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EP-4737559-A1 - SKIN MODEL FOR SYNCHRONIZING STUDIES OF PERMEATION AND MEMBRANE INTERACTIONS

EP4737559A1EP 4737559 A1EP4737559 A1EP 4737559A1EP-4737559-A1

Abstract

The present disclosure relates to an artificial skin model that mimics the intercellular lipid matrix of the stratum corneum in healthy or injured skin, including the short and long lamellar periodicity phases that are thought to be essential to the barrier function of the skin. In particular, the present disclosure relates to simple and cost-effective methods and processes for high throughput screening (HTS) of skin permeation assays and membrane interaction studies, particularly useful in the pharmaceutical, cosmetic, and chemical industries for the evaluation of topical formulations, transdermal delivery systems, and other skin-related treatments.

Inventors

  • BARBOSA FERNANDES, EDUARDA
  • DIONÍSIO LÚCIO, MARLENE SUSANA
  • FERNANDES CARDOSO, VANESSA
  • LANCEROS-MENDEZ, SENENTXU
  • MARTINS LOPES, CARLA

Assignees

  • Universidade do Minho
  • Universidade Do Porto
  • Fundação Ensino e Cultura Fernando Pessoa

Dates

Publication Date
20260506
Application Date
20251017

Claims (15)

  1. Artificial skin model comprising: a polymeric support layer; a lipid bilayer in the top of the polymeric support layer wherein the polymeric support layer supports the lipid bilayer; wherein the polymeric support layer has a porosity ratio between 20% - 95% with a pore size inferior to 650 nm; wherein the polymeric support layer is a protein-polymer composite; wherein the polymeric support layer is a polymeric scaffold.
  2. Artificial skin model according to the previous claim, wherein the polymeric support layer comprises a plurality of sublayers and wherein the sublayers are bound to each other.
  3. Artificial skin model according to any of the previous claims, wherein the protein-polymer composite comprises 1% - 50% (w/w protein-polymer ) of a protein and 50% - 99% (w/w protein-polymer ) of a polymer.
  4. Artificial skin model according to the previous claim, wherein the protein is keratin, or a keratin derivative extracted from woven and non-woven sources.
  5. Artificial skin model according to any of the previous claims 3-4, wherein the polymer is selected from a list consisting of: polyvinyl alcohol, polylactic-co-glycolic acid, polylactic acid, polycaprolactone, polyethylene oxide, polyvinylpyrrolidone; poly(methyl methacrylate), ethyl cellulose, or combinations thereof.
  6. Artificial skin model according to any of the previous claims, wherein the lipid bilayer comprises at least a lipid selected from the list consisting of: cholesterol, phosphatidylcholine, phosphatidylcholine diacyl derivatives, acylceramides, and a free fatty acid combination, or mixtures thereof; and/or wherein the lipid bilayer comprises phosphatidylcholine; or phosphatidylcholine diacyl derivative; or a mixture of: cholesterol and phosphatidylcholine; or cholesterol and phosphatidylcholine diacyl derivative; or cholesterol, phosphatidylcholine and phosphatidylcholine diacyl derivative.
  7. Artificial skin model according to any of the previous claims, comprising 0.001 - 35% (w/w lipid bilayer ) of cholesterol; preferably 1% (w/w lipid bilayer ) - 35% (w/w lipid bilayer ) of cholesterol; more preferably 5 % (w/w lipid bilayer ) - 33 % (w/w lipid bilayer ) of cholesterol; and/or comprising 65% - 100% (w/w lipid bilayer ) of phosphatidylcholine or phosphatidylcholine diacyl derivatives; preferably 67% - 95% (w/w lipid bilayer ) of phosphatidylcholine or phosphatidylcholine diacyl derivatives.
  8. Artificial skin model according to any of the previous claims 6-7, wherein the phosphatidylcholine diacyl derivatives are C(12) - C(24) fatty acyl substituted glycero-3-phosphocholines derivatives; preferably C(14) - C(20).
  9. Artificial skin model according to the previous claims 6, wherein the lipid bilayer comprises a mixture of an acylceramide combination, cholesterol, and at least two free fatty acids; preferably wherein the acylceramide combination is selected from a list consisting of: ceramide AP, ceramide EOS, or combinations thereof; and/or wherein the free fatty acid is selected from a list consisting of: arachidonic acid, behenic acid, lignoceric acid, hexacosanoic acid, myristic acid, or combinations thereof; preferably arachidonic acid, behenic acid, lignoceric acid, hexacosanoic acid, and myristic acid.
  10. Artificial skin model according to any of the previous claims 6 or 9, wherein the molar ratio between ceramide AP:ceramide EOS:cholesterol:arachidonic acid:behenic acid:lignoceric acid:hexacosanoic acid:myristic acid ranges from 23:10:33:3.3:17.7:10.6:0.9:0.5 to 23:10:33:2.7:14.3:13.4:1.1:1.5; more preferably is 23:10:33:3:16:12:1:1; and/or the molar ratio between ceramide AP: ceramide EOS: cholesterol: arachidonic acid:behenic acid:lignoceric acid: hexacosanoic acid:myristic acid ranges from 25:8:33:3:15:11.5:0.5:4 to 31:2:33:3:14:11:0:6, more preferably is 28:5:33:3:14.5:11.25:0.25:5.
  11. Artificial skin model according to any of the previous claims, wherein the lipid bilayer long periodicity phase thickness ranges from 10 nm - 16 nm; preferably 9 nm - 15 nm; more preferably 8 nm - 14 nm; and/or the lipid bilayer short periodicity phase thickness ranges from 5 nm - 7 nm; preferably 4 nm - 7 nm.
  12. Artificial skin model according to any of the previous claims, wherein the thickness of the polymeric scaffold ranges from 10 µm - 400 µm; preferably 10 µm - 100 µm; more preferably 10 µm - 60 µm.
  13. Artificial skin model according to any of the previous claims, for use in the screening of a pharmaceutical and/or a cosmetical active ingredient.
  14. Kit for use in the screening of pharmaceutical or cosmetical active ingredient comprising an artificial skin model described in any of the previous claims.
  15. Use of the artificial skin model according to any of the previous claims as a screener of a pharmaceutical and/or a cosmetical active ingredient; or as artificial healthy human skin model; or as an atopic dermatitis human skin model.

Description

TECHNICAL FIELD The present disclosure relates to an artificial skin model comprising a polymeric scaffold that supports a mixture that mimics the stratum corneum intercellular lipid matrix of healthy or injured skin. More specifically, the present disclosure relates to an artificial bioinspired skin model for skin permeation assay and for the study of molecular interactions and biophysical changes in the stratum corneum intercellular lipid matrix. BACKGROUND Skin barrier properties are vital in various research fields, including toxicology, risk assessment, and pharmaceutical and cosmetic product development. Delivering drugs or bioactive ingredients through the skin offers numerous advantages over traditional routes like oral or injectable [1]. However, the natural barrier function of the skin mainly due to the stratum corneum complex organization presents challenges to permeation. The stratum corneum consists of corneocytes embedded by an intercellular lipid matrix, in a 'brick-and-mortar' wall like structure (Fig. 1) [2, 3]. Lamellar and lateral organization is crucial for skin barrier function and integrity of the intercellular lipid matrix. Healthy intercellular lipid matrix of the stratum corneum is organized in a dense orthorhombic lateral packing with two lamellar phases (long and short lamellar periodicity phases) with repeat distances of 12-14 and 5-6 nm respectively (Fig. 1) [2, 4]. Injured skin (like atopic dermatitis skin) has impaired skin barrier due to changes in the intercellular lipid matrix from orthorhombic to a loosely hexagonal lateral packing [4]. Using skin models able to replicate the composition, structure and barrier properties of the intercellular lipid matrix is essential for assessing the percutaneous absorption, bioavailability, and safety of topically applied cosmetic ingredients and dermatological bioactive ingredients. The use of skin, whether human or animal, has long been regarded as the gold standard for assessing (trans)dermal permeability. Commercially available products like Transderm-Scop®, Androderm®, and Alora® are excised human skin used in permeation assays as ex vivo skin models. Yet, ethical, technical, and economic challenges have stalled its widespread use. Obtaining human skin poses hurdles due to ethical concerns, high variability, low throughput, and technical challenges, limiting its use [5, 6]. Similarly, obtaining animal skin is time-consuming, technically challenging, and costly, and excised skins do not accurately represent human skin physiology and structure [6]. Besides, both in vivo animal and human studies and ex vivo studies using excised skin models contribute significantly to the carbon footprint of pharmaceutical industries raising sustainability concerns. The pharmaceutical and industry/research sectors are increasingly inclined to adopt other in vitro testing methods to lessen reliance on in vivo or ex vivo models. This trend is further emphasized by the EU prohibition on animal testing for cosmetics and toxicology evaluations under Directive 76/768/EEC, which demands for alternative methods of skin permeation testing [7]. Models equivalent to the living skin developed by culturing human skin components such as keratinocytes and fibroblasts, and several human skin cell cultures are commercially available as skin models: EpiSkin™ model (Episkin, company of group L'Oréal, Lyon, France); EpiDerm™ model (MatTek Corporation, Ashland,MA, USA); SkinEthic™ (Episkin, company of group L'Oréal, Lyon, France); Labskin™ (Labskin, Lyon, France); EpiCS® model (CellSystems, Troisdorf, Germany); Straticell model (Straticell, Les Isnes, Belgium); and Labcyte model (Gamagori, Japan); Full-Thickness models such as a StrataTest® model (Stratatech, Madison, WI, USA); Phenion Full-Thickness Skin Model (Phenion, Düsseldorf, Germany); GraftSkin® (Apligraf; Organogenesis, MI, USA); EpiDermFT® (MatTek Corporation, Ashland, MA, USA); and Vitrolife-Skin™model (Kyoto, Japan). These models have been employed in Franz diffusion cells for permeation assessments or incorporated into microfluidic chips (known as "skin-on-chip"). However, they still fail to address several issues related to excised tissue, proving expensive, requiring specialized technical skills, and inadequately replicating human skin permeation, and results with these models frequently show large variations [8, 9]. Primarily utilized for skin irritation and toxicity assessments [10], these cellular models present challenges that surpass the conveniences offered by microfluidic analysis and screening purposes. Despite the potential benefits of microfluidic technology in enhancing screening processes, the integration of skin cell-based models presents obstacles to achieving high throughput screening (HTS). These include limitations in storage capacity of the cell-based models, lack of reproducibility, technical handling requirements, and elevated screening costs. Cell-free skin models have also been develope