Targeted Delivery of Zinc Pyrithione to Skin Epithelia.

Zinc pyrithione (ZnPT) is an anti-fungal drug delivered as a microparticle to skin epithelia. It is one of the most widely used ingredients worldwide in medicated shampoo for treating dandruff and seborrheic dermatitis (SD), a disorder with symptoms that include skin flaking, erythema and pruritus. SD is a multi-factorial disease driven by microbiol dysbiosis, primarily involving Malassezia yeast. Anti-fungal activity of ZnPT depends on the cutaneous availability of bioactive monomeric molecular species, occurring upon particle dissolution. The success of ZnPT as a topical therapeutic is underscored by the way it balances treatment efficacy with formulation safety. This review demonstrates how ZnPT achieves this balance, by integrating the current understanding of SD pathogenesis with an up-to-date analysis of ZnPT pharmacology, therapeutics and toxicology. ZnPT has anti-fungal activity with an average in vitro minimum inhibitory concentration of 10-15 ppm against the most abundant scalp skin Malassezia species (Malassezia globosa and Malassezia restrica). Efficacy is dependent on the targeted delivery of ZnPT to the skin sites where these yeasts reside, including the scalp surface and hair follicle infundibulum. Imaging and quantitative analysis tools have been fundamental for critically evaluating the therapeutic performance and safety of topical ZnPT formulations. Toxicologic investigations have focused on understanding the risk of local and systemic adverse effects following exposure from percutaneous penetration. Future research is expected to yield further advances in ZnPT formulations for SD and also include re-purposing towards a range of other dermatologic applications, which is likely to have significant clinical impact.

Human Epidermal Zinc Concentrations after Topical Application of ZnO Nanoparticles in Sunscreens.

Zinc oxide nanoparticle (ZnO NP)-based sunscreens are generally considered safe because the ZnO NPs do not penetrate through the outermost layer of the skin, the stratum corneum (SC). However, cytotoxicity of zinc ions in the viable epidermis (VE) after dissolution from ZnO NP and penetration into the VE is ill-defined. We therefore quantified the relative concentrations of endogenous and exogenous Zn using a rare stable zinc-67 isotope (67Zn) ZnO NP sunscreen applied to excised human skin and the cytotoxicity of human keratinocytes (HaCaT) using multiphoton microscopy, zinc-selective fluorescent sensing, and a laser-ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) methodology. Multiphoton microscopy with second harmonic generation imaging showed that 67ZnO NPs were retained on the surface or within the superficial layers of the SC. Zn fluorescence sensing revealed higher levels of labile and intracellular zinc in both the SC and VE relative to untreated skin, confirming that dissolved zinc species permeated across the SC into the VE as ionic Zn and significantly not as ZnO NPs. Importantly, the LA-ICP-MS estimated exogenous 67Zn concentrations in the VE of 1.0 ± 0.3 µg/mL are much lower than that estimated for endogenous VE zinc of 4.3 ± 0.7 µg/mL. Furthermore, their combined total zinc concentrations in the VE are much lower than the exogenous zinc concentration of 21 to 31 µg/mL causing VE cytotoxicity, as defined by the half-maximal inhibitory concentration of exogenous 67Zn found in human keratinocytes (HaCaT). This speaks strongly for the safety of ZnO NP sunscreens applied to intact human skin and the associated recent US FDA guidance.

Imaging the penetration and distribution of zinc and zinc species after topical application of zinc pyrithione to human skin.

Zinc pyrithione is an active component incorporated in an extensive range of topically applied commercial products that are used worldwide. Despite its prevalence, no published study has investigated the penetration of zinc from the zinc pyrithione complex into human skin. Zinc is crucial for healthy skin function however an elevated concentration of labile zinc is toxic outside a narrow concentration range. Synchrotron X-ray fluorescence microscopy in conjunction with X-ray absorption near edge structure spectroscopy was used to map the deposition of zinc, quantitate the amount of zinc within the skin and to identify a change in the chemical form of zinc after application. This study has demonstrated a ~3.8 fold increase in zinc concentration within the viable epidermis (VE) after 24 h topical application of zinc pyrithione that increased significantly by ~250 fold after 48 h when compared to control skin. Confocal microscopy using a labile zinc specific dye, ZinPyr-1, showed that zinc pyrithione disrupted the skin cells zinc homeostasis and significantly increased the intracellular zinc concentration leading to cell toxicity. Overall, this study demonstrates that topical application of zinc pyrithione formulations leads to an increase in zinc penetration in human skin, consequently, raising concerns for potential localised toxicity to occur.

Seborrheic dermatitis: topical therapeutics and formulation design.

Seborrheic dermatitis (SD) is a common dermatological disorder with symptoms that include skin flaking, erythema and pruritus. This review discusses the topical products available for treating SD, which target several aspects of disease pathobiology, including cutaneous microbial dysbiosis (driven by Malassezia yeast), inflammation, sebum production and skin barrier disruption. Among the various treatments available, zinc pyrithione (ZnPT) based products that exhibit anti-fungal action are the market leaders. A skin compartment approach is presented here for combining ZnPT exposure information with threshold levels for anti-fungal efficacy and toxicity, overall providing a comprehensive picture of ZnPT therapeutics and safety. While Malassezia yeast on the surface are effectively targeted, yeast residing beyond the superficial follicle may not receive adequate ZnPT for anti-fungal effect forming the basis for skin re-colonisation. Levels entering systemic circulation from topical delivery are well below toxic thresholds, however the elevated zinc levels within the viable epidermis warrants further investigation. Strategies to improve formulation design can be broadly classified as influencing 1) topical delivery, 2) therapeutic bioactivity, 3) skin mildness, and 4) sensory attributes. Successful SD treatment ultimately requires formulations that can balance efficacy, safety, and consumer appeal.

Multi-Modal Imaging to Assess the Follicular Delivery of Zinc Pyrithione.

Zinc pyrithione (ZnPT) is a widely used antifungal, usually applied as a microparticle suspension to facilitate delivery into the hair follicles, where it then dissociates into a soluble monomeric form that is bioactive against yeast and other microorganisms. In this study, we use multiphoton microscopy (MPM) and fluorescence lifetime imaging microscopy (FLIM) to characterise ZnPT formulations and map the delivery of particles into follicles within human skin. To simulate real-world conditions, it was applied using a massage or no-massage technique, while simultaneously assessing the dissolution using Zinpyr-1, a zinc labile fluorescent probe. ZnPT particles can be detected in a range of shampoo formulations using both MPM and FLIM, though FLIM is optimal for detection as it allows spectral and lifetime discrimination leading to increased selectivity and sensitivity. In aqueous suspensions, the ZnPT 7.2 µm particles could be detected up to 500 µm in the follicle. The ZnPT particles in formulations were finer (1.0-3.3 µm), resulting in rapid dissolution on the skin surface and within follicles, evidenced by a reduced particle signal at 24 h but enhanced Zinpyr-1 intensity in the follicular and surface epithelium. This study shows how MPM-FLIM multimodal imaging can be used as a useful tool to assess ZnPT delivery to skin and its subsequent dissolution.

Disposition and measured toxicity of zinc oxide nanoparticles and zinc ions against keratinocytes in cell culture and viable human epidermis.

Suspensions of the UV filter, zinc oxide nanoparticles (ZnO NP), are widely used in sunscreen products. This paper compared the relative disposition and local cytotoxicity of ZnO NP, and zinc ions formed on its dissolution, against keratinocyte cultures and in the human epidermis (ex vivo) after application of suspensions of ZnO NP. HaCaT keratinocyte cytotoxicities were found to be related to labile intra-cellular zinc but also total zinc and extra-cellular concentrations in cell culture media and to a degree ameliorated by the presence of a zinc chelating agent. Secondly, the zinc species were then dosed onto exposed ex vivo viable human epidermis and it was found that an increase in labile zinc level correlated with a shift in the metabolic state of the viable epidermis. This study highlights that excised viable skin acts as a more relevant model for determining cutaneous toxicity over keratinocyte monolayers in vitro.

Penetration of Zinc into Human Skin after Topical Application of Nano Zinc Oxide Used in Commercial Sunscreen Formulations.

Zinc oxide nanoparticles (ZnO NPs) are a key constituent of many commercial broad-spectrum sunscreens. Studies have shown that these NPs are retained on the superficial layers of the skins' barrier layer, the stratum corneum, and solubilized zinc species from the ZnO NPs have been shown qualitatively to penetrate intact human skin. The cytotoxicity of zinc is concentration- and species-dependent; however, to date, the amount of zinc permeating the skin strata is yet to be determined. Here, we applied commercial ZnO NPs to intact and impaired ex vivo barrier human skin. Artificial human sweat (to provide an electrolyte solution) and caprylic capric triglyceride (CCT; a common sunscreen formulation base) suspensions were applied to encompass potential "in-use" scenarios. A state-of-the-art multimodal approach analyzed zinc permeation. Our data show that elevated zinc concentrations within the skin are dependent on a number of variables, with barrier impairment and time being the most important factors followed by the vehicle, where sweat was more impactful than CCT. When ZnO NPs were applied to impaired barrier skin for 24 h, there was a 60-65-fold increase in zinc in the viable epidermis for both CCT and sweat compared to the control, increasing >100-fold after 48 h. Importantly, we identify that the localized cutaneous zinc concentration increase is not present as the nano ZnO that is used in sunscreens but only after dissolution and permeation as a different solubilized zinc species.

Antimicrobial efficacy and mechanism of action of poly(amidoamine) (PAMAM) dendrimers against opportunistic pathogens.

The aim of this study was to investigate a range of poly(amidoamine) (PAMAM) dendrimer generations against Gram-positive and Gram-negative skin pathogens and to determine any differences in antimicrobial potency for different generations, characterising how differences in physicochemical properties influence antimicrobial efficacy. A range of tests were carried out, including viable count assays to determine half maximal inhibitory concentration (IC50) values for each dendrimer, membrane integrity studies and an inner membrane permeabilisation assay. This is supported by scanning electron microscopy imaging of the interactions observed between dendrimers and bacteria. The results of this study indicate that the antimicrobial efficacy of native PAMAM dendrimers is dependent on generation, concentration and terminal functionalities, for example, the concentration at 50% growth inhibition (MIC50) (µg/mL), against Staphylococcus aureus was between 26.77 for the G2-PAMAM-NH2 dendrimer and 2.881 for the G5-PAMAM-NH2 dendrimer. There was a strong correlation between membrane disruption and the determined biocidal activity, making it a key contributing mechanism of action. This study demonstrates that selection of the type of PAMAM dendrimer is important as their inherent antimicrobial efficacy varies according to their individual physicochemical properties. This understanding may pave the way for the development of enhanced dendrimer-based antimicrobial formulations and drug-delivery systems.

Multiparameter toxicity screening on a chip: Effects of UV radiation and titanium dioxide nanoparticles on HaCaT cells.

Microfluidic screening is gaining attention as an efficient method for evaluating nanomaterial toxicity. Here, we consider a multiparameter treatment where nanomaterials interact with cells in the presence of a secondary exposure (UV radiation). The microfluidic device contains channels that permit immobilization of HaCaT cells (human skin cell line), delivery of titanium dioxide nanoparticles (TNPs), and exposure to a known dose of UV radiation. The effect of single-parameter exposures (UV or TNP) was first studied as a benchmark, and then multiparameter toxicity (UV and TNP) at different concentrations was explored. The results demonstrate a concentration-dependent protective effect of TNP when exposed to UV irradiation.

Optical Characterization of Zinc Pyrithione.

Zinc pyrithione is ubiquitous in commercial products particularly antidandruff shampoos. For the efficacy of zinc pyrithione therapeutic cleansers to be assessed accurately, the distribution of particles on the scalp needs to be visualized. Currently, no technique is available which provides the chemical specificity and sensitivity required. Here, we report application of fluorescence-lifetime imaging microscopy (FLIM) for high-contrast mapping of zinc pyrithione distribution on the scalp. Characterization of the zinc pyrithione emission by using both one-photon excitation at five specific wavelengths and two-photon excitation in the range of 740-820 nm revealed its FLIM fingerprint-a characteristic short average time-weighted emission lifetime of ΤZnPT = 250 ps. Bandpass-filtering FLIM signals at ΤZnPT enabled an efficient discrimination between the zinc pyrithione and major endogenous skin species in comparison with that of the conventional reflectance confocal microscopy. Our findings provide means for in vivo high-sensitivity assaying and high-contrast imaging of zinc pyrithione in biological systems.

Stimulus-Responsive Antibiotic Releasing Systems for the Treatment of Wound Infections.

There remains an unmet need for innovative treatments for chronic wound infections as they continue to be a financial and social burden on society. Because of the dynamic nature of wounds, this study investigated the utilization of stimulus-responsive plasma polymers for the development of pH- and thermoresponsive antibiotic delivery systems for the treatment of wound infections. Porous silicon films were loaded with the antibiotic levofloxacin (LVX) and subsequently coated with plasma polymer layers: first, poly(1,7-octadiene) (pOCT) for stability, followed by either the temperature-responsive polymer poly N,N-diethylacrylamide (pDEA) or the pH- responsive polymer poly 2-(diethylamino)ethyl methacrylate (pDEAEMA), to fabricate two delivery systems. The delivery systems were thoroughly characterized chemically and physically and tested in vitro through drug release and bacterial zone of inhibition studies. After a 16 h time point, the system containing pDEA achieved 3.2-fold greater release at 45 °C compared to 22 °C, whereas the system containing pDEAEMA achieved a 2.2-fold greater release when exposed to pH 8.5 media compared to pH 6.2 media. Furthermore, both systems retained their antimicrobial activity and demonstrated stimulus-responsive release to form zones of inhibition on relevant wound pathogens, Pseudomonas aeruginosa, Staphylococcus epidermidis and Staphylococcus aureus. Therefore, this proof-of-principle study confirms that stimulus-responsive porous silicon films can be utilized to deliver antibiotic when exposed to physiologically relevant stimuli such as pH and temperature with the potential to be applied to other pharmaceutics.

Dendrimer pre-treatment enhances the skin permeation of chlorhexidine digluconate: Characterisation by in vitro percutaneous absorption studies and Time-of-Flight Secondary Ion Mass Spectrometry.

Skin penetration and localisation of chlorhexidine digluconate (CHG) within the skin have been investigated in order to better understand and optimise the delivery using a nano polymeric delivery system of this topically-applied antimicrobial drug. Franz-type diffusion cell studies using in vitro porcine skin and tape stripping procedures were coupled with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to visualise the skin during various treatments with CHG and polyamidoamine dendrimers (PAMAM). Pre-treatment of the skin with PAMAM dendrimers significantly increased the amount and depth of permeation of CHG into the skin in vitro. The effect observed was not concentration dependant in the range 0.5-10mM PAMAM. This could be important in terms of the efficiency of treatment of bacterial infection in the skin. It appears that the mechanism of enhancement is due to the PAMAM dendrimer disrupting skin barrier lipid conformation or by occluding the skin surface. Franz-type diffusion cell experiments are complimented by the detailed visualisation offered by the semi-quantitative ToF-SIMS method which provides excellent benefits in terms of sensitivity and fragment ion specificity. This allows a more accurate depth profile of chlorhexidine permeation within the skin to be obtained and potentially affords the opportunity to map the co-localisation of permeants with skin structures, thus providing a greater ability to characterise skin absorption and to understand the mechanism of permeation, providing opportunities for new and more effective therapies.

Relative Penetration of Zinc Oxide and Zinc Ions into Human Skin after Application of Different Zinc Oxide Formulations.

Zinc oxide (ZnO) is frequently used in commercial sunscreen formulations to deliver their broad range of UV protection properties. Concern has been raised about the extent to which these ZnO particles (both micronized and nanoparticulate) penetrate the skin and their resultant toxicity. This work has explored the human epidermal skin penetration of zinc oxide and its labile zinc ion dissolution product that may potentially be formed after application of ZnO nanoparticles to human epidermis. Three ZnO nanoparticle formulations were used: a suspension in the oil, capric caprylic triglycerides (CCT), the base formulation commonly used in commercially available sunscreen products; an aqueous ZnO suspension at pH 6, similar to the natural skin surface pH; and an aqueous ZnO suspension at pH 9, a pH at which ZnO is stable and there is minimal pH-induced impairment of epidermal integrity. In each case, the ZnO in the formulations did not penetrate into the intact viable epidermis for any of the formulations but was associated with an enhanced increase in zinc ion fluorescence signal in both the stratum corneum and the viable epidermis. The highest labile zinc fluorescence was found for the ZnO suspension at pH 6. It is concluded that, while topically applied ZnO does not penetrate into the viable epidermis, these applications are associated with hydrolysis of ZnO on the skin surface, leading to an increase in zinc ion levels in the stratum corneum, thence in the viable epidermis and subsequently in the systemic circulation and the urine.

Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms, HaCaT keratinocytes and affects skin deposition.

The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC50 ranges from 31.25 to 125 µg/mL depending on the bacterial species) and HaCaT keratinocytes (IC50 78.65 µg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC50 of 7.85 µg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.