Porcine Ear Research Articles

Polyvinyl alcohol-based electrospun matrix as a delivery system for nanoemulsion containing chalcone against Leishmania (Leishmania) amazonensis

Cutaneous leishmaniasis is a worldwide public health problem. Conventional therapies, in addition to the high cost, have many adverse effects and cases of parasite’s resistance. Chalcones are secondary metabolites precursors in the flavonoid pathway and can be obtained naturally, but with low yield from plant raw material. Thus, the use of synthetic chalcones has been a promising strategy for the development of molecules with leishmanicidal activity. Thus, this work aimed to develop a controlled release system of two synthetic chalcone (trans-chalcones and 3'-(trifluormethyl)-chalcone) using polyvinyl alcohol nanofibers (PVA) as scaffold. The association of chalcones to the nanofibers was made by nanoemulsions (NE) thereof, i.e., a colloidal system on a nanometric scale, which allows compounds with opposite polarities to remain miscible and stable throughout their manipulation. Chalcone nanoemulsions were developed using the spontaneous emulsification technique. The NE were characterized regarding their particle size, polydispersion index (PDI), and zeta potential. The results showed NE with spherical shape, absolute values of zeta potential were higher than 30 mV and homogeneous distribution pattern (PDI < 0.3). Dynamics light scattering (DLS) analysis showed similar hydrodynamic rays, i.e., 180 nm (trans-chalcone NE) and 178 nm (NE containing 3′-(trifluormethyl)-chalcone, in addition to presenting encapsulation efficiency values close to 100 %. Subsequently, the NE were added to a polymeric solution of polyvinyl alcohol (PVA) and processed via the electrospinning technique affording a PVA matrix (15 %, w/v) nanofiber containing the chalcones NE at 1 mg.mL−1. In a follow-up experiment, the skin permeation assay of the PVA matrix-chalcone NE was performed in vitro using Franz type diffusion cells and porcine ear as biological model of study. The results showed that the treatments with the nanofibers containing the chalcone NE were retained mainly in the stratum corneum, while the NE suspensions containing chalcone were retained in the epidermis and dermis. This result is thought to be relevant, since parasites are located mainly in the dermis. Further, in vitro assay against the amastigote form of L. (L) amazonensis, showed IC50 values to trans-chalcone and 3'-(trifluormethyl)-chalcone of 24.42 ± 6.76 μg.mL−1 and 15.36 ± 4.61 μg.mL−1, respectively. In addition to improving the solubility of the compounds tested in culture medium without using organic solvents, chalcones in nano-emulsified form reduced the IC50 to 9.09 ± 1.24 μg.mL−1 (trans-chalcone) and 10.27 ± 2.27 μg.mL−1 (3'-(trifluormethyl)-chalcone) which confirmed the potential of the nanoemulsion containing chalcone for cutaneous leishmaniasis treatment.

In vitro Studies of Antitumor Effect, Toxicity/Cytotoxicity and Skin Permeation/Retention of a Green Fluorescence Pyrene-based Dye for PDT Application.

Photosensitizers (PS) are compounds that can generate reactive oxygen species under irradiation of appropriate light and are widely used in photodynamic therapy (PDT). Currently, topical PDT is an effective treatment for several skin diseases, including bacterial infections, fungal mycoses and psoriasis. In addition, PDT is also used to treat nonmelanoma skin cancer and can be a potential tool for melanoma, associated with other treatments. In this work, we evaluated the antitumor photoactivity of a new pyrene-based PS (TPPy) by using the murine melanoma cell line (B16F10). The in vitro permeation/retention tests in porcine ear skin were also performed in order to evaluate the potential application of the PS for topical use in skin cancer. Moreover, to determine the toxicity in vivo, we used the Galleria mellonella as an alternative animal model of study. The results showed that TPPy is a promising PS for application in PDT, with potential antitumor photoactivity (IC50 6.5μmol L-1 ), absence of toxicity in the G. mellonella model at higher concentration (70.0mmol L-1 ) and the accumulation tendency in the epidermis plus dermis sites (165.20±4.12ngcm-2 ).

Investigation of transfollicular caffeine penetration using microdialysis on ex vivo porcine ear skin

The aim of this study was to develop an ex vivo method that allows to quantify the transfollicular penetration of topically applied substances by combining microdialysis and selective follicular closure with varnish. An experimental setup with three skin areas on ex vivo intact porcine ear skin was designed (varnish on hair follicle, varnish next to hair follicle, no varnish). On each area, 10 µl/cm2 caffeine-hydroxyethyl-cellulose-gel was applied. Samples were collected for 22 h by microdialysis. After sampling, the skin layers were separated, homogenized and caffeine was quantified by high pressure liquid chromatography (HPLC) in all samples. Potential impact of the varnish placed next to the follicle by tension on the follicle during the drying process was monitored by a microscopic setup and could be excluded. The microdialysis and homogenization study showed a significantly reduced penetration of caffeine when the hair follicles were closed. In areas with open hair follicles caffeine was detected already in the first ten minutes after application. The reported novel combination of two methods is suitable to investigate ex vivo transfollicular penetration. Possible impact of the closure material in the control area can be ruled out by adjusting the design of the control area in future studies.

Hypochlorous acid-generating electrochemical scaffold eliminates Candida albicans biofilms.

Wound infections involving Candida albicans can be challenging to treat because of the fungus' ability to penetrate wound tissue and form biofilms. The goal of this study was to assess the activity of a hypochlorous acid (HOCl)-generating electrochemical scaffold (e-scaffold) against C. albicans biofilms in vitro and on porcine dermal explants (ex vivo). C. albicans biofilms were grown either on acrylic-bottom six-well plates (in vitro) or on skin tissue excised from porcine ears (ex vivo), and the polarized e-scaffold was used to generate a continuous supply of low concentration HOCl near biofilm surfaces. C. albicans biofilms grown in vitro were reduced to undetectable amounts within 24h of e-scaffold exposure, unlike control biofilms (5·28±0·034 log10 (CFU cm- 2 ); P<0·0001). C. albicans biofilms grown on porcine dermal explants were also reduced to undetectable amounts in 24h, unlike control explant biofilms (4·29±0·057 log10 (CFU cm- 2 ); P<0·0001). There was a decrease in the number of viable mammalian cells (35·6±6·4%) in uninfected porcine dermal explants exposed to continuous HOCl-generating e-scaffolds for 24h compared to explants exposed to nonpolarized e-scaffolds (not generating HOCl) (P<0·05). Our HOCl-generating e-scaffold is a potential antifungal-free strategy to treat C. albicans biofilms in chronic wounds. Wound infections caused by C. albicans are difficult to treat due to presence of biofilms in wound beds. Our HOCl producing e-scaffold provides a promising novel approach to treat wound infections caused by C. albicans.

Improving In Vitro Cartilage Generation by Co-Culturing Adipose-Derived Stem Cells and Chondrocytes on an Allograft Adipose Matrix Framework.

Microtia is an inherited condition that results in varying degrees of external ear deformities; the most extreme form is anotia. Effective surgical reconstruction techniques have been developed. However, these usually require multistage procedures and have other inherent disadvantages. Tissue engineering technologies offer new approaches in the field of external ear reconstruction. In this setting, chondrocytes are cultured in the laboratory with the aim of creating bioengineered cartilage matrices. However, cartilage engineering has many challenges, including difficulty in culturing sufficient chondrocytes. To overcome these hurdles, the authors propose a novel model of cartilage engineering that involves co-culturing chondrocytes and adipose-derived stem cells on an allograft adipose-derived extracellular matrix scaffold. Auricular chondrocytes from porcine ear were characterized. Adipose-derived stem cells were isolated and expanded from human lipoaspirate. Then, the auricular chondrocytes were cultured on the allograft adipose matrix either alone or with the adipose-derived stem cells at different ratios and examined histologically. Cartilage induction was most prominent when the cells were co-cultured on the allograft adipose matrix at a ratio of 1:9 (auricular chondrocyte-to-adipose-derived stem cell ratio). Furthermore, because of the xenogeneic nature of the experiment, the authors were able to determine that the adipose-derived stem cells contributed to chondrogenesis by means of a paracrine stimulation of the chondrocytes. In this situation, adipose-derived stem cells provide sufficient support to induce the formation of cartilage when the number of auricular chondrocytes available is limited. This novel model of cartilage engineering provides a setting for using the patient's own chondrocytes and adipose tissue to create a customized ear framework that could be further used for surgical reconstruction.

Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation.

There is heterogeneity among donor cells of the same source. Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer (SCNT). However, the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level. In this study, single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns. The results showed that 48 cells had similar gene expression patterns, whereas 4 cells (D11_1, D12_1, DW61_2, DW99_2) had significantly different gene expression patterns from those of other cells. There were no two cells with identical gene expression patterns. The gene expression patterns of D11_1, D12_1, DW61_2 and DW99_2 were analyzed, using the 48 cells with similar gene expression patterns as controls. Firstly, we used the R language statistics to select the differentially expressed genes in the 4 single cells, and identified the top 50 most significant differentially expressed genes. Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes. Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism, protein metabolism and cell response to stimulation. The main pathways from KEGG enrichment were related to cell cycle, cell metabolism, and DNA replication. Finally, based on the above results and in consideration with the SCNT research progress, we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation. This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells, thereby providing new ideas on improving the cloning efficiency of SCNT.

Modulated delivery of donepezil using a combination of skin microporation and iontophoresis.

The present study investigated the transdermal delivery of donepezil hydrochloride across dermatomed porcine ear skin using passive and physical enhancement techniques. In vitro permeation studies were performed on Franz diffusion cells. Microneedles were fabricated in the lab using a polymeric blend of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). The fabricated microneedles were characterized using SEM. Effect of PVA-PVP microneedles and ablative laser (P.L.E.A.S.E) alone, and in combination with anodal iontophoresis on the delivery of donepezil hydrochloride was investigated. Scanning electron microscopy, histology, methylene blue staining, and confocal laser microscopy were used to characterize the microchannels created in the skin. Permeation of donepezil after passive delivery was found to be 26.87±3.97µg/sq.cm. Microneedles, laser, and iontophoresis significantly increased the permeation to 282.23±8.28µg/sq.cm, 1562±231.8µg/sq.cm and 623.4±21.3µg/sq.cm. Also, a significantly higher permeation was achieved with microneedles and laser in combination with iontophoresis (1000±160.9µg/sq.cm and 1700.4±189.43µg/sq.cm respectively). A sharp increase in flux was observed with a combination of skin microporation and iontophoresis, however, the same was not observed for iontophoretic delivery alone. Thus, flux can be successfully tailored with a combination of skin microporation and iontophoresis to suit patient needs.

Hydrogels containing soybean isoflavone aglycones-rich fraction-loaded nanoemulsions for wound healing treatment – in vitro and in vivo studies

Soybean isoflavone aglycones have been investigated as potential wound healing compounds for topical application. The aim of this study was to evaluate the wound healing properties of a soybean isoflavone aglycones-rich fraction (IAF) when incorporated into lipid nanoemulsions dispersed in acrylic-acid hydrogels. Formulations exhibited a mean droplet size in the sub 200 nm range, negative ζ-potential (−60 mV), and displayed non-Newtonian pseudoplastic behavior. The addition of a gelling agent decreased the IAF release from formulations and improved the retention of these compounds in intact porcine ear skin when compared with a control propylene glycol solution. No IAF were detected in receptor fluid of Franz-type diffusion cells. However, increasing amounts of IAF were noticed in both skin layers and the receptor fluid when the tissue was partially injured (tape stripping), or when the epidermis was completely removed. In vitro studies showed that IAF elicits an increased proliferation and migration of keratinocytes (HaCaT cell line). Subsequently, the healing effect of the formulations was evaluated in a model of dorsal wounds in rats, by assessing the size of the lesions, histology, inflammatory markers, and antioxidant activity. Overall findings demonstrated the potential of IAF-loaded formulations to promote wound healing by increasing angiogenesis by ∼200 %, reducing the lipid oxidation (TBARS) by ∼52 % and the inflammation (TNFα) by ∼35 %, while increasing re-epithelialization by ∼500 %, visualized by the epithelium thickness.

Development, characterization and evaluation of nanocarrier based formulations of antipsoriatic drug “acitretin” for skin targeting

Acitretin, a 2nd generation retinoid, is a drug of choice for the treatment of severe psoriasis. Presently, only capsule formulation for oral administration is commercially available and associated with limitations of severe systemic side effects. In the present study, carrier elastic liposomal and ethosomal based topical gel formulations were developed, optimized, and characterized in-vitro, ex-vivo, and in-vivo to provide effective skin targeting of acitretin. In-vitro characterization exhibited nanometric vesicle size range (591 ± 16 and 360 ± 14 nm), high encapsulation efficacy (94.7 ± 2.1% and 91.2 ± 1.6%), optimum viscosity (1152 and 1320 cP), thixotropic behavior and good storage stability of the developed formulation. Ex-vivo skin permeation and deposition studies carried on four different biological membranes (rat, mice, porcine ear, and human cadaver skin) showed higher skin deposition of prepared carrier-based gels in comparison to plain acitretin gel. Better skin localization potential was also confirmed by mechanistic studies using Confocal Laser Scanning Microscopy and Scanning Electron Microscopy analysis. In-vivo antipsoriatic activity demonstrated the better therapeutic efficacy of carrier-based formulations. Thus, developed formulation seems to be a safe and promising alternative to oral route for effective topical delivery of acitretin with improved pharmacological activity and no systemic toxicity.

Generation of Marker-Free pbd-2 Knock-in Pigs Using the CRISPR/Cas9 and Cre/loxP Systems.

Porcine β-defensin 2 (PBD-2), expressed by different tissues of pigs, is a multifunctional cationic peptide with antimicrobial, immunomodulatory and growth-promoting abilities. As the latest generation of genome-editing tool, CRISPR/Cas9 system makes it possible to enhance the expression of PBD-2 in pigs by site-specific knock-in of pbd-2 gene into the pig genome. In this study, we aimed to generate marker-free pbd-2 knock-in pigs using the CRISPR/Cas9 and Cre/loxP systems. Two copies of pbd-2 gene linked by a T2A sequence were inserted into the porcine Rosa26 locus through CRISPR/Cas9-mediated homology-directed repair. The floxed selectable marker gene neoR, used for G418 screening of positive cell clones, was removed by cell-penetrating Cre recombinase with a recombination efficiency of 48.3%. Cloned piglets were produced via somatic cell nuclear transfer and correct insertion of pbd-2 genes was confirmed by PCR and Southern blot. Immunohistochemistry and immunofluorescence analyses indicated that expression levels of PBD-2 in different tissues of transgenic (TG) piglets were significantly higher than those of their wild-type (WT) littermates. Bactericidal assays demonstrated that there was a significant increase in the antimicrobial properties of the cell culture supernatants of porcine ear fibroblasts from the TG pigs in comparison to those from the WT pigs. Altogether, our study improved the protein expression level of PBD-2 in pigs by site-specific integration of pbd-2 into the pig genome, which not only provided an effective pig model to study the anti-infection mechanisms of PBD-2 but also a promising genetic material for the breeding of disease-resistant pigs.

Solid lipid microparticles for hair follicle targeting of adapalene and benzoyl peroxide - Release through targeted erosion

Sebum-dependent targeting of the upper hair follicle by solid lipid microparticles has been demonstrated for retinoids using adapalene as an example. An extension of this formulation principle to the combination of adapalene/benzoyl peroxide was achieved on the basis of congealed solid-in-oil dispersions of nanoparticulate benzoyl peroxide, which were incorporated into the solid lipid microparticles. To investigate the dermal distribution and release behaviour of the solid lipid microparticles after application, fluorescence microscopic ex vivo studies on porcine ear skin samples treated with the experimental vehicle were supplemented by calorimetric skin lipid interaction studies and release experiments on lipid-coated membranes, investigating the influence of sebum and stratum corneum lipids on the solid lipid microparticle matrix. Fluorescent microscopic images of the treated skin confirm the observation of preferential deposition in the upper hair follicle. In addition, the release experiments show a sebum-dependent release of the active ingredients, with no detectable release in the presence of stratum corneum lipids. The observed discrepancy is probably a result of the rapid disintegration of the particles on contact with sebum, as calorimetric investigations suggest. This behaviour, which is based on the similarity between the sebum components and the lipid matrix, allows a targeted release into sebum and thus increased follicular penetration.

Abstract 6239: A novel topical delivery system of carvedilol for skin cancer prevention

Abstract Solar ultraviolet (UV) light-induced non-melanoma skin cancer is the most common type of cancer diagnosed in the United States and remains one of the major public health concerns. There is an urgent need to develop effective and safe skin cancer chemopreventive agents. We have previously demonstrated that carvedilol, an FDA approved β-blocker for cardiovascular diseases, could prevent UV-induced skin cancer in vitro and in vivo. However, the β-adrenergic blockade effects of carvedilol might be a barrier for its repurposing as a cancer chemopreventive agent. In this study, we designed a novel topical formulation, namely carvedilol-loaded transfersomes (T-CAR), to maintain cancer preventive properties of carvedilol while avoiding systemic cardiovascular effects. T-CAR formulations were prepared using different phospholipids and surfactants at various ratios by the thin-film hydration method. The formulations were characterized in terms of particle size, zeta potential, and encapsulation efficiency. An optimal formulation was identified that composes carvedilol, soy phosphatidylcholine (SPC), and Tween 80 at a ratio of 1:3:0.5 with the particle size of 95.84 ± 3.8 nm, the zeta potential of +11.34 ± 0.7 mV, and the encapsulation efficiency of 93.7 ± 5.1%. The in vitro drug release and ex vivo skin permeation studies were performed using Pur-A-Lyzer mini dialysis kit and Franz diffusion cells, respectively. The optimal formulation showed a relative slow in vitro release profile and it could permeate through the porcine ear skin to the same degree as free carvedilol dissolved in acetone. 200 uL of 10 uM carvedilol dissolved in acetone, T-CAR containing the same amount of carvedilol as in acetone group, or the plain transfersome were topically applied to SKH-1 hairless mice 30 minutes before and immediately after single-dose UV irradiation. Compared to carvedilol solution, T-CAR demonstrated the same inhibitory effects against UV-induced epidermis thickening but exhibited increased inhibitory effects against UV-induced cyclobutane pyrimidine dimer (CPD) formation and mRNA expression of pro-inflammatory cytokines IL-1β and IL-6. In addition, carvedilol was not detectable by LC-MS in plasma after topical administration. These findings suggest that T-CAR could be a promising topical carvedilol formulation to prevent UV-associated skin cancer, with enhanced photoprotective effects but neglectable systemic cardiovascular effects. Citation Format: Mengbing Chen, Steven Yeung, Md Abdullah Shamim, Kaitlyn Coronel, Ayaz Shahid, Bradley Andresen, Jeffrey Wang, Ying Huang. A novel topical delivery system of carvedilol for skin cancer prevention [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6239.

Emulgels Containing Carbopol 934P and Different Vegetable Oils for Topical Propolis Delivery: Bioadhesion, Drug Release Profile, and Ex Vivo Skin Permeation Studies.

Topical administration can enable a more efficient therapy based on the improved bioavailability and patient compliance. Wounds and infections can lead to modifications of skin physiology and body protective function. Propolis (PRP) is utilized for skin protection and treatment. However, PRP extracts do not show suitable rheological characteristics and can cause irritation, pain, ulceration, and healing difficulties when they are administered on the harmed skin. Emulgels composed of Carbopol 934P (C934P) and different vegetable oils have been proposed for propolis extract release and may be a good strategy for topical delivery. The aim of this study was to investigate the bioadhesive properties, PRP release profile, skin permeation, and retention, by Franz's diffusion cell and photoacoustic spectroscopy (PS), of these emulgels. Formulations were composed of C934P and passion fruit oil (PF), sweet almond oil (SA), or andiroba oil (AO). PRP or by-product extracts were added to the systems, drug release profile was investigated, and porcine ear skin was utilized for analyses of bioadhesive properties, skin permeation, and retention. All formulations displayed similar bioadhesive force (0.05-0.07N); PRP release was modified (prolonged), dependent on formulation composition, and mainly governed by diffusion. PS and analysis using diffusion cell showed that the systems could provide dermal permeation and retention, which was more effective for formulations containing AO. Considering the importance of propolis for many skin therapies, the emulgels containing AO for PRP delivery are worthy of biological studies and further clinical evaluation.

Development of nanoemulsions containing penciclovir for herpes simplex treatment and a liquid chromatographic method to drug assessment in porcine skin layers

A successful formulation (penciclovir hydrogel nanoemulsion - HN) to be used in transdermal drug delivery route to treat herpes simplex virus was developed and an analytical method to quantify penciclovir (PCV) in porcine ear skin was stablished and validated. PCV nanoemulsions were prepared by high pressure homogenization and presented spherical mean droplet size 180 nm. The association efficiency and zeta potential were 87% and– 27 mV, respectively. The bioanalytical method developed showed specificity for the interferences from the skin matrixes (epidermis and dermis) and the linearity was in the range 0.1 – 25 μg/mL of drug, The mean recovery data for the three levels tested were 95.2% for the epidermis and 97.3% for the dermis and adequate results were obtained for repeatability and inter-day precision. In vitro percutaneous absorption studies with the PCV nanoemulsion and a market cream were conducted employing the porcine skin.The cumulative amounts of PCV permeated from cream and HN, 8 h after dosing, were 2.60 and 4.15 μg/cm², respectively, representing a quite higher flux and a much higher permeability coefficient for the developed formulation. It can be concluded that HN provide a good skin targeting effect and may be a promising carrier for topical delivery of penciclovir.

Validation study of an ex vivo porcine auricular model for simulation training in otoplasty.

To perform a validation assessment of a novel porcine ex vivo model for otoplasty training. A total of nine otolaryngology trainees performed a standard approach otoplasty on a porcine ear. They completed a series of tasks including posterior skin incision, anterior scoring, Mustardé suture placement and concha-mastoid suture placement. Trainees completed a post-task questionnaire assessing face validity, global content validity and task-specific content validity. Trainees' median scores for the porcine model were: 4 for face validity (interquartile range, 3-4), 5 for global content validity (interquartile range, 4-5) and 4 for task-specific content validity (interquartile range, 4-4). This study is the first to formally validate the ex vivo porcine auricular model as a useful tool for training in otoplasty. The model should be incorporated into simulation training for otoplasty in order to improve learning, enable acquisition of specific surgical skills and improve operative outcomes.

Tissue Levels of Flurbiprofen in the Rat Plantar Heel after Short-Duration Topical Iontophoresis are Sufficient to Induce Pharmacodynamic Responses to Local Pain Stimuli.

The objective of this study was to investigate the topical iontophoresis of flurbiprofen (FBF) as a means to enhance its local bioavailability and thereby provide an improved and targeted treatment of plantar heel pain. Initial in vitro experiments using porcine ear skin investigated iontophoretic transport of FBF under different conditions. Local FBF biodistribution in the rat paw in vivo was compared after topical or oral administration. Efficacy of pain management was investigated using a plantar incisional model by evaluating pharmacodynamic responses to local pain stimuli. The results demonstrated that iontophoresis of FBF significantly increased cutaneous deposition and transdermal permeation of FBF as compared to passive delivery—it also enabled drug input to be controlled by modulation of current density and drug concentration (r2 > 0.99). Topical iontophoresis of FBF in vivo enabled higher drug levels in skin and muscle in rat plantar aspect and superior pharmacodynamic responses to local pain stimuli, in comparison to oral and passive delivery. In conclusion, short-duration topical iontophoresis of FBF may better help to relieve plantar heel pain than oral or passive administration, which should be of clinical interest.

A stability-indicating ultra-fast liquid chromatography method for the assay of the main flavonoids of Achyrocline satureioides (Marcela) in porcine skin layers and nanoemulsions.

Achyrocline satureioides (marcela or macela) is a plant widely used in folk medicine in South America. Recently, there has been increasing interest for the development of skin care products containing A. satureoides extracts, due to its well-documented antioxidant, antiherpetic, and wound healing properties. The present study aimed to develop and validate a yet unexplored stability-indicating and robust ultra-fast liquid chromatography (UFLC) method for the simultaneous quantification of the main flavonoids of A. satureioides in extracts, nanoemulsions, and porcine skin layers. The chromatographic separation of flavonoids quercetin, luteolin, and 3-O-methylquercetin was performed on a Luna C18 analytical column (100 mm × 2.0 mm i.d.; particle size 2.5 μm) using isocratic elution with methanol/phosphoric acid 1% (48:52 v/v) with a flow rate of 0.3 mL/min at 40°C. The method was found to be specific, linear (R > 0.998), precise, accurate, and robust for all flavonoids assayed in A. satureioides extract, nanoemulsions, and porcine ear skin. A low matrix effect was noted for all complex matrices. The stability-indicating UFLC method was evaluated by submitting isolated flavonoids, a mixture of standards, and A. satureioides extract to acidic, alkaline, oxidative, UV-A/UV-C light, and thermal stress conditions. No peaks were found co-eluting with the flavonoids of interest in all matrices. The robustness of the method was confirmed using Plackett-Burman experimental design. The short run time (8 min) and reliability of the method could be useful for the determination of A. satureioides flavonoids in topical product development since extracts of this medicinal plant have been used to treat various skin disorders.

Ex Vivo and In Vivo Assessment of the Penetration of Topically Applied Anthocyanins Utilizing ATR-FTIR/PLS Regression Models and HPLC-PDA-MS.

Anthocyanins are natural colorants with antioxidant properties, shown to inhibit photoaging reactions and reduce symptoms of some skin diseases. However, little is known about their penetration through the stratum corneum, a prerequisite for bioactivity. The aim was to investigate anthocyanin penetration from lipophilic cosmetic formulations through the skin using a porcine ear model and human volunteers. ATR-FTIR/PLS regression and HPLC-PDA-MS were used to analyze anthocyanin permeation through the stratum corneum. Penetration of all anthocyanins was evident and correlated with molecular weight and hydrophilicity. Lower-molecular-weight (MW) anthocyanins from elderberry (449–581 Da) were more permeable within the skin in both ex vivo and in vivo models (Kp = 2.3–2.4 × 10−4 cm h−1) than the larger anthocyanins (933-1019 Da) from red radish (Kp = 2.0–2.1 × 10−4 cm h−1). Elderberry and red radish anthocyanins were found at all levels of the stratum corneum and at depths for activity as bioactive ingredients for skin health.

Bio-inspired apatite particles limit skin penetration of drugs for dermatology applications

Most treatments of skin pathologies involve local administration of active agents. One issue can however be the partial transcutaneous diffusion of the drug to blood circulation, leading to undesirable effects. In this work, the original use of submicron mineral particles based on bio-inspired calcium phosphate apatite was explored for the first time as drug carriers for favoring topical delivery. The permeation of a model drug across synthetic and biological membranes was investigated in both static and dynamic conditions. Our data show that adsorption of the drug on the apatite particles surface drastically limits its permeation, with lower effective diffusion coefficients (Peff) and smaller total released amounts. The retention of the apatite colloidal particles on porcine ear skin explants surface was demonstrated by combining histological observations and Raman confocal microscopy. All results converge to show that association of the drug to apatite particles favors skin surface effects. These findings point to the relevance of mineral-based particles as drug carriers for local delivery to the skin, and open the way to novel applications of bio-inspired apatites in dermatology. Statement of SignificanceCalcium phosphates (CaP) are major biomaterials in orthopedics and dentistry. Their resemblance to bone mineral allows new applications beyond bone repair, e.g. in nanomedicine. In 2018, a 14-page detailed review (M. Epple, Acta Biomaterialia 77 (2018) 1–14) provided clear facts in favor of the non-toxicity of nanosized CaP as an answer to discussions from EU and US study groups, thus clarifying the path to novel applications of nano CaP. In the present paper, bio-inspired apatite nanoparticles are used for the first time as drug carriers for dermatology for drastically limiting drug transcutaneous permeation and retaining a topical effect. We demonstrate this proof of concept via permeation cell tests, histology, Raman microscopy and photoluminescence after application on porcine ear skin.

N-acetylglucosamine microemulsions: Assessment of skin penetration, cytotoxicity, and anti-melanogenesis.

N-acetylglucosamine (NAG) is an amino sugar which can reduce melanin production. NAG has previously been formulated for topical use in many nanocarrier systems, excluding microemulsions (MEs). In this study, NAG was prepared in the form of MEs and assessed in terms of skin permeability, cytotoxicity, and effectiveness for cosmetic applications. To investigate the skin penetration, cytotoxicity, and anti-melanogenesis of N-acetylglucosamine loaded microemulsions (NAG-MEs). Two NAG-MEs (NME1 and NME2) were prepared. The in vitro penetration study of NAG-MEs was evaluated by modified Franz diffusion cells using full-thickness porcine ear skin as the membrane. The optimized formula was then selected for further assessment of cytotoxicity and efficiency. In vitro cytotoxicity was examined using human keratinocytes (HaCaT cells) and B16 melanoma cells. Anti-melanogenic activity was investigated by determination of melanin production of B16 melanoma cells. The cumulative amounts of NAG from NME1 and NME2 in the receptor fluid at 24hours were 1010.46±31.63 and 1260.99±100.19µg/cm2 and those accumulated in the skin membrane were 155.59±19.19 and 181.11±20.38µg/cm2 , respectively. NME2 and its blank counterpart (Blank-ME2) showed no adverse effects on the viability of both HaCaT and B16 melanoma cells. The anti-melanogenic activity data showed that the NME2 treated B16 cells exhibited a significant melanin reduction. NAG-MEs could allow NAG penetrate through and accumulate in full-thickness porcine ear skin. NME2 was safe for both normal human keratinocytes and melanoma cells. It also showed effectiveness on anti-melanogenic activity in B16 melanoma cells.