NL2023066B1 - Stabilized liposomes - Google Patents

Abstract

The present invention relates to a method for stabilizing liposomal vesicles dispersed in a first aqueous composition, the liposomal vesicles comprising a phospholipid bilayer enclosing a second aqueous composition. The liposomal vesicles further comprise one or more nutritional supplements selected from the group consisting of vitamins, minerals, enzymes, proteins, peptides, and/or herbal extracts. The method comprises adding ions into the first aqueous composition, the ions being chosen from Ca2+, Mgz”, and/or an’“. The invention furthermore relates to compositions obtainable by the method of the invention.

Description

P34086NL00/WZO Title: Stabilized liposomes Technical Field The present invention relates to a method for stabilizing liposomal vesicles in an aqueous dispersion, and to a composition for oral administration to a subject comprising the stabilized vesicles. Background Art A liposome is a spherical vesicle having at least one lipid bilayer. Liposomes are most often composed of phospholipids, but may also include other lipids. The lipid bilayer closely resembles human cell membranes, which is why liposomes can be more readily absorbed in the blood stream. Hydrophilic biologically active components may be dissolved in the core of a liposome, whereas lipophilic components associate with the bilayer or bilayers. A liposome can hence be loaded with hydrophobic and/or hydrophilic molecules. To deliver the molecules to a site of action, the lipid bilayers can fuse with other bilayers such as the cell membrane. Liposomes are most commonly used in the drug/medicine industry. Compared to conventional (i.e. non-liposomal) oral drug delivery methods, use of drug-loaded liposomes leads to an increased bioavailability of the drugs, and therefore an increased effectivity of the treatment. Use of liposomes for delivery of nutritional supplements is much less common than use of liposomes for medicinal applications.

In the art, liposomes loaded with drug molecules are commonly prepared as aqueous dispersions. A problem with these dispersions is that they only have limited physical stability. The liposomes can aggregate and precipitate as sediment. Additionally, on storage the biologically active compounds may be lost into the external aqueous phase. Furthermore, depending upon the type of lipid and biologically active compound present in the liposome, there is the potential for chemical degradation of the lipid components and/or the biologically active components in the aqueous dispersion.

Summary of Invention One object of the present invention is to provide an oral administration form of one or more nutritonal supplements. Another object of the present invention is to provide a good bioavailability of the nutritional supplements. A further object of the present invention is to provide a form that is stable during storage.

Thereto, the present invention provides a method for stabilizing liposomal vesicles dispersed in a first aqueous composition, the liposomal vesicles comprising a phospholipid bilayer enclosing a second aqueous composition, the liposomal vesicles further comprising one or more nutritional supplements selected from the group consisting of vitamins, minerals, enzymes, proteins, peptides, and/or herbal extracts, wherein the method comprises adding ions into the first aqueous composition, the ions being chosen from Ca2*, Mg?*, and/or Zn?*. The method of the invention can be carried out on existing liposomal formulations, i.e. liposomal formulations as known in the art. These existing formulations are commonly prepared by dissolving phospholipids and optional fat-soluble nutritional supplement(s) such as vitamins, minerals, enzymes, proteins, peptides, and/or herbal extracts in an organic solvent. A film of phospholipids is formed in a glass flask while the solvent is evaporated.

After complete evaporation of the solvent, an aqueous solution of nutritional supplement(s) is then added. The solution of nutritional supplement may be a physiological salt solution of the nutritional supplement. The solution of nutritional supplements is shaken together with the phospholipids. After further addition of water, the liposomal vesicles are formed. The nutritional supplements are then located either in the core of the liposome or in the wall, i.e.

within the bilayer, of the liposome, depending on whether the compounds are water-soluble or fat soluble, respectively.

Thus, the liposomal vesicles may be prepared by the following sequence of steps: a) dissolving phospholipids, e.g. from lecithin extracts, in an organic solvent to form a solution, b) simultaneously stirring and drying the solution in a vessel under reduced pressure, forming a dry film on the wall of the vessel, c) adding to the vessel an aqueous solution which may be a physiological salt solution, and agitating the solution, thereby removing the dry film from the wall of the vessel. The formation of vesicles is completed upon further addition of water.

In step a) fat-soluble nutritional supplement(s) may be added to the solution, and/or in step c} water-soluble nutritional supplement(s} may be added to the solution. The resulting suspension of liposomal vesicles may be filtered to result in a more uniform vesicle size. The aqueous solution of step c) is located inside the liposomal vesicles as the second aqueous composition. The further added water is located outside of the vesicles as the first aqueous composition.

Thus, this sequence of steps results in liposomal vesicles comprising a phospholipid bilayer, wherein the liposomal vesicles are dispersed in a first aqueous composition. The phospholipid bilayer encloses a second aqueous composition. The nutritional supplement(s) added in step a) are primarily located within the phospholipid bilayer, whereas the nutritional supplement(s) added in step (c) are primarily located inside of the liposomal vesicles, thus in the second aqueous composition.

Within the phospholipid bilayer of the liposomal vesicles, fat-soluble ingredients, i.e. vitamins, minerals, enzymes, proteins, peptides, and/or herbal extracts, may be comprised. Water- soluble ingredients will be comprised in the second aqueous composition. Ingredients which are both water- and fat-soluble may be comprised either in the second aqueous composition, within the phospholipid bilayers, or both.

Only after the liposomes comprising nutritional supplements in their core and/or in their wall have been formed, the ions Ca?*, Mg?*, and/or Zn?" are added. For example, the ions may be added as a solution of a salt of the ions. By adding the solution to the total liposomal composition, the salt is inherently added to the first aqueous composition. The second aqueous composition is shielded from the first aqueous composition by the phospholipid bilayer.

It is speculated that the ions are chelating with the phosphate groups of the phospholipids. The ions thereby effectively close off the liposomes, thereby preventing leakage of the nutritional supplements. The ions are divalent positive ions, such as Ca?’, Mg? and/or Zn?*. Calcium ions are preferred, as calcium can safely be added in relatively high amounts as compared to the other ions. The acceptable upper limit for daily intake from supplements and nutrition is 2.5 grams per day, as compared to 250 mg of magnesium from supplements, and 25 mg per day for Zinc.

Due to the method according to the invention, the ingredients within the liposomes, i.e.

the ingredients within the core as well as those within the bilayer are retained within the liposomes. There is less leakage of the ingredients/the biologically active compounds into the external aqueous phase, i.e. the first aqueous composition. Moreover, the size of the liposomes is reduced, thereby resulting in an increased uptake of the liposomes in the body after ingestion.

The invention also relates to a composition for oral administration to a subject, the composition comprising liposomal vesicles dispersed in a first aqueous composition, wherein the liposomal vesicles comprise one or more phospholipid bilayers enclosing a second aqueous composition, the liposomal vesicles further comprising one or more nutritional supplements selected from the group consisting of vitamins, minerals, enzymes, proteins, peptides, and/or herbal extracts, and wherein the combined concentration of Ca?*, Mg?*, and Zn? ions in the first aqueous composition is higher than 4 uM, preferably higher than 20 uM.

The composition of the invention may be discerned from prior art compositions in that the combined concentration of the ions Ca?*, Mg?*, and Zn?" ions in the first aqueous composition is higher than 4 uM, preferably higher than 20 uM. For example the concentration in the first aqueous compasition may be between 4 and 400 20 uM, preferably between 20 and 200 uM, more preferably between 20 and 80 20 uM. In prior art compositions, the combined amount of Ca?*, Mg?*, and Zn?" ions in the first aqueous composition, in other words: outside of the vesicles, is either non-existent or smaller than that.

If the nutritional supplements themselves do not comprise the mentioned ions, then the Ca?*, Mg?*, and Zn?" ions in the total composition, and therefore the ions in the first aqueous composition can only originate from the addition of the ions according to the method of the invention. In some cases however, the nutritional supplements may themselves be salts of Ca?*, Mg?*, and/or Zn?*. For example in the case of calcium ascorbate as a source of vitamin C. However, in this case the Ca? ions originating from the nutritional supplement will be located inside the liposomal vesicles. The ions Ca?*, Mg?*, and/or Zn?* outside of the liposomal vesicles, thus in the first aqueous composition, originate from the added ions, and the concentration in the first aqueous composition is higher than 4 uM, preferably higher than 20 uM. Even though some of the ions in prior art liposomal compositions may leak from within the liposomal vesicles - thus from the second aqueous composition - into the surrounding liquid - i.e. into the second aqueous composition - such concentrations of the ions are not obtained in prior art liposomal compositions.

The invention also relates to a composition for oral administration to a subject, the composition comprising liposomal vesicles dispersed in a first aqueous composition, wherein the liposomal vesicles comprise one or more phospholipid bilayers enclosing a second aqueous composition, the liposomal vesicles further comprising one or more nutritional supplements selected from the group consisting of vitamins, minerals, enzymes, proteins, peptides, and/or herbal extracts, and wherein the combined concentration of Ca?*, Mg?*, and Zn?" ions in the first aqueous composition is higher than the combined concentration of Ca?*, Mg?*, and Zn?" ions in the second aqueous composition. This is especially true when the nutritional supplements do not comprise calcium, magnesium or zinc.

The composition of the invention may be used as a food supplement. Description of embodiments Preferably, the ions are added in the form of a salt. The salt may be any salt that is soluble, at least to some extent, in water. Examples are citrate salts, ascorbate salts, and halide salts, such as chloride salts. An ascorbate salt provides additional nutritional value to the product.

Preferably the salt is a halide salt, such as a fluoride, chloride, bromide or iodide salt. For example, the salt may be calcium chloride (CaCl,). The advantage of halide salts is that they have an excellent solubility in water, thereby preventing the formation of precipitates.

Preferably, the salt is comprised in a third aqueous salt solution. Although the salt may be added to the first aqueous composition by adding the salt in its solid form, addition of an aqueous solution provides for better results.

Preferably the concentration of the salt in the third aqueous salt solution is between

0.01 and 1.0 % (w/v,) preferably between 0.05 and 0.5% (w/v), most preferably between 0.05 and 0.2% (w/v).

Preferably, the nutritional supplement is chosen from cannabidiol, vitamin C, vitamin 5 B12, curcumin and/or coenzyme Q10.

Cannabidiol (CBD) is one of the at least 113 cannabinoids identified in cannabis. It is a major phytocannabinoid, which accounts for 40% of the plant's extract. CBD does not appear to have any psychoactive effects such as those caused by tetrahydrocannabinol (THC). It is believed to have a downregulating impact on disordered thinking and anxiety. Potential uses are the subject of ongoing research. Cannabidiol is insoluble in water but soluble in organic solvents such as pentane and edible oils.

Vitamin C is also known as ascorbic acid or L-ascorbic acid. Instead of the pure vitamin it may be used in the form of salts like sodium ascorbate, potassium ascorbate and/or any other pharmaceutically acceptable salt. These salts have lesser pH values and are therefore result in lesser gastro-intestinal problems. Vitamin C and it salts are water soluble and an essential nutrient involved in the repair of tissue and the enzymatic production of certain neurotransmitters. Vitamin C is required for the functioning of several enzymes and is important for immune system function. It also functions as an antioxidant. High-dose vitamin C has been studied as a treatment for patients with cancer since the 1970s.

Vitamin B12 is a vitamin that plays a role in mammalian growth, haematopoiesis, production of epithelial cells, and maintenance of the nervous system. It is quite water-soluble and thus could be expected to be easily available to human subjects. However, the absorption from the gut of normal dietary amounts of vitamin B12 is believed to be dependent on gastric Intrinsic Factor (GIF), and the loss of Intrinsic Factor leads to vitamin B12 deficiency. The loss of ability to absorb vitamin B12 (B12) is the most common cause of adult B12 deficiency. Such a loss may, for example, be due to pernicious anaemia (with loss of Intrinsic Factor) or to a number of other conditions that decrease production of gastric acid, which also plays a part in absorption of B12 from foods. Deficiency is most significantly linked to inadequate absorption rather than low consumption, as those who consume high amounts of vitamin B12 may still experience deficiency as evidenced by a low blood concentration.

Curcumin is the principal curcuminoid of turmeric (Curcuma longa), a member of the ginger family, Zingiberaceae. Curcumin shows positive results in many drug discovery assays.

Coenzyme Q10 is a coenzyme that is ubiquitous in animals and most bacteria. It is a 1,4-benzoquinone, where Q refers to the quinone chemical group and 10 refers to the number of isoprenyl chemical subunits in its tail. It is a fat-soluble substance, which resembles a vitamin, and is present in all respiring eukaryotic cells, primarily in the mitochondria. It is a component of the electron transport chain and participates in aerobic cellular respiration. Coenzyme Q10 is widely used as a dietary supplement.

Preferably, the liposomal vesicles have size of between 50 — 500 nm for a good cellular uptake of the vesicles. More preferably the liposomal vesicles have size of between 50-250 nm, to provide for better cellular uptake. Most preferably the dehydrated liposomal vesicles have size of between 50 — 200 nm. Cellular uptake of such vesicles is optimal. The size of the vesicles is defined as the diameter of the smallest sphere that can enclose the vesicle.

Preferably, the composition comprises between 1 — 50 w/w% of nutritional supplement.

Preferably, the phospholipid bilayer comprises phospholipids chosen from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and/or glycerophosphocholine.

Preferably, concentration of phospholipids in the total composition is between 5 and 40 % (w/v), more preferably between 5 and 20 % (w/v), most preferably between 5 and 15 % (w/v).

As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the clauses and as a representative basis for teaching one skilled in the art to variously employ the present invention in any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting, but rather, to provide an understandable description of the invention.

The terms "a"/"an", as used herein, are defined as one or more than one. The terms including and/or having, as used herein, are defined as comprising (i.e., open language, not excluding other elements or steps). The mere fact that certain measures are recited in mutually different dependent clauses does not indicate that a combination of these measures cannot be used to advantage. All reaction conditions are under atmospheric pressure, unless otherwise indicated.

Brief description of the drawings Figure 1 is a schematic drawing of a liposome.

Figure 2a is a TEM image of liposomes according to comparative example 1.

Figure 2b is a TEM image of liposomes according to example 1.

Figure 3a shows DLS curves of liposomes according to comparative example 1.

Figure 3b shows DLS curves of liposomes according to example 1.

Detailed description of Figure 1 Figure 1 is a schematic drawing of a liposome 1. The liposome 1 comprises a bilayer 2 of phospholipids with a hydrophilic head 4 and hydrophobic tail 5. The liposome is surrounded by a first aqueous composition (no number) in which the liposome is suspended. The liposome comprises a second aqueous composition 3 enclosed by the bilayer 2. Instead of only one bilayer 2, a liposome may comprise multiple bilayers surrounding the second aqueous composition 3. Examples Comparative Example 1 50 g of Phospholipids were dissolved in a flask in 100 ml of an organic solvent after which the organic solvent was removed under reduced pressure while agitating, thereby forming a dry lipid film on the internal wall of the flask. Subsequently the material was diluted with 200 ml of a solution comprising 100 g vitamin C (a mixture of sodium ascorbate, potassium ascorbate and ascorbic acid). All materials were mixed under firm agitation, thereby creating a slurry. Then 250 ml of water was added to obtain a total of about 500 ml of a liposomal suspension. Nano sized particles of about 50 — 500 nm were subsequently obtained by sonification and/or by high pressure extrusion. The particles were sieved through a 0.2 micron sieve in order to separate the particles with a size of 200 nm or less from the larger particles and aggregates. After sieving, the particles were checked with light microscopy and laser diffraction. Figure 2a shows a TEM image of thus obtained liposomal vitamin C.

Example 1: Stabilized Liposomes 500 ml liposomal solution of Comparative Example 1 was taken in a 1 L beaker. 5 ml of a solution of 0.1 % (w/v) Calcium Citrate in water was added into it slowly. The mixture was stirred very well using a hand mixer at 1000 — 2000 rpm. During stirring, the temperature of the mixture was maintained around 40 °C. This process was continued for at least 25 — 30 minutes. After this, the mixture was allowed to settle down for 5 minutes. After filtration through a wide pore filter (> 1 micron) leaving out the sedimented particles, the mixture was subsequently homogenized using a high pressure homogeniser at a pressure of 300 bar. After the first pass (first homogenisation step) the resulting mixture was brought down to 30 °C. At this temperature, the homogenisation was repeated at the same pressure of 300 bar (second homogenisation step). Finally, the resulting solution was filtered through a 0.2 micron filter in order to exclude any sediments or bigger particles and stored at 25 to 30 °C. Figure 2b shows a TEM image of thus obtained stabilized liposomal vitamin C. Transition electron microscopic analysis (TEM) Figure 2 shows the electronic microscopic images of comparative example 1 (Fig. 2a) and stabilized liposomes (Fig. 2b), respectively. There is a notable difference in the structure, geometry and dispersion of particles. Fig. 2a indicates a non-homogeneous particle shape distribution. Fig. 2b however indicates a spherical core shell like structure. It is speculated that in the comparative example (Fig. 2a), water from within the liposomes tends to leak out since there is no specific bonding to hold the structure. It is speculated that due to this leakage, the particles do not have any well-organized structure. For the stabilized liposomes on the other hand, it can be seen that the particles are arranged in a well-organized globular pattern (figure 3b). It is speculated that this is the reason for non-leaking phenomena.

Dynamic light scattering analysis (DLS) Comparative example 1 and the stabilized liposomes were also measured with DLS to give an idea of the particle size distribution. It should be noted that with DLS the hydrodynamic volume of the suspended particles is measured, which results in different sizes than those that can be seen with TEM. Figure 3 (a and b) shows the intensity distribution values of comparative example 1 and stabilized liposomes, respectively. Both distributions were measured three times, and therefore the figures 3a and 3b each comprise 3 lines.

For the comparative example, most particles have a size of about 200 nm-300 nm. The curve is relatively wide, indicating inhomogeneity in sample geometry. In the case of stabilized liposomes, the curve is sharper due to spherical structures and consistency in the geometry. Most of the particles have a size of about 100 nm.

It can be concluded from the DLS data that, after the stabilization of the liposomes, the size of the particles decreases. This could be because of the formation of a more compact structure which is non-leaking. Repulsive forces are speculated to bind the particles tightly to form a core shell like structure as evident from figure 2b.

The decrease in particle size has two merits. (1) As the size of the particle decreases, bioavailability (the chance to adsorb the liposomes in the body) increases, and (2) it results in a more stable colloidal suspension due the increase in surface to volume ratio.

Zeta potential measurements The zeta potential of comparative example 1 was measured to be -25.6 mV and for the stabilized liposomes -25.7 mV. Thus, for the stabilized liposomes, there is a slight reduction in the zeta potential value. It is speculated that this may be because after the addition of calcium citrate salt, the multivalent cation Ca?* specifically absorbs on the liposomal surface and the sample as a whole tends to form a globular particle and reduces its zeta value. Conclusion of experiments Due to the addition of calcium ions, the liposomes tend to form a more spherical structure as evident from TEM micrographs. This is due to the non-leaking phenomenon from the modified liposomal structure. The size of material decreases after modification, which favourably enhances the bioavailability. Also, the zeta potential value slightly decreases confirming the formation of a more compact structure and better stability.

Vitamin C is a water soluble vitamin. However, the results are expected to be comparable for fat soluble ingredients such as cannabidiol and coenzyme Q10.

Claims (13)

-10 - CONCLUSIES-10 - CONCLUSIONS 1. Werkwijze voor het stabiliseren van liposomale blaasjes gedispergeerd in een eerste waterige samenstelling, waarbij de liposomale blaasjes een fosfolipide dubbellaag omvatten die een tweede waterige samenstelling insluit, waarbij de liposomale blaasjes verder één of meer voedingssupplementen omvatten gekozen uit de groep bestaande uit vitaminen, mineralen, enzymen, eiwitten, peptiden en/of kruidenextracten, waarbij de werkwijze het toevoegen van ionen aan de eerste waterige samenstelling omvat, waarbij de ionen worden gekozen uit Ca?*, Mg?’ en/of Zn?"A method of stabilizing liposomal vesicles dispersed in a first aqueous composition, wherein the liposomal vesicles comprise a phospholipid bilayer enclosing a second aqueous composition, the liposomal vesicles further comprising one or more nutritional supplements selected from the group consisting of vitamins, minerals , enzymes, proteins, peptides and / or herbal extracts, the method comprising adding ions to the first aqueous composition, the ions being selected from Ca? *, Mg? 'and / or Zn? " 2. Werkwijze volgens conclusie 1, waarbij ionen worden toegevoegd in de vorm van een zout.A method according to claim 1, wherein ions are added in the form of a salt. 3. Werkwijze volgens conclusie 1 of 2, waarbij het zout een halogenidezout is.A method according to claim 1 or 2, wherein the salt is a halide salt. 4. Werkwijze volgens willekeurig welke van de conclusies 2 - 3, waarbij het zout is omvat in een derde waterige zoutoplossing.The method of any of claims 2 to 3, wherein the salt is contained in a third aqueous saline solution. 5. Werkwijze volgens conclusie 4, waarbij de concentratie van het zout in de derde waterige zoutoplossing tussen 0,01 en 1,0 %, bij voorkeur tussen 0,05 en 0,5 %, met de meeste voorkeur tussen 0,05 en 0,2 % (w/v) ligt.A method according to claim 4, wherein the concentration of the salt in the third aqueous salt solution is between 0.01 and 1.0%, preferably between 0.05 and 0.5%, most preferably between 0.05 and 0 , 2% (w / v). 6. Samenstelling voor orale toediening aan een individu, waarbij de samenstelling liposomale blaasjes gedispergeerd in een eerste waterige samenstelling omvat, waarbij de liposomale blaasjes één of meer fosfolipidedubbellagen omvatten die een tweede waterige samenstelling insluiten, waarbij de liposomale blaasjes verder één of meer voedingssupplementen omvatten gekozen uit de groep bestaande uit vitaminen, mineralen, enzymen, eiwitten, peptiden en/of kruidenextracten, en waarbij de gecombineerde concentratie van Ca?*-, Mg?'- en Zn?*-ionen in de eerste waterige samenstelling hoger is dan 4 uM, bij voorkeur hoger dan 20 HM.A composition for oral administration to an individual, the composition comprising liposomal vesicles dispersed in a first aqueous composition, the liposomal vesicles comprising one or more phospholipid bilayers enclosing a second aqueous composition, the liposomal vesicles further comprising one or more nutritional supplements selected. from the group consisting of vitamins, minerals, enzymes, proteins, peptides and / or herbal extracts, and where the combined concentration of Ca? *, Mg? 'and Zn? * ions in the first aqueous composition is greater than 4 µM , preferably higher than 20 HM. 7. Samenstelling voor orale toediening aan een individu, waarbij de samenstelling liposomale blaasjes gedispergeerd in een eerste waterige samenstelling omvat, waarbij de liposomale blaasjes één of meer fosfolipide dubbellagen omvatten die een tweede waterige samenstelling insluiten, waarbij de liposomale blaasjes verder één of meer voedingssupplementen omvatten gekozen uit de groep bestaande uit vitaminen, mineralen, enzymen, eiwitten, peptiden en/of kruidenextracten, en waarbij de gecombineerde concentratie Ca?’-, Mg?'- en Zn?*-ionen in de eerste waterige samenstelling hoger is dan deA composition for oral administration to an individual, the composition comprising liposomal vesicles dispersed in a first aqueous composition, the liposomal vesicles comprising one or more phospholipid bilayers enclosing a second aqueous composition, the liposomal vesicles further comprising one or more nutritional supplements. selected from the group consisting of vitamins, minerals, enzymes, proteins, peptides and / or herbal extracts, and wherein the combined concentration of Ca? ', Mg?' and Zn? * ions in the first aqueous composition is higher than the -11 - gecombineerde concentratie van Ca?*-, Mg?*- en Zn?*-ionen in de tweede waterige samenstelling.-11 - combined concentration of Ca? * -, Mg? * - and Zn? * - ions in the second aqueous composition. 8. Samenstelling volgens conclusie 6 of 7, waarbij het voedingssupplement wordt gekozen uit cannabidiol, vitamine C, vitamine B12, curcumine en/of co-enzym Q10.Composition according to claim 6 or 7, wherein the nutritional supplement is selected from cannabidiol, vitamin C, vitamin B12, curcumin and / or coenzyme Q10. 9. Samenstelling volgens willekeurig welke van de conclusies 6 - 8, waarbij de liposomale blaasjes een grootte hebben van tussen 50 - 500 nm, bij voorkeur tussen 50 - 250 nm, met de meeste voorkeur tussen 50 - 200 nm.Composition according to any of claims 6-8, wherein the liposomal vesicles have a size of between 50-500 nm, preferably between 50-250 nm, most preferably between 50-200 nm. 10. Samenstelling volgens willekeurig welke van de conclusies 6 - 9, waarbij de samenstelling tussen 1 - 50 w/w% voedingssupplement omvat.The composition of any of claims 6-9, wherein the composition comprises between 1 - 50 w / w% nutritional supplement. 11. Samenstelling volgens willekeurig welke van de conclusies 6 - 10, waarbij de fosfolipide dubbellaag fosfolipiden omvat gekozen uit de groep bestaande uit fosfatidylcholine, fosfatidylethanolamine, fosfatidylserine, fosfatidylinositol en/of glycerofosfocholine.A composition according to any of claims 6-10, wherein the phospholipid bilayer comprises phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and / or glycerophosphocholine. 12. Samenstelling volgens willekeurig welke van de conclusies 8 - 11, waarbij de concentratie van fosfolipiden in de totale samenstelling tussen 5 en 40% (w/v), bij voorkeur tussen 5 en 20% (w/v), met meer voorkeur tussen 5 en 15% (w/v) ligt.Composition according to any of claims 8-11, wherein the concentration of phospholipids in the total composition is between 5 and 40% (w / v), preferably between 5 and 20% (w / v), more preferably between 5 and 15% (w / v). 13. Gebruik van een samenstelling volgens willekeurig welke van de conclusies 6 - 12 als voedingssupplement.Use of a composition according to any of claims 6-12 as a dietary supplement.