Protease-mediated Inflammation: An In Vitro Human Keratinocyte-based Screening Tool for Anti-inflammatory Drug Nanocarrier Systems

Abstract

Background: Refined encapsulation approaches in dermatotherapy gain increased interest. There is need of reproducible in vitro systems representing disease features to screen drug delivery systems for preclinical assessment. Inflammatory human skin diseases are commonly accompanied by abnormal epidermal differentiation and barrier impairment. Serine proteases (SPs) and their inhibitors play a critical role in such dysfunctional differentiation. SPs also initiate cellular pathways via activation of protease-activated receptors, which contribute to inflammation. Thus, function and activity of SPs should be considered for the design of new therapies of such disorders. Objectives: Herein, we established a novel simplified cell culture model, based on SP-mediated inflammation suitable to assess nanocarriers loaded with anti-inflammatory drugs. Methods: SP-mediated inflammation and the regulatory effect of free or encapsulated dexamethasone were determined by measuring interleukin-6 and interleukin-8 in culture medium of HaCaT (human adult low calcium temperature)-keratinocytes. Additionally, radical formation was analyzed by electron paramagnetic resonance spectroscopy. Cellular uptake of core-multishell nanocarriers was investigated by fluorescence microscopy. Cytotoxicity of all additives was determined by a viability assay. Results: SP-Stimulation of keratinocytes resulted in increased radical production and release of inflammatory cytokines without affecting cell viability. Induced inflammation was successfully downregulated by addition of free or encapsulated dexamethasone. Conclusion: SP-addition can be used as inflammatory stimulus in cell culture to mimic effects of aberrant enzymatic activities found in skin of atopic dermatitis patients. The set-up is appropriate as a preliminary test to examine the effectiveness of new molecules or delivery-systems to counteract serine protease-mediated inflammatory processes prior to skin studies.

Serine Protease-Mediated Cutaneous Inflammation: Characterization of an Ex Vivo Skin Model for the Assessment of Dexamethasone-Loaded Core Multishell-Nanocarriers

“…In previous investigations, however, Li et al elegantly demonstrated the role of plasmin as an amplifier of skin inflammation using intradermal injections of phosphate-buffered saline compared to plasmin in murine skin [41]. The overall observation of the combined SP treatment being a moderate inflammatory stimulus is in accordance with our own experiments using human keratinocyte cultures, which showed that the addition of low concentrations of trypsin and plasmin resulted in a mild-to-moderate radical formation, as assessed by electron paramagnetic resonance (EPR), while also increasing IL-6 and IL-8 levels without impairing the overall cell viability [54]. In vivo, increased TEWL values, as observed in the culture model, were positively correlated with an enhanced activity of plasmin and trypsin-like KLKs in the stratum corneum.…”

“…It was adapted from experimental protocols to make use of its proinflammatory properties. In cell cultures studies with keratinocytes exposed to 2.3 µg/mL trypsin, we identified this concentration as a moderate stimulus for the induction of the free radicals IL-6 and IL-8 [ 54 , 55 ].…”

Improved Skin Permeability after Topical Treatment with Serine Protease: Probing the Penetration of Rapamycin by Scanning Transmission X-ray Microscopy

“…Similar to previous work, this pretreatment was performed to model the enzymatic activity, cytokine environment, and barrier alteration typical of inflamed skin. 38 , 39 The samples were treated with a cotton swab for removing the formulation still sticking to the skin surface and subsequently prepared for STXM studies by fixation in 2.5% glutaraldehyde and 1% cacodylate buffer. Finally, they were embedded in EPON resin (Serva, Heidelberg, Germany) and cut into 200–300 nm slices with an area of typically 500 μm × 500 μm using an ultramicrotome, similar to previous work.…”

“…Trypsin applied in moderate concentrations capable of triggering inflammatory processes has been used as a stimulus in cell cultures to mimic enhanced serine protease activity, typically found in inflammatory skin diseases, such as atopic dermatitis. 38 , 39 It is also known that trypsin-like serine proteases in the SC are associated with desquamation. 40 These proteases are found to be more active in the outer SC than in the inner part of this skin layer.…”

Topical Delivery of Rapamycin by Means of Microenvironment-Sensitive Core-Multi-Shell Nanocarriers: Assessment of Anti-Inflammatory Activity in an ex vivo Skin/T Cell Co-Culture Model

“…The skin surface was pre-treated with serine protease (SP) to induce an impairment of the SC barrier, 32 , 33 activation of PAR2-mediated inflammatory cytokine release, 34–37 and free radical formation. 38 Furthermore, Jurkat cells were added to the system and activated with phytohaemagglutinin (PHA) and Th17 cytokines (IL-17A and IL-22) to reproduce the inflammatory environment typical for psoriasis. 39 This skin-cell co-culture set-up allowed us to measure the selectivity of drug release by oxidative-sensitive CMS nanocarriers as well as the effects of the delivered drug towards inflammatory T cells.…”