Characterization of an ester-based core-multishell (CMS) nanocarrier for the topical application at the oral mucosa.

OBJECTIVES: Topical drug administration is commonly applied to control oral inflammation. However, it requires sufficient drug adherence and a high degree of bioavailability. Here, we tested the hypothesis whether an ester-based core-multishell (CMS) nanocarrier is a suitable nontoxic drug-delivery system that penetrates efficiently to oral mucosal tissues, and thereby, increase the bioavailability of topically applied drugs. MATERIAL AND METHODS: To evaluate adhesion and penetration, the fluorescence-labeled CMS 10-E-15-350 nanocarrier was applied to ex vivo porcine masticatory and lining mucosa in a Franz cell diffusion assay and to an in vitro 3D model. In gingival epithelial cells, potential cytotoxicity and proliferative effects of the nanocarrier were determined by MTT and sulphorhodamine B assays, respectively. Transepithelial electrical resistance (TEER) was measured in presence and absence of CMS 10-E-15-350 using an Endohm-12 chamber and a volt-ohm-meter. Cellular nanocarrier uptake was analyzed by laser scanning microscopy. Inflammatory responses were determined by monitoring pro-inflammatory cytokines using real-time PCR and ELISA. RESULTS: CMS nanocarrier adhered to mucosal tissues within 5 min in an in vitro model and in ex vivo porcine tissues. The CMS nanocarrier exhibited no cytotoxic effects and induced no inflammatory responses. Furthermore, the physical barrier expressed by the TEER remained unaffected by the nanocarrier. CONCLUSIONS: CMS 10-E-15-350 adhered to the oral mucosa and adhesion increased over time which is a prerequisite for an efficient drug release. Since TEER is unaffected, CMS nanocarrier may enter the oral mucosa transcellularly. CLINICAL RELEVANCE: Nanocarrier technology is a novel and innovative approach for efficient topical drug delivery at the oral mucosa.

Breaking the Barrier - Potent Anti-Inflammatory Activity following Efficient Topical Delivery of Etanercept using Thermoresponsive Nanogels.

Topical administration permits targeted, sustained delivery of therapeutics to human skin. Delivery to the skin, however, is typically limited to lipophilic molecules with molecular weight of < 500 Da, capable of crossing the stratum corneum. Nevertheless, there are indications protein delivery may be possible in barrier deficient skin, a condition found in several inflammatory skin diseases such as psoriasis, using novel nanocarrier systems. METHODS: Water in water thermo-nanoprecipitation; dynamic light scattering; zeta potential measurement; nanoparticle tracking analysis; atomic force microscopy; cryogenic transmission electron microscopy; UV absorption; centrifugal separation membranes; bicinchoninic acid assay; circular dichroism; TNFα binding ELISA; inflammatory skin equivalent construction; human skin biopsies; immunohistochemistry; fluorescence microscopy; western blot; monocyte derived Langerhans cells; ELISA Results: Here, we report the novel synthesis of thermoresponsive nanogels (tNG) and the stable encapsulation of the anti-TNFα fusion protein etanercept (ETR) (~150 kDa) without alteration to its structure, as well as temperature triggered release from the tNGs. Novel tNG synthesis without the use of organic solvents was conducted, permitting in situ encapsulation of protein during assembly, something that holds great promise for easy manufacture and storage. Topical application of ETR loaded tNGs to inflammatory skin equivalents or tape striped human skin resulted in efficient ETR delivery throughout the SC and into the viable epidermis that correlated with clear anti-inflammatory effects. Notably, effective ETR delivery depended on temperature triggered release following topical application. CONCLUSION: Together these results indicate tNGs hold promise as a biocompatible and easy to manufacture vehicle for stable protein encapsulation and topical delivery into barrier-deficient skin.

Characterization of hyperbranched core-multishell nanocarriers as an innovative drug delivery system for the application at the oral mucosa.

BACKGROUND AND OBJECTIVES: In the oral cavity, the mucosal tissues may develop a number of different pathological conditions, such as inflammatory diseases (gingivitis, periodontitis) and autoimmune disorders (eg, oral lichen planus) that require therapy. The application of topical drugs is one common therapeutic approach. However, their efficacy is limited. Dilution effects due to saliva hinder the adherence and the penetration of drug formulations. Therefore, the bioavailability of oral topical drugs is insufficient, and patients may suffer from disease over years, if not life-long. MATERIAL AND METHODS: In the present study, we characterized core-multishell (CMS) nanocarriers for their potential use as drug delivery systems at oral mucosal tissues. For this purpose, we prepared porcine masticatory as well as buccal mucosa and performed Franz cell diffusion experiments. Penetration of fluorescently labeled CMS nanocarriers into the mucosal tissue was analyzed using confocal laser scanning microscopy. Upon exposure to CMS nanocarriers, the metabolic and proliferative activity of gingival epithelial cells was determined by MTT and sulforhodamine B assays, respectively. RESULTS: Here, we could show that the carriers penetrate into both mucosal tissues, while particles penetrate deeper into the masticatory mucosa. Electron paramagnetic resonance spectroscopy revealed that the 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy-labeled glucocorticoid dexamethasone loaded on to the CMS nanocarriers was released from the carriers in both mucosal tissues but with a higher efficiency in the buccal mucosa. The release from the nanocarriers is in both cases superior compared to the release from a conventional cream, which is normally used for the treatment of inflammatory conditions in the oral cavity. The CMS nanocarriers exhibited neither cytotoxic nor proliferative effects in vitro. CONCLUSION: These findings suggested that CMS nanocarriers might be an innovative approach for topical drug delivery in the treatment of oral inflammatory diseases.

Enhanced topical delivery of dexamethasone by ß-cyclodextrin decorated thermoresponsive nanogels.

Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, ß-cyclodextrin (ßCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of ßCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of ßCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: ∼2.5 fold in epidermis, ∼30 fold in dermis). Furthermore, DXM encapsulated in ßCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream.

Influence of the skin barrier on the penetration of topically-applied dexamethasone probed by soft X-ray spectromicroscopy.

The penetration of dexamethasone into human skin ex vivo is reported. X-ray microscopy is used for label-free probing of the drug and quantification of the local drug concentration with a spatial resolution reaching 70±5nm. This is accomplished by selective probing the dexamethasone by X-ray absorption. Varying the penetration time between 10min and 1000min provides detailed information on the penetration process. In addition, the stratum corneum has been damaged by tape-stripping in order to determine the importance of this barrier regarding temporally resolved drug penetration profiles. Dexamethasone concentrations distinctly vary, especially close to the border of the stratum corneum and the viable epidermis, where a local minimum in drug concentration is observed. Furthermore, near the basal membrane the drug concentration strongly drops. High spatial resolution studies along with a de-convolution procedure reveal the spatial distribution of dexamethasone in the interspaces between the corneocytes consisting of stratum corneum lipids. These results on local drug concentrations are interpreted in terms of barriers affecting the drug penetration in human skin.

Core-multishell nanocarriers: Transport and release of dexamethasone probed by soft X-ray spectromicroscopy.

Label-free detection of core-multishell (CMS) nanocarriers and the anti-inflammatory drug dexamethasone is reported. Selective excitation by tunable soft X-rays in the O 1s-regime is used for probing either the CMS nanocarrier or the drug. Furthermore, the drug loading efficiency into CMS nanocarriers is determined by X-ray spectroscopy. The drug-loaded nanocarriers were topically applied to human skin explants providing insights into the penetration and drug release processes. It is shown that the core-multishell nanocarriers remain in the stratum corneum when applied for 100min to 1000min. Dexamethasone, if applied topically to human ex vivo skin explants using different formulations, shows a vehicle-dependent penetration behavior. Highest local drug concentrations are found in the stratum corneum as well as in the viable epidermis. If the drug is loaded to core-multishell nanocarriers, the concentration of the free drug is low in the stratum corneum and is enhanced in the viable epidermis as compared to other drug formulations. The present results provide insights into the penetration of drug nanocarriers as well as the mechanisms of controlled drug release from CMS nanocarriers in human skin. They are also compared to related work using dye-labeled nanocarriers and dyes that were used as model drugs.

The effect of endothelialization on the epidermal differentiation in human three-dimensional skin constructs - A morphological study.

INTRODUCTION: Inducing vascularization in three-dimensional skin constructs continues to be difficult. In this study, two variations of human full-thickness skin constructs were examined. Type KCFB consists of keratinocytes (epidermal equivalent) and fibroblasts that were embedded in a collagen matrix (dermal equivalent). Type KCFB-EC consists of keratinocytes as well as fibroblasts and vascular endothelial cells. The epidermal equivalent of KCFB-EC constructs underwent cellular alterations in their differentiation possibly induced by the presence of endothelial cells. The objective of the study was to assess the effect of endothelial cells, i.e., endothelialization of the dermal equivalent on the differentiation of keratinocytes by comparing the morphology and ultrastructure of the two types of skin constructs, as well as to excised normal human skin. HYPOTHESIS: The differentiation of keratinocytes is influenced by the presence of endothelial cells. METHODS, PATIENTS, MATERIAL: KCFB constructs (keratinocytes, fibroblasts) and KCFB-EC skin constructs(kera-tinocytes, fibroblasts, endothelial cells) were prepared according to Küchler et al. [25]. After two weeks, the skin constructs were processed for analysis by light microscopy (LM) and electron microscopy (TEM), followed by quantitative, semi-quantitative as well as qualitative assessment. For comparison, analysis by LM and TEM of excised normal human skin was also performed. RESULTS: Both KCFB and KCFB-EC skin constructs and the human skin had all strata of stratified soft-cornified epidermis present. The comparison of the respective layers of the skin constructs brought the following characteristics to light: The KCFB-EC constructs had significantly more mitotic cells in the stratum spinosum, more cell layers in the stratum granulosum and more keratohyalin granules compared to KCFB skin constructs. Additionally, the epidermal architecture was unorganized in the endothelialized constructs and features of excessive epidermal differentiation appeared in KCFB-EC skin constructs. CONCLUSION: The endothelialization of the dermal equivalent caused changes in the differentiation of the epidermis of KCFB-EC skin constructs that may be interpreted as an unbalanced, i.e., uncontrolled or enhanced maturation process.

Selective Probing of the Penetration of Dexamethasone into Human Skin by Soft X-ray Spectromicroscopy.

Selective probing of dexamethasone in excised human skin using soft X-ray spectromicroscopy provides quantitative concentration profiles as well as two-dimensional drug distribution maps. Element- and site-selective excitation of dexamethasone at the oxygen K-edge with the lateral step width adjusted to 1 µm provides detailed information on the location of the drug in the different skin layers. The key of this work is to probe dexamethasone selectively at the carbonyl site (C3) by the O 1s → π* transition, providing also a most efficient way to quantify the drug concentration as a function of penetration depth in correlation with structural properties of the skin containing carboxyl and amide oxygen sites occurring at higher transition energy than dexamethasone. Following drug exposure for 4 h, the glucocorticoide is located in about equal amounts in the stratum corneum, the outermost horny layer of skin, and in the viable epidermis, whereas in the dermis no dexamethasone is detected. In the stratum corneum, most of the lipophilic drug is found in regions between corneocytes, where epidermal lipids are dominating.