Lipid encapsulated compositions

Abstract

The invention provides substantially dehydrated composition comprising an active agent, a lipid layer, and a network of fibres. The lipid layer encapsulates the network of fibres within the inner surface of the lipid layer. The network of fibres serves to inhibit deformation of the lipid layer. The substantially dehydrated composition can be hydrated for use. In keeping with this, there is also provided a lipid encapsulated composition comprising an active agent, a lipid layer and an aqueous core, wherein the lipid layer encapsulates the aqueous core, and the aqueous core comprises a solution comprising fibres. The lipid layer may be a liposome, and the lipid may be a phospholipid, for example lecithin. Fibres may comprise inulin, chitin, or chitosan. Active agents may be selected from a vitamin active agent, a mineral active agent, an extracellular matrix active agent, a herbal, fungal or botanic active agent, an oil or fatty acid active agent, a metabolically active agent, and a probiotic agent. Methods of manufacturing the compositions are also provided.

Description

[0001] LIPID ENCAPSULATED COMPOSITIONS

[0002] FIELD OF INVENTION

[0003] The present invention relates to compositions comprising lipid layers, such as liposomes, which encapsulate a network of fibres within an inner surface of the lipid layer. It also relates to compositions comprising lipid layers, such as liposomes, which encapsulate an aqueous core comprising a solution comprising fibres. The invention also relates to methods of manufacturing such compositions.

[0004] BACKGROUND

[0005] Lipid structures comprising a lipid layer, such as liposomes, represent popular vehicles for the delivery of active agents in a range of different contexts.

[0006] However, there are a number of drawbacks associated with known liposomal formulations. Among these, is liposomal leakage. This effect is frequently observed in respect of compositions using lipid encapsulation and involves loss of the liposomes’ payload overtime. This leakage thus adversely impacts stability of the compositions, and, as a consequence of the leakage occurring, such compositions may be subject to marked reduction in potency over time.

[0007] Furthermore, substantially dehydrated liposomal formulations also exhibit a number of drawbacks. During drying, and upon complete dehydration the liposome is often damaged or deformed, causing leakage. Similarly, to conventional non-dehydrated liposomal formulations, this effect involves the loss of the liposomes’ payload overtime and adversely impacts stability.

[0008] Lipid encapsulated formulations such as liposomal formulations of the prior art may also be subject to limitations in terms of the loading capacity and encapsulation efficiency that may be achieved and also the bioavailability of active agents from the compositions.

[0009] SUMMARY OF INVENTION

[0010] In a first aspect, the invention provides a substantially dehydrated composition comprising:

[0011] • an active agent;

[0012] • a lipid layer; and • a network of fibres; wherein the lipid layer encapsulates the network of fibres within the inner surface of the lipid layer.

[0013] In a second aspect, the invention provides a lipid encapsulated composition comprising:

[0014] • an active agent;

[0015] • a lipid layer; and

[0016] • an aqueous core; wherein the lipid layer encapsulates the aqueous core, and the aqueous core comprises a solution comprising fibres.

[0017] When used for the delivery of therapeutically effective active agents, compositions in accordance with the first or second aspect of the invention are suitable for use as medicaments.

[0018] In a third aspect, the invention provides a method of manufacturing a lipid encapsulated composition, the method comprising:

[0019] • combining:

[0020] • an aqueous solution comprising fibres;

[0021] • a lipid; and

[0022] • an active agent;

[0023] • forming a lipid layer from the lipid that encapsulates the aqueous solution comprising the fibres thereby forming a lipid encapsulated composition; and dehydrating the composition such that a network of fibres is formed within the lipid layer.

[0024] A method in accordance with the third aspect of the invention may suitably be used in manufacturing a composition in accordance with the first aspect of the invention.

[0025] Except for where context requires otherwise, any reference to a “composition of the invention” may be taken as referring to a composition in accordance with any embodiment of the first or second aspect of the invention, or a composition produced by the methods of the third aspect of the invention.

[0026] Except for where context requires otherwise, any reference to a “method of the invention” may be taken as referring to a method in accordance with any embodiment of the third aspect of the invention. As described further below, liposomes constitute particularly suitable forms of lipid layers for use in the compositions or methods of the invention.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows a flow diagram illustrating a method of the second aspect of the invention, useful in the production of a substantially dehydrated composition of the invention.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The inventors have developed new and advantageous lipid encapsulated compositions that can be used for the delivery of active agents. The new compositions offer particular advantages when they are substantially dehydrated. Substantially dehydrated compositions of the present invention can be taken by a recipient in their dehydrated form, or can be rehydrated prior to consumption by a recipient.

[0031] The dried compositions of the invention incorporate a network of fibres found within the inner surface of the lipid layer. In the case of lipid encapsulated compositions comprising a liposome as the lipid layer, the network of fibres may be present within the inner membrane of the liposome. In the hydrated form of the compositions of the invention, the fibres are present within the aqueous core of the encapsulating lipid layer. However, as the compositions are dried, and the aqueous core disappears, the fibres become ordered, and form into a stable network. The network of fibres provides a scaffold that effectively supports the lipid layer (for example a liposomal membrane), preventing deformation and damage.

[0032] This approach taken by the inventors confers a number of advantages on the compositions of the invention.

[0033] In particular, compositions of the invention have markedly advantageous stability as compared to dried lipid encapsulated compositions (such as dried liposomal compositions) of the prior art. Removal of water during drying of conventional lipid encapsulated compositions (such as liposomal formulations) often results in deformation of and damage to the lipid layers. This can compromise the integrity of the lipid layers leading to loss of the active agent and increasing incidences of leakage. The improved stability of the compositions of the invention, and support provided to the lipid layers (such as liposome membranes), reduces the tendency of the lipid encapsulated compositions to be damaged, and so reduces the likelihood of leakage from the compositions. Since the active agents are retained and protected within the intact lipid layers, the compositions of the invention also increase the bioavailability of active agents incorporated in the compositions.

[0034] The inventors have identified fibres that have characteristics that cause them to form networks within a lipid layer (such as a liposome) when a composition comprising the lipid structure is dehydrated. Without wishing to be bound by any hypothesis, the inventors believe that the formation of the network is driven by the action of van der Waals forces between the fibres. Selection of fires with appropriate properties is thus important in the production of the lipid encapsulated compositions of the invention.

[0035] The advantageous stability of the compositions of the invention may be demonstrated by any appropriate means. For example, the reduction in deformation of the lipid layer membranes (such as liposome membranes) within the compositions of the invention may be illustrated by appropriate electron microscopy techniques. Stability of the composition of the invention may also be demonstrated by the magnitude of their zeta potentials.

[0036] The compositions of the invention exhibit improved encapsulation efficiency and improved loading capacity as compared to compositions of the prior art. It will be appreciated that these improvements will provide benefits in a wide range of contexts and uses.

[0037] Advantageously, the fibres may have beneficial properties themselves. For example, inulin, which may be used as a suitable material for fibres in the compositions of the invention, can have probiotic activity that complement the properties of other active agents provided within the formulation.

[0038] Further advantages of the compositions of the invention will be apparent to the skilled reader of this disclosure.

[0039] The invention will now be further described, with reference to the definitions provided in the following pages.

[0040] Compositions of the invention

[0041] Except for where the context requires otherwise, references herein to “compositions of the invention” should be taken as directed to compositions in accordance with the first or second aspect of the invention, or to compositions that are the products of the methods of the third aspect of the invention.

[0042] Since the methods of the third aspect of the invention may be used to manufacture compositions in accordance with the first or second aspect of the invention, it will be appreciated that (unless context requires otherwise) features disclosed herein in connection with the compositions of the first or second aspect of the invention should also be taken as disclosed in connection with their use in the methods of the third aspect of the invention, and features disclosed in connection with the methods of the third aspect of the invention should also be taken as disclosed in connection with their use in the compositions of the first or second aspect of the invention.

[0043] Compositions of the present invention comprise lipid layers (such as liposomes) encapsulating fibres. These fibres form networks in dehydrated forms of the compositions, and are present in solutions in hydrated forms. Suitable examples of lipid layers may include lipid monolayers, or lipid bilayers, such as liposomes. Liposomes are structures comprising amphipathic molecules, such as phospholipids, which are characterised by their hydrophobic (waterrepelling) and hydrophilic (water-attracting) properties. In aqueous conditions, phospholipids are known to form layers which have an inner and outer membrane. In lipid layers (such as liposomes) found in compositions of the first aspect of the present invention the fibres form a network underlying the inner surface of the lipid, such as an inner membrane of a liposome.

[0044] In a suitable embodiment, a substantially dehydrated composition of the first aspect of the invention comprises:

[0045] • an active agent;

[0046] • a liposome; and

[0047] • a network of fibres; wherein the liposome encapsulates the network of fibres within the inner membrane of the liposome.

[0048] In a suitable embodiment, a composition of the second aspect of the invention provides a liposomal composition comprising:

[0049] • an active agent;

[0050] • a liposome; and

[0051] • an aqueous core; wherein the liposome encapsulates the aqueous core, and the aqueous core comprises a solution comprising fibres.

[0052] Compositions of the present invention can be in a substantially dehydrated form (in accordance with the first aspect of the invention) or in aqueous form (in accordance with the second aspect of the invention). Compositions in accordance with first aspect of the invention, or the products of the third aspect of the invention, can be rehydrated in an aqueous diluent, such as water, to provide a composition in accordance with the second aspect of the invention.

[0053] The compositions and methods of the invention may make use of a wide range of active agents, as considered further below. For example, the compositions and methods of the invention may comprise hydrophilic active agents, and / or hydrophobic active agents.

[0054] As will be appreciated, hydrophilic active agents will tend to be located in the aqueous core of compositions of the second aspect of the invention. When such composition are dehydrated, leading to the loss of the water in the aqueous core, a hydrophilic active agent will remain encapsulated within the lipid layer.

[0055] In contrast, hydrophobic active agents will tend to be located in the lipid layer, for example dissolved in the lipid. This distribution of a hydrophobic active agent will generally not change, irrespective of the hydration status of the composition.

[0056] In a suitable embodiment, a composition of the invention comprises a hydrophilic active agent. In such an embodiment of a composition of the first aspect of the invention, the hydrophilic active agent may be encapsulated within the lipid layer. For example, in the case of a composition of the first aspect of the invention comprising a liposome, the hydrophilic active agent may be encapsulated within the liposome. In such an embodiment of a composition of the second aspect of the invention, the hydrophilic active agent may be present in the aqueous core encapsulated within the lipid layer. For example, in the case of a composition of the second aspect of the invention comprising a liposome, the hydrophilic active agent may be present in the aqueous core encapsulated within the liposome.

[0057] In a suitable embodiment, a composition of the invention comprises a hydrophobic active agent. In such an embodiment of a composition of the first aspect of the invention, or of a composition of the second aspect of the invention, the hydrophobic active agent may be present in the lipid of the lipid layer. Suitably, in the case of a composition of the invention comprising a liposome, a hydrophobic active agent may be present in a membrane of the liposome. In a suitable embodiment, a hydrophobic active agent is present in the inner membrane of a liposome. In a suitable embodiment, a hydrophobic active agent is present in the outer membrane of a liposome. In a suitable embodiment, a hydrophobic active agent is present in both the inner and outer membranes of a liposome.

[0058] The compositions of the invention may be provided in any suitable form. For example, they may be provided for administration to a recipient in a substantially dry form. Suitable examples of such dried forms include tablets or powders comprising the compositions of the invention.

[0059] Alternatively, the compositions of the invention may be provided for administration to a recipient in a hydrated form. In particular, the composition may be hydrated with water, or another suitable aqueous diluent. Examples of such hydrated forms, include liquids, such as a syrups or gels.

[0060] The compositions of the invention may be further formulated in any required manner. Merely by way of example, powder or liquid forms of the compositions of the invention may be further formulated in gel capsules, or the like.

[0061] In a suitable embodiment, the composition of the invention is in the form of a nutritional supplement or foodstuff. Suitable active agents that may be incorporated in compositions of the invention in accordance with this embodiment are discussed in more detail elsewhere in the present specification.

[0062] “Encapsulation” as compared to “present in”

[0063] For the purposes of the present disclosure, a reference to “encapsulation”, or to a feature that “encapsulates” another or is “encapsulated within” another, should be taken as meaning that the encapsulating feature surrounds the encapsulated feature, but that the two remain distinct from one another. By way of example, a network of fibres or an aqueous core “encapsulated” by a lipid layer are surrounded by, but separate from, the lipid layer.

[0064] In contrast a feature that is stated to be “present in” a second feature is to be construed as being continuous with the second feature. For example, a hydrophobic active agent “present in” the lipid of a lipid layer may be dissolved within the lipid, or embedded in, the lipid. Dehydrating compositions

[0065] The skilled person will recognise that there are multiple common techniques that may be used to dehydrate a composition, and thus to generate a substantially dehydrated lipid encapsulated composition such as a substantially dehydrated liposomal composition. Suitable techniques that may be used to produce compositions of the first aspect of the invention, such as by the methods of the third aspect of the invention, include, but are not limited to: spray drying, freeze-drying, spray-freeze-drying or electro spraying.

[0066] Drying adjuvants are substances that protect the constituents of a composition during a drying process. It will be appreciated that drying adjuvants may usefully be employed in the compositions and / or methods of the invention. Thus, in a suitable embodiment, a composition of the of the invention further comprises a drying adjuvant. The drying adjuvant may be present in a composition in accordance with the first aspect of the invention. Suitably, a drying adjuvant may be present in a composition in accordance with the second aspect of the invention (for example, in a composition that is to substantially dehydrated, or a composition that has been formed on re-hydration of a previously dehydrated composition).

[0067] Suitably, gum Arabic is used as a drying adjuvant in a composition or method of the invention.

[0068] The term “substantially dehydrated”, used in the context of compositions of the invention, may be used to indicate that the composition comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% water w / w. Such a composition may comprise less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% water w / w.

[0069] Lipid layers suitable for use in the compositions or methods of the invention

[0070] The compositions of the invention comprise lipid layers, which are structures comprising lipids which encapsulate a mixture of fibres and optionally an active agent (in the case of composition of the invention comprising a hydrophilic active agent). In the case of compositions in accordance with the first aspect of the invention, the lipid encapsulates a network of fibres within the inner surface of the lipid layer, and optionally an active agent. In the case of compositions of the invention comprising a hydrophobic active agent, the active agent may be present in the lipid of the lipid layer. In a suitable embodiment, the lipid layers of a composition of the invention comprise a phospholipid.

[0071] In a suitable embodiment, the lipid layers of the composition comprise a monolayer, and the lipid comprises a phospholipid. Suitably in such an embodiment, the phospholipid may comprise a mixture of crude phospholipids comprising less than 50% phosphatidylcholines. Suitably, the phospholipid may comprise a mixture of crude phospholipids comprising approximately 10-15% phosphatidylcholines.

[0072] In a suitable embodiment, the lipid layers of the composition comprise a bilayer, and the lipid comprises a phospholipid. Suitably in such an embodiment, the phospholipid may comprise a mixture of crude phospholipids comprising more than 50% phosphatidylcholines. Suitably, the phospholipid may comprise a mixture of crude phospholipids comprising approximately 50-55% phosphatidylcholines.

[0073] In a suitable embodiment, the phospholipid is lecithin. Suitably, the lecithin may be derived from a source selected from the group consisting of: sunflower, grape seed, and soy beans.

[0074] In a suitable embodiment the lipid layer is a monolayer. In a suitable embodiment the lipid layer is a bilayer. A lipid monolayer refers to a single layer of lipid molecules. A lipid bilayer refers to a double layer of lipid molecules. Typically, lipid bilayers are formed by amphipathic molecules, such as phospholipids, with their hydrophobic tails facing inward and their hydrophilic heads facing outward toward the surrounding aqueous environments.

[0075] In the present disclosure, the lipid layers in the compositions of the invention encapsulate fibres, and may also encapsulate one or more hydrophilic active agents . In the case of compositions of the first aspect of the invention the fibres are incorporated in the form of a network of fibres located within the inner membrane of the lipid layer. In the case of compositions of the second aspect of the invention, the fibres are present in the aqueous core of the lipid layer.

[0076] The network of fibres within the lipid layer of dehydrated compositions of the invention supports the lipid layer (such as a liposome membrane) when dehydrated. The network of fibres thus reduces deformation of lipid layers within the compositions. This may be demonstrated by the shape of the lipid layers within these compositions. Suitably, the lipid layers within a dehydrated composition of the invention are non-flattened. For example, the lipid layers may be substantially spherical. In a suitable embodiment, the lipid layer comprises a liposome. Compositions of the invention comprising liposomes are discussed in more detail below.

[0077] Liposomes suitable for use in the compositions or methods of the invention

[0078] Suitably, compositions of the invention may comprise liposomes, which are structures comprising a lipid bilayer. When liposomes are hydrated the bilayer encapsulates an aqueous core.

[0079] In the present disclosure, liposomes in the compositions of the invention encapsulate fibres, and may also encapsulate one or more hydrophilic active agents . In the case of compositions of the first aspect of the invention the fibres are incorporated in the form of a network of fibres located within the inner membrane. In the case of compositions of the second aspect of the invention, the fibres are present in the aqueous core of the liposomes.

[0080] The network of fibres within the liposomes of dehydrated compositions of the invention supports the liposomal membrane when dehydrated. The network of fibres thus reduces deformation of liposomes within the compositions. This may be demonstrated by the shape of the liposomes within these compositions. Suitably, the liposomes within a dehydrated composition of the invention are non-flattened. For example, the liposomes may be substantially spherical.

[0081] In a suitable embodiment, the liposomes of a composition of the invention comprise a phospholipid. In a suitable embodiment, the phospholipid is lecithin. Suitably, the lecithin may be derived from a source selected from the group consisting of: sunflower, grape seed, and soy beans.

[0082] Benefits provided by fibres in compositions of the invention

[0083] A referred to herein, fibres are incorporated in the compositions of the invention, and are used in the methods of the third aspect of the invention. In the compositions of the first aspect of the invention, the fibres form a network within the inner surface of the lipid layer, such as within the inner membrane of a liposome. In the compositions of the second aspect of the invention, the fibres are present in the aqueous core encapsulated by the lipid layer. For the purposes of the present disclosure, a “network” of fibres is defined as any supportive structure composed of a suitable fibre. The networks found in the compositions of the first aspect of the invention may self-assemble on dehydration of the compositions. For the avoidance of doubt, reference to the network being “within” the inner surface of a lipid layer (such as within the inner membrane of a liposome) merely requires that the network is encapsulated by the lipid layer (such as a liposome membrane), rather than that the network is inserted into, or incorporate within, the lipid layer itself (for example between layers of liposome’s inner and outer membranes).

[0084] The presence of a network of fibres within the dehydrated compositions of the invention supports the lipid layers within these compositions, and thereby enhances their stability. When conventional lipid encapsulated compositions (such as liposomal compositions) are dried, the lipid layers within the compositions are typically damaged. This then leads to loss of the active agent from within the lipid structure (for example, from within a liposome), and hence adversely impacts the stability of the compositions.

[0085] In contrast, the presence of a network of fibres in dehydrated compositions of the invention provides mechanical reinforcement to the lipid layer (such as a liposomal membrane). The network of fibres is formed during the dehydration process, and acts as a physical scaffold that supports the inner surface of the lipid layer (for example the inner membrane of a liposome), thereby reinforcing the structure of the lipid layer, and preventing its collapse or deformation under external stress, such as shear forces, osmotic pressure, or temperature fluctuations. This reinforcement enhances stability of the lipid layers (such as liposomes), thereby increasing their retention of active agents and hence increasing stability of the compositions.

[0086] Without being bound by any hypothesis, the inventors also believe that specific properties of the fibres, such as their length or elasticity, contribute to the enhanced stability achieved. Examples of suitable parameters in respect of these properties are provided below.

[0087] Fibres suitable for use in the compositions or methods of the invention

[0088] Generally, any suitable fibre may be employed in the compositions or methods of the invention. In a suitable embodiment, fibres used in a composition or method of the invention comprise inulin, chitin, or chitosan. Merely by way of example, fibres of a composition of the invention may comprise inulin. Alternatively, fibres of a composition of the invention may comprise chitin. In a further example, fibres of a composition of the invention may comprise chitosan.

[0089] In a suitable embodiment, fibres of a composition of the invention are formed from inulin, chitin, or chitosan. For example, fibres of a composition of the invention may be formed from inulin. Alternatively, fibres of a composition of the invention may be formed from chitin. In a further example, fibres of a composition of the invention may be formed from chitosan.

[0090] Compositions of the invention may comprise a network formed of a single type of fibre. Merely by way of example, a composition or method of the invention may make use of only inulin fibres. Alternatively, a composition or method of the invention may make use of only chitin fibres. In a further example, a composition or method of the invention may make use of only chitosan fibres.

[0091] In a suitable embodiment, compositions of the invention may comprise of two or more types of fibres. For example, a composition or method of the invention may make use of both inulin and chitin fibres. Alternatively, a composition or method of the invention may make use of both inulin and chitosan fibres. In a further example, a composition or method of the invention may make use of both chitosan and chitin fibres.

[0092] The material from which the fibres are formed may contribute to the effectiveness of the compositions of the invention. Merely by way of example, inulin, which is a suitable example of a material from which suitable fibres may be formed, is known to function as a probiotic. Accordingly, individuals ingesting compositions of the invention comprising inulin fibres may receive a benefit from the probiotic effects of the inulin, in addition to the benefits provided by the active agents.

[0093] Merely byway of example, chitin, which is a suitable example of a material from which suitable fibres may be formed, is known to be biocompatible. Furthermore, chitin is also known to be biodegradable, non-toxic, and to have antimicrobial activity. Chitin is also known to have low immunogenicity. Accordingly, individuals ingesting compositions of the invention comprising chitin fibres may receive a benefit from one or all of these beneficial properties of chitin, in addition to the benefits provided by the active agents incorporated in the composition.

[0094] Chitosan represents another suitable example of a material from which fibres may be formed. Chitosan is also known to be biocompatible, biodegradable, and non-toxic. Furthermore, chitosan is known to chelate heavy metals, as well as having antimicrobial activity and low immunogenicity. Accordingly, individuals ingesting compositions of the invention comprising chitosan fibres may receive a benefit from one or all of these beneficial properties of chitosan, in addition to the benefits provided by the active agents.

[0095] In a suitable embodiment, the fibres within a network interact with one another via intermolecular forces. Examples of such forces include van der Waals forces. In a suitable embodiment, the fibres within a network are physically interwoven. In a suitable embodiment, the fibres within a network form a mesh. In a suitable embodiment, the fibres within a network are aligned with one another.

[0096] In a suitable embodiment, the interactions of the fibres present in a network in a composition of the first aspect of the invention are limited to interactions with other fibres. In such embodiments, the fibres do no interact directly with the active agent, and / or with the encapsulating lipid layer (for example with the inner or outer membrane of a liposome). Merely by way of example, the interactions of fibres present in a network in a composition of the invention may suitably be limited to interactions with other fibres via van der Waals forces.

[0097] Length of fibres suitable for use in the compositions or methods of the invention

[0098] In a suitable embodiment, the fibres of a composition of the invention have average lengths of between about 150nm and about 300nm. In a suitable embodiment, the fibres of a composition of the invention have a length of between about 150nm and about 300nm. Fibres with a length of between 150nm and 300nm have a higher surface area to volume ratio compared to longer fibres. The larger surface area of such fibres enables more van der Waals interactions between fibres. Such intermolecular bonding may contribute to improved stability of the liposomes, and hence improved stability of the compositions of the invention.

[0099] In a suitable example, fibres of a composition of the invention may have average lengths of between about 160nm and about 290nm, about 170nm and about 280nm, about 180nm and about 260nm, about 190nm and about 250nm, about 200nm and about 240nm or about 210nm and about 230nm. In a suitable example, fibres of a composition of the invention may have a length of between about 160nm and about 290nm, about 170nm and about 280nm, about 180nm and about 260nm, about 190nm and about 250nm, about 200nm and about 240nm or about 210nm and about 230nm.

[0100] In a suitable embodiment, the fibres of a composition of the invention have average lengths of 300nm or less. In a suitable example, fibres of a composition of the invention may have average lengths of 275nm or less, 250nm or less, 200nm or less, 150nm or less, 100nm or less or even 50nm or less. In a suitable embodiment, the fibres of a composition of the invention have a length of 300nm or less. In a suitable example, fibres of a composition of the invention may have a length of 275nm or less, 250nm or less, 200nm or less, 150nm or less, 100nm or less or even 50nm or less.

[0101] In a suitable embodiment, the fibres of a composition of the invention have average lengths of at least 50 nm. In a suitable example, fibres of a composition of the invention may have average lengths of at least 100nm, at least 150nm, at least 200nm, at least 250nm, at least 275nm, or at least 300nm. In a suitable embodiment, the fibres of a composition of the invention have a length of at least 50 nm. In a suitable example, fibres of a composition of the invention may have a length of at least 10Onm, at least 150nm, at least 200nm, at least 250nm, at least 275nm, or at least 300nm.

[0102] Suitably, the fibres of a composition of the invention have average lengths of about 150nm. In a suitable example, fibres of a composition of the invention may have average lengths of about 140nm, about 141 nm, about 142nm, about 143nm, about 144nm, about 145nm, about 146nm, about 147nm, about 148nm, about 149nm, about 150nm, about 151 nm, about 152nm, about 153nm, about 154nm, about 155nm, about 156nm, about 157nm, about 158nm, about 159nm or about 160nm. Suitably, the fibres of a composition of the invention have a length of about 150nm. In a suitable example, fibres of a composition of the invention may have a length of about 140nm, about 141 nm, about 142nm, about 143nm, about 144nm, about 145nm, about 146nm, about 147nm, about 148nm, about 149nm, about 150nm, about 151 nm, about 152nm, about 153nm, about 154nm, about 155nm, about 156nm, about 157nm, about 158nm, about 159nm or about 160nm.

[0103] Suitably, the length of the fibres may be demonstrated by assessment of high-resolution images, which may be produced via electron microscopy such as scanning electron microscopy (SEM) or transmission electron microscopy (TEM) or via Atomic Force Microscopy (AFM). Suitably, the length of fibres may be demonstrated by assessment of the fibre by use of a Shirley photoelectrical stapler, WIRA fibre length machine or the photoelectric Scanning method.

[0104] The inventors believe that the length of the fibres present in the compositions of the invention contribute to their beneficial properties, such as the magnitude of their zeta potential, discussed in more detail below. Diameter of fibres suitable for use in the compositions or methods of the invention

[0105] In a suitable embodiment, fibres of a composition of the invention have average diameters of between about 30nm and 40nm. In a suitable embodiment, fibres of a composition of the invention have a diameter between about 30nm and 40nm. Fibres according to a composition of the invention with a diameter of 30nm and 40nm again have a higher surface area to volume ratio compared to larger fibres, which again contributes to formation of inter-fibre molecular forces, thus enhancing the stability of the lipid layers.

[0106] In a suitable example, fibres of a composition of the invention may have average diameters of between about 30nm and about 40nm, about 31 nm and about 39nm, about 32nm and about 38nm, about 33nm and about 37nm or about 34nm and about 36nm. In a suitable example, fibres of a composition of the invention may have a diameter between about 30nm and about 40nm, about 31 nm and about 39nm, about 32nm and about 38nm, about 33nm and about 37nm or about 34nm and about 36nm.

[0107] Merely by way of example, fibres of a composition of the invention may have average diameters of about 30nm, about 31 nm, about 32nm, about 33nm, about 34nm, about 35nm, about 36nm, about 37nm, about 38nm, about 39nm or about 40nm. Merely by way of example, fibres of a composition of the invention may have a diameter of about 30nm, about 31 nm, about 32nm, about 33nm, about 34nm, about 35nm, about 36nm, about 37nm, about 38nm, about 39nm or about 40nm.

[0108] In a similar way to assessing the length of the fibres, the diameter of the fibres present in the compositions of the invention may be demonstrated by assessment of high-resolution images, which may be produced via electron microscopy such as scanning electron microscopy (SEM) or transmission electron microscopy (TEM) or via Atomic Force Microscopy (AFM).

[0109] The inventors believe that the diameter of the fibres present in the compositions of the invention contribute to their beneficial properties, such as the magnitude of their zeta potential, discussed in more detail below.

[0110] Aspect ratio of fibres suitable for use in the compositions or methods of the invention

[0111] In the context of the present invention, the aspect ratio of a fibre may be defined as the ratio of the length of a fibre to its diameter or width. The aspect ratio is an important parameter in characterizing fibres as it can affect their mechanical properties, such as elasticity, tensile strength, and surface area-to-volume ratio. Some of the interactions that may drive formation of networks between fibres, such as van der Waals forces, rely upon surface area, and so aspect ratio of fibres may play an important role in determining suitability for use in the compositions or methods of the invention.

[0112] In a suitable embodiment, fibres of a composition of the present invention have an aspect ratio of between 3 and 10. In a suitable example, fibres of a composition of the invention have an aspect ratio of between about 3 and about 10, of between about 4 and about 9, of between about 5 and about 8, of between about 6 and about 7, or between about 5 and about 6.

[0113] Merely by way of example, fibres of a composition of the present invention have an aspect ratio of about 3, of about 4, of about 5, of about 6, of about 7, of about 8, of about 9, or of about 10.

[0114] In a suitable embodiment, fibres of a composition of the present invention have an aspect ratio of between 5 and 6. In a suitable example, fibres of a composition of the invention have an aspect ratio of between about 5 and about 6, of between about 5.1 and about 5.9, of between about 5.2 and about 5.8, of between about 5.3 and about 5.7, or of between about 5.4 and about 5.6.

[0115] Merely by way of example, fibres of a composition of the present invention have an aspect ratio of about 5, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9 or about 6.0.

[0116] The inventors believe that the aspect ratio of the fibres present in the compositions of the invention contribute to their beneficial properties, such as the magnitude of their zeta potential, discussed in more detail below.

[0117] Tensile strength of fibres suitable for use in the compositions or methods of the invention

[0118] Tensile strength defines the maximum stress a material can withstand before it breaks or fails under tension. The tensile strength of a fibre depends on various factors, including its composition, structure, and manufacturing process.

[0119] In a suitable embodiment, fibres of a composition of the invention have a tensile strength of between 200MPa and 300MPa. Fibres of a composition of the invention with a tensile strength of between 200MPa and 300MPa are able to better reinforce the integrity of the liposome compared to lower strength fibres, preventing or reducing the leakage or release of encapsulated drugs. Merely by way of example, enhanced structural integrity associated with strength of the fibre may be important during storage and transportation of the lipid encapsulated compositions (such as liposomal compositions), ensuring that the drugs remain securely contained within the liposome until the desired release occurs.

[0120] Compositions with a higher tensile strength may not exhibit enough elasticity to allow sufficient deformability to travel through biological barriers such as capillaries. Furthermore, fibres with a higher tensile strength may exhibit reduced encapsulation efficiency. If the fibre within the lipid layer (such as a liposome) is too strong, it may hinder the encapsulation of an active agent within the lipid layer (such as a liposome).

[0121] In a suitable example, fibres of a composition of the invention may have a tensile strength of between about 200MPa and about 300MPa, about 210MPa and about 290MPa, about 220MPa and about 280MPa, about 230MPa and about 270MPa or about 240MPa and about 260MPa.

[0122] Merely by way of example, fibres of a composition of the invention may have a tensile strength of about 200MPa, about 205MPa, about 210MPa, about 215MPa, about 220MPa, about 225MPa, about 230MPa, about 235MPa, about 240MPa, about 245MPa, about 250MPa, about 255MPa, about 260MPa, about 265MPa, about 270MPa, about 275MPa, about 280MPa, about 285MPa, about 290MPa, about 295MPa or about 300MPa.

[0123] Elasticity of fibres suitable for use in the compositions or methods of the invention

[0124] Elasticity of a fibre may be defined as its ability to deform under stress and then return to its original shape once the stress is removed. It is a measure of the fibre's flexibility and resilience. The elasticity of a fibre is influenced by various factors, including its material composition, molecular structure, and processing conditions.

[0125] A skilled person will recognise that the elasticity of a fibre can be characterized using various parameters. Merely by way of example, the elasticity of a fibre may be characterized by calculating the Young’s modulus of the fibre. In a further example, the elasticity of a fibre could be characterized by calculating the elongation at break of the fibre. For the purposes of the present disclosure, the inventors have utilised Young’s modulus as the parameter to measure the elasticity of fibres of a composition of the invention. Young’s modulus may be defined as a measure of a material’s stiffness or resistance to elastic deformation under load.

[0126] In a suitable embodiment, fibres of a composition of the invention have a Young’s modulus of between 60GPa and 80GPa. Fibres with a Young’s modulus of between 60GPa and 80GPa are well suited to the formation of networks able to contribute advantageously to the structural integrity of liposomes. The elasticity of the fibres allows them to absorb mechanical stress or external forces, reducing deformation and rupture of the lipid layer (such as a liposomal membrane), and lowering the formation of aggregates between lipid layers (such as liposomes).

[0127] In a suitable example, fibres of a composition of the invention may have a Young’s modulus of between about 60GPa and about 80GPa, about 62GPa and about 78GPa or about 64GPa and about 76GPa, about 66GPa and about 74GPa or about 68GPa and about 72GPa.

[0128] Merely by way of example, fibres of a composition of the invention may have a Young’s modulus of about 60GPa, about 61 GPa, about 62GPa, about 63GPa, about 64GPa, about 65GPa, about 66GPa, about 67GPa, about 68GPa, about 69GPa, about 70GPa, about 71 GPa, about 72GPa, about 73GPa, about 74GPa, about 75GPa, about 76GPa, about 77GPa, about 78GPa, about 79GPa or about 80GPa.

[0129] Further advantages of the compositions of the invention

[0130] Overall, the enhanced stability of compositions of the present invention impact other potential advantages of these compositions namely, improved retention of active agents over time, improved shelf life and reduced leakage.

[0131] Increased loading capacity

[0132] The presence of a network of fibres within a lipid layer or liposome of a composition of the invention provides structural support and integrity to the encapsulating lipid layer (such as a liposome). This support helps prevent the lipid layer (such as a liposome) from collapsing or deforming under the increased weight or volume of the active agent, or in response to other forces, such as osmotic pressures. By supporting the structure of the liposomes, the network of fibres is able to increase the loading capacity of liposomes within the compositions of the invention, as compared to those present in suitable control compositions.

[0133] Retention of an active agent

[0134] As referred to elsewhere in this disclosure, in conventional lipid encapsulated compositions, such as liposomal compositions, retention of an active agent remains a key problem, due to leakage from the lipid encapsulating layer. This is exacerbated upon drying, or complete dehydration, of such compositions, where the encapsulating lipid layers (such as membranes of liposomes) are typically damaged leading to leakage and loss of active agents.

[0135] As discussed in more detail above, compositions of the present invention offer increased stability due to the properties of the incorporated fibres The increased stability improves the retention of an active agent through reduced leakage.

[0136] There also other advantages conferred by properties of the fibres which also contribute to improved retention of an active agent.

[0137] The networks of fibres found in the dehydrated compositions of the invention can also act as a protective barrier to other external factors that may otherwise contribute to degradation of the encapsulating lipid layers (such as liposomes) and leakage of active agents. Encapsulating lipid layers such as liposomes can be exposed to various external factors, such as pH changes, enzymatic degradation, or interactions with blood components. The networks of fibres found in the dehydrated compositions of the invention enhance the resistance of the encapsulating lipid layers’ to these external factors, thereby increasing retention of the active agents, and optimising their delivery.

[0138] Improved drug delivery and cellular uptake

[0139] Fibres with a length of between 150nm and 300nm can aid in the delivery of encapsulated drugs or therapeutic agents. The skilled person would appreciate that the small size allows the fibres to penetrate tissues more effectively, including crossing physiological barriers such as cell membranes or the blood-brain barrier. This enhanced penetration can improve the targeted delivery of drugs to specific sites, increasing their therapeutic efficacy.

[0140] Furthermore, smaller fibres can exhibit improved cellular uptake by target cells. The smaller size of fibres could allow for more efficient internalization by cells. This enhanced cellular uptake can be beneficial for applications such as targeted drug delivery to specific cell types or tissues.

[0141] Active agents suitable for use in the compositions or methods of the invention

[0142] The compositions of the invention comprise one or more active agents. These may be encapsulated within a lipid layer (such as a liposome), in the case of hydrophilic active agents, or present with the lipid of the lipid layer, in the case of hydrophobic active agents. The compositions of the invention may comprise any suitable active agent, including (but not limited to) those identified below.

[0143] Suitably, the compositions of the invention may comprise, orthe methods of the invention may comprise, more than one active agent. Merely by way of example, 2, 3, 4 or 5 different active agents may be used.

[0144] In a suitable embodiment, each encapsulating lipid layer (such as a liposome) present in a composition of the invention may comprise multiple active agents. These may be encapsulated within the lipid layer (in the case of hydrophilic active agents) and / or present within the lipid of the lipid layer (in the case of hydrophobic active agents).

[0145] In a suitable embodiment, the composition comprises a mixture of lipid layers, such as liposomes, comprising different active agents.

[0146] In some embodiments, the active agent (or agents) present in a composition of the invention is independently selected from the group consisting of: a vitamin active agent (such as vitamin C, vitamin E, vitamin D, vitamin K, or biotin); a mineral active agent (such as zinc, iron, magnesium or selenium); an extracellular matrix active agent (such as collagen or hyaluronic acid); a herbal, fungal or botanic active agent (such as fungal extracts or phytonutrients, for example curcumin or ashwagandha); an oil or fatty acid active agent (such as MCT oils, or omega-3 fatty acids, e.g. DHA); metabolically active agents (such as coenzyme Q10, nicotinamide mononucleotide, or glutathione); a pharmaceutical agent (small molecules such as insulin or antihistamines or therapeutic proteins such as peptides or antibodies) and / or probiotic agent (such as live bacterial strains or yeast). Vitamin or mineral active agents

[0147] Vitamins or minerals represent suitable active agents for incorporation in the compositions of the invention. Vitamin or mineral active agents are popularly used in nutritional supplements.

[0148] Examples of vitamin active agents that may be incorporated in the compositions of the invention include those selected from the group consisting of: vitamin C; vitamin E; vitamin D; vitamin K; and biotin.

[0149] Examples of mineral active agents that may be incorporated in the compositions of the invention include those selected from the group consisting of: zinc; iron; magnesium; and selenium.

[0150] Extracellular matrix active agents

[0151] Active agents may include one or more components present in the extracellular matrix. Examples of extracellular matrix active agents that may be incorporated in the compositions of the invention include those selected from the group consisting of: collagen; and hyaluronic acid.

[0152] Such extracellular matrix components to be used in a composition or method of the invention may be derived from any suitable source.

[0153] Herbal, fungal or botanic active agents

[0154] Compounds from herbal, fungal or botanic sources represent suitable active agents for incorporation in the compositions of the invention. Herbal, fungal or botanic active agents are popularly used in nutritional supplements.

[0155] Examples of herbal, fungal or botanic active agents that may be incorporated in the compositions of the invention include those selected from the group consisting of: fungal extracts; herbal extracts; botanic extracts, such as curcumin or ashwagandha; and phytonutrients. Oil or fatty acid active agents

[0156] Oils represent suitable active agents for incorporation in the compositions of the invention, as do other active agents comprising fatty acids. Examples of such active agents include those selected from the group consisting of: medium chain triglyceride (MCT) oils; and omega-3 fatty acids, such as docosahexaenoic acid (DHA).

[0157] Suitable sources of such active agents will be well known to those skilled in the art. Merely by way of example, suitable oil or fatty acid active agents may be derived from animal or vegetable sources.

[0158] Oil or fatty acid active agents will generally constitute hydrophobic active agents for the purpose of the present invention, as considered elsewhere in this disclosure.

[0159] Metabolically active agents

[0160] Metabolically active compounds represent suitable active agents for incorporation in the compositions of the invention. Metabolically active agents are popularly used in nutritional supplements.

[0161] Examples of metabolically active agents that may be incorporated in the compositions of the invention include those selected from the group consisting of: coenzyme Q10; nicotinamide mononucleotide; and glutathione.

[0162] Pharmaceutical active agent

[0163] Pharmaceutical compounds represent suitable active agents for incorporation in the compositions of the invention. Pharmaceutical active agents encompass a wide range of molecules relevant for inducing a therapeutic effect.

[0164] Examples of pharmaceutical active agents that may be incorporated in the compositions of the invention include small molecules such as those selected from the group consisting of: insulin; and antihistamines, or therapeutic proteins such as those selected from the group consisting of: peptides or antibodies. Probiotic active agents

[0165] Probiotics represent suitable active for incorporation in the compositions of the invention. Probiotic agents are increasingly used in nutritional supplements due to their proposed health benefits relating to the human microbiota.

[0166] Examples of probiotic active agents that may be incorporated in the compositions of the invention include live bacterial strains or yeast.

[0167] As mentioned above, fibres used in the compositions of the invention may also have probiotic properties, and these may complement or amplify the effects of probiotic active agents in such compositions.

[0168] Impact of polarity of active agents

[0169] The inventors have found that the polarity of active agents incorporated in compositions of the invention can influence various properties of the compositions. In particular, the polarity of an active agent may influence the loading capacity of the agent that can be achieved in a composition of the invention.

[0170] Hydrophilic active agents may achieve loading capacities in compositions of the invention that may be twice (or more) those that can be achieved in respect of hydrophobic active agents.

[0171] Furthermore, the polarity of an active agent may influence the solubility of the agent in compositions of the invention due to the active agent’s ability to form intermolecular interactions with solvent molecules. Finally, the polarity of an active agent may influence the size of the volume encapsulated by lipid layers (such as liposomes) present in compositions of the invention. Generally, polar active agents will result in the production of lipid layers (such as liposomes) that encapsulate smaller volumes, which may contribute to improved stability of compositions of the invention comprising such active agents.

[0172] In a suitable embodiment, a composition of the invention comprises lipid layers (such as liposomes) encapsulating a hydrophilic active agent. In a suitable embodiment, the only active agents in a composition of the invention are hydrophilic active agents.

[0173] As noted above, hydrophilic active agents encapsulated within a composition of the invention may be located in the aqueous core of compositions in accordance with the second aspect of the invention, or encapsulated within the lipid layer of compositions in accordance with the first aspect of the invention.

[0174] In a suitable embodiment, a composition of the invention comprises lipid layers (such as liposomes) in which a hydrophobic active agent is present. In a suitable embodiment, the only active agents in a composition of the invention are hydrophobic active agents.

[0175] In compositions of either the first or second aspect of the invention, hydrophobic active agents may be present in the lipid of a lipid layer, such as in a membrane of a liposome.

[0176] Suitably, in an embodiment, a composition of the invention comprises hydrophobic fibres.

[0177] In a suitable embodiment, a composition of the invention comprises hydrophilic fibres. Merely by way of example, fibres which comprise or are formed from inulin are hydrophilic in nature. Alternatively, fibres which comprise or are formed from chitin are hydrophilic in nature. In a further example, fibres which comprise or are formed from chitosan are hydrophilic in nature.

[0178] The inclusion of hydrophilic fibres may help to ensure that the active agent(s) are held in the core within an encapsulating lipid layer (such as a liposome), which assists in reducing leakage.

[0179] Alternatively, or additionally, the inclusion of hydrophilic fibres in the compositions of the invention may improve the stability of the encapsulating lipid layers (such as liposomes) within such compositions. The presence of hydrophilic constituents within the encapsulating lipid layers (such as liposomes) in the compositions of the invention reduces the surface energy of the lipid layers, in turn reducing the likelihood of fusion with other encapsulating lipid layers or with the surrounding medium. This results in increased stability of the lipid layers. In turn, this improved stability again results in reduced leakage. The improved stability also reduces degradation.

[0180] A skilled person wishing to practice the invention will readily be able to identify whether a particular active agent of interest is hydrophobic or hydrophilic. Based on this, they will be able to determine the loading levels that may be achieved in a composition of the invention comprising the active agent. Simply for guidance, examples of hydrophobic active agents include agents selected from the group consisting of: curcumin, PEA, DHA, Vitamin D3 and Vitamin K2, and oil or fatty acid active agents. Examples of hydrophilic active agents include agents selected from the group consisting of: Vitamin C, glutathione and ashwagandha. In a suitable embodiment, a composition of the invention is substantially alcohol-free. Suitably, such a composition may comprise 5000ppm or less of alcohol, for example, 4000ppm or less, 3000ppm or less, 2000ppm or less, 10OOppm or less, or 500ppm or less of alcohol.

[0181] Alcohol-based approaches are frequently used to increase solubility of active agents in compositions encapsulated in lipid layers. These approaches typically result in retention of approximately 5000ppm alcohol in a resultant composition. The presence of alcohol, such as ethanol, within a composition may be undesirable for a variety of reason including an unpleasant taste, the perceived incompatibility with products deemed ‘heath related’ or for persons of particular religions. The use of alcohol-based approaches to improve solubility also negatively impacts the stability of the lipid layers (such as liposomes). For example, ethanol-based approaches are known to erode lipid layers, such as liposomes, increasing leakage. The compositions of the invention are able to avoid such problems.

[0182] Properties of compositions of the invention

[0183] Lipid layers suitable for use in the compositions or methods of the invention

[0184] The compositions of the invention comprise lipid layers, which are structures comprising lipids having both hydrophobic (water-repelling) and hydrophilic (water-attracting) properties. In the present case, the lipid layers in the compositions of the invention encapsulate an emulsion that contains complexes in which one or more active agents is present.

[0185] In a suitable embodiment, the lipid layers of a composition of the invention comprise a phospholipid and / or a sphingolipid. Both phospholipids and sphingolipids are examples of lipids having the requisite hydrophobic and hydrophilic properties suitable for use in lipid layers (such as liposomes, discussed further below).

[0186] In a suitable embodiment, the phospholipid is lecithin. Suitably, the lecithin may be derived from a source selected from the group consisting of: sunflower, grape seed, and soy beans.

[0187] In a suitable embodiment the lipid layer is a monolayer. In a suitable embodiment the lipid layer is a bilayer. A lipid monolayer refers to a single layer of lipid molecules. A lipid bilayer refers to a double layer of lipid molecules. Typically, lipid bilayers are formed by amphipathic molecules, such as phospholipids, with their hydrophobic tails facing inward and their hydrophilic heads facing outward toward the surrounding aqueous environments. In a suitable example, composition according to the present invention comprising a lipid monolayer comprises a phospholipid. In such an embodiment, the phospholipid may comprise a mixture of crude phospholipids with 10-15% of the mixture comprising phosphatidylcholines.

[0188] In a suitable example, composition according to the present invention comprising a lipid bilayer comprises a phospholipid. In such an embodiment, the phospholipid may comprise a mixture of crude phospholipids with 50-55% of the mixture comprising phosphatidylcholines.

[0189] In a suitable embodiment, the lipid layer comprises a liposome. Phospholipids to be used in a liposome in such an embodiment may as considered for lipid bilayers above. Compositions of the invention comprising liposomes are discussed in more detail below.

[0190] In a suitable embodiment, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of less than 230nm. Suitably, in an embodiment, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of between about 170nm and about 230nm. For example, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of about 170nm, about 175nm, about 180nm, about 185nm, about 190nm, about 195nm, about 200nm, about 205nm, about 21 Onm, about 215nm, about 220nm, about 225nm or about 230nm. For example, the encapsulating lipid layers (such as liposomes) of a composition of the invention may have an average diameter of between about 170nm and about 230nm, such as between about 175nm and about 225nm, between about 180nm and about 220nm, between about 185nm and about 215nm, between about 190nm and about 210nm, or between about 195nm and about 205nm. In a suitable embodiment, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of about 200nm.

[0191] In a suitable embodiment, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of between about 70nm and about 230nm. For example, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of about 70nm, about 75nm, about 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 105nm, about 110nm, about 115nm, about 120nm, about 125nm, about 130nm, about 135nm, about 140nm, about 145nm, about 150nm, about 155nm, about 160nm, about 165nm, about 170nm, about 175nm, about 180nm, about 185nm, about 190nm, about 195nm, about 200nm, about 205nm, or about 210nm, In a suitable embodiment, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of between about 100nm and about 250nm. For example, the encapsulating lipid layers (such as liposomes) of a composition of the invention have an average diameter of about 100nm, about 105nm, about 110nm, about 115nm, about 120nm, about 125nm, about 130nm, about 135nm, about 140nm, about 145nm, about 150nm, about 155nm, about 160nm, about 165nm, about 170nm, about 175nm, about 180nm, about 185nm, about 190nm, about 195nm, about 200nm, about 205nm, about 210nm, about 215nm, about 220nm, about 225nm, about 230nm, about 235nm, about 240nm, about 245nm or about 250nm.

[0192] Encapsulating lipid layers (such as liposomes) with a diameter within the range of 70 nm to 230 nm, for example within the range of 170nm and 230nm are stable over time. The stability of the encapsulating lipid layers (such as liposomes) over time may be assessed using DLS to determine any changes in their size over a desired period of time. Larger encapsulating lipid layers (such as liposomes) are known to be more unstable due to increased incidences of self-aggregation of the lipid layer structures. Such self-aggregation may occur during longterm storage, or as a part of the manufacturing process. Physical instability results in the breakdown and short life of the encapsulating lipid layers, such as liposomes.

[0193] It will be appreciated that this increase in stability will provide advantages in terms of improved shelf-life of the compositions. Furthermore, though not wishing to be bound by any hypothesis, the inventors believe that the size of the encapsulating lipid layers (such as liposomes) present in the compositions of the invention contributes to their beneficial properties, such as the magnitude of their zeta potential, discussed in more detail below.

[0194] Encapsulating lipid layers (such as liposomes) produced by a method of the invention, or incorporated in a composition of the invention, may exhibit a narrow size distribution. This offers advantages, in that lipid layers (such as liposomes) exhibiting a narrower size distribution are more reproducible for commercial solutions.

[0195] Suitably, the narrow size distribution of the encapsulating lipid layers (such as liposomes) may be demonstrated by assessment of the polydispersity index (PDI), which may be investigated using a dynamic light scattering (DLS) approach, as described further in the Examples. A composition of the invention may have a PDI of less than 230nm (which may, for example, be measured by DLS), a value that would be considered beneficially low in respect of a composition incorporating lipid encapsulation (such as in liposomes). Liposomes suitable for use in the compositions or methods of the invention

[0196] Suitably, compositions of the invention may comprise liposomes, which prior to dehydration are structures comprising a lipid bilayer that encapsulates an aqueous core.

[0197] In a suitable embodiment, the liposomes of a composition of the invention comprise a phospholipid. In a suitable embodiment, the phospholipid is lecithin. Suitably, the lecithin may be derived from a source selected from the group consisting of: sunflower, grape seed, and soy beans.

[0198] In a suitable embodiment, the liposomes of a composition of the invention have an average liposomal diameter of less than 230nm. Suitably, in an embodiment, the liposomes of a composition of the invention have an average liposomal diameter of between about 170nm and about 230nm. For example, the liposomes of a composition of the invention have an average liposomal diameter of about 170nm, about 175nm, about 180nm, about 185nm, about 190nm, about 195nm, about 200nm, about 205nm, about 210nm, about 215nm, about 220nm, about 225nm or about 230nm. For example, the liposomes of a composition of the invention may have an average liposomal diameter of between about 170nm and about 230nm, such as between about 175nm and about 225nm, between about 180nm and about 220nm, between about 185nm and about 215nm, between about 190nm and about 210nm, or between about 195nm and about 205nm. In a suitable embodiment, the liposomes of a composition of the invention have an average liposomal diameter of about 200nm.

[0199] In a suitable embodiment, the liposomes of a composition of the invention have an average liposomal diameter of between about 70nm and about 230nm. For example, the liposomes of a composition of the invention may have an average liposomal diameter of about 70nm, about 75nm, about 80nm, about 85nm, about 90nm, about 95nm, about 10Onm, about 105nm, about 110nm, about 115nm, about 120nm, about 125nm, about 130nm, about 135nm, about 140nm, about 145nm, about 150nm, about 155nm, about 160nm, about 165nm, about 170nm, about 175nm, about 180nm, about 185nm, about 190nm, about 195nm, about 200nm, about 205nm, or about 210nm,

[0200] In a suitable embodiment, the liposomes of a composition of the invention have an average liposomal diameter of a composition of the invention have an average diameter of between about 100nm and about 250nm. For example, the liposomes of a composition of the invention may have an average liposomal diameter of about 10Onm, about 105nm, about 110nm, about 115nm, about 120nm, about 125nm, about 130nm, about 135nm, about 140nm, about 145nm, about 150nm, about 155nm, about 160nm, about 165nm, about 170nm, about 175nm, about 180nm, about 185nm, about 190nm, about 195nm, about 200nm, about 205nm, about 210nm, about 215nm, about 220nm, about 225nm, about 230nm, about 235nm, about 240nm, about 245nm or about 250nm.

[0201] Liposomes with a liposomal diameter within the range of 70 nm to 230 nm, for example within the range of 170nm and 230nm, are stable over time. The stability of the liposomes over time may be assessed using DLS to determine any changes in size of the liposomes over a desired period of time. Larger liposomes are known to be more unstable due to increased incidences of self-aggregation of liposomes. Such self-aggregation may occur during long-term storage, or as a part of the manufacturing process. Physical instability results in the breakdown and short life of the liposomes.

[0202] It will be appreciated that this increase in stability will provide advantages in terms of improved shelf-life of the compositions. Furthermore, though not wishing to be bound by any hypothesis, the inventors believe that the size of the liposomes present in the compositions of the invention contributes to their beneficial properties, such as the magnitude of their zeta potential, discussed in more detail below.

[0203] Liposomes produced by a method of the invention, or incorporated in a composition of the invention, may exhibit a narrow size distribution. This offers advantages, in that liposomes exhibiting a narrower size distribution are more reproducible for commercial solutions.

[0204] Suitably, the narrow size distribution of the liposomes may be demonstrated by assessment of the polydispersity index (PDI), which may be investigated using a dynamic light scattering (DLS) approach, as described further in the Examples. A composition of the invention may have a PDI of less than 230nm (which may, for example, be measured by DLS), a value that would be considered beneficially low in respect of liposomal compositions.

[0205] Beneficial stability of compositions of the invention

[0206] Compositions in accordance with the first aspect of the invention, or the products of a method in accordance with the third aspect of the invention, may exhibit a high level of stability, as compared to the compositions of the prior art. Without wishing to be bound by any hypothesis, the inventors believe that this high stability arises from a combination of the features present in the fibres within compositions of the invention, including the small length and diameter, the strength or elasticity of the fibres. The high stability of the compositions of the invention may be demonstrated by measurement of the magnitude of their zeta potential.

[0207] Previous publications have identified lipid layer formulations, such as liposomal formulations, having a zeta potential of less than -10 mV or greater than +10 mV as having advantageous stability. The compositions of the invention demonstrate zeta potentials of much greater magnitude, in turn indicating that their stability is much improved as compared to these earlier compositions.

[0208] In a suitable embodiment, the magnitude of the zeta potential of a composition of the invention is around -15 mV or less, or +15 mV or greater. For example, the magnitude of the zeta potential of a composition of the invention may be around -20 mV or less, or +20 mV or greater, or around -25 mV or less, or +25 mV or greater. In a suitable embodiment, the magnitude of the zeta potential of a composition of the invention is around -30 mV or less, or +30 mV or greater. In a suitable embodiment, the zeta potential of a composition of the invention is about -31 mV or +31 mV, about -32 mV or +32mV, or even about -33 mV or +33mV. In a suitable embodiment, the magnitude of the zeta potential of a composition of the invention is around - 35 mV or less, or +35 mV or greater. In a suitable embodiment, the zeta potential of a composition of the invention is about -36 mV or +36mV, about -37 mV or +37mV, about -38 mV or +38mV or even about -39 mV or +39mV. In a suitable embodiment, the magnitude of the zeta potential of a composition of the invention is around -40 mV or less, or +40 mV or greater. In a suitable embodiment, the zeta potential of a composition of the invention is about -41 mV or +41 mV, or even about -42 mV or +42mV.

[0209] A large positive or negative value of zeta potential, as demonstrated by the compositions of the invention, strongly indicates that they have excellent stability due to electrostatic repulsion between individual particles present within the composition. The zeta potentials observed in respect of compositions in accordance with the invention, results in a highly stable composition which contains non-agglomerated particles.

[0210] A zeta potential of very high or low magnitude (or example greater than 50mV or less than - 50mV) is disadvantageous in practice. In particular, a highly positive or negative zeta potential can increase the likelihood of agglomeration of the particles reducing overall stability. Furthermore, a highly positive or negative zeta potential can be associated with increased toxicity and reduced cellular uptake. The improved stability of the compositions in accordance with the invention prevents the loss of active agent (for example by liposomal leakage). Furthermore, the improved stability of the compositions in accordance with the invention reduces degradation of the encapsulating lipid layers, such as liposomes, within the compositions. Both the reduction in leakage and the reduction in degradation exhibited by the compositions in accordance with the invention contributes to further beneficial properties, including their high encapsulation efficiency, high loading capacity, and high bioavailability.

[0211] High encapsulation efficiency of compositions of the invention

[0212] Compositions in accordance with the first or second aspect of the invention or the products of methods in accordance with the third aspect of the invention may exhibit a high level of encapsulation efficiency, as compared to the compositions of the prior art. The encapsulation efficiency that the inventors have achieved in the compositions of the invention is greater than those previously reported in the prior art (as discussed below). It will be appreciated that the improvement in encapsulation efficiency that can be achieved in respect of the compositions of the invention will provide advantages in a wide range of contexts and uses.

[0213] The encapsulation efficiency is the percentage of an active agent present in a composition that is successfully encapsulated into the lipid layers (such as liposomes). It may be defined by the concentration of the incorporated material detected in the formulation over the initial concentration used to make the formulation.

[0214] A higher encapsulation efficiency directly reflects a higher concentration of the active agent within the lipid layer (such as a liposome) and hence a stronger therapeutic effect that may be achieved by the composition.

[0215] The increased stability of the compositions of the invention contributes to their high encapsulation efficiency, in that it reduces liposomal leakage and degradation.

[0216] The encapsulation efficiencies that may be achieved by the compositions of the invention are notably higher than those achieved by the prior art. For example, prior art compositions have previously been able to achieve encapsulation efficiencies of between 40-70%.

[0217] In contrast, in a suitable embodiment, the encapsulation efficiency of at least one active agent within a composition of the invention is 50% or more. The encapsulation efficiency may be 55% or more, 60% or more, 65% or more, or 70% or more. Indeed, in a suitable embodiment, the encapsulation efficiency of at least one active agent within a composition of the invention is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or even approximately 100%.

[0218] In a suitable embodiment, the encapsulation efficiency of at least one active agent within a composition of the invention is approximately 80%. For example, in a suitable embodiment, the encapsulation efficiency of at least one active agent within a composition of the invention is approximately 80.15%. In a suitable embodiment, the encapsulation efficiency of at least one active agent within a composition of the invention is approximately 80.4%.

[0219] In the case of compositions of the invention that comprise more than one active agent, two or more active agents provided in the composition may achieve the high encapsulation efficiency levels referred to in the preceding paragraphs. Indeed, each active agent present in a composition of the invention may achieve the high encapsulation efficiency levels referred to in the preceding paragraphs. For example, the encapsulation efficiency of each active agent within the liposomes of a composition of the invention may be 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, 75% or more, 80% or more (for example, approximately 80.4%), 85% or more, 90% or more, 95% or more, or even approximately 100%.

[0220] As demonstrated in the Examples, the compositions of the invention are able to maintain their high encapsulation efficiency with respect to incorporated active agents for extensive periods of time. It will immediately be appreciated that this provides advantages in respect of a product, in that is an indication of an increased shelf-life.

[0221] Suitably, a composition of the invention may maintain an encapsulation efficiency in respect of at least one active agent of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, or at least 85%, over a period of one month or more, two months or more, three months or more, four month or more, five months or more, six months or more, seven months or more, eight months or more, nine months or more, ten month or more, 11 months or more, or 12 months or more, Indeed, suitably such encapsulation efficiencies may be maintained for up to two years, up to three years, up to four years, or up to five years. High loading capacity of compositions of the invention

[0222] Compositions in accordance with the first or second aspect of the invention or the products of methods in accordance with the third aspect of the invention may exhibit a high loading capacity, as compared to the compositions of the prior art. The elevated stability of the compositions of the invention, leading to reduced leakage, and reduced degradation of encapsulating lipid layers, such as liposomes, may contribute to this advantageous property.

[0223] Loading capacity may be defined as the amount of an active agent that can be incorporated in a composition. Thus, it will be appreciated that a composition with a higher loading capacity contains more of a given active agent than does a composition with a lower loading capacity.

[0224] The loading capacity of the compositions of the invention is markedly improved as compared to those of the prior art. This advantageous property also offers benefits in a wide range of different applications of the compositions of the invention.

[0225] Compositions having a high loading capacity offer multiple advantages including, but not limited to, decreasing the dose volume that must be administered in order to achieve a desired extent of effect, reduce dosing time, and minimising the amount of excipient required in the composition.

[0226] A loading capacity of greater than 40% is notably high when compared to the values reported in respect of compositions of the prior art.

[0227] In a suitable embodiment, the loading capacity of a composition of the invention is 40% or more. For example, the loading capacity of a composition of the invention may be 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or approximately 100%.

[0228] In the case of compositions of the invention that comprise more than one active agent, two or more active agents provided in the composition may achieve the high loading capacity referred to in the preceding paragraphs. Indeed, each active agent present in a composition of the invention may achieve the high loading capacity referred to in the preceding paragraphs. High bioavailability of compositions of the invention

[0229] Compositions in accordance with the first or second aspect of the invention or the products of methods in accordance with the third aspect of the invention may exhibit a high bioavailability of an active agent incorporated therein, as compared to the compositions of the prior art.

[0230] This improvement in bioavailability offers many useful benefits. A given level of biological activity of an active agent may be achieved using a smaller quantity of the agent in the composition, thus decreasing the cost of manufacture of the composition. Alternatively, the compositions of the invention may allow an increased amount of a biological activity to be achieved in respect of a given quantity of an active agent incorporated in a composition of the invention, as compared to the same quantity of the active agent incorporated in a composition of the prior art.

[0231] Again, this advantageous property may, at least to some extent, arise from the elevated stability of the compositions of the invention, which leads to reduced leakage, and reduced degradation of lipid layers, such as liposomes. Furthermore, as noted above, the compositions in accordance with the invention contain lipid layers, such as liposomes, that encapsulate relatively small volumes. The smaller size of fibres within the lipid layers (such as liposomes) within the compositions of the invention allows easier penetration through biological barriers, which may also contribute to improved bioavailability.

[0232] It will be appreciated that an increase in bioavailability provided by a composition of the invention allows for a more efficient delivery system, as this will help a recipient to absorb more of the active agent, without the need to administer a higher dose of the composition.

[0233] The bioavailability of an active agent incorporated in a composition in the invention may be compared to any suitable control. Merely by way of example, the bioavailability of an active agent incorporated in a composition of the invention may be compared to the bioavailability of the same active agent in free solution.

[0234] In a suitable embodiment, an increase in bioavailability may be demonstrated by an increase in the area under the curve (AUC) as measured during a high-performance liquid chromatography (HPLC) or liquid Chromatography with tandem mass spectrometry (LC-MS- MS) assay. In a suitable embodiment, an increase in bioavailability may be demonstrated by an increase in the Cmax to Cmax peak value as measured during a high-performance liquid chromatography (HPLC) or liquid Chromatography with tandem mass spectrometry (LC-MS- MS) assay.

[0235] In a suitable embodiment, the bioavailability of an active agent incorporated in a composition of the invention may be increased 2-fold, or more, as compared to a suitable control. For example, the bioavailability of an active agent incorporated in a composition of the invention may be increased 3-fold, or more, 4-fold, or more, 5-fold, or more, 6-fold, or more, 7-fold, or more, 8-fold, or more, 9-fold, or more, or 10-fold, or more, as compared to a suitable control.

[0236] In a suitable embodiment, the bioavailability of an active agent incorporated in a composition of the invention may be increased 50%, or more, as compared to a suitable control. For example, the bioavailability of an active agent incorporated in a composition of the invention may be increased 50%, or more, 100%, or more, 150%, or more, 200%, or more, 250%, or more, 300%, or more, 350%, or more, or 400%, or more, as compared to a suitable control.

[0237] It will be appreciated that there are various parameters which may be assessed to determine an increase in bioavailability. Examples of such parameters are discussed further below.

[0238] In a suitable embodiment, an increase in cellular uptake may be used to indicate an increase in bioavailability. In a suitable embodiment, the cellular uptake of a composition of the invention, or of an active agent incorporated in such a composition, may be increased 2-fold, or more, as compared to a suitable control. For example, the cellular uptake may be increased 3-fold, or more, 4-fold, or more, 5-fold, or more, 6-fold, or more, 7-fold, or more, 8-fold, or more, 9-fold, or more, or 10-fold, or more, as compared to a suitable control.

[0239] In a suitable embodiment, cellular uptake may be assessed with reference to the uptake of fluorescently labelled compositions of the invention, for example compositions of the invention comprising fluorescently labelled active agents, into cells. In such an embodiment, an increase in cellular uptake may be demonstrated by an increase in fluorescence observed from fluorescent imaging. In a suitable embodiment, an increase in fluorescence observed from fluorescent imaging may be quantified by calculating the fluorescence intensity per cell or per unit area of a sample being imaged. The cells may, for example, be MCF7 cells.

[0240] In a suitable embodiment, an increase in cell permeability may be used to indicate an increase in bioavailability. In a suitable embodiment, the permeability of cells to a composition of the invention, or to an active agent incorporated in such a composition, may be increased 2-fold, or more, as compared to a suitable control. For example, permeability of cells may be increased 3-fold, or more, 4-fold, or more, 5-fold, or more, 6-fold, or more, 7-fold, or more, 8- fold, or more, 9-fold, or more, or 10-fold, or more, as compared to the permeability of the cells to a suitable control.

[0241] In a suitable embodiment, an increase in cell permeability may be measured using a Caco-2 permeability assay, an in vitro method used to assess the intestinal permeability of drugs or drug formulations. In a suitable embodiment, an increase in cell permeability may be demonstrated by an increase in the concentration of a composition of the invention, or of an active agent incorporated in such a composition, that has crossed the cell monolayer and appeared in the basolateral compartment. This concentration can be quantified using any appropriate analytical techniques (e.g., HPLC, LC-MS / MS).

[0242] In a suitable embodiment, an increase in adsorption of a composition of the invention may be used to indicate an increase in bioavailability. In a suitable embodiment, adsorption of a composition of the invention may be increased 2-fold, or more, as compared to a suitable control. For example, adsorption of a composition of the invention may be increased 3-fold, or more, 4-fold, or more, 5-fold, or more, 6-fold, or more, 7-fold, or more, 8-fold, or more, 9- fold, or more, or 10-fold, or more, as compared to a suitable control.

[0243] In a suitable embodiment, the rate of adsorption of a composition of the invention may be increased 2-fold, or more, as compared to a suitable control. For example, the rate of adsorption of a composition of the invention may be increased 3-fold, or more, 4-fold, or more, 5-fold, or more, 6-fold, or more, 7-fold, or more, 8-fold, or more, 9-fold, or more, or 10-fold, or more, as compared to a suitable control.

[0244] In a suitable embodiment, an increase in adsorption or increase in rate of adsorption of a composition of the invention may be measured from plasma samples at predetermined time points following liposome administration. In a suitable embodiment, analytical ultracentrifugation (AUC) may be used measure the adsorption or rate of adsorption of liposomes in plasma.

[0245] In the case of compositions of the invention that comprise more than one active agent, two or more active agents provided in the composition may achieve the increased levels of bioavailability referred to in the preceding paragraphs. Indeed, each active agent present in a composition of the invention may achieve the increased levels of bioavailability referred to in the preceding paragraphs. The inventors have noted that the increases in bioavailability that may be achieved are, to at least some extent, dependent on the nature of the active agent in question. In particular, the size of the active agent may increase bioavailability. Small active agents may be absorbed more readily and metabolised more effectively than larger active agents. Furthermore, the polarity of the active agent may increase bioavailability as hydrophilic active agents may exhibit difficulties crossing membranes.

[0246] For example, in the case of a composition in accordance with the present invention comprising vitamin C as an active agent in an increase in bioavailability of 3-fold or more may be achieved. In the case of a composition in accordance with the present invention comprising glutathione as an active agent in an increase in bioavailability of 4-fold or more may be achieved. In the case of a composition in accordance with the present invention comprising curcumin as an active agent in an increase in bioavailability of 5-fold or more may be achieved.

[0247] Reduced degradation of compositions of the invention

[0248] Compositions in accordance with the first or second aspects of the invention or the products of methods in accordance with the third aspect of the invention may exhibit reduced degradation, as compared to a suitable comparator or control.

[0249] It will be appreciated that compositions which exhibit reduced degradation, will also prevent the degradation of the active agent which is incorporated into the composition.

[0250] This reduction in degradation offers many useful benefits. Reduced degradation ensures that a higher concentration of the active agent reaches the target site, as compared to the same quantity of the active agent incorporated in a composition of the prior art. Alternatively, the compositions of the invention with reduced degradation may be less likely to be cleared rapidly from the bloodstream by the reticuloendothelial system (RES). This leads to an extended circulation time, allowing for a more sustained release of the active agent and improving its bioavailability, as compared to a suitable comparator or control.

[0251] Again, this advantageous property may, at least to some extent, arise from the elevated stability of the compositions of the invention.

[0252] It will be appreciated that a reduction of degradation provided by a composition of the invention allows for a more efficient delivery system, as this will help a recipient to receive a higher quantity of the active agent, without the need to administer a higher dose of the composition. The rate of degradation of a composition incorporating an active agent of the invention, or of an active agent incorporated in such a composition, may be compared to any suitable control.

[0253] In a suitable embodiment, a reduction in degradation may be demonstrated a lower percentage of burst degradation, optionally measured during in vitro digestion stability studies under simulated gastric, and intestinal conditions. In a suitable embodiment, dynamic light scattering (DLS) can be used to measure changes in liposome size before and after burst degradation to calculate a burst degradation percentage.

[0254] In a suitable embodiment, the burst degradation of a composition of the invention may be less than 15%, as compared to a suitable control. For example, the degradation of a composition of the invention may be less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% or even about 0%, as compared to a suitable control.

[0255] In a suitable embodiment, compositions of the invention may be more resistant to degradation when compared to a suitable control. For example, less than 85% of a composition of the invention may degrade. For example, less than 85%, less than 90% or even less than 95% of a composition of the invention may degrade.

[0256] Low cytotoxicity of compositions of the invention

[0257] Compositions in accordance with the first or second aspects of the invention or the products of methods in accordance with the third aspect of the invention may exhibit low cytotoxicity, as compared to the compositions of the prior art.

[0258] This low level of cytotoxicity offers many useful benefits. Compositions with low cytotoxicity are more biocompatible and less likely to cause adverse effects when administered to subjects. Additionally or alternatively, low cytotoxicity reduces non-specific interactions of compositions with cells and tissues, minimizing off-target effects. This improves the specificity and selectivity of the delivered active agent, enhancing its therapeutic efficacy while reducing side effects. Furthermore, compositions with low cytotoxicity exhibit prolonged circulation times in the bloodstream due to reduced clearance by the RES. This allows for sustained release of the encapsulated active agent and improved pharmacokinetic profiles, as compared to a composition of the prior art. Again, this advantageous property may, at least to some extent, arise from the elevated stability of the compositions of the invention. Additionally, this advantageous property may, at least to some extent, arise from the low zeta potential as low zeta potential particles exhibit decreased non-specific binding to cell membranes and reduced uptake by phagocytic cells, leading to lower cytotoxicity.

[0259] It will be appreciated that a composition of the invention which exhibits low cytotoxicity allows for a more biocompatible delivery system, as this will help minimize off-target effects.

[0260] The cytotoxicity a composition of the invention may be compared to any suitable control. Merely by way of example, the cytotoxicity of a composition of the invention may be compared to the rate of degradation of the same active agent in free solution.

[0261] In a suitable embodiment, lower cytotoxicity may be demonstrated by induction of a lower percentage of cell growth inhibition. In a suitable embodiment, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) analysis may be used to measure cytotoxicity. In a suitable embodiment, the percentage of cell viability using a MTT assay may be calculates using the following formula:

[0262] %Cell Viability = Abs samp Ze) / Abs (Control) xlOO where Abs (Sample) is absorption for the sample and Abs (Control) is absorption for the control

[0263] Alternatively, a IC50 values may be calculated from an MTT assay to represent the concentration of a compound required to inhibit 50% of cell viability or metabolic activity.

[0264] In a suitable embodiment, the increase in the cell growth inhibition of a composition of the invention may be less than about 45%, as compared to a suitable control. For example, the increase in the cell growth inhibition of a composition of the invention may be less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% or even about 0%.

[0265] In a suitable embodiment, the IC50 value of the composition of the invention, representing the concentration required to inhibit 50% of cell viability or metabolic activity, may be at least 4- fold lower than that of a suitable control. For example, the IC50 value of the composition of the invention, representing the concentration required to inhibit 50% of cell viability or metabolic activity, may be at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, at least 5-fold lower, at least 6-fold lower, at least 7-fold lower, at least 8-fold lower, at least 9- fold lower or even at least 10-fold lower than that of a suitable control.

[0266] Increased half-life of compositions of the invention

[0267] Compositions in accordance with the first or second aspects of the invention or the products of methods in accordance with the third aspect of the invention may exhibit a longer half-life, as compared to the compositions of the prior art.

[0268] This longer half-life offers many useful benefits. Compositions with a longer half-life remain in the bloodstream for an extended period, allowing more time for them to reach the target site as well as reducing the required dosing frequency. Alternatively, the compositions of the invention with a longer half-life may have a longer window of opportunity for active agent release, as compared to a composition of the prior art.

[0269] Again, this advantageous property may, at least to some extent, arise from the elevated stability of the compositions of the invention.

[0270] It will be appreciated that a longer half-life provided by a composition of the invention allows for or sustained drug release, extended circulation time, and reduced dosing frequency.

[0271] The half-life of a composition incorporating an active agent of the invention may be compared to any suitable control. Merely by way of example, the half-life of a composition of the invention incorporating an active agent may be compared to the half-life of the same active agent in free solution.

[0272] In a suitable embodiment, the half-life of a composition may be calculated using the following formula:

[0273] > 0.693 tl / 2 =Kel

[0274] Where keiis the elimination rate constant.

[0275] In a suitable embodiment, the half-life of a composition of the invention may be 5% longer or more, as compared to a suitable control. For example, the half-life of a composition of the invention may be 5% longer or more, 10% longer or more, 15% longer or more, 20% longer or more, 25% longer or more, 30% longer or more, 35% longer or more, 40% longer or more, 45% longer or more, 50% longer or more, 55% longer or more, 60% longer or more, 65% longer or more, 70% longer or more, 75% longer or more, 80% longer or more, 85% longer or more, 90% longer or more, 95% longer or more, or even 100% longer or more, as compared to a suitable control

[0276] In a suitable embodiment, the half-life of a composition of the invention may be 5 hours or more. For example, the half-life of a composition of the invention may be 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 24 hours or more, or even 48 hours or more.

[0277] Methods of manufacturing the compositions in accordance with the first aspect of the invention

[0278] The third aspect of the invention provides a method that may be used to manufacture a composition in accordance with the first aspect of the invention.

[0279] Specifically, the third aspect of the invention provides a method of forming a lipid encapsulated composition, the method comprising:

[0280] • combining:

[0281] • an aqueous solution comprising fibres;

[0282] • a lipid; and

[0283] • an active agent;

[0284] • forming a lipid layer from the lipid that encapsulates the aqueous solution comprising the fibres thereby forming a lipid encapsulated composition; and

[0285] • dehydrating the composition such that a network of fibres is formed within the lipid layer.

[0286] As the composition is dehydrated, the fibres present in the lipid encapsulated aqueous solution self-assemble to form a network within the inner surface of the lipid layer. In the case of a liposomal composition, the fibres self-assemble within the inner membrane of the liposome.

[0287] Generally, the fibres, lipids (for example phospholipids), and active agents may be as considered elsewhere in the specification, in connection with compositions of the invention. Particular considerations as to how the fibres, lipids and active agents may be used in the methods of the invention are set out below. Merely by way of example, the fibres may comprise, or be formed from, inulin, chitin, or chitosan. The physical properties of the fibres may be as discussed previously.

[0288] Similarly, active agents suitable for use in a method of the invention may be the same as those considered elsewhere in the present specification in the context of composition of the invention.

[0289] Suitable combinations of active agents and fibres to be incorporated in the compositions of the invention may also be as discussed previously in this disclosure.

[0290] As discussed further below, the hydrophilic or hydrophobic nature of active agents to be employed in the methods of the invention may influence the manner in which the active agent is combined with other constituents.

[0291] An aqueous solution comprising fibres

[0292] The methods of the third aspect of the invention involve utilise aqueous solutions comprising fibres. The fibres are dissolved in an aqueous diluent, such as water, to produce the required solution. This solution may then be combined with other constituents, to meet the requirements ofmethod of the invention.

[0293] The fibres used in the methods of the invention may comprise the same materials as considered in connection with the fibres used in the compositions of the invention. Similarly, the characteristics of fibres used in a method of the invention may be the same as those considered elsewhere in the present specification in the context of composition of the invention.

[0294] Forming fibres

[0295] Optionally, a method of the invention may comprise a further step of forming the required fibres prior to formation of the aqueous solution comprising the fibres. Again, the fibres formed may comprise the same materials as considered elsewhere. The fibres formed may have some (or all) of the characteristics described in relation to fibres used in compositions of the invention. In a suitable embodiment of such a method of the invention, the fibres are formed by a two part process involving hydrolysis of the required material (e.g. inulin, chitin or chitosan), to produce units of the material. These hydrolysed units may then be subject to size reduction in order to form fibres having the desired characteristics.

[0296] Merely by way of example, a suitable material from which fibres are to be formed may be hydrolysed to produce units that have a length of about 20pm, about 21 pm, about 22pm, about 23pm, about 24pm, about 25pm, about 26pm, about 27pm, about 28pm, about 29pm or about 30pm. Suitably in an embodiment, the material is hydrolysed to form units of a length of 25pm.

[0297] Units of a suitable material, may also be produced by methods other than hydrolysis. In either case, the units may be further reduced in size to form fibres to be used in the method of the invention.

[0298] A skilled person will recognise that there are various techniques that may be used to achieve the required reduction in size. Merely by way of example, mechanical milling may be used to further reduce the size and produce fibres suitable for use in a method of the invention. Such mechanical milling may be performed using a bead milling machine. Alternatively, electrospinning may be used to further reduce size and produce appropriate fibres. In a further example, chemical degradation may be used to further reduce size in the production of fibres.

[0299] In a suitable embodiment, hydrolysed material is further reduced in size by a bead milling machine to produce fibres for use in a method of the invention.

[0300] By way of example, bead milling suitable for reducing the size of hydrolysed material to produce fibres suitable for use in a method of the invention may comprise mixing at approximately 6000 rpm. Suitably, bead milling suitable for reducing the size of hydrolysed material to produce fibres suitable for use in a method of the invention may be performed for approximately 15 minutes.

[0301] A lipid

[0302] Suitably the lipid employed in accordance with a method of the invention is as considered elsewhere in the present specification. For example, the lipid maybe a phospholipid, such as lecithin. The lipid layer formed in a method of the invention may be a monolayer, or a bilayer (such as a liposome). The lipid, such as a phospholipid, to be employed in the method of the invention may be selected in light of the nature of the lipid layer to be formed.

[0303] In the case that a method of the invention is intended to produce a composition comprising a monolayer lipid layer, a suitable lipid may be a phospholipid comprising a mixture of crude phospholipids comprising less than 50% phosphatidylcholines. Suitably, the phospholipid may comprise a mixture of crude phospholipids comprising approximately 10-15% phosphatidylcholines.

[0304] In the case that a method of the invention is intended to produce a composition comprising a bilayer lipid layer, a suitable lipid may be a phospholipid comprising a mixture of crude phospholipids comprising more than 50% phosphatidylcholines. Suitably, the phospholipid may comprise a mixture of crude phospholipids comprising approximately 50-55% phosphatidylcholines.

[0305] An active agent

[0306] The active agents suitable for use in a method of the invention may be the same as those considered elsewhere in the present specification in the context of composition of the invention formed.

[0307] The way in which a solution comprising an active agent is formed may vary depending on whether a hydrophilic active agent, or a hydrophobic active agent, or a combination of hydrophilic and hydrophobic active agents, is to be used.

[0308] In the case of a hydrophilic active agent, the active agent may be dissolved in a polar diluent, suitably an aqueous diluent such as water, prior to being combined in a subsequent step of a method of the invention.

[0309] Alternatively, in the case of a hydrophobic active agent, the active agent may be dissolved in a hydrophobic diluent, prior to being combined in a subsequent step of a method of the invention. In a suitable embodiment, a hydrophobic active agent is dissolved in the lipid. Combining the aqueous solution comprising fibres, lipid, and active agent

[0310] The methods of the invention comprise a step of combining an aqueous solution comprising fibres (which will subsequently form a network of fibres in a composition), a lipid (which will form a lipid layer), and one or more active agents. These may be combined in any suitable manner, or simultaneously or sequentially, in order to produce a mixture comprising the aqueous solution and fibres, the lipid, and the active agent(s). It will be appreciated that specific permutations of the ways in which the various recited constituents may be combined may be selected with reference to the constituents that are being used.

[0311] For example, in the case that the method uses a hydrophilic active agent, the hydrophilic active agent may be dissolved in the aqueous solution comprising fibres, prior to combining this solution (comprising both fibres and a hydrophilic active agent) with the lipid.

[0312] Alternatively, a hydrophilic active agent may be dissolved in a polar solution, such as a further aqueous solution, and this polar solution combined with both the solution comprising fibres and the lipid. In an example of such an embodiment, the polar solution (such as a further aqueous solution), solution comprising fibres, and lipid may be combined simultaneously. Alternatively, the aqueous solution comprising the fibres and the lipid may be combined to produce a mixture comprising the fibres and the lipid, and this mixture combined with the polar solution (such as a further aqueous solution) comprising the hydrophilic active agent.

[0313] In the case that the method uses a hydrophobic active agent, the hydrophobic active agent may be dissolved in the lipid prior to combining with the aqueous solution comprising fibres. Alternatively, the hydrophobic active agent may be dissolved in a further hydrophobic solvent, and this mixture of hydrophobic active agent and hydrophobic solvent combined simultaneously or sequentially with the lipid and the aqueous solution comprising fibres.

[0314] Mixing a solution comprising fibres with a lipid

[0315] As referred to above, the methods of the third aspect of the invention may comprise mixing the solution comprising fibres with the lipid, such as lecithin. The lipid may comprise a hydrophobic active agent, and / or the solution comprising fibres may comprise a hydrophilic active agent. The lipid introduced in this mixing step forms the lipid layers (such as liposomes) present in the composition produced by the method. In a suitable embodiment, the lipid is combined with an aqueous solution that comprises fibres, but which does not comprise a hydrophilic active agent. The resultant mixture of fibres and lipid is then combined with an aqueous solution comprising a hydrophilic active agent in a later step.

[0316] In a suitable embodiment, mixing the solution of fibres with the lipid (for example a phospholipid, such as lecithin), is conducted by high pressure homogenization.

[0317] By way of example, high-pressure homogenization suitable for use in mixing a solution comprising fibres with a lipid in a method of the invention may comprise mixing at approximately 7000 rpm. Suitably, high-pressure homogenization suitable for use in mixing the solution comprising fibres with lipid in a method of the invention may be performed for approximately 5-10 minutes. Such protocols may be used in connection with phospholipids, such as lecithin.

[0318] Combining a mixture comprising fibres and lipid with a solution comprising the active agent

[0319] As referred to above, in a suitable embodiment a method of the third aspect of the invention involves combining a mixture comprising fibres and lipid with a solution comprising the active agent. The active agent may be a hydrophilic active agent, and the solution a polar solution, such as an aqueous solution.

[0320] In a suitable embodiment, mixing of the lipid-fibre mixture with the solution comprising an active agent is by high pressure homogenization.

[0321] By way of example, high-pressure homogenization suitable for use in combining the mixture comprising fibres and lipid (such as phospholipid) with the solution comprising the active agent in a method of the invention may comprise mixing at approximately 3000 rpm. Suitably, high- pressure homogenization suitable for use in combining the mixture comprising fibres and lipid with the solution comprising the active agent in a method of the invention may be performed for approximately 5 minutes. Forming lipid layers from the lipid that encapsulate the solution comprising the fibres and active agent

[0322] Once the aqueous solution comprising fibres, lipid and active agent (optionally provided in a further solution) have been combined, the combination formed will comprise both hydrophilic components (the aqueous solvent, and optionally any hydrophilic active agents) and hydrophobic components (the lipid, and optionally any hydrophobic active agent).

[0323] The hydrophilic and hydrophobic components of the combination enable the formation of lipid layers that will encapsulate portions of the aqueous solution (comprising the fibres, and optionally a hydrophilic active agent). Thus, a lipid encapsulated composition is formed. It will be appreciated that not all of the solution comprising the fibres (and optionally active agent) need be encapsulated by the lipid layers, such as liposomes, in this step.

[0324] In a suitable embodiment, the lipid layers are formed by high pressure homogenization of the combined mixture comprising the fibres, the lipid, and optionally a further solution comprising the active agent.

[0325] By way of example, high-pressure homogenization suitable for use in encapsulating the solution comprising the fibres and active agent within a lipid layer (such as a liposome) to form a composition in a method of the invention may comprise mixing at approximately 10000 - 12000 rpm. Suitably, high-pressure homogenization suitable for use in encapsulating the solution comprising the fibres and active agent within a lipid layer (such as a liposome) to form a composition in a method of the invention may be performed for approximately 10-15 minutes.

[0326] The mixtures produced as a result of such high-pressure homogenization may then be dried to form substantially dehydrated compositions.

[0327] Dehydrating the composition such that a network of fibres is formed within the liposomes

[0328] The methods of the third aspect of the invention involve dehydrating the lipid encapsulated composition (such as a liposomal composition), so that a network of fibres is formed within the lipid layers of the composition. As noted above, the fibres may self-assemble during dehydration to yield the required network. The dehydration may yield a substantially dehydrated lipid encapsulated composition (such as a substantially dehydrated liposome composition).

[0329] In a suitable embodiment, a method of the invention may further comprise adding a drying adjuvant to the lipid encapsulated composition (such as a liposome composition) prior to the dehydration step. Suitably, the drying adjuvant added is gum Arabic.

[0330] In a suitable embodiment, the drying adjuvant is mixed with the lipid encapsulated composition (such as a liposome composition) by high pressure homogenisation.

[0331] By way of example, high-pressure homogenization suitable for use in dehydrating the lipid encapsulated composition (such as a liposome composition) in a method of the invention may comprise mixing at approximately 10000 rpm. Suitably, high-pressure homogenization suitable for use in dehydrating the lipid encapsulated composition (such as a liposome composition) may be performed for approximately 5 minutes.

[0332] Suitably, in an embodiment, dehydration of the lipid encapsulated composition (such as a liposome composition) is by spray-drying. Suitably, in an embodiment spray-drying suitable for use in dehydrating a lipid encapsulated composition (such as a liposome composition) in a method of the invention is performed using an inlet temperature of approximately 180°C and an outlet temperature of approximately 90°C. For example, such spray-draying may be performed using an inlet temperature of about 175°C, about 180°C or about 185°C and an outlet temperature of about 85°C, about 90°C or about 95°C.

[0333] Substantially dehydrated compositions produced by the methods of the invention may be provided to a subject in their substantially dehydrated form, or may be dissolved in an aqueous diluent, such as water, before they are provided to a subject.

[0334] EXAMPLES

[0335] Example 1 : Preparation of an example composition of the invention.

[0336] Methods

[0337] Inulin extract was acid hydrolysed to obtain inulin fibres. The obtained inulin fibres were several micrometres in size 25pm). These fibres were introduced into a research lab bead milling machine for 15 minutes at an rpm of 6000 to obtain fibres of approximately 200-300 nm in size. From the obtained product, 50g of fibres were accurately weighed and added to a solution containing 100g phospholipids in water. The solution was then homogenized at 7000 rpm for 5- 10 minutes. 400g of vitamin C (the active molecule) was added and the solution was further homogenized for 5 minutes at 3000 rpm. A separate solution was made by mixing 400g of gum Arabic in water. The gum Arabic solution was homogenized well. The active loaded inulin lecithin complex was introduced into the gum Arabic solution and homogenized well at 10000 rpm for 5 minutes. The solution was then spray dried with an inlet temperature of 180°C and an outlet temperature of 90°c.

[0338] Results

[0339] A substantially dehydrated liposome composition encapsulating vitamin C was successfully produced.

[0340] Example 2: Analysis of structure, properties, and encapsulation efficacy of liposomes in a composition of the invention

[0341] A composition of the invention comprising glutathione as an active agent was manufactured, and its properties studied.

[0342] Zeta potential

[0343] The zeta potentials of dry liposomal glutathione powder were measured using a Nano-Zeta Potential Analyzer (Malvern Instruments Ltd., UK). Forthe comparison, results were compared with "Evaluation and clinical comparison studies on liposomal and non-liposomal ascorbic acid (vitamin C) and their enhanced bioavailability" as a reference.

[0344] The zeta potential of dry liposomal glutathione powder was -36.7 mV which indicates stability of the liposome. Compared to the reference, liposomal glutathione showed higher stability.

[0345] Particle size

[0346] Particle size of liposomal glutathione was measured using Dynamic Light Scattering (DLS). The results suggest that the maximum intensity of liposomes is around 200-250 nm. The results of particle size measurements by the DLS method indicated that liposomes have a homogenous size distribution. Encapsulation efficiency

[0347] High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, was used to measure encapsulation efficiency. The amount of a drug that is successfully trapped inside a micelle or nanoparticle is known as encapsulation efficiency. An encapsulation efficiency (EE, %) was carried out for liposomal glutathione Powder.

[0348] The HPLC method yielded an encapsulation efficiency of 80.15 % for liposomal glutathione and active content remain unaltered even after 3 months of storage. It will be appreciated that this result demonstrates both advantageous encapsulation efficiency and advantageous stability in respect of the compositions of the invention.

[0349] Example 3: Analysis of liposomes in a composition of the invention using in vitro models

[0350] The properties of the composition of the invention comprising glutathione (“liposomal glutathione”) were investigated further using in vitro models of the digestive tract.

[0351] Method of in vitro digestion model for pH stability of liposomal glutathione

[0352] The in vitro simulated digestion stability of the liposomal glutathione was analysed under simulated gastric and intestinal conditions to assess its degradation process. To understand the behaviour of glutathione in the gastrointestinal environments, in vitro simulated digestion stability study of pure glutathione and liposomal glutathione (glutathione incorporated in a composition of the invention) was performed in buffer pH 1.2, simulating the gastric pH, and buffer 6.8, simulating the intestinal pH.

[0353] A burst degradation of around 40% in simulated gastric fluid (SGF) and more than 50% in SIF conditions in the first 30 min was observed for glutathione (control), furthermore in simulated intestinal fluid (SIF) condition at pH 6.8, more than 75% of glutathione was degraded due to oxidation of glutathione. In contrast, the composition of the invention (liposomal glutathione) registered slow degradation (less than 10%) in both the SGF and SIF conditions. These results confirm the encapsulation of glutathione in the liposomal glutathione formulation, and the improved stability to digestion of the compositions of the invention. Particle size distribution study

[0354] The mean particle size (PS) for liposomal glutathione was measured by using the dynamic light scattering technique (Litesizer 500, Anton Paar, USA) at 25 °C.

[0355] The particle size of the glutathione was obtained in the range of 250 to 650 nm, whereas the particles in the liposomal glutathione composition of the invention registered in the range of 70 to 220 nm at the initial stage. The changes of particle size distribution of both glutathione and liposomal glutathione were analysed under both SGF and SIF conditions at different time intervals. The percentage of particle size distribution of liposomal glutathione registered is no considerable changes in either the SGF or SIF conditions, whereas glutathione registered a considerable change in the percentage of particle distribution. These results reveal that the liposomal glutathione composition of the invention protected the glutathione molecules in the liposomal formulation, as shown by the lack of any considerable degradation of the liposomally encapsulated glutathione in both the SGF and SIF conditions. The case of glutathione alone, which was not protected in liposomes of a composition in accordance with the invention, registered high reduction of percentage of particle distribution in both the SGF and SIF conditions, which indicates high degradation of the glutathione in this control formulation.

[0356] Example 4: Analysis of liposomes in a composition of the invention using different cellular models

[0357] Glutathione cytotoxicity

[0358] Glutathione cytotoxicity was measured using standard MTT assay protocols. The MTT assay for the liposomal glutathione and control glutathione formulation were tested against MCF-7 cell lines for various concentrations. The morphological change of MCF-7 cells after administration with control, glutathione and liposomal glutathione at different concentrations for 24 - 48 h were measured.

[0359] The results, set out in Table 1 , demonstrated that the incorporation of glutathione in a liposomal composition of the invention had no adverse impact on cytotoxicity, as compared to glutathione incorporated in a control formulation. Table 1. Percentage of cell viability of glutathione and liposomal glutathione by MTT assay

[0360] In vitro cellular uptake

[0361] Analysis of whether a product entered target cells, and its concentration, was undertaken using Coumarin-6 fluorescent probes. MCF-7 cells were cultured and treated with liposomal glutathione or glutathione, with or without Coumarin-6.

[0362] As observed from fluorescent imaging, cells incubated with the coumarin-6 loaded liposomal glutathione showed remarkably increased fluorescence, compared with cells incubated with coumarin-6 loaded glutathione control. This demonstrates that the coumarin-6-loaded liposomal glutathione compositions of the invention have significantly higher cellular uptake efficiency compared with the coumarin-6 loaded control glutathione formulation.

[0363] Ability to cross the intestinal barrier

[0364] To investigate the ability of the composition of the invention to cross the intestinal barrier, Caco-2 permeability assays were completed. Both liposomal glutathione compositions of the invention and control glutathione were tested. The permeability assay values of liposomal glutathione and glutathione were found to be 4.17 x 10-6 cm / s and 1.12 x 10-6 respectively at a concentration of 62.5 pg / ml. This result reveals that the liposomal glutathione composition of the invention has significantly higher permeability (increased 3.7-fold) as compared to the control glutathione formulation. Liposomal glutathione showed moderate permeability, whereas glutathione showed low permeability, which indicates that the formulation of glutathione in liposomal compositions of the invention increases its bioavailability, as compared to the control glutathione formulation.

[0365] Example 5: Analysis of liposomes in a composition of the invention in an in vivo adsorption Study

[0366] In this study, the oral bioavailability of an investigational product in accordance with the invention - Liposomal Glutathione (600-650 mg capsule containing NLT 250 mg of L- Glutathione / capsule), and a reference product (600-650 mg capsules which contains 250 mg of conventional Glutathione) was evaluated in a double blind, randomized, oral bioavailability comparison study of liposomal glutathione and non-liposomal glutathione in healthy adults under fasting conditions.

[0367] Twenty-four subjects enrolled in this study were administered both glutathione formulations, (a composition of the invention with liposome encapsulation of glutathione, or the control glutathione formulation) with sufficient interim washout period.

[0368] Over 48 hours, glutathione concentrations in venous blood were measured. The glutathione provided in a composition of the invention (Group A) showed significantly higher blood concentrations (56.63 ± 3.66 mg / dL) compared to non-liposomal glutathione (Group B; 15.16 ± 1.16 mg / dL). The relative bioavailability of liposomal glutathione was 359.30%, indicating increased bioavailability. Additionally, liposomal glutathione had a longer half-life (21.30 hours) compared to non-liposomal glutathione (17.73 hours).

[0369] In summary, this study demonstrates that oral delivery of glutathione encapsulated in a composition of the invention comprising liposomes (600-650 mg capsule containing NLT 250 mg glutathione) produced circulating concentrations of glutathione nearly 4-fold greater than those elicited by oral delivery of non-liposomal glutathione (250 mg capsule). The increased plasma concentration of glutathione was not associated with any adverse effects over the study period suggesting that this liposomal formulation provides a suitable means to enhance glutathione availability. SPECIFIC ASPECTS AND EMBODIMENTS OF THE INVENTION

[0370] The following paragraphs do not constitute claims, but do set out details of specific aspects, embodiments, and combinations of subject matter in respect of which the Applicant may wish to seek protection.

[0371] 1 . A substantially dehydrated composition comprising:

[0372] • an active agent;

[0373] • a lipid layer; and

[0374] • a network of fibres; wherein the lipid layer encapsulates the network of fibres within the inner surface of the lipid layer.

[0375] 2. A composition according to paragraph 1 , wherein the network of fibres inhibits deformation of the lipid layer.

[0376] 3. A lipid encapsulated composition comprising:

[0377] • an active agent;

[0378] • a lipid layer; and

[0379] • an aqueous core; wherein the lipid layer encapsulates the aqueous core, and the aqueous core comprises a solution comprising fibres.

[0380] 4. A composition according to any preceding paragraph, wherein the lipid layer comprises a lecithin.

[0381] 5. A composition according to any preceding paragraph, wherein the lipid layer comprises a bilayer in the form of a liposome.

[0382] 6. A composition according to any preceding paragraph, wherein the fibres have a length of about 150nm.

[0383] 7. A composition according to any preceding paragraph, wherein the fibres have a diameter between 30nm and 40nm. 8. A composition according to any preceding paragraph, wherein the fibres comprise inulin, chitin, or chitosan.

[0384] 9. A composition according to any preceding paragraph, comprising a hydrophilic active agent encapsulated within the lipid layer.

[0385] 10. A composition according to any preceding paragraph, comprising a hydrophobic active agent present in the lipid of the lipid layer.

[0386] 11. A composition according to any preceding paragraph, wherein an active agent is independently selected from the group consisting of: a vitamin active agent (such as vitamin C, vitamin E, vitamin D, vitamin K, or biotin); a mineral active agent (such as zinc, iron, magnesium or selenium); an extracellular matrix active agent (such as collagen or hyaluronic acid); a herbal, fungal or botanic active agent (such as fungal extracts or phytonutrients, for example, curcumin or ashwagandha); an oil or fatty acid active agent (such as MCT oils, or omega-3 fatty acids, e.g. DHA); metabolically active agents (such as coenzyme Q10, nicotinamide mononucleotide, or glutathione); a pharmaceutical agent (small molecules such as insulin or antihistamines or therapeutic proteins such as peptides or antibodies ) and / or probiotic agent (such as live bacterial strains or yeast).

[0387] 12. A composition according to any preceding paragraph, comprising less than 2% water by weight.

[0388] 13. A composition according to any preceding paragraph, wherein the zeta potential of the encapsulating lipid layers in the composition is less than -30 mV or greater than +30 mV.

[0389] 14. A composition according to any preceding paragraph, wherein the encapsulation efficiency of at least one active agent within the lipid layer is greater than 80%.

[0390] 15. A composition according to any preceding paragraph, wherein the loading capacity of the encapsulating lipid layers in the composition is greater than 40%

[0391] 16. A composition according to any preceding paragraph, wherein the composition exhibits at least a 2-fold increase in bioavailability of the active agent calculated in comparison with any compatible control. 17. A composition according to any preceding paragraph, wherein the composition is substantially alcohol-free.

[0392] 18. A method of manufacturing a lipid encapsulated composition, the method comprising:

[0393] • combining:

[0394] • an aqueous solution comprising fibres;

[0395] • a lipid; and

[0396] • an active agent;

[0397] • forming a lipid layer from the lipid that encapsulates the aqueous solution comprising the fibres thereby forming a lipid encapsulated composition; and dehydrating the composition such that a network of fibres is formed within the lipid layer.

[0398] 19. A method according to any of paragraphs 18, wherein the lipid comprises lecithin.

[0399] 20. A method according to paragraph 18 or paragraph 19, wherein the aqueous solution comprising fibres, the lipid, and the active agent are combined sequentially.

[0400] 21 . A method according to paragraph 20, wherein the aqueous solution comprising fibres and the lipid are combined to form a mixture, and this mixture is combined with a solution comprising an active agent.

[0401] 22. A method according to any of paragraphs 18 to 21 , comprising mixing the solution comprising fibres with the lipid, and / or combining of the lipid-fibre mixture with the solution of an active agent, and / or formation of the lipid layers are performed by high pressure homogenization.

[0402] 23. A method according to any of paragraphs 18 to 22, further comprising adding a drying adjuvant to the composition prior to the dehydrating step.

[0403] 24. A method according to any of paragraphs 18 to 23, wherein dehydration of the lipid encapsulated composition is by spray-drying.

[0404] 25. A method according to any of paragraphs 18 to 24, wherein the network of fibres is formed by self-assembly.

Claims

CLAIMS1 . A substantially dehydrated composition comprising:• an active agent;• a lipid layer; and• a network of fibres; wherein the lipid layer encapsulates the network of fibres within the inner surface of the lipid layer.

2. A composition according to claim 1 wherein the fibres in the network are associated with one another by van der Waal’s forces.

3. A composition according to any of the preceding claims, wherein the network of fibres provides a scaffold, supporting the inner membrane of the liposome.

4. A composition according to any of the preceding claims, wherein the network of fibres supports the lipid layer, thereby maintaining the lipid layer in a substantially spherical shape.

5. A composition according to any of the preceding claims, wherein the network of fibres inhibits deformation of the lipid layer.

6. A lipid encapsulated composition comprising:• an active agent;• a lipid layer; and• an aqueous core; wherein the lipid layer encapsulates the aqueous core, and the aqueous core comprises a solution comprising fibres.

7. A composition according to any one of the preceding claims, wherein the lipid layer comprises a phospholipid and / or a sphingolipid.

8. A composition according to claim 7, wherein said phospholipid is lecithin.

9. A composition according to claim 8, wherein said lecithin is derived from a source selected from the group consisting of: sunflower, grape seed, and soy beans10. A composition according to any one of the preceding claims, wherein the lipid layer is selected from the group consisting of: a monolayer; and a bilayer.

11. A composition according to claim 10, wherein the lipid layer comprises a bilayer in the form of a liposome.

12. A composition according to any of the preceding claims, wherein the fibres have a length of between 150nm and 300nm.

13. A composition according to any of the preceding claims, wherein the fibres have a length of less than 250nm.

14. A composition according to any of the preceding claims, wherein the fibres have a length of about 150nm.

15. A composition according to any of the preceding claims, wherein the fibres have a diameter between 30nm and 40nm.

16. A composition according to any of the preceding claims, wherein the fibres have an aspect ratio of between 3 and 10.

17. A composition according to any of the preceding claims, wherein the fibres have an aspect ratio of between 5 and 6.

18. A composition according to any of the preceding claims, wherein the fibres have a tensile strength of between 200MPa and 300MPa.

19. A composition according to any of the preceding claims, wherein the fibres have a Young’s modulus of between 60GPa and 80GPa.

20. A composition according to any of the preceding claims, wherein the fibres comprise inulin, chitin, or chitosan.

21. A composition according to claim 20, wherein the fibres consist of inulin, chitin, or chitosan.

22. A composition according to claim 21 , wherein the fibres consist of inulin.

23. A composition according to claim 21 , wherein the fibres consist of chitin.

24. A composition according to claim 21 , wherein the fibres consist of chitosan.

25. A composition according to any of the preceding claims, wherein the lipid layer is substantially spherical.

26. A composition according to any of the preceding claims, comprising a hydrophilic active agent.

27. A composition according to claim 26, wherein the hydrophilic active agent is encapsulated within the lipid layer.

28. A composition according to any of the preceding claims, comprising a hydrophobic active agent.

29. A composition according to claim 28, wherein the hydrophobic active agent is present in the lipid of the lipid layer.

30. A composition according to any one of the preceding claims, wherein an active agent is independently selected from the group consisting of: a vitamin active agent (such as vitamin C, vitamin E, vitamin D, vitamin K, or biotin); a mineral active agent (such as zinc, iron, magnesium or selenium); an extracellular matrix active agent (such as collagen or hyaluronic acid); a herbal, fungal or botanic active agent (such as fungal extracts or phytonutrients, for example, curcumin or ashwagandha); an oil or fatty acid active agent (such as MCT oils, or omega-3 fatty acids, e.g. DHA); metabolically active agents (such as coenzyme Q10, nicotinamide mononucleotide, or glutathione); a pharmaceutical agent (small molecules such as insulin or antihistamines or therapeutic proteins such as peptides or antibodies ) and / or probiotic agent (such as live bacterial strains or yeast).31 . A composition according to any of the preceding claims, wherein an encapsulating lipid layer comprises multiple active agents.

32. A composition according to any of the preceding claims, wherein the composition comprises a mixture of lipid layers comprising different active agents.

33. A composition according to any of the preceding claims, wherein the composition is in the form of a nutritional supplement or foodstuff.

34. A composition according to any preceding claim, further comprising a drying adjuvant.

35. A composition according to claim 34, wherein the drying adjuvant is gum Arabic.

36. A composition according to any of the preceding claims, comprising less than 5% water by weight.

37. A composition according to claim 36, comprising less than 4% water by weight.

38. A composition according to claim 37, comprising less than 3% water by weight39. A composition according to claim 38, comprising less than 2% water by weight40. A composition according to claim 39, comprising less than 1 % water by weight.

41. A composition according to any of the preceding claims, wherein the zeta potential of the encapsulating lipid layers in the composition is less than -30 mV or greater than +30 mV.

42. A composition according to claim 41 , wherein the zeta potential of the encapsulating lipid layers in the composition is about -39 mV or +39mV.

43. A composition according to any of the preceding claims, wherein the encapsulation efficiency of at least one active agent within the lipid layer is greater than 70%.

44. A composition according to claim 43, wherein the encapsulation efficiency of at least one active agent within the lipid layer is approximately 80%.

45. A composition according to claim 44, wherein the encapsulation efficiency of at least one active agent within the lipid layer is greater than 80%.

46. A composition according to any of claims 43 to 45, wherein the encapsulation efficiency of each active agent within the lipid layer is greater than 70%.

47. A composition according to any of the preceding claims, wherein the loading capacity of the encapsulating lipid layers in the composition is greater than 40%48. A composition according to any of the preceding claims, wherein the composition exhibits an increase in bioavailability of the active agent.

49. A composition according to any of the preceding claims, wherein the composition exhibits at least a 2-fold increase in bioavailability of at least one active agent.

50. A composition according to claim 49, wherein the composition exhibits at least a 3-fold increase in bioavailability of at least one active agent.

51. A composition according to claim 50, wherein the composition exhibits at least an approximately 4-fold increase in bioavailability of at least one active agent.

52. A composition according to claim 51 , wherein the composition exhibits at least an approximately 5-fold increase in bioavailability of at least one active agent.

53. A composition according to any of claims 49 to 52, wherein the fold increase in bioavailability is calculated in comparison with any compatible control.

54. A composition according to any of the preceding claims, wherein the composition maintains an encapsulation efficiency in respect of the active agent of at least 75% over a period of two months or more.

55. A composition according to any of the preceding claims, wherein the composition is substantially alcohol-free.

56. A method of manufacturing a lipid encapsulated composition, the method comprising:• combining:• an aqueous solution comprising fibres;• a lipid; and• an active agent;• forming a lipid layer from the lipid that encapsulates the aqueous solution comprising the fibres thereby forming a lipid encapsulated composition; and dehydrating the composition such that a network of fibres is formed within the lipid layer.

57. A method according to claim 56, further comprising forming the fibres by hydrolysis followed by size reduction.

58. A method according to claim 57, wherein hydrolysed fibres are further reduced in size by bead milling.

59. A method according to any of claims 56 to 58, wherein the lipid comprises lecithin.

60. A method according to any of claims 56 to 59, wherein the aqueous solution comprising fibres, the lipid, and the active agent are combined sequentially.

61. A method according to claim 60, wherein the aqueous solution comprising fibres and the lipid are combined to form a mixture, and this mixture is combined with a solution comprising an active agent.

62. A method according to claim 61 , wherein the mixture is combined with a polar solution comprising a hydrophilic active agent.

63. A method according to claim 62, wherein the polar solution is an aqueous solution.

64. A method according to any of claims 61 to 63, comprising mixing the solution comprising fibres with the lipid by high pressure homogenization.

65. A method according to any of claims 61 to 64, wherein combining of the lipid-fibre mixture with the solution of an active agent is by high pressure homogenization.

66. A method according to any of claims 56 to 65, wherein the lipid layers are formed by high pressure homogenization.

67. A method according to any of claims 56 to 66, further comprising adding a drying adjuvant to the composition prior to the dehydrating step.

68. A method according to claim 67, wherein the drying adjuvant is gum Arabic.

69. A method according to claim 67 or 68, wherein the drying adjuvant is mixed with the lipid encapsulated composition by high pressure homogenisation.

70. A method according to any of claims 56 to 69, wherein dehydration of the lipid encapsulated composition is by spray-drying.

71. A method according to claim 70, wherein spray-drying of the lipid encapsulated composition is performed using an inlet temperature of approximately 180°C and an outlet temperature of approximately 90°C.

72. A method according to any of claims 56 to 71 , wherein the network of fibres is formed by self-assembly.

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