Trivalent arsenicals induce skin toxicity through thiol depletion.

Arsenic, a widespread environmental contaminant, is highly toxic to human health. Arsenic exposure is associated with the occurrence of skin lesions and diseases. This study investigated the dermal toxicity of trivalent arsenicals (AsIII and MMAIII) and its underlying mechanism using human keratinocyte cell line and ex vivo porcine skin. AsIII and MMAIII induced concentration-dependent cell apoptosis and necrosis in HaCaT cells, which was confirmed in ex vivo porcine skin. AsIII and MMAIII increased reactive oxygen species generation and GSH depletion. Interestingly, radical scavenger antioxidants such as Vitamin C failed to mitigate arsenic-induced cytotoxicity, while thiol-containing compounds effectively alleviated it, suggesting a key role of thiol depletion in the trivalent arsenical-induced dermal toxicity. DMSA showed the strongest protective effects against AsIII and MMAIII-induced cytotoxicity in HaCaT cells. Of note, DMSA restored arsenical-induced tissue damage, and reduced the apoptosis in ex vivo porcine skin, highlighting its potential use to alleviate arsenic-induced skin lesions and diseases.

Tape Stripping Method in Dermatological and Pharmacological Research: Evaluating Fresh and Frozen-Thawed Porcine Skin.

Background The stratum corneum (SC) plays a crucial role in protecting the skin and regulating water loss. Tape stripping is a well-established method for studying skin barrier function and evaluating topical treatments. However, the behavior of fresh versus frozen-thawed skin during tape stripping has not been extensively compared. Objective This study aims to compare the removal of the stratum corneum from fresh and frozen-thawed porcine skin using tape stripping. It also aims to assess the reliability of tape weighing versus histological methods in quantifying SC removal. Methods Fresh and frozen-thawed porcine ears were obtained, cleaned, and subjected to tape stripping at varying numbers of strips from zero to 40. Tape weight and histological measurements were used to quantify SC removal. Statistical analyses were conducted to compare SC thickness and tape weight between the two types of skin. Results The study found that frozen-thawed skin exhibited a non-linear rate (r = 0.65) of SC removal per tape strip in the first five strips compared to a linear removal for fresh skin (r = 0.96). By the fifth tape strip, frozen-thawed samples had lost 80.6% of their SC, while fresh samples had only lost 33.5% (P < 0.03). Tape weighing and histological measurements showed strong correlations (r = 0.93 for fresh skin and r = 0.95 for frozen-thawed skin), indicating that tape weighing is a reliable alternative to histology for assessing SC removal on both sample types. Conclusions Fresh and frozen-thawed porcine skin respond differently to tape stripping, with frozen-thawed skin showing accelerated SC removal in the first five strips. The strong correlation between tape weighing and histological analysis supports the use of tape weighing as a practical tool for evaluating SC removal. These findings have implications for specimen selection and methodological standardization in dermatological and pharmacological research. Future research should explore alternative preservation and SC thickness measurement methods and their impact on tape stripping outcomes.

Gene Therapeutic Drug pCMV-VEGF165 Plasmid ('Neovasculgen') Promotes Gingiva Soft Tissue Augmentation in Rabbits.

Currently, an increasing number of patients are undergoing extensive surgeries to restore the mucosa of the gums in the area of recessions. The use of a connective tissue graft from the palate is the gold standard of such surgical treatment, but complications, especially in cases of extensive defects, have led to the development of approaches using xenogeneic collagen matrices and methods to stimulate their regenerative and vasculogenic potential. This study investigated the potential of a xenogeneic scaffold derived from porcine skin Mucoderm and injections of the pCMV-VEGF165 plasmid ('Neovasculgen') to enhance soft gingival tissue volume and vascularization in an experimental model in rabbits. In vitro studies demonstrated the biocompatibility of the matrix and plasmid with gingival mesenchymal stem cells, showing no toxic effects and supporting cell viability and metabolic activity. In the in vivo experiment, the combination of Mucoderm and the pCMV-VEGF165 plasmid (0.12 mg) synergistically promoted tissue proliferation and vascularization. The thickness of soft tissues at the implantation site significantly increased with the combined application (3257.8 ± 1093.5 µm). Meanwhile, in the control group, the thickness of the submucosa was 341.8 ± 65.6 µm, and after the implantation of only Mucoderm, the thickness of the submucosa was 2041.6 ± 496.8 µm. Furthermore, when using a combination of Mucoderm and the pCMV-VEGF165 plasmid, the density and diameter of blood vessels were notably augmented, with a mean value of 226.7 ± 45.9 per 1 mm2 of tissue, while in the control group, it was only 68.3 ± 17.2 per 1 mm2 of tissue. With the application of only Mucoderm, it was 131.7 ± 37.1 per 1 mm2 of tissue, and with only the pCMV-VEGF165 plasmid, it was 145 ± 37.82 per 1 mm2 of the sample. Thus, the use of the pCMV-VEGF165 plasmid ('Neovasculgen') in combination with the xenogeneic collagen matrix Mucoderm potentiated the pro-proliferative effect of the membrane and the pro-vascularization effect of the plasmid. These results indicate the promising potential of this innovative approach for clinical applications in regenerative medicine and dentistry.

In vitro skin puncture methodology for material characterization.

Quantifying the mechanical behavior of skin has been foundational in applications of cosmetics, surgical techniques, forensic science, and protective clothing development. However, previous puncture studies have lacked consistent and physiological boundary conditions of skin. To determine natural skin tension, excision of in situ porcine skin resulted in significantly different diameter reduction (shrinkage) in leg (19.5 %) and abdominal skin (38.4 %) compared to flank skin (28.5 %) (p = 0.047). To examine effects of initial tension and pre-conditioning, five conditions of initial tension (as percentage of diameter increase) and pre-conditioning were tested in quasistatic puncture with a 5 mm spherical impactor using an electrohydraulic load frame and custom clamping apparatus. Samples with less than 5 % initial tension resulted in significantly greater (p = 0.011) force at failure (279.2 N) compared to samples with greater than 25 % initial tension (195.1 N). Eight pre-conditioning cycles of 15 mm displacement reduced hysteresis by 45 %. The coefficient of variance was substantially reduced for force, force normalized by cutis thickness, displacement, stiffness, and strain energy up to 46 %. Pre-conditioned samples at physiological initial tension (14-25 %) resulted in significantly greater (p = 0.03) normalized forces at failure (278.3 N/mm) compared to non-conditioned samples of the same initial tension (234.4 N/mm). Pre-conditioned samples with 14-25 % initial tension, representing physiological boundary conditions, resulted in the most appropriate failure thresholds with the least variation. For in vitro puncture studies, the magnitude of applied initial tension should be defined based on anatomical location, through a shrinkage experimentation, to match natural tension of skin. Characterizing the biological behavior and tolerances of skin may be utilized in finite element models to aid in protective clothing development and forensic science analyses.

Percutaneous Absorption of Fireground Contaminants: Naphthalene, Phenanthrene, and Benzo[a]pyrene in Porcine Skin in an Artificial Sweat Vehicle.

Firefighters face significant risks of exposure to toxic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), during fire suppression activities. PAHs have been found in the air, on the gear and equipment, and in biological samples such as the skin, breath, urine, and blood of firefighters after fire response. However, the extent to which exposure occurs via inhalation, dermal absorption, or ingestion is unclear. In this study, three PAHs, naphthalene, phenanthrene, and benzo[a]pyrene, were applied to porcine skin in vitro in an artificial sweat solution to better gauge firefighters' dermal exposures while mimicking their sweaty skin conditions using an artificial sweat dosing vehicle. Multiple absorption characteristics were calculated, including cumulative absorption, percent dose absorbed, diffusivity, flux, lag time, and permeability. The absorption of the PAHs was greatly influenced by their molecular weight and solubility in the artificial sweat solution. Naphthalene had the greatest dose absorption efficiency (35.0 ± 4.6% dose), followed by phenanthrene (6.8 ± 3.2% dose), and lastly, benzo[a]pyrene, which had the lowest absorption (0.03 ± 0.04% dose). The lag times followed a similar trend. All chemicals had a lag time of approximately 60 min or longer, suggesting that chemical concentrations on the skin may be reduced by immediate skin cleansing practices after fire exposure.

Transdermal characteristic study of bovine sialoglycoproteins with anti-skin aging and accelerating skin wound healing.

BACKGROUND: Sialoglycoproteins play important roles in various biological processes, including cell adhesion, immune response, and cell signaling. Our previous studies indicated that the bovine sialoglycoproteins could be developed as a reagent against skin aging and as a new candidate for accelerating skin wound healing as well as inhibiting scar formation. However, transdermal characteristic of the bovine sialoglycoproteins is still unknown. AIMS: This study investigated the transdermal permeation of the bovine sialoglycoproteins through porcine skin using the Franz diffusion cell method. RESULTS: Our study showed that the bovine sialoglycoproteins could penetrate through the porcine skin with a linear permeation pattern described by the regression equation N% = 11.49 t-3.858, with a high coefficient of determination (R2 = 0.9903). The histochemical results demonstrated the widespread distribution of the bovine sialoglycoproteins between the epidermal and dermal layers, which suggesting parts of the bovine sialoglycoproteins had ability to traverse the epidermal barrier. The results of the lectin microarrays indicated highly enriched glycopatterns on the bovine sialoglycoproteins, which also appeared in permeated porcine skin. The LC-MS/MS analysis further showed that the bovine sialoglycoproteins were composed of approximately 100 proteins with molecular weight ranging from 748.4 kDa to 10 kDa, and there were 23 specific bovine sialoglycoproteins with molecular weight ranging from 69.2 kDa to 10 kDa to be characterized in permeated porcine skin. CONCLUSIONS: Parts of the bovine sialoglycoproteins with molecular weight less than 69.2 kDa had ability to traverse the epidermal barrier. Understanding the permeation characteristics of the bovine sialoglycoproteins for developing innovative formulations with therapeutic benefits, contributing to advancements in cosmetic and dermatological fields.

Physiologically-based pharmacokinetic model of in vitro porcine ear skin permeation for drug delivery research.

In silico techniques, such as physiologically based pharmacokinetic modeling (PBKP), are recently gaining importance. Computational methods in drug discovery and development and the generic drugs industry enhance research effectiveness by saving time and money and avoiding ethical issues. One key advantage is the ability to conduct toxicology studies without risking harm to living beings. This study aimed to repurpose the multi-phase multi-layer mechanistic dermal absorption (MPML MechDermA) PBPK model for simulation permeation through porcine ear skin under in vitro conditions. The work was divided into four steps: (1) the development of a pig ear skin model based on a previously collected dataset; (2) testing the model's ability to discriminate permeation between pig ear, human abdomen, and human back skin; (3) development of a caffeine permeation model; and (4) testing the caffeine model's performance against in vitro generated data sourced from the scientific literature. Data from 31 manuscripts were used for the development of the pig skin model. Based on these data, values specific to pig skin were found for 22 parameters of the MPML MechDermA model. The model was able to discriminate permeation between pig and human skin. A caffeine model was developed and used to simulate seven experiments identified in the literature. The model's performance was assessed by comparing simulated to observed results. Based on a visual check, all simulations were considered acceptable, whereas three out of seven experiments met the twofold difference criterion. The variability of the experimental data was considered the biggest challenge for reliable model assessment.

Mesoporous Silica-Based Membranes in Transdermal Drug Delivery: The Role of Drug Loss in the Skin.

Compared to other forms of drug administration, the use of Transdermal Drug Delivery Systems (TDDSs) offers significant advantages, including uniform drug release profiles that contribute to lower side effects and higher tolerability, avoidance of direct exposure to the gastrointestinal tract, better patient compliance due to their non-invasive means of application and others. Mesoporous silica membranes are of particular interest in this regard, due to their chemical stability and their tunable porous system, with adjustable pore sizes, pore volumes and surface chemistries. While this allows for fine-tuning and, thus, the development of optimized TDDSs with high loading capacities and the desired release profile of a given drug, its systemic availability also relies on skin penetration. In this paper, using a TDDS based on mesoporous silica membranes in Franz cell experiments on porcine skin, we demonstrate surprisingly substantial drug loss during skin penetration. Drug passage through porcine skin was found to be dependent on the age and pre-treatment of the skin. pH and temperature were major determinants of drug recovery rates as well, indicating drug loss in the skin by enzymatic metabolization. Regarding the TDDS, higher loading obtained by SO3H surface modification of the mesoporous silica membranes reduced drug loss. Still, high loss rates in the skin were determined for different drugs, including anastrozole, xylazine and imiquimod. We conclude that, beyond the fine-tuned drug release profiles from the mesoporous silica membrane TDDS, remarkably high drug loss in the skin is a major issue for achieving desired skin penetration and, thus, the systemic availability of drugs. This also poses critical requirements for defining an optimal TDDS based on mesoporous silica membranes.

Complex coacervation of low methoxy pectin with three types of gelatins for the encapsulation of fish oil.

Herein, the complex coacervation of low methoxy pectin (LMP) with three types of gelatins was explored to encapsulate fish oil. The fish oil@gelatin-LMP complex coacervates with good precipitation separation could be obtained at low gelatin concentrations (Fish gelatin, FG: 10-80 mg/mL; porcine skin gelatin, PSG: 10-40 mg/mL; bovine skin gelatin, BSG: 10-80 mg/mL), high gelatin: fish oil mass ratios (4:1-1:1), appropriate gelatin: LMP mass ratios (3:1-12:1 for FG and PSG, 6:1 for BSG), and appropriate pH (FG: 4.90-5.50; PSG: 4.80-5.40; BSG: 4.10-4.50). FG induced similar loading ability, lower encapsulation ability, and comparable peroxide values to the mammalian gelatins. FG induced higher or similar free fatty acid released percentages to mammalian gelatins in the in vitro gastrointestinal model at low gelatin concentrations (10-40 mg/mL). These results provided useful information to understand the protein-polysaccharide complex coacervation to encapsulate oil-based bioactive substances.

Evaluation of emergency skin decontamination protocols in response to an acid attack (vitreolage).

The incidence of "acid attacks" (vitreolage) is a global concern, with those affected often receiving lifelong medical care due to physical and psychological damage. The purpose of this study was to evaluate the effectiveness of several emergency skin decontamination approaches against concentrated (>99 %) sulphuric acid and to identify the effective window of opportunity for decontamination. The effects of four decontamination methods (dry, wet, combined dry & wet and cotton cloth) were assessed using an in vitro diffusion cell system containing dermatomed porcine skin. Sulphuric acid (H2SO4) was applied to the skin with decontamination protocols performed at 10 s, 30 s, 8 min, and 30 min post exposure. Skin damage was quantified by tritiated water (3H2O) penetration, receptor fluid pH and photometric stereo imaging (PSI), with quantification of residual sulphur (by SEM-EDS) to determine overall decontamination efficiency. Skin translucency (quantified by PSI) demonstrated a time-dependent loss of dermal tissue integrity from 10 s. Quantification of dermal sulphur content confirmed the rapid (exponential) decrease in decontamination efficiency with time. The pH of the water effluent indicated complete neutralisation of acid from the skin surface after 90 s of irrigation. Wet decontamination (either alone or immediately following dry decontamination) was the most effective intervention evaluated, although no decontamination technique was statistically effective after 30 s exposure to the acid. These data demonstrate the time-critical consequences of dermal exposure to concentrated sulphuric acid: we find no practical window of opportunity for acid decontamination, as physical damage is virtually instantaneous.

Nanocomposite Gels Loaded with Flurbiprofen: Characterization and Skin Permeability Assessment in Different Skin Species.

Nanocomposite gels consist of nanoparticles dispersed in a gel matrix. The main aim of this work was to develop nanocomposite gels for topical delivery of Flurbiprofen (FB) for humans and farm animals. Nanocomposite gels were prepared stemming from nanoparticles (NPs) freeze-dried with two different cryoprotectants, D-(+)-trehalose (NPs-TRE) and polyethylene glycol 3350 (NPs-PEG), sterilized by gamma (γ) irradiation, and gelled with Sepigel® 305. Nanocomposite gels with FB-NPs-TRE and FB-NPs-PEG were physiochemically characterized in terms of appearance, pH, morphological studies, porosity, swelling, degradation, extensibility, and rheological behavior. The drug release profile and kinetics were assessed, as well as, the ex vivo permeation of FB was assessed in human, porcine and bovine skin. In vivo studies in healthy human volunteers were tested without FB to assess the tolerance of the gels with nanoparticles. Physicochemical studies demonstrated the suitability of the gel formulations. The ex vivo skin permeation capacity of FB-NPs nanocomposite gels with different cryoprotectants allowed us to conclude that these formulations are suitable topical delivery systems for human and veterinary medicine. However, there were statistically significant differences in the permeation of each formulation depending on the skin. Results suggested that FB-NPs-PEG nanocomposite gel was most suitable for human and porcine skin, and the FB-NPs-TRE nanocomposite gel was most suitable for bovine skin.

The porcine skin microbiome exhibits broad fungal antagonism.

The skin and its microbiome function to protect the host from pathogen colonization and environmental stressors. In this study, using the Wisconsin Miniature Swine™ model, we characterize the porcine skin fungal and bacterial microbiomes, identify bacterial isolates displaying antifungal activity, and use whole-genome sequencing to identify biosynthetic gene clusters encoding for secondary metabolites that may be responsible for the antagonistic effects on fungi. Through this comprehensive approach of paired microbiome sequencing with culturomics, we report the discovery of novel species of Corynebacterium and Rothia. Further, this study represents the first comprehensive evaluation of the porcine skin mycobiome and the evaluation of bacterial-fungal interactions on this surface. Several diverse bacterial isolates exhibit potent antifungal properties against opportunistic fungal pathogens in vitro. Genomic analysis of inhibitory species revealed a diverse repertoire of uncharacterized biosynthetic gene clusters suggesting a reservoir of novel chemical and biological diversity. Collectively, the porcine skin microbiome represents a potential unique source of novel antifungals.

Parafilm® M and Strat-M® as skin simulants in in vitro permeation of dissolving microarray patches loaded with proteins.

In vitro permeation studies play a crucial role in early formulation optimisation before extensive animal model investigations. Biological membranes are typically used in these studies to mimic human skin conditions accurately. However, when focusing on protein and peptide transdermal delivery, utilising biological membranes can complicate analysis and quantification processes. This study aims to explore Parafilm®M and Strat-M® as alternatives to dermatomed porcine skin for evaluating protein delivery from dissolving microarray patch (MAP) platforms. Initially, various MAPs loaded with different model proteins (ovalbumin, bovine serum albumin and amniotic mesenchymal stem cell metabolite products) were prepared. These dissolving MAPs underwent evaluation for insertion properties and in vitro permeation profiles when combined with different membranes, dermatomed porcine skin, Parafilm®M, and Strat-M®. Insertion profiles indicated that both Parafilm®M and Strat-M® showed comparable insertion depths to dermatomed porcine skin (in range of 360-430 µm), suggesting promise as membrane substitutes for insertion studies. In in vitro permeation studies, synthetic membranes such as Parafilm®M and Strat-M® demonstrated the ability to bypass protein-derived skin interference, providing more reliable results compared to dermatomed neonatal porcine skin. Consequently, these findings present valuable tools for preliminary screening across various MAP formulations, especially in the transdermal delivery of proteins and peptides.

Effects of polyphenol and gelatin types on the physicochemical properties and emulsion stabilization of polyphenol-crosslinked gelatin conjugates.

Herein, six types of polyphenol-crosslinked gelatin conjugates (PGCs) with ≥ two gelatin molecules were prepared using a covalent crosslinking method with two types of polyphenols (tannic acid and caffeic acid) and three types of gelatins (bovine bone gelatin, cold water fish skin gelatin, and porcine skin gelatin) for the emulsion stabilization. The structural and functional properties of the PGCs were dependent on both polyphenol and gelatin types. The storage stability of the conjugate-stabilized emulsions was dependent on the polyphenol crosslinking, NaCl addition, and heating pretreatment. In particular, NaCl addition promoted the liquid-gel transition of the emulsions: 0.2 mol/L > 0.1 mol/L > 0.0 mol/L. Moreover, NaCl addition also increased the creaming stability of the emulsions stabilized by PGCs except tannic acid-crosslinked bovine bone gelatin conjugate. All the results provided useful knowledge on the effects of molecular modification and physical processing on the properties of gelatins.

Histological evaluation of monopolar and bipolar radiofrequency microneedling treatment in a porcine model.

BACKGROUND AND OBJECTIVE: Fractional radiofrequency microneedling (FRM) is widely used as an option for skin rejuvenation, however there is a lack of histological evidence for the various energy delivery systems available. The objective was to assess thermal denaturation of tissue and the wound healing response in monopolar mode versus bipolar mode. Histological analysis was performed to demonstrate the efficacy of automatic impedance feedback system in monopolar mode. STUDY DESIGN AND METHODS: In this study, the acute thermal effects caused by monopolar FRM treatment to the dorsal skin of pigs were assessed histologically by hematoxylin & eosin (H&E) staining. Then, one session of either monopolar or bipolar FRM was used to treat one or the other side of the pig using varying power levels and pulse widths. The acute and chronic tissue reactions were assessed using H&E, immunofluorescence, and western blot analysis at 0, 14, 30, and 90 days after treatment. The efficacy of the impedance feedback system was also monitored histologically. RESULTS: High-energy FRM treatment produced tissue loss and necrosis. The power level and pulse duration significantly affected the coagulation amount. Histopathology at 0, 14, 30, and 90 days showed that the skin tissue reaction was more pronounced for bipolar compared to monopolar FRM. Immunofluorescence showed the expression of TGF-ß, Ki67, MMP3, and elastin increased dramatically with both modes, but were higher in the bipolar FRM treated side. The automatic impedance feedback system could effectively adjust the output energy. CONCLUSIONS: We found that bipolar FRM produced greater thermal effects, more collagen coagulation, and more pronounced molecular changes compared with monopolar mode in a porcine animal model.

3D human foreskin model for testing topical formulations of sildenafil citrate.

Sildenafil citrate is an approved drug used for the treatment of erectile dysfunction and premature ejaculation. Despite a widespread application, sildenafil citrate shows numerous adverse cardiovascular effects in high-risk patients. Local transdermal drug delivery of this drug is therefore being explored as an interesting and noninvasive alternative administration method that avoids adverse effects arised from peak plasma drug concentrations. Although human and animal skin represents the most reliable models to perform penetration studies, they involve a series of ethical issues and restrictions. For these reasons new in vitro approaches based on artificially reconstructed human skin or "human skin equivalents" are being developed as possible alternatives for transdermal testing. There is little information, however, on the efficiency of such new in vitro methods on cutaneous penetration of active ingredients. The objective of the current study was to investigate the sildenafil citrate loaded in three commercial transdermal vehicles using 3D full-thickness skin equivalent and compare the results with the permeability experiments using porcine skin. Our results demonstrated that, while the formulation plays an imperative role in an appropriate dermal uptake of sildenafil citrate, the D coefficient results obtained by using the 3D skin equivalent are comparable to those obtained by using the porcine skin when a simple drug suspension is applied (1.17 × 10-10 ± 0.92 × 10-10 cm2/s vs 3.5 × 102 ± 3.3 × 102 cm2/s), suggesting that in such case, this 3D skin model can be a valid alternative for ex-vivo skin absorption experiments.

Complete genome sequence of the bacterium Desemzia incerta isolated from the dorsal skin of a Yucatan pig.

We have shown previously that an isolate of Desemzia incerta from porcine skin has antimicrobial activity against methicillin-resistant Staphylococcus aureus. We present here the complete D. incerta genome containing one circular chromosome and five circular plasmids.

Influence of Dose, Particle Size and Concentration on Dermal Penetration Efficacy of Curcumin.

The influence of size, particle concentration and applied dose (finite vs. infinite dose) on the dermal penetration efficacy of curcumin was investigated in this study. For this, curcumin suspensions with different particle sizes (approx. 20 µm and approx. 250 nm) were produced in different concentrations (0.625-5% (w/w)). The dermal penetration efficacy was determined semi-quantitatively on the ex vivo porcine ear model. The results demonstrated that the presence of particles increases the dermal penetration efficacy of the active compounds being dissolved in the water phase of the formulation. The reason for this is the formation of an aqueous meniscus that develops between particles and skin due to the partial evaporation of water from the vehicle after topical application. The aqueous meniscus contains dissolved active ingredients, and therefore creates a small local spot with a locally high concentration gradient that leads to improved dermal penetration. The increase in penetration efficacy depends on the number of particles in the vehicle, i.e., higher numbers of particles and longer contact times lead to higher penetration efficacy. Therefore, nanocrystals with a high particle concentration were found to be the most suitable formulation principle for efficient and deep dermal penetration of poorly water-soluble active ingredients.

Development of an ex vivo porcine skin model for the preclinical evaluation of subcutaneously injected biomacromolecules.

Subcutaneous administration is used to deliver systemically-acting biotherapeutics, e.g. antibodies, and locally-acting biomacromolecules, e.g. hyaluronic acid. However, few preclinical models are available to evaluate post-injection behaviour in the tissue microenvironment. In vivo animal studies are costly, time-consuming, and raise obvious ethical concerns. In vitro models are cost-efficient, high-throughput solutions, but cannot simulate complex skin structure and biological function. An ex vivo model (containing hypodermis) with an extended culture period that enabled longitudinal studies would be of great interest for both the pharmaceutical and cosmeceutical industries. We describe the development of one such ex vivo model, using viable full-thickness porcine skin. Structural integrity was evaluated using a histological scoring system: spongiosis and epidermal detachment were identified as discriminating parameters. Ki67 and Claudin-1 expression reported on epidermal cell proliferation and barrier function, respectively and their expression decreased as a function of incubation time. After optimization, the system was used to investigate the fate/impact of subcutaneously administered hyaluronic acid (HA) formulations. The results showed that HA was localized at the injection site and adjacent adipocytes were well preserved during 5 days' incubation and confirmed that the full-thickness ex vivo porcine skin model could provide a platform for preclinical evaluation of subcutaneously injected biomacromolecules.

The Development of Nonthermal Plasma and Tirapazamine as a Novel Combination Therapy to Treat Melanoma In Situ.

Although melanoma accounts for only 5.3% of skin cancer, it results in >75% of skin-cancer-related deaths. To avoid disfiguring surgeries on the head and neck associated with surgical excision, there is a clear unmet need for other strategies to selectively remove cutaneous melanoma lesions. Mohs surgery is the current treatment for cutaneous melanoma lesions and squamous and basal cell carcinoma. While Mohs surgery is an effective way to remove melanomas in situ, normal tissue is also excised to achieve histologically negative margins. This paper describes a novel combination therapy of nonthermal plasma (NTP) which emits a multitude of reactive oxygen species (ROS) and the injection of a pharmaceutical agent. We have shown that the effects of NTP are augmented by the DNA-damaging prodrug, tirapazamine (TPZ), which becomes a free radical only in conditions of hypoxemia, which is often enhanced in the tumor microenvironment. In this study, we demonstrate the efficacy of the combination therapy through experiments with B16-F10 and 1205 Lu metastatic melanoma cells both in vitro and in vivo. We also show the safety parameters of the therapy with no significant effects of the therapy when applied to porcine skin. We show the need for the intratumor delivery of TPZ in combination with the surface treatment of NTP and present a model of a medical device to deliver this combination therapy. The importance of functional gap junctions is indicated as a mechanism to promote the therapeutic effect. Collectively, the data support a novel therapeutic combination to treat melanoma and the development of a medical device to deliver the treatment in situ.