Vesicles based on glucose-derived surfactants and plant sterols

Abstract

Vesicles are provided that comprise at least one plant sterol and at least one glucose-derived surfactant.Furthermore, compositions are provided that comprise a plurality of vesicles as defined herein.Vesicles and compositions that comprise a plurality of vesicles are used in the preparation of suspensions, as delivery systems, or as drugs.Furthermore, methods for producing the vesicles described herein are also provided.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of vesicular systems. In particular, the present invention relates to vesicles based on plant sterols and glucose-derived surfactants. A method for obtaining vesicle systems based on the DELOS-susp method and their applications in the cosmetic and pharmaceutical fields are also provided. [Background technology]

[0002] Various delivery systems based on nano- and micro-technologies have been widely studied in the pharmaceutical industry, not only for therapeutic applications but also for bioimaging and diagnostics. Beyond medical applications, vesicular systems (or vesicles, VSs) are booming in the healthcare and cosmetic fields. The most widely studied and currently used system is liposomes, which allow for the protection of sensitive molecules and controlled penetration of active ingredients in target organs. However, several drawbacks regarding the physicochemical properties and stability of liposomes affect their reproducibility and limit their applications. Liposomes suffer from physical changes, i.e., instability, which can lead to increased permeability and ultimately to undesirable or rapid leakage rates. Furthermore, liposomes also undergo chemical changes related to hydrolysis, oxidation, and peroxidation of the constituent lipid molecules. Consequently, constraints such as the addition of cholesterol, low storage temperatures, or the addition of antioxidants are required to overcome these drawbacks and synthesize such systems while maintaining their stability and enabling their optimal storage.

[0003] Another type of drug delivery system that has attracted scientific interest is niosomes (nonionic surfactant vesicles). While they are considered liposome analogs with higher stability, some reports have suggested they are less stable due to issues with aggregation, swelling, fusion, and drug leakage. On the other hand, quatosomes, a special type of vesicles formed by the self-assembly of cholesterol molecules and quaternary ammonium surfactants (e.g., cetyltrimethylammonium bromide, myristalkonium chloride, cetylpyridinium chloride, stearalkonium chloride, etc.), are potential delivery systems for pharmaceutical and cosmetic ingredients due to their favorable physicochemical properties and long-term stability. However, the use of such surfactants in healthcare and pharmaceuticals is limited due to some reported skin irritation and other toxicological effects at high concentrations.

[0004] As a result, a new generation of ecologically and environmentally safe surfactants, glucose-derived surfactants (e.g., alkyl polyglucosides (APGs)), has recently begun to be investigated. However, few examples of vesicle formation involving this new generation of surfactants have been found in the art.

[0005] Furthermore, it is emphasized that the size and layer number of vesicles are important structural parameters that need to be controlled, as they are important factors that affect the performance of vesicles, for example, as drug carriers. Therefore, depending on their size and layer number, vesicle systems can be classified into small unilamellar vesicles (SUVs, d<200 nm), large unilamellar vesicles (LUVs, d>200 nm), and multilamellar vesicles (MLVs).

[0006] The vesicle membrane also plays an important role in terms of its stability, rigidity, permeability, functionalization, and responsiveness to external stimuli. Therefore, the performance of vesicles is greatly influenced not only by their uniformity in size and morphology, but also by the uniformity in membrane composition and supramolecular organization. The structural uniformity of vesicles is key to achieving a uniform and reproducible delivery response, along with the uniform release of the encapsulated active ingredient at the site of action.

[0007] Salim et al. (2015) reported the formation of Vs by thin film hydration (TFH) using octyl glucoside and octyl maltoside in combination with 20 mol% cholesterol and 8 mol% dicetyl phosphate (DCP). The stability of Vs was 3 months at room temperature (RT). However, storage at 4 °C resulted in the formation of a white solid precipitate likely caused by lipid-cholesterol phase separation. These systems have been used to encapsulate methylene blue and have a low encapsulation efficiency (EE) of 15-20%.

[0008] (2013) reported the formation of Vs with TFH using octyl glucoside, decyl glucoside, lauryl glucoside, and cholesterol in a 1:1 molar ratio in water. The stability of this system exceeded 12 months at room temperature. The encapsulation of methotrexate was studied and reported to have an EE of 80–95%.

[0009] Manconi, Vila et al. (2006) described the formation of multilamellar vesicles (MLVs) with TFH using different molar ratios of decyl glucoside and cholesterol, with particle sizes ranging from 1263.2 nm to 338.6 nm. The MLVs prepared in this study were large and polydisperse (PdI > 0.8). Encapsulation of methylene blue was tested, yielding EEs of 40–70%.

[0010] Manconi, Sinico et al. (2006) reported the formation of vesicles by TFH in PBS using decyl glucoside:cholesterol and caprylyl / capryl glucoside:cholesterol systems with dicetyl phosphate (DCP) or stearylamine as additives. Some of these systems were used to encapsulate the natural retinoid tretinoin, which resulted in EEs of 93–99.5%. The resulting tretinoin-loaded systems were primarily polydisperse MLVs with large particle sizes (>500 nm at saturated active concentrations and >264 nm at unsaturated active concentrations). Furthermore, unilamellar vesicles could only be obtained after several cycles of sonication.

[0011] As mentioned above, cholesterol is widely used in the formation of Vs and is generally They are used in health foods and personal care products. Nevertheless, growing awareness of the adverse effects resulting from the use of animal-derived products has prompted the need to develop new vesicle systems containing environmentally friendly, animal-free, and consumer-friendly ingredients. Summary of the Invention

[0012] One problem to be solved by the present invention is to provide a sustainable vesicle platform useful for the efficient encapsulation of agents, such as pharmaceutical and cosmetic agents.

[0013] The present invention generally relates to vesicles (Vs), vesicular systems, or other micro- or nano-material-containing particles containing plant sterols (e.g., β-sitosterol) and glucose-derived surfactants (e.g., alkyl polyglucoside surfactants). The particles developed in this invention are stable and can encapsulate agents, such as pharmaceuticals and cosmetic agents. Thus, the present invention provides a novel and versatile system that is not only consumer-friendly but also contributes to more sustainable manufacturing.

[0014] The inventors have developed a platform for generating homogeneous, single-layered Vs based on the use of plant-derived components and sugar-based surfactants, for example, by applying the DELOS-susp method (described in WO 2006 / 079889 A1). In this invention, plant sterols (e.g., β-sitosterol) are used as the main component of the vesicle membrane, replacing animal-derived sterols (e.g., cholesterol). This substitution, whether in whole or in part, is considered a clear improvement over the prior art from a sustainability perspective, since animal-derived products are well-known to have a significantly greater impact on environmental degradation, biodiversity loss, and climate change than plant-derived products. Furthermore, this new vesicle system is compatible with increasingly common practices, such as vegetarianism and other animal-free consumption, and therefore potentially reaches a larger audience in need, thereby broadening its scope of application.

[0015] Furthermore, the Vs of the present invention are also characterized by the use of glucose-derived surfactants, particularly alkyl polyglucosides (APGs), in addition to plant sterols. Glucose-derived surfactants are a new generation of environmentally friendly, biocompatible, and biodegradable surfactants that are commonly used in skin care and hair care cleansing products because they exhibit dermatological and ophthalmic safety, good wetting properties, and excellent foaming and cleaning capabilities. The use of glucose-derived surfactants for nanocarriers and microcarriers has been previously reported, but it has been little studied and developed. We believe that this is the first time that a vesicular system containing both a plant sterol, such as β-sitosterol (Sit), and a glucose-derived surfactant as an alternative to other surfactants is provided.

[0016] Surprisingly, the present inventors discovered that Vs containing plant sterols and glucose-derived surfactants (e.g., APG) require different conditions for formation than those previously disclosed. As described in the prior art, Vs containing animal-derived sterols (i.e., cholesterol) can be synthesized using APG as the surfactant with a chain length of 8, 10, or 12 carbons (Muzzalupo et al., 2013). Nevertheless, as shown in Example 1.2.1, only APGs with chain lengths of 12 or more carbons can be used to form Vs containing plant sterols (e.g., Sit). Thus, the present invention demonstrates that the type of sterol used determines which surfactant chain length leads to successful Vs formation, regardless of structural similarity. This effect of surfactant alkyl chain length on Vs formation is demonstrated herein, regardless of the synthetic method used (thin film hydration (TFH) or DELOS-susp). Overall, the inventors were able to determine and successfully implement the unexpected conditions required to synthesize this new vesicle system.

[0017] Furthermore, the present invention not only discloses the successful synthesis of such Vs, but also demonstrates their high-quality performance in terms of encapsulating numerous active ingredients, representing various types of drugs, including pharmaceutical and cosmetic molecules. As known to those skilled in the art of delivery systems, efficient vesicle formation and subsequent adequate encapsulation of active ingredients is a very challenging process that generally results in unsatisfactory results. These include, for example, the absence of Vs formation, insufficient Vs formation, including the formation of other undesirable structures, or the formation of vesicles that fail to efficiently encapsulate the desired active ingredient(s) or that exhibit poor colloidal stability. Therefore, the extraordinary versatility of the Vs of the present invention to encapsulate a variety of active ingredients would not have been anticipated by those skilled in the art.

[0018] The examples herein provide detailed experimental data demonstrating the formation of small, homogeneous Vs in aqueous media, consisting primarily of a combination of plant sterols and glucose-derived surfactants. The system provided herein is versatile and capable of forming neutral, positively, and negatively charged Vs. Furthermore, this platform is shown herein to be capable of incorporating numerous active compounds with diverse properties, including small lipophilic and hydrophilic active ingredients, proteins, and pigments. It therefore demonstrates clear potential as a sustainable delivery platform, particularly for healthcare and cosmetic applications.

[0019] Examples 1.2.1 and 1.2.2 demonstrate the successful formation of Vs containing plant sterol and glucose-derived surfactants (i.e., APGs) that arose only from the unexpected conditions of using surfactants with chain lengths of 12 carbons or greater. Thus, the surprising effect of surfactant alkyl chain length on the formation of Vs is demonstrated herein, regardless of whether the TFH or DELOS-susp method was used.

[0020] Example 1.2.3 demonstrates the synthesis of neutral, positively, and negatively charged Vs by self-assembly of plant sterols and APG (e.g., lauryl glucoside, LGL), among other components, using the DELOS-susp method. As disclosed in Example 1.2.4, both cosmetic-grade APG and highly purified APG (e.g., 98% LGL purity) were useful for the formation of neutral Vs. In terms of stability, longer than 12 months were achieved with Vs containing less purified LGL, and Vs containing highly purified LGL were also efficiently stabilized (see Section 1.2.5). These results demonstrate the broad range of potential applications of the above system in different fields, such as pharmaceuticals and cosmetics.

[0021] Example 1.2.6 also demonstrates the high versatility of the system provided herein through the synthesis of both negatively and positively charged Vs. Successful formation of Vs was achieved using three anionic additives: lauryl glucoside carboxylate (LGC), sodium laurate (SL), sodium lauroyl sarcosinate (SLS), and DC-cholesterol as the cationic additive. All forms of Vs exhibited remarkably high stability, ranging from more than six months to more than one year. These results support not only the versatility of this platform, but also its consistency.

[0022] Example 2 demonstrates that the vesicles of the present invention have excellent encapsulation capabilities for a variety of active ingredients. Negatively charged vesicles were able to efficiently encapsulate lipophilic small molecules (e.g., 7-dehydrocholesterol, tocopherol, cannabidiol) and small proteins with efficiencies exceeding 99%. Meanwhile, positively charged vesicles were able to encapsulate both lipophilic and hydrophilic active ingredients with unexpected efficiencies exceeding 88-99%. These results also demonstrate the versatility of the vesicle system of the present invention, demonstrating its excellent ability to encapsulate various types of active ingredients and, consequently, its wide range of potential applications. It is noteworthy that prior art documents disclosing the synthesis of similar vesicle systems do not disclose their ability to efficiently encapsulate such a wide variety of active ingredients.

[0023] Example 3 shows the specific activity assay of two different active molecules that were tested after being encapsulated in the newly developed Vs. The activity of the free and encapsulated active ingredients is compared to demonstrate that the molecules maintain their properties when loaded into the Vs.

[0024] In particular, Example 3.2.1 shows that the antioxidant capacity of tocopherol when dissolved in ethanol is similar to that exhibited when this active ingredient is encapsulated in negatively charged Vs consisting of Sit, LGL 98% purity, and LGC.

[0025] Example 3.2.2 shows that 7-dehydrocholesterol can be converted to cholecalciferol (vitamin D) upon irradiation after encapsulation in Vs. Cholecalciferol is a vitamin that plays a role in biochemical pathways in various cell types. This example shows that the ability of 7-dehydrocholesterol to be converted to the vitamin D form after encapsulation in Vs is maintained, and the conversion rate is the same as that observed when a suspension of the free active ingredient is irradiated.

[0026] Overall, the present inventors have developed a new sustainable system of vesicles containing plant sterols and glucose-derived surfactants, which exhibit highly desirable results in terms of composition and physicochemical properties, and can encapsulate various types of target active ingredients, such as proteins or hydrophilic / lipophilic small molecules. Therefore, the present invention is considered to be a good candidate system for vesicles to be applied in related fields, such as the pharmaceutical and cosmetic fields.

[0027] Accordingly, a first aspect of the present invention relates to vesicles comprising at least one plant sterol and at least one glucose-derived surfactant.

[0028] A vesicle or vesicular system comprises a sealed aqueous liquid compartment separated from its surroundings by one or more lipid bilayers.

[0029] A second aspect of the present invention relates to a composition comprising a plurality of vesicles as defined herein.

[0030] In another aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of Vs as defined herein and at least one pharmaceutically acceptable excipient, vehicle or carrier.

[0031] In another aspect, the present invention relates to a cosmetic composition comprising a cosmetically effective amount of a vesicle as defined herein and at least one cosmetically acceptable excipient, vehicle or carrier.

[0032] In another aspect, the present invention relates to a nutritional composition (e.g., a food supplement) comprising a nutritionally effective amount of a vesicle as defined herein and at least one nutritionally acceptable excipient, vehicle, or carrier.

[0033] In another aspect, the present invention relates to a food composition / food additive comprising an effective amount of a vesicle as defined herein and at least an acceptable excipient, vehicle or carrier.

[0034] In another aspect, the present invention relates to a detection or diagnostic composition comprising an effective amount of a vesicle as defined herein and at least an acceptable excipient, vehicle or carrier.

[0035] Another aspect of the invention relates to a vesicle as defined herein or a composition (particularly a pharmaceutical composition) comprising a vesicle, for use as a medicament.

[0036] Another aspect of the invention relates to the use of a vesicle or a composition comprising a vesicle as defined herein in the manufacture of a suspension, as a delivery system, or as a diagnostic or detection tool.

[0037] Finally, another aspect of the present invention relates to a method for the production of the vesicles described herein using the DELOS-susp method.

[0038] Throughout the specification and claims, the word "comprise" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the present invention. Furthermore, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein. The following examples and figures are provided herein for illustrative purposes and are not intended to limit the present invention. [Brief explanation of the drawings]

[0039] [Figure 1] Figure 1 shows a scheme of the vesicle preparation procedure by the DELOS-susp technique, including: (A) addition of organic solution; (B) expansion: addition of compressed CO2 and formation of a CO2-expanded solution; and (C) decompression into an aqueous solution. "OP" corresponds to "organic phase," "AP" corresponds to "aqueous phase," "ML" corresponds to "membrane lipid," "MS" corresponds to "membrane surfactant," "HOA" corresponds to "hydrophobic active ingredient," and "HIA" corresponds to "hydrophilic active ingredient." [Figure 2] The molecular structures of the sterols and surfactants used in the synthesis of the vesicle platform are shown. "MBB" corresponds to "main component," "CMC" corresponds to "charged membrane component," and "A '' corresponds to "additive." [Figure 3] (A) Macroscopic appearance, (B) micrographs taken with a Morphology G3 optical microscope at 50x magnification, (C) cryo-TEM images of the entire grid (top image, 50x magnification) and the Vs focused on the grid (bottom image, 3000x magnification), and (D) DLS comparison of the samples. "S" corresponds to "size" and "I" corresponds to "intensity." [Figure 4] Depiction of the composition of Vs obtained by the DELOS technique is shown: (A) Sit:LGL; (B) Sit:LGL:Chol-PEG600; (C) Sit:LGL:anionic additive (1:1:0.05-0.1); (D) Sit:LGL:anionic additive (1:0-0.8:0.2-1); (E) Sit:LGL:cationic additive (1:1:0.05); (F) Sit:LGL:cationic additive (0-0.8:1:0.2-1). "NEG" corresponds to "negatively charged molecule" and "POS" corresponds to "positively charged molecule." [Figure 5] Representative cryo-TEM images of neutral Sit:LGL Vs prepared with different qualities of LGL surfactant are shown: (A) Sit:LGL-mC12-16 (1:0.75); (B) Sit:LGL-mC12-16 (1:1); (C) Sit:LGL (LGL-p) (1:1). (D) Comparison of the three samples by DLS. "S" corresponds to "size" and "I" corresponds to "intensity." [Figure 6] (A) Representative cryo-TEM image of Sit:LGL-p:Chol-PEG600 Vs; (B) DLS depiction of particle size. "S" corresponds to "size" and "I" corresponds to "intensity." In this figure, LGL is LGL-p. [Figure 7] Representative cryo-TEM images of Sit:LGL Vs stabilized with ionic additives are shown, including (A) Sit:LGL:LGC (1:1:0.05); (B) Sit:LGL:SL (1:1:0.05); (C) Sit:LGL:SLS (1:1:0.05); and (D) Sit:LGL:DC-Chol (1:1:0.05). (E) shows a comparison of the samples by DLS. "S" corresponds to "size" and "I" corresponds to "intensity." In this figure, LGL is LGL-p. [Figure 8] (A) Representative cryo-TEM image of Sit:LGL:DC-Chol (0:1:1), and (B) physicochemical characterization of the positively charged Sit:LGL:DC-Chol Vs in terms of medium size (S), ζ-potential (ζ-Pot), and polydispersity index (PdI). In this figure, LGL is LGL-p. [Figure 9] (A) Representative cryo-TEM images of Sit:LGL:LGC (1:0.8:0.2 and 1:0:1), and (B) physicochemical characterization of positively charged Sit:LGL:LGC Vs in terms of mesomorphism (S), ζ-potential (ζ-Pot), and polydispersity index (PdI). In this figure, LGL is LGL-p. [Figure 10] Figure 2 shows a comparison of formulations loaded with the active ingredients Sit:LGC in terms of particle size (S), zeta potential (zeta-Pot), polydispersity index (PdI) and encapsulation efficiency (EE). 7-DHC, CBD, Dil / DiD, Dil, DiD, NCA, AG, b-FGF are mentioned in Example 2.1. [Figure 11]Figure 1 shows a comparison of Sit:LGL:LGC active ingredient-loaded formulations in terms of particle size (S), zeta potential (zeta-Pot), polydispersity index (PdI), and encapsulation efficiency (EE). 7-DHC, CBD, and TCP are referred to in Example 2.1. In this figure, LGL is LGL-p. [Figure 12] Representative cryo-TEM images of Sit:LGC Vs loaded with (A) 7-DHC, (B) CBD, (C) DiI / DiD, (D) NCA, and (E) AG are shown. 7-DHC, CBD, DiI / DiD, NCA, and AG are referred to in Example 2.1. [Figure 13] Comparison of Sit:LGL:DC-Chol active ingredient-loaded formulations in terms of particle size (S), zeta potential (ζ-Pot), polydispersity index (PdI), and encapsulation efficiency (EE). 7-DHC, CBD, TCP, MTX, HGH. In this figure, LGL is LGL-p. [Figure 14] Representative cryo-TEM images of Sit:LGC Vs loaded with (A) 7-DHC, (B) CBD, and (C) TCP are shown. In this figure, LGL is LGL-p. 7-DHC, CBD, and TCP are referred to in Example 2.1. [Figure 15] Figure 1 shows the conversion of 7-dehydrocholesterol (7DHC) to vitamin D upon irradiation of 7DHC-loaded vesicles compared to free 7DHC suspension, as quantified by HPLC. "7DHC" corresponds to "7-dehydrocholesterol," "C" corresponds to "concentration," "VD" corresponds to "vitamin D cholecalciferol," "F" corresponds to "free," "V" corresponds to "loaded vesicles," "BI" corresponds to "before irradiation," "30' I" corresponds to "30 min irradiation," and "1 h I" corresponds to "1 h irradiation." [Figure 16] (A): The structural differences between vesicles and emulsions (O / W and W / O); (B) Cryo-TEM images showing the differences between emulsions and vesicles due to their structural differences. "V" corresponds to "vesicles," "E" corresponds to "emulsion," "O / W" corresponds to "oil-in-water," "W / O" corresponds to "water-in-oil," "A" corresponds to "aqueous," and "O" corresponds to "oil-based." DETAILED DESCRIPTION OF THE INVENTION

[0040] definition Particle: The term "particle" refers to a material having at least one nano- or micro-dimension, such as inorganic particles, polymer particles, tubes, gels, or solid lipid particles, liposomes, or other types of vesicles. Particles can be called carriers when they encapsulate molecules as cargoes that can be delivered to specific tissues or protected from external agents. The terms "entity" and "capsule" can be used interchangeably herein with "particle."

[0041] Vesicle or Vesicular System: The terms "vesicle" and "vesicular system" are used interchangeably herein to refer to nano- or micro-particulate colloidal carriers that form spontaneously when certain lipids are hydrated in aqueous media, typically 0.02-5.0 μm in diameter. A vesicle or vesicular system contains an enclosed aqueous liquid compartment separated from its surroundings by one or more lipid bilayers.

[0042] Most self-assembling molecules are amphiphilic, i.e., they contain both hydrophilic and hydrophobic domains in their structure. Phospholipids, surfactants, and block copolymers, which are commonly used as self-assembling monomers for vesicle fabrication, typically consist of long hydrophobic tails and polar hydrophilic head groups. Under aqueous conditions, this dual hydrophobic and hydrophilic nature promotes their association through weak noncovalent interactions to form ordered aggregates with various morphologies and sizes ranging from nanometers to microns.

[0043] Vesicles are a type of lipid nanoparticle, but other types, such as emulsions, exist. However, they have different structures and properties. As mentioned above, vesicles have an enclosed aqueous liquid compartment separated from its surroundings by a lipid bilayer. In this sense, vesicles are surrounded by an aqueous environment from both the outside and the inside. In contrast, emulsions are dispersions of oil and water phases stabilized by surfactants (Figure 16(A)). Emulsions have a stabilized core phase surrounded by the other phase (Plaza-Oliver et al., 2021). These structural differences can also be seen using microscopy techniques such as cryo-TEM (Figure 16(B)). In cryo-TEM images, "oil-in-water" emulsions appear as solid black dots; they are oil droplets stabilized by an aqueous phase. On the other hand, the lipid bilayer of vesicles appears as a thin, black circular line in cryo-EM images, and the aqueous lumen within the vesicles is the same color as the external environment because both phases are aqueous.

[0044] There are other types of vesicular systems used for drug delivery, including liposomes (composed primarily of phospholipids), vesicles containing nonionic surfactants (niosomes), vesicles containing cationic surfactants (cationic vesicles), or vesicles containing both cationic and anionic surfactants (catanionic vesicles).

[0045] Liposome: The term "liposome" refers to a self-assembling structure containing one or more membranes comprising lipid bilayers, each membrane containing two monolayers of oppositely oriented amphiphilic lipid molecules. Liposomes can have a single bilayer membrane (small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs)) or multiple bilayer membranes (large multilamellar vesicles (MLVs)).

[0046] Phytosterols or Plant Sterols: As used herein, the terms "phytosterols," "plant sterols," or "plant-derived sterols" are used interchangeably and refer to a group of naturally occurring molecules found in plants that can be classified as sterols and stanols depending on the presence or absence of a double bond in the sterol ring. Naturally occurring sterols are biosynthetically derived from squalene and are structurally similar to cholesterol, a sterol exclusive to animals. The core structure of sterols consists of four fused rings: three six-membered cyclohexane rings and one five-membered cyclopentane ring, with a hydroxyl group at the 3-position of the A ring.

[0047] Glucose-derived surfactants: The term "glucose-derived surfactants" refers to surfactants derived from glucose molecules, including alkyl polyglucosides and their derivatives (e.g., methyl glucoside esters), and fatty acid glucamides. Glucose-derived surfactants are considered to have exceptional product safety in terms of ecological, toxicological, and dermatological properties. Glucose-derived surfactants are used in detergents and dishwashing detergents, among other applications.

[0048] Stabilizer: The term "stabilizing agent" or "stabilizer" refers to an ingredient or compound that is added to a vesicle system to increase its colloidal stability.

[0049] Therapeutic Agent: The term "therapeutic agent" is used herein to refer to any drug or substance that can produce an effect in the body or area to which it is administered. As described herein, the term "therapeutic agent" can be used to refer, for example, to a "pharmaceutical agent" or a "cosmetic agent."

[0050] vesicular system One aspect of the present invention relates to vesicles comprising at least one plant sterol and at least one glucose-derived surfactant.

[0051] In some embodiments, the plant sterol comprises at least one plant sterol selected from the group consisting of β-sitosterol (Sit), β-sitostanol, campesterol, campestanol, stigmasterol, brassicasterol, stigasterol, ergosterol, Δ5-avenasterol, and combinations thereof. In some embodiments, the plant sterol is a combination of plant sterols. In some embodiments, the plant sterol comprises Sit. In particular embodiments, the plant sterol comprises a combination of Sit and at least one other plant sterol. In more particular embodiments, the combination comprises greater than 70% Sit. In other embodiments, the plant sterol comprises 70% to 95% Sit. In particular embodiments, the plant sterol comprises 70%, 75%, 80%, 85%, 90%, or 95% Sit.

[0052] In some embodiments, the at least one glucose-derived surfactant is selected from the group consisting of alkyl polyglucoside surfactants (APGs), fatty acid glucamides, and methyl glucoside esters. More particularly, the glucose-derived surfactant is an APG or a derivative thereof described herein. In more specific embodiments, the APG or a derivative thereof has a carbon chain length of at least 12 carbons. In another embodiment, the APG or a derivative thereof has a carbon chain length of 12, 14, 16 carbons, or a combination thereof. In another embodiment, the at least one glucose-derived surfactant is an APG. In particular, the alkyl polyglucoside surfactant is lauryl glucoside (LGL).

[0053] In some embodiments, the APG is an alkyl polyglucoside derivative, particularly an alkyl polyglucoside derivative selected from the group consisting of carboxylates, carbonates, butyl ethers, ethoxylates, isethionates, ethers, sulfates, epoxide adducts, phosphates, sulfosuccinates, esters, and glycerol ethers, and inorganic salts.

[0054] In another embodiment, the alkyl polyglucoside surfactant or derivative thereof is selected from the group consisting of octyl glucoside (OGL), decyl glucoside (DGL), LGL, tetradecyl glucoside, hexadecyl glucoside, tetradecyl D-glucoside (14C), hexadecyl β-D-glucopyranoside (16C), octadecyl D-glucoside (18C), arachidyl glucoside (20C), and mixtures of C12-20 alkyl glucosides. , cetearyl glucoside (a mixture of C18-20 alkyl glucosides), C20-22 alkyl glucosides, coco-glucoside (an alkyl chain residue of a fatty alcohol derived from coconut acid), isostearyl glucoside (branched), octyldodecyl glucoside (branched), phosphorus derivatives of alkyl polyglucosides, and lauryl glucoside carboxylate (LGC).

[0055] In some embodiments, the APG is selected from the group consisting of LGL, tetradecyl glucoside, hexadecyl glucoside, or a combination thereof. In certain embodiments, the APG further comprises OGL and / or DGL. In certain embodiments, the APG is LGL. In other embodiments, the APG is LGL in combination with one or more other APGs. In certain embodiments, the APG comprises a combination of LGL, tetradecyl glucoside, and hexadecyl glucoside. In some embodiments, the APG comprises a combination of OGL, DGL, LGL, tetradecyl glucoside, and / or hexadecyl glucoside.

[0056] In some embodiments, the glucose-derived surfactant comprises fatty acid glucamide.Particularly, the glucamide is selected from the group consisting of lauroyl methyl glucamide, myristoyl methyl glucamide, cocoyl methyl glucamide, sunflower oil methyl glucamide, glucamine oxide and betaine, anionic glucamide, bifunctional glucamide and combinations thereof.In some embodiments, the glucose-derived surfactant comprises fatty acid glucamide combined with at least one other glucose-derived surfactant.

[0057] In some embodiments, the vesicles further comprise at least one additive. In certain embodiments, the additive is a surfactant. More specifically, the vesicles further comprise at least one surfactant selected from the group consisting of sodium lauroyl sarcosinate (SLS), sodium laurate (SL), sodium dodecyl sulfate (SDS), sodium lauroyl glycinate, N3-lauroyl lysine methyl ester hydrochloride, and combinations thereof. In some embodiments, the vesicles comprise a glucose-derived surfactant and further comprise at least one compound selected from the group consisting of LGC, SLS, SL, SDS, sodium lauroyl glycinate, N3-lauroyl lysine methyl ester hydrochloride, and combinations thereof. In certain embodiments, the glucose-derived surfactant comprises at least one APG. More specifically, the APG is LGL, and the vesicles further comprise at least one compound selected from the group consisting of LGC, SLS, SL, SDS, sodium lauroyl glycinate, N3-lauroyl lysine methyl ester hydrochloride, and combinations thereof.

[0058] As shown in the examples of the present invention, glucose-derived surfactants (e.g., APG or LGL) can be substituted, in whole or in part, with other compounds, such as other APGs (e.g., LGC) or other compounds considered as additives (e.g., SLS or SL). Thus, in some embodiments, glucose-derived surfactants (e.g., APG) are substituted, in whole or in part, with other compounds. In some embodiments, glucose-derived surfactants (e.g., APG) are partially substituted with a compound selected from the group consisting of LGC, SLS, SL, SDS, sodium lauroyl glycinate, N3-lauroyl lysine methyl ester hydrochloride, and combinations thereof. In certain embodiments, the glucose-derived surfactant is APG and is partially substituted with other compounds. In more specific embodiments, the APG is LGL and is partially substituted with at least one compound selected from the group consisting of LGC, SL, and SLS.

[0059] In another embodiment, the glucose-derived surfactant (e.g., APG) is completely replaced by another compound. In some embodiments, the glucose-derived surfactant (e.g., APG) is completely replaced by at least one compound selected from the group consisting of LGC, SLS, SL, SDS, sodium lauroyl glycinate, N3-lauroyl lysine methyl ester hydrochloride, and combinations thereof. In a specific embodiment, the glucose-derived surfactant (e.g., APG) is completely replaced by LGC. In a more specific embodiment, the glucose-derived surfactant is APG, and the APG is completely replaced by LGC. More particularly, the APG is LGL, and the APG is completely replaced by LGC.

[0060] Alternatively, the present invention relates to vesicles comprising a plant sterol and at least one glucose-derived surfactant, wherein the at least one glucose-derived surfactant is an APG selected from LGL and / or LGC. In some embodiments, the APG is a combination of LGL and LGC. In some embodiments, the APG is LGL. In other embodiments, the APG is LGC. In some embodiments, the vesicles further comprise an additive selected from SL and / or SLS.

[0061] In another embodiment, the vesicles comprise at least one plant sterol and at least one glucose-derived surfactant, wherein the surfactant has a purity selected from at least 80%, 90%, 95%, 96%, 97%, 98%, and 99%. More particularly, the surfactant has a purity of at least 98%. In another embodiment, the vesicles comprise at least one plant sterol and an APG. In some embodiments, the APG is an LGL. In particular, the LGL has a purity selected from at least 80%, 90%, 95%, 96%, 97%, 98%, and 99%. More particularly, the LGL has a purity of at least 98%. In a specific embodiment, the at least one plant sterol comprises Sit.

[0062] In another embodiment, the vesicles comprise at least one plant sterol and at least one glucose-derived surfactant, wherein the molar ratio of plant sterol to glucose-derived surfactant is 0.05-1:0.2-2. In particular embodiments, the molar ratio of plant sterol to glucose-derived surfactant is 0.2-1:0.2-2. In particular, the molar ratio of plant sterol to glucose-derived surfactant is 1:0.75-2. More particularly, the molar ratio of plant sterol to glucose-derived surfactant is 1:1. In another embodiment, the glucose-derived surfactant is APG, wherein the molar ratio of plant sterol to APG is 0.05-1:0.2-2, particularly 0.2-1:0.2-2. In particular embodiments, the molar ratio of plant sterol to APG is 1:0.75-2, more particularly 1:1. In some embodiments, the APG is LGL. In other embodiments, the plant sterol includes Sit.

[0063] In some embodiments, the vesicles are for use in at least one composition selected from the group consisting of pharmaceutical compositions, cosmetic compositions, nutraceutical compositions (e.g., food supplements), food compositions, food additives, and detection and diagnostic compositions.

[0064] Stabilizer: Neutral additive Vesicles (Vs) are modified to increase their colloidal stability by the addition of molecules that can enhance such stability without compromising other desirable properties of the Vs. Molecules used to increase such stability are considered stabilizers.

[0065] In some embodiments, the vesicles further comprise a stabilizing agent, hi certain embodiments, the stabilizing agent is selected from the group consisting of a neutral additive, a cationic additive, and an anionic additive.

[0066] In certain embodiments, the neutral additive is selected from the group consisting of polyethylene glycol (PEG) moieties, PEGylated fatty acids, PEGylated phospholipids, polysaccharides, zwitterionic polypeptides, polyamino acid-based polymers, poly(2-oxazoline)-based polymers, polyvinylpyrrolidine, other PEGylated membrane components, and non-ionic surfactants. In certain embodiments, the polysaccharide is chitosan. In certain embodiments, the polyamino acid-based polymer is selected from the group consisting of polyglutamic acid, poly(hydroxyethyl-L-asparagine), and poly(hydroxylethyl-L-glutamine). In certain embodiments, the PEGylated membrane component is a PEGylated aliphatic amine. In certain embodiments, the non-ionic surfactant is Tween 80.

[0067] In certain embodiments, the neutral additive is PEGylated fatty acid. Particularly, the PEGylated fatty acid is selected from the group consisting of PEGylated cholesterol, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000). In more particular embodiments, the PEGylated fatty acid is PEGylated cholesterol.

[0068] Pegylation is a process by which molecules or macrostructures are modified by conjugation with PEG molecules or their derivatives, which are non-toxic and non-immunogenic polymers. PEG and its derivatives that can be used for such modifications are considered PEGylation agents. In other embodiments, the PEGylation agent is selected from the group consisting of PEG, PEG monostearate, PEG stearate, and PEG-4 stearate. In certain embodiments, the PEGylation agent is PEG.

[0069] PEGylated cholesterol is a conjugate comprising a cholesterol moiety (Chol) and a PEG moiety. In certain embodiments, the cholesterol moiety and the PEG moiety are covalently attached. In particular, the conjugate has the structure Chol-PEG n-X, where n is the number of PEG monomers in the PEG moiety. More particularly, n is selected from 50 to 2000. In some embodiments, n is selected from 200 to 1000. In particular, n is selected from the group consisting of 200, 300, 400, 500, 600, 700, 800, 900, and 1000. More particularly, n is 600. In another embodiment, X is selected from the group consisting of -SH, -OH, -CHO, -OCH, -NH, -NH, -CH, -N, -COOH, -maleimide, a peptide, an antibody, and a sugar.

[0070] In some embodiments, the vesicles comprise at least one plant sterol and at least one glucose-derived surfactant, and further comprise a neutral additive. In particular, the neutral additive is a PEGylated fatty acid. In some embodiments, the ratio of neutral additive to plant sterol is 1 to 20%. In other embodiments, the ratio of neutral additive to plant sterol is selected from the group consisting of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. In particular embodiments, the ratio of neutral additive to plant sterol is 5 to 15%. In more particular embodiments, the ratio of neutral additive to plant sterol is 6 to 10%. In even more particular embodiments, the ratio of neutral additive to plant sterol is 7%. In another embodiment, the ratio of plant sterol:glucose-derived surfactant:neutral additive is 0.90-0.99:1:0.01-0.1. In certain embodiments, the ratio of plant sterol:glucose-derived surfactant:neutral additive is selected from the group consisting of 0.90:1:0.1, 0.91:1:0.09, 0.92:1:0.08, 0.93:1:0.07, and 0.94:1:0.06. In certain embodiments, the neutral additive is a PEGylated fatty acid, particularly PEGylated cholesterol. In another embodiment, the plant sterol comprises Sit. In another embodiment, the glucose-derived surfactant is APG.

[0071] Stabilizer: Charged additive Vesicles can be modified to enhance their stability by adding charged molecules, i.e., charged additives, which can enhance the stability of Vs without impairing other desirable properties of Vs. Thus, charged additives are a type of stabilizer used in the present invention. The addition of such charged molecules is used to change the charge of Vs.

[0072] Vesicles containing a positively charged additive, i.e., a cationic additive, are ultimately positively charged. Conversely, Vs containing a negatively charged additive, i.e., an anionic additive, are ultimately negatively charged. Adjusting the charge of Vs by adding a charged additive results in higher encapsulation efficiency of certain molecules, which have a higher encapsulation capacity in charged Vs, regardless of whether they are positive or negative, than electrically neutral vesicles. In some embodiments, Vs that further contain an anionic additive and are thus negatively charged are useful for encapsulating positively charged molecules. Similarly, Vs that further contain a cationic additive and are thus positively charged are useful for encapsulating negatively charged molecules.

[0073] Thus, in some embodiments, the vesicles further comprise a stabilizer, particularly a charged additive. In certain embodiments, the charged additive is selected from an anionic additive and a cationic additive. In other embodiments, the additive is present in the membrane of the vesicle. Alternatively, the additive is located inside the vesicle. In some embodiments, the additive is bound to the membrane of the vesicle. In other embodiments, the additive interacts with the vesicle via surface charges.

[0074] In certain embodiments, the additive is an anionic additive (i.e., negatively charged). In some embodiments, the vesicles further comprise an anionic additive, and the vesicles are used in a cosmetic composition. In another embodiment, the anionic additive is present in the membrane of the vesicle. Alternatively, the anionic additive is disposed inside the vesicle. In certain embodiments, the anionic additive is selected from the group consisting of LGC, SLS, SL, SDS, disodium citrate glucoside, sodium tartrate glucoside, disodium sulfosuccinate glucoside, amino acid-derived surfactants, anionic phospholipids, anionic lipids, and combinations thereof. In certain embodiments, the anionic additive is selected from the group consisting of LGC, SLS, SL, and combinations thereof. In another specific embodiment, the anionic phospholipid is 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). In another specific embodiment, the anionic lipid is selected from cholesterol sulfate, cholesterol phosphate, and cholesteryl acetate.

[0075] In another embodiment, the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:1:0.05-0.1. In certain embodiments, the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:1:0.05, 1:1:0.08, or 1:1:0.1. In certain embodiments, the anionic additive is selected from the group consisting of LGC, SLS, SL, and combinations thereof, and the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:1:0.05-0.1. In certain embodiments, the anionic additive is LGC, and the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:1:0.05. In another embodiment, the anionic additive is SLS, and the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:1:0.08. In another embodiment, the anionic additive is SL, and the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:1:0.1. In some embodiments, the plant sterol comprises Sit. In some embodiments, the glucose-derived surfactant is APG, particularly LGL.

[0076] In another embodiment, the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:0-0.8:0.2-1. In particular embodiments, the ratio of plant sterol:glucose-derived surfactant:anionic additive is selected from the group consisting of 1:0:1, 1:0.2:0.8, 1:0.5:0.5, and 1:0.8:0.2. In more particular embodiments, the anionic additive is LGC, and the ratio of plant sterol:glucose-derived surfactant:anionic additive is 1:0-0.8:0.2-1. In some embodiments, the plant sterol comprises Sit. In some embodiments, the glucose-derived surfactant is APG, particularly LGL.

[0077] In some embodiments, the additive is a positively charged cationic additive. In certain embodiments, the vesicle further comprises a cationic additive, and the vesicle is used in a pharmaceutical composition. In another embodiment, the cationic additive is disposed inside the vesicle. Alternatively, the anionic additive is present in the membrane of the vesicle. In certain embodiments, the cationic additive is selected from the group consisting of cationic lipids, cationic phospholipids, surfactants containing quaternary amines, and amino acid-derived surfactants. In certain embodiments, the cationic lipid is DC-cholesterol (3α-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol hydrochloride, DC-Chol). In another specific embodiment, the surfactant containing a quaternary amine is selected from the group consisting of stearalkonium chloride, dicetyldimonium chloride, behentrimonium chloride, and cetrimide.

[0078] In particular embodiments, the ratio of plant sterol:glucose-derived surfactant:cationic additive is 0-1:1:0.05-1. In more particular embodiments, the ratio of plant sterol:glucose-derived surfactant:cationic additive is 0-0.8:1:0.2-1. In another particular embodiment, the ratio of plant sterol:glucose-derived surfactant:cationic additive is selected from the group consisting of 1:1:0.5, 0.8:1:2, 0.5:1:0.5, 0.2:1:0.8, and 0:1:1. In particular, the cationic additive is DC-Chol. In some embodiments, the plant sterol includes Sit. In some embodiments, the glucose-derived surfactant is APG, in particular LGL.

[0079] Shape and Size In some embodiments, the vesicles have a round or similar shape, or in some embodiments, the vesicles have an oval or similar shape.

[0080] In some embodiments, the vesicles have an average diameter of at least 20 nm. In another embodiment, the vesicles have an average diameter of at least 50 nm. In another embodiment, the vesicles have an average diameter between 20 nm and 5 μm. In certain embodiments, the vesicles have an average diameter between 50 and 800 nm. In certain embodiments, the average diameter is selected from the group consisting of 50-200 nm, 100-300 nm, and 200-800 nm. In another embodiment, the vesicles are positively charged and have an average diameter of 50-200 nm. In another embodiment, the vesicles are negatively charged and have an average diameter of 100-300 nm. In another embodiment, the vesicles are neutral and have an average diameter of 200-800 nm.

[0081] Drug encapsulation In some embodiments, the vesicles can encapsulate a drug, also referred to herein as an active ingredient. In one embodiment, the vesicles can encapsulate at least one drug selected from the group consisting of pharmaceuticals, cosmetics, food additives, vitamins, diagnostic agents, and detection agents. In some embodiments, the vesicles can encapsulate at least one drug selected from the group consisting of small chemical molecules, biomolecules, and complexes thereof. In certain embodiments, the biomolecules are selected from the group consisting of peptides, hormones, metabolites, antibodies, proteins, enzymes, oligonucleotides, nucleic acids, carbohydrates, and lipids. In certain embodiments, the drug is encapsulated inside the vesicle. In other embodiments, the drug is encapsulated in the membrane of the vesicle. In certain embodiments, the drug is bound to the membrane of the vesicle. In other embodiments, the drug is In another embodiment, the vesicle further comprises a drug, particularly a drug selected from the group consisting of pharmaceuticals, cosmetic agents, food additives, vitamins, diagnostic agents, and detection agents.

[0082] In some embodiments, the vesicle further comprises a pharmaceutical agent. In certain embodiments, the pharmaceutical agent is lipophilic. In other embodiments, the pharmaceutical agent is hydrophilic. In other particular embodiments, the pharmaceutical agent is hydrophilic. In other embodiments, the pharmaceutical agent is selected from the group consisting of small molecules, peptides, hormones, metabolites, antibodies, proteins, enzymes, oligonucleotides, nucleic acids, carbohydrates, lipids, and complexes thereof.

[0083] In some embodiments, the vesicles further comprise a cosmetic agent. In certain embodiments, the cosmetic agent is lipophilic. In other embodiments, the cosmetic agent is hydrophilic. In other embodiments, the cosmetic agent is selected from the group consisting of small molecules, peptides, hormones, metabolites, antibodies, proteins, enzymes, oligonucleotides, nucleic acids, carbohydrates, lipids, and complexes thereof. In certain embodiments, the cosmetic agent is a small molecule. In other embodiments, the cosmetic agent is a peptide.

[0084] In some embodiments, the vesicles further comprise a food additive. In certain embodiments, the food additive is selected from the group consisting of small chemical molecules, biomolecules, and complexes thereof. In certain embodiments, the biomolecule is selected from the group consisting of peptides, hormones, metabolites, antibodies, proteins, enzymes, oligonucleotides, nucleic acids, carbohydrates, and lipids. In another embodiment, the food additive is selected from vitamins and minerals.

[0085] In some embodiments, the vesicles further comprise a diagnostic or detection agent. Diagnostic or detection agents may also be referred to as tracking or labeling agents. These agents can be used to label the vesicles (e.g., using a fluorescent dye as the tracking / labeling agent) to track the distribution of Vs and, optionally, the delivery of a drug / active ingredient (e.g., a pharmaceutical or cosmetic agent of interest). In certain embodiments, the diagnostic or detection agent is selected from the group consisting of dyes, reagents, and biomarkers. More particularly, the diagnostic or detection agent is a dye, particularly a fluorescent dye. In some embodiments, the vesicles are capable of detecting a specific molecule. In certain embodiments, the molecule is selected from the group consisting of a peptide, hormone, metabolite, antibody, protein, enzyme, oligonucleotide, nucleic acid, carbohydrate, and lipid.

[0086] In another embodiment, the vesicles comprise a diagnostic or detection agent, and the vesicles are used as a bioimaging tool to track the delivery of the agent. In another embodiment, the vesicles comprise a labeling agent, a targeting ligand, and a therapeutic agent. In particular, the vesicles comprise a labeling dye, a targeting ligand for site-specific labeling, and a therapeutic agent to be delivered. Thus, in some embodiments, the present invention relates to the use of the vesicles described herein as a bioimaging tool.

[0087] In some embodiments, the vesicles can encapsulate at least one lipophilic drug. In some embodiments, the lipophilic drug is a lipophilic small molecule. In certain embodiments, the lipophilic small molecule is selected from the group consisting of 7-dehydrocholesterol, cannabidiol (CBD), α-tocopherol (TCP), and carbocyanine dyes. In some embodiments, the vesicles can encapsulate at least one hydrophilic drug. In some embodiments, the hydrophilic drug is a hydrophilic vitamin. In certain embodiments, the hydrophilic small molecule is selected from the group consisting of niacinamide or vitamin B3 and ascorbyl glucoside (AG). In some embodiments, the vesicles can encapsulate at least one small protein. In certain embodiments, the small protein is basic fibroblast growth factor (bFGF).

[0088] composition In another aspect, the present invention also relates to a composition comprising a plurality of vesicles (Vs) as defined herein, i.e., vesicles (Vs) comprising at least one plant sterol (e.g., β-sitosterol (Sit)) and at least one glucose-derived surfactant. In particular, the Vs comprise Sit and at least one alkyl polyglucoside surfactant (APG).

[0089] In some embodiments, the composition is selected from the group consisting of a pharmaceutical composition, a cosmetic composition, a nutraceutical composition (e.g., a food supplement), a food composition, a food additive, and a detection or diagnostic composition.

[0090] Accordingly, another aspect of the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of Vs, as defined herein, and at least one pharmaceutically acceptable excipient, vehicle, or carrier. In some embodiments, the at least one acceptable excipient, vehicle, or carrier is selected from the group consisting of hydroxypropyl methylcellulose, polymethacrylate-based copolymers, polyethylene glycol, polyvinylpyrrolidone, polyvinyl methyl ether / maleic acid, ethanol, isopropyl alcohol, linolin, alginic acid, starch, hyaluronic acid, cellulose, water, wax, agar, pectin, sucrose, maltose, lactose, trehalose, and inorganic salts.

[0091] In another aspect, the present invention relates to a cosmetic composition comprising a cosmetically effective amount of Vs, as defined herein, and at least one cosmetically acceptable excipient, vehicle, or carrier. In some embodiments, the at least one acceptable excipient, vehicle, or carrier is selected from the group consisting of hydroxypropyl methylcellulose, carbomer, alginic acid, water, myristyl myristate, paraffin, glycerin, mineral oil, polyethylene glycol, butylene glycol, xanthan gum, lanolin, cetyl alcohol, cetearyl alcohol, sodium benzoate, potassium sorbate, phenoxyethanol, benzyl alcohol, trehalose, and sucrose.

[0092] In some embodiments, the cosmetic composition is formulated in the form of a hydrogel. In particular, the composition is formulated in the form of a hydrogel and comprises at least one acceptable excipient, vehicle, or carrier selected from the group consisting of hydroxypropyl methylcellulose, carbomer, and alginic acid. In another embodiment, the cosmetic composition is formulated in the form of an emulsion. In particular, compositions formulated in the form of an emulsion comprise at least one acceptable excipient, vehicle, or carrier selected from the group consisting of water, myristyl myristate, paraffin, glycerin, mineral oil, polyethylene glycol, butylene glycol, xanthan gum, lanolin, cetyl alcohol, and cetearyl alcohol. In another embodiment, the cosmetic composition is formulated in the form of a semi-solid composition. In particular, compositions formulated in the form of a semi-solid composition comprise at least one acceptable excipient, vehicle, or carrier selected from the group consisting of sodium benzoate, potassium sorbate, phenoxyethanol, and benzyl alcohol. In some embodiments, the cosmetic composition is formulated in the form of a liquid composition. In particular, compositions formulated in the form of liquid compositions contain at least one acceptable excipient, vehicle, or carrier selected from the group consisting of sodium benzoate, potassium sorbate, phenoxyethanol, and benzyl alcohol. These four compounds (i.e., sodium benzoate, potassium sorbate, phenoxyethanol, and benzyl alcohol) are used as preservatives for semi-solid and liquid compositions. In some embodiments, the cosmetic composition is formulated in the form of a solid composition. In particular, compositions formulated in the form of a solid composition contain at least one acceptable excipient, vehicle, or carrier selected from trehalose and sucrose.

[0093] In another aspect, the present invention relates to a nutraceutical composition (e.g., a food supplement) comprising a nutraceutically effective amount of Vs, as defined herein, and at least one nutraceutically acceptable excipient, vehicle, or carrier. In some embodiments, the at least one acceptable excipient, vehicle, or carrier is selected from the group consisting of trehalose, sucrose, mono- and diglycerides of fatty acids, starch and corn-based polymers, alginic acid, and gum bases.

[0094] In another aspect, the present invention relates to a food composition / food additive comprising an effective amount of Vs, as defined herein, and at least one acceptable excipient, vehicle, or carrier, in some embodiments, the at least one acceptable excipient, vehicle, or carrier is selected from the group consisting of trehalose, sucrose, mono- and diglycerides of fatty acids, starch and corn-based polymers, alginic acid, and gum bases.

[0095] In another aspect, the present invention relates to a detection or diagnostic composition comprising an effective amount of Vs as defined herein and at least one acceptable excipient, vehicle, or carrier, in some embodiments, the at least one acceptable excipient, vehicle, or carrier is a dye, particularly a fluorescent dye.

[0096] In some embodiments, the composition is administered topically, orally, intravenously, via inhalation, parenterally, via mucosal administration, subcutaneously, or ophthalmically. In particular, the composition is administered topically.

[0097] Application of the vesicles / composition In another aspect, vesicles (Vs) or compositions comprising the Vs described herein are used in the preparation of a suspension. Alternatively, this aspect relates to the use of the Vs or compositions described herein for the preparation of a suspension. In some embodiments, the suspension is for topical, oral, intravenous, inhalation, parenteral, mucosal, subcutaneous, or ocular administration.

[0098] In another aspect, the Vs or compositions described herein are used as a delivery system. Alternatively, this aspect relates to the use of the Vs or compositions described herein as a delivery system. In certain embodiments, the composition is used as a delivery system for at least one active ingredient. In some embodiments, the active ingredient is selected from the group consisting of pharmaceuticals, cosmetic agents, food additives, vitamins, diagnostic agents, and detection agents. More particularly, the active ingredient is a pharmaceutical or cosmetic agent. In some embodiments, it is a pharmaceutical or cosmetic agent.

[0099] Another embodiment relates to a Vs or composition as defined herein for use as a medicament. In another embodiment, the Vs or composition as described herein is for use in the treatment, diagnosis or prevention of a disease. This embodiment relates to a Vs or composition as defined herein. Alternatively, the Vs or compositions may be formulated as a method for the treatment, diagnosis or prevention of disease, comprising administering the Vs or compositions to a subject in need thereof.

[0100] In some embodiments, the Vs or compositions described herein are used as diagnostic or detection tools, particularly bioimaging tools. Alternatively, this aspect relates to the use of the Vs or compositions described herein as diagnostic or detection tools, particularly bioimaging tools. In particular, the compositions include a diagnostic or detection agent (also referred to herein as a tracking or labeling agent). More particularly, the compositions include a diagnostic or detection agent, a targeting ligand, and an active ingredient to be delivered. Alternatively, the compositions include a diagnostic or detection agent, a targeting ligand, and a therapeutic agent to be delivered.

[0101] In some embodiments, the Vs or compositions described herein can penetrate cells or detect specific cell types, tissues, or molecules. Thus, in some embodiments, the composition further comprises a cell-penetrating or cell-targeting moiety. In certain embodiments, the moiety is a peptide.

[0102] In some embodiments, the cell-entering or cell-targeting moiety is selected from the group consisting of arginylglycylaspartic acid (RGD), palmitoyl tripeptide-1 (Pal-GHK), melanostatin DM (HRAWFK), AHK copper (AHK2Cu), and transferrin. In some embodiments, a composition comprising Vs or Vs can contribute to cell adhesion, cell spreading, actin cytoskeleton formation, and / or focal adhesion formation, stimulate the release of specific proteins, cross the blood-brain barrier, and / or have anti-aging effects. In certain embodiments, a composition comprising Vs or Vs further comprises RGD and can contribute to cell adhesion, cell spreading, actin cytoskeleton formation, and / or focal adhesion formation. In certain embodiments, a composition comprising Vs or Vs further comprises Pal-GHK and can stimulate the release of collagen and decorin. In certain embodiments, a composition comprising Vs or Vs further comprises transferrin and can cross the blood-brain barrier. In certain embodiments, Vs or a composition comprising Vs further comprises HRAWFK and / or AHK2Cu and has an anti-aging effect.

[0103] method Another aspect of the present invention relates to a method for producing the above vesicles using the DELOS-SUSP method. The CO2-based DELOS-SUSP method ensures robustness and reproducible scale-up of Vs production.

[0104] In some embodiments, the present invention relates to a method for producing vesicles using the DELOS-susp method, the method comprising: a) preparing an organic solution containing the components of the vesicles to be formed, i.e. at least one plant sterol, and optionally at least one glucose-derived surfactant and / or stabilizer, and optionally a lipophilic active ingredient, solubilized in an organic solvent, for example ethanol, and filling said solution into a container at atmospheric pressure; b) CO mole fraction X between 0.1 and 0.7 CO2adding liquid compressed CO2 to the vessel at an operating temperature of 30-60°C and an operating pressure of 85-115 bar and forming a CO2 expanded solution containing all dissolved components; and c) Depressurizing the CO2-expanded solution into an aqueous solution optionally containing at least one glucose-derived surfactant and / or hydrophilic active ingredient by applying a flow of N2 at a working pressure of 85-135 bar and keeping the pressure in the vessel constant. Thus, glucose-derived surfactants can be solubilized in organic or aqueous solutions.

[0105] In some embodiments, the membrane components of the organic solution in step (a) include a plant sterol, a glucose-derived surfactant, and / or a stabilizer. In certain embodiments, the stabilizer is selected from a neutral additive and a charged additive. More specifically, the stabilizer is selected from the group consisting of a PEGylated fatty acid, a cationic additive, an anionic additive, or a combination thereof. In some embodiments, the stabilizer is selected from the group consisting of Chol-PEG, DC-Chol, SLS, SL, and combinations thereof. In some embodiments, the plant sterol includes Sit. In another embodiment, the glucose-derived surfactant is APG, particularly LGL.

[0106] The surfactant can be added in the organic solution added in step (a), when CO2 is added in step (b), or in the aqueous solution used in step (c). Thus, in some embodiments, the organic solution in step (a) comprises at least one glucose-derived surfactant. In another embodiment, step (b) of the method further comprises adding at least one glucose-derived surfactant in a vessel. Furthermore, in some embodiments, the aqueous solution in step (c) comprises water and / or a buffer. In particular, the aqueous solution in step (c) further comprises at least one glucose-derived surfactant. In some embodiments, the at least one glucose-derived surfactant is APG, particularly LGL.

[0107] In some embodiments, the CO mole fraction (X CO2 ) is 0.1 to 0.7. In certain embodiments, the CO mole fraction (X CO2 ) is 0.3 to 0.5. More specifically, the CO mole fraction (X CO2 ) is 0.3 or 0.5.

[0108] In some embodiments, the operating temperature is between 30°C and 60°C. In certain embodiments, the operating temperature is 40°C. In other embodiments, the operating temperature is 60°C.

[0109] In some embodiments, the method comprises increasing the temperature in step (c) to 60°C.

[0110] In some embodiments, the N2 flow is applied at an operating pressure of 85 to 135 bar. In particular, the N2 flow is applied at an operating pressure of 100 to 135 bar.

[0111] In some embodiments, the present invention relates to a method of producing vesicles using a thin film hydration (TFH) method, the method comprising: a) preparing an organic solution containing the components of the vesicles to be formed, i.e. at least one plant sterol, at least one glucose-derived surfactant and / or optionally a stabilizer, and optionally a lipophilic active ingredient, solubilized in an organic solvent, for example chloroform; b) evaporation of the solvent to form a thin film having lipophilic membrane components; and c) Hydration of the thin film with an aqueous solution optionally containing a hydrophilic active ingredient.

[0112] Example Example 1: Development of a novel carrier platform The present inventors have developed a platform of homogeneous vesicles prepared using plant-derived components and sugar-based surfactants (i.e., glucose-derived surfactants). Various vesicle systems were prepared by applying the DELOS-susp method and were thoroughly characterized in terms of size, stability, and morphology.

[0113] 1.1 Materials and methods material Phytopin DERMexpert, a mixture of pine plant sterols containing 79.7% β-sitosterol (Sit), was obtained from Purextract. Plantacare® 1200 UP (LGL-mC 12-16 ), Plantacare® 2000 UP (DGL-mC 8-16 ), Plantacare® 810 UP (DGL-mC 8-10 ) and Plantapon® LGC Sorb (LGC) were provided by BASF. Dodecyl β-D-glucopyranoside (LGL-p) and decyl β-D-glucopyranoside (DGL-p) were obtained from Carbosynth Ltd. Sodium laurate (SL) was provided by TCI Europe NV. 3α-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol hydrochloride (DC-Chol) and cholesterol-polyethylene glycol-600 (Chol-PEG600) were purchased from Merck. Ethanol HPLC grade was purchased from Scharlab. Carbon dioxide and nitrogen were supplied by Carburos Metalicos SA. The water used was pretreated with a MilliQ Advantage A10 water purification system (Millipore). N-octyl-β-D-glucopyranoside (OGL-p) and N-lauroylsarcosine sodium salt (SLS) were purchased from Alfa Aesar. Decyl β-D-glucopyranoside was purchased from Carbobsynth.

[0114] Method of producing the vesicle system i) DELOS-susp device configuration The system consists of a 6-50 mL high-pressure vessel. The temperature is maintained using an external fluid heating jacket, and the temperature and pressure are controlled by a temperature controller and a pressure-indicating regulator. CO2 is pumped into the reactor through a thermostatic syringe pump (Model 260D, ISCO Inc., Lincoln, US) and introduced into the vessel through two valves until the working pressure is reached. A variable-speed agitator ensures homogeneity of the mixture in the volume-expanded phase. Furthermore, using a pressure-reducing micrometric valve, the expanded solution contained in the vessel is decompressed into the aqueous phase placed in the collector at atmospheric pressure. Simultaneously, nitrogen pressure is regulated by a pressure-regulating valve and introduced directly from a pressurized reservoir into the vessel through two valves.

[0115] ii) Preparation of vesicles: Decompression of expanded liquid organic solutions (DELOS-susp) As shown in FIG. 1, the procedure involves: (a) filling a vessel with an organic solution containing membrane components (e.g., β-sitosterol, LGL-p, DGL-p, OGL-p, Chol-PEG, DC-Chol, SLS, and / or SL) solubilized in ethanol at atmospheric pressure; (b) X CO2(c) Addition of liquid compressed CO2 at a CO2 mole fraction of 0.3–0.5, Tw = 40 °C, and a working pressure of Pw = 85–115 bar to form a CO2-swollen solution in which all membrane components were dissolved; and (d) decompression of the CO2-swollen solution into an aqueous solution containing a water-soluble surfactant (i.e., a mixture of glucose-derived surfactants (i.e., APG)) at the desired concentration. A nitrogen (N2) flow at a working pressure of Pw = 100–135 bar was used to pump the CO2-swollen solution from the reactor, maintaining constant pressure in the vessel during decompression. The average time per experiment was 30 min. All samples were filtered through a 0.45 μm pore size polyethersulfone (PES) membrane syringe filter to remove impurities derived from the starting material (mainly a mixture of Sit and APG). As a result, a uniform, opaque colloidal dispersion of Vs was obtained in water containing 15% (vol / vol) ethanol. The vesicles (Vs) were stored at 4 °C until further characterization. The surfactant may be added in the organic solution added in step (a), when CO2 is added in step (b), or in the aqueous solution formed during step (c). The molecular structures of the sterols and surfactants used herein are shown in Figure 2.

[0116] iii) Preparation of vesicles by thin film hydration (TFH) The TFH procedure included: (a) dissolution of lipophilic membrane components (e.g., β-sitosterol, LGL-p, DGL-p, and / or OGL-p) in chloroform; (b) evaporation of the organic solvent using a rotary evaporator to form a thin film consisting of the membrane components; and (c) hydration of the thin film with 10 mL of aqueous solution at 60°C with magnetic stirring for 30 min. Finally, one day after preparation, the samples were sonicated at 60°C for 30 min.

[0117] Vesicle characterization i) Characterization of size, polydispersity index, and zeta potential The mean particle size, particle size distribution (or polydispersity index, PdI), and apparent zeta potential of all produced vesicles were measured using a dynamic light scattering (DLS) and electrophoretic light scattering (ELS) analyzer (Malvern Zetasizer Ultra, Malvern Instruments, UK) coupled with noninvasive backscattering technology (NIBS). The reported zeta potential values ​​correspond to the apparent zeta potential calculated using the Helmholtz-Smoluchowski approximation. All reported values ​​were the average of three consecutive measurements of the same sample at 25 °C using the Zetasizer software 7 days after vesicle production (except for DC-Chol-containing VS, which require 2 months for stabilization). Size data are based on the intensity size distribution and correspond to the z-average of the three measurements. In some cases, three replicates of the same formulation were prepared (as specified in the corresponding tables) to ensure the robustness of the results. In these cases, size data correspond to the z-average ± standard deviation of the three replicates. ii) Morphological characterization by cryo-transmission electron microscopy (cryo-TEM) Preparations were measured by cryo-TEM to directly analyze the vesicle morphology and investigate their homogeneity (heterogeneity) and coexistence of structures. Information on size, shape, number of bilayers, and bilayer distribution was collected. Vitrified samples were prepared several days after VS production. Samples were vitrified in a controlled sample preparation chamber according to well-established procedures and examined at cryogenic temperatures with a T12 G2 Tecnai (FEI) and a Talos F200C (Thermo Fisher) microscope. Ted Pella perforated grids were used; the temperature of the vitrified samples was always kept below -170°C. Images were recorded using a Gatan UltraScan 2kx2k CCD camera or a Ceta camera in low-dose operation. Images were recorded at various magnifications (8.8K to 53K) to adequately capture all structures, i.e., at various length scales ranging from a few nanometers to several hundred nanometers. No image processing was performed, except for background subtraction.

[0118] 1.2 Results 1.2.1 Synthesis of Vs: Effect of alkyl chain length of surfactant Vesicles containing the plant sterol Sit in combination with glucose-derived surfactants were prepared using the DELOS-susp method. The effects of surfactants with different carbon chain lengths (pure lauryl glucoside (LGL-p), pure decyl glucoside (DGL-p), and pure octyl glucoside (OGL-p)) were compared by keeping the glucose polar head group of the surfactant constant. Compositions containing a mixture of OGL-p and DGL-p, and C8-C8 16 A composition containing a mixture of alkyl glucosides was also compared.

[0119] As shown in Table 1, Vs were formed only when the surfactant carbon chain length was 12 carbons or longer. The best conditions found for the synthesis of Sit:LGL-p (1:1) were used to evaluate the formation of Sit:DGL-p and Sit:OGL-p. However, Vs were not obtained in this process. With the use of DGL-p and OGL-p, Vs were also not formed when the aqueous phase was heated to 60 °C during the decompression step, nor when additives were used to stabilize such formation, i.e., after the addition of 3 wt / wt% of the anionic additives LGC or SLS (see Samples #4-6 and #9 in Table 1).

[0120] Furthermore, C 8-10 Sugar-based surfactants containing a mixture of alkyl glucosides of different chain lengths (DGL-m C8-10 When sample #7 in Table 1 was used, Vs was not obtained as well. However, 16 Alkyl glucoside mixture (DGL-m C8-16 When sugar-based surfactants containing ), plant sterol-based Vs were formed (see sample #8 in Table 1).

[0121] These results confirm that not only does the surfactant structure play an important role in the formation of plant sterol-based Vs, but the sterol side chains also influence the self-assembly of membrane components. [Table 1]

[0122] 1.2.2 Synthesis of Vs: Effect of method on Vs formation: Comparison of DELOS-susp and LGL thin film hydration (TFH) Vesicles containing APG were previously obtained by combining cholesterol with either OGL-p, DGL-p, or LGL-p using the TFH method (Muzzalupo et al., 2013). However, as shown above (Example 1.2.1), it is disclosed herein that vesicles containing plant sterols instead of cholesterol are formed only in combination with sugar-based surfactants with carbon chain lengths of 12 or more (e.g., LGL-p). These results therefore suggest that the substitution of Chol by Sit may have unexpected effects on the self-assembly of membrane components.

[0123] To confirm that these unexpected results were independent of the method used to form Vs, additional studies were conducted to confirm the relationship between surfactant carbon chain length and Vs formation with sterols using the TFH method (as described in Muzzalupo et al. 2013). The results were then compared with those obtained by the DELOS-susp method (shown in Example 1.2.1). Again, three different surfactants, OGL-p, DGL-p, and LGL-p, were used to evaluate Vs formation with Sit.

[0124] The macroscopic appearance of the three samples clearly shows the difference between the use of OGL-p and DGL-p compared to the use of LGL-p, as shown in Figure 3(A). The Sit:LGL-p (1:1) batches showed a uniform dispersion, while the Sit / OGL-p (1:1) and Sit / DGL-p (1:1) batches appear to be completely sedimented.

[0125] Furthermore, the samples were observed under a Morphology G3 optical microscope, as shown in Figure 3(B), and they showed different types of particles. In the case of the Sit:OGL-p system, the precipitates were aggregates with a crystalline appearance. In the case of the Sit:DGL-p system, two different particle populations were observed: one with a crystalline appearance and the other spherical particles. Finally, in the case of the Sit:LGL-p system, only a spherical particle population was observed; there were no crystalline precipitates in the sample.

[0126] The samples were then observed using cryo-TEM, as shown in Figure 3(C), top panel. The Sit / OGL-p and Sit / DGL-p samples clearly showed large aggregates covering the grid quadrant where the samples were analyzed. On the other hand, the Sit / LGL-p system showed a uniform distribution on the grid. These results were consistent with their macroscopic appearance: the samples that appeared to have precipitated (Sit / DGL-p and Sit / OGL-p) showed large aggregates that were impossible to analyze by cryo-TEM, whereas the Sit / LGL-p system, which was uniform on the macroscopic scale, also showed a uniform appearance on the nanometer scale.

[0127] As shown in the bottom panel of Figure 3(C), when the samples were analyzed at higher resolution using cryo-TEM, only the Sit / LGL-p system could be analyzed because the large aggregates in the Sit / DGL-p and Sit / OGL-p systems did not allow for this analysis. For the Sit / LGL-p system, a large amount of Vs was observed, all of which were homogeneously distributed and not aggregated. These results are consistent with the macroscopic appearance of the samples discussed earlier: the aggregates in the Sit / DGL-p Vs formed unstable aggregates and precipitated, whereas the Sit / LGL-p Vs remained in suspension because they were homogeneously dispersed.

[0128] Finally, the samples were analyzed by dynamic light scattering (DLS) using a Zetasizer Ultra instrument, as shown in Figure 3(D). For Sit / OGL-p (particle size 4041 nm, PdI 1.60) and Sit / DGL-p (particle size 8376 nm, PdI 1.61), the samples were highly inhomogeneous, meaning the measurements did not pass the instrument's quality standards and the data were unreliable. On the other hand, Sit / LGL-p Vs showed a particle size of 250 nm and a PdI of 0.23 ± 0.06, which was considered reliable.

[0129] These results confirmed that the synthesis of plant sterol-containing Vs depends on the alkyl chain length of the surfactant, regardless of the method used. For example, Vs containing Sit are only formed when APGs with 12 or more carbon atoms are used. The use of OGL or DGL does not allow the formation of plant sterol-containing Vs, unlike cholesterol-containing Vs, as described in Muzzalupo et al. (2013) (see Table 2). Thus, surprisingly, it appears that the substitution of cholesterol for plant sterols affects the relationship between the APG used and the successful formation of Vs, regardless of the method used. [Table 2]

[0130] 1.2.3 Synthesis of Sit:LGL Vs Considering the results of Examples 1.2.1 and 1.2.2, the DELOS-susp method was applied to prepare various vesicle systems containing plant sterols, including β-sitosterol, and LGL as a glucose-derived surfactant. The Vs formed by the DELOS-susp method comprise neutral, positive, and negative delivery systems formed primarily by the self-assembly of Sit and LGL (see Table 3 and Figure 4 ).

[0131] Neutral carriers were large unilamellar vesicles with particle sizes ranging from 217 to 683 nm and colloidal stabilities ranging from several weeks to more than 12 months, depending on the purity of the APG surfactant used. Negatively and positively charged carriers based on glucose-derived surfactants were both small unilamellar vesicles. Negatively charged carriers composed of plant sterols, LGL, and LGC ranged from 148 to 193 nm, depending on the molar ratio of LGC to surfactant, and they maintained their physicochemical properties for more than a year. Meanwhile, positive Vs were obtained by incorporating different molar ratios of DC-cholesterol (DC-Chol). The size of these Vs ranged from 64 to 104 nm, and they were stable for more than 12 months. [Table 3]

[0132] 1.2.4 Synthesis of neutral Vs containing Sit / LGL To evaluate the formation of neutral Vs, LGL molecules of different quality and purity were used. Mixtures of alkyl glucosides with different carbon chain lengths (e.g., LGL-m C12-16 When cosmetic-grade reagents, known to contain HCl and have lower purity, were used, Vs were formed at all of the Sit:LGL molar ratios tested. However, other structures such as ribbons, large MLVs, and solid particles were also observed in cryo-TEM images and particle size measurements of the best-performing DELOS sample, as shown in Figure 5(A-B).

[0133] In these methods, the formation of Vs using 98% pure LGL (LGL-p) was also evaluated. Neutral Sit:LGL-p (1:1) Vs were obtained by increasing the aqueous phase temperature to 60 °C during the decompression step, as shown in Figure 5(C). The neutral Sit / LGL Vs produced with LGL-p were larger and more polydisperse (particle size 683 ± 45 nm, PdI 0.43 ± 0.11) than those produced with the APG mixture (particle size 217 ± 11 nm, PdI 0.22 ± 0.01) (see Figure 5(D)).

[0134] 1.2.5 Chol-PEG 600 Stabilization of neutral Vs by addition of PEGylated cholesterol was used to enhance the colloidal stability of the neutral Sit / LGL-p carrier. PEGylated cholesterol (Chol-PEG) was added at 7.4% w / w. 600 ) was added to a membrane of neutral Vs. The stability of Vs increased to over 3 months, as shown in FIG. Notably, such additives avoid the use of high temperatures during Vs formation, which is an important condition for encapsulating heat-labile active molecules such as proteins. PEGylation of Vs membranes helps improve the colloidal stability of Vs due to steric hindrance caused by the long carbon chains of PEG molecules and by the formation of hydrogen bonds with the solvent, thus avoiding vesicle aggregation.

[0135] 1.2.6 Synthesis of positively and negatively charged Vs containing Sit / LGL The synthesis of charged Vs was achieved by adding charged molecules, both anionic and cationic, to films of neutral Vs, which resulted in improved colloidal stability due to charge repulsion. 3% w / w of charged molecules were added to the membrane components. 12 Anionic surfactants such as LGC, SL, and SLS, derived from lauryl alcohol chains, enabled the formation of Vs, as shown in Figure 7(A-C). Next, DC-Chol, a sterol-like molecule, was added to the Sit / LGL membrane as a cationic additive, which also enabled Vs formation, as shown in Figure 7(D). The resulting charged Vs exhibited high stability, up to more than 12 months. Notably, such additives avoid the need for high temperatures during Vs formation, which is an important condition for encapsulating heat-labile active molecules such as proteins. These results demonstrate the ability of this novel sugar-based Vs platform to form both positively and negatively charged Vs, thus supporting the many potential applications that can result from this platform. For example, DC-Chol is a pH-sensitive molecule that may be useful for intracellular drug delivery.

[0136] 1.2.7 Screening for LGL:LGC and Sit:DC-Chol ratios The versatility of the new sugar-based Vs platform was further explored by screening different ratios of charged membrane components, ranging from the complete replacement of Sit by DC-Chol and the complete replacement of LGL-p by LGC, as shown in Figures 8 and 9, respectively. The average particle size of Vs decreased from 104 nm to 64 nm with increasing DC-Chol ratio, while the opposite behavior was observed in the ζ potential. This is because the greater the amount of positively charged surfactant, the greater the surface charge of the particles. Similar behavior was observed when different amounts of LGC were screened (see Figure 9). In both cases, the greatest change in particle size was observed between the LGL-p:LGC or Sit:DC-Chol molar ratios of (0.8:0.2) and (0.5:0.5), while increasing the LGC or DC-Chol molar ratio beyond 0.5 did not significantly change the physicochemical properties of the Vs. No significant changes in ζ-potential were observed between Vs containing different molar ratios of LGC and DC-Chol, but the ζ-potential values ​​were higher in absolute value for the neutral Sit / LGL Vs.

[0137] Example 2. Encapsulation of active ingredients in glucose-derived Vs platforms The encapsulation of various active ingredients was evaluated to demonstrate the potential applications (e.g., pharmaceutical and cosmetic applications) of the Vs platform disclosed herein. A number of molecules / compounds representing three different types of active ingredients, including small biomolecules, proteins, and dyes, were selected and screened in the negative and positive LGL vesicle systems according to the physicochemical properties of the molecules to be encapsulated.

[0138] 2.1. Materials and Methods material The materials used to synthesize the Vs of the present invention are as disclosed in Example 1, Section 1.1 Materials. Additionally, materials corresponding to the active ingredients used were added. These were as follows: 7-dehydrocholesterol (7-DHC), methotrexate (MTX), and 2-OaD-glucopyranosyl-L-ascorbic acid (ascorbyl glucoside, AG) were purchased from Merck. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD) and 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were obtained from Invitrogen and Thermo Fisher Scientific. Niacinamide PC (NCA) and DL-α-tocopherol (TCP) were obtained from DSM. Pure cannabidiol (CBD) isolate was obtained from CBD Cure. PnP b-FGF (basic fibroblast growth factor) was obtained from PnP Biopharm. Human growth hormone (HGH) was purchased from Prospec. HPLC grade ethanol was purchased from Scharlab.

[0139] DELOS-susp method i) Synthesis of Vs containing active ingredients The synthesis of Vs was carried out as disclosed in Example 1, section 1.1 DELOS-susp method. Additionally, active ingredients were added at specific steps of the process depending on their properties: lipophilic active ingredients (7-dehydrocholesterol, tocopherol, cannabidiol, DiI, and DiD) were added together with the membrane components in step (a) (at atmospheric pressure in the vessel), while hydrophilic active ingredients (niacinamide, ascorbyl glucoside, methotrexate, b-FGF, and HGH) were added during step (b) in aqueous solutions containing water-soluble surfactants.

[0140] ii) Encapsulation efficiency (EE) by UV-visible spectroscopy The encapsulation efficiency (EE) of each active ingredient was measured by UV and visible light absorbance using a UV-Visible spectrophotometer (Thermo Scientific™ Evolution™ 201 UV-Visible Spectrophotometers, Thermo Scientific). For lipophilic active ingredients (7-DHC, CBD, TCP, DiD, and DiI), the VS membrane was dissociated to release the active ingredient by diluting the sample in 80% (vol / vol) ethanol until an absorbance below 1 was obtained within the calibration curve. The concentration of each active ingredient was determined using the calibration curve in the same medium as the corresponding VS system. The active ingredient was quantified before and after filtration, and both values ​​were used to calculate the EE using Equation 1 (described herein). Because lipophilic molecules are not soluble in aqueous media, filtered samples were considered to contain only the incorporated active ingredient, since any precipitated, unincorporated active ingredient would remain in the filter. formula 1

number

[0141] A different procedure was performed for the hydrophilic active agents (NCA, AG, MTX), where after the Vs filtration step, a separate step was applied to separate the unincorporated active ingredients. For NCA and AG, an ultracentrifugation step of the filtered samples was performed (6 h, 4 °C, 600,000 g) (Sorvall Discovery M150 Micro-Ultracentrifuge, Thermo Scientific). In the case of MTX, separation of unincorporated active ingredient from Vs was performed by tangential flow filtration (TFF) (KrosFlo® Research IIi TFF diafiltration system (KR2i) equipped with an mPES filter hollow fiber column cut-off 300 KDa (C04-E100-05-N, Spectrum Labs, SL). The supernatant in the case of centrifuged samples and the permeate fraction in the case of TFF filtered samples were collected and diluted to an absorbance value less than 1 and within the calibration curve. The concentration was calculated using the calibration curve with the same medium of the corresponding Vs system. The encapsulation efficiency was then calculated using equation 2, as described herein: formula 2

number

[0142] iii) Encapsulation efficiency (EE) by fluorescence spectroscopy: The encapsulation efficiency (EE) of both proteins was measured by fluorescence spectroscopy using a Varian Cary Eclipse (Agilent Technologies, Santa Clara, USA). In both cases, separation of unincorporated and incorporated proteins into the Vs was performed using a TFF (KrosFlo® Research IIi TFF Diafiltration System (KR2i) equipped with an mPES filter open-column cutoff 300 kDa (C04-E100-05-N, Spectrum Labs, SL). The permeate fraction was then collected and analyzed by fluorescence excitation at 278 nm. Concentrations were calculated using a calibration curve using the same medium as the corresponding Vs system. The encapsulation efficiency was then calculated using Equation 2, as described herein.

[0143] 2.2 Results 2.2.1 Sit:LGC and Sit:LGL:LGC systems The Sit:LGC (1:1) system was used to incorporate both lipophilic and hydrophilic active ingredients, while Sit:LGL:LGC (1:0.8:0.2) was selected to test the encapsulation of only lipophilic molecules, as shown in Table 4. The following molecules were used: - Lipophilic small molecules: 7-dehydrocholesterol (7DHC), which is photochemically converted to vitamin D3 in the skin; cannabidiol (CBD), a phytocannabinoid found in the cannabis plant that has been studied for many medical uses; α-tocopherol (TCP), a form of vitamin E; and carbocyanine dyes (DiI and DiD) as fluorescent markers. -Hydrophilic vitamins: niacinamide (NCA) or vitamin B3, and ascorbyl glucoside (AG), a vitamin C derivative. -Small protein: basic fibroblast growth factor (b-FGF).

[0144] Both systems (Sit:LGC and Sit:LGL:LGC) showed high encapsulation efficiencies (EE) (49–77%) for lipophilic active ingredients, whereas Sit:LGC showed low EE (8–20%) for hydrophilic active ingredients, except for CBD (22–33%) (Table 4, Figures 10–12). This is consistent with the fact that lipophilic active ingredients can be inserted into the Vs membrane, which is also lipophilic, while hydrophilic active ingredients are less likely to be incorporated into the aqueous lumen of the Vs. In the case of b-FGF, it was encapsulated very efficiently (over 99%) in the Sit / LGC carrier due to electrostatic interactions between the positively charged amino acids of the protein and the negatively charged Vs at the working pH. [Table 4]

[0145] 2.2.2. Sit:LGL-p:DC-Chol series The Sit:LGL-p:DC-Chol (0.8:1:0.2) system, which has cationic properties provided by the protonated amine groups of DC-Chol, was also used to incorporate lipophilic and hydrophilic molecules. The lipophilic active ingredients selected were: 7-DHC, CBD, and TCP. For hydrophilic active ingredients, they were selected according to their charge at the working pH to favor electrostatic interactions with the positively charged Vs. Methotrexate (MTX), a chemotherapeutic agent and immune system suppressant, was selected as an example of a hydrophilic small molecule, while human growth hormone (HGH) was selected as an example of a small protein.

[0146] Similar results were obtained as in the above system. The encapsulation efficiency of lipophilic active ingredients was very high (all above 90%), even higher than the EE of negatively charged Vs. Surprisingly, MTX was also encapsulated with high efficiency (EE=88%), despite being a hydrophilic molecule. Finally, for the protein HGH, it also showed an extremely high EE of over 99%. These results are shown in Table 5 and Figures 13-14. [Table 5]

[0147] Example 3 Functional testing of active ingredients incorporated into glucose-derived Vs platforms After demonstrating the encapsulation of the active ingredients, their functional activity after incorporation into the VS was evaluated. It was important to demonstrate the preservation of function after formulation. Two different active ingredients were tested using the same vesicle system, and specific assays were performed for each to examine their specific functionality.

[0148] 3.1. Materials and Methods material Materials used in the synthesis of Vs were as disclosed in Example 1, Section 1.1 Materials and Example 2, Section 2.1 Materials. Additionally, additional materials and reagents were used to perform the functional assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) and cholecalciferol were purchased from Merck. HPLC-grade solvents methanol (MeOH) were purchased from Alco (Barcelona, ​​Spain), isopropanol (IPA) from Fischer (New Hampshire, United States), and formic acid (HCOOH) from Sigma-Aldrich (Missouri, United States).

[0149] DELOS-susp method i) Synthesis of Vs containing active ingredients The synthesis of Vs was carried out as disclosed in Example 1, section 1.1 DELOS-susp method and Example 2, section 1.1 DELOS-susp method. TCP-loaded Vs were prepared using Sit, LGL-p, and LGC in a molar ratio of 1:0.8:0.2 with TCP at a concentration of 0.75 mg / ml (dispersion medium HO / ETOH 15% (v / v)). 7DHC-loaded Vs were prepared using Sit, LGL-p, LGC, and 7DHC in a molar ratio of 0.2:0.8:0.2:0.8.

[0150] Functional assay for α-tocopherol (TCP): Evaluation of antioxidant capacity i) Antioxidant capacity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay The DPPH assay is a well-known and established method for measuring the antioxidant capacity of molecules. DPPH is a lipid-soluble, stable free radical that is easily reduced in the presence of antioxidants. DPPH is purple in its oxidized state, which changes to yellow / white upon reduction. This color change can be followed using UV-visible spectroscopy. Assays were performed in ethanol, always maintaining a DPPH:TCP molar ratio of 1:0.3. To obtain a final volume of 3 mL, DPPH was diluted with ethanol to a final concentration of 63.4 μM. Free TCP and TCP DELOS-NV were diluted to a final TCP concentration of 19 μM. Each solution was then mixed and kept at room temperature in the dark for 30 minutes. The absorbance of the solution was measured at 517 nm using a Thermo Scientific™ Evolution™ 201 / 220 UV-Visible Spectrophotometer (Thermo Scientific). The percentage of TCP antioxidant capacity was calculated using Equation 3, where Abs 対照 is the absorbance at 517 nm of DPPH in ethanol, and Abs 試料 is the absorbance at 517 nm of the samples (free TCP or TCP DELOS-NV with DPPH) prepared as described. formula 3

number

[0151] Functional assay for 7-dehydrocholesterol (7DHC): Conversion of 7DHC to vitamin D i) Characterization by UV-Vis spectroscopy before irradiation The concentration of 7DHC loaded in the Vs was previously characterized by UV-visible spectroscopy as disclosed in Section 2.1 Materials and Methods of Example 2.

[0152] ii) Irradiation Assay 7DHC is a molecule present in human skin keratinocytes and is converted to cholecalciferol (vitamin D) upon exposure to sunlight. The purpose of this functional study was to demonstrate that both free 7DHC and 7DHC encapsulated in new VSs have the same conversion ratio to cholecalciferol in suspension. To this end, VSs consisting of Sit, LGL-p, LGC, and 7DHC were prepared in a molar ratio of 0.2:0.8:0.2:0.8. Additionally, a 15% (v / v) suspension of 7DHC in water / EtOH was also prepared at the same concentration as the 7DHC in the VS suspension. Next, 1 mL of each suspension was placed in a well of a 12-well plate (n=3). The samples were irradiated with a UV lamp (λ=302 nm) for 30 minutes and 1 hour. Finally, the samples were incubated in the dark at room temperature for 48 hours and then analyzed by HPLC.

[0153] iii) Measurement of the conversion of 7DHC to vitamin D by HPLC analysis The concentrations of 7DHC and vitamin D (cholecalciferol) after 30 minutes and 1 hour of UV irradiation were quantified by HPLC. Results were compared with the initial concentrations before irradiation, measured by UV-visible spectroscopy. Samples of free 7DHC and 7DHC-loaded Vs were analyzed. Stock standard solutions of 7DHC and vitamin D were prepared in methanol, and lower concentration standard solutions were obtained by dilution of the stock solutions in methanol. To prepare the samples, 1 mL of each sample was dissolved in 4 mL of methanol. 10 μL of each sample was injected into the HPLC system. Analysis was performed using a Waters liquid chromatograph system (Milford, MA, USA) coupled to a Waters PDA detector type HPLC 2998. Empower 3 software was used for instrument control and data analysis. Detection was performed at 282 nm for 7DHC and 264 nm for cholecalciferol.

[0154] An Atlantis Premier BEH C18 AX 2.5µm 4.6x150mm (Waters) column was used to separate sample components before detection. The column temperature was set at 40°C. Two solvents, A: water and B: methanol: isopropyl alcohol: formic acid (94.9:5:0.1, v:v:v), were used in isocratic elution mode. Solvent B was vacuum filtered through a PVDF membrane (0.45µm pore size). The mobile phase flow rate was 2ml / min. An isocratic flow of 10% mobile phase A + 90% mobile phase B was used, with a total elution time of 23 minutes. Quantitation was performed by integrating the peak area of ​​the corresponding analyte and interpolating the peak area of ​​the 7-DHC or cholecalciferol standard curve.

[0155] 3.2 Results 3.2.1 Functional Assays for TCP: Antioxidant Capacity Assessment The antioxidant capacity of TCP was evaluated after its incorporation into NV to confirm that the molecule remained active after encapsulation. Antioxidant capacity was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. DPPH is a stable free radical that can be reduced in the presence of hydrogen-donating antioxidants, such as TCP. The activity of encapsulated TCP was compared with that of free TCP dissolved in ethanol. The assay was performed at a DPPH:TCP molar ratio of 1:0.3. Considering that 1 mole of TCP reduces 2 moles of DPPH, the expected antioxidant capacity for this assay was 60%. After analysis, both samples, free TCP and TCP encapsulated in DELOS-NV, possessed the expected antioxidant (OX) capacity, indicating that the encapsulation of TCP in NV does not affect the AOX function of the molecule (Table 6). Therefore, it can be concluded that lipophilic vitamins can be loaded into aqueous formulations of NV while maintaining their antioxidant capacity. [Table 6]

[0156] 3.2.2 Functional Assays for 7DHC: Conversion to Vitamin D To assess whether 7DHC could be converted to vitamin D (VitD) after incorporation of the molecule into Vs, an irradiation assay was performed. Free 7DHC in water / EtOH 15% (vol / vol) and 7DHC loaded into Vs were irradiated with a UV lamp (λ = 302 nm) for 30 minutes or 1 hour (n = 3). The conversion of 7DHC to VitD was quantified by HPLC. A decrease in 7-DHC concentration in irradiated samples was expected, while VitD was expected to appear in the samples, indicating that 7DHC was converted to VitD.

[0157] As can be seen in Figure 15 and Table 7, there is a decrease in 7DHC concentration for either free or Vs-loaded 7DHC. This decrease is similar in both cases (from 832 μg / mL to 389 ± 4 μg / mL and 350 ± 12 μg / mL, respectively, after 1 hour), indicating that loading 7DHC in vesicles does not affect its conversion to Vit D. Regarding Vit D production, levels after 1 hour were also similar for the free and Vs-loaded 7DHC samples (15 ± 1 μg / mL and 17 ± 4 μg / mL). These results allow us to conclude that after encapsulation, 7DHC maintains its ability to convert to vitamin D, a molecule that plays an active role in cellular function. [Table 7]

[0158] References Non-patent literature: Salim, M., Abou-Zied, OK, Udani Kulathunga, H., Baskaran, A., Kuppusamy, UR & Hashim, R. (2015). Alkyl Mono- and Di-Glucoside Sugar Vesicles as Potential Drug Delivery Vehicles: Detecting Drug Release Using Fluorescence. RSC Adv., 5(68), 55536-55543. Muzzalupo, R., Tavano, L. & La Mesa, C. (2013). Alkyl Glucopyranoside-Based Niosomes Containing Methotrexate for Pharmaceutical Applications: Evaluation of Physico-Chemical and Biological Properties. Int. J. Pharm., 458(1), 224-229. Manconi, M., Vila, AO, Sinico, C., Figueruelo, J., Molina, F. & Fadda, AM (2006). Theoretical and Experimental Evaluation of Decypolyglucoside Vesicles as Potential Drug Delivery Systems. J. Drug Deliv. Sci. Technol., 16(2), 141-146. Manconi, M., Sinico, C., Valenti, D., Lai, F. & Fadda, A. M. (2006). Niosomes as carriers of tretinoin: III. A Study into the in Vitro Cutaneous Delivery of Vesicle-Incorporated Tretinoin. Int. J. Pharm., 311(1-2), 11-19. Plaza-Oliver, M., Santander-Ortega, MJ, Lozano, MV (2021).Current approaches in lipid-based nanocarriers for oral drug delivery,Drug Deliv.Transl.Res.11 471-497. Patent documents: International Publication No. 2006 / 079889A1

Claims

1. A vesicle comprising at least one plant sterol and at least one glucose-derived surfactant.

2. 2. The vesicle of claim 1, wherein at least one of the glucose-derived surfactants is an alkyl polyglucoside surfactant or a derivative thereof, and the alkyl polyglucoside surfactant or the derivative thereof has a carbon chain length of at least 12 carbons.

3. 3. The vesicle of claim 2, wherein the alkyl polyglucoside surfactant or the derivative thereof is selected from the group consisting of octyl glucoside, decyl glucoside, lauryl glucoside, tetradecyl glucoside, hexadecyl glucoside, tetradecyl D-glucoside, hexadecyl β-D-glucopyranoside, octadecyl D-glucoside, arachidyl glucoside, a mixture of C12-20 alkyl glucosides, cetearyl glucoside, C20-22 alkyl glucosides, coco glucoside, isostearyl glucoside, octyldodecyl glucoside, phosphorus derivatives of alkyl polyglucosides, and lauryl glucoside carboxylate.

4. 4. The vesicle of claim 3, wherein the alkyl polyglucoside surfactant is lauryl glucoside, tetradecyl glucoside, hexadecyl glucoside, or a combination thereof.

5. 5. The vesicle of claim 4, wherein the alkyl polyglucoside surfactant is lauryl glucoside.

6. The vesicle of any one of claims 1 to 5, wherein the plant sterol comprises β-sitosterol.

7. The vesicle of any one of claims 1 to 6, wherein the vesicle further comprises a stabilizer selected from the group consisting of a neutral additive, a cationic additive, and an anionic additive.

8. The vesicle of claim 7, wherein the stabilizer is a neutral additive that is a complex comprising covalently attached cholesterol and polyethylene glycol moieties.

9. 8. The vesicle of claim 7, wherein the stabilizer is an anionic additive selected from the group consisting of lauryl glucose carboxylate, sodium lauroyl sarcosinate, and sodium laurate, and combinations thereof.

10. The vesicle of claim 7, wherein the stabilizing agent is a cationic excipient that is DC-cholesterol.

11. 11. The vesicle of any one of claims 1 to 10, further comprising an active ingredient selected from the group consisting of pharmaceutical agents, cosmetic agents, food additives or supplements, vitamins, diagnostic agents and detection agents.

12. A vesicle according to any one of claims 1 to 11 for use as a drug.

13. 12. Use of a vesicle as defined in any one of claims 1 to 11 as a delivery system or as a diagnostic or detection tool.

14. 12. Use of vesicles as defined in any one of claims 1 to 11 for the preparation of a suspension for topical, oral, intravenous, inhalation, parenteral, mucosal, subcutaneous or ocular administration.

15. 12. A composition comprising a plurality of vesicles as defined in any one of claims 1 to 11 selected from the group consisting of a pharmaceutical composition, a cosmetic composition, a nutraceutical composition, a food composition, a food additive or food supplement and a detection or diagnostic composition.

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