Compound having properties of protection from UV radiation and method for its obtainment

Abstract

Compound based on titanium dioxide or a derivative thereof or generated from a precursor thereof functionalized and / or complexed with a surfactant and / or a lipopeptide, method for obtaining it and use as a UV radiation protective agent and as a sunscreen in creams and cosmetic formulations for topical use, as well as in medical devices.

Description

[0001] “COMPOUND HAVING PROPERTIES OF PROTECTION FROM UV RADIATION AND METHOD FOR ITS OBTAINMENT”

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to a compound having sunscreen properties for protection against UV radiation and a method for obtaining it, wherein said compound comprises a physical sunscreen, such as titanium dioxide or a derivative or a precursor of titanium dioxide, in combination with a surfactant and / or a lipopeptide and / or a pharmaceutically acceptable salt thereof.

[0004] TECHNICAL BACKGROUND

[0005] The skin is the largest organ in the human body and it is very important to ensure adequate protection from external aggressions, including environmental pollution and UV radiation.

[0006] The latter, based on wavelength, are divided into UVB (290-320 nm) - responsible for erythema, sunburn, and direct damage to DNA - and UVA (320-400 nm) - which promotes skin aging by inducing reactive oxygen species (ROS).

[0007] It follows that photoprotection is the main preventive strategy against skin aging and photo-induced and / or photo-mediated diseases, the most serious of which is skin cancer, using molecules capable of absorbing, reflecting, or dispersing the radiation incident on the skin itself.

[0008] To implement photoprotection, sunscreen products or filters are used, which must ensure effectiveness, pleasantness and safety in use: they, in fact, must at the same time providing high protection performance, reduced skin penetration, easy and pleasant application, and high photostability (both in terms of inertia towards the other components of the sunscreen product and resistance against the deteriorating action of sunlight).

[0009] Sunscreens are divided into two broad categories based on their mechanism of action: physical sunscreens filters and chemical sunscreens filters.

[0010] Chemical (or organic) filters are generally aromatic compounds conjugated with otherfunctional groups in ortho and para to promote electron delocalization. This general structure allows the molecule to act as exogenous chromophores capable of absorbing high-energy UV photons (UVB), passing from a lower energy electronic state (ground state) to a higher energy excited state, and then release a lower amount of energy that is less harmful to the skin.

[0011] Physical (or inorganic) filters are naturally occurring mineral powders that reflect and scatter incident UV radiation, making the skin "impervious" to sunlight. They offer protection from both UVA and UVB rays, as well as being chemically inert, non-irritating, and having low skin permeability.

[0012] Biosurfactants are amphiphilic compounds that possess significant surface and / or interfacial activity due to the presence of both hydrophobic and hydrophilic groups in a single molecule. Microbial biosurfactants can be produced as secondary metabolites that remain attached to the surface of the microbial cell or are secreted outside the cells. Currently, most surfactants are derived from the petrochemical industry, but as this form of production is no longer considered sustainable, interest has shifted towards biosurfactants in order to suitably replace their synthetically produced counterparts. The main characteristics of biosurfactants are non-toxicity, biodegradability, bioavailability, biocompatibility, ecological acceptability, high selectivity, and environmental compatibility. In addition, they may have lower critical micelle concentration (CMC) values than synthetic surfactants, improving their efficiency in various applications.

[0013] Microbial biosurfactants can be classified based on molecular weight, chemical composition, or microbial source.

[0014] Titanium dioxide (TiO₂) nanoparticles are suitable for use in cosmetics and polymer films because they effectively absorb UV light while remaining transparent to visible light. Their widespread use, however, requires the development of methods that are capable of as much as possible reduce their potential risks to humans and the environment.

[0015] The international application WO90 / 11067 describes a composition of sunscreens comprising a mixture of different particle sizes of titanium dioxide. The lecithin mentioned in this document serves exclusively to improve the physical stability of the suspension and the sensory appeal of the sunscreen, in order to obtain a stable formulationwith titanium and lecithin. However, this is not a hybrid system, and lecithin is a particular molecular entity (phospholipid).

[0016] The patent application JPH11171541 describes a composition comprising titanium dioxide particles and mentions sodium stearate as a surfactant.

[0017] Lecithin and sodium stearate are examples of classic chemical surfactants with purely dispersing functions.

[0018] The patent application CN 109589274 describes a dispersion of titanium dioxide in cosmetic formulations. Again, the compounds involved concern the mere physical stability of the suspension, and the method describes a dispersion of the compounds, not their complexation or functionalization.

[0019] The patent application JP2010270073 describes titanium dioxide coated with cationic surfactants and a method for dispersing the compounds, not their complexation or functionalization.

[0020] Cationic surfactants have undesirable toxicity and biocompatibility profiles, making such compounds unsuitable for the purposes of the present invention.

[0021] There is therefore a need for a compound with UV radiation protection properties and an innovative method for obtaining it, which is easy to implement and offers advantages in terms of effectiveness and low environmental impact.

[0022] OBJECTS OF THE INVENTION

[0023] One purpose of the present invention is to improve on the prior art.

[0024] Another purpose of the present invention is to provide a compound with UV radiation protection properties that is effective and safe.

[0025] A further purpose of the present invention is to provide a compound with UV radiation protection properties that has a low environmental impact, since it is of a bio-inspired and skin-compatible sunscreen.

[0026] Another purpose of the present invention is to provide a method for obtaining a compound with UV radiation protection properties that is easy and quick.

[0027] A further purpose of the present invention is to provide a method for obtaining a compound with UV radiation protection properties that has a low environmental impact. In accordance with one aspect of the present invention, a compound and a method areprovided according to the attached independent claims.

[0028] The dependent claims refer to preferred and advantageous embodiments of the invention.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Further features and advantages of the present invention will become more apparent from the detailed description of a preferred, but not exclusive, embodiment of the invention, illustrated by way of example, but not limitation, in the accompanying drawings, in which:

[0031] Figure 1 is a graph illustrating the zeta potential curves (on the y-axis, measured in mV) versus pH (on the x-axis) of P25, SS, and P25@SS (1:1),

[0032] Figure 2 illustrates various samples in a test whose purpose is to evaluate the stability of the dispersion over time of various formulations, including EU, SS, P25, and combinations such as SS@EU, SS@P25, P25@SS. The images were taken at three different times: immediately after preparation (t = 0), after 8 hours (t = 8h), and after 3 days (t = 3d). Specifically, P25 means titanium dioxide powder having particles of the specified size, for example obtained from the commercial product called Aeroxide® P25 (Evonik Industries AG, Essen, Germany), EU means titanium dioxide powder, for example obtained from the commercial product called Eusolex® T-AVO (Merk KGaA, Darmstadt, Germany), SS means sodium salt of surfactin (Sodium Surfactin), and the undissociated form of SS (SCOOH) is obtained by treating SS with a cation exchange resin called Dowex® 50Wx8 (Merk KGaA, Darmstadt, Germany),

[0033] Figure 3 shows a sequence of FTIR-ATR spectra obtained on the powders of the various dried samples including P25, EU, SS, P25 functionalized with SS (P25@SS) and EU functionalized with SS (EU@SS). In particular, the analysis was conducted by comparing the starting materials with A) P25@SS and B) EU@SS in the range 550-4000 cm-1,

[0034] Figure 4 shows SEM images of the morphologies of the starting materials (P25, EU) and TiO₂ modified with SS (P25@SS and EU@SS) compared to the physical mixture EU+SS or P25+SS, at different magnifications (Mag);

[0035] Figure 5 shows some TEM images of the TiO₂-based sample (P25@SS) on a copper grid with resin inclusion (A and C) and without resin inclusion (B and D);Figure 6 shows a thermal analysis of the pure or as-are materials: A) P25; B) EU; C) SS and optimized samples modified with SS: D) P25@SS (1:1) and E) EU@SS. The TGA measurement (light gray line) and DSC curve (dark gray line) were performed at a heating rate of 10°C / min, with a temperature range between 24°C and 900°C;

[0036] Figure 7 shows the results of cell proliferation (% cell viability) of BEAS-2B cells treated with increasing concentrations of particles for A) 6h, B) 24h. The gray bar shows the negative control, and as the positive control 10% DMSO was used, indicated as light gray;

[0037] Figure 8 shows the results of cell proliferation (% cell viability) of RAW264.7 cells treated with increasing concentrations of particles for A) 6h, B) 24h. The dark gray bar shows the negative control, and 10% DMSO, indicated as light gray, was used as the positive control;

[0038] Figure 9 shows the results of cell proliferation (% cell viability) of HSF cells treated with increasing concentrations of particles for 24 hours.

[0039] EMBODIMENTS OF THE INVENTION

[0040] The present invention relates to a compound having properties as a sunscreen, i.e., protection from UV radiation, and a method for obtaining it, wherein said compound comprises a physical sunscreen filter, such as titanium dioxide or a derivative or precursor of titanium dioxide, in combination with a surfactant and / or a lipopeptide and / or a pharmaceutically acceptable salt thereof.

[0041] In particular, thanks to this invention, it is possible to obtain an isolated compound or complex, in powder form, which has improved properties (for example, superior by at least one third or at least 20%), particularly with regard to the sun protection factor (SPF), compared to the individual isolated components. With this invention, therefore, it has been possible to achieve a booster effect for the SPF of the indicated compound.

[0042] This differs from known solutions in the prior art, in which the inorganic sunscreen is simply dispersed by a biopolymer in a solvent, without any complexation between the various components.

[0043] The compound in question is based on particles, or preferably nanoparticles, of TiO₂ or a derivative of titanium dioxide or a precursor of titanium dioxide, in which these particlesare functionalized and / or complexed with a biosurfactant, for example based on lipopeptides, such as surfactin, or a sodium salt of surfactin (Sodium Surfactin, SS), or at least one of the other lipopeptides or surfactants listed below, or a salt of the surfactant or lipopeptide.

[0044] The biosurfactant in the present invention, and in particular surfactin, is a commercial product, standardized with an SDS (Safety Data Sheet), and therefore has standard characteristics of purity and composition.

[0045] This differs from other similar but non-commercial compounds, for which purity and composition cannot be guaranteed in a standard way.

[0046] Functionalization and / or complexation using surfactants has proven essential for manage UV-induced photoactivity and significantly reduce in vitro ecotoxicity, while improving the photo stability and UV filtering performance of the physical filter involved. It follows that the compound according to the present invention acts as an effective and safe sunscreen, which can be incorporated into a cosmetic formulation or medical device, as it has UV radiation protection properties for the human body, and in in particular for the skin of the human body.

[0047] The present invention relates to the creation of a functional complex that optimizes the UV protection index, not simply the miscibility of the respective components.

[0048] The object of this invention is not generic TiCh, but rather the specific synergy between the mineral lattice of titanium dioxide (or its precursor or derivative) and the surfactant, specifically the biosurfactant, i.e., the lipopeptide surfactant and, more specifically, surfactin. This synergy and / or this surfactin and / or this lipopeptide surfactant acts as an SPF booster, as indicated below.

[0049] Therefore, the results obtained also revealed the functionality and effectiveness of the colloidal process that is the object of this invention.

[0050] The application as a sunscreen of the compound according to the present invention is therefore a new technical property, linked to specific photoprotective activity, for a purpose not previously described in the prior art.

[0051] Furthermore, this application was not obvious to a person skilled in the art based solely on the chemical synthesis of the compounds involved, as will be described in more detail below.According to a first version of the invention, the particles of the compound in question are surface-functionalized with the surfactant. This results in the formation of a coating based on surfactants and / or lipopeptides (determined by functionalization) of the TiO₂ particles or the particles of the titanium dioxide derivative or the precursor of titanium dioxide.

[0052] According to a second version, the particles of the compound in question are functionalized and / or complexed superficially with TiO₂. This determines that the TiO₂ or the derivative or precursor of TiO₂ is external to the compound in question, i.e., adhered externally compared to the surfactant. For example, this can preferably occur in the case of titanium dioxide commercially known as P25.

[0053] This may vary depending on the conditions of the production method.

[0054] As for TiO₂ particles, they may preferably be nanometric in size, thus falling within the definition of nanoparticles. Nanoparticles (NPs) of titanium dioxide (TiO₂) have desirable properties in various applications, namely their high surface area to volume ratio, as well as their photoelectric and photochemical properties. However, these aspects also contribute to their potential toxicity, both to humans and the environment. One of the ways in which TiO₂ nanoparticles can cause toxicity is through the production of reactive oxygen species (ROS) or the adsorption and transport bioactive molecules, which can lead to oxidative stress, inflammation, genetic damage, metabolic alterations, and eventually cancer. The ability to form ROS is crucial for photocatalytic activity, but it is also important for dark catalysis. On the other hand, TiO₂ nanoparticles have a positive impact on health by mitigating exposure to ultraviolet (UV) radiation and the resulting skin alterations.

[0055] The method according to the present invention, as anticipated, by functionalizing and / or complexing the (nano)particles of TiO₂ or its derivative or its precursor with a surfactant (e.g., a lipopeptide), allows the photocatalytic activity of these particles to be suppressed and their dispersion to be improved, for example, in a cosmetic matrix, in addition to improving the environmental and health profiles of TiO₂ NPs, while preserving their functional properties.

[0056] The titanium dioxide used in the present invention may be in powder form, for example called P25, or in the form of silica-coated TiO₂ particles, for example calledEUSOLEXRT-AVO (INCI: Titanium dioxide, silica, is a silica-coated titanium dioxide powder) or generated in situ in the presence of a surfactant and / or a lipopeptide, starting from a derivative or precursor of titanium dioxide, such as for example starting from isopropoxide (Ti[OCH(CH₃)₂]₄). The latter is greener or more environmentally friendly than other forms of titanium.

[0057] P25 titanium is a form of titanium dioxide powder consisting of nanoparticles with size around 25 nanometers or around 21 nanometers or less than these values.

[0058] Among lipopeptides, a preferred example is surfactin or the sodium salt of surfactin (Sodium Surfactin, SS).

[0059] If desired, lipopeptides are produced by Bacillus ssp.

[0060] Surfattin (also known as surfactin) is a lipopeptide with an amphiphilic structure, with an oligopeptide ring as a hydrophilic group and a fatty acid chain as a hydrophobic group. Specifically, it is a lipopeptide.

[0061] In detail, surfactin consists of a cyclic peptide group with seven amino acids (L-Glu, L-Leu, D-Leu, L-Val, L-Asp, D-Leu, and L-Leu) and a chain of β-hydroxy fatty acids of variable length (C-C). The first and last amino acids, L-Glu and L-Leu, form the lactone ring through an ester bond.

[0062] In the present invention, surfactin and / or lipopeptide biosurfactant confer unique biological and stability properties to the compound of the invention.

[0063] Surfactin is considered an extremely effective surfactant molecule; in fact, at a concentration of 20 pM, it decreases the surface tension of water from 72 to 27 mN / m. This value is significantly lower than the surface tensions of most known biosurfactants. An example of the structure of the cyclic lipopeptide surfactin is shown below (Formula I).

[0064]

[0065] However, surfactin can also have other structures, and differences in the structure of the molecule (e.g., in the hydrocarbon tail — length and structure — and in the amino acid composition) influence the properties of the surfactin itself. For example, with the elongation of the chain, the surface and interfacial activity of the surfactant and its ability to bind other molecules such as TiO₂ and also to penetrate the phospholipid cell membrane, are improved.

[0066] The cyclic structure of the peptide portion of surfactin is also fundamental to its adsorption and penetration into the phospholipid membrane. Disruption of the lactone ring leads to a reduction in surface activity. Amino acids also play a role in the biological activity of surfactin: surface activity increases, while the critical micelle concentration decreases when amino acids are replaced with more hydrophobic ones. Furthermore, if the negatively charged amino acids Glu and Asp are methylated or amidated, the ability of surfactin to lower surface tension increases, while modifying the same amino acids with aminomethane sulfonic acid drastically decreases the surface activity of surfactin due to electrostatic and steric factors.

[0067] Fengicin is another cyclic lipopeptide that can be used in the present invention. In particular, fengicin consists of 10 amino acids (Glu, Orn, Tyr, Thr, Glu, Ala, Pro, Gln, Tyr, and Ile) and a chain of β-hydroxy fatty acids (C14-C18) attached to the N-terminal end of the molecule.The peptide portion of the lipopeptide fengicin consists of a decapeptide chain, of which 8 amino acids (Tyr, Thr, Glu, Ala, Pro, Gln, Tyr, and Ile) are involved in the formation of an octapeptide ring through a lactone bond between the phenolic-OH group of the of Tyr3 and the C-terminal-COOH group of IlelO.

[0068] An example of the structure of the cyclic lipopeptide fengicin is shown below (Formula II). <?,

[0069]

[0070] The heterogeneity of the members of the fengicin family is linked to the sixth position of the peptide portion and the length of the P-hydroxy fatty acid chain. Based on the variation of the single amino acid in position 6 of the peptide ring, fengicins are classified into two variants, namely fengicin A and fengicin B. In particular, the latter has d-Val in position 6 instead of d-Ala.

[0071] The lipopeptide plipastatin also belongs to the same class. While fengicin A has d-Tyr in position 3 and 1-Tyr in position 9, plipastatin A has 1-Tyr and d-Tyr, respectively.

[0072] Like surfactin, fengicin can also be produced by the NRPS (non-ribosomal peptide synthetases) enzyme, and its potential is mainly linked to its strong widespread antifungal activity.

[0073] Among the possible surface modifiers of titanium dioxide particles, the lipopeptide cyclic Sodium Surfactin (SS) is a multifaceted bio-metabolite.

[0074] It is preferably derived from sustainable sources (e.g., food waste, agricultural by-products, or the agri-food industry). This lipopeptide has a wide range of beneficial activities, linked both to its easy biodegradability and its ecological nature of the substance.

[0075] Other examples of lipopeptides that can be used in this invention are iturin, viscosin, lichenisin, gramicidin, polymyxin, megovalcin, etc.

[0076] The choice of lipopeptide surfactant in general, and surfactin in particular, is not obvious for the purpose of improving a sunscreen, such as titanium dioxide (or its derivative or precursor), and in fact, the prior art uses standard surfactants for purely process purposes, which do not actually provide an improvement in SPF.

[0077] On the contrary, as indicated, the lipopeptide surfactant, and surfactin in particular, are capable of improving the SPF in the compound according to the present invention, thanks to their complexation and / or functionalization with titanium dioxide particles (or their precursor or derivative).

[0078] Other possible surfactants that can be used in the present invention for the functionalization and / or complexation of titanium dioxide particles or their precursors or derivatives are: glycolipids, such as ramnolipids, trehalose lipids, sophorolipids, lipids of mannosyl erythritol, cellobiolipids, or fatty acids or phospholipids or neutral lipids, such as corinomycolic acid, spiculisporic acid, phosphatidylethanolamines, etc., or a particulate biosurfactant such as vesicles, whole microbial cells, a polymeric biosurfactant, Emulsan, Liposano, Alasano, a biodispersant, a polysaccharide protein complex, mannoproteins, etc.

[0079] In general, according to one version, the surfactants used in the method of the present invention are derived from sustainable sources (e.g., food waste, agricultural by-products, or agro-food industry by-products).

[0080] The method according to the present invention, for obtaining the compound having the above-mentioned anti-UV properties, is therefore capable of synthesizing a compound comprising the physical filter physical filter based on TiO₂ or its precursor or derivative by functionalizing it and / or complexing it with a surfactant and / or a lipopeptide, such as surfactin or the sodium salt of surfactin (SS) or another surfactant and / or lipopeptide indicated above, creating a complex with improved properties.

[0081] According to at least a version of the invention, such method takes place via hetero-coagulation or, in any case, is based on a hetero-coagulated colloidal approach.

[0082] In particular, this process may comprise a step of providing (nano)particles of titanium dioxide or a precursor or derivative thereof, a step of providing a solution of surfactant and / or lipopeptide, and a step of adding the solution of surfactant and / or lipopeptide to the particles of titanium dioxide or its precursor or derivative obtaining a functionalization of the latter (and in particular of the titanium dioxide) by means of the surfactant and / or lipopeptide of the solution provided.

[0083] According to a first embodiment, the step of providing (nano)particles of titanium dioxide or its precursor or derivative may be carried out by sol-gel synthesis of of titanium dioxide (phase A).

[0084] Phase A may comprise the following (sub)phases:

[0085] preparing a solution of water and hydrochloric acid, e.g., fuming, if desired with a surfactant such as TritonX-100. For example, this step may comprise in a flask, e.g., with a nominal volume of 250 ml, add 46.4 g of ultrapure water and 0.250 g of fuming HC1 in the presence or absence of 0.02 g of TritonX-100 at 2% v / v;

[0086] gently shaking the solution to avoid the formation of bubbles and heat to 50°C or at least 40°C.

[0087] when the above temperature is reached, adding to the heated solution a powder of titanium dioxide or its derivative or precursor and mix for example vigorously. For example, this step involves adding 3.2 g of TiO₂ P25 powder or 3.3 g of titanium isopropoxide to the heated solution under vigorous stirring and maintaining the mixture at reflux for 24 h.

[0088] A gel is obtained from phase A.

[0089] The step of providing a surfactant and / or lipopeptide solution, according to a first embodiment, consists of solubilizing the surfactant and / or lipopeptide in water (phase B). For example, this phase B may comprise solubilizing 2 g of lipopeptide (surfactant) in the form of sodium salt in 10 ml of ultrapure water.

[0090] The step of adding the surfactant and / or lipopeptide solution to the titanium dioxide particles to achieve functionalization and / or complexation of the latter by means of the surfactant and / or lipopeptide in the solution provided, according to a first embodiment, comprises an aggregation phase (phase C).Phase C may comprise the following (sub)phases: adding the gel obtained from phase A to the solution obtained from phase B and mixing while heating to a temperature of 40°C, for example for 24 hours.

[0091] This phase C comprises, for example, adding 40 ml of phase A to 8 ml of phase B, maintaining agitation at 500 rpm at 40°C for 24 hours under reflux.

[0092] After 24 hours, a milky white suspension forms from phase C, which separates with difficulty if left undisturbed. This suspension is characterized by a dense surface foam. The pH of the solution is between 5 and 6.

[0093] The process according to the present invention may also comprise a phase D comprising the following steps: centrifuging the suspension obtained from phase C, for example at 8000 rpm for 20 minutes, washing the pellet obtained from the centrifugation step with water (removing the excess supernatant), for example with 3x1 volumes of ultra-pure water, and centrifuging, for example under the same conditions as the first centrifugation. The washing and centrifugation steps may be repeated several times.

[0094] Finally, the solid pellet is collected, for example in a 250 ml borosilicate glass flask, frozen in liquid nitrogen and freeze-dried for 48 hours.

[0095] This yields a powder of the compound according to the present invention, in which the particles of TiO₂ or its derivative or precursor form a complex with the surfactant and / or lipopeptide.

[0096] According to a second embodiment of the present invention (which exploits a colloidal self-assembly approach), the method may involve the preparation of nanoparticles modified by functionalization, according to the following steps:

[0097] the step of providing the titanium dioxide particles comprises a step of dispersing the (nano)particles of titanium dioxide or its derivative or its precursor in ultrapure water or ethanol (EtOH, concentration from 10 to 30 mM) by sonication to separate the loosely agglomerated particles and obtain a particle suspension (phase A'),

[0098] the step of supplying a surfactant and / or lipopeptide solution takes place, for example, by supplying a solution of surfactant or lipopeptide or SS or SCOOH (unsalted surfactin), referred to as phase B',

[0099] mixing the surfactant and / or lipopeptide solution with the particle suspension to obtain a reaction mixture which is sonicated, for example at a frequency of 39 kHz, forexample for 30 minutes,

[0100] keeping under agitation, for example magnetic, at 60°C (phase C) for example for 24 hours, if desired under reflux conditions, obtaining a TiO₂ -lipopeptide mixture or a TiO₂- surfactant mixture.

[0101] Preferably, the step of obtaining a reaction mixture, and if desired the subsequent steps, take / s place in the dark, for example by wrapping the container in aluminum foil to avoid photocatalytic reactions.

[0102] At the end of the reaction, the TiO₂- lipopeptide mixture or the TiO₂ -surfactant mixture (e.g. SS) is centrifuged (phase D, e.g. at 6000 rpm for 30 minutes at +4°C) to promote pellet deposition, and the pellet is washed with water, e.g., 2x1 volume of ultrapure water, removing any unbound surfactant or lipopeptide residues. This is followed by a lyophilization step (e.g., for 72 hours).

[0103] In order to arrive at the present invention, various options were studied (e.g., varying the source of TiO₂, the order of addition of the various components, the weight ratio of TiO₂ / lipopeptide or TiO₂ / surfactant or TiO₂ / SS or titanium isopropoxide / SS) and the resulting properties were compared, for example, by measuring the capacity of filtration UV, of photoreactivity, of the index of dustiness and of the end-point ecotoxicological. This has made it possible to obtain a method that is optimized in terms of both safety and sustainability.

[0104] The present invention therefore also relates to the cosmetic use of the compound according to the present invention, or of the compound obtained by the process of the present invention, for topical use, as a UV radiation filter or, in general, as a UV radiation protective component in creams and cosmetic formulations, or medical devices.

[0105] This use occurs with the compound present in an amount between 1 and 200 μg / ml. The present invention also relates to a cream or cosmetic formulation or a medical device for topical use comprising the compound according to the present invention or obtained according to the method of the present invention, wherein the compound acts as a filter against UV radiation.

[0106] Examples of implementation:

[0107] The resulting samples, depending on the order of addition (first the component in whichit was dripped the second), were named P25@SS or SS@P25 and EU@SS or SS@EU, respectively, and the respective data are shown in Table 1 below.

[0108] Table 1: Details of the reaction variables applied for each compound in the optimization phase of the colloidal approach.

[0109] a)

[0110] Modified Dropped Reaction Solvent Stoichiometry [mM]TiO2 [mM]ligand materials Solution

[0111] .

[0112] P25@SS. ss. Mi'lliQ. i'-'i 15?8 mM. (1:1)

[0113] i'"'j^.

[0114] S'S"@'P25. P25 Mi'lliQ. i'-'i 15JmM. (1:1)

[0115] SS MiiliQ 30"mM 15 mM (2:1)

[0116] SS@P25 P25 MiiliQ 15 mM 3O'mM (2:1)

[0117] — —

[0118] P25@SS SS EtOH i'-'i T5?8 mM (1:1)

[0119] — —

[0120] SS"@P25 P25 EtOH i'-'i l'5'JmM (1:1)

[0121] i-i.....................................

[0122] EU@SS Milii^ nJ'mM

[0123] (1:1)

[0124] iyj'^'.

[0125] SS@EU. EU Miii'iQ. i'-'i 1'74'mM. (1:1)

[0126] UE@SS. SS. EtOH i'-'i i'74 mM. 1'74'mM. (1:1)

[0127] _____ iyj-^

[0128] EU EtOH 1'74'mM (1:1)

[0129]

[0130] T5"SS@EU. EU EtOH 16:1 9.4 mM 94.4 mM (10:1)

[0131] l?i.....................................

[0132] P2T@SCO''''''' SC 'OOH. I'ss 'mM 15JmM OH (1:1)

[0133] 1?1.....................................

[0134] SC66H@P™ P25. 15?8'mM 15 ImM 25 (1:1)

[0135] b)

[0136] Optimized Dropped Reaction Solvent Weight Ratio [mM]TiO2[mM]ligand Materials Solution

[0137] 7^1................................

[0138] P25@SS. 3T6 mM 1.6 mM (13.2wt%)

[0139] fiO2@SS. MilliQ'”'''^^

[0140]

[0141] In the preliminary phase (indicated with a), different stoichiometric ratios were applied and then the optimized material was considered (indicated with b), for example having the weight ratio best suited to describe the experimental conditions and trigger a ligand exchange reaction between the hydroxylated surface of the titanium dioxide particles and the ligands, i.e., the surfactant or lipopeptide, and / or optimized in that it demonstrates improved UV radiation filtering properties.

[0142] Examples of characterization and analysis of the properties of the colloidal compounds obtained: Evaluation of the in vitro sun protection effectiveness of the samples analyzed Preparation of the emulsion

[0143] To evaluate the UV or solar radiation filtration parameters, a cosmetic formulation scheme was designed for an example of an oil-in-water (O / W) emulsion on which the P25@SS or TiO₂@SS compound (e.g., TiO₂ derived from the titanium isopropoxide precursor) was tested compared to P25 or TiO₂ and SS alone.

[0144] In particular, the emulsions were designed taking into account the functionalization dataof the P25@SS or TiO2@SS product obtained by TGA measurement (weight loss of approximately 10 wt%), starting from a total amount of UV filter in the formulation of 10% to obtain the same concentration of P25 in both formulations.

[0145] In practice, the aqueous phase (Phase I) and the oil phase (Phase II) were prepared separately. Phase II was heated to approximately 65°C to facilitate the melting of the components, while Phase I involved preheating the ultrapure water (UW, 70°C); at this point the components were added, starting with the solvation of xanthan gum, for using, for example, a Silverson® L5M-A Turboemulsifier (Silverson®, Evry, France), until completely dispersed. When both phases were homogeneous and at the appropriate temperature of approximately 70°C, emulsification was carried out by slowly pouring Phase I into Phase II while stirring, for example using the Silverson® Turboemulsifier L5M-A, until a homogeneous product was obtained. Subsequently, this product was left to reach room temperature under gentle stirring and, once it reached 30-40°C, it was completed by adding phase IV containing the preservatives and finally phase III containing the sunscreen with the aid of a homogenizer, for example the Polytron™ PT1200E portable homogenizer (Kinematica, Malters, Switzerland) 57.5 illlll 57.5 Illlll Illlll D-Panthenol PANTHENOL Moisturizer 0.5 0.5 0.5 0.5 0.5

[0146] Rhodicare T XANTHAN GUM Viscosity enhancer 0.3 0.3 0.3 0.3 0.3

[0147] DDI AQUA 56.7 56.7 56.7 56.7 56.7 PHASE II 21.5 21.5

[0148]

[0149] ■Illi liilll IlllllBrij 72 STEARETH-2 Emulsifier 3 3 3 3 3 Brij S721- PAG- STEARETH-21 Emulsifier 2 2 2 2 2 (SG)

[0150] GLYCERYL GMS STEARATE Emulsifier 1.5 1.5 1.5 1.5 1.5 Cetearyl CETEARYL

[0151] alcohol ALCOHOL Emollient 5 5 5 5 5

[0152] COCO- Cetiol C5 CAPRYLATE Emollient 10 10 10 10 10 PHASE III 10 19 11 20 20

[0153] TITATIUM

[0154] P25 DIOXIDE UV filter - 9 - - 9

[0155] SODIUM SS SURFACTIN - - 1 - 1

[0156] TITANIUM

[0157] P25@SS DIOXIDE, UV filter - - - 10 -

[0158] rTiO2@SS SURFACTIN

[0159] Cetiol C5 COCO- Emollient 10 10 10 10 10

[0160] CAPRYLATE PHASE IV iiiiiii BENZYL

[0161] Bioscontrol ALCOHOL, Preservative 1 1 1 1 1 Sinergy BAS DEHYDROACET

[0162] IC ACID, AQUA

[0163] NaOH 0.05% SODIUM pH regulator Up to pH 6.4

[0164] HYDROXIDE

[0165]

[0166] Determination of sun protection factor (SPF)

[0167] The in vitro SPF (Sun Protection Factor) analysis of the formulation was performed using a method proposed for the ISO 24443:2012 standard for the determination of in vitro UVA protection, and adapting it to UVB (Dimitrovska Cvetkovska A, Manfredini S, Ziosi P, Molesini S, Dissette V, Magri I, Scapoli C, Carrieri A, Durini E, and Vertuani S 2017 Factors affecting SPF in vitro measurement and correlation with in vivo results Int. J. Cosmet. Sci. 39 310-9), also taking into account the requirements of European Recommendation EC 647 / 2006 of September 22, 2006 on the evaluation of the efficacy of sunscreen products (C(2006) 4089) Off. J. Eur. Union 26539-43).

[0168] The method is based on spectrophotometric evaluation of UV absorbance (calculated from transmittance) using a spectrophotometer, such as SHIMADZU UV-2600, equipped with a 60 mm ISR 2600 integrating sphere and coupled with SPF calculation software (SPF calculator software version 2.1, Shimadzu, Milan, Italy), with wavelengths emission from 290 to 400 nm and increments of 1 nm.

[0169] Each sample was irradiated using the Atlas SUNTEST CPS+ solar simulator (Suntest CPS; Atlas, Linsengericht, Germany), equipped with a xenon lamp, an optical filter to cut wavelengths below 290 nm, and an IR-block filter to avoid the thermal effect, and set to operate between 40-200 W / m2in accordance with ISO 24443:2012.

[0170] Four plates with 5 measurements acquired were prepared for the investigation. Results with a covariance of less than 3% were accepted, and after irradiation, the respective values were obtained.

[0171] The SPF (Sun Protection Factor), relating to radiation that induces erythema, is calculated as described in the following equation (1):

[0172] In vitro SPF

[0173]

[0174] where E(λ) is the erythema action spectrum (CIE-1987) at a wavelength λ, I(λ) is the irradiance spectrum received from the UV source at λ, A(λ) indicates the monochromatic absorbance per plate before UV exposure of the product layer under test at λ, and d(λ) is the λ step (1nm).i. SPF label

[0175] ii. UVAPF (UVA Protection Factor), described by the absorption spectra after irradiation using the following equation (2):

[0176] UVAPF =

[0177]

[0178] 2)P^> 10 ” W <1)

[0179] where I(λ), A(λ), d(λ) are defined by the equation, P(λ) is the PPD (Persistent Pigment Darkening) spectrum, and C is the adjustment coefficient of the SPF value calculated in vitro to the labeled SPF value (in vivo) (recommended between 0.8 and 1.2).

[0180] The UVA protection factor (UVAPF) of each sunscreen formulation was verified instrumentally before and after a period of controlled UV irradiation.

[0181] iii. label SPF / UVAPF ratio, based on the formula's performance in in the UVA range, compared to the overall SPF value declared on the label. Similar to critical λ, this ratio provides an assessment of the breadth of protection across UV spectra without considering the extent of filtering activity. Values close to 1 are indicative of broadspectrum activity.

[0182] iv. Critical λ, which describes the breadth of protection across all UV spectra (280-400 nm). It is the wavelength at which 90% of the area under the absorbance curve (AUC) is reached starting from 290 nm.

[0183] Colloidal behavior: DLS and ELS

[0184] The colloidal properties of the sample suspensions were studied in terms of hydrodynamic diameter (dDLS) and Zeta potential ( -potELS), determined respectively by dynamic and electrophoretic light scattering measurements using a Zetasizer nano ZSP (model ZEN5600, Malvern Instruments, UK).

[0185] Both analyses on colloidal samples were performed at 25°C on powders dispersed in ultrapure water (UW) with 10 minutes of ultrasonic mixing to obtain a final concentration of 320 pg / mL (320 ppm). After a 2-minute temperature equilibration step, 1 ml of the samples for DLS and 700 pL of the samples for ELS were measured consecutively three times, and the data were obtained by calculating the average of the three measurements. To evaluate the isoelectric point (pHi.e.p), acid-base titrations were performed. Titrantsused were 0.1 M KOH and 0.1 M HC1 solutions on suspensions diluted with distilled water.

[0186] With regard to the aforementioned physicochemical characterization techniques, and in particular the evaluation of colloidal properties (to assess colloidal stability, the hydrodynamic diameter (dDLS), the Zeta potential ( -potELS) at natural pH, and the pHi.e.p values of both pure or as-is materials and TiO2-modified samples) were measured in UW. The results are reported in Table 2 below.

[0187] Table 2: Results of the colloidal characterization of the starting and modified materials diluted in UW at 25°C. The composite samples are named by first indicating the component into which the second component was dripped, with the weight ratio between the two components in parentheses. The table shows the data in terms of hydrodynamic diameter (dDLS) in nm, Pdl, Zeta potential ((^-potELS) in mV, working pH, and pH i.e.p. for both the starting materials or as-is and the TiO2-based nanosuspensions.

[0188] Starting Reaction

[0189] materials Solvent dDLS [nm] Pdl ^-potELS [mV] pH pH i.e.p.

[0190] Peroxide (P25) 599 + 50 0.5 + 41.1 3.7 6.13 Eusolex T-AVO 724 ±30 0.4 -42.3 7.8 2.98 (EU)

[0191] Sodium Surfactin 1852 + 131 0.8 -48.5 6.4 < 2 (SS)

[0192] Surfactant

[0193] (SCOOH) 1653 + 36 n.a. 0 5 2.4 Modified

[0194] materials

[0195] (molar ratio)

[0196] P25@SS (1:1) MilliQ 241 + 8 0.3 -30.3 6.2 3.07 SS@P25 (1:1) MilliQ 289 + 39 0.4 -37.8 5.9 2.75 P25@SS (2:1) MilliQ 173.7 + 19 0.3 -24.0 5 2.99 SS@P25 (2:1) MilliQ 843 + 357 0.7 -47.0 5.5 < 2

[0197]

[0198] P25@SS (1:1) EtOH 2049 + 687 1 -28.7 6.1 n.a. SS@P25 (1:1) EtOH 2368 + 422 0.5 -21.6 5.5 n.a. EU@SS (1:1) MilliQ 224 + 6 0.3 -27.2 4.9 3.1 SS@EU (1:1) MilliQ 259 + 59 0.3 -39.5 5.7 3.07 EU@SS (1:1) EtOH 274 + 4 0.3 -29.3 9.1 n.a. SS@EU (1:1) EtOH 227 + 3 0.3 -39.1 7 n.a. SS@EU (10:l) EtOH 890 + 50 0.4 -63.0 6.5 3.43 P25@SCOOH MilliQ 1334 + 108 n.a. -51.0 5 1.9 (1:1)

[0199] SCOOH@P25 MilliQ 2560 + 881 n.a. -24.0 5.5 2.1 (1:1)

[0200] Optimized

[0201] materials

[0202] (weight ratio)

[0203] P25@SS MilliQ 315 + 8 0.4 -24.8 6.8 4.3 (13.2 wt%)

[0204] TiO2@SS MilliQ 300+6 0.41 -23.2 6.6 4.1

[0205]

[0206] In particular, DLS analysis of suspended TiCh P25 NPs showed a particle size distribution of approximately 600 nm, highlighting the strong tendency of TiO2 to form large agglomerates in aqueous media.

[0207] The data shown in Table 2 above reveal the following.

[0208] The TiO2@SS (1:1) composites dispersed in water showed smaller hydrodynamic diameters than the pure P25 and EU powders, regardless of the order of addition, probably due to steric hindrance and electrostatic stabilization due to the sufficiently high negative C, potential provided by the SS component. The P25@SS samples in EtOH or excess SS are characterized by large agglomerates, probably due to the compression of the electric double layer in a non-aqueous solvent or a coagulation effect due to depletion caused by excess SS.

[0209] By reducing the amount of SS (data not shown), the minimum amount of SS (10%) thatprovides good colloidal stability was identified, and the P25@SS or TiO2@SS sample, with a weight content of not less than 10% (e.g., 13.2 wt.% or 13%) of SS compared to TiO2, was considered the optimized sample.

[0210] Preferably, according to at least one embodiment of the present invention, the compound comprises at least 10% by weight of surfactant or lipopeptide relative to the weight of the titanium dioxide particles or a weight percentage content preferably of about 13% or 13.2% or between 10% and 15% relative to the weight of the titanium dioxide particles. In general, in the compound according to the present invention, the surfactant and the physical filter particles are in a ratio of 1: 1 or in a ratio of 7,6: 1 or in a ratio of 7: 1 or in a ratio between 1:1 and 10:1, or said surfactant is present in a percentage by weight of 13.2% or 13% or in a percentage of at least 10% or at least 5% or at least 1% or between 10% and 15% or between 5% and 30% or between 1% and 90% or between 5% and 80% or between 5% and 50% with respect to the weight of the particles.

[0211] According to a further version, in the compound according to the present invention, the particles of the physical filter and the surfactant are in a ratio of 1: 1 or in a ratio of 7,6:1 or in a ratio of 7:1 or in a ratio between 1:1 and 10:1, or such particles are present in a percentage by weight of 13.2% or 13% or in a percentage of at least 10% or at least 5% or at least 1% or between 10% and 15% or between 5% and 30% or between 1% and 90% or between 5% and 80% or between 5% and 50% relative to the weight of the surfactant. The increase in hydrodynamic diameter is observed in SCOOH-coated samples obtained by the self-assembly method with unassociated surfactants. This is due to the absence of electrostatic stabilization provided by the uncharged SCOOH surfactant. By comparing the pH (i.e., the zero charge point) of P25 and SS (Figure 1), a pH range in which the two components have opposite charges (from 2 to 6), has been observed, favoring heterocoagulation between the two compounds. Confirming the presence of SS around the TiO2 surface, a shift in the pH of SS towards a more acidic pH was observed.

[0212] In summary, the above data show that electrostatic interactions (zeta potential) are the main mechanism underlying the formation of compounds according to the present invention. In this regard, the main driving force was observed in the P25@SS sample, obtained through an electrostatic interaction between negatively charged SS and positively charged titanium nanoparticles, facilitated by mixing the sols (e.g., in well-defined ratios) and by the reaction that took place during agitation, e.g., magnetic, preferably for 24 hours.

[0213] The coating of SS on TiO2 surfaces (or vice versa) has therefore proved very promising, exploiting the opposite charge of the particles involved.

[0214] The P25@SS prototype was obtained by dispersing 13.2 wt% of SS in water, for example ultra-pure water, compared to P25, showing a zeta potential (^-potELS) value of -24.8mV, pHi.e.p. of 4.3, and dDLS of 315 ±8 nm (Pdl 0.4).

[0215] As demonstrated by the data reported (hydrodynamic diameter (dDLS) and zeta potential measurements), the surfactant or lipopeptide or SS coating stabilizes the colloidal behavior of titanium in aqueous suspension, resulting in a reduction in dimensional values compared to pure or as-is P25 (599+50 nm and Pdl 0.5) and a different surface reactivity (^-potELS of P25 is +41.1 mV with a pHi.e.p. of 6.13).

[0216] As shown in Figure 2, the stability of particle dispersion was evaluated for 3 days; all samples, including the control, show relatively homogeneous dispersion, indicating good initial mixing of particles or emulsions. TheSS@EU and P25@SS or TIO2@SS samples, in particular, show good initial dispersion. P25 and SS appear as clear solutions, demonstrating that these substances are initially well dispersed. After 8 hours, SS@EU and P25 show good stability, with no visible phase separation or sedimentation. This suggests that these formulations can maintain their dispersion state for a moderate period of time. SS and P25@SS, however, begin to show signs of slight sedimentation or aggregation, indicating the onset of instability in these samples within 3 days. The TiO2@SS sample behaves similarly. P25 and SS @EU maintain a clear and homogeneous appearance, suggesting excellent long-term dispersion stability for 3 days. This indicates that P25, probably in its nanoparticulate form, is stable in the medium and does not aggregate or even after prolonged exposure to UV rays.

[0217] Taking all the data into account, for practical reasons only, subsequent characterizations were continued on two samples, P25@SS (SS 13.2 wt%) and EU@SS (1:1), both dispersed in ultrapure MilliQ water (hereinafter simply referred to as P25@SS and EU@SS), which represent the optimal compromise between a lower tendency to form aggregates and greater colloidal stability compared to P25 and EU, respectively.However, this does not constitute a limitation for the present invention but rather an advantage in terms of the stability of the sunscreen product composed of them.

[0218] Structural characterization: FTIR-ATR spectroscopy

[0219] The structural analysis of the samples obtained was performed using a Fourier transform infrared spectrometer equipped with a total reflection attenuated reflection accessory (FTIR-ATR).

[0220] The prepared samples (P25@SS (1:1) and EU@SS (1:1), as well as P25 and EU and SS alone, are were analyzed in dry form. The ATR spectra were recorded in transmission mode, performing a total of 8 scans for the powder samples at room temperature (25°C) with a spectral resolution of 4 cm-1, using Resolution Pro version 2.5.5 software (Agilent Technologies, USA). The scan was performed in the range 4000-550 cm-1range to identify the main functional groups.

[0221] Structural analysis and assignment of basic peaks were performed on the FTIR-ATR spectrum to characterize the "coating" on the SS and nanopowder samples, in particular to investigate any changes occurring in the secondary structure of SS.

[0222] Figure 3 shows a sequence of spectra obtained on P25, EU, SS, P25@SS, and EU@SS powders. In particular, it is possible to observe and assign the contributions of TiO2, the lipopeptide skeleton, and the lipopeptide head group. Since all the spectra of each group of SS-treated samples have similar characteristics, a representative example is shown for P25@SS (Fig. 3A) and one for EU@SS (Fig. 3B).

[0223] As for the IR spectrum of SS, it clearly shows the characteristic peaks of peptides at 3290 cm-1(N-H stretching mode), 1535 cm ’(N-H bending and C=O stretching mode for Glul and Asp5) and at 1645 cm resulting from the CO-N bond stretching mode. Furthermore, the bands at 2956-2927 cm and 1467-1368 cm are representative of C-H group vibrations, confirming the presence of aliphatic chains (CEE; CH2) with symmetric stretching at 2871 cm ’. As for the lactone ring, a characteristic peak at 1736 cm-1as C=O stretching (carbonyl group of the cyclic ester). The IR analysis pattern confirms the cyclic nature of the lipopeptide and the characteristics of the lipopeptide biosurfactant described above.

[0224] As regards the EU@SS product (Fig. 3B), the EU spectrum (black line) is is mainlycharacterized by the peak at 1084 cm attributable to Si-O-Si stretching, since EU is commercially described as SiO2 coated with TiO2, while the band at 577 cm ’is representative of the TiO2 matrix. EU also shows an additional band at approximately 950 cm ’ for the stretching of Si-O- and Si-O-Ti species. Unlike P25, the broad band at approximately 3350 cm ’ for O-H stretches is less pronounced, reduced by interaction with the silica shell.

[0225] In the EU@SS spectrum (light gray line in Fig. 3B), there is a significant decrease in the Si-O-Si signal at 1084 cm ’ (characteristic of the pre-coating), in favor of the new type of external coating, for example based on surfactant. In fact, the characteristic SS peaks can be observed but slightly shifted towards lower frequencies, such as 1641 cm ’ 1520cm’, with the disappearance of the peak at 1389 cm ’attributable to C-H stretching.

[0226] From this, it can be deduced that the surfactant, for example SS, "displaces" the silica coating of EU.

[0227] Morphological characterization: SEM-EDS and TEM analysis

[0228] The chemical composition and surface morphologies of the particles were analyzed using scanning electron microscopy (SEM) (model EVO 40, DE) coupled with an energy dispersive X-ray spectroscopy (EDS) detector (Inca Energy 300, Oxford Instruments). The samples were examined at an acceleration voltage of 15-20 kV.

[0229] To improve the surface conductivity of the samples and consequently the resolution of the images, a gold sputtering deposition treatment can be applied by vacuum evaporation. The images were collected at high vacuum and with a high electron beam voltage to achieve maximum resolution. EDS system analysis was used to verify the presence of the coating, e.g., organic, through elemental / compo sition analysis, in a "spot mode" in which the beam was located on a single manually selected area within the field of view.

[0230] As for the morphology of the particles of the selected sample modified with SS, abbreviated as P25@SS and EU@SS, Figure 4 shows some SEM images of the compounds. These images are visibly different when compared to the pure physical mixtures pure or as-is TiO2 or TiO2+SS. The typical roughness of titanium dioxide is attenuated in the TiO2@SS composites, while in physical mixtures the presence of the two separate phases is still evident.EDS analysis (elemental composition)

[0231] The TEM images of the analyzed titanium dioxide powders are shown in Figure 5. The analyzed samples were subjected to the normal resin inclusion process (A and C) and without resin (B and D). Therefore, P25 nanoparticles have a spheroidal morphology, with typical primary particle diameters of 20-30 nm. P25@SS composites showed better dispersion, with poorly defined nanostructures. The TEM images of P25@SS confirmed that the TiCh nanoparticles were almost incorporated into the SS matrix, in accordance with the above-mentioned heterocoagulation process. However, in order to exclude the influence of the resin used in the preparation of the samples, samples prepared with and without resin were compared, which proved to be essential for keeping the particles well separated in the case of sample P25@SS.

[0232] From this, it can be deduced that the method according to the present invention produces a complex and not a physical mixture, unlike what is known in the prior art, where SS has been recognized as having a dispersing function.

[0233] Therefore, from a morphological point of view, using SEM, the pure or as is P25 nanoparticles were observed to be spherical in shape; however, after functionalization, a structural variation was noted.

[0234] There is therefore a transition from a spherical shape to an amorphous shape, demonstrating the complexation that has taken place, which is better clarified by TEM. Therefore, the component according to the present invention has an amorphous shape.

[0235] Thermal analysis: TGA and DSC

[0236] Thermal behavior was studied using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), for example using a Netzsch STA 449 F3 Jupiter® instrument. The measurements were performed on sample powders weighing approximately 6 mg, placed in a platinum / rhodium crucible using alumina (Al2O3) for internal calibration. The experiment was conducted in an air / Ni mixture (40 ml / min of air and 80 ml / min of N2) with a heating rate of 5°C / min from room temperature to 900°C. The weight losses of the target samples (at 900°C) are shown in Table 3. As far as P25 is concerned, the minimum weight loss observed refers to water evaporation (0.91%). Thiswas slightly higher for EU, as it is a commercial product with a SiO2 coating and a presumably higher concentration of adsorbed water. In contrast, the thermal behavior of the organic molecule alone (SS) is characterized by significant decomposition of the biosurfactant up to a weight loss of 90.55%. In the samples modified with SS, the difference in in TGA mass compared to the respective pure or as-is materials is attributed to the amount of SS adsorbed (approximately 10 wt%), in accordance with the estimated amount needed to stabilize the P25 sample (13.2 wt%). Otherwise, the weight loss observed in the EU@SS sample (approximately 4%) suggests that the selected EU@SS (1:1) sample, containing 100% of the SS content compared to EU, indicates that this sample has an excess of SS that is not adsorbed on the surface.

[0237] The TGA analysis coupled with the DSC technique is shown in Figure 6.

[0238] Table 3. Weight loss (%) detected at 900°C by TGA analysis. Weight loss (%) of each sample is simply the difference between the weight of the sample before and after the heating process.

[0239] Sample Weight loss (%)

[0240] P25 0.91 +0.02

[0241] EU 4.04 + 0.03

[0242] SS 90.55 + 0.09

[0243] P25@SS 13.70 + 0.03

[0244] EU@SS 4.19 + 0.01

[0245]

[0246] The DSC curves of P25 are characterized by an exothermic peak with a Tm of 414°C, attributed to the phase transformation from anatase to rutile. The maximum peak temperature (Tm) of a broad fusion is the most significant and is therefore used in practice to distinguish between different forms of nano- TiOi.

[0247] As for EU, the greater weight loss compared to P25 can be attributed to greater desorption of physically absorbed water, as confirmed by the corresponding endothermic peak in the DSC. This is due to the fact that SS is a hygroscopic material, therefore capable of absorbing water from the surrounding environment during handling.The thermogram of P25@SS shows significant differences compared to that of P25. The main characteristics that differentiate the peaks are (i) the peak area proportional to the enthalpy of the process, (ii) the direction of the peak (endothermic or exothermic process), (iii) the shape (defined by onset, peak temperature, endpoint, and inflection), all characteristics that depend on the quality of the surface coating analyzed. After the initial dehydration phase (at a temperature below 100°C), the heat treatment of P25@SS is accompanied by two endothermic peaks (Tml 323°C, Tm2423°C), proportional to a high heat demand. This second phase corresponds to the degradation of organic matter (SS) from the sample. The two distinct peaks observed in the DSC can be associated with the denaturation of the skeleton chain, which leads to release of the trapped biosurfactant and its thermal decomposition, together with the breaking of various types of bonds (functionalization and complexation) on the surface of TiO2. However, the same considerations cannot be made when observing the EU@SS adduct. Following functionalization, in fact, no different thermal profile is observed compared to to pure or as-is EU. The first thermal domain characterized by an endothermic peak (Tml at 381°C) does not differ significantly from the corresponding peak of TiO2 EU (Tml of 373°C), as does the post-transition baseline temperature at 566°C, most likely because the excess SS masks the information related to the processes occurring at the substrate-coating interfaces for EU@SS.

[0248] In summary, TGA analysis of pure or as-is starting materials and TiO2 NPs modified, conducted in the temperature range 24°C-900°C, in an air / Ni mixture (40mL / min of air and 80mL / min of N2) with a heating rate of 5°C / min, was performed to estimate the surface coverage rate of the NPs for each ligand (surfactant and / or lipopeptide). The TGA of pure P25 showed no mass loss in the temperature range examined, confirming the purity of the starting material previously observed by FT-IR analysis. As for the functionalized P25 NPs, a mass loss of about 10% was observed for SS-functionalized P25 NPs, but only a minimal amount for EU@SS compared to pure or as-is EU. Furthermore, DCS analysis corroborated the chemisorption of the SS ligand on P25, demonstrating the endothermic processes that occur approximately between 300°C and 500°C, associated with the decomposition of the attached organic fraction.Photocatalysis - Photocatalytic activity (safety test)

[0249] The photocatalytic activity of TiO2-based samples was determined by adapting a method previously reported in the literature (van Driel BA, Kooyman PJ, van den Berg KJ, et al (2016) A quick assessment of the photocatalytic activity of TiO2 pigments - From lab to conservation study, Microchemical Journal 126:162-171. https: / / doi. Org / 10.1016 / J. MICROC.2015.ll.048), monitoring the degradation of a synthetic water-soluble food dye used in the analyses as an indicator (AB9), after UV radiation. The dye degradation test was prepared by adding 10 mg of photocatalytic material to a 7.7 pM ethanol solution of AB9, to a final volume of 100 ml, and sonicated for 10 minutes to disperse the powder evenly in the AB9 solution. The experiment was conducted under magnetic stirring and at room temperature in a UV box equipped with a UV-A lamp (Osram, L BLUE UVA 18W / 78, wavelength) at a distance of 20 cm from the sample. The wavelength of the emitted light is in the UVA range 315-400 nm, with an irradiation power of 4.7 W and an intensity of 7800 cd. The same set-up was used to evaluate the shielding effects of TiO2-based materials.

[0250] After exposure, the samples (2 ml) were centrifuged at 8500 rpm for 5 minutes at room temperature and filtered (0.45 pm) to separate the powder from the dye solution; they were then analyzed by UV-Vis spectrophotometry. The analysis was performed by measuring the AB9 solution at kmax= 630 nm with a UV-vis spectrophotometer (UV-6300PC, VWR, Milan, Italy). The concentration of AB9 was evaluated based on a calibration curve, and the results are expressed in terms of dye depletion efficiency (Dye depletion efficiency, %) using equation 1, which indicates the ratio between the amount of reagent consumed (depleted dye) and the amount of reagent initially present in the reaction environment, according to Lambert-Beer's law, where A0 is the initial absorbance and Af is the final absorbance:

[0251] Eq. 1 Dye depletion efficiency (%) = (A0 - Af) / A0 xlOO

[0252] The photocatalytic activity of TiO2-based samples was examined and linked to the potential risks associated with photogenerated reactive oxygen species (ROS). Therefore, the depletion of AB 9 dye under visible light irradiation was studied, following the methodology described above. The dye depletion efficiency of the modified TiO2 samples modified with and without UV light activation is shown in Table 4. As expected, TiO2P25 was modified in such a way that it could be used as a dye. As expected, TiO2P25 with anatase crystal structure showed the highest depletion efficiency (70.84%), thanks to its high photocatalytic reactivity, in addition to the contribution of dye adsorption in the dark, evidenced by a slight discoloration of AB9 (4.76%). In contrast, TiO2EU showed no photoresponsivity, confirming that the rutile phase is less active and could, in this case, also be inhibited by the silica shell. The slight improvement in efficiency shown by the EU@SS sample (1.78%) is most likely due to the increased dispersion of EU in the presence of SS, which can increase the number of available photoactive surface groups. None of the TiO2-SS samples analyzed, regardless of the options selected, showed significant depletion of the AB9 dye, confirming the effective ability of SS, intimately bound to the TiO2surface, to suppress photocatalytic activity. However, P25@SS (13.2 wt%) is confirmed as the best sample in terms of photo stability, showing the lowest reactivity. To assess how the intimate interaction between TiO2and SS in the heterocoagulated sample affected photo stability, the reactivity of the physical mixture (P25+SS Mix, 9:1) was also evaluated, confirming that, in this case, the SS matrix is not as effective in suppressing photocatalytic efficiency (approximately 17%).

[0253] Table 4. Depletion of AB9 dye expressed as a percentage of dye depleted relative to the initial concentration (depletion efficiency %) under "dark" or " UV" conditions Sample Analysis conditions Photocatalytic Efficiency (%)

[0254] AB9 Dark 0.00

[0255] UV 0.00

[0256] P25 Dark 4.76

[0257] UV 70.84

[0258] EU Dark 0.26

[0259] UV 0.64

[0260] SS Dark 0.04

[0261] UV 0.10

[0262]

[0263] P25+SS Dark 0.33

[0264] (Physical UV 17.35

[0265] mixture)

[0266] P25@SS Dark 0.54

[0267] (13.2 wt%) UV 0.70

[0268] MilliQ

[0269] P25@SS(1:1) Dark 0.82

[0270] MilliQ UV 1.19

[0271] SS@P25(1:1) Dark 0.47

[0272] MilliQ UV 2.63

[0273] EU@SS (1:1) Dark 0.12

[0274] MilliQ UV 1.78

[0275]

[0276] Dustiness index - Vortex Shaker method

[0277] The dustiness of TiO2-based powders was studied using the high-emission Vortex Shaker method to simulate real-life handling scenarios.

[0278] The data were evaluated using two parameters derived from sampling the breathable filters and CPC (Condensation Particle Counter) measurements:

[0279] 1) Mass dust index (DIRM) measured in mass (mg / kg).

[0280] It is defined as the mass Amf of particles collected on a respirable sampling filter divided by the massM 0 of dust placed in the cylindrical tube (Equation 2)

[0281] Eq. 2 〚DI〛_RM=(Δmf) / M_0

[0282] 2) Numerical dustiness index (DIRN) measured in number (# / mg). It is defined as the number of particles emitted during the vibration period (tV) from the massM 0 of dust placed in the cylindrical tube (Equation 3).

[0283] Eq 3 〚DI〛_RN=1 / M_0 ∑_0^T (C_CPC (t)*Q_VS* 〚Δt〛_CPC) / t_V

[0284] Adapting a test protocol previously reported in the literature (Dazon C, Witschger O, BauS, et al (2017) Pulverization of 14 carbon nanotubes using the vortex mixer method. J Phys Conf Ser 838:012005. https: / / doi.org / 10.1088 / 1742- 6596 / 838 / 1 / 012005), test samples were prepared by pouring 0.5 cm3of powder into a microtest tube. After filling, the test tubes were weighed to an accuracy of to the nearest 10 pg. The samples were conditioned for at least 24 hours prior to dustiness testing in a laboratory chamber with controlled humidity at 50 ± 5 % RH (Relative Humidity). After cleaning the entire test bench and / or, if necessary, changing the tubes and connections, and after equipping the cyclone with a pre-weighed filter for gravimetric analysis, the air flows were checked using a flow calibrator.

[0285] Air cleaning through the test bench was then evaluated based on CPC measurements. The mass of test sample M0 was obtained from the difference between the mass of the filled micro tube and the mass of the micro tube after emptying.

[0286] The vortex shaker method is also capable of determining the specific surface areas of s of dusts with a single point using the Brunauer-Emmett-Teller (BET) analysis method, in which the samples were pre-treated under vacuum at 120°C.

[0287] The dustiness of a powder is an important physical-chemical indicator of worker exposure and contributes to the definition of health and safety aspects in the production and processing of chemical products / materials, particularly relevant for defining the safety profile of ultrafine powders. The number-based dustiness index (DIRN) and mass-based dustiness index (DIRM), together with the results of preliminary data from the determination of the dustiness index, such as specific surface area and bulk density, are shown in Table 5.

[0288] Comparing the samples in terms of BET, no significant differences are found.

[0289] The only parameter that shows a clear difference is the apparent density, with P25 showing a lower density due to the presence of many aggregates and the resulting interparticle voids. In terms of the dustiness index, the comparison between P25@SS and pure or as-is P25 showed a narrow range of values, with no significant improvements. Therefore, it is noted that, unexpectedly, functionalization with SS did not lead to an increase in emission potential, despite the greater dispersibility of TiO2in the SS matrix.Table 5. Results obtained from the Vortex mixer - Respirable fraction (<4 pm).

[0290] Specific

[0291] Material DI RM DIRN surface area Bulk density Median Mode 1

[0292] (mg / kg) CV(%) (# / mg) CV(%) (m2 / g) (g / cm3 j CV(%) (pm) (pm)

[0293] P25 6.16E+0 3.01E+01 1.03E+05 3.04E+0 4.87E+01 1.64E- 4.81E+ 1.17E+00 1.84E+0

[0294] 3 1 01 0 0

[0295] P25@SS 2.46E+0 5.24E+01 2.00E+05 3.32E+0 4.46E+01 4.05E- 5.24E+ 8.10E-01 1.84E+0

[0296] 3 1 01 0 0

[0297] EU 1.04E+0 3.52E+01 1.28E+06 6.88E+0 4.95E+01 9.37E- 6.49E+ 8.70E-01 1.16E+0

[0298] 5 0 02 0 0

[0299]

[0300] Ecotoxicity

[0301] The study to evaluate the toxicity of the materials according to the present invention was carried out by performing an algae test and a ZET test. The materials were characterized and their behavior was analyzed in fresh water as a test medium.

[0302] Preliminary algae test: Freshwater algae and cyanobacteria, OECD 201 growth inhibition test The ecotoxicity of the various nanomaterials was tested using the OECD TG 201 method on Selenastrum capricornutum. The culture medium used for algae growth and during the test was that specified by the EPA 1003.0 method.

[0303] The materials under study (also known as Enhanced Nano Materials) were dispersed in artificial fresh water, the medium in which the organism grows. The dispersion conditions were optimized and compared with materials dispersed in ultra-pure water (modified and unmodified) and based on the NANoREG dispersion method. No stabilizers were used.

[0304] The dispersion method consists of estimating the volume of ultra-pure water to re-disperse the nanomaterial at the desired concentration. Then, the powder must be weighed in aselected, well-cleaned vial / bottle and dispersed using the ultra-pure water estimated in the first step to achieve the desired concentration (it is not recommended to exceed 2.56 mg / ml). To achieve optimal dispersion, 1 ml of ultra-pure water under agitation for 1 minute. After 1 minute of incubation, the rest of the volume was added under agitation until the desired concentration was reached. Finally, the vial / bottle was placed in a container with ice water and the dispersion was sonicated for at least 5 minutes. To prepare the ice water mixture, a 250 ml glass beaker was filled with ice and placed upside down in an insulated box. As soon as the volume of ice reached 10-15% of the beaker, the vial was placed inside the beaker to keep the dispersion cold during sonication. The sonication time and conditions must be adjusted according to the sonicator available in each laboratory, in accordance with the NANoREG protocol.

[0305] Three replicates of the algae were exposed for a period of 72 hours to dispersed nanomaterials in fresh water. The nominal concentrations were 100, 10, 1, 0.1, 0.01, and 0 mg / L. Algae growth was estimated using optical density at 670 nm (OD670). The OD of the algae suspensions was measured during the 3 days of the test, i.e., after 24 hours, 48 hours, and 72 hours. Data processing was performed using the MOSAIC tool for ecotoxicologists and regulators designed by the University of Lyon. A material was considered toxic when the EC50, i.e., the concentration at which the population is halved, is less than 100 ppm. In the case of algae, EC50 will be considered to be the population in which growth is halved.

[0306] Firstly, the algae test is a preliminary test to determine the acceptability or otherwise of the nanoparticle in the specific context of use and regulatory standards. It is a test of the inhibition of the growth of aquatic algae and cyanobacteria.

[0307] To assess whether this value is acceptable, it is typically compared to regulatory thresholds or guidelines established by the relevant authorities. These thresholds may vary depending on the jurisdiction and the specific application of the substance. For example, some regulatory bodies may have established permissible limits for the ecotoxicity of substances in water bodies or other environmental compartments.

[0308] In this case, the concentration of particles that may be toxic to algae has been determined. The objective of this test is to examine the toxicity of different samples. The detailed data obtained to determine the acceptability of the materials are summarized in Table 6.Table 6: Preliminary results of the algae test. The data in the table express the parts per million (ppm) or mg / L of particles present in the aquatic environment. The EC50 represents the concentration of titanium nanoparticles that leads to a 50% reduction in growth or in the vitality of the algae population studied.

[0309] EC50........................................

[0310] Sample EC50 Algae 95% interval 6 / 780..........................................

[0311] TiO2P25 Unmodified

[0312] 25 1.000 0.496; 5.586

[0313] ............................

[0314] 50 1,453

[0315] Biosurfactant (e.g., SS) 10 0.058 0.003; 0.462

[0316] .............................

[0317] 25 L215

[0318] 50 24.9888 10; Inf

[0319] TiO2P25 modified 10 11,139 2,690; 65,431

[0320] ___

[0321] with biosurfactant (e.g., 25 13,574

[0322] SS) 50 18,063 10,282; 72,766

[0323]

[0324] It is clear that the modification of the surface of the titanium nanoparticle by the biomolecule, i.e., the surfactant and / or lipopeptide, facilitates the reduction of the ecotoxic effect on algae. In this case, the functionalization of nanoparticles increases the effective concentration 50 (EC 50) from 1.4 to 18 mg / L, by a factor of ten (10 times more). The incorporation of the biomolecule represents a promising alternative for reducing the ecotoxicity of the nanoparticle.

[0325] The EC50 (Effective Concentration 50) is a measure commonly used in ecotoxicology to quantify the concentration of a substance that causes a certain effect in 50% of a test population within a given period of time. In the context of ecotoxicity studies of titanium nanoparticles on algae, EC50 represents the concentration of titanium nanoparticles that leads to a 50% reduction in the growth or vitality of the algae population studied. Consequently, the lower the concentration, the more toxic the nanoparticles are, as demonstrated by the fact that a lower concentration is required to reduce algae growth by 50%. Conversely, increasing the concentration makes the nanoparticles less toxic.

[0326] It is very important to emphasize that the result obtained is based on an amount of SSadsorbed, equal to only 13.2% by weight on TiCh. It should be noted that this type of test is not capable of measuring other effects, such as chronic effects, and is therefore only capable of measuring effects on growth or vitality.

[0327] Acute fish embryo toxicity (AFE) and / or zebrafish embryo toxicity (ZET) tests

[0328] In this ecotoxicity test, the toxicity of nanomaterials on zebrafish embryos was assessed by following and adapting OECD 236 (zebrafish embryo test) for freshwater organisms. Zebrafish egg deposition and sorting were performed as previously described (https: / / doi.org / 10.3390 / nanol3142112). Embryotoxic effects are evaluated evaluated in different stages of development of fish zebra (https: / / anatomypubs.onlinelibrary.wiley.com / doi / abs / 10.1002 / aja.1002030302), in an adaptation of OECD guideline 236. Ten viable zygotes per replicate (well) were arbitrarily transferred to a 24-well plate and exposed in water (in a semi-static regime) to nominal concentrations diluted in series 0, 0.01, 0.1, 1, 10, and 100 mg / L of NPs and 13.2 mg / L of the biomolecule (SS), SS concentration in a dispersion of 100 mg / L of NPs, up to 80 hours post-fertilization (hpf). Pre-filtered freshwater was defined as the experimental control. Four replicates of test concentrations were examined in two independent experiments. Two ml per well (replicate) were used. Particle stability was characterized using a size analyzer (SZ-100 device, Horiba, ABX SAS, Amadora, Portugal).

[0329] The particles were dispersed in the test culture medium (freshwater) and compared with a dispersion in ultrapure water. Dispersion stability was studied immediately after dispersion (Oh) and at the maximum incubation time during the ZET assay (24h). The SS@P25 NPs were dispersed at a concentration of 100 mg / L using a 37 Hz ultrasonic bath at 100% power for 30 minutes. The P25 NPs were dispersed using a 1:1 ratio of humic acid as a stabilizer and 30 minutes of sonication of the probe at 50% amplitude with pulses of 30 s on and 10 s off. The test concentrations of NPs were first dispersed and the pH was verified at the nominal concentrations lower and higher to ensure a tolerable environment for normal embryonic development of zebrafish (doi: 10.1002 / aja.1002030302). NP concentrations were preheated to 26 ± 1°C each day before testing, and the plates were exposed to the same photoperiod as the breeding tank.An experiment was recognized as "valid" at an embryonic lethality threshold set at 10% compared to the experimental control. At 8, 32, 56, and 80 hpf hours post-fertilization) diverse events of embryonic development corresponding to their age have been studied. The embryos were observed and photographed, if necessary, using a Nikon Eclipse Ts2 inverted microscope coupled with a Nikon DS-Fi3 camera. Survival was checked at all hpf. Tests on fish embryonic stages were conducted at the International Iberian Nanotechnology Laboratory (INL) (Braga, Portugal), as an adaptation of the FET test (OECD Guideline 236), as defined by the Council Directive 86 / 609 / EEC on the protection of animals used for experimental purposes, which sets the regulatory exposure limit at the free-living stage (i.e., at the end of embryogenesis). Since the last endpoint tested was prior to this stage of development, the provisions of the directive do not apply and therefore no ethical approval was required.

[0330] 100 mg / L was considered the maximum (nominal) test concentration of NPs, based on OECD Guideline 203, which sets the limit for testing chemicals at this range.

[0331] SS@P25 did not exert acute toxicity on zebrafish embryos (no lethal or sub-lethal effect at the endpoints measured in the test). Some sub-lethal parameters were recorded only up to a maximum concentration of 10 mg / L, because at the highest concentration (100 mg / L) the embryos were completely covered by a layer of NPs that prevented observation of the embryos under the microscope.

[0332] Even unmodified TiO2P25 shows no acute toxicity to zebrafish embryos. Therefore, these data lead to the conclusion that functionalization does not increase toxicity. In light of the results of the eco-toxicological test, no potential acute toxic effect in freshwater was observed for SS-modified titanium dioxide in aquatic organisms.

[0333] In vitro evaluation: Cell viability assay on BEAS-2B and RAW264.7 and HSF (Human Skin Fibroblast cells) cell lines

[0334] Cell viability was measured using the Cell Counting Kit-8 (WST-8) and Alamar Blue assays on human bronchial epithelial cells (BEAS-2B), on the mouse alveolar macrophage cell line (RAW264.7) from a tumor induced by the Abelson murine leukemia virus, and on human skin fibroblasts (HSF). In the WST-8 assay, the amount of the formed formazan dye is directly related to the metabolic activity of the cells. Similar to the WST-8 assay, Alamar Blue (resazurin assay) evaluates cell proliferation by measuring dehydrogenase activity within cells. When detected, resazurin, a low-fluorescence blue dye, is reduced to resorufin, a highly fluorescent red dye fluorescent dye. The tests were performed as described in the manufacturer's instructions.

[0335] Prior to cell viability studies, NP dispersion studies were conducted in various aqueous media, including ultra-pure water (UW), phosphate-buffered saline (PBS), and Dulbecco's Modified Eagle Medium (DMEM), using an ultrasonic bath at increasing time intervals in order to find conditions of good dispersion without damaging the structure of the NPs. Consequently, DMEM was found to be the best choice for use with cells. The NPs were dispersed in DMEM at a higher concentration and diluted to lower concentrations for in vitro analysis. The cells were seeded in transparent 96-well plates and exposed to NPs at increasing concentrations (1-10-100-200 μg / ml) at different times (6-24 hours). The cells were then washed twice with IX PBS and incubated in fresh medium with 10% WST-8 reagent for 3 hours. Subsequently, 80 pL aliquots of the color-developed medium were transferred to new 96-well plates to eliminate interference from NPs and NPs absorbed by the cells (80 pL instead of 100 pL was transferred to avoid the formation of bubbles that interfere with absorbance measurement). The absorbance of each well containing the WST-8 reagent was measured at 450 nm (690 nm was used as the reference wavelength and subtracted) in a microplate reader (iMark™, Bio-Rad, UK). The results are reported as relative WST-8 activity, where 1.0 corresponds to the absorbance measured in the control cultures.

[0336] As indicated, preliminary evaluation of the in vitro cytotoxicity of P25, SS, and P25@SS was conducted using BEAS-2B, RAW264.7, and HSF cell lines. Cells were exposed to concentrations ranging from 1 to 200 pg / mL for 24 hours.

[0337] The results of cell proliferation on human bronchial epithelial cells BEAS-2B of the powders are shown in Figure 7. The starting materials (P25 and SS) were used as controls for comparison. TiO2-NP does not cause any cytotoxicity in lung epithelial cells at concentrations below 10 pg / ml, even at longer exposure times. However, at higher concentrations, a toxic effect is observed at 6 hours, which disappears after 24 hours. Interestingly, however, there is a significant reduction in cytotoxicity observed in cultures exposed to P25@SS in the same critical concentration / exposure conditionsfor uncontaminated P25. A slight increase in BEAS-2B cell viability can be observed after 6 hours of treatment with P25 @SS particles at 1 and 10 pg / ml. As for SS, it has been found to exert dose-dependent cytotoxicity on BEAS-2B cells. For 6 hours of treatment, 1 pg / ml and 10 pg / ml of native SS does not show cytotoxicity, but concentrations of 100-200 μg / ml cause a decrease in cell viability, with behavior similar to the positive control. A similar situation is observed after 24 hours for higher concentrations, but surprisingly, concentrations below 1.0 pg / ml actually lead to an increase in cell viability, as typically observed in nanomaterial viability tests.

[0338] However, although the toxicity values at 100 and 200 μg / ml represent significant experimental evidence, these are very different circumstances from the concentrations actually used. In fact, in the functionalization product where the weight ratio of SS is reduced (approximately 10%), the same trend is not observed.

[0339] In addition, Figure 8 shows the cytotoxicity data for the powders determined by the WST-8 assay on the RAW264.7 mouse alveolar macrophage cell line. The RAW264.7 macrophage cell has long been used as an in vitro model to study the response of inflammatory molecules to various synthetic stimuli. Macrophages are the first cells of the immune response and represent the innate immune system; their response is essential for the survival of cells, tissues, organs, and systems. Therefore, from the perspective of the possible risk of NP inhalation, the effects of dust on the viability of immunomodulators were evaluated based on dose and exposure time.

[0340] As shown in Figure 8, the cell viability of RAW264.7 decreases sharply at P25@SS concentrations of 100 and 200 μg / ml after both 6 and 24 hours, compared to the control. At lower concentrations, cytotoxicity was less pronounced but remained higher than that of pure or untreated TiO2. However, this evidence can be corroborated by observing the cytotoxicity data on macrophage cells exposed to SS. In fact, with reference to SS treatment alone, the cell viability of inflammatory cells recorded the lowest value with increasing concentration starting at 100 pg / ml. However, once again, and similar to what was observed in BEAS 2B, SS at lower concentrations (1 and 10 pg / ml) has the best beneficial impact on cell viability.

[0341] Finally, examining the data collected from treatment with pure P25 or as-is, no difference was observed in the concentration-dependent effects on cell viability n RAW264.7 cellsat both exposure time points, with the exception of a favorable effect at 200 μg / ml after 24 hours.

[0342] The cytotoxicity results of human skin fibroblast cells treated with NP at different concentrations provide important insights into concentration-dependent effects. As seen in Figure 9, at the lowest concentration (1 μg / ml), all treatments - P25, SS, and P25@SS - maintained or slightly increased cell viability compared to the negative control (NC), indicating the absence of cytotoxic effects at this concentration. When the concentration was increased to 10 μg / ml and 100 μg / ml, cell viability remained consistently above 100%, particularly in the P25 and P25@SS treatments, demonstrating a dose-independent response in this range. This suggests that these substances, even at higher concentrations, are well tolerated by fibroblasts, without causing a significant reduction in viability. At the highest concentration (200 μg / ml), there was no marked decrease in cell viability, indicating that both P25 and SS, individually or in combination, exhibit strong biocompatibility even at high doses. Interestingly, the P25@SS compound did not introduced any additional cytotoxic effects compared to P25 or SS alone, suggesting that the combined formulation is as safe as the individual components. These results highlight that the concentration range tested (1-200 μg / ml) has no negative impact on fibroblast cell viability, making these substances very suitable for use in topical applications such as sunscreens, where high concentrations of active ingredients are often required for effective UV protection. The concentration-independent viability in all treatments further underscores their potential for safe, long-term use in skin care formulations.

[0343] In summary, the side effects of TiO₂ NPs in terms of photoreactivity have been studied both at the acellular level (Blue Acid 9 assay) and intracellularly on ex vivo human skin. The Blue Acid 9 photodegradation model under visible light irradiation shows the highest photocatalytic efficiency (70.84%) for P25 with anatase crystal structures, compared to P25@SS (0.70%). The ability of SS to act as a detoxifying agent was evident, assuming a dual mechanism of electron scavenging phenomena electrons and reducing the formation of ROS. The results demonstrate that the prepared P25@SS not only significantly attenuates the negative reactivity of P25, reducing its toxicity, but also confirms the effectiveness of the coating process compared to a simple physical mixture (characterized by a photocatalytic efficiency of approximately 17.35% after UVirradiation). Subsequent in vitro and ex vivo toxicological studies have further investigated the toxicity of nanometric P25 in the absence of UV radiation and its potential harmful effects when exposed to UV rays. With regard to biological interactions, the actual biocompatibility of the nanocomposite was discussed on the mouse alveolar macrophage cell line (RAW264.7) and on human bronchial epithelial cells (BEAS 2B) were discussed. Overall, preliminary data on cell viability (WST-8 assay) of BEAS 2B and RAW264.3 cells treated with NPs for 24 hours revealed a similar trend. P25 and SS themselves showed dose-dependent cytotoxicity after 6 hours of treatment at higher concentrations (100-200 μg / mL), with a decrease after 24 hours, while concentrations below 10 μg / mL were not cytotoxic. Particularly, it is interesting to note the significant reduction in cytotoxicity observed in cultures exposed to P25@SS under the same critical concentration / exposure conditions as for pure or untreated P25.

[0344] Photoprotective efficacy

[0345] Using spectrophotometric measurements, the amount of UV radiation (calculated from transmittance) passing through the product film was evaluated on the set of oil-in-water (O / W) formulations prepared as indicated above. Photo stability studies were conducted using a solar simulation device and in vitro SPF analysis was performed according to ISO 24443:2012.

[0346] It was observed that all the formulations tested were photostable and the UV filtration parameters are shown in Table 7.

[0347] Table 7: UV filtration parameters obtained from the in vitro SPF analysis of each formulation

[0348] Formulation Description SPF UVA-PF SPF SPF Critical Label Label / UVA PF λ (nm) Formula A Emulsion base 1.05 nd 6 nd 291 Formula B Emulsion with 6.45 2.79 6 2.15 375

[0349] P25

[0350]

[0351] Formula C Emulsion with SS 1.01 nd 6 nd 290 Formula D Emulsion with 8.52 2.75 6 2.19 374

[0352] P25@SS

[0353]

[0354] As regards the P25@SS formula, there is an increase in terms of SPF (8.52) compared to the P25-based formula (SPF of 6.45). This totally unexpected result suggests that functionalization with the surfactant and / or lipopeptide does not negatively affect negatively on UV filtration parameters and, on the contrary, allows for an increase of 23%. It can therefore be deduced that the coating of TiO₂ particles introduces a variable in the optimal dispersion of the UV filter, increasing its effectiveness.

[0355] The compound according to the present invention, therefore, in its complexed form with the surfactant and / or lipopeptide, has an increased UV filtering capacity of t least 20% compared to titanium dioxide as is.

[0356] Furthermore, the compound according to the present invention has the additional advantages described herein.

[0357] Therefore, it can be seen that TiO₂ nanoparticles stabilized and functionalized with surfactants and / or lipopeptides have fulfilled the above-mentioned purposes, resulting in the creation of effective biocompatible inorganic UV filters.

[0358] Furthermore, this compound is particularly effective in mitigating the side effects of inorganic filters, such as photoreactivity. It is known that the behavior of single and aggregated TiO₂ nanoparticles in the presence of environmental compounds is mainly regulated by the complex interaction between attractive and repulsive electrostatic interactions, steric interactions, and van der Waals interactions. Consequently, with the present invention, TiO₂ NPs have been stabilized with respect to aggregation, sedimentation, and other destabilizing phenomena by using the indicated surfactant, for example, surfactin, preferably Sodium Surfactin, SS, which acts as a stabilizing agent. Furthermore, this molecule has a low environmental impact, as it is obtained from waste or by-products of the agri-food industry, for for example, by fermentation.

[0359] Furthermore, the most effective method for obtaining this compound has proven to be the one described above, which exploits the colloidal approach in the two variants illustrated. The examples above concern the functionalization with SS of two different types ofnanometric titanium dioxide particles (P25 and EU), but similar results can be obtained with nanometric titanium dioxide particles (P25 and EU) with SS, but similar results can be obtained with surfactin as is or with the other indicated surfactants or lipopeptides. The present invention, which combines the coating of surfactant and / or lipopeptide on TiO₂ (nano)particles (or vice versa) using the method indicated, has proven to be effective to modulate the photoreactivity of titanium dioxide without altering its UV filter properties.

[0360] The strategy of trapping TiO₂ NPs by coupling them with surfactant or preferably with lipopeptide (SS) neutralized photocatalytic (phototoxic) activity while improving photoprotection and photo stability. P25@SS has been shown to be safe, determining its applicability as surface-modified nanometric TiO₂ for preventing oxidative stress and inflammation caused by UV radiation. These significant advantages of the compound according to the present invention, such as P25@SS, some results of which have been reported in the previous section, compared to commonly available inorganic TiO₂-based filters, highlight its potential as a safe and sustainable solution in various applications. In conclusion, the modification of TiO₂ nanoparticles or its derivative or its precursor with a surfactant, for example a lipopeptide, such as surfactin, particularly in the form TiO₂@SS or P25@SS > 10% (e.g., 13.7%), effectively increases colloidal stability and reduces potential toxicity without compromising functional properties. The success of this approach hinges on the greater safety of the indicated nanoparticles, using a compound and a process that address environmental and human health issues.

[0361] It has thus been seen that the present invention relates to a compound based on titanium dioxide or a derivative or precursor thereof functionalized and / or complexed with a surfactant and / or a lipopeptide, as well as a method for obtaining it, suitable for use as a UV radiation protection agent and as a sunscreen in creams or cosmetic formulations for topical use, as well as in medical devices.

Claims

CLAIMS1. Compound having UV radiation protection properties for the human body, for example capable of providing UV radiation protection for at least one composition containing it, wherein said compound comprises particles of a physical filter, comprising titanium dioxide or a derivative of titanium dioxide or generated from a precursor of titanium dioxide, and a surfactant, wherein said particles of titanium dioxide or its derivative or precursor comprise or consist of a powder of titanium dioxide, or are in the form of silica-coated TiO₂ particles, or are obtained from a derived or precursor of titanium dioxide, such as isopropoxide of titanium (Ti[OCH(CH₃)₂]₄), characterized in that said particles of titanium dioxide or titanium dioxide derivative or generated from the titanium dioxide precursor are functionalized and / or complexed with said surfactant.

2. Compound according to claim 1, wherein said compound is a sunscreen and / or said particles of titanium dioxide or titanium dioxide derivative or generated by titanium dioxide precursor have a surface coating comprising said surfactant or form a surface coating of said surfactant.

3. Compound according to claim 1 or 2, wherein said surfactant is a lipopeptide comprising or consisting of surfactin or a sodium salt of surfactin (Sodium Surfactin or SS) or fengicin or plipastatin or iturin, viscosin, lichenisin, gramicidin, polymyxin, megovalcin.

4. A compound according to any of the preceding claims, wherein said surfactant is a lipopeptide derived from sustainable sources, such as food waste or refuse, agricultural or agri-food industry by-products, and / or fermentation.

5. Compound according to claim 1 or 2, wherein said surfactant comprises or consists of glycolipids, such as ramnolipids, trehalose lipids, sophorolipids, mannosyl erythritol lipids, cellobiolipids, or fatty acids or phospholipids or neutral lipids, such as corinomycolic acid, spiculisporic acid, phosphatidylethanolamines, or a particulate biosurfactant such as vesicles, whole microbial cells, a polymeric bio surfactant, a protein complex, mannoproteins.

6. Composition according to any of the preceding claims, wherein said particles of said physical filter consist of titanium dioxide nanoparticles functionalized and / or complexed with said surfactant and / or with said lipopeptide.Composition according to claim 3, wherein said surfactin has a cyclic structure according to the following Formula I:or wherein said fengicin has a cyclic structure according to the following Formula II:

8. Compound according to any of the preceding claims, wherein said physical filter particles and said surfactant are in a ratio of 1: 1 or in a ratio of 7,6:1 or in a ratio of 7:1 or in a ratio between 1: 1 and 10:1 or in a ratio between 1: 1 and 1: 10 or in which said surfactant is present in a percentage by weight of 13.2 wt% or 13% or in a percentage of at least 10% or at least 5% or at least 1% or between 10% and 15% orbetween 5% and 30% or between 1% and 90% or between 5% and 80% or between 5% and 50% with respect to the weight of said particles or vice versa, wherein said surfactant and said particles of a physical filter are in a ratio 1: 1 or in a ratio of 7,6:1 or in a ratio of 7:1 or in a ratio between 1: 1 and 10: 1 or in a ratio between 1: 1 and 1: 10 or in which said particles are present in a percentage by weight equal to 13.2wt% or 13% or in a percentage of at least 10% or at least 5% or at least 1% or between 10% and 15% or between 5% and 30% or between 1% and 90% or between 5% and 80% or between 5% and 50% with respect to weight of said surfactant.

9. Method for obtaining a compound with UV radiation protection properties for the human body according to one or more of the preceding claims, wherein said method comprises the following steps:providing particles of titanium dioxide or a derivative of titanium dioxide or generated from a precursor of titanium dioxide (step A or step A'), wherein said particles of titanium dioxide or its derivative or precursor comprise or consist of a titanium dioxide powder, or are in the form of TiO₂ particles coated with silica, or are obtained from a derivative or precursor of titanium dioxide, such as titanium isopropoxide (Ti[OCH(CH₃)₂]₄), providing an aqueous solution of surfactant (phase B or phase B'), wherein said surfactant is a lipopeptide, for example derived from sustainable sources, such as food waste or refuse, agricultural or agri-food industry by-products, and / or from fermentation, adding said particles of titanium dioxide or its derivative or precursor to said aqueous surfactant solution, obtaining functionalization and / or complexation of said particles by said surfactant,wherein said functionalizing and / or complexing step comprises mixing while heating at a temperature of 40°C (phase C) or 60°C (phase C) or above 40°C, for example for 24 hours, obtaining a TiO₂-surfactant mixture,centrifuging said TiO₂-surfactant mixture and washing the resulting pellet in water (phase D),collecting the pellet resulting after having centrifuged the solution obtained from said washing, obtaining a powder of said compound in which said particles of TiO₂ or its precursor or its derivative form a complex with said surfactant and / or are functionalized with said surfactant, as the latter functionalizes the outer surface of each titanium dioxideparticle.

10. Method according to the previous claim, wherein said step of providing particles of titanate is carried out by sol-gel synthesis of said titanium dioxide particles from said titanium dioxide or said derivative or said precursor of titanium dioxide, obtaining a gel (phase A).

11. Method according to the preceding claim, wherein said step A comprises the following steps:preparing a solution of water and hydrochloric acid, e.g., fuming, optionally with a surfactant,gently stirring said solution to avoid the formation of bubbles and heating to 50°C or at least 40°C,when said temperature is reached, adding to the heated solution a powder of titanium dioxide or its precursor or derivative to the heated solution and mix vigorously, for example.

12. Method according to the preceding claim, wherein said step comprises adding water, for example ultrapure water, for example 46.4 g of ultrapure water, and HC1, e.g., 0.250 g of fuming HC1, in the presence or absence of said surfactant, e.g. 0.02 g of TritonX-100 at 2% v / v, and subsequently adding TiO₂ powder or the precursor titanium isopropoxide, e.g. 3.2 g of TiO₂ powder or 3.3 g of titanium isopropoxide (precursor), under vigorous stirring and maintaining the mixture at reflux for 24 h.

13. Method according to claim 9, wherein said step of providing a surfactant solution comprises a step of solubilizing said surfactant (step B), for example wherein said step B comprises solubilizing said surfactant or lipopeptide in water, for example 2 g of said surfactant or lipopeptide, for example in the form of sodium salt, in ultrapure water, for example 10 ml of ultrapure water.

14. Method according to any of claims 9 to 13, wherein said step C comprises an aggregation step comprising, for example, the following steps: adding said step A, for example 40 ml of said step A, to said step B, for example 8 ml of said step B, maintaining under agitation, for example at 500 rpm at 40°C for 24 h under reflux.

15. Method according to any of claims 9 to 14, wherein said method is a heterocoagulated colloidal method.

16. Method according to claim 9, wherein said step of providing particles of titanium dioxide or its derivative or generated from its precursor is carried out by dispersing said particles in water, for example ultrapure water, or ethanol (EtOH), for example at a concentration of 10 to 30 mM, by sonication to separate the loosely agglomerated particles and obtain a suspension of said particles (phase A').

17. Method according to claim 9, wherein before said phase C there is a step of mixing said surfactant or lipopeptide solution with the suspension of said particles to obtain a reaction mixture which is sonicated, for example for 30 minutes.

18. Method according to claim 9 and any of claims 16 and 17, wherein said method is a colloidal self-assembly method.19 Method according to claim 9, wherein said step of collecting the pellet includes collecting said pellet, optionally freezing it in liquid nitrogen, and freeze-drying said pellet.

20. Topical use of the compound according to any of claims 1 to 8 as a sunscreen and UV radiation protective component in creams or cosmetic formulations.

21. Use of the compound according to the preceding claim, wherein said compound is present in an amount of between 1 and 200 μg / ml.

22. Cosmetic cream or formulation or medical device for topical use comprising said compound according to one or more of claims 1 to 8, wherein said compound acts as a filter against UV radiation.

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