RESUMO
Anionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid anionic liposomes to deliver tolerogenic adjuvants and antigen peptides has potential applications for the treatment of autoimmune and inflammatory diseases. However, the preparation of these highly rigid anionic liposomes using traditional methods such as lipid film hydration presents problems in terms of scalability and loading efficiency of some costly tolerogenic adjuvants like 1-α,25-dihydroxyvitaminD3. Here we propose the use of an off-the-shelf staggered herringbone micromixer for the preparation of these formulations and performed a systematic study on the effect of temperature and flow conditions on the size and polydispersity index of the formulations. Furthermore, we show that the system allows for the encapsulation of a wide variety of peptides and significantly higher loading efficiency of 1-α,25-dihydroxyvitaminD3 compared to the traditional lipid film hydration method, without compromising their non-inflammatory interaction with dendritic cells. Therefore, the microfluidics method presented here is a valuable tool for the preparation of highly rigid tolerogenic liposomes in a fast, size-tuneable and scalable manner.
Assuntos
Lipossomos , Microfluídica , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos , Lipídeos/química , Lipossomos/química , Microfluídica/métodos , PeptídeosRESUMO
Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI-/-) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI-/- epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI-/- mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/deficiência , Epiderme/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Ácido 4-Aminobenzoico/farmacocinética , Animais , Apolipoproteínas B/metabolismo , Antígenos CD36/genética , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Ésteres do Colesterol/sangue , Ésteres do Colesterol/metabolismo , Epiderme/patologia , Ácidos Graxos Insaturados/metabolismo , Feminino , Lecitinas/genética , Lecitinas/metabolismo , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Full thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a reduced skin barrier. The most pronounced differences in lipid composition are a reduction in lipid chain length and increased monounsaturated lipids. The liver-X-receptor (LXR) activates the monounsaturated lipid synthesis via stearoyl-CoA desaturase-1 (SCD-1). Therefore, the aim was to improve the SC lipid synthesis of FTMs by LXR deactivation. This was achieved by supplementing culture medium with LXR antagonist GSK2033. LXR agonist T0901317 was added for comparison. Subsequently, epidermal morphogenesis, lipid composition, lipid organization and the barrier functionality of these FTMs were assessed. We demonstrate that LXR deactivation resulted in a lipid composition with increased overall chain lengths and reduced levels of monounsaturation, whereas LXR activation increased the amount of monounsaturated lipids and led to a reduction in the overall chain length. However, these changes did not affect the barrier functionality. In conclusion, LXR deactivation led to the development of FTMs with improved lipid properties, which mimic the lipid composition of NHS more closely. These novel findings may contribute to design interventions to normalize SC lipid composition of atopic dermatitis patients.
Assuntos
Meios de Cultura/farmacologia , Receptores X do Fígado/antagonistas & inibidores , Cultura Primária de Células/métodos , Pele/efeitos dos fármacos , Sulfonamidas/farmacologia , Ceramidas/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ácidos Graxos não Esterificados , Humanos , Hidrocarbonetos Fluorados/farmacologia , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Morfogênese/efeitos dos fármacos , Pele/crescimento & desenvolvimento , Pele/metabolismo , Estearoil-CoA Dessaturase/metabolismoRESUMO
The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 µM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Lipogênese/efeitos dos fármacos , Ácido Palmítico/farmacologia , Pele Artificial , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Células Epidérmicas/metabolismo , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/biossíntese , Lipídeos/química , Morfogênese/efeitos dos fármacos , Ácido Palmítico/química , Pele/química , Pele/efeitos dos fármacos , Pele/metabolismoRESUMO
Human skin equivalents (HSEs) are three dimensional models resembling native human skin (NHS) in many aspects. Despite the manifold similarities to NHS, a restriction in its applications is the altered in vitro lipid barrier formation, which compromises the barrier functionality. This could be induced by suboptimal cell culturing conditions, which amongst others is the diminished activation of the vitamin D receptor (VDR) signalling pathway. The active metabolite of this signalling pathway is 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). An interacting role in the formation of the skin barrier has been ascribed to this pathway, although it remains unresolved to which extent this pathway contributes to the (mal-)formation of the epidermal barrier in HSEs. Our aim is to study whether cell culture medium enriched with 1,25(OH)2D3 affects epidermal morphogenesis and lipid barrier formation in HSEs. Addition of 20 nM 1,25(OH)2D3 resulted in activation of the VDR signalling pathway by inducing transcription of VDR target genes (CYP24A and LL37) in keratinocyte monocultures and in HSEs. Characterization of HSEs supplemented with 1,25(OH)2D3 using immunohistochemical analyses revealed a high similarity in epidermal morphogenesis and in expression of lipid processing enzymes. The barrier formation was assessed using state-of-the art techniques analysing lipid composition and organization. Addition of 1,25(OH)2D3 did not alter the composition of ceramides. Additionally, the lateral and lamellar organization of the lipids was similar, irrespective of supplementation. In conclusion, epidermal morphogenesis and barrier formation in HSEs generated in presence or absence of 1,25(OH)2D3 leads to a similar morphogenesis and comparable barrier formation in vitro.
Assuntos
Calcitriol/farmacologia , Epiderme/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Vitaminas/farmacologia , Calcitriol/metabolismo , Células Cultivadas , Epiderme/metabolismo , Epiderme/ultraestrutura , Humanos , Receptores de Calcitriol/metabolismo , Transdução de Sinais/efeitos dos fármacos , Engenharia Tecidual , Vitaminas/metabolismoRESUMO
Epidermal ß-glucocerebrosidase (GBA1), an acid ß-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: i) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-ß-D-glucopyranoside or resorufin-ß-D-glucopyranoside; and ii) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various ß-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.
Assuntos
Ensaios Enzimáticos , Corantes Fluorescentes/química , Glucosilceramidase/análise , Pele/enzimologia , Coloração e Rotulagem/métodos , Benzoxazinas/química , Compostos de Boro/química , Cicloexanóis/química , Compostos de Epóxi/química , Expressão Gênica , Glucosídeos/química , Glucosilceramidase/metabolismo , Humanos , Himecromona/análogos & derivados , Himecromona/química , Técnicas de Cultura de TecidosRESUMO
Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.
Assuntos
Meios de Cultura/farmacologia , Epiderme/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/análise , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/farmacologia , Células Cultivadas , Ácidos Eicosanoicos/metabolismo , Ácidos Eicosanoicos/farmacologia , Epiderme/química , Epiderme/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Humanos , Queratinócitos , Modelos Biológicos , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Pele Artificial , Técnicas de Cultura de TecidosRESUMO
Treatment strategies for superficial mycosis caused by the dermatophyte Trichophyton rubrum consist of the use of topical or oral antifungal preparations. We have recently discovered that T. rubrum is susceptible to photodynamic treatment (PDT), with 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) as a photosensitizer. The susceptibility appeared to depend on the fungal growth stage, with PDT efficacy higher with microconidia when compared to mycelia. The aim of this study was to investigate, with the use of scanning electron microscopy, the morphological changes caused by a lethal PDT dose to T. rubrum when grown on isolated human stratum corneum. Corresponding dark treatment and light treatment without photosensitizer were used as controls. A sub-lethal PDT dose was also included in this investigation The morphologic changes were followed at various time points after the treatment of different fungal growth stages. Normal fungal growth was characterized by a fiber-like appearance of the surface of the hyphae and microconidia with the exception of the hyphal tips in full mycelia and the microconidia shortly after attachment to the stratum corneum. Here, densely packed globular structures were observed. The light dose (108 J/cm2) in the absence of Sylsens B, or the application of the photosensitizer in the absence of light, caused reversible fungal wall deformations and bulge formation. However, after a lethal PDT, a sequence of severe disruptions and deformations of both microconidia and the mycelium were observed leading to extrusion of cell material and emptied fungal elements. In case of a non-lethal PDT, fungal re-growth started on the remnants of the treated mycelium.
Assuntos
Fotoquimioterapia , Trichophyton/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Escuridão , Epiderme/microbiologia , Humanos , Hifas/efeitos dos fármacos , Hifas/ultraestrutura , Microscopia Eletrônica de Varredura , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Compostos de Piridínio/farmacologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/ultraestrutura , Tempo , Trichophyton/efeitos dos fármacosRESUMO
BACKGROUND: Photodynamic treatment (PDT) refers to a treatment with light-activated agents (photosensitizers) in combination with visible light and molecular oxygen. Recently, we have demonstrated that the porphyrins, 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) are excellent photosensitizers to be used against Trichophyton rubrum both in vitro and ex vivo. OBJECTIVES AND METHODS: The objective of this study was to investigate the key factors involved in PDT efficacy of both photosensitizers in an ex vivo situation during different fungal growth stages using a recently developed ex vivo model. The study focused on the influence of pH and ion strength of incubation media, photochemical properties of the photosensitizers (spectra and singlet oxygen production), and the effect of several scavengers of reactive oxygen species (sodium azide, histidine, mannitol) and phenylmethylsulphonylfluoride (keratinase inhibitor) on the PDT efficacy. RESULTS AND CONCLUSIONS: The results show that an optimal pH and low concentrations of calcium are crucial for a selective binding of Sylsens B to the fungus, leading to an increased PDT efficacy. This selective binding to T. rubrum cannot be accomplished for DP mme. It can be concluded that the prerequisite for successful treatment is a use of a low molarity solution of pH 5, supplemented with a chelating agent and a keratinase activity-repressing agent. Under these conditions, PDT with Sylsens B inactivates, initially via singlet oxygen, effectively the fungus in different fungal growth stages.