A platform for mimicking biological air-interfaces

Abstract

The present invention generally relates to synthetic biological model systems and biomimetic platforms More specifically, the invention relates to cell-free platforms comprising lipid assemblies arranged on substrates that mimic the organisation of biological air-interfaces, such as the tear-film lipid layer of the eye, the stratum corneum of the skin, and the pulmonary surfactant film lining the alveoli of the lung. The invention further relates to methods of manufacturing such platforms and their use in diagnosing eye health and disease, screening compounds and formulations for ocular treatment and other cosmetic formulations, sensing environmental contaminants, and toxicological applications.

Description

[0001] A PLATFORM FOR MIMICKING BIOLOGICAL AIR-INTERFACES

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003]

[0001] This application claims the benefit of priority of United States Patent Application No. 63 / 710,043 filed 22 October 2024, the content of which being hereby incorporated by reference in its entirety for all purposes.

[0004] FIELD OF INVENTION

[0005]

[0002] The present invention generally relates to synthetic biological model systems and biomimetic platforms. More specifically, the invention relates to cell-free platforms comprising lipid assemblies arranged on substrates that mimic the organisation of biological air-interfaces, such as the tear-film lipid layer of the eye, the stratum corneum of the skin, and the pulmonary surfactant film lining the alveoli of the lung. The invention further relates to methods of manufacturing such platforms and their use in diagnosing eye health and disease, screening compounds and formulations for ocular treatment and other cosmetic formulations, sensing environmental contaminants, and toxicological applications.

[0006] BACKGROUND

[0007]

[0003] On December 29, 2022, U.S. President Joseph Biden, Jr. signed into law the FDA Modernization Act 2.0. The bill countermands the long-held mandate, first enacted under the 1938 Federal Food, Drug, and Cosmetics Act, requiring animal testing for every single, new drug development protocol. This dramatic reversal in practice is enabled by the recognition that adequate and reliable alternatives can be put in place. This confidence stems in large part from the spectacular advances in the molecular-level understanding of (1) human and animal biology and (2) mechanisms by which exogenous environmental substances (e.g., drugs, devices, pollutants, cosmetic ingredients, viruses, bacteria, microplastics, et cetera) interact with the living beings. Since the passing of the FDAMA 2.0 merely eighteen months ago, the act has already gained substantial societal-level, public support, the latter fueled both by the animal welfare issues and by the growing understanding of the limited scope of the relevance of animal studies to the human biology.

[0008]

[0004] A significant consequence of these developments is a rapidly growing demand for animal-free (ex vivo, in-vitro, all-synthetic, and in-silico) alternatives across multiple market sectors. Major market sectors affected by the FDAMA 2.0 include (1) generic and biosimilar drugs, which have the clinically same active ingredients and work mechanistically in the same way as their FDA-approved trade name counterparts; (2) cosmetic derivatives, which require toxicology studies for each different formulation; and (3) selected ocular injectables, which require irritancy tests.

[0009]

[0005] The regulatory approval for the classification of irritants currently requires the in vivo Draize rabbit eye irritation test. Several ex vivo alternatives have been proposed. These include (1) organotypic (full thickness corneal) models, (2) reconstructed human cornea-like epithelium assays, and (3) cytotoxicity & cell-function assays (epithelium based). Although useful, none of these tests fully cover the entire irritation severity spectrum, and no single test, combination of tests, or testing strategy is currently poised to replace the Draize eye test. In vitro cell- or organ-free molecular assays currently employ either liposomal formulations or use bulk lipid mixtures. The formal, liposomal assays, do not recapitulate the lamellar organisation of the TFLL and they often do not use the non-polar lipids.

[0010]

[0006] An alternative to liposomal formulation is a patented technology by Lebrun (US10041922B2). These in vitro formulations rest on the premise that the ocular irritancy is caused by the disruption of the molecular integrity of the ocular tissue and that this disruption can be modelled simply by using molecular constituents in a water-soluble, water-insoluble, or amphiphilic matrices. For example, the water soluble matrix utilizes a proprietary (undisclosed composition) of macromolecules, including amino acids, peptides, proteins, lipids, glycoproteins, carbohydrates or natural or synthetic polymers. These components are derived as cell or tissue extracts. The model does not mimic the TFLL organisation, may underrepresent TFLL components, and measures only bulk level changes to assay irritancy.

[0011]

[0007] Artificial tears and molecularly tailored lipid mixtures can in principle, be used to reconstitute the lipid composition and organisation of the tear film. However, there are no reports of the deliberate use of these technologies for assaying tear film irritants. Furthermore, none of the currently available in-vitro alternatives are also high-throughput or multiplexed methods.

[0012]

[0008] In view of the above, there is a need to develop in vitro platforms of the air interfaces for organs such as the skin, lung, and eye to evaluate the interactions with exogenous substances occurring at the air interfaces. In particular, there is a need for the provision of effective, low-cost in vitro assays with rapid screening capability for eye irritancy of substances.

[0013] SUMMARY

[0014]

[0009] In one aspect, there is provided a platform for mimicking a biological air-interface, comprises: a substrate; and a lipid assembly deposited on the substrate, wherein the lipid assembly mimics the lamellar, stratified, or monolayer organisation and optionally polarity gradient, of the biological airinterface, and wherein the lipid assembly comprises an amphiphilic lipid phase, and / or a non-polar lipid phase.

[0015]

[0010] In various embodiments, the substrate comprises a glass substrate, silicon substrate, gold substrate, metal oxide substrate, polymeric substrate, transducer surface, liquid droplet, hydrogel, or other soft material, preferably the substrate is a planar glass substrate or glass bead comprising a hydrophobic coating, more preferably trimethylsilyl (TMS) beads.

[0016]

[0011] In various embodiments, the lipid assembly mimics the stratified lamellar arrangement and polarity gradient of the tear-film lipid layer (TFLL) of the eye, and comprises or consists of a non-polar lipid phase and an amphiphilic lipid phase comprising one or more interfacial amphiphilic lipids and one or more amphiphilic phospholipids.

[0017]

[0012] In various embodiments, the non-polar lipid phase comprises cholesteryl esters (CEs) and wax esters (WEs), and the amphiphilic lipid phase comprises (O-acyl)-co-hydroxy fatty acids (OAHFAs), glycerophospholipids, lysophospholipids and sphingomyelin.

[0018]

[0013] In various embodiments, the lipid assembly comprises cholesteryl lignocerate, cholesteryl nervonate and behenyl oleate, preferably comprises about 10-30 mol% of cholesteryl lignocerate, about 10-30 mol% of cholesteryl nervonate and about 35-55 mol% of behenyl oleate based on total lipid content.

[0019]

[0014] In various embodiments, the amphiphilic lipid phase comprises 5-(palmitoyloxy)stearic acid (5-PAHSA), 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1 -palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, 1 -oleoyl-sn-glycero-3-phosphocholine, 1 -oleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin derived from chicken egg, preferably the lipid assembly comprises about 2-6 mol% of 5-(palmitoyloxy)stearic acid (5-PAHSA), about 1-4 mol% of 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine, about 0.5-2 mol% of 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, about 0.5-2 mol% of 1 -oleoyl-sn-glycero-3-phosphocholine, about 1-4 mol% of 1-oleoyl-sn-glycero-3-phosphoethanolamine and about 2-6 mol% of sphingomyelin derived from chicken egg based on total lipid content.

[0020]

[0015] In various embodiments, the thickness of the amphiphilic lipid phase is about 3-5 nm, and the thickness of the non-polar lipid phase is about 30-200 nm.

[0021]

[0016] In various embodiments, the lipid assembly mimics the repeating lamellar strata and periodic polarity gradient of the stratum corneum (SC) lipid barrier of the skin, wherein each lamellar stratum comprises a non-polar lipid phase and an amphiphilic lipid phase comprising one or more interfacial amphiphilic lipids.

[0022]

[0017] In various embodiments, the amphiphilic lipid phase comprises ceramides (Cer), free fatty acids (FFAs), cholesterol and cholesterol sulfate, preferably the amphiphilic lipid phase comprises about 40-80 mol % of the lipid assembly, and the non-polar lipid phase comprises about 5-30 mol % of the total lipid assembly.

[0023]

[0018] In various embodiments, the lipid assembly mimics the monolayer organisation and polarity gradient of the alveolar surfactant (AS) layer of the lung, and comprises an amphiphilic lipid phase comprising one or more amphiphilic lipids, and optionally surfactant-associated proteins to form a lipid-protein assembly.

[0019] In various embodiments, the amphiphilic lipid phase comprises dipalmitoylphosphatidylcholine (DPPC), preferably the amphiphilic lipid phase comprises about 80-95 mol % of the lipid assembly, and optionally the lipid assembly comprises about 5-20 mol % of surfactant-associated proteins.

[0024]

[0020] In various embodiments, the platform further comprises an aqueous phase, wherein the aqueous phase is in direct contact with, or contiguous to, the amphiphilic lipid phase so as to simulate the aqueous phase of the native biological air-interface.

[0025]

[0021] In various embodiments, the lipid assembly is arranged in an inverted “upside-down” configuration relative to the native biological air-interface.

[0026]

[0022] In various embodiments, the platform is a cell-free platform.

[0027]

[0023] In another aspect, there is provided a method of manufacturing the cell-free platform disclosed herein, comprising the steps of: providing a substrate; and depositing a lipid assembly onto the substrate, wherein the lipid assembly comprises an amphiphilic lipid phase, and / or a non-polar lipid phase, wherein depositing the lipid assembly comprises sequential or co-deposition of one or more lipid compositions forming the amphiphilic lipid phase and / or non-polar phase, wherein the lipid assembly mimics the lamellar, stratified, or monolayer organisation and optionally polarity gradient, of the biological air-interface.

[0028]

[0024] In another aspect, there is provided a biosensor device comprising the platform disclosed herein.

[0029]

[0025] In another aspect, there is provided the use of the platform, or biosensor device disclosed herein for screening or assaying a candidate substance.

[0030]

[0026] In another aspect, there is provided a method for screening or assaying a candidate substance, comprising the steps of: contacting the candidate substance to the platform disclosed herein; and measuring one or more biophysical properties of the platform to obtain one or more biophysical properties values, wherein the one or more biophysical values are indicative of the interaction between the candidate substance and the platform.

[0031]

[0027] In various embodiments, the one or more biophysical values of the platform is used to predict the toxicity or irritancy of the candidate substance.

[0032]

[0028] In various embodiments, the one or more biophysical properties values are compared to reference values, wherein a differential value is indicative of the interaction between the candidate substance and the lipid assembly.

[0029] In various embodiments, the one or more biophysical properties is selected from morphology, uniformity and contiguity of the lipid phases, fluidity and dynamics, and integrity.

[0033] BRIEF DESCRIPTION OF DRAWINGS

[0034]

[0030] FIG. 1 shows the tear-film lipid layer (TFLL) is stratified, consisting of outermost layer of nonpolar lipids (-30-200 nm) and a small interfacial layer, 3-5 nm thick, consisting of amphiphilic lipids.

[0035]

[0031] FIG. 2 shows the stratified organisation of the entire tear film, consisting of outer oil (or lipid) layer, middle aqueous layer, and inner mucin layer.

[0036]

[0032] FIG. 3 shows a schematic of the spatial organisation of the amphiphilic lipids, interfacial lipids, and non-polar lipids in a representative platform disclosed herein that mimics the tear-film lipid layer.

[0037]

[0033] FIG. 4 shows stratified molecular organisation of lipids of TFLL. CE = cholesteryl esters, WE = Wax esters, TG = triglycerides and PL = phospholipids.

[0038]

[0034] FIG. 5 shows a representative microscopy image of the TFLL. Non-polar lipids were stained in green with Bodipy (left panel) whereas the amphiphilic lipids were stained in red (middle panel) with Rhodamine-PE (red). The combined image is shown on the right panel. Scale bar = 200 pm.

[0039]

[0035] FIG. 6 shows lipid fluidity of the TFLL using fluorescence recovery after photobleaching. Fluorescence recovery was evident by 5 min in both non-polar (top row) and amphiphilic (bottom row) lipid layer. The photobleached region was highlighted. Scale bar = 100 pm.

[0040]

[0036] FIG.7 shows biophysical changes of TFLL after incubation with irritants: Benzalkonium chloride and sodium salicylate are used as representative irritants for Category 1 and 2 eye irritants, respectively. Scale bar = 100 pm.

[0041]

[0037] FIG. 8 shows the TFLL-on-bead with a schematic of a hydrophobic silica bead coated with the non-polar and amphiphilic lipid layers, suspended in aqueous medium for the assay. Non-polar lipids were stained in green with Bodipy (middle panel) whereas the amphiphilic lipids were stained in red (bottom panel) with Rhodamine-PE (red).

[0042]

[0038] FIG.9 shows TFLL-on-bead irritation assay. The fluorescence micrographs show fluorescence intensity changes of the BODIPY 493 / 503 and Rhodamine-PE fluorophores when the lipid-coated beads are incubated with glycerol (No Category eye irritant) and sodium lauryl sulfate (Category 1 eye irritant). DETAILED DESCRIPTION

[0043]

[0039] It will be appreciated by a person skilled in the art that numerous variations and / or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive. Also, the invention includes any combination of features described for different embodiments, including in the summary section, even if the feature or combination of features is not explicitly specified in the summary section or the detailed description of the present embodiments.

[0044]

[0040] The native tear film lipid layer (TFLL) is the outermost, lipid-laden oily layer of the tear film secreted by the meibomian glands located in the tarsal plates of the upper and lower eyelids. It is a multicomponent, multifunctional, cell-free film at the interface between the eye and the environment (i.e., air). As the first line of defense, it plays many important roles in the protection, lubrication, and the normal health of the eye. Some of the major roles include providing optical transparency, reducing the surface tension, retarding water evaporation, facilitating re-spreading following blinks, and conferring antimicrobial resistance. This multifunctionality results from the multi-component chemical composition and a complex and dynamic molecular organisation of the TFLL. Previous work has established that the TFLL is organised as a bi-lamellar structure consisting of two compositionally different layers: (1 ) a thin amphiphilic sub-layer (2-5 nm), residing as a polar, amphiphilic, multicomponent phospholipid monolayer at the aqueous boundary of the tear-film and (2) a thicker bulk (30-200 nm thick), composed of a mixture of many different non-polar, hydrophobic lipids, which contacts the surrounding air (FIG.

[0045] 1).

[0046]

[0041] The entirety of the tear film is also a lamellar structure. Beginning with the outermost TFLL (FIG. 1), the tear film has an intermediate aqueous layer and the inner mucus layer, which contacts the corneal epithelial cells (FIG.2). Even subtle changes in the composition of the tear film can compromise the structural and functional integrity of the TFLL. These changes can be induced due to pathological conditions that impair vision (e.g., dry eye disease and blepharitis) or may be induced when environmental interferents (e.g., molecules, particles, and microbes) interact with the TFLL. This is perhaps most common in the case of many consumer products (such as lotions, hair products, and other cosmetics). These products often contain molecules and particles that, upon contact with the tear film, become trapped in the hydrophobic or the amphiphilic layers of the TFLL. Similar complications may arise when polluted water, microbes and molecules from the environment contact the eye. The contaminants may be dissolved substances, particulate matters, or vapor phase molecules or particles.

[0047]

[0042] In all of these cases, either prompted by regulatory approvals or by public health hazard mitigation procedures, there is a continuous demand for the toxicology assays. Current approaches in many cases (e.g., cosmetics) rely on animal testing data for toxicology assessment and validation before they can be approved and released to the market.

[0043] Accordingly, the present inventors have developed a platform that mimics the molecular makeup and organization of the lamellar lipid arrangement of the TFLL for use in in vitro assays to test substances and their effects on the TFLL. The platform disclosed herein may be termed as an in vitro platform.

[0048]

[0044] It will be appreciated that the illustrative TFLL platform exemplified herein, may also be adapted for other biological interfaces, including but not limited to, skin (stratum corneum) and lung (alveolar interface). Molecularly defined interfaces, particularly those that are composed largely of lipid molecules, can be assembled following similar approaches. These assemblies may then confer similar advantages as above for the assessment of molecular interactions and biophysical impact on the assemblies.

[0049]

[0045] In this regard, the stratum corneum lipid barrier is understood to be primarily composed of ceramides, cholesterol, and free fatty acids, which together form multilamellar stacks exhibiting long and / or short periodicity phases. Within this composition, non-polar lipids are present only as minor components, whereas interfacial amphiphilic lipids represent the major constituents. These lipids selforganise into repeating lamellar structures characterised by alternating amphiphilic and non-polar regions, thereby establishing a periodic polarity gradient across the lamellae and providing the skin’s principal permeability barrier.

[0050]

[0046] Further, the alveolar air-liquid interface is understood to be principally formed by pulmonary surfactant, a lipid-protein complex dominated by phospholipids. Within this composition, amphiphilic phospholipids represent the major constituents. At the air-interface, the amphiphilic phospholipids orient to form a surface-active monolayer in which hydrophobic acyl chains extend toward the air phase and amphiphilic headgroups face the aqueous phase, thereby establishing a unidirectional polarity gradient across the monolayer that is essential for lowering surface tension, preventing alveolar collapse, and maintaining pulmonary compliance. The alveolar air-liquid interface may be referred to the pulmonary surfactant at the alveolar interface, or the pulmonary surfactant film lining the alveoli.

[0051]

[0047] The present invention therefore provides a platform that mimics biological interfaces such as, but not limited to, the air exposed interfaces of the eye, skin, or lung. The term “biological interfaces”, in this context, may refer to the air-exposed epithelial boundary layers of tissues that form the first line of interaction with the external environment. These interfaces are composed primarily of lipid-rich films organised at the air-liquid or air-tissue boundary, and serve to maintain homeostasis, regulate permeability, and protect underlying tissues. In various embodiments, the biological air-interface is substantially composed of lipids, meaning that lipids constitute the dominant molecular fraction responsible for the physical and functional characteristics of the interface. In this context, “substantially composed of lipids” may refer to interfaces in which lipids account for at least about 70%, preferably at least about 80%, and more preferably at least about 90% of the structural and functional composition of the surface film. Proteins, carbohydrates, and other biomolecules may be absent, incidental, or present only in minor amounts that do not materially affect the lipid-driven organisation and properties of the interface.

[0052]

[0048] In various embodiments, the platform disclosed herein may mimic the air-exposed biological surface of a tissue, and more particularly, models and mimics a biological air-interface.

[0053]

[0049] In various embodiments, the biological air-interface may be selected from the tear-film lipid layer (TFLL) of the eye, the stratum corneum lipid barrier of the skin, and the alveolar surfactant layer of the lung. In this context, the platform disclosed herein may function as a synthetic model of these interfaces, thereby enabling screening, sensing, or therapeutic evaluation without the need for intact cells or tissues.

[0054]

[0050] In various embodiments, the platform provides a reproducible model that mimics the lamellar, stratified, or monolayer organisation of the naturally occurring biological air- interface. As used herein, the phrase “mimicking the lamellar, stratified, or monolayer organisation of a biological air-interface” may refer to the reproduction, emulation, or functional replication of the structural architectures observed at naturally occurring biological air-liquid interfaces. Lamellar organisation may refer to the periodic, multilayered stacking of lipids into bilayer or multilamellar phases, as exemplified by the extracellular lipid lamellae of the stratum corneum of the skin, in which alternating amphiphilic and nonpolar regions create a repeating polarity gradient that confers barrier and protective functions. Stratified organisation may refer to a discrete, sheet-like arrangement of compositionally distinct lipid layers or phases, such as the tear film lipid layer of the eye, where a non-polar lipid layer overlays an amphiphilic lipid layer comprising an interfacial amphiphilic phase to establish a polarity gradient. Monolayer organisation may refer to the orientation of amphiphilic lipids into a single-molecule-thick film at the airliquid boundary, as exemplified by pulmonary surfactant at the alveolar interface (i.e. pulmonary surfactant film lining the alveoli), wherein lipid acyl chains extend toward the air phase and amphiphilic headgroups face the aqueous phase to generate a unidirectional polarity gradient. In all cases, mimicking does not require absolute identity of molecular constituents with the natural interface, but rather the reproduction of its organisational and / or functional performance, to a degree sufficient for use in screening, sensing, analytical, or therapeutic applications. In various embodiments, the platform mimics the lamellar stratified organisation of the biological air-interface, as per the TFLL.

[0055]

[0051] In various embodiments, the platform may refer to a synthetic, cell-free construct or model comprising lipids and optionally one or more additional molecular constituents (e.g., proteins, polymers, surfactants, or combinations thereof) arranged in association with a supporting substrate in a defined architecture. In various embodiments, the platform may be a cell-free platform that does not require or include living cells or tissues.

[0056]

[0052] Accordingly, there is provided a cell-free platform for mimicking a biological air- interface, comprising or consisting of a substrate and a lipid assembly deposited on the substrate. The lipid assembly is configured to mimic the lamellar, stratified, or monolayer organisation of the biological airinterface.

[0057]

[0053] In various embodiments, the substrate may be a hydrophilic substrate or a hydrophobic substrate. In various embodiments, the substrate may be modified to be either hydrophobic or hydrophilic, or exhibit inherent hydrophobic or hydrophilic properties. In various embodiments, a substrate may be chemically or physically modified to impart either hydrophobicity or hydrophilicity.

[0058]

[0054] In various embodiments, the substrate may be a hydrophilic substrate. The hydrophilic substrate may refer to a support whose surface exhibits an affinity for water and amphiphilic molecules, typically through the presence of hydroxyl, carboxyl, amine, or other amphiphilic functional groups. Hydrophilic substrates facilitate the orientation of lipid headgroups toward the substrate and may be employed to promote the assembly of amphiphilic lipid strata in a stratified lipid assembly. Exemplary hydrophilic solid substrates include untreated glass, oxidized silicon, plasma- or UV / ozone-treated glass, and metal oxides such as TiOs or AI2O3. Hydrophilic modifications include plasma oxidation, UV / ozone treatment, silanization with hydrophilic silanes (e.g., aminopropyltriethoxysilane, carboxyethylsilanes), coating with hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylic acid (PAA), or bioinspired coatings such as polydopamine. These treatments increase surface energy and provide functional groups that interact with amphiphilic lipid headgroups.

[0059]

[0055] In various embodiments, the substrate may be a hydrophobic substrate. The hydrophobic substrate may refer to a surface material that exhibits water-repelling (non-wetting) properties. Hydrophobicity may be inherent to the material or imparted through surface treatment, chemical modification, or coating. In various embodiments, the hydrophobic substrate may be any material so long as the hydrophobic surface induces spontaneous stratification of non-polar lipids and subsequently amphiphilic liquids onto the hydrophobized surface. Hydrophobic modifications include silanization with alkylsilanes (e.g., octadecyltrichlorosilane, trimethylsilyl silanes), fluorosilanes, deposition of hydrophobic polymers (e.g., polytetrafluoroethylene, fluoropolymers), or hydrocarbon coatings. These treatments lower surface energy, favouring interaction with lipid acyl chains and promoting non-polar lipid assembly.

[0060]

[0056] In various embodiments, the substrate provides a stable surface for lipid self-assembly or deposition. The substrate may comprise a glass substrate, silicon substrate, gold substrate, metal oxide substrate, polymeric substrate, transducer surface, liquid droplet, hydrogel, or soft material. The substrate may be planar or structured, including smooth, patterned, porous, or micro- or nano-textured formats. Soft or fluid substrates, such as aqueous droplets, hydrogel supports, or elastomeric materials, may also be employed to model dynamic or deformable interfaces. Collectively, these substrates provide tunable physicochemical environments that facilitate ordered lipid deposition and mimic the biological air-interfaces.

[0057] In various embodiments, the substrate may be a solid substrate, which as used herein refers to any non-fluid, rigid or semi-rigid material capable of supporting a lipid assembly, lamellar assembly, or other biomolecular layer on its surface. The term “solid substrate” encompasses inorganic and organic materials including, but not limited to, glass, silicon, metals, metal oxides, ceramics, and polymeric materials, as well as composite or layered materials thereof. The solid substrate may be planar or structured, for example smooth, patterned, porous, or micro- or nano-textured.

[0061]

[0058] In various embodiments, the substrate may be a hydrophobic solid substrate. In various embodiments, the hydrophobic solid substrate may be selected from glass, silicon, gold, metal oxides, polymeric surfaces, or transducer surfaces, including surfaces that have been modified to enhance hydrophobicity (e.g., silanized glass, fluoropolymer-coated metals, or hydrophobized sensor chips). Such substrates provide a stable platform for the deposition, assembly, or analysis of lamellar lipid layers and other biomolecular films in vitro.

[0062]

[0059] In various embodiments, the hydrophobic solid substrate may be a glass substrate comprising a hydrophobic coating. In various embodiments, the hydrophobic coating may be octadecyltrichlorosilane (OTS).

[0063]

[0060] In various embodiments, the hydrophobic solid substrate may be a planar solid substrate selected from glass, silicon, gold, metal oxide, polymeric, or transducer surfaces, optionally coated with a hydrophobic silane, fluoropolymer, or other hydrophobizing agent.

[0064]

[0061] In various embodiments, the hydrophobic solid substrate may comprise microspherical particles, including silica beads, glass beads, or polymeric beads, optionally silanized or otherwise surface-modified to impart hydrophobicity. In various embodiments, the hydrophobic solid substrate may comprise trimethylsilyl (TMS) beads or other silica-based particles that have been rendered hydrophobic by surface modification. Such substrates provide a three-dimensional, particulate surface for lipid deposition, enabling the formation of lamellar stratified lipid layers in a spherical or irregular geometry. The use of TMS beads may facilitate suspension-based assays, increase surface area for lipid assembly, or incorporation into microfluidic, sensing, or diagnostic devices.

[0065]

[0062] As used herein, the term “deposited on” refers to the spatial arrangement or assembly of one phase or layer in physical association with another phase or substrate, such that the overlying material is supported by, contiguous with, or positioned relative to the underlying phase or substrate. The association may occur through physical adsorption, selective or preferential molecular diffusion, hydrophobic or hydrophilic interactions, electrostatic interactions, van der Waals forces, or covalent / chemical modification. In the context of the lipid platform, “deposited on” encompasses not only sequential or stepwise placement but also spontaneous or induced molecular self-assembly, wherein adjacent lipid phases are in continuous contact through cohesive or interdigitated molecular interactions. Thus, the term may describe layers that are contiguous and integrated into a continuous interfacial structure, rather than distinct or separated strata. In various embodiments, “deposited on” includes assembly upon, supported by, or in molecular continuity with a substrate or another lipid phase, optionally comprising one or more lamellar arrangements of the corresponding lipid class.

[0066]

[0063] As used herein, the term “lipid assembly” refers to a structured arrangement of lipid molecules that mimics the lamellar, stratified, or monolayer organisation of a biological air-interface, thereby reproducing its functional properties, and optionally polarity gradient. The lipid assembly may optionally comprise distinct non-polar, and amphiphilic lipid phases arranged in strata, domains, or layers, and encompasses lipid compositions organised in ordered formats including multilamellar constructs, lamellar phases, stratified lipid films, or monolayer films at the air-liquid interface. Such lipid assemblies functionally replicate the organisation of naturally occurring air-interfaces, including the lamellar architecture of the stratum corneum, the lamellar stratified arrangement of the tear film lipid layer, and the monolayer organisation of pulmonary surfactant.

[0067]

[0064] In various embodiments, the lipid assembly may be organised to establish a polarity gradient across its layers, domains or phases. Such an organisation may create a progressive transition from hydrophobic to amphiphilic to hydrophilic polarity within the assembly, or a repeating periodic polarity as in multilamellar systems, thereby mimicking the structural and functional organisation of naturally occurring air-exposed biological interfaces.

[0068]

[0065] In various embodiments, the lipid assembly may comprise or consist of a non-polar lipid phase, and / or a amphiphilic lipid phase. The terms “layer”, “phase” and “fraction” may thus be employed interchangeably to describe the arrangement of lipid classes, and may be used synonymously unless otherwise specified. As used herein, the term “fraction” refers to a compositionally or functionally distinguishable portion of the lipid assembly that is enriched in one or more lipid classes, yet remains contiguous and in molecular continuity with adjoining portions. In this regard, the term “layer” may refer to these ordered lamellar phases, which collectively recapitulate the compositional makeup and organisation of air-exposed biological interfaces, and is not restricted to a single molecular monolayer. In various embodiments, each “layer” may correspond to a lamellar phase comprising one or more molecular strata of the designated lipid class. Thus, the non-polar lipid phase may also be referred to as the non-polar lipid layer or non-polar lipid fraction, and the amphiphilic lipid phase as the amphiphilic lipid layer or amphiphilic lipid fraction.

[0069]

[0066] The non-polar lipids refer to a class of hydrophobic, neutral lipid molecules that lack strongly amphiphilic or ionizable headgroups. These lipids are predominantly localized within hydrophobic strata or cores and function to provide impermeability, lubrication, and low surface energy at biological airinterfaces. Non-polar lipids may include, without limitation, cholesteryl esters (CEs), wax esters (WEs), triglycerides (TGs), diacylglycerols (DAGs), long-chain hydrocarbons such as squalene, alkyl esters, and other long-chain neutral lipids characteristic of air-exposed epithelial surfaces such as the stratum corneum, ocular surface, and pulmonary alveolar interface. Cholesteryl esters are fully hydrophobic sterol derivatives formed by esterification of cholesterol at the C-3 hydroxyl group and are characteristic storage or barrier lipids within non-polar compartments. Triglycerides (TGs), composed of triesters of glycerol and long-chain fatty acids (e.g., palmitic, stearic, oleic, lignoceric), serve as neutral hydrophobic lipids that may be partially hydrolyzed to diacylglycerols (DAGs) under physiological conditions. Wax esters (WEs) are long-chain fatty acid-fatty alcohol esters that are fully hydrophobic and contribute to surface impermeability and lubrication; exemplary WEs include oleyl palmitate, palmitoyl stearate, myristyl palmitate, cetyl palmitate, behenyl oleate, and stearyl arachidate, typically ranging from C32 to C48 in total chain length.

[0070]

[0067] In various embodiments, the non-polar lipids may comprise cholesteryl esters (CEs) such as CE(24:0) (cholesteryl lignocerate), CE(24:1 (15Z)) (cholesteryl nervonate), CE(26:0), CE(20:0), or mixtures thereof.

[0071]

[0068] In various embodiments, the non-polar lipid phase may comprise orconsist of one or more nonpolar lipids, more particularly 1 , 2, 3, 4, 5 or 6 non-polar lipids.

[0072]

[0069] As used herein, the term “amphiphilic lipids" refers to a class of lipid molecules that contain both hydrophilic and hydrophobic moieties, enabling spontaneous orientation at interfaces between polar and non-polar phases. Amphiphilic lipids may self-assemble into lamellar, monolayer, or multilayer structures that establish a polarity gradient characteristic of biological air-interfaces, including the tearfilm lipid layer, stratum corneum, and pulmonary surfactant. Amphiphilic lipids may include, for example, phospholipids, lysophospholipids, sphingolipids, glycolipids, or other lipids bearing polar or ionic headgroups.

[0073]

[0070] In various embodiments, the amphiphilic lipids may comprise lipids possessing strongly polar or ionic headgroups, typically derived from glycerophospholipids, lysophospholipids or sphingolipids. Such lipids orient with their hydrophilic headgroups toward aqueous phases and hydrophobic tails toward adjacent lipid regions, forming stable monolayers or bilayers. Amphiphilic lipids may include, without limitation, phosphatidylcholines (PC); phosphatidylethanolamines (PE); phosphatidylserines (PS); phosphatidylglycerols (PG); phosphatidylinositols (PI); lysophosphatidylcholine (LPC); lysophosphatidylethanolamine (LPE); PC(16:0 / 18:1 (9Z)) (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, POPC); PE(16:0 / 18:1 (9Z)) (1 -palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, POPE); PC(18:1(9Z) / 0:0) (1 -oleoyl-sn-glycero-3-phosphocholine, LPC analogue); PE(18:1 (9Z) / 0:0) (1 -oleoyl-sn-glycero-3-phosphoethanolamine, LPE analogue); dipalmitoylphosphatidylcholine (DPPC); phosphatidylglycerol (PG; e.g., dioleoyl-PG or dipalmitoyl-PG); phosphatidylinositol (PI; e.g., dioleoyl-Pl); phosphatidylethanolamine (PE; e.g., dioleoyl-PE); and sphingomyelin, including naturally sourced sphingomyelin such as Egg SM (sphingomyelin derived from chicken egg).

[0074]

[0071] In various embodiments, the amphiphilic lipids may refer to a subset of amphiphilic lipids enriched at the boundary between non-polar and polar lipid phases (i.e. interfacial amphiphilic lipids). These molecules typically possess long hydrophobic chains and moderately polar or weakly ionizable headgroups, allowing partial insertion into non-polar strata while maintaining interactions with polar or aqueous environments. In various embodiments, interfacial amphiphilic lipids may include, without limitation, (O-acyl)-w-hydroxy fatty acids (OAHFAs) such as 5-PAHSA (5-(palmitoyloxy)stearic acid), 9-PAHSA, 12-PAHSA, and related positional isomers; w-hydroxy fatty acids and their esters (other than OAHFAs); long-chain amphiphilic fatty alcohols; acylated sterols such as hydroxylated or acylsubstituted cholesterol derivatives; monoglycerides and diglycerides with long-chain acyl groups; ceramides (including non-hydroxy, a-hydroxy, and w-hydroxy species); free fatty acids; cholesterol and cholesterol sulfate, which may function as weakly amphiphilic interfacial sterols. In various embodiments, the interfacial amphiphilic lipids may refer to, and include, OAHFAs, cholesteryl OAHFAs (Chl-OAHFAs), type 1 w-hydroxy wax diesters (type 1 w-WdiE) and type 2 w-hydroxy wax diesters (type 2 w-WdiE).

[0075]

[0072] Accordingly, the amphiphilic lipid phase of the lipid assembly may comprise one or more of the above amphiphilic lipid species, optionally in relative proportions selected to mimic the composition and biophysical properties of the targeted biological air-interface. In various embodiments, the amphiphilic lipid phase of the lipid assembly may comprise one or more interfacial amphiphilic lipids and one or more other amphiphilic lipids outlined above. In various embodiments, the interfacial amphiphilic lipids and other amphiphilic lipids together define a continuous amphiphilic phase that is contiguous with the non-polar lipid phase, thereby forming a cohesive and polarity-graded lipid architecture analogous to natural biological air-interfaces. In various embodiments, an interfacial amphiphilic lipid phase may be present in between, at the interface of, the non-polar lipid phase and amphiphilic lipid phase.

[0076]

[0073] It will be appreciated that the composition of the lipid assembly may be defined with respect to the relative proportion of each lipid phase and its constituent lipids. In this regard, the lipid assembly may comprise varying proportions of non-polar lipids, and amphiphilic lipids. In various embodiments, the lipid assembly comprises one or more lipid classes or phases, each defined in terms of its molar proportion relative to the total lipid content of the assembly (% mol). As used herein, “mol%” refers to the mole fraction of a given lipid species or phase expressed as a percentage of the total lipid content of the lipid assembly. Unless otherwise specified, all % mol values herein refer to the molar fraction of a given lipid or lipid class based on the total moles of lipids present in the assembly. The relative content of each lipid class or phase may typically range from about 5 mol% to 95 mol% of the total lipid assembly, for example from about 10-80 mol%, or about 15-70 mol%, as appropriate to reproduce the polarity gradient, structural organization, and functional characteristics of the corresponding natural interface.

[0077]

[0074] In various embodiments, the relative thicknesses of the phases may be varied depending on the biological air-interface to be modelled (e.g., eye, skin, or lung). Thus, the thickness of the lipid phases of the lipid assembly may be selected to approximate the dimensions of the natural biological air-interfaces. For example, the amphiphilic lipid phase may have a thickness of about 2-15 nm, and the non-polar lipid phase a thickness of about 5-200 nm. In various embodiments, the amphiphilic lipid phase may have a thickness of about 2-5 nm, reflecting the approximate molecular dimensions of phospholipid or glycolipid monolayers. In various embodiments, the non-polar lipid phase may have a thickness of about 5-50nm.

[0078]

[0075] In various embodiments, non-polar lipids may orient toward the environment (air), amphiphilic lipids may orient toward the substrate, wherein the amphiphilic lipids at the interface (i.e. interfacial amphiphilic lipids) provide molecular continuity such that the non-polar and amphiphilic layers are contiguous and seamlessly integrated. In various embodiments, amphiphilic lipids may orient toward the environment (air), non-polar lipids orient toward the substrate, wherein the amphiphilic lipids at the interface provide molecular continuity such that the non-polar and amphiphilic layers are contiguous and seamlessly integrated.

[0079]

[0076] In various embodiments, the platform recapitulates the natural biological air-interface in an “upside-down” arrangement, with the substrate substituting for the air interface in vivo. In this configuration, the lipid phase is oriented such that lipids, which in vivo face the external air, are instead deposited adjacent to the substrate. In this context, the organisation of the lipid assembly may be oriented in an inverted fashion relative to the native biological surface, as well as the overall polarity gradient being inverted.

[0080]

[0077] In various embodiments, the lipid assembly is deposited on the substrate such that the arrangement of the lipid phases or layering are inverted relative to the natural orientation of air-exposed biological interfaces (i.e. an “upside-down” arrangement).

[0081]

[0078] In various embodiments, the platform recapitulates the polarity gradient, or periodic polarity, of the natural biological air-interface in an “upside-down” arrangement. By reproducing this continuous polarity gradient or lamellar polarity pattern, the platform provides a controllable and biomimetic model system suitable for screening, analytical, and interfacial studies of lipid organization and function.

[0082]

[0079] In various embodiments, the lipid assembly comprises layers of lipid classes that are in an “upside-down” arrangement to mimic the polarity gradient of the biological air-interface. In particular, in this “upside-down” arrangement, the relative orientation of lipid phases is inverted with respect to the native in vivo organisation, such that the polarity gradient is also reversed relative to the air-aqueous boundary. This configuration recapitulates the lamellar stratified organisation of natural air-exposed interfaces in an inverted (“upside-down”) orientation, enabling the lipid assembly to serve as a cell-free, in vitro platform for modelling barrier and functional properties of the eye, skin, and lung. This approach can be adapted for the mimicking of the stratified lamellar arrangement of the TFLL, the lamellar lipid stacks of the stratum corneum barrier of the skin, and the monolayer lipid-protein film of the pulmonary surfactant of the lung.

[0080] In other embodiments, the lipid assembly may be arranged in a “right-side-up” configuration, wherein the orientation of the lipid phases corresponds to that found in the natural biological airinterface. Such a “right-side-up” configuration reproduces the natural directionality of lipid stratification present in air-exposed tissues, including the tear-film lipid layer (TFLL), the stratum corneum barrier of the skin, and the pulmonary surfactant film lining the alveoli, thereby providing a physiologically relevant in vitro model for studies of surface tension, permeability, adsorption, and lipid-protein interactions.

[0083]

[0081] In various embodiments where the lipid assembly comprises an arrangement of lipid layers, the lipid layers may be defined relative to their positioning on the substrate. The inner layer may refer to the basal lipid stratum in direct contact with the substrate, the sub-layer (or intermediate layer) refers to a lipid stratum deposited between the inner and outer layers, and the outer layer refers to the lipid stratum positioned most distal to the substrate and in contact with the surrounding environment (e.g., medium). In various embodiments, the inner layer comprises non-polar lipids, the sub-layer comprises interfacial amphiphilic lipids, and the outer layer comprises amphiphilic lipids.

[0084]

[0082] In various embodiments, the lipid assembly comprises lipids alone, while in other embodiments, the lipid assembly may comprise proteins that are naturally associated with the biological air-interface being mimicked, thereby forming a proteolipid assembly. For example, when the platform mimics the tear-film lipid layer (TFLL) of the eye, proteins such as lipocalins, lactoferrin, lysozyme, and mucins may be incorporated to influence lipid layer stability, spreading, and antimicrobial protection. When mimicking the stratum corneum lipid barrier of the skin, proteins such as keratin and filaggrin degradation products (natural moisturizing factor, NMF), as well as lipid-processing enzymes including ceramidases and lipases, may be included to replicate barrier function. When mimicking the alveolar surfactant layer of the lung, surfactant-associated proteins SP-A, SP-B, SP-C, and SP-D may be incorporated, as they are essential for lowering surface tension, stabilizing phospholipid monolayers, and mediating innate immune defense. In these embodiments, proteins may be admixed with lipid fractions prior to deposition or allowed to adsorb onto pre-formed lipid strata, thereby enhancing the functional mimicry of the platform.

[0085]

[0083] In various embodiments, the amphiphilic lipid phase may be in direct contact with the substrate and the non-polar lipid phase is exposed to the environment. In various embodiments, a non-polar lipid phase may be deposited on the substrate, and the amphiphilic lipid phase is deposited on the non-polar lipid phase, such that the non-polar face of the amphiphilic lipid phase is in direct contact with the nonpolar lipid phase and the amphiphilic face of the amphiphilic lipid phase is exposed to the environment.

[0086]

[0084] In various embodiments, the lipid assembly may further comprise an aqueous phase, wherein the aqueous phase is in contact with the non-polar lipid layer if the amphiphilic lipid phase is in direct contact with the substrate, or the aqueous phase is in contact with the amphiphilic lipid phase if the nonpolar lipid phase is in direct contact with the solid substrate. -Tear film lipid layer (TFLL)

[0087]

[0085] The platform disclosed herein may be a TFLL platform that mimics the stratified lamellar arrangement of the TFLL.

[0088]

[0086] In various embodiments, the lipid assembly may comprise a non-polar lipid phase, and an amphiphilic lipid phase. That is, the non-polar lipid phase, and an amphiphilic lipid phase are lamellar phases organised in a stratified arrangement.

[0089]

[0087] In various embodiments, the amphiphilic lipid phase comprises interfacial amphiphilic lipid phase. Since the TFLL comprises a non-polar lipid, air-exposed outer layer, the phases of the platform may be, in order from the substrate: non-polar -> interfacial amphiphilic -» amphiphilic lipid. In this regard, the lamellar phases comprise one or more lipid strata, establishing a hydrophobic-to-hydrophilic polarity gradient that recapitulates the native interface.

[0090]

[0088] In various embodiments, the non-polar lipid phase may comprise cholesteryl esters (CEs) such as CE(24:0) (cholesteryl lignocerate), CE(24:1 (15Z)) (cholesteryl nervonate), CE(26:0), CE(20:0), or mixtures thereof. In various embodiments, the non-polar lipid phase may comprise wax esters (WEs) such as WE(22:0 / 18:1(9Z)) (behenyl oleate), WE(24:0), WE(24:1), WE(26:0), WE(20:0), or mixtures thereof.

[0091]

[0089] In various embodiments, the non-polar lipid phase may comprise about 30 mol% to about 90 mol% of the lipid assembly. That is, the non-polar lipid phase constitutes about 30-90 mol% of the total lipid assembly. In various embodiments, the non-polar lipid phase may comprise about 35-90 mol%, about 40-70 mol%, about 70-85 mol%, about 75-90 %mol, or about 80-85 mol% of the lipid assembly. In various embodiments, the non-polar lipid phase may comprise about 84 mol% of the lipid assembly.

[0092]

[0090] In various embodiments, the non-polar lipid phase comprises cholesteryl esters and wax esters. In various embodiments, the lipid assembly may comprise cholesteryl esters at about 35-45 mol% and wax esters at about 40-50 mol% of the lipid assembly.

[0093]

[0091] In various embodiments, the non-polar lipid phase comprises cholesteryl lignocerate, cholesteryl nervonate, and behenyl oleate. In various embodiments, the lipid assembly comprises about 10-30 mol%, preferably 20 mol% of cholesteryl lignocerate, about 10-30 mol%, preferably 20 mol% of cholesteryl nervonate and about 35-55 mol%, preferably about 44 mol% of behenyl oleate of the lipid assembly.

[0094]

[0092] In various embodiments, the non-polar lipid phase may constitute approximately 80-90 mol% of the overall lipid assembly of the platform. For example, the TFLL may be modelled using about 35-45 mol% cholesteryl esters, comprising an equimolar mixture of CE(24:0) and CE(24:1 (15Z)), together with about 40-50 mol% wax esters, consisting of WE(22:0 / 18:1(9Z)). The relative proportions of cholesteryl esters and wax esters may be varied within these ranges to mimic the lamellar stratified organisation of the TFLL or various conditions or diseased state of the TFLL.

[0095]

[0093] In various embodiments, the amphiphilic lipid phase may comprise or consist of one or more amphiphilic lipids, more particularly at least 1 , 2, 3, 4, 5, 6 or 7 amphiphilic lipids.

[0096]

[0094] In various embodiments, the amphiphilic lipid phase may comprise of one (1 ) interfacial amphiphilic lipid.

[0097]

[0095] In various embodiments, the interfacial amphiphilic lipid may comprise about 1 mol% to about 20 mol% of the lipid assembly. That is, the interfacial amphiphilic lipid constitutes about 1-20 mol% of the total lipid assembly. In various embodiments, the interfacial amphiphilic lipids may comprise about 2-15 mol%, about 2-10 mol%, about 2-8 mol%, about 2-6 %mol, or about 3-5 mol%of the lipid assembly. In various embodiments, the interfacial amphiphilic lipids may comprise about 4 mol% of the lipid assembly.

[0098]

[0096] In various embodiments, the interfacial amphiphilic lipid may comprise (O-acyl)-co-hydroxy fatty acids (OAHFAs), preferably 5-PAHSA (5-(palmitoyloxy)stearic acid). In various embodiments, the lipid assembly may comprise about 2-6 mol%, preferably 4 mol%, OAHFA, preferably 5-PAHSA. The relative proportions of OAHFAs may be varied within to mimic the lamellar stratified organisation of the TFLL or various conditions or diseased state of the TFLL.

[0099]

[0097] In various embodiments, the amphiphilic lipid phase may comprise one or more interfacial amphiphilic lipids and a mixture of three (3), four (4), or five (5) other amphiphilic lipids. In various embodiments, the amphiphilic lipid phase may comprise a mixture of five (5) amphiphilic lipids.

[0100]

[0098] In various embodiments, the amphiphilic lipids may comprise about 2 mol% to about 25 mol% of the lipid assembly. That is, the amphiphilic lipids constitute about 2-25 mol% of the total lipid assembly.

[0101]

[0099] In various embodiments, the amphiphilic lipids may comprise about 5-20 mol%, about 5-15 mol%, about 8-15 mol%, about 10-15 %mol, or about 11-13 mol% of the lipid assembly. In various embodiments, the amphiphilic lipids may comprise about 12 mol% of the lipid assembly.

[0102]

[0100] In various embodiments, the amphiphilic lipids may comprise glycerophospholipids. In various embodiments, the amphiphilic phospholipids comprise a mixture of glycerophospholipids selected from PC(16:0 / 18:1 (9Z)) (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and PE(16:0 / 18:1 (9Z)) (1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine).

[0101] In various embodiments, the amphiphilic lipids comprise lysophospholipids. In various embodiments, the amphiphilic lipids comprise a mixture of PC(18:1 (9Z) / 0:0) (1 -oleoyl-sn-glycero-3-phosphocholine), and PE(18:1 (9Z) / 0:0) (1 -oleoyl-sn-glycero-3-phosphoethanolamine).

[0103]

[0102] In various embodiments, the amphiphilic lipids comprise sphingomyelin, including naturally sourced sphingomyelin such as Egg SM (sphingomyelin derived from chicken egg).

[0104]

[0103] In various embodiments, the amphiphilic lipid phase comprises a mixture of 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1 -palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, 1 -oleoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin derived from chicken egg.

[0105]

[0104] In various embodiments, the amphiphilic lipids may comprise glycerophospholipids, lysophospholipids and sphingomyelin in substantially equal proportions, corresponding to a molar ratio of about 1 :1 :1 (glycerophospholipid:lysophospholipid:sphingomyelin) of the total amphiphilic lipid phase.

[0106]

[0105] In various embodiments, the lipid assembly may comprise about 2-6 mol%, preferably about 4 mol% of glycerophospholipids, about 2-6 mol%, preferably about 4 mol%, of lysophospholipids and about 2-6 mol%, preferably about 4 mol%, of sphingomyelin, of the lipid assembly. In various embodiments, the lipid assembly may comprise about 1-4 mol%, preferably about 3 mol% of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, about 0.5-2 mol%, preferably about 1 mol%, of 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, about 0.5-2 mol%, preferably about 1 mol%, of 1-oleoyl-sn-glycero-3-phosphocholine, about 1-4 mol%, preferably about 3 mol% of 1 -oleoyl-sn-glycero-3-phosphoethanolamine and about 2-6 mol%, preferably about 4 mol%, of sphingomyelin derived from chicken egg, of the lipid assembly.

[0107]

[0106] In various embodiments, the lipid assembly comprises or consists of: a non-polar lipid phase comprising or consisting of cholesteryl lignocerate, cholesteryl nervonate and behenyl oleate; and an amphiphilic lipid phase comprising an interfacial amphiphilic lipid comprising or consisting of 5-PAHSA and a mixture of amphiphilic phospholipids comprising or consisting of 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1 -palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, 1 -oleoyl-sn-glycero-3-phosphocholine, 1 -oleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin derived from chicken egg.

[0108]

[0107] In various embodiments, the lipid assembly comprises or consists of about 20 mol% of cholesteryl lignocerate, about 20 mol% of cholesteryl nervonate and about 44 mol% of behenyl oleate, about 4 mol% of 5-PAHSA, about 3 mol% of 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine, about 1 mol% of 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, about 1 mol% of 1 -oleoyl-sn-glycero-3- phosphocholine, about 3 mol% of 1-oleoyl-sn-glycero-3-phosphoethanolamine and about 4 mol% of sphingomyelin derived from chicken egg.

[0109]

[0108] In various embodiments, the TFLL platform may further comprise an aqueous phase deposited on the amphiphilic lipid phase. In this regard, in the context of mimicking the TFLL the overlying aqueous phase may simulate the tear aqueous subphase. The aqueous phase may have a thickness of about 2-50 pm, preferably 5-20 pm, and more preferably 8-15 pm, thereby approximating physiological tearfilm thickness while ensuring adequate hydration and interfacial stability of the lipid assembly.

[0110]

[0109] In various embodiments, the amphiphilic lipid phase may be a thickness of about 5-20nm, or about 5-15nm, or about 3-5 nm, and the non-polar lipid phase may be a comparatively thicker phase of about 30-200 nm. In various embodiments, the thickness of the amphiphilic lipid phase is about 3-5 nm, and the thickness of the non-polar lipid phase is about 30-200 nm.

[0111]

[0110] In various embodiments, the platform may comprise a hydrophobic substrate, whereby the nonpolar lipid phase (i.e. first layer or phase) is deposited on the hydrophobic substrate, and an amphiphilic lipid phase (i.e. second layer or phase) is deposited on the non-polar lipid phase. Collectively, this organisation may create a progressive polarity gradient from hydrophobic to hydrophilic across the platform that mimics the TFLL.

[0112]

[0111] In various embodiments, the lipid assembly is deposited on the substrate such that the relative arrangement of the lipid phases is inverted with respect to the natural orientation of the TFLL at the ocular surface. That is, the organization of the lipid strata is established in an “upside-down” arrangement, wherein the inside and outside are inverted. In other words, the inner aqueous phase of the ocular interface (FIG. 2) is the outermost aqueous environment away from the substrate, and the outermost non-polar lipid layer at the air boundary is buried close to the hydrophobic substrate of the platform (FIG. 3).

[0113]

[0112] In various embodiments, the platform recapitulates the polarity gradient characteristic of the natural TFLL in this inverted orientation. In the native TFLL, the non-polar lipids (e.g., wax esters and cholesteryl esters) form the outer air-exposed surface, while amphiphilic lipids (e.g., (O-acyl)-w-hydroxy fatty acids and phospholipids) align toward the underlying aqueous tear phase. In the in vitro “upsidedown” configuration, this arrangement is reproduced in reverse, with the non-polar lipids contacting the substrate and the amphiphilic lipids directed outward toward an aqueous medium. This inverted polarity reproduces the interfacial energy balance, stratified organisation, and surface-active properties of the native tear-film lipid layer in a cell-free format.

[0114]

[0113] Accordingly, in the “upside-down” configuration of the TFLL platform, the non-polar lipid phase forms the inner hydrophobic stratum contiguous with the substrate, while the amphiphilic lipid phase forms the outermost hydrophilic-facing stratum interfacing with an aqueous phase. This configuration preserves the functional polarity gradient of the TFLL, enabling the platform to model the spreading dynamics, surface tension regulation, and stability of the native tear film under controlled in vitro conditions.

[0115] -Stratum corneum lipid barrier of the skin

[0116]

[0114] The platform disclosed herein may be a stratum corneum (SC) platform that mimics the multilamellar lipid barrier architecture of the SC of the skin. The SC platform may reproduce the alternating amphiphilic and non-polar strata characteristic of the native SC, thereby recapitulating its polarity periodicity, barrier function, and permeability -controlling properties.

[0117]

[0115] In various embodiments, the lipid assembly may comprise a non-polar lipid phase and an amphiphilic lipid phase organised in repeating lamellar strata. That is, the non-polar lipid phase and the amphiphilic lipid phase together form an ordered, stratified arrangement analogous to the lamellae of the SC.

[0118]

[0116] In various embodiments, the amphiphilic lipid phase may comprise or consist of one or more amphiphilic lipids, more particularly at least 3 to 6 amphiphilic lipids representative of the principal SC lipid classes.

[0119]

[0117] In various embodiments, the amphiphilic lipid phase may comprise ceramides (Cer), free fatty acids (FFAs), cholesterol, and cholesterol sulfate. The ceramides may include non-hydroxy, a-hydroxy, and w-hydroxy subclasses, optionally N-acylated with saturated or monounsaturated fatty acids of Cie-C26 chain length. Selection of ceramide subclass and free-fatty-acid chain length / unsaturation modulates lamellar repeat distance, packing lattice (orthorhombic / hexagonal), transition temperatures, and permeability; a-hydroxylated ceramides with saturated FFAs enhance hydrogen-bond networks and reduce permeability, whereas unsaturated chains (e.g., oleate) fluidize the matrix, expand repeat distances, and increase permeability.

[0120]

[0118] In various embodiments, the amphiphilic lipid phase comprises interfacial amphiphilic lipids, and optionally other amphiphilic lipids. Because the native SC comprises alternating hydrophobic and amphiphilic strata, the phases of the SC platform may be, in order from the substrate: non-polar — • amphiphilic. Within each lamellar, these phases establish a repeating hydrophobic-to-hydrophilic polarity gradient that reproduces the periodic polarity of the natural SC barrier.

[0121]

[0119] In various embodiments, the lipid assembly may comprise about 30 mol % ceramides, about 20 mol % free fatty acids, and about 10 mol % cholesterol and cholesterol sulfate. The relative proportions of ceramide subclasses and FFAs may be varied to mimic the lamellar periodicity and permeability properties of the SC.

[0120] In various embodiments, the amphiphilic lipid phase may form ordered lamellar strata of about 5-15 nm in thickness, while the contiguous non-polar lipid phase may form hydrophobic layers of about 10-50 nm, consistent with the intercellular lamellar architecture of the SC (repeat spacing approximately 10-30 nm).

[0122]

[0121] In various embodiments, the amphiphilic lipid phase of the SC platform may self-organise into lamellar structures corresponding to the short-periodicity phase (SPP) and / or the long-periodicity phase (LPP) of the native stratum corneum. These two lamellar motifs differ in repeat spacing, molecular composition, and packing order, and together define the barrier’s mechanical integrity and permeability profile.

[0123]

[0122] The SPP typically exhibits a repeat distance of about 50-60 A (5-6 nm) and consists of relatively simple bilayer-like arrangements of ceramides, free fatty acids, and cholesterol. In this phase, the ceramide acyl chains are closely packed in an orthorhombic lattice, with hydrogen-bonded headgroups forming a narrow interfacial zone. The SPP is associated with shorter ceramide species and contributes to the dense, rigid component of the SC barrier. Within the platform, SPP lamellae may form spontaneously upon co-assembly of saturated ceramides with stearic or lignoceric acids and cholesterol.

[0124]

[0123] The LPP exhibits a repeat distance of about 120-130 A (12-13 nm) and displays a more complex multi-layered morphology in which ceramide molecules adopt an extended or hairpin conformation spanning multiple bilayer equivalents. The LPP is enriched in w-hydroxy ceramides esterified to long-chain fatty acids, together with cholesterol and free fatty acids that fill interstitial spaces and stabilize the lattice. The LPP contributes to long-range continuity and flexibility of the lipid barrier and dominates the water-diffusion resistance of the SC. Within the biomimetic platform, formation of the LPP may be promoted by inclusion of co-hydroxy ceramides with C30-C34 acyl chains and by controlled hydration or annealing to enable extended-chain packing.

[0125]

[0124] In various embodiments, the platform may be configured to form either the SPP, the LPP, or a mixed SPP / LPP morphology, depending on the desired permeability and mechanical characteristics. Accordingly, in certain embodiments the amphiphilic lipid phase comprises one or more lamellar strata organized as short-periodicity lamellae (~5-6 nm) and / or long-periodicity lamellae (~12-13 nm), together defining a repeating hydrophobic-hydrophilic polarity pattern analogous to that of the native stratum corneum. The relative proportion of SPP to LPP within the platform may be selected to modulate permeability, flexibility, and stability, thereby enabling precise modelling of normal, dry, or diseased skin conditions.

[0126]

[0125] In various embodiments, the lipid assembly mimicking the SC comprises or consists of repeating lamellar strata formed by alternating non-polar and amphiphilic lipid phases. Each lamella comprises a non-polar lipid phase containing cholesterol, and optionally cholesteryl lignocerate, cholesteryl nervonate, and long-chain hydrocarbons, contiguous with an amphiphilic lipid phase comprising an amphiphilic lipid mixture of ceramides, free fatty acids, cholesterol and cholesterol sulfate. The lamellae are periodically repeated along the normal axis of the film, producing a shortperiodicity lamellar repeat of about 5-6 nm and / or a long-periodicity lamellar repeat of about 12-13 nm, analogous to the multilamellar architecture of the native SC. This repeating organisation establishes a periodic hydrophobic-hydrophilic polarity gradient and reproduces the barrier continuity and mechanical cohesion characteristic of the epidermal lipid matrix.

[0127]

[0126] In various embodiments, the repeating lamellar strata are present in a number sufficient to establish the short- and / or long-periodicity spacing characteristic of the native SC. Accordingly, the lipid assembly may comprise at least two (2) lamellar repeats, preferably 3-10 repeats, more preferably 5-20 repeats, and in certain embodiments 20-50 or more repeats when increased barrier thickness or mechanical robustness is desired. By way of example, 5-20 repeats correspond to an overall lamellar thickness of approximately 25-100 nm for short-periodicity lamellae (-5-6 nm repeat) or 60-260 nm for long-periodicity lamellae (-12-13 nm repeat), which is representative for in-vitro SC barrier models while remaining compatible with supported-film and bead-coating formats.

[0128]

[0127] In various embodiments, the SC platform may further comprise an aqueous phase deposited on an amphiphilic lipid phase, simulating the intercellular aqueous domains of the SC or the hydration layer present in vivo.

[0129]

[0128] In various embodiments, the SC platform may comprise a hydrophobic substrate upon which a repeating lamellar lipid assembly is formed. The lipid assembly comprises alternating non-polar lipid phases and amphiphilic lipid phases arranged in a stratified, multilamellar architecture, whereby the non-polar lipid phase (first layer) is deposited or self-assembled on the substrate, and the amphiphilic lipid phase (second layer) is assembled on the non-polar lipid phase. This alternating sequence of nonpolar and amphiphilic strata is periodically repeated, thereby generating a repeating hydrophobic-hydrophilic polarity gradient consistent with the SPP and LPP lamellae observed in the native stratum corneum. Collectively, this organisation creates a repeating polarity gradient, thereby mimicking the lamellar architecture, periodic polarity, and barrier properties of the natural stratum corneum.

[0130]

[0129] In various embodiments, the lipid assembly is deposited or self-assembled on the substrate such that the orientation of the lipid strata is inverted relative to the natural arrangement of the stratum corneum (SC). That is, the lipid phases are organised in an “upside-down" configuration, wherein the orientation of the hydrophobic and hydrophilic domains is reversed with respect to the in vivo epidermal barrier.

[0131]

[0130] In various embodiments, the SC platform recapitulates the repeating periodic polarity gradient characteristic of the SC lipid matrix in this inverted orientation. In the native SC, alternating amphiphilic and non-polar strata are oriented such that hydrophilic headgroups of amphiphilic lipids with intercellular aqueous regions, while hydrophobic tails form interdigitated non-polar domains between adjacent lamellae. In the in vitro “upside-down” configuration, this arrangement is reproduced in reverse, with the non-polar lipid components oriented toward and in contact with the substrate, and the amphiphilic components directed outward toward the environment or aqueous phase.

[0132]

[0131] This inverted organisation preserves the periodic lamellar polarity of the native stratum corneum while allowing the polarity gradient to extend along the substrate normal in reverse orientation (hydrophobic-to-hydrophilic from substrate to aqueous phase).

[0133]

[0132] In various embodiments, the “brick and mortar” structure of the stratum corneum may be mimicked by reconstituting the lipid lamellae with cells isolated from human or animal skins or by coassembling the lipid layers with synthetic cells. Thus, in various embodiments, rather than being a cell-free platform, the platform and lipid assembly may comprise cells isolated from human or animal skins, or synthetic cells.

[0134]

[0133] Such cells may include keratinocytes, corneocytes, melanocytes, Langerhans cells, or fibroblasts. Keratinocytes may be primary or immortalized cells capable of differentiating into corneocytes, which upon terminal differentiation form flattened, anucleate cell remnants surrounded by cross-linked protein envelopes. Corneocytes may be isolated from human or porcine epidermis and reconstituted within the lipid lamellae, thereby recapitulating the natural “brick-and-mortar” barrier. Fibroblasts may be included beneath the lipid assembly to form a dermal equivalent or supportive layer. Melanocytes or Langerhans cells may be optionally co-cultured to reproduce pigmentation or immune-related features of the epidermis. The synthetic or engineered cells may be designed to structurally or functionally emulate skin cells. Such synthetic cells may include giant unilamellar vesicles (GUVs), liposomes, polymersomes, or hybrid lipid-polymer vesicles. These vesicular structures may be embedded within or beneath the lipid lamellae, forming deformable “bricks” dispersed in the lipid “mortar.” Alternatively, the synthetic cells may comprise hydrogel microcapsules or protocells coated with one or more lipid monolayers or bilayers, optionally containing biological or synthetic cargo, to provide structural rigidity or biosensing capability.

[0135] -Alveolar surfactant layer of the lung

[0136]

[0134] The platform disclosed herein may be an alveolar surfactant (AS) platform that mimics the monolayer organisation of the pulmonary surfactant layer lining the alveoli of the lung. The platform may reproduce the unidirectional polarity gradient and surface-tension-reducing behaviour of the native alveolar surfactant film, thereby recapitulating its structural, interfacial, and mechanical functions in maintaining alveolar stability and pulmonary compliance.

[0137]

[0135] In various embodiments, the lipid assembly may comprise an amphiphilic lipid phase forming a monomolecular layer (i.e. a monolayer), supported by, or contiguous with, a non-polar lipid phase. That is, the amphiphilic lipid phase and non-polar lipid phase together establish an ordered, unidirectional stratification analogous to the air-liquid interfacial organisation of the native pulmonary surfactant.

[0138]

[0136] In various embodiments, the lipid assembly comprises amphiphilic lipids, optionally together with one or more surfactant-associated proteins (Surfactant Protein A (SP-A), Surfactant Protein B (SP-B), Surfactant Protein C (SP-C), or Surfactant Protein D (SP-D)) to form a lipid-protein assembly. In various embodiments, the lipid assembly comprises one or more surfactant-associated proteins selected from SP-A, SP-B, SP-C, or SP-D.

[0139]

[0137] In the native pulmonary surfactant, the amphiphilic lipids orient with their hydrophilic headgroups facing the aqueous alveolar subphase and hydrophobic acyl chains directed toward the air phase, establishing a unidirectional polarity gradient normal to the interface. In the AS platform, the lipid assembly comprises a monomolecular layer of amphiphilic lipids containing minor proportions of nonpolar lipids that may modulate fluidity and packing. The amphiphilic lipids are organized as a unidirectional monolayer in which the hydrophobic acyl chains of the amphiphilic lipids are oriented toward the underlying hydrophobic substrate or lipid phase, while the polar headgroups face the overlying aqueous medium. This arrangement establishes a single, continuous hydrophobic-to-hydrophilic polarity gradient normal to the surface, faithfully reproducing the air-liquid interfacial polarity and surface-tension-reducing activity characteristic of the native pulmonary surfactant film.

[0140]

[0138] In various embodiments, the surfactant-associated protein fraction may comprise about 5 mol % to about 20 mol % of the total lipid-protein assembly, preferably about 10-15 mol %. That is, the surfactant-associated protein fraction constitutes about 5-20 mol% of the total lipid assembly.

[0141]

[0139] In various embodiments, the amphiphilic lipid phase may comprise or consist of one or more amphiphilic lipids.

[0142]

[0140] In various embodiments, the amphiphilic lipid phase may comprise phosphatidylcholines (PC) such as dipalmitoylphosphatidylcholine (DPPC) as the dominant constituent, optionally together with phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). DPPC may constitute about 40-60 mol % of the total lipid assembly, providing the principal surface-active component responsible for lowering surface tension during the respiratory cycle.

[0143]

[0141] In various embodiments, the amphiphilic lipid phase may further comprise minor fractions of unsaturated phospholipids, such as 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine (POPE), and cholesterol, which modulate fluidity, respreading, and phase coexistence within the monolayer. The amphiphilic lipid phase may also comprise sphingomyelin (SM) and phosphatidylinositol (PI) in trace amounts, contributing to the interfacial stability and charge distribution.

[0142] In various embodiments, the lipid assembly mimicking the alveolar surfactant comprises or consists of a reservoir lipid phase, contiguous with an amphiphilic lipid phase one or more other amphiphilic lipids, including saturated and unsaturated phospholipids, such as dipalmitoylphosphatidylcholine (DPPC). This arrangement establishes a unidirectional hydrophobic-to-hydrophilic polarity gradient, analogous to that of the native pulmonary surfactant at the alveolar airliquid interface.

[0144]

[0143] In various embodiments, the amphiphilic lipid phase may comprise about 80-95 mol % of the total lipid assembly, preferably about 85-90 mol %, with a representative composition of the lipid assembly including about 50-60 mol % DPPC, about 10-15 mol % PG, about 5-10 mol % PE, about 5 mol % PI, and about 5-10 mol % cholesterol. In various embodiments, the lipid assembly comprises or consists of about 10 mol % cholesterol, about 5 mol % cholesteryl esters, about 50 mol % DPPC, about 15 mol % PG, about 10 mol % PE, and about 5 mol % PI, of the total lipid assembly. The relative proportions of these components may be varied to tune film compressibility, collapse pressure, and surface tension to replicate physiological or pathological surfactant states. Variation in phospholipid acyl-chain saturation, cholesterol content, and associated surfactant proteins modulates monolayer packing density, collapse pressure, and re-spreading dynamics, enabling reversible reduction of surface tension during the respiratory cycle to maintain alveolar stability and pulmonary compliance.

[0145]

[0144] In various embodiments, the non-polar lipid phase may form a layer of about 5-20 nm in thickness, contiguous with the overlying amphiphilic lipid monolayer, and functioning as a lipid reservoir that regulates the phase transitions, spreading, and adsorption kinetics of the surfactant film. In various embodiments, the amphiphilic lipid monolayer may form a thin surface-active layer of about 2-5 nm, with or without integration with surfactant-associated proteins. The combined lipid-protein assembly may thus define a total structural thickness of approximately 10-25 nm, consistent with the natural alveolar surfactant film observed in vivo.

[0146]

[0145] In various embodiments, the AS platform may further comprise an aqueous subphase underlying the non-polar lipid phase, representing the alveolar lining fluid. The aqueous layer may have a thickness of about 2-50 pm, preferably 5-20 pm, approximating the physiological alveolar lining depth while ensuring sufficient hydration for interfacial stability and surface activity.

[0147]

[0146] In various embodiments, the AS platform may comprise a hydrophobic substrate, upon which the unsaturated lipid phase (first layer) is deposited or self-assembled, followed by formation of the amphiphilic saturated phospholipid phase, namely DPPC, with or without surfactant-associated proteins (second layer) on the unsaturated lipid phase. Collectively, this organisation creates a unidirectional polarity gradient extending from hydrophobic to hydrophilic regions along the substrate normal, thereby mimicking the interfacial architecture, polarity distribution, and dynamic surface-active behavior of the native alveolar surfactant. The resulting structure mimics the interfacial layer of the saturated lipid in contiguity with the buried region consisting of more unsaturated lipids (e.g., DOPC). The resulting structure is distinct from the multilamellar lipid matrices of epidermal stratum corneum barriers and the stratified polarity gradient of the TFLL.

[0148]

[0147] In various embodiments, the lipid assembly may be formed in an “upside-down” arrangement relative to its natural orientation at the air-liquid interface, such that the non-polar lipid phase contacts the substrate and the amphiphilic phospholipid phase is directed outward toward the aqueous phase. This inverted configuration reproduces the functional polarity, adsorption kinetics, and surface-tension-modulating properties of the native pulmonary surfactant film in a stable, cell-free in vitro format.

[0149] - Platforms mimicking conditions or disease states

[0150]

[0148] It is also contemplated that the platform disclosed herein may be tailored to mimic specific diseased states or ageing stages of the biological air-interfaces by systematically varying the composition, abundance, thickness or organisation of lipids within each stratum of the lipid assembly. For example, in a model of the tear-film lipid layer (TFLL), altered ratios of cholesteryl esters to wax esters, or reductions in (O-acyl)-w-hydroxy fatty acids, may be introduced to recapitulate lipid deficiencies observed in conditions such as dry eye disease or meibomian gland dysfunction. Similarly, the stratum corneum lipid barrier of the skin may be modified by reducing ceramide subclasses or cholesterol content to reflect the barrier impairment seen in atopic dermatitis or psoriasis. In the alveolar surfactant model, perturbations such as a decreased dipalmitoylphosphatidylcholine (DPPC) fraction or altered surfactant protein content may be incorporated to reproduce the dysfunctional surfactant composition observed in acute respiratory distress syndrome (ARDS) or chronic obstructive pulmonary disease (COPD). These variations may enable the platform to serve as a versatile in vitro model for assaying the effects of candidate substances under pathological conditions.

[0151]

[0149] In various embodiments, the platform may be adapted to reflect lipid compositional changes associated with ageing. For instance, age-related alterations in tear film composition, such as reduced wax ester chain length or increased lipid peroxidation, may be introduced into the TFLL mimic. The skin platform may be adjusted to reflect the decline in ceramide subclasses and increase in free fatty acid heterogeneity that occurs with chronological ageing, resulting in diminished barrier function and increased susceptibility to irritants. The lung platform may be modified to reflect decreased phospholipid turnover or accumulation of oxidised lipids associated with ageing-related pulmonary decline. By incorporating these disease- and age-specific lipid alterations, the platform allows systematic assessment of how candidate substances interact with compromised or altered lipid interfaces, as well as the functional contribution of individual lipid constituents that are altered in abundance during disease progression or ageing.

[0152]

[0150] Thus, in various embodiments, the platform, and more particularly the lipid assembly and lamellar, stratified, or monolayer organisation of the biological air-interface, may be designed to mimic a disease or condition associated with the biological air-interface being mimicked. -Method of Manufacture

[0153]

[0151] It is also contemplated that there are provided methods of manufacturing the platform disclosed herein. All embodiments disclosed above in relation to the platform disclosed herein, similarly apply to the method of its manufacture, and vice versa.

[0154]

[0152] Accordingly, in another aspect, there is provided a method of manufacturing the platform disclosed herein, comprising the steps of:

[0155] providing a substrate; and

[0156] depositing a lipid assembly disclosed herein on the solid substrate, wherein the lipid assembly mimics the lamellar, stratified, or monolayer organisation, and optionally polarity gradient, of a biological air-interface.

[0157]

[0153] In various embodiments, the substrate provided may be modified to be either hydrophobic or hydrophilic such that its surface energy biases lipid orientation. For example, a hydrophobic silane / SAM may be used for non-polar lipid phase first contact, or a hydrophilic oxide may be used for vesicle fusion.

[0158]

[0154] In various embodiments, lipids may be provided and formulated into compositions suitable for deposition. In various embodiments, the lipid assembly may be prepared by providing one or more lipid compositions comprising lipid classes selected from non-polar lipids, interfacial amphiphilic lipids, and / or amphiphilic lipids, in a solvent, dispersion, micelle, or vesicle form suitable for deposition and self-assembly.

[0159]

[0155] In various embodiments, the lipids used in the method of manufacture may be provided in any suitable form that enables deposition onto the substrate or underlying lipid phase and assembly into the lipid assembly.

[0160]

[0156] In various embodiments, non-polar lipids may be dissolved in organic solvents (e.g., hexane, isooctane, chloroform, chloroform:methanol 2:1 v / v) to provide a non-polar lipid composition for depositing, followed by controlled solvent evaporation. For example, non-polar lipids such as wax esters or cholesteryl esters may be dissolved in an organic solvent and applied by spreading, drop-casting, incubation, or immersion, followed by drying to form a uniform lipid layer.

[0161]

[0157] In various embodiments, the interfacial amphiphilic lipids may be provided in organic solutions or mixed solvent systems (e.g., chloroform:methanol) or as fine dispersions / emulsions to promote insertion at interfaces. Interfacial amphiphilic lipids, including species such as (O-acyl)-w-hydroxy fatty acids, may be provided either in an organic solvent solution or in an aqueous suspension or emulsion formulated to promote their incorporation between the polar amphiphilic and non-polar strata.

[0162]

[0158] In various embodiments, the amphiphilic lipids may be provided as vesicle (SUV / LUV) suspensions, micelles, or lipid solutions for Langmuir transfer: vesicles may adsorb / fuse to yield continuous monolayers / bilayers depending on platform. Solvent and dispersion choice is selected per lipid solubility and target architecture. Amphiphilic lipids, such as glycerophospholipids, lysophospholipids, or sphingomyelin, may be provided in the form of aqueous dispersions, micelles, or vesicle suspensions that can adsorb or fuse onto the interfacial lipid stratum.

[0163]

[0159] In various embodiments, mixed lipid preparations or compositions containing two or more lipid classes may be employed to enable cooperative or hierarchical self-assembly into ordered lamellar phases. The choice of solvent, suspension, or dispersion medium may be selected according to lipid solubility and the desired method of deposition.

[0164]

[0160] In various embodiments, the depositing step of each lipid class may be achieved by any suitable method that results in the association of the lipid molecules with the substrate surface or respective lipid phases, including but not limited to spreading, coating, drop-casting, incubating, immersing, contacting with a lipid solution, contacting with a lipid vesicle suspension, drying, rinsing, or selfassembly under aqueous conditions. In this regard, depositing encompasses both sequential application of discrete lipid fractions (e.g., applying a non-polar lipid phase, followed by an interfacial lipid phase, followed by a polar amphiphilic lipid phase) and cooperative or hierarchical self-assembly in which stratification occurs spontaneously when lipid mixtures are contacted with the substrate.

[0165]

[0161] In various embodiments, the lipid phases may be deposited and formed by sequential selfassembly of non-polar, and / or amphiphilic lipids. In various embodiments, amphiphilic lipids and interfacial amphiphilic lipids may be combined in a mixed vesicle suspension and deposited onto the underlying lipid phase or substrate.

[0166]

[0162] In various embodiments, the lipid phases may be deposited and formed by one-step cooperative or hierarchical self-assembly of non-polar, and / or amphiphilic lipids.

[0167]

[0163] In various embodiments, co-deposition may be employed, whereby the interfacial amphiphilic lipids may be mixed with the amphiphilic lipids and / or non-polar lipids in a single preparation or composition and deposited to drive cooperative or hierarchical self-assembly into ordered lamellae.

[0168]

[0164] In various embodiments, the amounts of lipid compositions deposited onto the substrate may be selected to achieve uniform coverage and reproducible stratification while avoiding excess aggregation or phase separation. The lipid mixtures may be prepared at concentrations ranging from about 0.01 mg / mL to about 10 mg / mL, depending on lipid type and solvent system, and may be applied in volumes ranging from about 1 pL to about 100 pL per cm2of substrate surface area. For bead-based substrates, lipid solutions or suspensions may be added in volumes sufficient to provide a thin coating layer. For slide-based substrates, defined droplets or spreads of lipid mixtures, such as about 5-50 pL, may be deposited per slide or per patterned corral area. Amphiphilic lipids are often provided in the form of vesicle suspensions, which may be prepared at concentrations of about 0.1-10 mM total lipid, and deposited in volumes of about 5-50 pL with contact times ranging from seconds to minutes to facilitate adsorption or fusion. Interfacial amphiphilic lipids may be combined with non-polar lipids, with the relative molar ratios adjusted to reflect the desired model system, for example ranging from 1 mol% to 20 mol% of the total lipid composition. After deposition, excess or unbound lipid may be removed by washing with aqueous buffer or high-resistivity water to ensure that only the structured lipid strata remain associated with the substrate surface.

[0169]

[0165] In various embodiments, the step of depositing the lipid assembly may comprise depositing the lipid classes, or lipid compositions, onto the substrate to form a target architecture selected from: (i) a stratified lamellar assembly comprising a non-polar lipid phase contiguous with one or more amphiphilic phases, representative of the tear-film lipid layer (TFLL); (ii) a periodically repeating lamellar assembly comprising alternating non-polar and amphiphilic phases, representative of the stratum corneum (SC); or (iii) an amphiphilic lipid monolayer containing minor non-polar lipid constituents, optionally with surfactant-associated proteins, that self-assembles into a unidirectional monolayer representative of the alveolar surfactant (AS) film.

[0170]

[0166] In embodiments where the TFLL platform disclosed herein is to be prepared, the method may comprise depositing a non-polar lipid composition, and an amphiphilic lipid composition on the substrate to form the lipid assembly. In various embodiments, the method may comprise depositing, on the substrate, a non-polar lipid composition, an interfacial amphiphilic lipid composition, and an amphiphilic lipid composition, to form a stratified lamellar assembly. In various embodiments, the method comprises depositing either a non-polar lipid composition the substrate to form a first phase, and subsequently depositing an interfacial amphiphilic lipid composition on the first phase to form a second phase, and then depositing an amphiphilic lipid composition on the second phase to form a third phase. In this regard, the deposition steps result in the formation of a lipid stratum and the desired arrangement of the first phase and the third phase within the platform, wherein one stratum is enriched in non-polar lipids and the other stratum is enriched in amphiphilic lipids. These strata exhibit contrasting molecular orientations, such that one presents a predominantly hydrophobic environment and the other presents a predominantly hydrophilic environment, thereby establishing the polarity gradient that characterises the lamellar stratified organisation of the platform. In various embodiments, the amphiphilic lipids and interfacial amphiphilic lipids may be combined in a mixed composition and deposited simultaneously onto the underlying lipid layer or substrate for co-deposition.

[0171]

[0167] In embodiments where the TFLL platform disclosed herein is to be prepared, the substrate may comprise hydrophobically coated glass, and the method may comprise spreading a non-polar lipid composition onto a hydrophobically coated surface of the substrate, for example an octadecyltrichlorosilane (OTS)-treated glass surface, to form a first layer. Subsequently, the substrate coated with the first layer may be inverted onto a drop of an amphiphilic lipid vesicle suspension, also comprising the interfacial amphiphilic lipids and amphiphilic lipids, allowing incubation for a defined contact time, to facilitate deposition of the amphiphilic lipids as a second and third layer. In this regard, the interfacial amphiphilic lipids and amphiphilic lipids are deposited together. After incubation, the substrate may be rinsed in water, to remove unbound or loosely associated lipid, thereby forming a stratified lipid construct on the glass substrate that mimics the lamellar organisation of a biological airinterface.

[0172]

[0168] In embodiments where the TFLL platform disclosed herein is to be prepared, the solid substrate may comprise TMS beads, and the method may comprise incubating the TMS beads with a solution or suspension of non-polar lipids in an organic solvent, followed by evaporation of the solvent and drying under nitrogen or vacuum to form beads coated with the first layer. Subsequently, the beads coated with the first layer may be contacted with a solution or suspension of interfacial amphiphilic lipids and amphiphilic lipids to form a second and third layers respectively, followed by washing to remove unbound lipids, thereby forming a layered lipid construct on the bead surface (FIG. 3). In various embodiments, the beads coated with the first layer may be contacted with a solution or suspension of interfacial amphiphilic lipids and amphiphilic lipids to form the second and third layers.

[0173]

[0169] In embodiments where the SC platform disclosed herein is to be prepared, the method may comprise depositing, on the substrate, a non-polar lipid composition and an amphiphilic lipid composition organised in repeating lamellar strata to form a multilamellar lipid assembly. In various embodiments, the method may comprise depositing a non-polar lipid composition on the substrate to form a first phase, followed by deposition of an amphiphilic lipid composition comprising interfacial amphiphilic lipids, to form a second phase contiguous therewith. These deposition steps may be repeated to generate alternating non-polar and amphiphilic strata along the substrate normal, thereby reproducing the periodic polarity gradient characteristic of the native stratum corneum. This alternating organisation establishes a repeating hydrophobic-to-hydrophilic polarity gradient that underlies the barrier and permeability-controlling properties of the SC platform.

[0174]

[0170] In various embodiments, the SC lipid assembly may be co-assembled or in contact with cells isolated from human or animal skin. The cells may be cultured, fixed, or desiccated, and may be embedded within or beneath the lipid lamellae such that the overall assembly mimics the “brick-and-mortar” structure of the stratum corneum. In other embodiments, the platform comprises synthetic or artificial cells, including lipid vesicles, polymersomes, hydrogel microcapsules, or minimal cells comprising lipid membranes encapsulating cell-free expression systems. The synthetic cells may be coated, embedded, or layered within the lipid assembly, optionally at an air-liquid interface, thereby providing a structurally and functionally representative model of the stratum corneum barrier.

[0175]

[0171] Cells may be incorporated into the SC lipid assembly by deposition onto a hydrophobic or amphiphilic substrate prior to lipid assembly formation, by embedding within successive lipid strata, or by coating individual cells with amphiphilic or interfacial lipids. In some embodiments, keratinocytes or synthetic vesicular cells are positioned on the substrate and overlaid with lamellar lipid films comprising ceramides, cholesterol, and free fatty acids, thereby forming a multilayered composite that reproduces the natural organization of the epidermal barrier. In other embodiments, corneocytes or vesicular cells are dispersed among pre-formed lipid lamellae to create a three-dimensional, cell-lipid composite.

[0176]

[0172] In embodiments where the SC platform disclosed herein is to be prepared, the substrate may comprise hydrophobically coated glass, and the method may comprise spreading a non-polar lipid composition onto a hydrophobically coated surface of the substrate, for example an octadecyltrichlorosilane (OTS)-treated glass surface, to form a first layer representing the non-polar lipid phase. Subsequently, the substrate coated with the first layer may be contacted or inverted onto a dispersion or vesicle suspension containing amphiphilic lipids, allowing incubation for a defined contact time to facilitate deposition of the amphiphilic lipids as a second lamellar stratum. These deposition steps may be repeated sequentially to generate multiple alternating non-polar and amphiphilic strata, thereby forming a multilamellar lipid construct that mimics the repeating lamellar organisation and polarity gradient of the stratum corneum. After incubation, the substrate may be rinsed with water or aqueous buffer to remove unbound or loosely associated lipid, yielding a structurally ordered lamellar lipid assembly.

[0177]

[0173] In embodiments where the SC platform disclosed herein is to be prepared, the solid substrate may comprise trimethylsilyl (TMS)-functionalised silica beads, and the method may comprise incubating the beads with a solution or suspension of non-polar lipids in an organic solvent, followed by solvent evaporation and drying under nitrogen or vacuum to form beads coated with the first non-polar layer. Subsequently, the beads coated with the first layer may be contacted with an aqueous or organic dispersion containing amphiphilic lipids, to form an amphiphilic stratum contiguous with the underlying non-polar layer. Optionally, sequential repetitions of non-polar and amphiphilic lipid deposition may be performed to establish a repeating lamellar structure on the bead surface. After incubation, the beads may be washed with aqueous buffer to remove unbound or loosely associated lipid, thereby forming a multilamellar lipid construct on the bead surface that recapitulates the lamellar architecture and barrier properties of the native stratum corneum.

[0178]

[0174] In embodiments where the AS platform disclosed herein is to be prepared, the method may comprise depositing a non-polar lipid composition and an amphiphilic lipid composition on the substrate to form a unidirectional monolayer lipid assembly. In various embodiments, the method may comprise depositing, on the substrate, a non-polar lipid composition to form a first phase functioning as a hydrophobic sublayer or lipid reservoir, and subsequently depositing an amphiphilic lipid composition to form a second phase as a continuous monomolecular layer. In various embodiments, the non-polar and amphiphilic lipid compositions may be co-deposited or spread sequentially by solvent-casting, vesicle fusion, or Langmuir-Blodgett transfer to promote uniform monolayer formation and surface-tension-reducing behaviour.

[0179]

[0175] In embodiments where the AS platform disclosed herein is to be prepared, the substrate may comprise hydrophobically coated glass, and the method may comprise spreading a non-polar lipid composition onto the hydrophobic surface of the substrate, for example an OTS-treated glass slide, to form a first lipid sublayer. Subsequently, the substrate coated with the first layer may be contacted with a suspension or vesicle dispersion containing amphiphilic lipids, and optionally minor surfactant-associated proteins. Incubation for a defined contact time facilitates the adsorption and spreading of the amphiphilic lipids into a monomolecular amphiphilic layer over the non-polar subphase. After incubation, the substrate may be gently rinsed to remove excess lipid, thereby forming a unidirectional monolayer assembly that reproduces the air-liquid interfacial polarity and surface-tension-reducing properties of the alveolar surfactant film.

[0180]

[0176] In embodiments where the AS platform disclosed herein is to be prepared, the solid substrate may comprise TMS-functionalised silica beads, and the method may comprise incubating the beads with a solution of non-polar lipids in an organic solvent, followed by drying under nitrogen or vacuum to produce beads coated with the first hydrophobic layer. The coated beads may then be contacted with a vesicle suspension of amphiphilic lipids, optionally containing minor surfactant proteins, to form a continuous amphiphilic lipid monolayer over the non-polar surface. Following incubation, the beads may be rinsed in water or buffer to remove unbound lipid, producing a monolayer-coated construct that models the structural polarity and dynamic surface activity of the native alveolar surfactant at the airliquid interface.

[0181]

[0177] In various embodiments, there is provided a platform produced by the method of manufacture disclosed herein.

[0182]

[0178] In various embodiments, the method may further comprise a step of analysing the lipid assembly, at one or more steps and stages of manufacture, to verify the organisation of the lipid layers. The analysis may comprise obtaining one or more structural, chemical, or functional readouts indicative of (i) the presence of the lipid phases and (ii) their positional order on the substrate.

[0183]

[0179] The analysis may include, without limitation, incorporation of lipid class-specific fluorescent probes into the lipid fractions and acquisition of fluorescence images, such as widefield or confocal micrographs, to confirm the presence and distribution of each stratum. For example, a non-polar lipid layer may be verified using BODIPY 493 / 503, an interfacial amphiphilic lipid layer may be confirmed using a labelled (O-acyl)-w-hydroxy fatty acid analogue, and an amphiphilic lipid layer may be imaged using Rhodamine-PE or NBD-PC. Optical metrology techniques such as spectroscopic ellipsometry or interferometric reflectance may be performed to measure incremental thickness after each deposition, with values consistent with a thin amphiphilic layer, an interfacial layer, and a thicker non-polar layer. In further embodiments, surface property changes such as contact angle or surface tension may be recorded sequentially to evidence the establishment of a polarity gradient across the assembly. Chemical mapping methods such as time-of-flight secondary ion mass spectrometry (ToF-SIMS), angle-resolved X-ray photoelectron spectroscopy (XPS), or ATR-FTIR may be applied after each step to verify enrichment of lipid-specific fragments or headgroups. On transducer substrates, quartz crystal microbalance with dissipation monitoring (QCM-D) or surface plasmon resonance (SPR) may be employed to track sequential mass and viscoelastic changes. Together, these analyses enable validation that each deposited lipid stratum is present, properly ordered, and collectively mimics the lamellar stratified organisation of the biological air-interface.

[0184]

[0180] In various embodiments, the analysis step may be carried out after each lipid class or phase is deposited, in order to validate the organisation and arrangement of the lipid assembly.

[0185] -Methods of Use

[0186]

[0181] As will be appreciated, the platform disclosed herein enables in vitro modelling of the biological air-interface, and thus may be used to assess interactions with exogenous agents such as irritants or allergens.

[0187]

[0182] Thus, it is also contemplated that there are provided methods of using the platform disclosed herein for in vitro assays. All embodiments disclosed above in relation to the platform, and method of manufacture, disclosed herein, similarly apply to the methods of use, and vice versa. The methods of use disclosed herein may be defined as in vitro methods.

[0188]

[0183] The platform disclosed herein may be used for assaying interactions between a candidate substance and a biological air-interface. As used herein, “assaying” encompasses detecting, measuring, screening, evaluating, or otherwise characterising the interaction of the candidate substance at a biological air-interface.

[0189]

[0184] In this context, the “interaction” to be assayed or evaluated may refer to any physical, chemical, or biological effect that a candidate substance exerts upon the lipid assembly. Interactions may include, without limitation, physicochemical changes such as alteration of surface tension, polarity gradients, wettability, or lipid layer organization; structural disruption of the lamellar stratified organisation, including destabilisation, thinning, or phase separation of the lipid layers; biophysical responses such as altered permeability, barrier integrity, or hydration properties of the lipid construct; and functional outcomes relevant to toxicology and safety testing, such as detection of lipid layer collapse, increased surface instability, or markers of irritation.

[0190]

[0185] In various embodiments, the candidate substance may refer to any compound, mixture, formulation, or agent whose interaction with a biological air-interface is to be tested or evaluated using the disclosed platform.

[0191]

[0186] In various embodiments, the candidate substance may include, without limitation, exogenous substances such as environmental irritants, airborne particulates, pollutants, allergens, and microbial products; pharmaceutical or ophthalmic agents including drug formulations, excipients, preservatives, and delivery vehicles intended for topical or systemic administration; cosmetic and personal care products such as creams, lotions, cleansers, surfactants, and emulsifiers applied to the eye, skin, or respiratory surfaces; industrial and household chemicals including solvents, detergents, and volatile organic compounds; and novel test compounds under development for therapeutic, cosmetic, or diagnostic applications where safety, tolerance, or compatibility with epithelial barrier layers is to be assessed. In various embodiments, the candidate substance may be a known irritant.

[0192]

[0187] In various embodiments, where the platform mimics the tear-film lipid layer (TFLL) of the eye, candidate substances may include ophthalmic formulations, preservatives, and environmental irritants. Interactions may be assayed in terms of tear film stability, surface tension, and lipid layer integrity, thereby supporting toxicology assessment, irritancy screening, and validation of the suitability of substances for human use.

[0193]

[0188] Accordingly, it is also contemplated that the platform may be used as a biosensor device or as part of a biosensor device. The biosensor device may be used as a detection system configured to assay interactions between a candidate substance and the biological air-interface.

[0194]

[0189] In various embodiments, the platform comprised in the biosensor device may be composed of a transducer surface acting as the solid support with the lipid assembly deposited thereon. The device may include an optical, electrical, acoustic, or mechanical transducer operably coupled to the platform to detect changes in physical properties such as fluorescence, surface tension, refractive index, conductivity, capacitance, or mass loading.

[0195]

[0190] In various embodiments, the biosensor device may include, without limitation, optical biosensors, in which the platform is integrated with a fluorescence microscope, confocal system, or waveguide sensor and interactions are measured by fluorescence micrographs, spectral shifts, or optical interference patterns; electrochemical biosensors, in which the lipid assembly are deposited on a conductive or electrode-modified substrate such as gold or indium tin oxide and changes in capacitance, impedance, or current are detected following candidate substance exposure; acoustic biosensors, in which the platform is deposited on a quartz crystal microbalance (QCM) or surface acoustic wave (SAW) device and interaction events are measured as changes in frequency or dissipation; and mechanical biosensors, in which the platform is integrated into a cantilever or microfluidic channel and changes in lipid properties are detected as alterations in mechanical deflection, flow, or pressure.

[0196]

[0191] In various embodiments, the biosensor device may comprise an array of sensing elements, each incorporating the platform disclosed herein. Candidate substances may be assayed across the array simultaneously, enabling multiplexed detection of toxicity, irritancy, or lipid-specific effects.

[0197]

[0192] In various embodiments, the biosensor device comprising the platform may be configured such that the platform is divided into individual sensing elements (for example, micropatterned corrals on a substrate, discrete wells, or lipid-coated microspheres), each of which serves as a discretised measurement site. Each element may function independently as a biosensor, providing a single readout of the interaction between a candidate substance and the lipid assembly. By integrating multiple such elements into a common device, the biosensor may thereby operate as an array of biosensors, in which many discrete measurements can be obtained in parallel. This configuration enables parallel or massively parallel measurements, allowing high-throughput detection of irritancy, toxicity, or modulatory effects across a large number of sensing sites.

[0198]

[0193] Accordingly, in various embodiments, there is provided a biosensor array comprising a plurality of platforms disclosed herein, wherein the platforms are discrete from each other.

[0199]

[0194] In various embodiments, the platform may be used in an assay or any experimental, analytical, or diagnostic procedure in which the disclosed platform is used to detect, measure, or evaluate changes in the lipid assembly in response to a candidate substance. Assays may be qualitative or quantitative and may employ optical, spectroscopic, imaging, or biophysical readouts to monitor alterations in the structural, chemical, or functional properties of the lipid construct. Such assays may include, without limitation, surface tension measurements, fluorescence or microscopy-based visualisation of lipid organisation, permeability testing, stability assessments, or functional evaluations of barrier properties. In this context, the platform serves as a reproducible in vitro model of the biological air- interface, enabling direct measurement of lipid assembly responses to exogenous or applied substances.

[0200]

[0195] In various embodiments, the platform may be divided into a plurality of discrete measurement regions (e.g., corrals, compartments, wells, or bead surfaces) comprising a lipid assembly deposited thereon. In various embodiments, the number of discrete regions may exceed the number of effective interaction events produced by the candidate substance, such that statistical analysis of positive versus negative responses across the ensemble provides a digital readout of irritancy or toxicity. This digital format enables highly sensitive detection of rare or low-abundance interaction events and permits quantification of the probability of disruption, permeability change, or morphological alteration per region.

[0201]

[0196] In various embodiments, the platform may comprise a micropatterned substrate or a collection of microspherical beads, each coated with the lipid assembly. A candidate substance may be contacted with the platform at a concentration lower than the total number of measurement regions, such that not all regions are affected. The fraction of regions showing a measurable effect (e.g., lipid collapse, fluorescence loss, permeability increase) relative to the total number of regions is then analysed using Poissonian statistics to determine the probability distribution of interaction events. For example, the presence of lipid disruption in 20% of 1 ,000 discrete corrals may correspond to a defined interaction probability that reflects the irritancy level of the candidate substance.

[0197] In various embodiments, the platform may be used for digital assays configured for toxicology assessment by classifying single-region responses into severity bins (e.g., mild, moderate, severe disruption) and aggregating the results across the platform. In various embodiments, this approach may be extended to multiplexed formats, wherein corrals or beads coated with different lipid assemblies are mixed together, allowing simultaneous digital assaying of candidate substance effects across multiple biological interfaces.

[0202]

[0198] In this regard, it is also contemplated that there is provided a method of assaying interactions between a candidate substance and the platform disclosed herein. This method may thus allow for predicting the effects, such as toxicity or irritancy, of the candidate substance to the biological airinterface that the platform is designed to mimic.

[0203]

[0199] In various embodiments, the platform may be used to assay interactions between a candidate substance and the lipid assembly by measuring one or more structural, physicochemical, or functional properties of the construct, which may be termed as biophysical properties.

[0204]

[0200] In various embodiments, the biophysical properties may include, without limitation, morphology (e.g., lamellar spacing, thickness, and surface topography), uniformity and contiguity of the lipid strata (e.g., continuous versus patchy coverage of the substrate), fluidity and dynamics (e.g., lipid chain mobility, lateral diffusion, or microviscosity), and integrity (e.g., barrier stability, resistance to collapse, or phase separation). Additional measurable parameters may include surface tension, wettability, polarity gradients, hydration state, and permeability. These biophysical properties may be evaluated using quantitative and qualitative optical readouts, including but not limited to fluorescence microscopy, confocal imaging, fluorescence recovery after photobleaching (FRAP), anisotropy measurements, and interferometry, as well as other spectroscopic or imaging-based methods. Changes in these properties provide an indication of the extent and nature of the interaction between the candidate substance and the lipid assembly, thereby enabling assessment of the potential effects of the substance on the biological air-interface being mimicked.

[0205]

[0201] In this regard, the biophysical properties measured in the platform act as proxies for the corresponding properties of the native biological air-interface. Accordingly, properties such as lipid layer uniformity, continuity, fluidity, wettability, and surface tension, when measured in the platform, correspond to the same classes of properties that govern barrier integrity, hydration control, and surface stability in vivo. Thus, changes observed in the platform in response to a candidate substance can be directly interpreted as indicative of how the candidate substance would interact with, or perturb, the corresponding biological air-interface. In this way, the platform provides a practical and reproducible surrogate system in which biophysical measurements on the model are used as proxy indicators of biological function.

[0202] Accordingly, in various embodiments, there is provided a method for screening or assaying a candidate substance, comprising the steps of:

[0206] contacting the candidate substance to the platform disclosed herein; and

[0207] measuring one or more biophysical properties of the platform to obtain one or more biophysical properties values, wherein the measured one or more biophysical values of the platform are indicative of the interaction between the candidate substance and the platform.

[0208]

[0203] In various embodiments, the one or more biophysical properties comprise uniformity and contiguity, fluidity and dynamics of the lipid assembly.

[0209]

[0204] The contacting step may refer to any step of exposing the platform or lipid assembly to a candidate substance under conditions that allow physical, chemical, or biological interaction between the substance and the lipid strata. Contacting may be carried out in any manner suitable to enable interaction at the air-interface of the platform and may be continuous or transient, static or dynamic. In various embodiments, the contacting step may comprise incubating the platform with the candidate substance in solution or suspension for a predetermined period, while in other embodiments, contacting may involve deposition, immersion, or exposure to the candidate substance in liquid, aerosol, or vapor form. The mode of contacting may be selected according to the physical properties of the candidate substance and the biological air-interface being mimicked.

[0210]

[0205] In various embodiments, the measuring step may comprise obtaining quantitative and qualitative optical readouts of the one or more biophysical properties.

[0211]

[0206] In various embodiments, the measuring step may comprise obtaining quantitative and / or qualitative fluorescence readouts of the one or more biophysical properties of the lipid assembly. Fluorescence readouts, as used herein, encompass the collection and analysis of data derived from fluorescently labelled lipid components, probes, or markers incorporated into or associated with the lipid strata, and may include visualisation, imaging, or spectroscopic detection of emitted light under excitation at defined wavelengths. Such readouts may be obtained using fluorescence microscopy, confocal laser scanning microscopy, widefield epifluorescence microscopy, or fluorescence spectroscopy.

[0212]

[0207] In various embodiments, a fluorescence micrograph may be obtained to visualise the spatial distribution, morphology, uniformity, and continuity of the lipid layers. In another embodiment, quantitative fluorescence techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence anisotropy, or ratiometric fluorescence measurements may be employed to assess lipid fluidity, lateral diffusion, and microviscosity within the strata. In further embodiments, changes in fluorescence intensity, spectral shift, or probe localization may be used to determine interactions of a candidate substance with the lipid assembly, such as disruption, permeabilization, or phase separation.

[0208] In various embodiments, the measured one or more biophysical properties values may be compared to reference values, whereby a differential value of the one or more physical properties is indicative of the interaction between the candidate substance and the lipid assembly

[0213]

[0209] As used herein, the term “reference value” may refer to a baseline or control measurement of one or more physical properties of the lipid assembly in the absence of the candidate substance, or under conditions known to maintain the integrity of the lipid strata. Reference values may include, without limitation, quantitative or qualitative measurements of morphology, uniformity, contiguity, fluidity, dynamics, surface tension, polarity gradient, wettability, or permeability obtained from an untreated platform. In some embodiments, reference values may also be derived from exposure of the platform to a non-interacting control substance (e.g., buffer, isotonic solution, or inert carrier), or from averaged measurements across multiple replicate control assays.

[0214]

[0210] As used herein, the term “differential value" may refer to the magnitude and / or direction of change between the measured property of a lipid assembly after exposure to a candidate substance and the corresponding reference value. A differential value maybe expressed as a numerical difference, a ratio, or a relative percentage change. For example, a decrease in fluorescence recovery rate compared to the reference value may indicate reduced lipid fluidity; an increase in surface roughness or heterogeneity compared to reference morphology may indicate disruption of layer continuity; and a change in contact angle compared to reference wettability may indicate altered surface polarity.

[0215]

[0211] A differential value that deviates from the reference value is indicative of an adverse or beneficial interaction between the candidate substance and the lipid assembly. Such interactions may include disruptive effects such as thinning, collapse, or phase separation of lipid strata, which may indicate destabilisation of the biological air-interface; permeabilizing effects such as increased solute or tracer diffusion, which may correlate with loss of barrier integrity; destabilizing effects such as reduced uniformity or continuity of the lipid assembly, altered wettability, or increased surface roughness, which may reflect structural compromise of the interface; and modulatory effects such as altered lipid fluidity, viscosity, or dynamics, which may modify the protective function of the interface. In various embodiments, the observed differential values are directly indicative of adverse effects relevant to toxicology, including irritancy (for example, disruption of tear film stability, induction of barrier irregularities in the stratum corneum, or destabilisation of surfactant layers in the lung) and toxicity (for example, irreversible damage to lipid organisation, persistent barrier dysfunction, or collapse of protective lamellae). In this context, the platform provides a functional assay system for detecting and quantifying the irritancy and toxicity potential of candidate substances on lipid-based biological airinterfaces such as the tear film lipid layer, the stratum corneum of the skin, and the alveolar surfactant of the lung.

[0216]

[0212] In various embodiments, the measured one or more biophysical properties of the platform may be used to predict the toxicity or irritancy of the candidate substance.

[0213] Accordingly, in various embodiments, there is provided a method for predicting toxicity or irritancy of a candidate substance, comprising the steps of:

[0217] contacting the candidate substance to the platform disclosed herein; and

[0218] measuring one or more biophysical properties of the platform to obtain one or more biophysical properties values,

[0219] wherein the measured one or more biophysical values of the platform are indicative of the toxicity or irritancy of the candidate substance.

[0220]

[0214] In various embodiments, the level or degree of toxicity or irritancy of the candidate substance may be predicted. The term “level” or “degree" of toxicity or irritancy may refer to the extent, severity, or magnitude of adverse interaction between a candidate substance and the lipid platform, as determined by changes in one or more measurable biophysical properties of the lipid assembly relative to a reference value. The level may be expressed qualitatively, for example as negligible, mild, moderate, or severe, or quantitatively, for example as a percent change, fold difference, or threshold value. In various embodiments, a mild level of irritancy may correspond to a small but detectable change in fluorescence intensity or surface tension (e.g., <10% deviation from reference values) without gross disruption of lipid continuity, a moderate level may correspond to measurable reductions in lipid fluidity, continuity, or wettability (e.g., 10-30% deviation), partial phase separation, or increased permeability to tracers, and a severe level may correspond to significant collapse, thinning, or fragmentation of the lipid strata (e.g., >30% deviation from reference), loss of uniformity or contiguity across more than 50% of the surface, or complete loss of barrier function as indicated by tracer penetration. In certain embodiments, quantitative thresholds may include, without limitation, a fluorescence recovery after photobleaching (FRAP) reduction of >25%, a contact angle shift of >15 degrees, or a surface tension change exceeding 5 mN / m, each being indicative of at least moderate irritancy, while severe toxicity may be predicted where structural analysis demonstrates collapse or irreversible disruption of the lamellar stratified organisation.

[0221]

[0215] A threshold value or level may be associated with a statistic, whereby a threshold value or level is a value or level above or below which the difference in the measured property is assigned significance (i.e. via a statistical method), for example a p-value <0.05 may be considered as statistically significant.

[0222] EXAMPLES

[0223]

[0216] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the platforms, and uses disclosed herein may be carried out and applied, and are intended to be purely exemplary and are not intended to limit the disclosure.

[0224] EXAMPLE 1 : The Ocular Interface

[0217] A representative platform configuration for the model TFLL system includes a hydrophobized solid surface (e.g., glass, silicon, gold, other transducers) (FIG. 3). Tear film organisation on these hydrophobic surfaces can be reconstituted in several ways. These include (1) sequential self-assembly of non-polar and amphiphilic lipid layers. (2) One-step cooperative / hierarchical self-assembly of nonpolar and then the amphiphilic lipids. (3) Application of native tear films from human or animal sources. (4) Commercially sourced artificial tear film formulations. In all of these cases, the underlying hydrophobic surface induces spontaneous stratification of first the non-polar and then the amphiphilic lipids onto the hydrophobized surface. This then recapitulates the molecular organisation of the TFLL-air interface in an “upside-down” organisation.

[0225]

[0218] External irritants, or perturbants, or test molecules that may interfere with the TFLL can be introduced to the platform from solid, liquid or gas phases, to evaluate their effects on either the nonpolar and / or the amphiphilic layer, both of which contribute to the biophysical properties of the tear-film covering the cornea.

[0226]

[0219] The platform disclosed herein differs from current in vitro designs. First, the platform reconstitutes both the amphiphilic and non-polar components of the tear film, which is a significant improvement over the current liposomal models, which neither incorporate the non-polar layer nor mimics the lamellar stratified organisation. Second, although the bulk lipid matrix model (See Patent: Lebrun, US10041922B2) incorporates both nonpolar and amphiphilic layers, it suffers from the lack of control over the relative sizes of the two layers. Neither of these previous methods support parallelisation or imaging-based measurements.

[0227]

[0220] Because both nonpolar and amphiphilic layers are readily influenced by extraneous agents and because both contribute sensitively to the tear-film stability and function, the platform takes a leap forward in accurately modelling the ocular surface circumventing the need to use animal models.

[0228]

[0221] The platform has the following features:

[0229] a) Multicomponent. Various predetermined mixtures of amphiphilic and nonpolar lipids can be used. These include those that recapitulate the dominant components of healthy eyes, diseased eyes, human and animal eyes, including compositional diversity, if any, of various human and animal demographics. In one rendition, we use nine lipids (five amphiphilic lipids, three non-polar lipids, and one interfacial lipid) to capture the polarity gradients.

[0230] b) Molecularly tailored or formulations-based models and patient-derived tears. The platform may be molecular constructed bottom up with synthetic lipids, or may employed commercial formulations or patient-derived tears. The platform may be adapted for molecular level control over the tear composition and thus can mimic diseased conditions.

[0231] c) Stratified and gradient organization. The TFLL is composed of a thin (3-5 nm) amphiphilic lipid layer, which resides as a monolayer at the underlying aqueous boundary of the tear film, and a thicker bulk, 30-40 nm thick outermost layer of non-polar lipids. Based on Miyamoto, et.al. (2020) eLife, TFLL, a stratified, multi-component lipid organization, exhibits a lipid polarity gradient across the non-polar, and amphiphilic layers(FIG. 4). In this organization, it suggests that cholesteryl esters (CEs) and wax esters (WEs) in the nonpolar layer have the lowest polarity among meibum lipids. (O-acyl)-w-hydroxy fatty acids (OAHFAs), phospholipids (PLs) and cholesterol form the amphiphilic lipid sublayer that contacts the aqueous layer. Cholesteryl-OAHFAs and wax diesters (WdiEs) are located at the interface (interfacial layer) between these two sublayers and have a role in connecting the amphiphilic and non-polar lipid sub-layers. Our experimental configuration allows recapitulation of the stratified organization. d) High throughput. The platform can be configured to present ordered or unordered arrays of discrete elements / corrals with each element presenting engineered TFLL stratified layers. In another rendition, the platform can be localized on three-dimensional surfaces of microspherical particles / beads as well as other geometries (ellipsoid / ovoid, rod-shaped, cube, etc). In all of these cases, individual elements / particles allow for discretized measurements thereby enabling parallel or massively parallel measurements.

[0232] e) Digital assay. In many of the configurations above, the number of corrals or microbeads can exceed irritant concentration thus allowing for Poissonian statistics and digital assaying.

[0233]

[0222] The TFLL platform is the only in vitro assay that allows “smart” assessment of irritants that affect the TFLL. TFLL is composed of predetermined mixtures of lipids that recapitulate the TFLL in vivo. Effects of irritants on TFLL can be rationalised based on the biophysical changes observed on TFLL platform.

[0234]

[0223] The TFLL platform allows for complete control over lipid composition, whereby the bottom-up approach allows for mimicking of specific diseased states or ageing stages and for assessment of the effect of specific lipid constituents (e.g. those that show altered abundance in diseased states or ageing stages).

[0235]

[0224] The TFLL platform can be prepared in a high-throughput format with multiple, independent data points can be collected concurrently for each test compound.

[0236]

[0225] The use of the TFLL platform in vitro may allow for rapid assessment (within minutes) of the effects of test compounds, since the in vitro models are sensitive to exogenous perturbant.

[0237] EXAMPLE 2: TFLL and Assay Design

[0238]

[0226] The core of the assay consists of following main steps: a) lipid selection for TFLL, b) TFLL reconstitution, c) measurements including quantitative analysis of morphology, fluidity, and continuity of TFLL mimetic layers, d) presentation of irritants by direct incubation, microfluidic flows or vapor phase infusion processes, and (e) differential measurements of the changes of TFLL after incubation with irritants.

[0239]

[0227] In what follows, workflow for one illustrated, non-limiting, design is presented.

[0240] a) Lipid selection for TFLL

[0241] i. Non-polar lipids

[0242]

[0228] Human tear is -30-45 mol% cholesterol esters (CE) and ~30-50mol% of wax esters. The acyl chains of these are generally long (mainly C22:1 - C34:1), with the most abundant CEs possessing 26:0, 24:0, 24:1 and 20:0, while the most abundant WEs are those with 24:0, 24:1 , 26:0, and 20:0. Largely, 40 mol% for CE and 44 mol% for WE are selected for the TFLL model. The 40 mol% CE is comprised of equimolar of CE(24:0) and CE(24:1 (15Z)), whereas the 44 mol% of WE consists exclusively of WE(22:0 / 18:1 (9Z)).

[0243] ii. Amphiphilic lipids

[0244]

[0229] The phospholipids in the tear are more of equal abundance: glycerophospholipids, lysophospholipids and sphingomyelin. The most common mammalian lipid species with C16:0 and C18:1 acyl chain lengths are selected for the glycerophospholipids and lysophospholipids, i.e. PC(16:0 / 18:1 (9Z)), PE(16:0 / 18:1 (9Z)), PC(18:1 (9Z) / 0:0) and PE(18:1 (9Z) / 0:0). For sphingomyelin, the natural lipid from chicken egg, which has a dominant C16:0 acyl chain moiety, is selected. These are integrated into our TFLL model.

[0245]

[0230] An important class of amphiphilic molecules found in the meibum and tear are the (O-acyl)-OJ- hydroxy fatty acids (OAHFAs). The presence of monoesters of co-hydroxy fatty acids with fatty acids give the interfacial OAHFAs, and it is now recognised that these comprise a significant fraction (-4 mol%) of meibomian lipids. 5-PAHSA is selected, which is partly based on commercially available lipids.

[0246]

[0231] Table 1. TFLL lipid composition

[0247] Layer Lipid type Lipid (abbrev.) Lipid full name Mol%

[0248] PC(16:0 / 18:1(9Z)) 1-palmitoyl-2-oleoyl-glycero- 3-phosphocholine 3 PE(16:0 / 18:1 (9Z)) 1-palmitoyl-2-oleoyl-glycero- 3-phosphoethan 1 Amphiphilic olamine

[0249] lipid Glycerophospholipid PC(18:1 (9Z) / 0:0) 1 -oleoyl-sn-glycero-3- phosphocholine 1 PE(18:1(9Z) / 0:0) 1 -oleoyl-sn-glycero-3- phosphoethanolamine 3 Egg SMaSphingomyelin (egg, chicken) 4 Amphiphilic 5-(palmitoyloxy)stearic acid Interfacial OAHFAb5-PAHSA 4 lipid

[0250] Non-polar Cholesteryl ester CE(24:0) cholesteryl lignocerate 20 lipid CE(24:1 (15Z)) cholesteryl nervonate 20

[0251]

[0252] Wax ester WE(22:0 / 18:1(9Z)) behenyl oleate 44 Note: “This Natural Lipid is a mixture and with 16:0 being the dominant fatty acid moiety.bOAHFA refers to (O-acyl)-hydroxy fatty acid.

[0253] b) TFLL reconstitution

[0254]

[0232] 1. Cleaned microscopy glass slides (Schott D263®, 22 mm x 22 mm) were stored in a beaker filled with 18 £2 Milli-Q water before dried with N2 in a fume hood.

[0255]

[0233] 2. OTS coating was applied to the glass slide by submerging the glass slide sequentially in OTS solution (1 :1000 in Toluene, 15 min), chloroform (rinse) and acetone (3 min sonication at 37 kHz, 100% power using ElmaP water bath sonicator), before dried with N2.

[0256]

[0234] 3. Photomask was placed on the OTS coated glass slides, before etching by UV ozone using Digital UV Ozone System (Novascan) for 30 min.

[0257]

[0235] 4. The glass slides were coated with non-polar lipid layer, supplemented with the interfacial amphiphilic lipid. Twenty microliters of the non-polar lipid mixture were spread onto the OTS-coated surface using a 18G needle.

[0258]

[0236] 5. The non-polar lipid layer was imaged using AxioObserver.ZI inverted widefield fluorescence microscope (Zeiss) under 20x magnification lens and using Alexa Fluor 488 filter.

[0259]

[0237] 6. The glass slides were inverted onto a drop of amphiphilic lipid vesicle suspension to allow a contact time of 2 min. and rinse in a beaker filled with 18 £2 Milli-Q water.

[0260]

[0238] 7. Without exposing to air, the glass slide was transferred to a new petri dish filled with 18 £2 Milli-Q water and the amphiphilic and non-polar lipid layer was imaged using AxioObserver.ZI inverted widefield fluorescence microscope (Zeiss) using Alexa Fluor 568 and 488 filter, respectively. A representative image is shown in FIG. 5.

[0261]

[0239] 8. For the reconstitution of TFLL on microspherial beads, the beads were incubated sequentially with non-polar lipids, dried, followed by incubation with the amphiphilic lipid vesicle suspension for 30 min. In another rendition, the reconstitution of TFLL may be done in a single step using lipid vesicle composing of non-polar and amphiphilic lipids.

[0262] c) Measurements including quantitative analysis of morphology, fluidity, and continuity of TFLL mimetic layers

[0263]

[0240] Biological lipid molecules are freely diffusible within the lipid layers. To access if the reconstructed TFLL exhibits similar fluidity properties, fluorescence recovery after photobleaching (FRAP) was performed on the non-polar lipid layer and amphiphilic lipid layer, separately. A small region was selectively photobleached using full laser intensity under 63x magnification lens with smallest diaphragm for 5 min. A reduction in the fluorescence signal was observed in the photobleached area. The recovery of the fluorescence intensity was monitored every minute and was evident after five minutes recovery (FIG. 6). This demonstrates that the fluidity properties were retained in the TFLL reconstructed in the method presented. d) Presentation of irritants by direct incubation, and differential measurements of the changes of TFLL after incubation with irritants

[0264]

[0241] When TFLL was subjected to benzalkonium chloride and sodium salicylate, which are representative irritants for Category 1 and 2A eye irritants, respectively, both the amphiphilic and nonpolar lipid layers were deteriorated (FIG. 7). The rate of deterioration for benzalkonium chloride was higher than that for sodium salicylate. This indicates that the platform can differentiate chemicals with different categories of irritancy.

[0265]

[0242] In another design, hydrophobic microspherical trimethylsilyl silanized (TMS) silica beads (TMS-beads) coated with the nonpolar and amphiphilic lipid layers sequentially were prepared using the composition in Table 1. Briefly, the TMS-beads were first incubated with the desired amount of nonpolar lipid dissolved in chloroform (section iii.b.4), mixed and dried. Next, amphiphilic lipid vesicle suspension (section ii.b.6) was added, washed, and imaged under a confocal fluorescence microscope. The fluorescence micrograph indicates the presence of both the nonpolar (BODIPY 493 / 503) and amphiphilic (Rhodamine-PE) lipid layers (FIG. 8). Moreover, beads coated with different lipid TFLL lipid compositions can be mixed to allow for multiplex measurements.

[0266]

[0243] The lipid-coated beads were presented with irritants by direct incubation and differential measurements of the changes of TFLL after incubation with irritants were measured. When TFLL was subjected to glycerol and sodium lauryl sulfate (SLS), which are representative irritants for No Category and Category 1 eye irritants, respectively, the fluorescence intensities of the two fluorophores were reduced, suggesting that both the amphiphilic and non-polar lipid layers were deteriorated (FIG.9). The rate of deterioration for SLS was higher than that for glycerol, confirming that the platform can differentiate chemicals with different categories of irritancy.

[0267]

[0244] Ocular toxicity testing is part of the growing global ophthalmology market due to the changing demographics to an aging population. According to World Report on Vision 2019, at least 2.2 billion people have a vision impairment and by conservative estimation, at least 1 billion people have a vision impairment that could have been prevented or has yet to be addressed. This translates to a global ophthalmology market size of USD 55 billion in 2021 and is projected to grow at a CAGR of 6.6% to reach USD 77 billion by 2027. Market sizes for the ophthalmic drug, drug delivery system and implants, are each valued at USD 39 billion, 14 billion and 13 billion, in 2021 respectively with a CAGR of 5.8-6.8% for the next 7-10 years.

[0268]

[0245] Toxicology testing is a critical component of safety evaluation in the personal care products, drug formulations, environment, and water industries. The global in vitro toxicology testing market size is valued at USD 13.7 billion in 2024 and it is estimated to reach USD 28.2 billion by 2031 , with a CAGR of 10.8% (Straits Research). Ocular toxicity segment accounts for about 12% of the total global market size (Global Market Insights). North America, Europe and Asia Pacific regions are the leading in vitro toxicology market shareholders.

[0246] Accordingly, the present invention finds utility and is of importance in respect of (1) the ocular testing service providers, who are key players propelling the rapid growth of the toxicology testing market and (2) the producers of consumers products that have direct and indirect contacts with the eyes, namely liquid lotions, artificial tears, contact lens lotions. The global markets for each of the three products are valued on average at about USD 10 billion in 2024, with a CAGR of about 8%.

[0269]

[0247] Liquid lotion: This platform will transform the way formulations are optimized in a lowcost, rapid, animal-free manner. The cost and time required for existing eye irritancy assays is substantial in view of the large number of chemicals and formulations required to be tested iteratively during early product development stage. A balance between the test accuracy, cost, and time spent is crucial when deciding the type of eye irritancy assay to use. Existing methods are not optimal and novel solutions are highly sought after.

[0270]

[0248] Artificial tears: The platform serves as a toolkit for identifying and optimizing artificial tear formulations that are stable against various desiccating conditions (such as changing relative humidity and temperature extremes) and environmental pollutants (such as tobacco smoke exposure). These environmental factors promote the development of dry eye syndrome (DED) - a multifactorial disorder associated with the presence of an abnormal tear film that leads to ocular surface irritation and inflammation, as well as contact lens users - in normal individuals and worsen DED in individuals with his disorder.

[0271]

[0249] Contact lens: The choice of materials used for contact lens is vital for long term wear of contact lens which can lead to depletion or stimulation of tear film components. Our platform allows the biophysical and biochemical process at the contact lens-tear film lipid layer to be characterized, therefore allowing modification of optimal contact lens materials as well as the contact lens lotions for maximum comfort.

[0272] EXAMPLE 3: Chemical (GHS) Testing

[0273]

[0250] Testing was performed for additional chemicals. Table 2 shows the comparison of results obtained from the bead assay format with benchmark GHS category. Assignment of predicted GHS category and within-category rank ordering of tested chemicals based on quantitative fluorescence decay data. Each of the chemicals is tested at four different concentrations. The raw fluorescence intensity values are converted to predicted GHS categories based on an internal scoring metric. Excellent correlation was obtained.

[0274]

[0251] Table 2. Benchmarking of 22 chemicals with predefined GHS categories.

[0275] GHS Concentration (%, v / v) Total Predicted category 0.50% 1% 2% 4% Score Category

[0276]

[0277] Glycerol Cat NC 90.9 87.8 95.2 85.7 0 Cat NC NaCI Cat NC 98.0 93.3 94.0 94.0 0 Cat NC Propylene Glycol Cat NC 84.6 73.9 74.5 66.1 3 Cat 2 cellosolve acetate Cat NC 78.3 80.2 78.9 81.7 0 Cat NC Hexanol Cat 2 111.5 114.1 121 .7 89.5 0 Cat NC Methyl acetate Cat 2 64.1 75.3 55.5 61.6 3 Cat 2 Ethylacetate Cat 2 88.2 82.6 72.8 78.7 1 Cat 2 Isopropanol Cat 2 62.0 70.8 68.1 55.5 4 Cat 2 Sodium Benzoate Cat 2 82.0 70.8 55.2 63.0 3 Cat 2 potassium cyanate Cat 2 84.2 84.4 84.6 57.3 1 Cat 2 triazole Cat 2 59.1 91.1 69.4 2.2 6 Cat 1 Sodium salicylate Cat 2 65.4 70.7 50.3 34.4 7 Cat 1 Methylresorcinol Cat 1 30.7 24.7 18.7 25.9 16 Cat 1 NaOH Cat 1 85.2 82.1 80.7 44.7 4 Cat 2 Acetic acid Cat 1 75.7 66.4 45.9 39.2 9 Cat 1 Butanol Cat 1 65.9 51.6 47.1 43.9 10 Cat 1 Butrylactone Cat 1 80.6 67.5 41.4 42.5 9 Cat 1 Benzalkonium Cat 1

[0278] chloride 44.5 26.9 11.1 5.3 16 Cat 1 Imidazole Cat 1 60.0 44.5 38.8 63.1 10 Cat 1 Isobutanol Cat 1 79.6 50.8 44.6 71.9 6 Cat 1 Sodium Lauryl Cat 1

[0279] Sulfate 3.5 1.4 2.0 2.2 16 Cat 1 Trichloroacetic acid Cat 1

[0280]

[0281] 89.1 81.0 50.0 33.5 8 Cat 1

[0282]

[0252] Table 3 shows the comparison of results obtained from the chip and the bead assays. Both assays produce comparable outcome, demonstrating the robustness of the assays in response to chemicals spanning the three GHS eye irritation categories. Both geometries allow for differentiation of same-GHS-category substances.

[0283]

[0253] Table 3. Benchmarking of 22 chemicals with predefined GHS categories (Cat 1 , Cat 2, Cat NC) with both chip and bead assay format.

[0284] GHS Category Chemical Chip (0.5% Irritans) Beads (0.5% Irritants) NC Toluene 53.49 54.06

[0285] NC Glycerol 94.56 80.57

[0286] NC Sodium oxalate 66.39 75.58

[0287] NC Propylene glycol 85.39 88.77

[0288] NC Olive Oil 87.43 80.57

[0289] NC Tween 20 38.81 30.87

[0290] Cat 2A Sodium Benzoate 41.04 102.47

[0291] Cat 2A Methyl acetate 87.65 71.07

[0292] Cat 2A Sodium Salicylate 62.64 117.43

[0293] Cat 2A Isopropanol 81.03 77.48

[0294]

[0295] Cat 2A Hexanol 78.93 90.02

[0296] Cat 2A ethyl acetate 74.54 64.40

[0297] Cat 2A Pyridine 78.60 92.64

[0298] Cat 2A N-laurylsacrosine 19.97 21.57

[0299] Cat 1 Acetic acid 64.16 80.99

[0300] Cat 1 Imidazole 87.08 69.22

[0301] Cat 1 NaOH 48.41 37.52

[0302] Cat 1 Butanol 66.51 72.54

[0303] Cat 1 Isobutanol 61.81 54.53

[0304] Cat 1 5-methylresorcinol 31.27 32.08

[0305] Cat 1 SDS 51.02 6.53

[0306] Cat 1 Benzalkonium chloride 31.16 26.34

[0307]

[0308]

[0254] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims.

[0309]

[0255] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The platforms, methods, and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0310]

[0256] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

Claims

CLAIMSWhat is claimed is:

1. A platform for mimicking a biological air-interface, comprising:a substrate; anda lipid assembly deposited on the substrate,wherein the lipid assembly mimics the lamellar, stratified, or monolayer organisation and optionally polarity gradient, of the biological air-interface, andwherein the lipid assembly comprises an amphiphilic lipid phase, and / or a non-polar lipid phase.

2. The platform of claim 1 , wherein the substrate comprises a glass substrate, silicon substrate, gold substrate, metal oxide substrate, polymeric substrate, transducer surface, liquid droplet, hydrogel, or other soft material, preferably the substrate is a planar glass substrate or glass bead comprising a hydrophobic coating, more preferably trimethylsilyl (TMS) beads.

3. The platform of claim 1 or 2, wherein the lipid assembly mimics the stratified lamellar arrangement and polarity gradient of the tear-film lipid layer (TFLL) of the eye, and comprises or consists of a non-polar lipid phase and an amphiphilic lipid phase comprising one or more interfacial amphiphilic lipids and one or more other amphiphilic lipids.

4. The platform of claim 3, wherein the non-polar lipid phase comprises cholesteryl esters (CEs) and wax esters (WEs), and the amphiphilic lipid phase comprises (O-acyl)-w-hydroxy fatty acids (OAHFAs), glycerophospholipids, lysophospholipids and sphingomyelin.

5. The platform of claim 4, wherein the lipid assembly comprises cholesteryl lignocerate, cholesteryl nervonate and behenyl oleate, preferably comprises about 10-30 mol% of cholesteryl lignocerate, about 10-30 mol% of cholesteryl nervonate and about 35-55 mol% of behenyl oleate based on total lipid content.

6. The platform of claim 4 or 5, wherein the amphiphilic lipid phase comprises 5-(palmitoyloxy)stearic acid (5-PAHSA), 1 -palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1 -palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, 1 -oleoyl-sn-glycero-3-phosphocholine, 1 -oleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin derived from chicken egg, preferably the lipid assembly comprises about 2-6 mol% of 5-(palmitoyloxy)stearic acid (5-PAHSA), about 1-4 mol% of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, about 0.5-2 mol% of 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine, about 0.5-2 mol% of 1 -oleoyl-sn-glycero-3-phosphocholine, about 1-4 mol% of 1-oleoyl-sn-glycero-3-phosphoethanolamine and about 2-6 mol% of sphingomyelin derived from chicken egg based on total lipid content.

7. The platform of any one of claims 3-6, wherein the thickness of the amphiphilic lipid phase is about 3-5 nm, and the thickness of the non-polar lipid phase is about 30-200 nm.

8. The platform of claim 1 or 2, wherein the lipid assembly mimics the repeating lamellar strata and periodic polarity gradient of the stratum corneum (SC) lipid barrier of the skin, wherein each lamellar stratum comprises a non-polar lipid phase and an amphiphilic lipid phase comprising one or more interfacial amphiphilic lipids.

9. The platform of claim 8, wherein the amphiphilic lipid phase comprises ceramides (Cer), free fatty acids (FFAs), cholesterol and cholesterol sulfate, preferably the amphiphilic lipid phase comprises about 40-80 mol % of the lipid assembly, and the non-polar lipid phase comprises about 5-30 mol % of the total lipid assembly.

10. The platform of claim 1 or 2, wherein the lipid assembly mimics the monolayer organisation and polarity gradient of the alveolar surfactant (AS) layer of the lung, and comprises an amphiphilic lipid phase comprising one or more amphiphilic lipids, and optionally one or more surfactant-associated proteins to form a lipid-protein assembly.

11. The platform of claim 10, wherein the amphiphilic lipid phase comprises dipalmitoylphosphatidylcholine (DPPC), preferably the amphiphilic lipid phase comprises about 80-95 mol % of the lipid assembly, and optionally the lipid assembly comprises about 5-20 mol % of surfactant-associated proteins.

12. The platform of any one of claims 1 -11 , further comprising an aqueous phase, wherein the aqueous phase is in direct contact with, or contiguous to, the amphiphilic lipid phase so as to simulate the aqueous phase of the native biological air-interface.

13. The platform of any one of claims 1 -12, wherein the lipid assembly is arranged in an inverted “upside-down” configuration relative to the native biological air-interface.

14. The platform of any one of claims 1 -13, wherein the platform is a cell-free platform.

15. A method of manufacturing the cell-free platform of any one of claims 1-14, comprising the steps of:providing a substrate;depositing a lipid assembly onto the substrate, wherein the lipid assembly comprises an amphiphilic lipid phase, and / or a non-polar lipid phase, wherein depositing the lipid assembly comprises sequential or co-deposition of one or more lipid compositions forming the amphiphilic lipid phase and / or non-polar phase,wherein the lipid assembly mimics the lamellar, stratified, or monolayer organisation and optionally polarity gradient, of the biological air-interface.

16. A biosensor device comprising the platform of any one of claims 1 -14.

17. Use of the platform of any one of claims 1 -14, or biosensor device of claim 15 for screening or assaying a candidate substance.

18. A method for screening or assaying a candidate substance, comprising the steps of:contacting the candidate substance to the platform of any one of claims 1-14; and measuring one or more biophysical properties of the platform to obtain one or more biophysical properties values,wherein the one or more biophysical values are indicative of the interaction between the candidate substance and the platform.

19. The method of claim 18, wherein the one or more biophysical values of the platform is used to predict the toxicity or irritancy of the candidate substance.

20. The method of claim 18, wherein the one or more biophysical properties values are compared to reference values, wherein a differential value is indicative of the interaction between the candidate substance and the lipid assembly.

21. The method of claim 18, wherein the one or more biophysical properties is selected from morphology, uniformity and contiguity of the lipid phases, fluidity and dynamics, and integrity of the lipid assembly.

Images