for both surfactants in saliva buffer pH 6.8. Spectrophotometric analyses, in turn, indicated chemical interactions between INH and SDS in both pH values (2.0 and 6.8) whereas no interaction between the drug and ST was observed. Despite the interaction between SDS and drug, this surfactant increased the buccal transport rate of INH by approximately 11 times when compared with the control. In contrast, ST did not increase the drug permeability. The INH retention in SDS-treated mucosa was significantly higher when compared with the control and an effect on intercellular lipids was suggested. In vivo studies are needed to confirm the high INH absorption found here. Grapical abstract.

Transbuccal delivery of metal complexes of isoniazid as an alternative to overcome antimicrobial resistance problems.

In isolated isoniazid (INH)-resistant strains, deletion or mutations in thekatGgene have been identified, which result in loss of catalase-peroxidase activity. This enzyme plays a key role in the activation of this prodrug. As an alternative, the coordination of the INH to metal complexes has been purposed to activate it regardless of enzyme functionality. Although pentacyanido(isoniazid)ferrate(II) complexes have shown to be effective against resistant strains of Mycobacterium tuberculosis, low oral bioavailability was found. In this context, buccal mucosa was selected as an alternative route to the metal complex delivery. Moreover, oral manifestations of tuberculosis(TB) have been observed in some patients, particularly when resistant strains are present, and no therapeutic options are currently available on the market. Pentacyanidoferrate (PCF-INH) and Prussian-blue (PB-INH) complexes were initially prepared and characterized, followed by buccal permeability studies in Franz-type diffusion cells. The electrochemical potential of the complexes demonstrated their ability to self-activate. Job's method suggested the presence of structural defects in PB-INH complexes, which was correlated with permeability results. In fact, PB-INH showed a higher dissociation rate in salt-rich aqueous medium and thus a high transport rate of INH through the buccal mucosa. Its passage through the tissue would not be possible due to the high molecular size. PCF-INH, in turn, presented a lower dissociation rate in the salt-rich aqueous medium, justifying its slower transport rate through the tissue. Taken together, these results suggest that INH-based metal complexes may be efficiently administered through the buccal route, impacting on both oral bioavailability and microbial resistance.

Development of curcumin-loaded chitosan/pluronic membranes for wound healing applications.

The emergence of new materials with improved antibacterial, anti-inflammatory and healing properties compared to conventional wound dressings has both social and economic appeal. In this study, novel chitosan-based (CTS) membranes containing curcumin (CUR) incorporated in Pluronic (PLU) copolymers were developed and characterized to obtain suitable properties for applications as a wound healing dressing. The mechanical, thermal, swelling, wettability, release and permeation properties were evaluated by DSC, TGA, water contact angle measurements, FTIR, fluorescence and microscopic techniques. Membranes containing PLU and CUR presented wettability close to the ideal range for interaction with cellular components (contact angle ~40-70°), improved mechanical properties, higher thermal stability, high swelling degree (>800%) and CUR release (~60%) compared to samples without PLU addition. A higher retention of CUR in the epidermis than in the dermis layer was observed, which also was confirmed by confocal microscopy. Furthermore, the CTS-PLU membranes loaded with CUR showed to be active against Staphylococcus aureus and Pseudomonas aeruginosa (MIC = 25 and 100 mg mL-1, respectively), the microbial species most present in chronic wounds. Overall, the CTS-PLU-CUR membranes presented suitable properties to act as a new wound healing dressing formulation and in vivo studies should be performed to confirm these benefits.

Transbuccal delivery of benznidazole associated with monoterpenes: permeation studies and mechanistic insights.

Benznidazole (BZN) represents the only drug currently available for the treatment of Chagas disease in most endemic countries. When administered orally, high doses are required due to its extensive hepatic metabolism and its toxicity represents the main reason for treatment withdrawals. Because of these complications, transbuccal administration of BZN was investigated. This route avoids the first-pass hepatic metabolism and presents high permeability, with direct access to the systemic circulation. BZN was applied on porcine buccal mucosa after pretreatment with pure eugenol, carvacrol or limonene. Thermal (DSC) and spectroscopic (FT-IR) analyzes were performed to investigate the mechanisms of drug absorption enhancement. The permeability coefficient values of BZN increased 2.6, 2.9 and 4.9-fold after pretreatment with eugenol, carvacrol and limonene, respectively. The lag time, in turn, was shortened in the pretreated samples. The DSC and FT-IR analyzes suggested that transport of BZN through the buccal mucosa is associated with log P and size of monoterpenes. Limonene, the most effective absorption enhancer, contributed to greater interaction with non-polar domains of the buccal epithelium. Overall, BZN showed to be efficiently transported through the buccal route, but in vivo pharmacokinetic studies should be performed to confirm these findings.

Coumestrol/hydroxypropyl-ß-cyclodextrin association incorporated in hydroxypropyl methylcellulose hydrogel exhibits wound healing effect: in vitro and in vivo study.

Several beneficial effects on the skin have been reported for coumestrol (COU), such as protection against photoaging and improvement of skin elasticity and thickness in postmenopausal women. However no reports on the effect of COU on wound healing were found. Nevertheless, COU has low aqueous solubility, which is a crucial limitation for biological tests. The present study was designed as a two-step experiment to evaluate the wound healing effect of COU. First, we used fibroblasts and the experimental in vitro artificial wound model, scratch assay, to compare the effects of COU free, dissolved in dimethyl sulfoxide (DMSO) or Dulbecco's modified Eagle's medium (DMEM), or associated with hydroxypropyl-ß-cyclodextrin (HPßCD). The 50 µM (66.1%) and 10 µM (56.3%) COU/HPßCD association induced cell proliferation and migration in inflicted wounds. Subsequently, the in vivo wound healing experimental model (Wistar rats) revealed that COU/HPßCD incorporated into hypromellose (HPMC) hydrogel had similar efficacy in wound healing in comparison to the positive control (Dersani®), with the advantage that 50% wound healing was achieved within a shorter period. In summary, the results successfully demonstrated, for the first time, the wound healing effect of COU/HPßCD incorporated into HPMC hydrogel and describe the feasibility of the biological tests with the use of HPßCD instead DMSO.

Potential use of secondary products of the agri-food industry for topical formulations and comparative analysis of antioxidant activity of grape leaf polyphenols.

The aim of the present study was to develop a phytocosmetic using Vitis waste by-products, for use as a topical formulation for skin protection against ultraviolet radiation damage. The study also evaluates the free radical scavenger activity of the crude extracts of dried leaves of Vitis vinifera and Vitis labrusca, as well as the anthocyanins, flavonoid fraction and isolated compounds. Next, release and permeation studies of hydrogels were performed using Franz-type diffusion cells. Flavonoid acted more intensively in TRAP and conjugated dienes antioxidant assays, whereas anthocyanins had higher antioxidant activity in hydroxyl and nitric oxide assay. Only quercetin-3-O-glucuronide (5) was released from hydrogels, and the flavonoid retention in porcine ear skin after eight hours of permeation was below of limit of quantification for this compound. The polyphenols present in Vitis are capable of absorbing UV and visible light, justifying their potential as sunscreens for the development of a phytocosmetic.

Topical Delivery of Coumestrol from Lipid Nanoemulsions Thickened with Hydroxyethylcellulose for Antiherpes Treatment.

We have recently shown that coumestrol, an isoflavonoid-like compound naturally occurring in soybeans, alfafa, and red clover, inhibited Herpes Simplex Virus types 1 (HSV-1) and 2 (HSV-2) replication. In this study, we designed coumestrol formulations in an attempt to enable its topical delivery to mucosa tissues. Physicochemical and microscopic examinations suggested that coumestrol was efficiently incorporated in positively-charged nanoemulsions dispersed in a hydroxyethylcellulose gel. The higher coumestrol flux through excised porcine esophageal mucosa was detected from nanoemulsions composed by a fluid phospholipid (dioleylphosphocholine, DOPC) in comparison with that of a rigid one (distearoylphosphocholine, DSPC) in two mucosa conditions (intact and injured). Such results were supported by confocal fluorescence images. Furthermore, the low IC50 values demonstrated an increasement in the antiviral inhibition against HSV-1 and HSV-2 after incorporation of coumestrol into nanoemulsions containing DOPC. Overall, coumestrol-loaded nanoemulsions proved to be beneficial for herpes simplex treatment.

Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis: optimization using a full factorial design.

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 2(2) full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant - soybean lecithin or sorbitan monooleate and type of co-surfactants - polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential -39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 µg·g(-1)) - the main response of interest - confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 µM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment.

Antiherpes Activity and Skin/Mucosa Distribution of Flavonoids from Achyrocline satureioides Extract Incorporated into Topical Nanoemulsions.

This study investigated the inhibitory effects of Achyrocline satureioides extract (ASE) incorporated into a topical nanoemulsion on Herpes Simplex Virus type 1 (HSV-1/KOS strain) replication, as well as the distribution of the main ASE flavonoids (quercetin, luteolin, and 3-O-methylquercetin) in porcine skin and mucosa. The ASE-loaded nanoemulsion showed more pronounced effects against HSV-1 replication when compared to the ASE or pure quercetin, as determined by the viral plaque number reduction assay. All flavonoids were detected in the skin epidermis (2.2 µg/cm(2)) and the mucosa upper layers (3.0 µg/cm(2)) from ASE-loaded nanoemulsion until 8 h after topical application. A higher amount of flavonoids was detected when these tissues were impaired, especially in deeper mucosa layers (up to 7-fold). Flavonoids were detected in the receptor fluid only when the mucosa was injured. Such results were supported by confocal microscopy images. Overall, these findings suggest that the tested ASE-loaded nanoemulsion has potential to be used topically for herpes infections.