EP4069184A1

EP4069184A1 - Lipid vesicles as oxidative stress sensors

Info

Publication number: EP4069184A1
Application number: EP20816518.3A
Authority: EP
Inventors: Florian Gruber; Marie-Sophie NARZT; Christopher KREMSLEHNER; Johannes Grillari
Original assignee: Medizinische Universitaet Wien; Universitaet fuer Bodenkultur Wien BOKU
Current assignee: Medizinische Universitaet Wien; Universitaet fuer Bodenkultur Wien BOKU
Priority date: 2019-12-04
Filing date: 2020-12-04
Publication date: 2022-10-12
Also published as: WO2021110979A1

Abstract

The present invention relates to a lipid vesicle comprising at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid, the use of said lipid vesicle in a method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, preferably wherein said oxidative stress is associated with skin damage and/or leads to skin damage, and the use of said lipid vesicle in a method for selecting or producing a compound or composition which prevents and/or protects from said oxidative stress. Furthermore, the invention relates to medical and non-medical uses of a compound or composition obtained by said production method, in particular for protecting the skin from said oxidative stress, and/or for preventing, ameliorating and/or treating a skin damage and/or a skin disorder.

Description

Lipid vesicles as oxidative stress sensors

The negative effects of exposure of the skin to ultraviolet (“UV”) light are well-known. Prolonged exposure to sunlight, mainly UVA and UVB, causes oxidative stress within the skin and skin damage. When skin is, i.e. repeatedly, exposed to UV light having a wavelength of from about 280 nm to about 400 nm for long time, this can lead to serious conditions such as inter alia skin erythema, inflammation, skin aging or skin cancer.

Especially UVA and UVB account for most of the UV radiation that penetrates the skin. The main detrimental effect of UVB is direct DNA damage (leading to a sunburn), whereas the main detrimental effect of UVA is oxidative stress. Of note, oxidative stress leads to indirect damage of the DNA and other cellular and extracellular molecules in the skin, i.e. lipids, and may also contribute to a sunburn (Stahl (2003), Mol Aspects Med. 24(6):345-51).

Moreover, urban and domestic pollution, with external and internal exposure routes, further promotes skin aging and tissue damage (Krutmann (2017), J Dermatol Sci. 85(3): 152-161; Vierkotter (2010), J. Invest. Dermatol. 130: 2719-2726 and Li (2015), J. Dermatol. Sci. 79: 148-154). Furthermore, it is thought that pollution and UV light, i.e. UVA, cause oxidative stress and skin damage in a synergistic way (Marrot (2018), Curr Med Chem.25(40):5469- 5486; Soeur (2017), Dermatol Sci. 86(2): 162-169; Araviiskaia (2019), J Eur Acad Dermatol Venereol. 33(8): 1496-1505). In this regard, the physical oxidative destruction of barrier lipids and oxidative damage to cells in the lower epidermis and dermis has been observed.

Oxidative stress is a biological state that occurs when a cell's antioxidant capacity is overwhelmed by reactive oxygen species (ROS), e.g. elicited by UVA, causing a redox imbalance. The oxidative stress caused by UVA in the skin is mainly mediated by singlet oxygen (Tyrrell (2004), Antioxid Redox Signal. 6(5):835-40). Singlet oxygen, in turn, activates enzymes which generate ROS including radicals (Gruber (2012), J Lipid Res. 53(6): 1232-42). It is well known how ROS oxidize phospholipids of the skin and how oxidized phospholipids relate to skin damage, i.e. cell membrane damage.

Oxidized phospholipids are thought to modify the function and physical properties of cell membranes, for example, the water penetration. Moreover, radicals attack DNA, membrane lipids and proteins, generating carbon radicals. These in turn react with oxygen to produce a peroxyl radical that can attack adjacent fatty acids to generate new carbon radicals. This cascade leads to a chain reaction producing lipid peroxidation products, in particular within cell membranes. Thus, oxidative stress leads to damage of the cell membrane which may result in dysregulated membrane structure and cell permeability (Walton (2006), J Lipid Res. 47(9): 1967-74), decreased ability to excrete or detoxify waste products (Faghiri (2006), Invest Ophthalmol Vis Sci. 47(l):397-404) and accumulation of intracellular lipids that are toxic (Ramprecht (2015), Chem Phys Lipids. 189:39-47). In addition, collagen fibers may be damaged. Eventually, oxidative stress may thus lead to skin wrinkles, uneven skin tone, loss of skin thickness and elasticity, and other signs of skin aging. This process is commonly referred to as photoaging. Moreover, oxidative stress may adversely affect the immune system or lead to skin cancer, for example, melanoma.

Numerous studies have described the relationships between UV light, oxidative stress, skin damage, skin ageing, skin disorders and how a protection therefrom might be achieved. Reference is made, for example, to Solis-Calero (2015), Oxid Med Cell Longev. 2015:319505; Dupont (2013), Int. J. Cosmet. Sci. 35: 224-232; Krutmann (2006), Skin Aging. Springer Heidelberg, New York; 2006: 33-43; Krutmann (2012), J. Invest. Dermatol. 132: 976-984; Marionnet (2015), Int. J. Mol. Sci. 16: 68-90; and Marionnet (2014), PLoS One. 9: el05263.

To protect skin from photodamage that may result from exposure to UV light, sunscreens or compositions with sunscreen active material have been developed. However, many sunscreens do not sufficiently block UVA despite the detrimental role of UVA in skin ageing and cancer. Moreover, protection from UVA seems to play a major role in the prevention of UV light induced immunosuppression. There is thus a large interest in developing improved sunscreens which effectively protect from both UVA and UVB light.

Of note, under certain circumstances, UV light and the associated oxidative stress also have therapeutic effects, for example, UV phototherapy is used for the treatment of graft-versus- host disease, atopic dermatitis, psoriasis, vitiligo, and acne vulgaris (Rodenbeck (2016), Clin Dermatol. 34(5):607-13; Lim (2015), J Am Acad Dermatol. 72(6): 1078-80; York (2010), Int J Dermatol. 49(6):623-30).

However, appropriate means and methods to determine or differentiate the effects of UV light on the skin, are currently lacking. Furthermore, the information about the protective effect of a sunscreen or a cosmetic developers and customers currently obtain is insufficient, because it is based on crude methods, foremost the sun protection factor (SPF). The SPF is an FDA approved in vivo method that is based on the minimal erythemal dose (MED). The MED is defined as the lowest time at a defined fluence of UV irradiation produce measurable erythema on an individual. The SPF is calculated from the MED on skin that is protected by a test substance divided by the MED on the unprotected skin. However, as already indicated above, the erythema is mainly caused as a reaction to direct DNA damage by UVB irradiation. Thus, the SPF does not reliable indicate the protection from UV light induced oxidative stress. Moreover, the SPF does not correlate with the protection from UV light induced immunosuppression (Poon (2003), J Invest Dermatol. 121(1): 184-90). Moreover, sunscreens which are developed by ignoring the effects of UVA, may be harmful to the user. For example, it was found that after 60 minutes of sunscreen treatment, the amount of absorbed sunscreen was so high that the amount of ROS was higher in the sunscreen-treated skin than in the untreated skin (Hansen (2006), Free Radic Biol Med. 41(8): 1205-12).

Thus, an in vitro method has been developed to determine the protection from UVA which is based on vitro UV substrate spectrophotometry (Matts (2010), Int J Cosmet Sci. 32(l):35-46). Such a method, however, only allows to assess the blocking of UVA, but not the protection from oxidative stress caused by UV light. In other words, measuring the physical absorption properties of sunscreens does not provide very useful information about the protection from harmful effects caused by UV light, i.e. in combination with other agents such as pollutants. For example, a composition comprising particulate matter may block UVA to some extent, but still increase oxidative stress in the skin due to synergistic effects with the remaining UV light.

Using “bad” or, more precisely, wrongly tested sunscreens may even cause harm to the users. For example, it has been proposed that usage of sunscreen may cause, and not prevent, melanoma (see e.g. Garland (1992), Am J Public Health, 82 (4): 614-5; Westerdahl (2000), International Journal of Cancer, 87 (1) 145-150; Autier (1995), Int. J. Cancer 61 (6): 749- 755; Weinstock (1999), Journal of Investigative Dermatology Symposium Proceedings, 4 (1): 97-100; and Vainio and Bianchini (2000), Scand J. of Work Environment and Health, 26: 529-531.).

Thus, there is still a need for means and methods for determining the effects UV light has on skin, identifying agents which enhance said effects, and identifying compositions or compounds which protect from such effects.

Accordingly, the invention relates to a method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, and wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid.

Further provided herein is a lipid vesicle comprising at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid.

The inventors have surprisingly found an in vitro method for testing certain effects UV light has on skin without employing living beings, actual skin, skin cells or cell extracts. As illustrated in the appended Examples, the inventors have generated lipid vesicles as cell mimics which allowed to reproduce certain (bio)chemical reactions occurring in the skin of humans when exposed to UV light or UV light in combination with a pollutant such as, inter alia , Diesel particulate matter (DPM). However, said lipid vesicles can be also used to determine the oxidative stress caused by UV light in combination with other pollutants or agents which may be even comprised in sunscreens, for example heavy metals, inter alia zinc oxide (Ma (2011), Environ Pollut. 159(6): 1473-80). The lipid vesicle of the invention can be regarded as a cell mimic, at least, because it is, similarly as a cell, a spherical particle whereof the outer layer consists, at least to a large extent, of lipids. Moreover, the cells of a vertebrate, in particular of a mammal such as a human, which are most exposed to the sun and hence UV light, are skin cells. Thus, the lipid vesicle of the invention can be regarded in particular as a skin mimic or a skin cell mimic.

Surprisingly, it has been found that oxidative stress caused by UV light within the skin could be also generated within said lipid vesicles when they are exposed to UV light, i.e. UVA. Thus, the lipid vesicle of the invention which is treated and/or contacted with UV light or UV light in combination with an additional agent as provided herein, can be used as a surrogate to determine at least some effects said UV light or UV light in combination with an additional agent has on skin cells.

This further allowed the inventors to determine alterations of skin components, in particular the oxidation of certain membrane lipids of skin cells.

Thus, the invention further relates to a method for determining the damage of a skin component caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid. Preferably herein, said skin component is a lipid, preferably a cell lipid, very preferably a cell membrane lipid.

As illustrated in the appended Examples, lipid vesicles comprising an oxidizable cell membrane lipid, such as inter alia PAPC, may be further particularly suitable for determining oxidative stress that is caused by UV light or UV light in combination with an additional agent and which is associated with and/or leads to skin damage because at least some of the oxidation products of said cell membrane lipid are not merely markers for oxidative stress but have inherent biological functions, e.g. inter alia in adaptive immunity and cellular stress reactions (Bochkov (2010), Antioxid Redox Signal. 12(8): 1009-59). For example, hydroperoxides of PAPC (PAPC-OOH) are agonists for the transcription factors Nrf2 and Atf4.

Assessing the effects of UV light in an in vitro system is highly beneficial as it circumvents the ethically problematic testing on humans or animals as is still done for the Sun Protection Factor. Moreover, the present invention allows to determine the oxidative stress caused by UV light, i.e. UVA, which is often neglected although meanwhile well known for having detrimental consequences on the appearance of the skin and leading to the development of severe diseases such as skin cancer.

Thus, preferably in context of this invention, the oxidative stress is associated with skin damage and/or leads to skin damage. Said oxidative stress may lead to skin damage, and eventually to skin disorders or a cancerous modification of the skin.

To reach at this highly useful in vitro method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, the inventors first demonstrated that lipid vesicles comprising an oxidizable sensor lipid and a non-oxidizable reference lipid could be formed and said sensor lipid could be oxidized by UV light (see e.g. the appended Examples 1, 2 and 5). It was further surprisingly found that the oxidation products of said sensor lipid could be detected by liquid chromatography in combination with mass spectrometry (i.e HPLC-MS/MS) and that the quantification of said oxidation products was surprisingly precise and robust, i.e. because normalization with the reference lipid was possible (see e.g. the appended Examples 5 and 6). It has been further discovered that 1- palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) was particularly suitable as sensor lipid and l,2-dipalmitoyl-.s?z-glycero-3-phosphocholine (DPPC) was particularly suitable as the respective reference lipid.

Thus, in a particularly preferred aspect, the invention relates to a method for determining oxidative stress caused by UV light, preferably UVA, or said UV light in combination with a pollutant, in particular Diesel particulate matter, wherein said method comprises a step of treating and/or contacting a lipid vesicle with said UV light or said UV light in combination with said pollutant, wherein said lipid vesicle comprises preferably PAPC as oxidizable sensor lipid and preferably DPPC as non-oxidizable reference lipid, wherein the oxidative stress is determined by normalizing the amount of at least one oxidation product of said sensor lipid by the amount of said reference lipid, and preferably wherein said amounts are determined by liquid chromatography in combination with mass spectrometry.

Moreover, it was a surprising finding, as illustrated in the appended Examples, that the oxidative stress could be quantified so precisely that the synergistic increase of oxidative stress caused by UV light in combination with Diesel particulate matter could be reproduced by quantifying the oxidation products of the sensor lipid contained in the lipid vesicles according to the invention.

Moreover, the method and lipid vesicles as described above and as illustrated in the appended Examples, can be used for selecting a compound or composition which prevents and/or protects from oxidative stress caused by UV light or UV light in combination with an additional agent. In particular, it has been surprisingly found that the oxidation of the sensor lipid contained in lipid vesicles comprising an oxidizable sensor lipid and a non-oxidizable reference lipid was reduced when said lipid vesicles were covered with a cosmetic composition, i.e. a make-up, that is advertised for its protective effect against UV light (see e.g. the appended Examples 5 and 6). It was surprising, that this method allowed to reproduce the protective effect of the cosmetic composition against UV light.

As the means and methods provided herein allow determining if and how much a certain compound or composition prevents and/or protects from oxidative stress caused by UV light, said means and methods are particularly useful for producing improved sunscreens and cosmetics such as make-up which are applied on the skin to prevent skin ageing and skin diseases. As the means and methods according to the present invention allow to determine oxidative stress caused by UVA, said means and methods are further particularly useful for evaluating the protective effect of a compound or composition against UVA radiation. Because an actual cosmetically and clinically relevant effect of UVA is measured with the means and methods provided herein, the invention is advantageous over other methods which solely consider the blocking, absorption and/or reflection of UVA. Moreover, since the determination of oxidative stress is performed with cell and skin mimics according to the invention, the oxidative stress determined is particularly closely related to the biochemical processes taking place within the skin that is exposed to UV light, which his comprised i.e. in the sun light. Furthermore, as the means and methods according to the present invention even allow to quantify the synergistic effects of UV light in combination with pollutants such as Diesel particulate matter, said means and methods are an excellent proxy for the oxidative stress occurring in the skin of animals or humans in their daily life, for example when staying on a sunny day at a road with high traffic in a city. It is thus expected that the preventive and/or protective effect of a compound or composition against UV light as provided herein is particularly realistic as it mimics aspects of real-world situations. Thus, it is also expected that compounds or compositions selected according to the invention may have a superior performance in preventing skin ageing and skin diseases.

Thus, the invention further relates to a method for selecting a compound or composition which prevents and/or protects from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said selection comprises determining the efficacy of said compound or composition in preventing and/or protecting from said oxidative stress, and wherein determining said efficacy comprises treating, contacting and/or covering the lipid vesicle with said compound or composition prior to and/or during treating and/or contacting said lipid vesicle with the UV light or the UV light in combination with the additional agent.

Further provided herein is a method for producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of selecting said compound or composition according to the method for selecting a compound or composition as provided herein.

Also provided herein is a compound or composition obtainable by the method for producing a compound or a composition as provided herein.

Moreover, said compound or composition is also provided herein for use in preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent.

In addition, the invention relates to the use of said compound or composition for protecting the skin from oxidative stress caused by UV light or UV light in combination with an additional agent.

Furthermore, the invention relates to the use of the lipid vesicle as provided herein in any of the methods of the invention.

As illustrated in the appended Examples, Diesel particulate matter - a known enhancer of oxidative stress caused by UV light - strongly increased the oxidative stress, i.e. the amount of lipid oxidation products within the lipid vesicles according to the invention when treated with UV light, i.e. UVA. Thus, the means and methods provided herein can be also used for identifying agents which enhance certain effects of UV light, in particular oxidative stress. The identification of such agents is highly useful for at least two reasons. First, is critical for the health of humans and animals to avoid agents which enhance the oxidative stress of sunlight that may lead to skin ageing, skin damage, a cancerous modification of the skin and/or skin diseases. Evidently, to avoid such agents, they first have to be identified.

Furthermore, it may be important to determine agents which enhance the oxidative stress of UV light, in particular UV light of certain wavelengths such as UVA, for their use in phototherapy. For example, oxidative stress may be exploited inter alia to cause directed damage to malign cells such as cancer cells or undesired immune cells in the skin. It is also conceivable that the generation of ROS may be exploited for the destruction of toxins, e.g. after a bite of an animal.

Thus, the invention further relates to a method for determining the enhancement of oxidative stress caused by an agent in combination with UV light, wherein said method comprises the steps of

(a) treating and/or contacting a lipid vesicle with the UV light,

(b) treating and/or contacting a lipid vesicle with the UV light and said agent, and

(c) comparing the oxidative stress caused by (a) and (b), wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non- oxidizable reference lipid. Furthermore, the invention relates to a method for producing an agent according to the invention, wherein said method comprises a step of selecting said agent, and wherein the selection comprises a step of determining the enhancement of oxidative stress caused by said agent in combination with UV light as provided herein.

Thus, the invention also relates to an agent obtainable by said method for producing an agent.

In detail, the invention relates to a method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, and wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid.

Preferably herein, the oxidative stress according to the invention is caused by UV light or UV light in combination with an additional agent. However, in certain embodiments, said UV light may be replaced by or supplemented with another trigger.

A trigger, as used herein, refers to a means which by itself can cause oxidative stress according to the invention. In contrast, a means which, dependent on the context, is either known to enhance said oxidative stress caused by said trigger or is suspected to enhance said oxidative stress caused by said trigger, is termed herein an “agent”. Both, a trigger or an agent can be, as used herein, any physical or chemical means such as a compound, a particle, a composition and/or a radiation, as long as it does not lead to the destruction of the lipid vesicle of the invention or any means required to carry out the invention. The skilled person can judge if a means may be suitable as a trigger or agent of not. For example, it is evident that inter alia strong heat such as a fire, or very reactive or corrosive chemicals are not suitable to detect the comparably mild effect of lipid oxidation. Evidently, oxidative stress or lipid oxidation in the context of the invention does thus not relate to combustion of lipids but rather to mild lipid oxidation or peroxidation. Lipid peroxidation can be described, as further explained below, as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (Ayala (2014), Oxid Med Cell Longev. 2014:360438).

The preferred trigger to be employed in the means and methods of the present invention is UV light. Alternatively, or in addition to UV light, a trigger may be a further means which generates singlet oxygen such as Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'- tetraiodofluorescein) in combination with white light, or hydrogen peroxide in combination with iron (Fe²⁺). Thus, the term “UV light” may be replaced by the terms “Rose Bengal in combination with white light” and/or “hydrogen peroxide in combination with iron” in context of the methods as provided herein and in cases wherein it is evident for the skilled artisan that the UV light functions merely as the “trigger” of the oxidative stress in the herein employed inventive test systems. Accordingly, in context of this invention, said “UV light trigger” may be replaced in certain embodiments by other “triggers”, like “Rose Bengal in combination with white light” or “hydrogen peroxide in combination with iron”. Other embodiments are also envisaged. Accordingly, the qualification of a substance and/or a combination of substances as a “trigger” is that said substance and/or said combination of substances are characterized by generating singlet oxygen and/or said substance and/or said combination of substances is a photosensitizer which produces a chemical change in another molecule in a photochemical process. In context of the “trigger” definition provided herein, the term “light” also refers to a substance since it is to be considered as a “radiation agent”.

Accordingly, UV light is the preferred means causing oxidative stress in context of the methods of this invention. UV light is considered as a major cause of oxidative stress acting on the skin, and/or being involved in skin damage/skin diseases. Moreover, humans and animals are often exposed to UV light and seek protection from the adverse effects of UV light. In addition, UV light is used in phototherapies, and further development and refinement of such therapies is desirable. Thus, the means and methods employing UV light as a trigger of oxidative stress as provided herein are particularly useful for satisfying those desires. However, the means and methods according to the invention are not restricted to the employment of UV light but also allow to determine oxidative stress caused by another trigger as long as said trigger leads to the generation of singlet oxygen which is, as explained above, a major mediator of oxidative stress within the skin. For example, the skin of humans or animals may come into contact with certain chemicals causing oxidative stress and/or skin damage, and Rose Bengal in combination with white light or hydrogen peroxide in combination with iron (Fe²⁺) may be comprised in or at least mimic such chemicals. It is contemplated that the means and methods according to the invention are useful for identifying agents which enhance the oxidative stress of such chemicals, for example, inter alia a hair bleach comprising hydrogen peroxide. Similarly, the means and methods according to the invention may be useful for identifying compounds and or compositions which prevent and/or protect from the oxidative stress caused by such chemicals, for example, inter alia a hair bleach.

The term “skin” as used herein, refers to the soft outer tissue covering of vertebrates and/or a tissue that consists of an outer epidermal layer and an inner dermal layer which are connected by a basement membrane, whereas the terms “skin mimic”, “skin cell mimic” or “skin surrogate” refer to artificial compositions which do not occur as such in animals, much less in vertebrates, but which have at least one aspect in common with skin or skin cells. Preferably, the common aspect comprises at least a lipid, preferably a cell lipid, very preferably a cell membrane lipid.

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Herein, said imbalance is, unless otherwise indicated, caused by UV light or UV light in combination with an additional agent. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA, and potentially also extracellular components, for example extracellular matrix proteins such as collagen fibers. Herein, the damage caused by oxidative stress, is preferably skin damage, more preferably the damage to skin cells, and the damaged component of the cell is, unless indicated otherwise, a lipid, preferably a cell lipid, very preferably a cell membrane lipid. Preferably herein, the damage of the lipid relates to the oxidation of said lipid. A major mediator of oxidative stress caused by UV light or UV light in combination with an additional agent, in particular in the skin, is singlet oxygen. Without being bound by theory, singlet oxygen activates enzymes which generate ROS including radicals.

However, it is immediately clear to the skilled person that a condition which leads to damage of lipids, in particular cell lipids, i.e. their oxidation due to oxidative stress, very likely also causes further damage to the cell and its environment. For example, said oxidative stress comprising increased levels of reactive oxygen species (ROS), may cause inter alia DNA base damage, as well as strand breaks in DNA. Furthermore, some reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signaling.

Singlet oxygen, systematically named dioxygen(singlet) and dioxidene, is a gaseous inorganic chemical with the formula 0=0 (also written as ¹ [O2] or ¹02), which is in a quantum state where all electrons are spin paired. It is kinetically unstable at ambient temperature, however the rate of decay is slow. In terms of its chemical reactivity, singlet oxygen is far more reactive toward organic compounds more prevalent triplet ground state of O2. Singlet oxygen is responsible for the photodegradation of many materials including lipids such as cell membrane lipids, but it may be still used in phototherapy.

Reactive oxygen species (ROS) are chemically reactive chemical species containing oxygen, for example, inter alia peroxides, superoxide, hydroxyl radical, singlet oxygen and alpha- oxygen. The reduction of molecular oxygen (O2) produces superoxide (Ό2-) and is the precursor of most other reactive oxygen species. Dismutation of superoxide produces hydrogen peroxide (H2O2). Hydrogen peroxide in turn may be partially reduced to hydroxyl radical (ΌH).

Some cell lipids are polyunsaturated fatty acids which are primary targets for free radical and singlet oxygen oxidations, particularly arachidonic acid and linoleic acid. For example, singlet oxygen might attack linoleic acid to produce 13-hydroxy-9Z,l lE-octadecadienoic acid, 9- hydroxy-10E,12-Z-octadecadienoic acid, 10-hydroxy-8E,12Z-octadecadienoic acid, and 12- hydroxy-9Z-13-E-octadecadienoic. Similar attacks on arachidonic acid produce a large set of products including various isoprostanes, hydroperoxy- and hydroxy- eicosatetraenoates, and 4-hydroxyalkenals. Suitable markers for lipid peroxidation, and thus oxidative stress, are, for example, inter alia linoleic and arachidonic acid products, and in particular, F2-isoprostanes (Forman et ak, Free Radic Biol Med. 2015 Jan;78:233-5).

Lipid peroxidation, lipid oxidation, and the generation of lipid oxidation products or oxidized lipids, as used herein, refer to the oxidative degradation of lipids which is the process in which (free) radicals remove electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which lie methylene bridges (-CH2-) that possess especially reactive hydrogen atoms. A methylene bridge, is any part of a molecule with formula -CH2-, namely, a carbon atom bound to two hydrogen atoms and connected by single bonds to two other distinct atoms in the rest of the molecule. As with any radical reaction, the reaction consists of three major steps: initiation, propagation, and termination. The chemical products of this oxidation are known as lipid peroxides or lipid oxidation products. Initiation is the step in which a fatty acid radical is produced. The most notable initiators in living cells are reactive oxygen species (ROS) which combines with a hydrogen atom to make water and a fatty acid radical. The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl-fatty acid radical. This radical is also an unstable species that reacts with another free fatty acid, producing a different fatty acid radical and a lipid peroxide, or a cyclic peroxide if it had reacted with itself. This cycle continues, as the new fatty acid radical reacts in the same way. When a radical reacts with a non-radical, it always produces another radical, which is why the process is called a "chain reaction mechanism". The radical reaction stops when two radicals react and produce a non-radical species. This happens only when the concentration of radical species is high enough for there to be a high probability of collision of two radicals.

Oxidative stress which is associated with skin damage and/or which leads to skin damage, especially affects dermal fibroblasts and basal keratinocytes (Marionnet et ak, PLoS One. 2014; 9: el05263). Said oxidative stress may not only cause lipid peroxidation, but also apoptosis of the cells. Furthermore, oxidative stress may alter collagen and elastic fibers. Overall, those effects lead to skin ageing. Since the trigger is, unless otherwise indicated, UV light, said skin ageing usually also refers herein to photoaging.

Skin ageing, as used herein, further refers to skin drying, thickening, sagging and/or wrinkling of the skin, and/or a leathery appearance of the skin.

Preferably, the oxidative stress according to the invention is associated with and/or leads to lipid oxidation, lipid peroxidation, photodamage (damage of biomolecules by light), skin ageing, the formation of reactive lipid species, formation of lipid adducts to proteins, protein crosslinking, modifications of proteins by advanced lipoxidation products, formation of protein aggregates, reactive oxygen species (ROS), mitochondrial dysfunction, cellular senescence associated ROS, fenton reaction of lipids, a lipid hydroperoxide chain reaction, and/or sunburn.

In conclusion, determining the oxidation of a sensor lipid comprised in the lipid vesicle of the invention which mimics aspects of the skin, i.e. of skin cells, by a trigger as provided herein, allows to determine both, the oxidative stress which is associated with skin damage and/or which leads to skin damage, and the damage of a skin component by oxidative stress. Herein, said trigger is, unless otherwise indicated, UV light. Of note, determining the oxidation of said sensor lipid according to the invention is preferably done in vitro, preferably in a cell free system.

Thus, the means and methods provided herein further allow determining, or at least estimating, how much an agent enhances UV light induced the skin damage or how much a compound or composition prevents and/or protects from UV light induced skin damage (without or without enhancement by an additional agent). Notably, this is achieved without actually measuring the damage to true skin, but the damage to a cell or skin mimic, i.e. the oxidation of the sensor lipid comprised in the lipid vesicle provided herein.

Thus, the invention also relates to a method for determining or estimating the skin damaging activity of UV light or UV light in combination with an additional agent, wherein said method comprises a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid. Preferably herein, said skin component is a lipid, preferably a cell lipid, very preferably a cell membrane lipid. In particular, said method for determining or estimating the skin damaging activity of UV light or UV light in combination with an additional agent can further comprise a step of determining if or how much said agent enhances said skin damaging activity caused by the UV light, preferably the oxidative stress. Preferably, the skin damaging activity of said agent is determined by comparing the oxidative stress caused by said UV light without said agent to the oxidative stress caused by said UV light in combination with said agent.

As mentioned above, said skin damaging activity refers to the damage UV light or UV light in combination with an additional agent generates in a cell or skin mimic, which in turn refers in particular to the oxidation of the sensor lipid comprised in the lipid vesicle provided herein. Thus, the skin damaging activity is herein preferably mediated by the oxidative stress described herein.

When reference is made herein to “any of the methods of the invention” or “any of the methods according to the invention”, the same applies to the method for determining oxidative stress caused by UV light or UV light in combination with an additional agent according to the invention and as provided herein; the method for selecting a compound or composition which prevents and/or protects from the oxidative stress caused by the UV light or the UV light in combination with the additional agent according to the invention and as provided herein; the method for producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent according to the invention and as provided herein; the method for determining the damage of a skin component caused by UV light or UV light in combination with an additional agent according to the invention and as provided herein; the method for determining the enhancement of oxidative stress caused by an agent in combination with UV light according to the invention and as provided herein; and the method for determining or estimating the skin damaging activity of UV light or UV light in combination with an additional agent according to the invention and as provided herein.

Any of said methods of the invention comprise at least a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non- oxidizable reference lipid.

In particular, all embodiments as provided herein specifying the lipid vesicle and/or any of the lipids comprised therein, the UV light and/or the agent, may be employed in any of said methods of the invention, unless indicated otherwise. If a specific link between one of the methods of the invention and an embodiment is made, said embodiment is preferred and/or particularly useful for said method.

Preferably, any of the methods of the invention as provided herein is an in vitro method. The term “m vitro ”, as used herein, refers to a condition or environment outside of a living organism, preferably outside of a cell. Preferably, said in vitro condition or environment is cell free. Preferably, said cell free in vitro condition or environment does not comprise a crude cell extract.

The terms “UV”, “ultraviolet”, “UV light”, “UV irradiation” and “UV radiation” are used interchangeably herein and the same is true for the subgroups of UV light such as UVA or UVB. The terms “UV-A” “UVA”, with or without further specification as “light”, “irradiation” or “radiation”, are used interchangeably herein.

UV light, as used herein, refers to ultraviolet light, which is an electromagnetic radiation with a wavelength from 10 nm to 400 nm - which is shorter than that of visible light but longer than X-rays. UV radiation is, for example, present in sunlight, and contributes about 10% of the total electromagnetic radiation output from the Sun. Of the ultraviolet radiation that reaches the Earth's surface, more than 95% is UVA, and the rest is UVB. There is essentially no UVC.

Herein, the UV light according to the invention is capable of oxidizing the at least one oxidizable sensor lipid comprised in the lipid vesicle of the invention. In other words, the sensor lipid and the UV must be selected such that said UV light can oxidize said sensor lipid within a reasonable time, e.g. 1 min, 10 min, 1 hour or 1 day to an extent that oxidation products of said lipid sensor lipid are detectable by methods known in the art, in particular by mass spectrometry, preferably by HPLC/MS-MS.

Preferably herein, the UV light is longwave UV light, UVA and/or has a wavelength between 315 nm and 400 nm. In particular, as used herein, UVA has a wavelength between 315 nm and 400 nm.

The oxidative stress, as used herein, in particular the oxidative stress which is associated with and/or which leads to skin damage, is caused in particular by UVA.

Moreover, the compound or composition obtainable by the method for producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent according to present invention, preferably prevents and/or protects from oxidative stress caused by UVA or UVA in combination with an additional agent, preferably because said UVA is employed in said method and/or in the selection method according to the invention.

Preferably herein, the UV light, preferably the UVA, has a fluency of 1 J/cm² to 200 J/cm², preferably of 10 J/cm² to 30 J/cm². Fluency, as used herein, refers to the radiant exposure or radiant fluence which is the radiant energy received by a surface per unit area, or equivalently irradiance of a surface integrated over time of irradiation and has, for example, the unit J/cm².

Preferably herein, the agent of the invention comprises at least one pollutant.

Pollutants are, for example, as classified by the Environmental Protection Agency (EPA) of the EISA inter alia: lead (i.e. from metal & industrial processing plants), particulate matter (i.e. soot, exhaust from industry), nitrogen oxide (i.e. from car exhaust), sulphur oxide (i.e. from industrial plants) and ozone. Preferably herein, a pollutant contributes to air pollution, and is thus selected from particulate matter (PM), which are commonly referred to as fine (PM2.5, PM1₀) or coarse particles, and gases (O3, CO2, CO, SO2, NO2) or volatile organic compounds. Small particles are typically produced by combustion and the larger ones by mechanical processes that create and then suspend dust particles in the wind. Furthermore, a pollutant herein can comprise a secondary pollutant, preferably a secondary pollutant which contributes to smog (i.e. in the troposphere), for example, inter alia a peroxyacetyl nitrate. Secondary pollutants may arise from photochemical reactions between the pollutants as described above, heat and UV radiation.

Preferably herein, the pollutant comprises Diesel particulate matter (DPM), and/or at least one polycyclic aromatic hydrocarbon (PAH) and/or at least one nitrated form thereof (nitro- substituted PAHs; nitro-PAHs). In particular, any compound or combination of compounds comprised in DPM may be used as a pollutant herein. Reference is made to the Standard Reference Material 2975 of the National Institute of Standards and Technology of the USA, and the DPM (NIST2975) of Sigma- Aldrich.

In preferred embodiments, the agent as provided herein is capable of inducing and/or enhancing oxidative stress. Preferably, said agent is capable of enhancing the oxidative stress caused by UV light. In this case, an agent is preferred which has been demonstrated to have a synergistic effect with UV light, in particular with UVA. Diesel particulate matter is particularly suitable as such an agent because it is known to enhance the oxidative stress caused by UV light. Further suitable agents may be heavy metals, such as inter alia zinc, in particular zinc oxide, or psoralens.

In certain embodiments, the agent as provided herein is suspected of inducing and/or enhancing oxidative stress. Preferably, said agent is suspected of enhancing the oxidative stress caused by UV light. In this case, the agent is tested if or to what extent it enhances the effect caused by the UV light and/or the skin damaging activity of the UV light. As it is unknown in this case if said agent enhances said oxidative stress before performing an experiment according to invention, the agents to be screened are not particularly limited. However, preferably an agent suspected of enhancing the oxidative stress by UV light is a pollutant as described herein, in particular a pollutant which exists outside of buildings and thus could possibly functionally interact with the UV light comprised in sun light.

Preferably herein, the effect caused by the UV light or the synergistic effect of the UV light and the agent comprises oxidative stress and/or damage of a skin component, preferably oxidative stress.

In certain embodiments, the agent enhances a beneficial and/or therapeutic effect of the UV light. A beneficial effect of UV light is, for example, inter alia the production on Vitamin D or serotonin. A therapeutic effect is, in particular, an effect exploited in phototherapy which is for example used inter alia for the treatment of certain skin conditions such as psoriasis, eczema, jaundice, vitiligo, atopic dermatitis, and localized scleroderma.

In phototherapy, light therapy or heliotherapy, as used herein, a subject is exposed to daylight or light of certain wavelengths, in particular UV light by using artificial light sources such as polychromatic polarized light, lasers, light-emitting diodes, fluorescent lamps, dichroic lamps or very bright, full-spectrum light. The light is administered for a prescribed amount of time and, in some cases, at a specific time of the day. A suitable agent for phototherapy is, for example, inter alia a psoralen.

Photodynamic therapy is a form of phototherapy using nontoxic light-sensitive compounds that are exposed selectively to light, whereupon they become toxic to targeted malignant and other diseased cells. Malignant or diseases cell are, for example, inter alia cancer cells, tumor cells, cells infected by a virus or a bacterium, and/or immune cells contributing to an autoimmune response.

Thus, in certain embodiments, the agent becomes toxic in combination with UV light. Preferably, said toxicity is mediated by oxidative stress. Such an agent may be used for photodynamic therapy.

As described above, the lipid vesicle of the invention can be regarded as a cell mimic, in particular a skin cell mimic. As illustrated in the Examples, the inventors have further surprisingly found a skin mimic which not only mimics skin cells but also the extracellular matrix of the skin (see e.g. Example 3) by embedding said lipid vesicle in a collagen matrix. This allows to reproduce the biochemical reactions occurring in the skin exposed to UV light even more realistically. Thus, it is expected that the employment of such a skin mimic according to the invention in any of the methods of the invention may allow to even better determine the oxidative stress which is associated with and/or which leads to skin damage, and which is caused by UV light and or an UV light in combination with an additional agent. Thus, agents which are identified according to the invention wherein a skin mimic is employed may be particularly useful for their use in phototherapy and/or may be particularly hazardous agents which lead to skin ageing, skin damage and/or a skin disease in combination with UV light. Moreover, compounds or compositions which are identified according to the invention wherein a skin mimic is employed may be particularly useful for their use in preventing and/or protecting from said oxidative stress, since said compounds or compositions are preferably applied on the skin.

Thus, preferably herein, the lipid vesicle of the invention is contained in a medium, wherein said medium comprises a matrix, a gel or a solution.

Thus, the invention also relates to a medium containing the lipid vesicle of the invention, i.e., wherein said medium comprises a matrix, gel or a solution, as described herein,

In other words, preferably herein, the lipid vesicle of the invention is embedded in said medium. Preferably, the lipid vesicles of the invention are dispersed in said medium or a layer of said medium. Preferably said lipid vesicles are homogenously dispersed in said medium. Preferably, said medium enforces a certain type of localization of said lipid vesicles, in particular a homogenous dispersion. Thus, said medium is very preferably a gel-like matrix. Embedding the lipid vesicle of the invention in a medium is furthermore particularly preferred in any of the methods of the invention, when the UV light is employed in combination with an additional agent and/or when the efficacy of a compound or composition in preventing and/or protecting from oxidative stress is assayed, because said agent and/or said compound or composition may be also contained or embedded in said medium, or cover said medium. A medium which is a gel-like matrix is particularly useful when said agent or said compound or composition covers said medium, or in other words, is applied to the outside of said medium. Preferably, said agent is embedded in the medium according to the invention. Preferably said agent is Diesel particulate matter. Preferably the DPM concentration in said medium is 20 - 50 pg/ml, preferably 30 pg/ml, and/or 20 - 50 pg/mg, preferably 30 pg/mg.

Thus, in certain embodiments, the medium wherein the lipid vesicle of the invention is contained further comprises an agent according to the invention. Preferably, said agent comprises at least one pollutant according to the invention.

In preferred embodiments, the medium comprises at least one protein of the extracellular matrix such as inter alia collagen, a glycoprotein, fibronectin, laminin and/or a proteoglycan, preferably collagen. Preferably, said protein forms fibers or fibrils such as collagen fibrils, microfibrils, and elastic fibers, preferably collagen fibrils.

In certain embodiments, the medium further comprises hyaluronan.

Preferably, said medium comprising said protein(s) is a gel-like matrix. Of note, said proteins may be extracted from tissue and/or be recombinant proteins.

Preferably, said medium does not comprise cells or a crude cell extract. A crude cell extract does not include a highly purified cell extract, or isolated proteins or a combination of isolated proteins.

A lipid vesicle which is contained or embedded in a medium comprising at least one protein of the extracellular matrix, or a medium containing the lipid vesicle of the invention and at least one protein of the extracellular matrix is particularly useful as a skin mimic. As already indicated above, in such a skin mimic, the lipid vesicle is a cell mimic, and the medium mimics the extracellular matrix of the skin.

In preferred embodiments, the medium is covered by a UV light permeable membrane, preferably a nylon mesh. Preferably, said membrane contacts the medium. Covering the medium with a membrane is particularly useful, when an agent and/or a compound or composition is applied at the outside of the medium as it may prevent mixing of the medium and said agent and/or compound or composition. Many cosmetic compositions or cosmetics, as described herein, are creamy or gel-like, for example inter alia a make-up as illustrated in the appended Examples, and thus are preferably applied at the outside of said medium. Thus, a UV light permeable membrane is particularly useful when the effects of cosmetic compositions such as make-up, are determined according to the invention. Preferably, the cosmetic composition is applied on the outside of the medium in a quantity which reflects the ordinary cosmetic use of said composition. In particular, as illustrated in the appended Examples, 1-2 mg/cm² of a creamy composition such as a make-up may be applied at the outside of the medium, preferably on the UV light permeable membrane.

Preferably, said UV light permeable membrane is permeable to UVA. Preferably, said permeable membrane is a mesh or a lattice.

In preferred embodiments, said compound or composition is contacting said UV light permeable membrane.

In preferred embodiments, said UV light permeable membrane and/or said compound or composition is facing towards a UV light source.

As described above, the UV light according to the invention is capable of oxidizing the at least one oxidizable sensor lipid comprised in the lipid vesicle of the invention. Thus, said at least one oxidizable sensor lipid of the invention is oxidizable by the UV light according to the invention, unless indicated differently.

In certain embodiments, the lipid vesicle of the invention comprises more oxidizable sensor lipid(s) than non-oxidizable reference lipid(s) in weight. Preferably, said lipid vesicle comprises at least 1.2-times more oxidizable sensor lipid(s) than non-oxidizable reference lipid(s) in weight. Preferably, said lipid vesicles comprises at most 99 times, preferably at most 90 times more oxidizable sensor lipid(s) than non-oxidizable reference lipid(s) in weight. In particular, the ratio of the weight of the at least one oxidizable sensor lipid over the weight of the at least one non-oxidizable reference lipid may be 1.2, 1.5, 2.0, 5.0, 9.0 or 99.0. Preferably, said weight ratio is between 1.5 and 4.0, preferably 2.3 +/- 10%.

The lipid vesicle of the invention may be a liposome or a micelle. A micelle in aqueous solution typically forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic single-tail regions in the micelle center. A micelle is thus composed of a lipid monolayer. A liposome is a spherical vesicle having at least one lipid bilayer. A lipid bilayer is a thin polar membrane made of two layers of lipid molecules. A liposome may be an unilamellar liposome or a multilamellar liposome. An unilamellar liposome is a spherical vesicle, bounded by a single bilayer of an amphiphilic lipid or a mixture of such lipids, containing aqueous solution inside the chamber. A multilamellar liposome has several layers of lipid bilayers between which there is an aqueous solution. Preferably, the lipid vesicle of the invention is a liposome.

In certain embodiments, the lipid vesicle of the invention has a mean vesicle diameter between 30 nm and 1000 nm, preferably between 50 nm and 600 nm, preferably between 100 nm and 300 nm. In particular, the diameter refers to the hydrodynamic diameter that relates to the hydrodynamic size which is obtained, for example, by Dynamic Light Scattering and is defined as the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured (see e.g. the INFORM WHITE PAPER 2011, Malvern Instruments Limited). In a plurality of lipid vesicles, which is, for example, typically used for any of the methods of the invention, individual lipid vesicles may have a diameter which is above or below said mean vesicle diameter. Methods to determine the size of a lipid vesicle are known in the art, for example, but not limited to Nanoparticle Tracking Analysis. Particularly suitable is imaging of the vesicles with the Nanosight technology (Malvern instruments), preferably with Nanosight 300.

As illustrated in the appended Examples, it was surprising that a lipid vesicle according to the invention could be produced.

Thus, the invention further relates to a method for producing a lipid vesicle, said method comprising the steps of

(a) mixing at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid,

(b) drying said mixture,

(c) resuspending the dried mixture, and

(d) agitating, preferably vortexing, the resuspended mixture.

Preferably, in step (b) the mixture of (a) is dried as a thin film. Preferably, in step (c), the dried mixture of (b) is resuspended with a buffer, preferably a lx Hanks Balanced Salt Solution (HBSS). Preferably, the vortexing in step (d) is performed with an ordinary vortexing device at full speed, preferably for 2 minutes.

In certain embodiments, the resuspended mixture is sonicated in step (d) with or without agitating.

As regards the lipid vesicle produced by the production method provided herein, the same applies as described herein in the context of the lipid vesicle of the invention or any of the methods of the inventions wherein said lipid vesicle is used.

As already mentioned above, in particularly preferred embodiments, the lipid vesicle of the invention is contained in a medium, preferably a gel-like matrix which preferably comprises collagen.

Thus, the method for producing a lipid vesicle as provided herein preferably further comprises the steps of

(e) adding the lipid vesicles to an aqueous solution comprising an extracellular matrix protein,

(f) incubating said solution containing said lipid vesicles, and (g) applying pressure to said solution containing said lipid vesicles.

Preferably, in step (e), the extracellular matrix protein is collagen, preferably at a concentration of 2 - 4 mg/ml, preferably 3 mg/ml. Preferably, the aqueous solution comprises a buffer, preferably HBSS. Preferably, in step (f), the solution is incubated at 30 - 40 °C, preferably at 37°C, preferably for 2 hours. Preferably, in step (g), the pressure is applied by applying a weight of 40 - 55 g, preferably 47 g, per 3.6 cm² of the incubated solution. Preferably said pressure is applied for 90 min.

In case, Diesel particulate matter (DPM) is used as an agent which enhances the oxidative stress of UV light, DPM is added to the aqueous solution in step (e) of the method for producing a lipid vesicle as provided herein, preferably at a concentration of 20 - 50 pg/ml, preferably 30 pg/ml.

It has been surprisingly discovered, as illustrated e.g. in the appended Example 4, that the lipid vesicles maintain their integrity, in particular their size, in the collage matrix.

Thus, preferably herein, the lipid vesicle maintains its integrity, in particular its size, in the medium according to the invention.

As lipid, as used herein, refers to a biomolecule that is soluble in nonpolar solvents and amphiphilic. Thus, although not well dissolvable in an aqueous solution, a lipid, as used herein, has both hydrophilic (water-loving, polar) and lipophilic (fat-loving) properties. Lipids are, for example, inter alia fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids and prenol lipids. The term lipid also comprises oil and fat, such as cholesterol, and preferably, phospholipids and triglycerides. A triglyceride is an ester derived from glycerol and three fatty acids.

Herein, the lipid usually comprises a fatty acid. A fatty acid, as used herein, is a carboxylic acid with an aliphatic chain. In particular, fatty acids have 4 to 28 carbon atoms, which are usually aligned in an unbranched chain. Usually, the number of carbon atoms is an even number. However, the length of the fatty acid as used herein must allow the formation of a lipid vesicle. Thus, if a fatty acid is the only fatty acid comprised in the lipid according to the invention, said fatty acid may suitably have a length between 8 and 18 carbon atoms. When the lipid according to the invention comprises two fatty acids, each of the fatty acid may suitably have a length of at least 14 carbon atoms.

As a minimum, the fatty acid according to the invention thus comprises at least 8 carbon atoms, preferably at least 14 carbon atoms. In particular, the number of carbon atoms of a fatty acid, as used herein, denotes the length of the aliphatic chain of said fatty acid.

Very preferably herein, the lipid is a phospholipid. Phospholipids are a class of lipids that are a major component of all cell membranes. They can form lipid bilayers because of their amphiphilic characteristic. The structure of the phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are preferably joined together by a glycerol molecule. The phosphate groups may be modified with simple organic molecules such as choline, ethanolamine or serine. The hydrophilic phospholipid head preferably contains a negatively charged phosphate group and glycerol. The hydrophobic phospholipid tails preferably consist of two long fatty acid chains which avoid interactions with water. When placed in aqueous solutions, phospholipids are driven by hydrophobic interactions that result in the fatty acid tails aggregating to minimize interactions with water molecules. A phospholipid is, for example, but not limited to a glycerophospholipid such as inter alia phosphatidic acid (phosphatidate), phosphatidylethanolamine (cephalin), phosphatidylcholine, phosphatidylserine, phosphoinositides, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate, or a phosphosphingolipids such as inter alia sphingomyelin, ceramide phosphorylcholine, ceramide phosphorylethanolamine and ceramide phosphoryllipid.

Preferably herein, the phospholipid is a glycerophospholipid (phosphoglyceride) which refers to a glycerol-based phospholipid. Glycerophospholipid are the main component of biological membranes. Specifically, the term glycerophospholipid signifies any derivative of glycerophosphoric acid that contains at least one O-acyl, or O-alkyl, or O-alk-T- enyl residue attached to the glycerol moiety. The alcohol here is glycerol, to which two fatty acids and a phosphoric acid are attached as esters. Glycerophospholipids consists of various diverse species which usually differ slightly in structure. A glycerophospholipid is, for example, inter alia a plasmalogen or a phosphatidate.

Plasmalogens are a type of phosphoglyceride. The first carbon of glycerol has a hydrocarbon chain attached via an ether, not ester, linkage. The linkages are more resistant to chemical attack than ester linkages are. The second (central) carbon atom has a fatty acid linked by an ester. The third carbon links to an ethanolamine or choline by means of a phosphate ester.

Phosphatidates are lipids in which the first two carbon atoms of the glycerol are fatty acid esters, and the third is a phosphate ester. The phosphate serves as a link to another alcohol- usually ethanolamine, choline, serine, or a carbohydrate. The identity of the alcohol determines the subcategory of the phosphatidate. There is a negative charge on the phosphate and, in the case of choline or serine, a positive quaternary ammonium ion. (Serine also has a negative carboxylate group.) The presence of charges give a "head" with an overall charge. The phosphate ester portion ("head") is hydrophilic, whereas the remainder of the molecule, the fatty acid "tail", is hydrophobic. These are important components for the formation of lipid bilayers. Phosphatidates are, for example, inter alia phosphatidylethanoamines or phosphatidylcholines.

Preferably herein, the phosphatidate is a phosphatidylcholine. Phosphatidylcholines may be also lecithins. Choline is the alcohol, with a positively charged quaternary ammonium, bound to the phosphate, with a negative charge. Lecithins are present in all living organisms. Lecithinis designates any group of yellow-brownish fatty substances occurring in animal and plant tissues which are amphiphilic. Lecithins comprise, for example, inter alia glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidyl serine, and phosphatidic acid.

Thus, in certain embodiments, the lipid is a lecithin.

Preferably herein, the lipid is a cell membrane lipid, for example, but not limited to a phospholipid and/or a glycolipid. Membrane lipids are a group of lipids which form the double-layered surface of all cells (lipid bilayer). The terms “cell membrane lipid” and “membrane lipid” are used interchangeably herein.

In preferred embodiments, the cell membrane lipid is a phospholipid, preferably a glycerophospholipid, more preferably a phosphatidate, most preferably a phosphatidylcholine.

In certain embodiments, the cell membrane lipid is a glycolipid.

Glycolipids are lipids with a carbohydrate attached by a glycosidic (covalent) bond. Glycolipids are found on the surface of all eukaryotic cell membranes, where they extend from the phospholipid bilayer into the extracellular environment. The essential feature of a glycolipid is the presence of a monosaccharide or oligosaccharide bound to a lipid moiety. The saccharides that are attached to the polar head groups on the outside of the cell are the ligand components of glycolipids, and are likewise polar, allowing them to be soluble in the aqueous environment surrounding the cell. The lipid and the saccharide form a gly coconjugate through a glycosidic bond, which is a covalent bond. The anomeric carbon of the sugar binds to a free hydroxyl group on the lipid backbone. The structure of these saccharides varies depending on the structure of the molecules to which they bind. Glycolipids are, for example, but not limited to glyceroglycolipids, galactolipids, sulfolipids, glycosphingolipids, cerebrosides, galactocerebrosides, glucocerebrosides, sulfatides, gangliosides, globosides, glycophosphosphingolipids, glycophosphatidylinositols.

In certain embodiments, the cell membrane lipid is a sphingolipid. Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine.

Of note, a lipid, as used herein, may be at same time, for example, inter alia a cell membrane lipid, a phospholipid, a glycolipid, a sphingolipid, fat and/or a lecithin.

The lipids and/or fatty acids, in particular, the unsaturated fatty acids described herein, can be synthesized and/or extracted from a natural source. Suitable extracts are disclosed, for example, in W02008119556.

The definitions and preferences regarding the lipids as described herein, i.e. above, apply similarly to the unsaturated and saturated lipids as provided herein, except for the features which distinguish unsaturated lipids from saturated lipids. The distinguishing features are well known in the part, and least partly, described herein, in particular in the following.

A saturated compound is a chemical compound that resists addition reactions such as inter alia hydrogenation, oxidative addition, and binding of a Lewis base.

An unsaturated lipid, as used herein, comprises an unsaturated fatty acid. An unsaturated fatty acid refers herein to a fatty acid chain with at least one double bond. Preferably herein, the unsaturated lipid comprises a polyunsaturated fatty acid. A polyunsaturated fatty acid refers herein to a fatty acid chain with at least two double bonds. The term "unsaturated" refers to the fact that the molecule contains less than the maximum amount of hydrogen (if there were no double bonds). These materials exist as cis or trans isomers depending on the geometry of the double bond. The hydrocarbon chains in trans fats align more readily than those in cis fats, but less well than those in saturated fats. The position of the carbon-carbon double bonds in carboxylic acid chains in fats is designated by Greek letters. The carbon atom closest to the carboxyl group is the alpha carbon, the next carbon is the beta carbon and so on. In fatty acids the carbon atom of the methyl group at the end of the hydrocarbon chain is called the omega carbon because omega is the last letter of the Greek alphabet. Omega-3 fatty acids have a double bond three carbons away from the methyl carbon, whereas omega-6 fatty acids have a double bond six carbons away from the methyl carbon. A saturated lipid, as used herein, does not comprise any unsaturated fatty acid, but usually at least one saturated fatty acid. Thus, all fatty acids comprised in a saturated lipid are saturated. In particular, in a saturated fatty acid, essentially all carbon (C) atoms are linked by a single bond and not by a double bond.

Thus, the lipid as provided herein usually comprises a fatty acid which is either saturated or unsaturated as indicated. Evidently, the fatty acid comprised in a lipid is bound to the rest of said lipid. Preferably said bond is an ether or ester bond. The skilled person immediately understands which modifications occur during such binding, e.g. the removal of hydrogen and/or oxygen atoms from the fatty acid.

Herein, the sensor lipid comprised in the lipid vesicle of the invention is unsaturated. In particular, at least one of the fatty acids comprised in said sensor lipid is unsaturated.

The terms “reference lipid” and “calibrator lipid” are used interchangeably herein.

Preferably herein, the reference (calibrator) lipid comprised in the lipid vesicle of the invention is saturated. In particular, all of the fatty acids comprised in said reference lipid are saturated.

Herein, the sensor lipid and the reference lipid must be capable of forming a lipid vesicle according to the invention, in particular in an aqueous solution and/or in the medium as provided herein.

Herein, the sensor lipid according to the invention is oxidizable by UV light. Herein, the reference lipid according to the invention it not oxidizable by UV light (non-oxidizable).

Thus, preferably herein, the oxidizable sensor lipid comprises an unsaturated fatty acid.

Preferably herein, the unsaturated fatty acid is a polyunsaturated fatty acid. The greater the degree of unsaturation in a fatty acid, in particular the more double bonds in the fatty acid, the more vulnerable it is to lipid peroxidation. Thus, said polyunsaturated fatty acid comprises preferably at least four double bonds, preferably exactly four double bonds.

Preferably herein, the double bonds of the polyunsaturated fatty acid are arranged in a certain way. In particular, the double bonds are separated by methylene bridges (-CH2-) that possess especially reactive hydrogen atoms as described above in the context of oxidative stress and lipid peroxidation. A carbon atom is herein also denoted by a “C”.

In certain embodiments, the unsaturated fatty acid comprises 16, 18, 20 or 22 C atoms, preferably 20 C atoms.

In preferred embodiments, the unsaturated fatty acid is an omega-3 fatty acid or an omega-6 fatty acid. Preferably, the unsaturated fatty acid is an omega-6 fatty acid.

Omega-3 fatty acids are, for example, inter alia hexadecatrienoic acid, a-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, clupanodonic acid, docosahexaenoic acid, tetracosapentaenoic acid, and tetracosahexaenoic acid. Omega-6 fatty acids are, for example, inter alia linoleic acid, gamma-linolenic acid, calendic acid, eicosadienoic acid, dihomo- gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid, and tetracosapentaenoic acid.

In very preferred embodiments, the unsaturated fatty acid is arachidonic acid.

In the most preferred embodiments, the oxidizable sensor lipid is a phosphatidylcholine comprising an arachidonic acid. Preferably, said oxidizable sensor lipid further comprises a palmitic acid. Most preferably, said oxidizable sensor lipid is l-palmitoyl-2-arachidonoyl-sn- glycero-3-phosphorylcholine (PAPC; also known inter alia as 2-Arachidonoyl-l-palmitoyl- sn-glycero-3-phosphocholine or (2R)-2-[(5Z,8Z,l 1Z,14Z)-5,8,1 l,14-Icosatetraenoyloxy]-3- (palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate):

Of note, the skilled person recognizes immediately when the name of a compound has to change slightly based on the context. For example, the term “arachidonic acid” becomes “arachidonoyl” is the context of PAPC, and palmitic acid becomes “palmitoyl”. Thus, the terms “arachidonic acid” and “arachidonoyl”, for example a considered equivalent. Similar considerations apply to the other molecules described herein, i.e. the lipids and/or fatty acids. Preferably herein, the non-oxidizable reference lipid comprises a saturated fatty acid and does not comprise any unsaturated fatty acid.

Saturated acids are, for example, inter alia propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid.

In certain embodiments, the saturated fatty acid comprises 14, 16, 18, 20 or 22 C atoms, preferably 16 C atoms.

In preferred embodiments, the saturated fatty acid is palmitic acid.

In the most preferred embodiments, the non-oxidizable reference lipid is a phosphatidylcholine comprising a palmitic acid. Preferably, said oxidizable sensor lipid comprises two palmitic acids. Most preferably, said oxidizable sensor lipid is dipalmitoylphosphatidyl choline (DPPC; also known inter alia as 1 ,2-dipalmitoyl-.sn-glycero- 3-phosphocholine or [(2R)-2,3-di(hexadecanoyloxy)propyl] 2-(trimethylazaniumyl)ethyl phosphate): The use of DPPC in lipid vesicles if further particularly preferred because DPPC is one of the most abundant components of eukaryotic cell membranes. Thus, lipid vesicles comprising DPPC may be particularly suitable as cell mimics as described herein.

As described above, the treatment and/or contacting with UV light or UV light in combination with an additional agent leads to oxidation of the oxidizable sensor lipid.

Thus, preferably herein, at least one oxidation product of the oxidizable sensor lipid(s) comprises at least one residue comprising at least one oxygen in place of at least one double bond comprised in said oxidizable sensor lipid(s). Preferably, said residue(s) are in place of the double bond(s) of the (poly)unsaturated fatty acid(s) comprised in said sensor lipid(s). The residue resulting from oxidation in place of a double bond is, for example, inter alia a hydroperoxide, a hydroxy group, an epoxide, a cyclopentanone, an aldehyde, an isoprostane, an isolevuglandin or an isothromboxane. Further oxidation products are described, for example, in Bochkov (2010), Antioxid Redox Signal. 12(8): 1009-59.

In preferred embodiments, the at least one residue comprising at least one oxygen in place of at least one double bond comprised in said oxidizable sensor lipid(s), comprises a ketone, an aldehyde, a hydroperoxide and/or an isoprostane.

In very preferred embodiments, the lipid vesicle according to the invention comprises an oxidizable sensor lipid and a non-oxidizable reference lipid, wherein said sensor lipid is a phosphatidylcholine comprising an arachidonic acid, wherein said reference lipid is a phosphatidylcholine comprising a palmitic acid, wherein said reference lipid does not comprise an unsaturated fatty acid, wherein said sensor lipid is oxidizable by UV light, and wherein the oxidation product of said sensor lipid comprises in place of the double bond(s) of said arachidonic acid a ketone, an aldehyde, a hydroperoxide and/or an isoprostane. Preferably said lipid vesicle is in a medium which is a gel-like matrix comprising collagen.

Herein, at least one oxidation product of the sensor lipid and at least one reference lipid, comprised in the lipid vesicle according to the invention, must be quantifiable by mass spectrometry.

Preferably herein, the mass spectrometry is tandem mass spectrometry (MS/MS). Preferably herein, the mass spectrometry is combined with high-performance liquid chromatography (HPLC). Thus, very preferably herein, the mass spectrometry is HPLC-MS/MS.

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. Tandem mass spectrometry is a technique in instrumental analysis where two or more mass spectrometers are coupled together using an additional reaction step to increase their abilities to analyze chemical samples, in particular biomolecules. The molecules of a given sample are ionized and the first spectrometer (designated MSI) separates these ions by their mass-to-charge ratio (often given as m/z or m/Q). Ions of a particular m/z-ratio coming from MSI are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ion-molecule reaction, or photodissociation. These fragments are then introduced into the second mass spectrometer (MS2), which in turn separates the fragments by their m/z- ratio and detects them. The fragmentation step makes it possible to identify and separate ions that have very similar m/z-ratios in regular mass spectrometers. HPLC is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

Liquid chromatography-mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography such as HPLC with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides structural identity of the individual components with high molecular specificity and detection sensitivity. In addition to the liquid chromatography and mass spectrometry devices, an LC-MS system contains an interface that efficiently transfers the separated components from the LC column into the MS ion source. Nowadays, most extensively applied LC-MS interfaces are based on atmospheric pressure ionization (API) strategies like electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI).

Herein, preferably a hybrid mass spectrometer is used for mass spectrometry of the lipids comprised in the lipid vesicle, in particular after treatment and/or contacting with UV light or UV light in combination with an additional agent. A hybrid mass spectrometer is a device for tandem mass spectrometry that consists of a combination of two or more m/z separation devices of different types.

The hybrid mass spectrometer comprises, for example, inter alia a quadrupole mass analyzer or a time-of-flight mass spectrometer. Preferably herein, the mass spectrometer comprises a quadrupole mass analyzer, preferably a QTRAP® mass spectrometers. Preferably herein, the mass spectrometer is equipped with an electro-spray ion source.

In case, the lipid is a phosphatidylcholine, the detection of said lipid is preferably done in positive ion mode, by selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) of MS/MS transitions, and/or by using a phosphatidylcholine specific product ion (i m/z 184) which corresponds to the cleaved phosphocholine residue.

Preferably herein, the amount of oxidative stress is determined by determining the amount of oxidation of the at least one oxidizable sensor lipid comprised in the lipid vesicle according to the invention.

It has been surprisingly found that the quantification of oxidative stress is particularly robust, when the amount of oxidation products of the sensor lipid extracted from the lipid vesicles upon contacting and/or treating with the UV light or the UV light in combination with an additional agent is normalized by the amount of the reference lipid(s) extracted from the same lipid vesicles (see e.g. the appended Examples 5 and 6). Said normalization allows to control, in particular, for much of the variance in sample quantity and sample handling which occurs before the individual lipids are actually quantified, in particular by mass spectrometry. Thus, said normalization allows to determine said oxidative stress with a reduced variability. Therefore, fewer replicate measurements are required for obtaining a reliable quantification of the oxidative stress.

Thus, preferably herein, any of the methods of the invention further comprises a step of determining the amount of oxidation of the at least one oxidizable sensor lipid. Preferably, determining said amount of oxidation comprises the steps of (a) determining the amount of at least one oxidation product of the oxidizable sensor lipid(s), (b) determining the amount of the at least one non-oxidizable reference lipid, and (c) normalizing the amount of the at least one oxidation product of the oxidizable sensor lipid(s) by the amount of at least one of said reference lipid(s).

Preferably herein, said amounts of the at least one oxidation product of the oxidizable sensor lipid(s) and the at least one non-oxidizable reference lipid are determined by mass spectrometry, preferably by liquid chromatography combined with mass spectrometry, very preferably by HPLC-MS/MS.

In case the lipid vesicles of the invention are contained in a medium, said lipid vesicles are preferably isolated from said medium before they are subjected to mass spectrometry as described herein. In certain embodiments, the isolation of lipid vesicles from the medium, in particular a gel like matrix, comprises the steps of

(a) homogenizing the medium comprising the lipid vesicles in a mix comprising methanol, and acetic acid,

(b) centrifuging the homogenate,

(c) purifying the supernatant by hexane liquid-liquid extraction, and

(d) extracting the lipid vesicle from the purified supernatant comprising a mix of formic acid and chloroform.

Preferably, butylhydroxytoluene is employed additionally in said homogenizing, purification and/or extraction steps. Further details on the isolation of lipid vesicles are provided herein in the appended Example 5 and Gruber et al., J Lipid Res. 2012 Jun;53(6): 1232-42.

Moreover, in certain embodiments, the amount of oxidation of the at least one oxidizable sensor lipid treated and/or contacted with the UV light or the UV light in combination with the additional agent is compared to a control not treated and/or contacted with said UV light or said UV light in combination with said additional agent. Although, the normalization as described above already controls for many sources that affect the quantification of the lipids, a further negative control as described in the previous sentence is useful since further parameters, e.g. of the measurement itself may affect the quantification of oxidative stress.

In certain embodiments, any of the methods of the invention further comprises a step of selecting a compound or composition which prevents and/or protects from the oxidative stress caused by the UV light or the UV light in combination with the additional agent, wherein said selection comprises determining the efficacy of said compound or composition in preventing and/or protecting from said oxidative stress, and wherein determining said efficacy comprises treating, contacting and/or covering the lipid vesicle with said compound or composition prior to and/or during treating and/or contacting said lipid vesicle with the UV light or the UV light in combination with the additional agent.

In certain embodiments, any of the methods of the invention further comprises a step of producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of selecting said compound or composition according to the invention.

As already mentioned above, the means and methods according to the invention are particularly useful for selecting and/or producing a compound or composition which prevents and/or protects from oxidative stress caused by UV light or UV light in combination with an additional agent. As oxidative stress may lead to skin ageing, skin damage, a cancerous modification of the skin, and/or a skin disease, therapeutic uses and non-therapeutic uses of said compound or composition are provided herein. In particular, said compound or composition is a medicament and/or a pharmaceutical composition when it is used for a method of treating a disease. In particular, said compound or composition is a cosmetic composition and/or a cosmetic when it is used for cosmetic, non-therapeutic purposes only. However, a compound or a composition as provided herein may be both, a pharmaceutical and a cosmetic composition, and/or be used for therapeutic uses and non-therapeutic uses.

In certain embodiments, the compound or composition according to the invention is a pharmaceutical composition. Preferably, said pharmaceutical composition comprises a skin cream, a sunscreen, and/or an ointment. Preferably, the pharmaceutical composition is applied on the skin.

If the compound or composition according to the invention is a cream, a gel or an ointment, it preferably covers the medium comprising the lipid vesicle according to the invention in any of the methods of the invention, preferably by contacting the UV permeable membrane according to the invention. If the compound or composition according to the invention can be dissolved or suspended in an aqueous solution and/or is a hydrophilic liquid, it is preferably contained in the medium comprising the lipid vesicle according to the invention in any of the methods of the invention.

Cosmetics, or a cosmetic composition, as used herein, refers to a composition for enhancing or altering the appearance of the face or texture of the body. A make-up, as used herein, refers to a cosmetic. In particular, a make-up is a cosmetic composition designed for applying to the face. Thus, a make-up is for example, inter alia a lipstick, eye shadow, primer, concealer, foundation, face powder, blush.

Lipsticks are intended to add color and texture to the lips. Lip stains have a water or gel base and may contain alcohol to help the product stay on leaving a matte look. Lip sticks may be further used to moisturize, tint, and protect the lips. Primer is used to set the face before make-up is applied. This creates another layer between the skin to prevent acne and makeup clogging up pores. Primer creates an even tone throughout the skin and makes makeup last longer. Primer is applied throughout the face including eyes, lips, and lashes. This product has a creamy texture and applies smoothly. A concealer covers imperfections of the skin. Concealer is often used for any extra coverage needed to cover acne/pimple blemishes, undereye circles, and other imperfections. Concealer is often thicker and more solid than foundation, and provides longer lasting, more detailed coverage as well as creating a fresh clean base for all the rest of the makeup. This product also brightens up the skin and applying under the foundation can remove blemishes and discoloration because of acne scars. Foundation is used to smooth out the face by covering spots, acne, blemishes, or uneven skin tone. These are sold in a liquid, cream, or powder, or more recently in a mousse. Foundation provides sheer, matte, dewy or full coverage. Foundation primer is applied before foundation to fill out pores, create a dewy look or create a smoother finish. They usually come in cream formulas to be applied before foundation as a base. Face powder sets the foundation and under eye concealer, giving it a matte finish while also concealing small flaws or blemishes. It can also be used to bake the foundation, so that it stays on longer and create a matte finish. Rouge, blush, or blusher is cheek coloring to bring out the color in the cheeks and make the cheekbones appear more defined. Rouge comes in powder, cream, and liquid forms.

Hair care products, as used herein, are for example, inter alia shampoos or conditioners.

Skin care products, as used herein, are for example, inter alia moisturizers, cleansers, exfoliators and anti-aging treatments. In particular, said skin care products are applied on the skin, for example, to moisturize and/or clean the skin.

Thus, in preferred embodiments, the compound or composition according to the invention is a cosmetic composition. Preferably, said cosmetic composition comprises a make-up, a skin care product, a hair care and/or a sunscreen. Preferably, said skin care product is a skin cream.

In very preferred embodiments, the compound or composition according to the invention is a make-up.

Sunscreen, also known as sunblock, is a lotion, spray, gel, foam (such as an expanded foam lotion or whipped lotion), stick or other topical product that absorbs or reflects some of the sun's ultraviolet (UV) radiation and thus primarily helps protect against sunburn. Diligent use of sunscreen might also slow or temporarily prevent the development of wrinkles, dark spots and sagging skin. Depending on the mode of action, sunscreens can be classified into physical sunscreens (/.<?., zinc oxide and titanium dioxide, which stay on the surface of the skin and mainly deflect the sunlight) or chemical sunscreens (/.<?., UV organic filters, which absorb the UV light). In addition to moisturizers and other inactive ingredients, sunscreens contain one or more of the following active ingredients, which are either chemical or mineral in nature: organic chemical compounds that absorb ultraviolet light, inorganic particulates that reflect, scatter, and absorb UV light (such as inter alia titanium dioxide, zinc oxide, or a combination of both), organic particulates that mostly absorb UV light like organic chemical compounds, but contain multiple chromophores that reflect and scatter a fraction of light like inorganic particulates. An example is Tinosorb M. The mode of action is about 90% by absorption and 10% by scattering. The principal active ingredients in sunscreens are usually aromatic molecules conjugated with carbonyl groups. This general structure allows the molecule to absorb high-energy ultraviolet rays and release the energy as lower-energy rays, thereby preventing the skin-damaging ultraviolet rays from reaching the skin. So, upon exposure to UV light, most of the ingredients (with the notable exception of avobenzone) do not undergo significant chemical change, allowing these ingredients to retain the UV-absorbing potency without significant photodegradation. A chemical stabilizer is included in some sunscreens containing avobenzone to slow its breakdown. The stability of avobenzone can also be improved by bemotrizinol, octocrylene and various other photostabilisers.

Active compounds of sunscreens are, for example, inter alia p-Aminobenzoic acid, Padimate O, Phenylbenzimidazole sulfonic acid, Cinoxate Dioxybenzone, Oxybenzone, Homosalate, Menthyl anthranilate, Octocrylene, Octyl methoxycinnamate, Octyl salicylate, Sulisobenzone, Trolamine salicylate, Avobenzone, Ecamsule, Titanium dioxide, Zinc oxide, 4- Methylbenzylidene camphor, Parsol Max, Tinosorb M, Parsol Shield, Tinosorb S, Neo Heliopan AP Mexoryl XL, Benzophenone-9, Uvinul T 150, Uvinul A Plus, Uvasorb HEB, Parsol SLX, and Amiloxate.

Sunscreens or active compounds thereof may be also comprised in some hair care products. Currently, benzophenone-4 and ethylhexyl methoxycinnamate are the two sunscreen compounds most commonly used in hair products.

Thus, make-up and/or skin care products as provided herein may further comprise sunscreen or at least one active compound thereof, as described herein.

Preferably herein, the compound or composition further comprises a skin care active compound.

Preferably herein, the compound or composition further comprises at least one typical ingredient of cosmetics such as a modified natural oil or fat, iron oxide, talc, zinc oxide, bismuth oxychloride, a porous mineral and/or a pigment/colorant.

Since the lipid vesicle according to the invention is used in

(i) the method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, (ii) the method for determining the damage of a skin component caused by UV light or UV light in combination with an additional agent, and/or

(iii) the method for determining or estimating the skin damaging activity of UV light or UV light in combination with an additional agent, said lipid vesicle and said methods can be further used for determining the efficacy of a compound or composition in preventing and/or protecting from

(i) said oxidative stress, (ii) said damage of a skin component, and/or (iii) said skin damaging activity.

Thus, any of the methods of the invention can be used for identifying and/or producing a compound or composition which prevents and/or protects from oxidative stress, damage of a skin component and/or the skin damaging activity, of UV light or UV light in combination with an additional agent. Thus, the compound or the composition according to the invention can preferably be applied on the skin of an individual for therapeutic and/or non-therapeutic uses. Since the compound or composition prevents and/or protects from adverse effects of UV light or UV light in combination with an additional agent such as a pollutant, for example traffic and industry exhausts, said compound of composition is preferably a sunscreen, and or a make-up or skin care product intended for use as a sunscreen.

Herein, the protective and/or preventive effect determined for a compound or composition may comprise the chemical or physical capacity to block UV light, and/or prevent and/or protect from UV light induced oxidative modifications of biomolecules, in particular cell membrane lipids. In other words, the protective and/or preventive effect of the compound or composition according to the invention refers to the prevention of, protection from and/or reduction of oxidative stress, and/or damage of a skin component. Said prevention, protection and/or reduction may be achieved, for example, inter alia by reflecting and/or absorbing UV light, and/or the elimination of radicals. Thus, in contrast to at least most prior art methods, any of the methods of the invention allows to determine the effects of UV light or UV light in combination with an additional agent, and the prevention thereof and/or the protection therefrom, in particular the oxidative stress which is associated with skin damage and/or which leads to skin damage, without relying on assumptions how said effects or said prevention thereof and/or protection therefrom are achieved.

As described above, the oxidative stress and the skin damage according to the invention are preferably closely linked. As also already mentioned, said oxidative stress and skin damage may lead to skin ageing.

Thus, the compound or composition according to the invention is preferably used for protecting the skin from oxidative stress caused by UV light or UV light in combination with an additional agent. Preferably, said compound or composition is used for the prevention or retardation of skin ageing.

Preferably herein, the skin damage or oxidative stress according to the invention is further associated with and/or leads to a skin disorder and/or a cancerous modification of the skin.

If said skin damage and/or oxidative stress is associated with a skin disorder or a cancerous modification of the skin, it refers to the oxidative stress and/or the damage to a cell membrane lipid, for example lipid peroxidation, which is caused by and/or which causes said skin disorder or cancerous modification of the skin. If said skin damage and/or oxidative stress is caused by said skin disorder or cancerous modification of the skin, said skin damage and/or oxidative stress refers to a phenotype of said skin disorder or cancerous modification of the skin, in particular to a phenotype which concerns a cell membrane lipid, for example a certain oxidation product of a membrane lipid.

Thus, the skin disorder herein is, for example, but not limited to eczema, a photodermatose, an inflammatory skin disease, psoriasis, atopic dermatitis, contact dermatitis, actinic keratosis, an occupational dermatosis with UV exposure or chemical exposure, graft versus host disease, acne, a modification of the skin due to skin transplantation, a lipid storage disease, and/or sunburn.

If said skin damage and/or oxidative stress leads to a skin disorder or a cancerous modification of the skin, it refers to an adverse outcome of said skin damage and/or oxidative stress if said skin damage and/or oxidative stress is not prevented or treated and/or no protection from said oxidative stress has been applied.

Thus, the compound or composition according to the invention may be used for preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent. Preferably, said skin damage is caused by oxidative stress, and preferably, said skin disorder is caused by oxidative stress or said skin damage.

In a further aspect, the invention relates to a compound or composition as provided herein for use in preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent. Preferably, said skin damage is caused by oxidative stress, and preferably, said skin disorder is caused by oxidative stress or said skin damage.

Hence, the invention also relates to a method for preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said method comprises a step of administering the compound or composition according to the invention to an individual, and wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent. Preferably, said skin damage is caused by oxidative stress, and preferably, said skin disorder is caused by oxidative stress or said skin damage.

Herein, the skin disorder which may be prevented, ameliorated or treated by the compound or composition according to the invention, is preferably a photodermatose, actinic keratosis, cancer, polymorphic light eruption, juvenile spring eruption, actinic prurigo, hydroa vacciniforme, solar urticaria, chronic actinic dermatitis, a genodermatose/DNA repair deficient disorder, a cornification disorder, the Smith-Lemli-Opitz syndrome, porphyria, lupus erythematosus, erythema multiforme, atopic eczema, psoriasis, viral exanthemata, pemphigus, dermatitis herpetiformis, rosacea. Preferably, the cancer herein is skin cancer, in particular basal-cell skin cancer, squamous-cell skin cancer and/or melanoma. The compound or composition according to the invention may be particularly useful for immunosuppressed patients, which are, inter alia , more sensitive to UV light induced cancers.

In a further aspect, the invention relates to an agent according to the invention for use in the treatment of a skin disorder, wherein said agent enhances the oxidative stress caused by UV light.

Herein, the skin disorder which may be prevented, ameliorated or treated by the agent according to the invention, is selected from graft-versus-host disease, psoriasis, vitiligo, cancer, i.e. head and/or neck cancer, eczema, atopic dermatitis, polymorphous light eruption, cutaneous T-cell lymphoma, lichen planus, i.e. oral lichen planus, a bacterial, viral or fungus infection, acne, wet age-related macular degeneration, atherosclerosis and oral leukoplakia.

As already indicated above, it is expected that the invention as provided herein allows to identify improved compounds and composition for preventing or slowing down skin ageing, preventing skin damage, and/or preventing a cancerous modification of the skin and/or a skin disease. Moreover, it is also expected that the invention as provided herein allows to identify improved agents for phototherapy, and/or better categorize the hazardous properties of agents, in particular with respect to skin damage, a cancerous modification of the skin and/or a skin disease.

Furthermore, the present invention relates to a kit comprising the lipid vesicle of the invention. Said kit may further comprise a medium as provided herein in the context of the invention, preferably wherein said medium is suitable for preparing a skin mimic as described herein. Thus, the kit of the invention may comprise a medium containing the lipid vesicle of the invention. Furthermore, the medium containing said lipid vesicle may be covered by a UV light permeable membrane.

Furthermore, the kit of the invention may comprise an additional agent as provided herein in the context of the present invention. Furthermore, the kit of the invention may comprise an UV light source that is suitable for providing UV light as described herein in the context of the invention.

In particular, the kit of the invention may be used in a method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, a method for selecting or producing a compound or composition which prevents and/or protects from said oxidative stress, and/or in a method of the present invention.

Thus, the kit of the invention may further comprise a brochure or leaflet with instructions for carrying out at least one of said methods, i.e. at least one method of the present invention.

The present invention relates in particular to the following items:

1. A method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, and wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid.

2. The method of item 1, wherein the oxidative stress is associated with skin damage and/or leads to skin damage, preferably wherein said skin damage is associated with and/or leads to a skin disorder and/or a cancerous modification of the skin.

3. The method of items 1 or 2, wherein the oxidative stress is associated with and/or leads to lipid oxidation, lipid peroxidation, photodamage (damage of biomolecules by light), skin ageing, the formation of reactive lipid species, formation of lipid adducts to proteins, protein crosslinking, modifications of proteins by advanced lipoxidation products, formation of protein aggregates, reactive oxygen species (ROS), mitochondrial dysfunction, cellular senescence associated ROS, fenton reaction of lipids, a lipid hydroperoxide chain reaction, and/or sunburn. The method of any of items 1 to 3, wherein the oxidative stress is associated with and/or leads to a skin disorder and/or a cancerous modification of the skin. The method of any one of items 2 to 4, wherein said skin disorder is selected from the group consisting of eczema, a photodermatose, an inflammatory skin disease, psoriasis, atopic dermatitis, contact dermatitis, actinic keratosis, an occupational dermatosis with UV exposure or chemical exposure, graft versus host disease, acne, a modification of the skin due to skin transplantation, a lipid storage disease, and/or sunburn. The method of any of items 1 to 5, wherein the amount of oxidative stress is determined by determining the amount of oxidation of the at least one oxidizable sensor lipid. The method of item 6, wherein determining the amount of oxidation of the at least one oxidizable sensor lipid comprises determining the amount of at least one oxidation product of the oxidizable sensor lipid(s), determining the amount of the at least one non-oxidizable reference lipid, and normalizing the amount of the at least one oxidation product of the oxidizable sensor lipid(s) by the amount of said reference lipid. The method of item 7, wherein the amounts of the at least one oxidation product of the oxidizable sensor lipid(s) and the at least one non-oxidizable reference lipid are determined by liquid chromatography combined with mass spectrometry, preferably by HPLC-MS/MS. The method of any of items 6 to 8, wherein the amount of oxidation of the at least one oxidizable sensor lipid treated and/or contacted with the UV light or the UV light in combination with the additional agent is compared to a control not treated and/or contacted with said UV light or said UV light in combination with said additional agent.

The method of any of items 1 to 9, wherein the agent comprises at least one pollutant. 11. The method of item 10, wherein the at least one pollutant is capable of inducing and/or enhancing oxidative stress.

12. The method of items 10 or 11, wherein the at least one pollutant comprises diesel particulate matter (DPM), and/or at least one polycyclic aromatic hydrocarbon and/or at least one nitrated form thereof.

13. The method of any of the preceding items, wherein the UV light is capable of oxidizing the at least one oxidizable sensor lipid comprised in the lipid vesicle.

14. The method of any of the preceding items, wherein the UV light is longwave UV light.

15. The method of any of the preceding items, wherein the UV light is UVA.

16. The method of any of the preceding items, wherein the UV light has a wavelength between 315 and 400 nm.

17. The method of any of the preceding items, wherein the UV light has a fluency of 1 J/cm² to 200 J/cm², preferably of 10 J/cm² to 30 J/cm²

18. A method for selecting a compound or composition which prevents and/or protects from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein selecting said compound or composition comprises determining the efficacy of said compound or composition in preventing and/or protecting from said oxidative stress, wherein said oxidative stress is determined by the method of any of claims 1 to 17, and wherein determining said efficacy comprises treating, contacting and/or covering the lipid vesicle with said compound or composition prior to and/or during treating and/or contacting said lipid vesicle with the UV light or the UV light in combination with the additional agent.

19. A method for producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of selecting said compound or composition according to the method of item 18.

20. A compound or composition obtainable by the method of item 19. The method of items 18 or 19 or the compound or composition of item 20, wherein the compound or composition is a cosmetic composition. The method or the compound or composition of item 21, wherein the cosmetic composition comprises a make-up, a skin care product, a hair care, and/or a sunscreen, wherein the skin care product is preferably a skin cream. The method of any of items 18 to 22 or the compound or composition of any of items 20 to 22, wherein the compound or composition is a pharmaceutical composition. The method or the compound or composition of item 23, wherein the pharmaceutical composition comprises a skin cream, a sunscreen, and/or an ointment. The method of any of items 18 to 24 or the compound or composition of any of items 20 to 24, wherein the compound or composition comprises a skin care active compound. The method of any of items 18 to 25 or the compound or composition of any of items 20 to 25, wherein the efficacy of said compound or composition in preventing and/or protecting from said oxidative stress comprises the chemical or physical capacity to block UV light, and/or prevent and/or protect from UV light induced oxidative modifications of biomolecules, in particular cell membrane lipids. The compound or composition of any of items 20 to 26 for use in preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent, preferably wherein said skin disorder is a photodermatose, actinic keratosis, cancer, and/or cancerpolymorphic light eruption, juvenile spring eruption, actinic prurigo, hydroa vacciniforme, solar urticaria, chronic actinic dermatitis, a genodermatose/ DNA repair deficient disorder, a cornification disorder, the Smith- Lemli-Opitz syndrome, porphyria, lupus erythematosus, erythema multiforme, atopic eczema, psoriasis, viral exanthemata, pemphigus, dermatitis herpetiformis, rosacea. Use of the compound or composition of any of items 20 to 26 for protecting the skin from oxidative stress caused by UV light or UV light in combination with an additional agent. A lipid vesicle comprising at least one oxidizable sensor lipid and at least one non- oxidizable reference lipid. The method of any of the preceding items or the lipid vesicle of item 29, wherein the lipid vesicle is contained in a medium, and wherein said medium comprises a matrix, a gel or a solution, preferably a gel-like matrix. The method or the lipid vesicle of item 30, wherein the medium comprises collagen. The method or the lipid vesicle of items 30 or 31, wherein the medium comprises an agent, wherein said agent preferably comprises at least one pollutant. The method or the lipid vesicle of any of items 30 to 32, wherein the medium is covered by a UV light permeable membrane, preferably a nylon mesh. The method or the lipid vesicle of item 33, wherein said UV light permeable membrane is in contact with a compound or composition, and/or wherein said UV light permeable membrane is facing towards a UV light source. The method of any of the preceding items or the lipid vesicle of any of items 29 to 34, wherein the at least one oxidizable sensor lipid is oxidizable by UV light. The method of any of the preceding items or the lipid vesicle of any of items 29 to 35, wherein the lipid vesicle comprises more oxidizable sensor lipid(s) than non- oxidizable reference lipid(s) in weight. The method of any of the preceding items or the lipid vesicle of any of items 29 to 36, wherein the lipid vesicle comprises at least 1.2-times more oxidizable sensor lipid(s) than non-oxidizable reference lipid(s) in weight. The method of any of the preceding items or the lipid vesicle of any of items 29 to 37, wherein the lipid vesicle has a mean vesicle diameter between 30 nm and 1000 nm, preferably between 50 nm and 600 nm, preferably between 100 nm and 300 nm. The method of any of the preceding items or the lipid vesicle of any of items 29 to 38, wherein the oxidizable sensor lipid comprises an unsaturated fatty acid. The method or the lipid vesicle of item 39, wherein the unsaturated fatty acid comprises at least two double bonds. The method or lipid the vesicle of items 39 or 40, wherein the unsaturated fatty acid comprises at least four double bonds, preferably exactly four double bonds. The method or the lipid vesicle of items 40 or 41, wherein the double bonds of the unsaturated fatty acid are separated by methylene bridges. The method or the lipid vesicle of any of items 39 to 42, wherein the unsaturated fatty acid comprises 16, 18, 20 or 22 C atoms, preferably 20 C atoms. The method or the lipid vesicle of any of items 39 to 43, wherein the unsaturated fatty acid is an omega-6 fatty acid, preferably an arachidonic acid. The method of any of the preceding items or the lipid vesicle of any of items 29 to 44, wherein the oxidizable sensor lipid is a cell membrane lipid. The method of any of the preceding items or the lipid vesicle of any of items 29 to 45, wherein the oxidizable sensor lipid comprises a fatty acid chain that is ether bound or esterified. The method of any of the preceding items or the lipid vesicle of any of items 29 to 46, wherein the oxidizable sensor lipid is a phospholipid, preferably a phosphatidylcholine. The method of any of the preceding items or the lipid vesicle of any of items 29 to 47, wherein the oxidizable sensor lipid comprises l-palmitoyl-2-arachidonoyl-sn-glycero- 3-phosphorylcholine (PAPC). The method of any of the preceding items or the lipid vesicle of any of items 29 to 48, wherein the non-oxidizable reference lipid comprises a saturated fatty acid and does not comprise any unsaturated fatty acid. The method or the lipid vesicle of item 49, wherein the saturated fatty acid comprises 14, 16, 18, 20 or 22 C atoms, preferably 16 C atoms. 51. The method of any of the preceding items or the lipid vesicle of any of items 29 to 50, wherein the non-oxidizable reference lipid comprises a palmitic acid.

52. The method of any of the preceding items or the lipid vesicle of any of items 29 to 51, wherein the non-oxidizable reference lipid is a cell membrane lipid and/or a phospholipid, preferably a phosphatidylcholine.

53. The method of any of the preceding items or the lipid vesicle of any of items 29 to 52, wherein the non-oxidizable reference lipid comprises l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC).

54. The method or the lipid vesicle of any of items 40 to 53, wherein the oxidizable sensor lipid can form at least one oxidation product, e.g. upon oxidative stress, wherein the oxidation product comprises at least one residue comprising at least one oxygen in place of at least one double bond comprised in an unsaturated fatty acid comprised in said oxidizable sensor lipid.

55. The method or the lipid vesicle of item 54, wherein the at least one residue comprises a ketone or an aldehyde.

56. The method or the lipid vesicle of items 54 or 55, wherein the at least one residue comprises a hydroperoxide.

57. The method or the lipid vesicle of any of items 54 to 56, wherein the at least one residue comprises an isoprostane.

58. The method of any of the preceding items or the lipid vesicle of any of items 29 to 57, wherein the lipid vesicle is a liposome or a micelle.

Accordingly, the invention, inter alia , relates to the following specific embodiments:

I. A method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of treating and/or contacting a lipid vesicle with the UV light or the UV light in combination with the additional agent, and wherein said lipid vesicle comprises at least one oxidizable sensor lipid and at least one non-oxidizable reference lipid, preferably wherein the oxidative stress is associated with skin damage and/or leads to skin damage.

II. A method for producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of selecting said compound or composition, wherein said selection comprises determining the efficacy of said compound or composition in preventing and/or protecting from said oxidative stress, and wherein determining said efficacy comprises treating, contacting and/or covering the lipid vesicle with said compound or composition prior to and/or during treating and/or contacting said lipid vesicle with the UV light or the UV light in combination with the additional agent, preferably wherein the oxidative stress is associated with skin damage and/or leads to skin damage.

III. The method of embodiment I or II further comprising a step of determining the amount of oxidation of the at least one oxidizable sensor lipid, wherein determining said amount of oxidation comprises the steps of

(a) determining the amount of at least one oxidation product of the oxidizable sensor lipid(s),

(b) determining the amount of the at least one non-oxidizable reference lipid, and

(c) normalizing the amount of the at least one oxidation product of the oxidizable sensor lipid(s) by the amount of at least one of said reference lipid(s), preferably wherein the amounts of said oxidation product(s) and said reference lipid(s) are determined by liquid chromatography combined with mass spectrometry, preferably by HPLC-MS/MS.

IV. A lipid vesicle comprising at least one oxidizable sensor lipid and at least one non- oxidizable reference lipid.

V. The method of any of embodiments I to III, or the lipid vesicle of embodiment IV, wherein the at least one oxidizable sensor lipid is oxidizable by UV light, and wherein the at least one non-oxidizable reference lipid is not oxidizable by UV light.

VI. The method of any of embodiments I to III or V, or the lipid vesicle of embodiments IV or V, wherein said sensor lipid(s) and/or said reference lipid(s) comprise a cell membrane lipid and/or a phospholipid, preferably a phosphatidylcholine. VII. The method of any of embodiments I to III or V or VI, or the lipid vesicle of any of embodiments IV to VI, wherein said sensor lipid(s) comprises an unsaturated fatty acid, wherein said reference lipid(s) comprises a saturated fatty acid, and wherein said reference lipid(s) does not comprise any unsaturated fatty acid, preferably wherein the at least one oxidizable sensor lipid comprises l-palmitoyl-2-arachidonoyl-sn-glycero- 3-phosphorylcholine, and preferably wherein the at least non-oxidizable reference lipid comprises dipalmitoylphosphatidylcholine.

VIII. The method of any of embodiments I to III, or V to VII, or the lipid vesicle of any of embodiments IV to VII, wherein the lipid vesicle is contained in a medium, and wherein said medium comprises a matrix, a gel or a solution, preferably a gel-like matrix which preferably comprises collagen.

IX. The method of any of embodiments I to III or V to VIII, or the lipid vesicle of any of embodiments IV to VIII wherein at least one oxidation product of the oxidizable sensor lipid(s) comprises at least one residue comprising at least one oxygen in place of at least one double bond comprised in said oxidizable sensor lipid(s), preferably wherein said residue comprises a ketone, an aldehyde, a hydroperoxide and/or an isoprostane.

X. A compound or composition for use in preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said compound or composition is obtainable by the method of any of embodiments II, III or V to IX, wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent, preferably wherein said skin disorder is eczema, a photodermatose, an inflammatory skin disease, psoriasis, atopic dermatitis, contact dermatitis, actinic keratosis, an occupational dermatosis with UV exposure or chemical exposure, graft versus host disease, acne, a modification of the skin due to skin transplantation, a lipid storage disease, and/or sunburn.

XI. Use of a compound or composition obtainable by the method of any of embodiments II, III or V to IX for protecting the skin from oxidative stress caused by UV light or UV light in combination with an additional agent.

XII. The method of any of embodiments I to III or V to IX, the compound or composition for use according to embodiment X, or the use of embodiment XI, wherein the oxidative stress is associated with skin damage and/or leads to skin damage.

XIII. The method of any of embodiments I to III, V to IX or XII, the compound or composition for use according to embodiments X or XII, or the use of embodiments XI or XII, wherein the oxidative stress is associated with and/or leads to lipid oxidation, lipid peroxidation, photodamage, skin ageing, the formation of reactive lipid species, formation of lipid adducts to proteins, protein crosslinking, modifications of proteins by advanced lipoxidation products, formation of protein aggregates, reactive oxygen species (ROS), mitochondrial dysfunction, cellular senescence associated ROS, fenton reaction of lipids, a lipid hydroperoxide chain reaction, and/or sunburn.

XIV. The method of any of embodiments I to III, V to IX, XII or XIII, the lipid vesicle of any of embodiments V to IX, the compound for use according to any of embodiments X, XII or XIII, or the use of any of embodiments XI to XIII, wherein the UV light is longwave UV light, UVA and/or has a wavelength between 315 nm and 400 nm.

XV. The method of any of embodiments I to III, V to IX, or XII to XIV, the compound for use according to any of embodiments X, XII to XIV, or the use of any of embodiments XI to XIV, wherein the agent comprises at least one pollutant, preferably wherein the at least one pollutant comprises diesel particulate matter (DPM), and/or at least one polycyclic aromatic hydrocarbon and/or at least one nitrated form thereof.

The invention is also characterized by the following figures, figure legends and the following non-limiting examples.

Brief description of the Figures

Figure 1: The principle of the sensor/calibrator vesicles. Lipid vesicles containing an oxidizable sensor lipid such as PAPC and a non-oxidizable calibrator (reference) lipid such as

DPPC, preferably in a defined ratio, may be embedded in a matrix. When exposed to UV light, in particular UVA, the sensor lipid, but not the calibrator lipid, can be oxidized ( e.g . to oxPAPC). The presence of a pollutant such as diesel particulate matter may further enhance the oxidative effect of the UV light. Upon application of the UV light, all lipids can be extracted in one isolation step and then be analyzed using HPLC-MS/MS. The system can be further used for testing to what extent a compound or composition such as a make-up protects from the UV light induced oxidation of the sensor lipids. For example, the composition can be applied on top of a UV permeable lattice or mesh covering the matrix before or during application of the UV light.

Figure 2: Size distribution of sensor vesicles determined with Nanoparticle Tracking Analysis a) Photomicrograph of sensor vesicles imaged with Nanosight 300 technology b) Hydrodynamic size and particle concentration determination of irradiated and sham-trated sensor vesicles (DPPC/PAPC) both displaying a mean size of 214 nm. Of note, the buffer alone (HBSS, light grey) yielded virtually no countable particles c) The majority of the vesicles was attributed to the hydrodynamic size classe from 100 to 299 nm, regardless of UV irradiation.

Figure 3: Photograph 1 shows the pressuring process. Photograph 2 shows the collagen matrix skin mimics after the squeezing procedure (on the left a native collagen matrix with lipid vesicles, on the right such a matrix supplemented with 75 pg of Diesel particulate matter). Photograph 3 shows samples that are covered with a UVA-light permeable nylon mesh.

Figure 4: Size distribution of vesicles within the liquid recovered after squeezing the collagen matrix that was either supplemented with vesicles or not. The hydrodynamic size determination was performed with nanoparticle tracking analysis.

Figure 5: Normalized intensity of the peaks (area of m/z 769 (PAPC-keto) divided by area of m/z 734 (DPPC) multiplied by 10.000). Data points of the replicates of each of the four independent experiments share the same label. Ves.: Lipid vesicles; DPM: Diesel particulate matter; UVA: UV-A light.

Figure 6: Phospholipid hydroperoxide analysis a): Extracted ion chromatograms (XIC) of the respective multiple reaction monitoring (MRM) transitions (-> 184) with a mass/charge ratio (m/z) of 814 and a diagnostic product ion of m/z 184. The area of the peak with an elution time of 5,5-8 min corresponds to the isobaric species of the phospholipid hydroperoxide PAPC-OOH. The black line corresponds to the amount of PAPC-OOH in vesicles, the gray line to the amount of PAPC-OOH in vesicles incubated with DPM followed by UVA-irradiation. b) and c) The peak area that corresponds to PAPC-OOH was normalized to the peak area of DPPC and values were multiplied with 10.000. Data from two independent experiments with 2 brands of make-up are shown (b: n = 3; error bars indicate SD c: n=l). Asterisks indicate significant differences (*P < 0.05; ** P < 0.01) determined by ANOVA.

Figure 7: Reactive carbonyl lipid analysis a) Extracted ion chromatograms (XIC) of the respective multiple reaction monitoring (MRM) transitions with a mass/charge ratio of m/z 796. The area of the peak with an elution time of 6-8 min corresponds to PAPC-keto. The black line corresponds to the amount of PAPC-keto in vesicles, the gray line to the amount of PAPC-keto in vesicles incubated with DPM followed by UVA-irradiation. b) and c): The peak area that corresponds to PAPC-keto was normalized to the one of DPPC and multiplied with 10.000. Data from two independent experiments with 2 brands of make-up are shown (b: n = 3; error bars indicate SD c: n=l). Asterisks indicate significant differences (*P < 0.05; ** P < 0.01) determined by ANOVA.

Figure 8: Phospholipids with Isoprostanoid modifications a) Extracted ion chromatograms (XIC) of the respective multiple reaction monitoring (MRM) transitions with a mass/charge ratio (m/z) of 832. The area of the peak with an elution time of 5-7 min corresponds to isoPGF2a-PPC. The black line corresponds to the amount of isoPGF2a-PPC in vesicles, the gray line to the amount of isoPGF2a-PPC in vesicles incubated with DPM followed by UVA- irradiation. b) and c): The peak area that corresponds to isoPGF2a-PPC was normalized to the peak area of DPPC and multiplied with 10.000. Data from two independent experiments with 2 brands of make-up are shown (b: n = 3; error bars indicate SD c: n=l). Asterisks indicate significant differences (*P < 0.05; ** P < 0.01) determined by ANOVA.

Figure 9: Recovery of sensor and calibrator lipids. HPLC-MS/MS quantification of raw (not normalized) data of the original vesicle components DPPC and PAPC retrieved from the collagen matrix after a typical experiment including pollution, UV radiation and topical make-up (n = 3; error bars indicate SD). Significant differences were determined by ANOVA.

Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

Examples

Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

Example 1. Preparation of lipid vesicles as cell mimics

Lipid vesicles consisting of cell membrane lipids which mimicked some aspects of cells were prepared. The lipid vesicles were prepared from a total of 200 pg phospholipid mixture containing 70 % w/w of di-palmitoyl-phosphatidylcholine and 30 % of l-palmitoyl-2- arachidonoyl-sn-glycero-3-phosphorylcholine (both from Avanti Lipids, Alabaster, Alabama). The mixture was dried as thin film in a 4 ml glass vial. For the preparation of the vesicles the dried lipid film was vortexed at full speed for 2 minutes with 500 mΐ of IX Hanks’ Balanced Salt solution (HBSS) buffer. The solution was either irradiated with 20J/cm² of UVA-1 or not irradiated to serve as sham treated control as indicated; see also Figure 2.

Example 2. Lipid vesicle characterization

To determine the size distribution and concentration of these vesicles, the “Nanoparticle Tracking Analysis” of the Nanosight 300 Analyzer (Malvern) was performed. From the results, the mean vesicle size and concentration was calculated. A video camera was simultaneously tracking the laser illumination scatter from individual particles in suspension. The artificial vesicles containing DPPC and PAPC were subsequently diluted (1:100) in particle-free PBS. Experiment videos () (7 videos of 20s per measurement and a minimum of 333 frames per sample; snapshot in Figure 2a) were analyzed using NTA 3.1 build 3.1.54 software (Malvern). Both, non-irradiated and UV-irradiated sensor vesicles displayed a mean size distribution of 214 nm (Fig. 2b). The majority (51% or 56%) of vesicles was in the size range from 100 nm to 199 nm, and 24% or 26% were in the size range from 200 nm to 299 nm, without or with irradiation, respectively. (Figure 2c).

Example 3. Preparation of a collagen matrix with vesicles as skin mimic. A collagen matrix containing lipid vesicles, and thus mimicking some aspects of skin, was prepared. A Collagen solution (3mg/ml; PureCol Typel Bovine Collagen; Advanced BioMatrix) was mixed with lOx HBSS and H2O (8:1:1; V:V:V). Per sample, 2,5 ml collagen solution was prepared, each containing 400 pg of the lipid vesicles as prepared in Example 1. To this matrix solution 30 pg/ml (in total 75 pg) of diesel particulate matter standard reference material (NIST® SRM® 2975, obtained from Sigma) was added when indicated. The collagen solution was incubated for 2 h at 37°C in ambient air and afterwards the collagen was condensed by applying pressure by applying a weight of 46,5 g to the surface (3,6 cm²) for 90 minutes (photographs 1, 2 of Figure 3). In photograph 3 of Figure 3, the collagen matrix was covered with a UVA-light permeable nylon mesh before UVA exposure. When indicated, 6 mg of the make-up test formulation was applied on the nylon mesh.

Example 4. The Lipid vesicles retain their integrity and properties in the collagen matrix.

It was assumed that the liquid recovered after applying the pressure to the collagen matrix as prepared in Example 3 contained some of the lipid vesicles. Thus, the recovered liquid was again analyzed with “Nanoparticle Tracking Analysis”. This revealed that the lipid vesicles retained their integrity and properties within the collagen matrix. The recovered vesicles were intact and had a size distribution comparable to the vesicles before integration into the matrix, with a mean hydrodynamic size of 241 nm and the majority of vesicles between 100 nm and 299 nm in size. Native bovine collagen did not contain comparable intrinsic vesicles or lipids at a level that would influence interpretation of results (Figure 4).

Example 5. An in vitro assay for determining the efficacy of a cosmetic composition in preventing or protecting from oxidative stress or cell membrane damage caused by UV light and/or a pollutant. a) The assay setup

Lipid vesicles were prepared as described in Example 1 and formulated into a collagen matrix, further containing, where indicated, 30 pg/ml Diesel particulate matter as described in

Example 3. The collagen matrix containing the lipid vesicles was covered with a 40 pm pore size nylon mesh (Falcon cell strainer). On top of the 3,6 cm² area of the mesh, 6 mg of a make-up test formulation was applied homogenously. Make-up 1 was: CHANEL Le Blanc

Fluide B20 Lot: 3307043021789 and make-up 2 was: MANHATTAN Endless perfection, breathable, Lot: 7225. Then, the indicated samples were exposed to UV light (UVA; 20J/cm²) or to sham irradiation for the controls with the make-up pointing towards the light source. b) Isolation of the lipid vesicles from the collagen matrix

One half of each sample as prepared in a) (corresponding to a surface area of 1,8 cm²) was homogenized in Precellys bead homogenizer tubes containing a methanol (97% v/v)/acetic acid (3% v/v) that was supplemented with butylhydroxytoluene (BHT, 0.01% w/v). Ten nanograms of internal standard 1 ,2-dinanoyl-.s»-glycero-3-phosphocholine (DNPC m/z: 538) (Avanti Lipids, Alabaster, Alabama) were added and homogenization was performed at 5800 rpm for 20 seconds on samples that were cooled on ice before. The homogenate was centrifuged for 5 min, 1000 ref at 4°C and the supernatant was purified by the hexane liquid- liquid extraction (LLE) procedure described in Gruber et al., J Lipid Res. 2012 Jun;53(6): 1232-42. ). In brief, the samples were washed three times by a procedure where 4 ml hexan/BHT (0.01%) were added to the samples, followed by vortexing and aspiration of the hexan layer. After the third wash, 1.5 ml HCOOH (0,7M) and chloroform/BHT (0.01%) were added and vortexed. The lower, organic phase was transferred into a new glass tube, dried under argon and stored at -20°C until the mass spectrometric analysis. c) Mass spectrometric analysis of the lipid vesicles (HPLC-MS/MS)

The samples (argon-dried organic phase as described in b)) were reconstituted in 85% aqueous methanol containing 5 mM ammonium formate and 0,1% formic acid. For injection into a core-shell-type VI 8 column (Kinetex 2.6pm, 50mm x 3.0 mm ID; Phenometex, Torrance, CA) the samples were kept at 20°C and using a 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany), which was coupled to a 4000 QTrap triple quadrupole linear ion trap hybrid mass spectrometer system equipped with a Turbo V electro-spray ion source (Applied Biosystems, Foster City, CA). The detection of the phospholipids was carried out in positive ion mode by selected reaction monitoring (RSM) of 99 distinct MS/MS transitions using a PC- specific product ion ( m/z 184) which corresponds to the cleaved phosphocholine residue.

Data acquisition was performed with the Analyst software, version 1.6.3 from Applied Biosystems. For quantification of each lipid oxidation product the area under the elution peaks for each specific mass transition was integrated as described in Gruber et al., J Lipid Res. 2012 Jun;53(6): 1232-42 and these values were either shown as raw data or normalized to l,2-dipalmitoyl-5?z-glycero-3 -phosphocholine (DPPC m/z. 734), the calibrator (reference) lipid which was contained in the lipid vesicles. d) Reproducibility of the assay

To evaluate the reproducibility of the assay, the normalized peak intensities of a ketone- containing oxidation product of PAPC (PAPC-keto m/z 796) were calculated from four experiments wherein the samples as prepared in a) had been exposed to UVA (20 J/cm²) (Figure 5).

Example 6. Quantification of oxidation products of PAPC by mass spectrometric analyses. a) Hydroperoxides of PAPC (PAPC-OOH)

UVA exposure led to a significant increase in the levels of hydroperoxides of PAPC (PAPC- OOH m/z 814) extracted from the collagen matrix as shown by PAPC-OOH data that were normalized to the co-extracted calibrator lipid DPPC (m/z: 734). In the presence of Diesel Particulate Matter (DPM) the formation of PAPC-OOH was further significantly increased. The presence of 6 mg / 3.6 cm² make-up on the nylon mesh significantly reduced both the UV- induced lipid oxidation, and the oxidation resulting from the combination of DPM and UV light exposure (Figure 6). b) PAPC-keto

Highly reactive carbonyl group containing phospholipids are potentially dangerous side products of lipid oxidation. A significant induction of a keto-modified oxidation product of PAPC (m/z: 796) was detected upon UVA exposure which was amplified in presence of DPM in the sensor vesicle derived lipid extracts (Figures 5 and 7). Significant reduction of DPM/UV light induced keto-PAPC was found when the make-up 1 had been applied in the test system. c) PAPC-F-isoprostanes

F-isoprostanes quantified with HPLC are regarded the gold standard analytes for non- enzymatic redox stress quantification in biological systems. A phospholipid oxidation product that corresponds to an F-Isoprostane esterified to the phospholipid backbone (which resulted from oxidation of the arachidonic acid chain at the sn-2 position of PAPC) could be quantified. In the example, the combination of DPM and UV led to a significant increase in a lipid species corresponding to isoPGF2a-PPC, (m/z 832). Formation of this lipid species was significantly inhibited by presence of make-up 1 during irradiation (Figure 8).

Of note, the experiments with make-up 2 were not repeated (Figures 6 to 8), merely because of the low amount of make-up 2 available to the inventors when the experiments were performed.

Claims

2. The method of claim 1, wherein the oxidative stress is associated with skin damage and/or leads to skin damage, preferably wherein said skin damage is associated with and/or leads to a skin disorder and/or a cancerous modification of the skin.

3. The method of claims 1 or 2, wherein the oxidative stress is associated with and/or leads to lipid oxidation, lipid peroxidation, photodamage (damage of biomolecules by light), skin ageing, the formation of reactive lipid species, formation of lipid adducts to proteins, protein crosslinking, modifications of proteins by advanced lipoxidation products, formation of protein aggregates, reactive oxygen species (ROS), mitochondrial dysfunction, cellular senescence associated ROS, fenton reaction of lipids, a lipid hydroperoxide chain reaction, and/or sunburn.

4. The method of any of claims 1 to 3, wherein the oxidative stress is associated with and/or leads to a skin disorder and/or a cancerous modification of the skin.

5. The method of any one of claims 2 to 4, wherein said skin disorder is selected from the group consisting of eczema, a photodermatose, an inflammatory skin disease, psoriasis, atopic dermatitis, contact dermatitis, actinic keratosis, an occupational dermatosis with UV exposure or chemical exposure, graft versus host disease, acne, a modification of the skin due to skin transplantation, a lipid storage disease, and/or sunburn.

6. The method of any of claims 1 to 5, wherein the amount of oxidative stress is determined by determining the amount of oxidation of the at least one oxidizable sensor lipid.

7. The method of claim 6, wherein determining the amount of oxidation of the at least one oxidizable sensor lipid comprises determining the amount of at least one oxidation product of the oxidizable sensor lipid(s), determining the amount of the at least one non-oxidizable reference lipid, and normalizing the amount of the at least one oxidation product of the oxidizable sensor lipid(s) by the amount of said reference lipid.

8. The method of claim 7, wherein the amounts of the at least one oxidation product of the oxidizable sensor lipid(s) and the at least one non-oxidizable reference lipid are determined by liquid chromatography combined with mass spectrometry, preferably by HPLC-MS/MS.

9. The method of any of claims 6 to 8, wherein the amount of oxidation of the at least one oxidizable sensor lipid treated and/or contacted with the UV light or the UV light in combination with the additional agent is compared to a control not treated and/or contacted with said UV light or said UV light in combination with said additional agent.

10. The method of any of claims 1 to 9, wherein the agent comprises at least one pollutant.

11. The method of claim 10, wherein the at least one pollutant is capable of inducing and/or enhancing oxidative stress.

12. The method of claims 10 or 11, wherein the at least one pollutant comprises diesel particulate matter (DPM), and/or at least one polycyclic aromatic hydrocarbon and/or at least one nitrated form thereof.

13. The method of any of the preceding claims, wherein the UV light is capable of oxidizing the at least one oxidizable sensor lipid comprised in the lipid vesicle.

14. The method of any of the preceding claims, wherein the UV light is longwave UV light.

15. The method of any of the preceding claims, wherein the UV light is UVA.

16. The method of any of the preceding claims, wherein the UV light has a wavelength between 315 and 400 nm.

17. The method of any of the preceding claims, wherein the UV light has a fluency of 1 J/cm² to 200 J/cm², preferably of 10 J/cm² to 30 J/cm²

19. A method for producing a compound or a composition which is effective in preventing and/or protecting from oxidative stress caused by UV light or UV light in combination with an additional agent, wherein said method comprises a step of selecting said compound or composition according to the method of claim 18.

20. A compound or composition obtainable by the method of claim 19.

21. The method of claims 18 or 19 or the compound or composition of claim 20, wherein the compound or composition is a cosmetic composition.

22. The method or the compound or composition of claim 21, wherein the cosmetic composition comprises a make-up, a skin care product, a hair care, and/or a sunscreen, wherein the skin care product is preferably a skin cream.

23. The method of any of claims 18 to 22 or the compound or composition of any of claims 20 to 22, wherein the compound or composition is a pharmaceutical composition.

24. The method or the compound or composition of claim 23, wherein the pharmaceutical composition comprises a skin cream, a sunscreen, and/or an ointment.

25. The method of any of claims 18 to 24 or the compound or composition of any of claims 20 to 24, wherein the compound or composition comprises a skin care active compound.

26. The method of any of claims 18 to 25 or the compound or composition of any of claims 20 to 25, wherein the efficacy of said compound or composition in preventing and/or protecting from said oxidative stress comprises the chemical or physical capacity to block UV light, and/or prevent and/or protect from UV light induced oxidative modifications of biomolecules, in particular cell membrane lipids.

27. The compound or composition of any of claims 20 to 26 for use in preventing, ameliorating and/or treating a skin damage and/or a skin disorder, wherein said skin damage is associated with and/or caused by oxidative stress, wherein said skin disorder is associated with and/or caused by oxidative stress or said skin damage, and wherein said oxidative stress is caused by UV light or UV light in combination with an additional agent, preferably wherein said skin disorder is a photodermatose, actinic keratosis, cancer, and/or cancerpolymorphic light eruption, juvenile spring eruption, actinic prurigo, hydroa vacciniforme, solar urticaria, chronic actinic dermatitis, a genodermatose/ DNA repair deficient disorder, a cornification disorder, the Smith- Lemli-Opitz syndrome, porphyria, lupus erythematosus, erythema multiforme, atopic eczema, psoriasis, viral exanthemata, pemphigus, dermatitis herpetiformis, rosacea.

28. Use of the compound or composition of any of claims 20 to 26 for protecting the skin from oxidative stress caused by UV light or UV light in combination with an additional agent.

29. A lipid vesicle comprising at least one oxidizable sensor lipid and at least one non- oxidizable reference lipid.

30. The method of any of the preceding claims or the lipid vesicle of claim 29, wherein the lipid vesicle is contained in a medium, and wherein said medium comprises a matrix, a gel or a solution, preferably a gel-like matrix.

31. The method or the lipid vesicle of claim 30, wherein the medium comprises collagen.

32. The method or the lipid vesicle of claims 30 or 31, wherein the medium comprises an agent, wherein said agent preferably comprises at least one pollutant.

33. The method or the lipid vesicle of any of claims 30 to 32, wherein the medium is covered by a UV light permeable membrane, preferably a nylon mesh.

34 The method or the lipid vesicle of claim 33, wherein said UV light permeable membrane is in contact with a compound or composition, and/or wherein said UV light permeable membrane is facing towards a UV light source.

35. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

34, wherein the at least one oxidizable sensor lipid is oxidizable by UV light.

36. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

35, wherein the lipid vesicle comprises more oxidizable sensor lipid(s) than non- oxidizable reference lipid(s) in weight.

37. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

36, wherein the lipid vesicle comprises at least 1.2-times more oxidizable sensor lipid(s) than non-oxidizable reference lipid(s) in weight.

38. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

37, wherein the lipid vesicle has a mean vesicle diameter between 30 nm and 1000 nm, preferably between 50 nm and 600 nm, preferably between 100 nm and 300 nm.

39. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

38, wherein the oxidizable sensor lipid comprises an unsaturated fatty acid.

40. The method or the lipid vesicle of claim 39, wherein the unsaturated fatty acid comprises at least two double bonds.

41. The method or lipid the vesicle of claims 39 or 40, wherein the unsaturated fatty acid comprises at least four double bonds, preferably exactly four double bonds.

42. The method or the lipid vesicle of claims 40 or 41, wherein the double bonds of the unsaturated fatty acid are separated by methylene bridges.

43. The method or the lipid vesicle of any of claims 39 to 42, wherein the unsaturated fatty acid comprises 16, 18, 20 or 22 C atoms, preferably 20 C atoms.

44. The method or the lipid vesicle of any of claims 39 to 43, wherein the unsaturated fatty acid is an omega-6 fatty acid, preferably an arachidonic acid.

45. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to 44, wherein the oxidizable sensor lipid is a cell membrane lipid.

46. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to claim45, wherein the oxidizable sensor lipid comprises a fatty acid chain that is ether bound or esterified.

47. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

46, wherein the oxidizable sensor lipid is a phospholipid, preferably a phosphatidylcholine.

48. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

47, wherein the oxidizable sensor lipid comprises l-palmitoyl-2-arachidonoyl-sn- gly cero-3 -phosphorylcholine (P APC) .

49. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

48, wherein the non-oxidizable reference lipid comprises a saturated fatty acid and does not comprise any unsaturated fatty acid.

50. The method or the lipid vesicle of claim 49, wherein the saturated fatty acid comprises 14, 16, 18, 20 or 22 C atoms, preferably 16 C atoms.

51. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

50, wherein the non-oxidizable reference lipid comprises a palmitic acid.

52. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

51, wherein the non-oxidizable reference lipid is a cell membrane lipid and/or a phospholipid, preferably a phosphatidylcholine.

53. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to

52, wherein the non-oxidizable reference lipid comprises 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC).

54. The method or the lipid vesicle of any of claims 40 to 53, wherein the oxidizable sensor lipid can form at least one oxidation product, e.g. upon oxidative stress, wherein the oxidation product comprises at least one residue comprising at least one oxygen in place of at least one double bond comprised in an unsaturated fatty acid comprised in said oxidizable sensor lipid.

55. The method or the lipid vesicle of claim 54, wherein the at least one residue comprises a ketone or an aldehyde.

56. The method or the lipid vesicle of claims 54 or 55, wherein the at least one residue comprises a hydroperoxide.

57. The method or the lipid vesicle of any of claims 54 to 56, wherein the at least one residue comprises an isoprostane.

58. The method of any of the preceding claims or the lipid vesicle of any of claims 29 to 57, wherein the lipid vesicle is a liposome or a micelle.

59. The lipid vesicle of any of claims of claims 29 to 58 for use in a method for determining oxidative stress caused by UV light or UV light in combination with an additional agent and/or in a method for selecting or producing a compound or composition which prevents and/or protects from said oxidative stress, preferably a method according to any of the preceding claims.

60. A kit comprising the lipid vesicle of any of claims of claims 29 to 59.

61. The kit of claim 60 further comprising an additional agent according to any one of claims 10 to 12.

62. The kit of claims 60 or 61 for use in a method for determining oxidative stress caused by UV light or UV light in combination with an additional agent, a method for selecting or producing a compound or composition which prevents and/or protects from said oxidative stress, preferably a method according to any of the preceding claims.

63. The kit of any of claims 60 to 62, wherein said kit further comprises a brochure or leaflet with instructions for carrying out a method of any of the preceding claims.

64. A method for producing a lipid vesicle, said method comprising the steps of

(b) drying said mixture,

(c) resuspending the dried mixture, and

(d) agitating, preferably vortexing, the resuspended mixture, thereby forming a lipid vesicle.

65. The method of claim 65, wherein in step (b) the mixture of (a) is dried as a thin film.

66. The method of claims 64 or 65, wherein the at least one oxidizable sensor lipid is oxidizable by UV light, and wherein the at least one non-oxidizable reference lipid is not oxidizable by UV light.

67. The method of any of claims 64 to 66, wherein said sensor lipid(s) and/or said reference lipid(s) comprise(s) a cell membrane lipid and/or a phospholipid, preferably a phosphatidylcholine.

68. The method of any of claims 64 to 67, wherein said sensor lipid(s) comprise(s) an unsaturated fatty acid, wherein said reference lipid(s) comprises a saturated fatty acid, and wherein said reference lipid(s) does not comprise any unsaturated fatty acid.

69. The method of any of claims 64 to 68, wherein the at least one oxidizable sensor lipid comprise(s) l-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine.

70. The method of any of claims 64 to 69, wherein the at least one non-oxidizable reference lipid comprises dipalmitoylphosphatidylcholine.

71. The method of any of claims 64 to 70, further comprising the steps of

(e) adding the lipid vesicle(s) to an aqueous solution comprising an extracellular matrix protein, preferably a collagen,

(f) incubating said solution containing said lipid vesicle(s), and

(g) applying pressure to said solution containing said lipid vesicle(s), in particular, thereby forming a medium comprising said lipid vesicle(s) and said extracellular matrix protein, e.g. a skin mimic.

72. The method of claim 71, wherein in step (f), the solution is incubated at 30 to 40 °C, and/or in step (g), the pressure is applied by applying a weight of 40 to 55 g per 3.6 cm² of the incubated solution.

73. The method of claims 71 or 72, wherein an agent that enhances the oxidative stress of UV light, e.g. Diesel particulate matter (DPM), is added to the aqueous solution in step (e).