Formulations comprising phospholipid nanomicelles loaded with resveratrol and / or rutin

Abstract

The present application discloses formulations comprising polyphenol- loaded phospholipid nanomicelles. The described phospholipid nanomicelles are loaded with resveratrol and / or rutin, which are particularly suitable for the delivery of these polyphenols. The formulations herein described is also suitable for skin diseases or conditions which can be prevented or treated with the administration of resveratrol and / or rutin.

Description

[0001] FORMULATIONS COMPRISING PHOSPHOLIPID NANOMICELLES LOADED WITH RESVERATROL AND / OR RUTIN

[0002] Technical field

[0003] This application relates to formulations comprising phospholipid nanomicelles loaded with resveratrol and / or rutin.

[0004] Background art

[0005] Natural-derived molecules are receiving significant attention by pharmaceutical industry, and have been explored for a broad spectrum of applications, because of their adaptability, safety and affordability (Chaachouay and Zidane, 2024) . Although several conventional synthetic drugs are effective for the treatment of skin conditions, they often have side effects, making phytoconstituents excellent alternatives (Gorain et al. , 2022) . To address the current problems of conventional skin dosage forms, new drug formulations based on natural compounds, e.g. , plant- or fruit-derived chemicals, with antioxidant, antimicrobial and anti-inflammatory activities are being considered (Shah and Arnini- Nik, 2017) .

[0006] The advances in nanotechnology field have led to the design of different nanodelivery systems (or nanocarriers) for incorporating / encapsulating unstable, labile or / and poor water-soluble compounds, paving the way for new possibilities and opportunities in pharmaceutical and cosmetic sectors (Joye et al. , 2014) . Nanodelivery systems improve the bioavailability of compounds by increasing their absorption and penetration through biological barriers (Kaur and Kesharwani, 2021) . The effective delivery to specific sites, controlled release, modification of organoleptic properties, and prevention of interactions with other antagonistic components are additional advantages of nanodelivery systems (Pool et al. , 2013; Rein et al. , 2013) . Poorly water-soluble drugs can be effectively delivered to several regions of the body using nanocarriers composed of lipid raw materials. There are several lipid-based nanocarriers currently being considered for pharmaceutics and cosmetics, such as solid lipid nanoparticles and nanostructured lipid carriers (Fathi et al., 2024a, b) , self- (micro) emulsifying drug delivery systems (Prajapat et al. , 2017) , micro and nanoemulsions (Parikh and Patel, 2016) , and hybrids nanocarriers that combine lipids and polymers, such as polymeric-lipid nanocapsules (Jakubiak et al. , 2017) and polymeric-lipid micelles (Kumari et al. , 2017) . These nanocarriers have several benefits, including biocompatibility and high loading capacity due to the strong affinity for poorly water-soluble drugs, which ultimately contribute to increase membrane permeation to loaded drugs (Macedo et al. , 2014) .

[0007] Micelles are supramolecular aggregates of surfactant molecules dispersed in a liquid, forming nanocolloids with diameter between 5 and 100 nm (Bose et al. , 2021) . Phospholipid micelles, or normal-phase micelles, are generated when amphiphilic molecules — which contain two regions of varying affinity to water — self-assemble when in contact with water. In these amphiphilic molecules, the region facing the external surface of the micelle is called the hydrophilic region and the hydrophobic region of the molecules forms the core (Schroeder et al., 2012) . Because of this property, the micellar surface of phospholipid micelles attracts polar molecules, whereas the inner phase of the micelle can carry lipophilic compounds. Thus, they are often suggested as delivery systems for poorly water-soluble or water-insoluble molecules (Bachu et al. , 2018; Bose et al. , 2021) .

[0008] Besides increasing the drugs' solubility and bioavailability, other concerns posed by the manufacturing of such nanocarriers include the need to respond to the growing demand and technological advancements as nanotechnology develops and attracts increasing attention in various fields. As a result, issues related to scaling up and mass production begin to emerge, raising concerns about the potential effects on the planet's health, besides person's physical, mental, and social wellbeing (Bartolozzi et al. , 2020; Halada and Orlov, 2018; Jensen and Lewinski, 2018) .

[0009] The treatment of skin disorders includes a variety of drugs and products, such as UV protectors, antioxidants, anti-inflammatories, antibiotics, moisturizing agents, healing agents, and analgesics, that can be used either externally or internally (Soleymani et al. , 2019) . However, these conventional therapies are often insufficient to treat more difficult conditions, e.g. , inflammation and proliferation of skin wounds (Wang et al. , 2019) . As an alternative and / or complementary approach, plants and fruits are two of the natural sources of pharmacologically active ingredients, such as flavonoids and polyphenolic compounds, with several beneficial properties for enhancing skin integrity and health ( Bahramsoltani et al. , 2019) .

[0010] Given their beneficial effects in a range of biological systems, polyphenolic compounds are becoming increasingly popular in pharmaceutical, cosmeceutical and nutraceutical products. They are a category of plant-based functional components characterized by diverse chemical structures and a range of biological actions, with antioxidant, anti-inflammatory, and anticancer effects. Nevertheless, numerous polyphenolic compounds have limited use in pharmaceutical products because of their poorly water-soluble character.

[0011] Rutin, also known as Vitamin P, is a polyphenolic flavonoid found in a variety of plants, including citrus fruits, buckwheat, betula leaves, passionflower, green tea, and apples ( Ganeshpurkar and Saluja, 2017; Hosseinzadeh and Nassiri-Asl, 2014; Sikder et al. , 2014; Yadav et al. , 2016) . It is known to be a harmless bioactive ingredient with potential uses for biological and pharmacological applications (Shahi et al. , 2019) . Numerous studies have demonstrated that this flavonoid has antiinflammatory, anti-allergic, antibacterial, anti-cancer, anti-viral, anti-wrinkle, wound-healing, and neuroprotective properties (Ahmed et al. , 2022; Elsawy et al. , 2019; Ganeshpurkar and Saluja, 2017; Memar et al. , 2022; Seo et al., 2016) . Among the different pharmacological effects (Macedo et al. , 2014; Neto et al. , 2021) , rutin is particularly noted due to its preventive effect against several oxidative stress- related disorders (Ganeshpurkar and Saluja, 2017) , because it decreases the level of reactive oxygen species (ROS) produced in macrophages and in other skin cells (Gao et al. , 2013; Gggotek et al. , 2019; Javed et al. , 2012) . Owing to its ROS scavenging capacity, rutin has been proposed as a smart ingredient in functional foods (Wahed et al., 2023) . Rutin is also used in dermocosmetic products, e.g. for anti-aging, allergic and atopic skin, mainly because of its antioxidative properties (Choi and Kim, 2013; Choi et al. , 2016) . However, although rutin exhibits remarkable pharmacological properties, the efficacy of this flavonoid is limited due to its poor water solubility and limited stability, which consequently affect its bioavailability (Gullon et al. , 2017; Xiao et al. , 2016) . For the same reasons, rutin shows poor skin penetration rate and, thus, cannot be efficiently incorporated in dermal products to treat or control skin disorders (Sionkowska et al. , 2023) . To overcome these problems, new strategies to enhance the beneficial effect of rutin on the skin, such as drug delivery systems, have been extensively explored .

[0012] Resveratrol is a polyphenolic non-flavonoid bioactive and also a natural phytoalexin that is produced by plants during pathogen attack (Jeandet et al. , 2002) , and can be found in grapes (skin and seeds) , nuts, berries and red wine (Lucarini et al. , 2018) .

[0013] Resveratrol was found to activate phase II drug-metabolizing enzymes, to inhibit cyclooxygenase and hydroperoxidase enzyme activity, exhibiting anti-inflammatory properties, and showing antiprogression effects via promoting cell differentiation in promyelocytic leukemia cells (Wang et al. , 2012) . Resveratrol may also mitigate the multidrug resistance of cancer cells and, when used with therapeutically approved chemotherapeutics, resveratrol can be effective as anti-cancer drug, improving the pharmacokinetic profile of classical anti-neoplastic drugs (Arabzadeh et al. , 2021) .

[0014] Resveratrol has numerous beneficial properties, including antiinflammatory and immunomodulatory activity, promoting wound healing (Cassano et al. , 2020; Huang et al. , 2019; Svajger and Jeras, 2012; Zhao et al. , 2020) . It is also used in cosmetics as a photoprotector and anti-aging, due to its collagen-stimulating and antioxidant properties (Patricia Farris et al. , 2013; Ratz-Lyko and Arct, 2019) . Resveratrol has been widely studied in recent years for its antimicrobial activity against a wide range of microorganisms and successful treatments of various skin diseases, such as wounds (Berce et al., 2018) , acne (Fabbrocini et al. , 2011) and melanoma (Gong and Xia, 2020) .

[0015] Although this polyphenolic compound is relatively well absorbed orally, it undergoes a first-pass hepatic metabolism, which results in low oral bioavailability and, in addition, it has a very short half-life, around 8 to 14 minutes (Francioso et al. , 2014; Walle et al. , 2004) . Therefore, the topical administration of resveratrol is considered a suitable alternative over the oral route for the treatment of skin injuries, since its metabolism in the skin is significantly lower than in the liver and, consequently, a higher amount of bioactive may be delivered to the site of interest (Murakami et al. , 2014) . Furthermore, compared to its oral administration, topical treatments with resveratrol are associated with a lower risk of side effects, and potential skin irritations are reported to be low (Moyano-Mendez et al., 2014; Tsai et al. , 2016) . However, due to its hydrophobicity and the difficulties in controlling fate in the skin, topical administration of resveratrol is still a challenge (Shrotriya et al. , 2017) .

[0016] In order to widen the use of resveratrol and rutin in pharmaceutical and cosmetic formulations, nanotechnology is an approach to be considered, aiming at increasing both physicochemical stability and solubility of bioactives ( Szulc-Musiol and Sarecka-Hu j ar , 2021) . The low solubility and low chemical stability of resveratrol and rutin are limitations that can be overcome when loaded into lipid nanocarriers, as these latter provide higher chemical stability, controlled release, and may act as skin permeation enhancers (Summerlin et al. , 2015) .

[0017] Hydrogels are semi-solid dosage forms, composed of a hydrophilic polymeric three-dimensional network, with the ability to retain a high amount of water. Hydrogels can be used as vehicles for drugs alone, but also for drugs-loaded into nanocarriers to improve the consistency of the formulation aimed for skin application. Hydrogels can be used in wound healing because they simultaneously absorb the exudates and keep the wound moisturised. Since they are non-irritating, they do not react with biological tissue and are permeable to metabolites, hydrogels are appropriate for all stages of the skin healing process, namely, haemostasis, inflammation, cell migration / proliferation and maturation. Thus, hydrogel dressings are being widely studied for accelerated wound healing. To expedite wound treatment, a large variety of hydrogel dressings can be applied to several types of skin wounds, depending on the need. This resulted in the rapid introduction of a range of dressing materials into the pharmaceutical market, such as sheets, saturated gauze, and gels. With the development of nanotechnology, nanoparticles loaded with bioactives of interest are being combined with other biomaterials to create innovative hybrid systems for new applications. Following this evolution, the addition of nanocarriers to hydrogels is gaining even more interest.

[0018] Before a bioactive ingredient is able to show its health-beneficial effects, it undergoes a variety of processes until it reaches its target. It needs to become bioavailable and bioacces sible , attributes that are mostly af fected by the biofunctional properties of the bioactive ingredients . Regarding s kin delivery of antioxidants , such as resveratrol and rutin, it is expected that they go deeper into the s kin layers yet without reaching the systemic circulation . The degree of permeation of the s kin and controlled penetration of bioactive ingredients for dermopharmaceutical use can be governed by the use of speci fic delivery systems . Among these , lipid delivery sys tems play a relevant role because they act as permeation enhancers . Phospholipid micelles are a typical example of success ful delivery systems selected to improve the solubility of poorly water-soluble drugs / bioactives for that specific purpose , besides allowing the modi fication of the release profile of the payloads and targeting them to speci fic locations and thus reducing the ris k of side ef fects from systemic distribution of drugs / bioactives .

[0019] Olive pomace is a semi-solid residue with a high amount of water in its constitution ( 60% ) and has low pH . Olive pomace also contains a high percentage of phenolic compounds and retains most of the phenolic content of the olive , whereas only 1-2% of the phenolic content is present in olive oil . However, as a by-product, olive pomace becomes phytotoxic and non-biodegradable , but it is of great interest to several industries due to its varied bioactivities and health-promoting properties , and new forms of reuse are being proposed . It has been demonstrated that olive pomace , as well as its bioactive phenolic compounds , show antiinflammatory, antioxidant and photoprotective activities . Furthermore , its application has also been reported in cosmetics and in dermatological products for the treatment of s kin diseases using di fferent delivery systems . Hydrogels based on olive pomace are one example of these delivery systems . Therefore , to value these olive by-products , their sustainable recovery has been highlighted in several research studies .

[0020] Summary

[0021] In one embodiment , the patent application relates to a formulation of phospholipid nanomicelles loaded with at least one polyphenol comprising :

[0022] L-a-Phosphatidyl choline in a concentration between 0 . 01 and 10 % w / w; at least one surfactant in a concentration between 0.01 and

[0023] 5 % w / w; water in a concentration between 80 and 99.97 % w / w; at least one polyphenol in a concentration between 0.01 and 5 % w / w, wherein the polyphenol is selected from resveratrol and / or rutin.

[0024] In one embodiment, the surfactant is selected from Polysorbate 80, Sorbitan monooleate, or mixtures thereof.

[0025] In one embodiment, L-a-Phosphatidyl choline is obtained from soy lecithin .

[0026] In one embodiment, the nanomicelles comprise a surface charge between ±20 and ±40 mV.

[0027] In one embodiment, the nanomicelles comprise an average particle size below 100 nm.

[0028] In one embodiment, the nanomicelles comprise an average particle size between 70 and 90 nm.

[0029] In one embodiment, the formulation comprises an encapsulation efficiency between 90 and 100%.

[0030] In one embodiment, the formulation comprises a loading capacity between 0.01 and 5% .

[0031] In one embodiment, the patent application relates to a hydrogel formulation with phospholipid nanomicelles loaded with at least one type of polyphenol, comprising:

[0032] L-a-Phosphatidyl choline micelles loaded with at least one polyphenol in a concentration between 0.005 and 5 % w / w; polyacrylic acid in a concentration between 0.1 and 5 % w / w; propylparaben in a concentration between 0.005 and 2.5 % w / w; olive pomace in a concentration between 0.05 and 5 % w / w; wherein the at least one polyphenol encapsulated in the micelles is selected from resveratrol and / or rutin. In one embodiment, the formulation or the hydrogel formulation are for use in the prevention or treatment of skin conditions or diseases.

[0033] In one embodiment, the skin conditions or diseases are selected from dehydration, aging, inflammation, rash, dermatitis, eczema, hives, urticaria, allergic reactions, psoriasis, wounds.

[0034] In one embodiment, the formulation or hydrogel formulation are for topical application.

[0035] In one embodiment, the patent application relates to a method to obtain the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol, comprising the steps of: heating a solution comprising: resveratrol and / or rutin in a concentration between 0.01 and 5 % w / w, and

[0036] L-a-Phosphatidyl choline in a concentration between 0.01 and 10 % w / w, at a temperature between 70 and 95°C under stirring between 500 and 5000 rpm, for a time between 1 and 5 minutes;

[0037] Adding to the previous solution an aqueous solution of at least one surfactant in a concentration between 0.01 and 5 % w / w;

[0038] Heating the mixture at a temperature between 75 and 95°C under stirring between 500 and 5000 rpm, for a time between 1 and 10 minutes;

[0039] Stirring the mixture between 5000 and 10000 rpm for a time between 3 and 10 min to form a pre-emulsion;

[0040] Sonicating the pre-emulsion with a probe for a time between 1 and 10 min and between 60 and 80% amplitude to obtain the formulation.

[0041] In one embodiment, the surfactant is selected from Polysorbate 80, Sorbitan monooleate, or mixtures thereof.

[0042] In one embodiment, the patent application relates to a method to obtain a hydrogel formulation with phospholipid nanomicelles loaded with at least one polyphenol, comprising the steps of:

[0043] Preparing a 1-10 w / w solution of polyacrylic acid by mixing polyacrylic acid in a concentration between 1 and 10 % w / w, water and propylparaben in a concentration between 0.01 and 5 % w / w; Adjusting the pH of the solution between 6.5 and 7, under stirring between 500 and 5000 rpm, for a time between 1 and 10 minutes, to obtain a hydrogel;

[0044] Storing the resultant hydrogel at a temperature between 0 and 8°C, for a time between 5 and 15 h;

[0045] Adding phospholipid nanomicelles loaded with at least one polyphenol to the hydrogel in a ratio between 0.001:10 and 5:1 mass, wherein the polyphenol is selected from resveratrol and / or rutin;

[0046] Adding olive pomace in a concentration between 0.1 and 10% w / w to the hydrogel .

[0047] In one embodiment, the nanomicelles formulation or the hydrogel formulation are cosmetic formulations or dermopharmaceutical formulations .

[0048] General description

[0049] The present patent application relates to a formulation comprising phospholipid nanomicelles loaded with at least one type of polyphenol, more specifically polyphenols such as resveratrol and / or rutin.

[0050] In one aspect of the present application, it is described the preparation of a formulation comprising phospholipid nanomicelles loaded with rutin ( Example 1 ) .

[0051] In another aspect of the present application, it is described the preparation of a formulation comprising phospholipid nanomicelles loaded with resveratrol (Example 2) .

[0052] In another aspect of the present application, it is described the preparation of a formulation comprising phospholipid nanomicelles loaded with resveratrol and rutin - dual loading (Example 2) .

[0053] In yet another aspect of the present application, it is described the preparation of a hydrogel formulation comprising phospholipid nanomicelles loaded with both resveratrol and rutin (Example 3) .

[0054] The formulation herein described is particularly suitable for the treatment of skin diseases or conditions, which can be prevented or treated with resveratrol and / or rutin. Herein it is also des cribed a method to obtain the formulation comprising polyphenol-loaded phospholipid nanomicelles , that is environmentally safe by using generally recogni zed as safe ( GRAS ) materials and avoiding organic solvents . The method allows to obtain nanomicelles for the loading of the rutin and / or resveratrol , to improve their poorly-water soluble profiles and potentially enhance s kin permeation to polyphenols .

[0055] This patent application also dis close s the dual loading o f rutin and resveratrol and their improved the physicochemical stability, encapsulation parameters and rheological profiles .

[0056] Furthermore , this application also dis closes further developments that enable greater stability in the encapsulation of resveratrol and rutin, in combination with the reuse of the by-product olive pomace .

[0057] One of the aims of the present application is to dis close a hydrogel formulation that can be used as a cosmetic or dermopharmaceutical product capable of effectively delivering resveratrol and / or rutin with appealing properties to the consumer, such as smoothnes s and an eco- friendly character .

[0058] Olive pomace hydrogels comprising the nanomicelles formulation loaded with rutin and / or resveratrol are suitable to nourish and hydrate the s kin, offering healing, antioxidant , and antiaging properties due to the presence of rutin and / or resveratrol . Their composition incorporates bioactive ingredients from natural sources with beneficial skin effects , along with the by-product olive pomace .

[0059] The raw materials used in this formulation are all safe , green, easily found in nature , and respond to the environmental concerns .

[0060] Therefore , the formulation herein dis closed aims to achieve several ob j ectives :

[0061] - to load rutin and / or resveratrol into biocompatible nanomicelles ;

[0062] - to obtain an innovative s kin care product from natural source s and safe ingredients ;

[0063] - to develop a low-cost product with multiple s kin-beneficial properties ; to address economic, environmental and social sustainability challenges by the use of by-products as a new source for a hydrogel formulation with the ultimate aim to achieve zero waste;

[0064] - to develop an innovative green technology-based product prioritizing human health and beauty.

[0065] In summary, the loading of rutin and / or resveratrol into nanomicelles promotes their protection and facilitates their permeation into the skin when applied topically, enhancing their biological action. Additionally, the use of olive pomace in hydrogel formulations, which is rich in skin- related bioactive ingredients and vitamins, nourishes the skin and provides a green hydrogel-based vehicle.

[0066] Brief description of drawings

[0067] For easier understanding of this application, figures that represent the preferred forms of implementation, which nevertheless are not intended to limit the technique disclosed herein, are attached in the annex.

[0068] Figure 1 shows the mean hydrodynamic diameter (Z-Ave) (nm) , polydispersity index (PDI) , and zeta potential (ZP) (mV) of blank and rutin-loaded micelles stored at 25 °C (A, C and E) and at 4 °C (B, D and F) , recorded one day after production (day 1) and after 7, 15, 30, 45 and 60 days. Results are expressed as mean ± SD (n = 3) . Significant differences between day 1 and the other times for the same micelles (e.g. , blank vs. blank; rutin-micelles vs. rutin-micelles) are represented by (*) . Significant differences between blank micelles and micelles loaded with rutin for the same time are represented by (**) , p < 0.05 (two-way ANOVA, followed by Tukey' s test) .

[0069] Figure 2 shows the Transmission electron microscopy (TEM) analysis of rutin-loaded micelles in lOOOOOx magnification. ( - , 100 nm) .

[0070] Figure 3 shows Differential scanning calorimetry (DSC) analysis of bulk rutin .

[0071] Figure 4 shows the DSC analysis of blank and rutin-loaded micelles, stored at 25°C and at 4°C, for 24h after production (left-hand side) and 60 days (right-hand side) .

[0072] Figure 5 shows the rheological analysis of blank and rutin-loaded micelles stored at 4°C and at 25°C for 24h after production.

[0073] Figure 6 shows the rheological analysis of blank and rutin-loaded micelles stored at 4°C and at 25°C for 60 days after production. Figure 7 shows the mean hydrodynamic diameter (Z-Ave) (nm) , polydispersity index (PDI) , and zeta potential (ZP) (mV) of blank, resveratrol-loaded micelles, and resveratrol-rutin-loaded micelles stored at 25 °C (A, C and E) and at 4 °C (B, D and F) , recorded one day (day 1) after production and after 7, 15, 30, 45 and 60 days. Results are expressed as mean ± SD (n = 3) . Significant differences between blank micelles and micelles loaded with resveratrol (RES) and with resveratrol and rutin (RES-RU) for the same time are represented by (*) ; Significant differences between RES-micelles and RES-RU micelles for the same period are represented by (#) , p < 0.05 (two-way ANOVA, followed by Tukey' s test) .

[0074] Figure 8 shows the TEM image of resveratrol-loaded micelles (left-hand side) and rutin-resveratrol-loaded micelles (right-hand side) .

[0075] Figure 9 shows the DSC analysis of resveratrol. Figure 10 shows the DSC analysis of resveratrol-loaded micelles, resveratrol-rutin-loaded micelles and blank micelles stored at 4°C and 25°C for 24 h after production (left-hand side) and 2 months (right-hand side) .

[0076] Figure 11 shows the rheological analysis of blank, resveratrol-loaded micelles and resveratrol-rutin-loaded micelles stored at 4°C and at 25°C for 24h after production.

[0077] Figure 12 shows the rheological analysis of blank, resveratrol-loaded micelles and resveratrol-rutin-loaded micelles stored at 4°C and at 25°C for 2 months after production.

[0078] Figure 13 shows the DSC analysis of the hydrogels A (with olive pomace) and hydrogels B (without olive pomace) and of fresh olive pomace.

[0079] Figure 14 shows the oscillation frequency sweep test of Hydrogels A (hydrogels containing olive pomace) , comparing the profiles between blank (without micelles) and with micelles (Mcl, Mc2, Mc3) .

[0080] Figure 15 shows the oscillation frequency sweep test of Hydrogels B (hydrogels without olive pomace) , comparing the profiles between blank (without micelles) and with micelles (Mcl, Mc2, Mc3) .

[0081] Detailed description of embodiments

[0082] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application. The present application relates to a formulation comprising phospholipid nanomicelles loaded with at least one type of polyphenol.

[0083] In one embodiment, the phospholipid nanomicelles are loaded with resveratrol and / or rutin.

[0084] In one embodiment, the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol comprises:

[0085] L-a-Phosphatidyl choline in a concentration between 0.01 and 10 % w / w; at least one surfactant in a concentration between 0.01 and 5 % w / w; water in a concentration between 80 and 99.97 % w / w; at least one polyphenol in a concentration between 0.01 and 5 % w / w, wherein the polyphenol is selected from resveratrol and / or rutin .

[0086] In one embodiment, the surfactant is selected from, but not limited to, Polysorbate 80 (Tween 80) , Sorbitan monooleate (Span 80) , or mixtures thereof .

[0087] In one embodiment, L-a-Phosphatidyl choline is obtained from soy lecithin .

[0088] In one embodiment, the L-a-Phosphatidyl choline nanomicelles comprise a surface charge between ±20 and ±40 mV.

[0089] In one embodiment, the L-a-Phosphatidyl choline nanomicelles comprise an average particle size below 100 nm.

[0090] In one embodiment, the L-a-Phosphatidyl choline nanomicelles comprise an average particle size between 70 and 90 nm.

[0091] In one embodiment, the formulation comprises an encapsulation efficiency between 90 and 100%.

[0092] In one embodiment, the formulation comprises a loading capacity between 0.01 and 5% . In one embodiment, the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol is used for the prevention or treatment of skin conditions or diseases.

[0093] In another embodiment, the conditions or diseases are selected from, but not limited to, dehydration, aging, inflammation, rash, dermatitis, eczema, hives, urticaria, allergic reactions, psoriasis, wounds.

[0094] In one embodiment, the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol is in the form of a serum, a lotion, a cream, a milk, an ointment, a paste, a foam, an emulsion, a hydrogel, a shower gel, a mask, a stick, a patch.

[0095] In one embodiment, the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol is suitable for topical use.

[0096] In one embodiment, a hydrogel formulation comprising phospholipid nanomicelles loaded with at least one type of polyphenol, comprises:

[0097] L-a-Phosphatidyl choline micelles loaded with at least one polyphenol in a concentration between 0.01 and 5 % w / w; polyacrylic acid in a concentration between 0.1 and 5 % w / w; propylparaben in a concentration between 0.005 and 5 % w / w; olive pomace in a concentration between 0.05 and 5 % w / w; wherein the at least one polyphenol encapsulated in the micelles is selected from resveratrol and / or rutin.

[0098] In one embodiment, the composition of the phospholipid nanomicelles loaded with at least one polyphenol is the same as described above in the embodiments for the formulation comprising the phospholipid nanomicelles loaded with at least one polyphenol.

[0099] In one embodiment, the polyacrylic acid is carbomer 940.

[0100] In one embodiment, the hydrogel formulation comprising phospholipid nanomicelles loaded with at least one type of polyphenol is a cosmetic hydrogel formulation. In one embodiment , the hydrogel formulation comprising phospholipid nanomicelles loaded with at least one type of polyphenol is a dermopharmaceutical hydrogel formulation .

[0101] In one embodiment , the hydrogel formulation is used for the prevention or treatment of s kin conditions or diseases .

[0102] In one embodiment , the hydrogel formulation is for topical use .

[0103] In one embodiment , the method to obtain the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol comprises the steps of : heating a solution comprising : resveratrol and / or rutin in a concentration between 0 . 01 and 5 % w / w, and L-a-Phosphatidyl choline in a concentration between 0 . 01 and 10 % w / w, at a temperature between 70 and 95 ° C under stirring between 500 and 5000 rpm, for a time between 1 and 5 minutes ;

[0104] Adding to the previous solution an aqueous solution of at least one surfactant in a concentration between 0 . 01 and 5 % w / w; Heating the mixture at a temperature between 75 and 95 ° C under stirring between 500 and 5000 rpm, for a time between 1 and 10 minutes ;

[0105] Stirring the mixture between 5000 and 10000 rpm for a time between 3 and 10 min to form a pre-emulsion ;

[0106] Sonicating the pre-emulsion with a probe for a time between 1 and 10 min and between 60 and 80% amplitude to obtain the formulation .

[0107] In one embodiment , the surfactant is selected from, but not limited to , Polysorbate 80 ( Tween 80 ) , Sorbitan monooleate ( Span 80 ) , or mixtures thereof .

[0108] In one embodiment , L-a-Phosphatidyl choline is obtained from soy lecithin .

[0109] In another embodiment , the method to obtain a hydrogel formulation comprising phospholipid nanomicelles loaded with at least one polyphenol comprises the steps of : Preparing a 1-10 w / w solution of polyacrylic acid by mixing polyacrylic acid in a concentration between 1 and 10 % w / w, water and propylparaben in a concentration between 0.01 and 5 % w / w; Adjusting the pH of the solution between 6.5 and 7, under stirring between 500 and 5000 rpm, for a time between 1 and 10 minutes, to obtain a hydrogel;

[0110] Storing the resultant hydrogel at a temperature between 0 and 8°C, for a time between 5 and 15 h;

[0111] Adding phospholipid nanomicelles loaded with at least one polyphenol to the hydrogel in a ratio between 0.001:10 and 5:1 mass, wherein the polyphenol is selected from resveratrol and / or rutin ;

[0112] Adding olive pomace in a concentration between 0.1 and 10% w / w to the hydrogel .

[0113] Example 1 - production and characterization of rutin-loaded nanomicelles for pharmaceutical applications 1. Materials and methods

[0114] 1.1 Materials

[0115] Rutin was purchased from Acros Organics (Geel, Belgium) . Polysorbate (Tween®80) and Sorbitan monooleate (Span®80) were obtained from Acofarma (Barcelona, Spain) . Soy Lecithin ( L-oc-Phosphatidyl choline ) was purchased from Sigma-Aldrich (St. Louis, MO, USA) . For all analyzes, Milli-Q water was used (water filtered through the Millipore system (Merk, Darmstadt, Germany) .

[0116] 1.2 Production of nanomicelles

[0117] Hot shear homogenisation and ultrasonication were applied in the production of the micelles to promote the encapsulation of lipophilic drugs. The lipid phase, composed of rutin (0.1% w / w) and soy lecithin (4.9 %w / w) , was heated on a heating plate (IKA C-MAG HS7, Staufen, Germany) , under constant mechanic stirring with a glass rod, until a homogeneous mixture was formed. The aqueous phase contained 94% (w / w) of ultrapurified water and 1% surfactant (Tween®80 and Span®80) . The composition of the prepared micelles is shown in Table 1. The aqueous phase was added to the lipid phase, resulting in a mixture that was heated on a heating plate under constant mechanic stirring until boiling. The resulting dispersion was subjected to high-speed stirring (7000 rpm) using an Ultra-Turrax (IKA T25, Staufen, Germany) for 5 min. Then, this pre-emulsion was subjected to a probe sonication (VCX 130, Sonics & Materials INC. , Newtown CT, USA) for 5 min and 70% amplitude. The obtained formulation was split into two batches and stored at different temperatures (25 °C and 4 °C) , both under dark for a period of 60 days, and physicochemically characterized. For comparison purposes, a blank formulation (without rutin) was also prepared following the same procedure without adding the drug to the inner lipid phase.

[0118] Table 1: Composition of the phospholipid micelles with and without rutin.

[0119] 1.3 Hydrodynamic average size, polydispersity index and zeta potential of micelles

[0120] A Zetasizer (Malvern Instruments, Malvern, UK) , dynamic light scattering (DLS) was used to record the micelles average size (Z-Ave) and their size distribution measured as polydispersity index (PDI) . Prior to the measurements, all samples were diluted (1:10) with ultrapurified water. All samples were analysed in triplicate (10 runs per measurement) . Data were analysed at a temperature of 25 °C and expressed as the arithmetic mean ± standard deviation (SD) . The zeta potential (ZP) , which is an indirect measure of the surface electrical charge of the micelles, was determined by measuring the electrophoretic mobility using the same Zetasizer. Samples were also diluted (1:10) with Milli-Q water previously adjusting its conductivity to 50 pS / cm, and subsequently analysed at 25 °C, and the average of six runs, each one with ten cycles, was obtained.

[0121] 1.4 Transmission electron microscopy analysis

[0122] Transmission electron microscopy (TEM) was used to observe the morphological characteristics of micelles. A volume of 20 mL of each sample was placed for two min on a carbon film-coated 300 mesh copper grid. Filter paper was used to absorb the leftovers. Each sample was negatively stained with 2% w / v uranyl acetate for a maximum of two min. The grid was then allowed to dry under air exposure. A Zeiss EM900 transmission electron microscope (Oberkochen, Baden-Wurttemberg, Germany) running at an 80 kV acceleration voltage was utilized for the analysis .

[0123] 1.5 Differential Scanning Calorimetry

[0124] The thermal properties of the prepared micelles were analyzed by differential scanning calorimetry (DSC) using a DSC 200 F3 Maia ® (NETZSCH, Selb, Germany) . This equipment includes a twin oven with two settings. One position is taken by the crucible holding the sample for analysis. The empty reference crucible is positioned in the opposite location. Approximately, 5 mg of each sample were weighed in an aluminium crucible. After being closed, the crucible was placed in the oven and set to a temperature programme from 10 °C to 70 °C at a heating rate of 10 ’C / min. Bulk rutin was subjected to the same conditions, yet up to a maximum temperature of 280 °C and with nitrogen flow at a rate of 30 ml / min for the cooling. The thermograms were generated, and the melting onset temperature, enthalpy and melting point were recorded using the Proteus® 6.1.0B Thermal Analysis software (NETZSCH, Selb, Germany) .

[0125] 1.6 Encapsulation parameters (EE% and LC%)

[0126] Encapsulation efficiency (EE%) of rutin-loaded micelles was determined by calculating the ratio between the loaded flavonoid and the total amount of bioactive initially weighed to be incorporated into the micelles. The loading capacity (LC%) was determined by calculating the ratio between the loaded flavonoid and the total amount of lipids initially weighed to produce the micelles, using the following equations (Fathi et al. , 2024a) : total amount of rutin — free amount of rutin EE(%~) = - — - — -:- - - x 100 total amount of rutin total amount of rutin — free amount of rutin LC(° / o) = - - — - - - - - x 100 total amount of lipid

[0127] In order to determine the amount of free rutin, micelle dispersions were placed into an ultrafiltration unit (lOKDa Arnicon Ultra; Merk Millipore®, Carrigtwohill , Ireland) and centrifugated at 3500 rpm for 30 min (Centrifuge Eppendorf, AG 5804, Hamburg, Germany) . After centrifugation, a clear solution was obtained and 1 ml of the supernatant was collected, representing the non-encapsulated drug (free rutin) . The supernatant volume was diluted with 1 ml of methanol. A UV-Vis detector (Jasco V- 650 Spectrophotometer, Tokyo, Japan) was used to quantify the rutin by determining the absorbance of the sample at 206 nm and quantified against a calibration curve.

[0128] 1.7 Rheological analysis

[0129] Using a cone-and-plate test geometry (plate diameter of 20 mm) and the Kinexus Lab + Rheometer from Malvern (Malvern, Worcestershire, UK) , rheological study of micelle dispersions was performed under the oscillation frequency sweep test. The analysed frequency range was from 0 to 10 Hz, and the stress amplitude was kept at 1 Pa. The upper geometry was then subjected to a torque, which increased the shear stress. The formulations underwent analysis at a temperature of 25 °C ± 1 °C, with a gap of 1 mm between the plates. A frequency sweep was performed in the oscillation mode, ranging from 0.1 to 10.0 Hz, with a strain of 1%. The rSpace for Kinexus Lab + program (version 1.75 Malvern Instruments, Worcestershire, UK) was utilized to collect the data.

[0130] 1.8 Statistical analysis

[0131] All results are expressed as the mean ± standard deviation (SD) . Data obtained for the Z-Ave, PDI, and ZP of micelles were analyzed by two- way ANOVA and Tukey's multiple comparisons test was used to compare the differences between the groups at a significance level of p-value < 0.05. The statistical analysis was performed by GraphPad Prism 8.4.3.

[0132] 2. Results and Discussion

[0133] The nanomicelles were produced using polysorbate Tween 80 and sorbitan monooleate (Span 80) as emulsifying agents that stabilize the nanocarriers, in which the mixture of both exhibited the best synergistic effect, as evidenced by ( Fu et al. , 2010; Yousefi et al. , 2023) . These surfactants were added to the aqueous phase prior to the dispersion of the lipid phase, soy lecithin (containing 94% of phosphatidylcholine) as the primary lipid component mixed with rutin. Soy lecithin (phosphatidylcholine) enhances the stability, solubility, and bioavailability of drugs in pharmaceutical formulations including nanoemulsions and nanomicelles, acting as both a lipid phase and a natural surfactant, it facilitates the formation of stable nanocarriers, improving drug encapsulation and membrane permeability. Its amphiphilic properties aid in solubilizing poorly water-soluble drugs and enabling controlled release, while ensuring biocompatibility and safety for effective drug delivery (Fathi et al. , 2021a; Rupp et al. , 2010) . Furthermore, the emulsification process that combines shear homogenisation and ultrasonication ensures uniform dispersion of the lipid phase within the aqueous medium (Fathi et al. , 2021a) , facilitating the formation of stable micelles with encapsulated rutin. In this study, the mixture of Tween 80, Span 80, and soy lecithin exhibited the best physicochemical properties.

[0134] The Z-Ave and PDI of produced nanomicelles were evaluated, as these parameters serve as indicators of the quality of the lipid nanocarriers. A lower PDI correlates with increased stability (Fathi et al. , 2022; Fathi et al. , 2021b) , as it reduces the risk of Ostwald ripening by promoting strong repulsion between particles. This prevents water- soluble compounds from diffusing from smaller droplets to larger ones during Ostwald ripening. The Z-Ave and PDI of both blank and rutin- loaded nanomicelles were characterized by DLS . The Z-Ave and PDI of all prepared formulations (rutin-free and rutin-loaded micelles) were determined over time on day 1, and then 7 days, 15 days, 30 days, 45 days and 60 days after production. The Z-Ave of the blank micelles ranged from 77.95 ± 0.35 nm to 83.75 ± 1.05 nm, and for the rutin-loaded micelles from 72.6 ± 0.31 nm to 87.10 ± 6.38 nm over the timeframe of 60 days at 25 and 4 °C. The results are presented in Figure 1.

[0135] From the results shown in Figure 1, the influence of the storage temperature on the Z-Ave, PDI and ZP of micelles is noted. With the aging, at 25 °C, there was no significant increase in the Z-Ave of the blank micelles (Figure 1A, left) (p>0.05) , but a significant increase in the Z-Ave of rutin-loaded micelles was observed by day 60 (87.10 ± 6.38 nm) compared to the values obtained for the same micelles on day 1 (78.99 ± 0.86) (Figure 1A, right) when stored at 4 °C. The increase of z-Ave by day 60 of rutin-loaded micelles stored at room temperature is attributed to the dynamic bahavior of lipid vesicles when dispersed in water (Walde and Ichikawa, 2021) . When lipid vesicles are dispersed in water, they can fuse, leading to an increase in their size. Likewise, vesicles can aggregate for various reasons, e.g. , the presence of a high ionic strength, pH variation and lipid composition (Walde and Ichikawa, 2021) . Additionally, fusion and aggregation events can create a broader size distribution, which leads to an increase in PDI, which was also observed . In rutin-loaded micelles after 60 days of production, the PDI value increased statistically when compared to that recorded on day 1 (Figure 1C, right) . After incorporating rutin into micelles, no significant differences in Z-Ave were observed when compared to the blank micelles at all timepoints .

[0136] According to ZP data, all micelles showed a negative surface charge with ZP values above I 30 | mV at two storage temperatures, indicating high electrochemical stability of micelles (Clogston and Patri, 2011; Lukhele et al. , 2023) . However, at 25 °C, rutin-loaded micelles depicted the lowest ZP values (in modulus) on day 45 and day 60 compared to day 1 (Figure IE, right) , and the incorporation of rutin into micelles also decreased the ZP (in modulus) significantly after 7, 30, 45 and 60 days of production compared to blank micelles at the same time (Figure IE) .

[0137] When incorporating rutin into the micelles, there is a reduction in repelling forces between the micelles, reducing the value of the zeta potential. These interactions can lead to possible aggregation between the micelles, resulting in lower stability of the formulation (Abdelbary et al. , 2013; Gao et al. , 2023; Singla et al. , 2023) .

[0138] In contrast to the data obtained at 25 °C, when stored at 4 °C both the blank and rutin-loaded micelles kept the size parameters constant and a high surface electrical charge (Figures IB and ID) , rending them longterm stability. Micelles are thermodynamically sensitive, i.e. , temperature significantly affects their structural integrity. At room temperature (between 20 and 30°C) , micelles are generally more dynamic and their surfactant molecules undergo faster movements that can result in the dissociation, aggregation or fusion of micelles (Espinosa and Grigera, 2015; Sauer and Moraru, 2012) . Higher temperatures also tend to destabilize the packing of surfactant molecules on the micellar surface, leading to a decrease in encapsulation efficiency and, in some cases, the release of the drug from the micellar core (Sauer and Moraru, 2012) . On the contrary, at 4 °C there are less interactions between the different constituents, leading to more stable micelles.

[0139] Although the agglomeration of micelles was favored by the increased temperature (25°) , these results from the evaluation of micelles stability indicated that, in general, both micelles were stable over time at all time points . The PDI maintained a relatively low value and without major variations over the eight weeks, offering a good prognosis for the long-term stability of the micelles, as the prepared micelles can then be considered monodisperse. High values of ZP, whether positive or negative, due to electrostatic repulsion, predict the long-term stability of the nanocarriers (Honary and Zahir, 2013; Zielihska et al. , 2019) .

[0140] TEM images shown in Figure 2 depict the morphology of micelles loaded with 0.1% rutin, showing a spherical shape and particle size lower than 100 nm. In the DLS technique, the hydrodynamic diameters of micelles are recorded, while the TEM analysis the data represent the size of the micelles in the dry state. The results suggest no aggregation of micelles after their loading with rutin, corroborating the results obtained by DLS .

[0141] Figure 3 shows the DSC profile of the bulk rutin. Rutin shows three endothermic peaks, which indicate several phase transitions: the melting point and decomposition process (Ahmad et al., 2016; da Costa et al. , 2002) . Rutin shows a major endothermic peak with onset temperature at 162 °C, end set temperature at 212.3 °C and a sharp peak at 177.2 °C. The sharp endothermal peak corresponds to its melting point and indicates the loss of the crystalline structure (Asfour and Mohsen, 2018) . Figure 4 shows the thermograms of the blank and rutin-loaded micelles stored at the two different temperatures. The thermograms depicted in Figure 4 are typical of vesicles made of phospholipids, without endothermic peaks within the analysed temperature range. These profiles also confirm no chemical interactions between rutin and the micelles, as both samples (blank and rutin-loaded) have similar DSC profiles. No remarkable influence of the storage temperature was also registered, with the calorimetric behaviour being very similar among the samples stored at different temperatures.

[0142] The encapsulation efficiency (EE%) and loading capacity (LC%) were determined as quantitative and qualitative parameters of the newly prepared loaded micelles. The EE reached 92.09% and the LC was 1.96%, indicating that rutin was successfully incorporated into micelles. The rheological profiles of the prepared formulations recorded 24 hours after production are shown in Figure 5. Figure 6 shows the rheological profiles of samples recorded 2 months after production. For comparison purposes between blank micelles and rutin-loaded micelles, the storage modulus (G' ) , loss modulus (G") and shear viscosity were recorded at two different times, 24h after production and 60 days after production. The storage modulus (G' ) or elastic component quantifies the energy stored within the material during deformation and subsequently recovered after stress release, i.e. , it resembles the amount of energy that the dispersion needs to be distorted, while the loss modulus (G") or viscous component translates the energy lost during deformation. Storage modulus and loss modulus are key parameters used to characterize the viscoelastic behaviour of materials (Jaishankar and McKinley, 2014) .

[0143] As seen in all the profiles, the elastic component was higher than the viscous component in the applied frequency range, both after 24h of production and after 60 days, regardless the storage temperature. When that happens, it means that the sample exhibits more elastic behaviour than viscous behaviour (Budai et al. , 2023) . When the frequency is low, the shear rate is also low. This indicates that dispersions have a great capacity to preserve the medium's initial strength (Wei et al. , 2022) . When the frequency range increases, the shear rate increases as well, requiring more energy and raising the viscous and elastic components in the process. The G" showed a lower dependence on the applied force, while the G' increased significantly in the frequency range. This same profile could be observed in the shear viscosity, which also increases as the frequency increases. Comparing the viscosity of all formulations, it can be stated that, 24h after production, samples stored at 4 °C have higher values than samples stored at 25 °C. However, the shear viscosity values after 24 hours, compared to those after 2 months of storage, are different. In rutin-loaded micelles, the shear viscosity values increased after 60 days of storage, compared to the values measured after 24h. The opposite occurred with the blank samples; the shear viscosity values decreased over time. The highest shear viscosity values were recorded for the blank micelles at a storage temperature of 4 °C, for 24h. The obtained results from the rheological analysis corroborate the assumption of the micelles stability over time. Nor the time or the temperature of storage induced a significant change behaviour of the prepared micelles. 3. Conclusions

[0144] In this example, rutin-loaded micelles were successfully prepared using an ecofriendly methodology. Rutin-loaded nanomicelles with appropriate size, high ZP values, high encapsulation efficiency and loading capacity, and high stability were obtained. The Z-Ave and ZP of the micelles remained practically the same over 60 days of storage. However, the storage temperature influenced these physicochemical parameters. Rutin- loaded micelles had a significant increase in size and a decrease in the ZP value when stored at 25 °C. In the rheological analysis, rutin-loaded micelles exhibited an increase in the shear viscosity values after 60 days of storage, compared to the values measured after 24h after production of the micelles. The opposite occurred with the blank micelles; the shear viscosity values decreased over time, suggesting that the presence of the bioactive compound influences the viscosity of the sample. The high EE and LC of rutin confirm the added-value of the method to overcome the problem of poorly-water soluble drugs.

[0145] Example 2 - physicochemical characterization and rheological behaviour of nanomicelles for dual delivery of resveratrol and rutin

[0146] 1. Materials and Methods

[0147] 1.1 Materials

[0148] The materials used in this example were obtained as previously described and resveratrol was purchased from Fagron, Barcelona, Spain.

[0149] 1.2 Production of nanomicelles

[0150] In the production of aqueous micelle dispersions, the hot shear homogenisation technique, followed by ultrasonication method, was used as described above in example 1. Resveratrol-loaded nanomicelles, resveratrol-rutin-loaded nanomicelles (dual-loading) were produced and compared to blank micelles (without any bioactive ingredient) . The composition of the prepared micelles is shown in Table 2.

[0151] Table 2: Composition of the prepared resveratrol-loaded micelles and resveratrol-rutin-loaded micelles .

[0152] To prepare nanomicelles, the aqueous phase contained 94% (w / w) of ultrapurified water and 1% surfactant (Tween®80) was added to the lipid phase, and both were heated under constant stirring, until the mixture boiled. After that, the mixture was subjected to high-speed stirring using an Ultra-Turrax (IKA T25, Staufen, Germany) for 5 minutes at 7000 rpm and then, subjected to a sonicator for 5 minutes with an amplitude of 70% ( (Sonics Vibracell, Newtown, CT, USA) using Qsonica 4435 Q55 Sonicator Microprobe, 1 / 4 ' ', with 0.635 cm of tip diameter) . The resulting dispersions were split in two batches for storage at two different temperatures, 25°C and 4°C, until further use. For the loaded micelles, the bioactive ingredients were added to the lipid phase prior to mixing it with the water phase. The dispersions were monitored and characterized over a period of 60 days, as described in the following sections .

[0153] 1.3 Average size, polydispersity index and zeta potential

[0154] Average size (Z-Ave) , size distribution (polydispersity index, PDI) and zeta potential (ZP) of blank and loaded micelles were determined by the dynamic light scattering (DLS) method. The measurements were taken as described in the example 1 above.

[0155] 1.4 Transmission electron microscopy

[0156] The morphology and size of the prepared micelles in their dry state were analysed using transmission electron microscopy and the measurements were carried out as described in example 1.

[0157] 1.5 Differential Scanning Calorimetry

[0158] Differential Scanning Calorimetry (DSC) analysis of the nanomicelle samples prepared was carried out using the DSC 200 F3 Maia ® equipment (NETZSCH, Selb, Germany) and the analysis was carried out as described in the example 1 above.

[0159] In position 0, an empty crucible was placed, representing the reference. The bulk resveratrol sample was heated from 10°C to 290°C (20°C above the melting point) and the bulk rutin sample was heated from 10°C to 280°C (20°C above the melting point) . The heating rate of both was 10°C per minute. Sample cells were purged with nitrogen (flow 30 ml / min) . The micelles were subjected to the same conditions, but up to a maximum temperature of 70°C.

[0160] 1.6 Encapsulation efficiency and loading capacity

[0161] The encapsulation efficiency (EE%) and loading capacity (LC%) of nanomicelles containing resveratrol were determined after separation, by centrifugation, of the non-incorporated drug. The sample was placed in an ultrafiltration unit (lOKDa Arnicon; Merk Millipore®, Carrigtwohill , Ireland) and centrifugation (Centrifuge Eppendorf, AG 5804, Hamburg, Germany) occurred at 3500 rpm for 30 minutes. Then, 1 mL of the clear supernatant was diluted with 1 mL of methanol and quantified against a calibration curve to determine the resveratrol concentration. A UV-Vis detector (Jasco V-650 Spectrophotometer, Tokyo, Japan) was used to measure the RES content at 306 nm. The same method was used for dualloaded micelles (i.e. , containing resveratrol and rutin) , quantified against the respective calibration curve of each bioactive. Rutin was recorded at 355 nm. The EE% and LC% were then calculated according to the following equations : 100 where M(T) is the total amount of resveratrol or rutin in micelles; M ( / ) is the amount of free resveratrol or rutin in micelles dispersion and M(lipicT) is the total amount of lipid in micelles.

[0162] 1.7 Rheological analysis

[0163] Rheological analysis of the prepared micelles was carried out using a Kinexus Lab + rheometer (Malvern, Worcestershire, UK) , equipped with a 20 mm plate diameter probe. The test applied for the analysis was the oscillation frequency sweep test at a constant stress amplitude of 1 Pa. The frequency range used was from 0 to 10 Hz. The samples were measured at 25°C, with a gap of 1 mm between the equipment plate and the probe. A small portion of the sample was placed on the equipment plate and shear stress was applied. After analysis, data were collected using the rSpace for Kinexus Lab + software (version 1.75 Malvern Instruments) . 1.8. Statistical analysis

[0164] All results are expressed as mean ± standard deviation (SD) . Data obtained for the average size, PDI, and ZP of samples were analysed by two-way ANOVA and Tukey's multiple comparisons test was used to compare the differences between the groups at a significance level of p-value < 0.05. The statistical analysis was performed by GraphPad Prism 8.4.3.

[0165] 2. Results and Discussion

[0166] All loaded micelles were produced with 4.9% ( w / w) of soy lecithin and 0.1% (w / w) of active compound representing the lipid phase of the nanocarriers (Table 2) . The aqueous phase comprised 94% (w / w) of water and 1% (w / w) of surfactant (polysorbate 80) . For blank micelles, the composition was the same, but without the bioactive.

[0167] To evaluate the stability of the formulations, the Z-Ave, PDI and ZP of all prepared formulations were determined on day 1, day 7, day 15, day 30, day 45 and day 60 after production. Results recorded 24 hours after the production (day 1) reached a mean size of 64.34 ± 0.23 nm with PDI 0.272 ± 0.004 for blank micelles at 25°C and 65.37 ± 0.41 nm with PDI 0.278 ± 0.002 for blank micelles at 4°C. Micelles loaded with bioactive ingredients showed higher particle size values than the unloaded ones. Resveratrol-loaded micelles had in Z-Ave 70.87 ± 1.05 nm with PDI 0.267 ± 0.004 when stored at 25°C and 69.47 ± 0.66 nm with PDI 0.273 ± 0.006 when stored at 4°C. Dual-loaded micelles had a particle size of 68.78 ± 0.41 nm with PDI 0.2737 ± 0.008 when stored at 25°C and 70.41 ± 1.16 nm with PDI 0.276 ± 0.004 when stored at 4°C. As both samples presented a low PDI (below 0.300) , the formulations were considered monodisperse.

[0168] Figure 7 shows the mean size parameters and ZP values over storage time at different temperatures. The Z-Ave values of resveratrol-loaded micelles and resveratrol-rutin-loaded micelles significantly increased compared to blank micelles in almost timepoints at both storage temperatures (Figure 7A and 7B) . However, the Z-Ave of micelles containing both bioactive ingredients significantly increased after day 60 at 25°C (Figure 7A) and after day 15 and 30 at 4°C (Figure 7B) compared to micelles containing only resveratrol.

[0169] According to PDI data, at 25°C, the values obtained for micelles loaded with resveratrol significantly increased over the course of two months, compared to blank micelles (Figure 7C) . The same occurred when micelles containing resveratrol were compared with the dual loaded micelles stored at 25°C.

[0170] A high ZP value, such as |± 30 mV| , is considered suitable to promote repulsive forces between micelles to prevent their aggregation (Zielihska et al. , 2020) . In our work, the results obtained were within the range of values described as being optimal for this parameter, i.e. , in the blank samples and in the micelles loaded with bioactive ingredients, recording negative values of -41.96 and -64.18 mV. In general, over two months, ZP values remained within the range of acceptable values considered to be a good indicator of formulation stability .

[0171] A significant difference was noted in the ZP values between the two micelle formulations stored at distinct temperatures. Those stored at 25°C and containing resveratrol; there was a higher variation in values, and, in general, the ZP significantly decreased (in modulus) from day 30 compared to blank micelles (Figure 7E) . The same pattern occurred for micelles containing both resveratrol and rutin from day 7 to day 45 after production. However, ZP values obtained for dual-loaded micelles significantly increased after 60 days of storage compared to those having only resveratrol (Figure 7E) .

[0172] For the loaded micelles stored at 4°C, the ZP remained significantly higher (Figure 7F) , confirming the high electrostatic stability of the micelles. The storage temperature of the samples is thus a governing factor for micelles stability.

[0173] TEM analysis was used to obtain information about the morphology and shape of the prepared micelles. The images from the analysis showed micelles of spherical shape with diameters below 100 nm (Figure 8) . The micelle sizes recorded in the TEM technique were slightly smaller than the values recorded in the DLS technique.

[0174] Figure 9 shows the DSC analysis of resveratrol.

[0175] The thermal analysis of blank micelles, resveratrol-loaded and resveratrol-rutin-loaded micelles showed similar DSC profiles, which indicates that there was no interaction between polyphenols and soy lecithin and surfactants composing the micelles (Figure 10) . The DSC results also confirm that both polyphenols are loaded within the micelles, and no solid matrix was seen in the nanocarriers. No influence of the temperature and time of storage was seen for the prepared micelles .

[0176] EE and LC were calculated to estimate the amount of resveratrol and rutin encapsulated in the micelles. The EE reached 98.69% and the LC was 2.11% for resveratrol-micelles . For resveratrol-rutin micelles, the same parameters were calculated to estimate the amount of resveratrol and rutin that were encapsulated in the micelles. For resveratrol, the EE was 99.86% and the LC was 1.01%; for rutin, the EE was 98.31% and the LC was 0.99%. These results corroborate those of DSC confirming the successful loading of both polyphenols in micelles.

[0177] Rheological properties of semi-solids and liquids aimed at topical administration strongly influence their physical and sensory properties. Figure 11 shows the rheological analysis, recorded 24 hours after production, of blank micelles, micelles loaded with resveratrol, and micelles loaded with rutin and resveratrol, and Figure 12 shows the same test 2 months after production. The storage modulus G' or elastic component, the loss modulus G" or viscous component and the shear viscosity are presented. As seen, in general, G' was higher than G" in the measured frequency range, as shown in Figure 11 and Figure 12, indicating a sample with more elastic characteristics. The G" showed less dependency on the frequency, while the G' increased significantly in the frequency range from 0 to 10 Hz, highly depending on it. The rheological analysis of the micelles showed a behaviour in which the shear viscosity increased with frequency.

[0178] Based on the results shown in Figure 11 when stored at a temperature of 4°C, both samples loaded with polyphenols registered higher values of shear viscosity and G' than those stored at 25°C. Blank micelles stored at 4°C recorded the highest shear viscosity and G' values after 24 hours of production. These results suggest a certain influence of storage temperature on the viscoelastic behaviour of the prepared micelles, but only 24 h after production. After 2 months of storage, the results changed. In both samples, the shear viscosity values increased compared to the results recorded after 24h. Two months after production, resveratrol-loaded micelles showed higher shear viscosity and higher G' values, than 24 h after production and the same happened for the dualloaded micelles. The presence of a second polyphenol within the micelles induced the increase of shear viscosity, which was also increased over storage time, offering appropriate rheological behaviour for topical administration .

[0179] 3. Conclusions

[0180] In this example, resveratrol-loaded micelles and resveratrol-rutin- loaded micelles were successfully produced with suitable Z-Ave, PDI and ZP, and no considerable differences were recorded between the two types of prepared micelles. For both types of micelles, the encapsulation efficiency and loading capacity were very high, indicating that both polyphenols were successfully loaded into the micelles. Regarding the rheology analysis, remarkable differences in results between the prepared micelles were found. Two months after production, the shear viscosity of micelles loaded with resveratrol and rutin was found to be much higher than micelles loaded with resveratrol only, suggesting that the presence of more than one polyphenol increases the viscosity of the micelles. From the obtained results, one can consider the dual loading of polyphenols into micelles an interesting innovative delivery approach for antioxidants for topical administration.

[0181] Example 3 - hydrogels comprising the phospholipid nanomicelles for topical administration of resveratrol and rutin

[0182] 1. Materials and Methods

[0183] 1.1 Materials

[0184] The materials used in this example included Polysorbate 80 (Acofarma, Barcelona, Spain) , Sorbitan monostearate 80 (Acofarma, Barcelona, Spain) , L-a-Phosphatidyl choline (Sigma-Aldrich, St. Louis, MO, USA) , Resveratrol (Fagron, Barcelona, Spain) , Rutin (Acros Organics, Geel, Belgium) , Carbomer 940 (Fagron, Barcelona, Spain) , Sodium Hydroxide (Sigma-Aldrich, St. Louis, MO, USA) , Propylparaben (Acofarma, Barcelona, Spain) and Propylene glycol (Acofarma, Barcelona, Spain) . Olive pomace was obtained from local producers. Deionised water (Milli-Q water home supplied) was used throughout all experiments.

[0185] 1.2 Production of Resveratrol -Rutin-Loaded Micelles

[0186] For the production of nanomicelles, the hot shear homogenisation technique followed by the ultrasonication method was used as previously described, with adaptations. The compositions of the different micelles are shown in Table 4. Briefly, for all micelles (Mcl - resveratrol, Mc2 - rutin, and Mc3 - resveratrol and rutin) , the active compound concentration was 0.1% (w / w) . This, mixed with soy lecithin (L-a- Phosphatidyl choline ) , which is a phospholipid, formed the lipid phase of the system. The aqueous phase comprised non-ionic surfactants (Polysorbate 80 and / or Span 80) dissolved in ultra-purified water. The aqueous phase was added to the lipid phase, and the obtained mixture was heated under constant stirring until boiling. The resulting dispersion was stirred at high speed (7000 rpm) using an Ultra-Turrax (IKA, Staufen, Germany) for 5 min. This mixture was subsequently subjected to sonication with a probe for 5 min and an amplitude of 70%. The obtained micelles were then stored at room temperature until further use.

[0187] Table 4. Composition of resveratrol-loaded micelles, rutin-loaded micelles and resveratrol-rutin-loaded micelles.

[0188] 1.3 Production of hydrogels comprising resveratrol -loaded micelles, rutin-loaded micelles, and with resveratrol -rutin loaded-micelles

[0189] For the acceptance of a topical product, there are several important parameters to be considered, such as its easy application to the skin, the sensorial properties (brightness and amount of residue) and the appearance of the product. As micelle dispersions do not have an adequate consistency for application to the skin, as they are liquid systems, their transformation into semi-solid systems becomes an attractive solution for product acceptability, besides improving the stability of micelles by reducing the risk of forming aggregates over the shelf-life. Thus, hydrogels based on micelles (Hydrogel A and Hydrogel B) were also prepared .

[0190] Carbomer was used to improve the viscosity of the system and obtain suitable consistency levels for topical application. First, a 2% (w / v) carbomer 940 gel was prepared with the aid of the Unguator® mixer (Gako Deutschland GmbH, Schesslitz, Germany) . Carbomer 940, water and propylparaben were weighed and mixed at a speed of 2400 rpm for 30 s. Propylparaben was used as a preservative, and a 10% (w / v) solution of sodium hydroxide (0.1 M) was used to adjust the pH value of the carbomer up to 6.5-7. The solution of sodium hydroxide was added to the mixture dropwise, under gentle stirring of 600 rpm for 8 min and 30 s, to induce polymer gelation until reaching pH 6.5. The obtained hydrogel was kept at rest for the following 12 h to eliminate the presence of any existing air bubbles at a controlled temperature of 2-8 °C for further studies. For the final preparation of the hydrogel loaded with micelles, the previously prepared 2% carbomer gel was used.

[0191] In hydrogel A, micelles were incorporated into the hydrogel. In hydrogel B, micelles were incorporated into the hydrogel along with olive pomace. Both semi-solids (hydrogels A and hydrogels B) were then homogenized using an Unguator® at 600 rpm for 1 minute. The final formulation comprised 50% of 2% carbomer gel, 49.7% of micelles and 0.3% of olive pomace (hydrogel A) and 50% of 2% carbomer gel and 50% of micelles (hydrogel B) . The composition of all prepared hydrogel formulations is represented in Tables 5 and 6.

[0192] Table 5. Composition of 100 g of 2% carbomer 940 gel.

[0193] Table 6. Composition of hydrogels A and hydrogels B.

[0194] 1.4 Differential Scanning Calorimetry

[0195] Differential Scanning Calorimetry analysis was used to ascertain the physical state and thermal properties of the micelles-loaded hydrogels. This task was carried out on a DSC 200 F3 Maia® (NETZSCH, Selb, Germany) . The equipment consists of an oven with two different chambers. One chamber houses the crucible that contains the sample, and the other houses the reference crucible (position 0) . The samples were weighed (approximately 5 mg) in an aluminium crucible that was subsequently closed. The analysis consists of a temperature program between 20 °C and 70 °C, at a heating rate of 10 °C / min, to which the samples are subjected to record the corresponding thermogram. Data analysis was performed using the thermal analysis software "Proteus® 6.1.0B" software (NETZSCH, Selb, Germany) .

[0196] 1.5 Rheological Analysis

[0197] The viscoelastic properties of the prepared hydrogels were evaluated using a Rheometer Kinexus Lab + (Malvern, Worcestershire, UK) . The hydrogels were measured at a temperature of 25 °C with a spacing of 1 mm gap between plates. A portion of each sample under analysis was placed on the lower plate of the equipment, where a torque was subsequently applied, which promoted the shear stress. The frequency used in the analysis was from 0.1 to 10.0 Hz, in oscillation mode and 1% deformation. Data were compiled and collected using the software "rSpace for Kinexus Lab +" (version 1.75 Malvern Instruments) .

[0198] 1.6 Texture Analysis

[0199] To analyse the texture of the different samples, a texture analyser TA- XT2i© (Stable Micro Systems, Godaiming, UK) and the software "Exponent" (version 6.1.12.0) were used. The sample was placed in the equipment, and a P / 0.5 Dia Delrin Aoacque probe penetrated it with a 5 Kg load cell. The firmness and cohesiveness were evaluated at room temperature (22-25 °C) for all samples. Data acquisition and analysis were performed using the software "Texture Expert®" (version 6.1.12.0) . 2. Results and Discussion

[0200] DSC was carried out to analyse the thermal properties of hydrogels A and B before (blank) and after dispersing the loaded micelles, in comparison to olive pomace at the same temperature range (20-65 °C) and the results are presented in Figure 13. All samples presented similar endothermic profiles. No peaks were recorded in the analysis of the different A and B hydrogel samples, corroborating the idea that the systems do not have a solid matrix.

[0201] Some hydrogels produced comprised carbomer, which is a well-known high molecular weight, hydrophilic, crosslinked polymer of polyacrylic acid. Carbomer creates a three-dimensional polymer network that absorbs water and exhibits transitory, reversible interchain entanglements making it more versatile and robust than other chemical hydrogels. Its rheological properties, whether dispersed in water or in water / glycerol mixtures, have been extensively characterised. This example used the polymer dissolved in water to obtain the semi-solid base for the dispersion of our micelles. The oscillatory study (Figures 14 and 15) was carried out to compare the rheological profile of the obtained micelles -containing hydrogels with and without the presence of olive pomace. This test describes the system response as a function of frequency at constant shear strain and provides information on the storage modulus (elastic component, G' ) , loss modulus (viscous component, G") and shear viscosity. The G' resembles the amount of energy that the dispersion needs to be distorted, while the G" reflects the energy lost during deformation. As the frequency range increases, the shear rate also increases, thus requiring more energy and consequently increasing G" and G' . This test is relevant to be run in the final product to assess whether it has the appropriate rheological properties to be administered on the skin. This example used polymer (2%, m / v) dissolved in water, neutralised with NaOH 0.1 M until reaching pH 6.5, in which micelles containing the bioactive ingredients were dispersed. The results show that, for all tested formulations depicted in Figures 14 and 15, the G' modulus (the storage or elastic modulus) was always much higher than the G" modulus (the loss or viscous modulus) throughout the entire frequency range (0.1-10 Hz) . These results indicate that the elastic properties dominate over the viscous behaviour. These data point out to the presence of a gel-like structure and indicate that the system is more elastic than viscous, which is a characteristic of viscoelastic systems, wherein the microstructure retains energy from oscillations and relaxes adequately to discharge a portion of that energy through microstructural rearrangements. When a viscoelastic material experiences stress, its response consists of elastic deformation (which stores energy) and viscous flow (which loses energy) , confirming the suitability of the developed semi-solids for topical administration. It can also be observed that the G' and G" of all samples are strongly dependent on the frequency, indicating short relaxation times for the microstructures within the applied frequency range. Viscosity was also found to be frequencydependent as its values decreased significantly with increasing frequency, particularly when starting the test. This behaviour is typical of viscoelastic semi-solids and can be found for standard topical dosage forms indicating that the formulations can be easily rubbed onto the skin. When comparing blank hydrogels to those containing bioactives- loaded micelles, the presence of these latter increased the viscosity of the semi-solids in general terms but did not compromise the firmness and cohesiveness of the hydrogels (Table 7) . The viscosity of the hydrogels A and B was also strongly dependent on the presence of olive pomace. In hydrogel A (Figure 14) , containing olive pomace, the viscosity is considerably lower in all samples (A Mcl, A Mc2 and A Mc3) compared to hydrogel B formulations (Figure 15) , showing that olive pomace reduces the viscosity of the systems. It was also seen that in hydrogels B, a greater distance was recorded between the moduli G' and G" for each formulation compared to hydrogels A, showing a greater capacity to retain energy (storage modulus) than to lose energy (loss modulus) .

[0202] Table 7. Textural properties of the hydrogels.

[0203] The first negative force that is recorded in the texture analysis is related to the cohesiveness of the gel. The sample A Mc3, the one based on the hydrogels comprising dual loaded-nanomicelles and containing olive pomace, was found to be the least cohesive hydrogel with the force closest to zero (0.1302 N) . In general, hydrogels B were found to be more cohesive than hydrogels A, except for sample B Mcl.

[0204] In summary, the results indicate that dispersing micelles into hydrogels may improve both rheological properties and the texture of semi-solid hydrogels, whereas the presence of olive pomace does not compromise the firmness and cohesiveness of the systems as they were recorded within the same range of values . This by-product can further be exploited for its emollient, moisturiser and nourisher properties. These findings highlight the interest in the use of olive pomace as a new ingredient of hydrogel formulations for skin administration.

[0205] 3. Conclusions

[0206] The aim of this example was to prepare a new eco-friendly hydrogel loaded with different natural antioxidants, with the reuse of a by-product (olive pomace) . Three different types of hydrogels, namely, one containing resveratrol-loaded micelles , a second containing rutin-loaded micelles, and a third containing resveratrol-rutin-loaded micelles, were successfully produced with and without olive pomace. The hydrogels presented G' values much higher than the G" values across the whole frequency range, indicating a gel-like structure and showing that the systems were more elastic than viscous, confirming this key attribute for topical administration. It was also found that the viscosity of hydrogels A and B was strongly dependent on the presence of micelles and olive pomace; while the presence of micelles increased the viscosity of the gels, when adding olive pomace, the system showed lower viscosity yet without compromising firmness and cohesiveness. From the texture analysis, hydrogels containing olive pomace have a more rigid, firm and cohesive matrix than those hydrogels based only on carbomer. The results of this analysis agree with those of the rheological analysis and corroborate the interest in using by-products in pharmaceutical and cosmetic formulations.

[0207] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims.

Claims

1. CLAIMS1. A formulation comprising phospholipid nanomicelles loaded with at least one polyphenol comprising:L-a-Phosphatidyl choline in a concentration between 0.01 and 10 % w / w; at least one surfactant in a concentration between 0.01 and 5 % w / w; water in a concentration between 80 and 99.97 % w / w; at least one polyphenol in a concentration between 0.01 and 5 % w / w, wherein the polyphenol is selected from resveratrol and / or rutin.

2. The formulation according to the previous claim, wherein the surfactant is selected from Polysorbate 80, Sorbitan monooleate, or mixtures thereof.

3. The formulation according to any of the previous claims, wherein L- a-Phosphatidyl choline is obtained from soy lecithin.

4. The formulation according to any of the previous claims, wherein the nanomicelles comprise a surface charge between ±20 and ±40 mV.

5. The formulation according to any of the previous claims, wherein the nanomicelles comprise an average particle size below 100 nm.

6. The formulation according to the previous, wherein the nanomicelles comprise an average particle size between 70 and 90 nm.

7. The formulation according to any of the previous claims, wherein the formulation comprises an encapsulation efficiency between 90 and 100%.

8. The formulation according to any of the previous claims, wherein the formulation comprises a loading capacity between 0.01 and 5%.

9. Hydrogel formulation comprising phospholipid nanomicelles loaded with at least one type of polyphenol, comprising:L-a-Phosphatidyl choline micelles loaded with at least one polyphenol in a concentration between 0.005 and 5 % w / w; polyacrylic acid in a concentration between 0.1 and 5 % w / w;propylparaben in a concentration between 0.005 and 2.5 % w / w; olive pomace in a concentration between 0.05 and 5 % w / w; wherein the at least one polyphenol encapsulated in the micelles is selected from resveratrol and / or rutin.

9. Use of the formulation described in any of the claims 1 to 8 or the hydrogel formulation described in claim 9 for the prevention or treatment of skin conditions or diseases.

10. Use of the formulation described in any of the claims 1 to 8 or the hydrogel formulation described in claim 9, according to the previous claim, wherein the skin conditions or diseases are selected from dehydration, aging, inflammation, rash, dermatitis, eczema, hives, urticaria, allergic reactions, psoriasis, wounds.

11. Use of the formulation described in any of the claims 1 to 8 or the hydrogel formulation described in claim 9 for topical application.

12. The formulation described in any of the claims 1 to 8 or the hydrogel formulation described in claim 9, wherein the formulation is a cosmetic formulation or a dermopharmaceutical formulation.

13. Method to obtain the formulation comprising phospholipid nanomicelles loaded with at least one polyphenol described in any of the claims 1 to 8, comprising the steps of: heating a solution comprising: resveratrol and / or rutin in a concentration between 0.01 and 5 % w / w, andL-a-Phosphatidyl choline in a concentration between 0.01 and 10 % w / w, at a temperature between 70 and 95°C under stirring between 500 and 5000 rpm, for a time between 1 and 5 minutes;Adding to the previous solution an aqueous solution of at least one surfactant in a concentration between 0.01 and 5 % w / w;Heating the mixture at a temperature between 75 and 95°C under stirring between 500 and 5000 rpm, for a time between 1 andlO minutes;Stirring the mixture between 5000 and 10000 rpm for a time between 3 and 10 min to form a pre-emulsion;Sonicating the pre-emulsion with a probe for a time between 1 and 10 min and between 60 and 80% amplitude to obtain the formulation.

14. Method according to the previous claim wherein the surfactant is selected from Polysorbate 80, Sorbitan monooleate, or mixtures thereof.

15. Method to obtain a hydrogel formulation comprising phospholipid nanomicelles loaded with at least one polyphenol described in claim 9, comprising the steps of:Preparing a 1-10 w / w solution of polyacrylic acid by mixing polyacrylic acid in a concentration between 1 and 10 % w / w, water and propylparaben in a concentration between 0.01 and 5 % w / w;Adjusting the pH of the solution between 6.5 and 7, under stirring between 500 and 5000 rpm, for a time between 1 and 10 minutes, to obtain a hydrogel;Storing the resultant hydrogel at a temperature between 0 and 8°C, for a time between 5 and 15 h;Adding phospholipid nanomicelles loaded with at least one polyphenol to the hydrogel in a ratio between 0.001:10 and 5:1 mass, wherein the polyphenol is selected from resveratrol and / or rutin;Adding olive pomace in a concentration between 0.1 and 10% w / w to the hydrogel .

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