NL2022082B1

NL2022082B1 - Transport vessels for facilitating trans-membrane transport

Info

Publication number: NL2022082B1
Application number: NL2022082A
Authority: NL
Inventors: James Taylor Glen; Hendrik Van Der Westhuizen Jan; John Davis Henry
Original assignee: Univ Free State
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2020-06-25
Also published as: ZA201907859B

Abstract

The present invention relates to transport vessels for facilitating the transport of a biologically active substance across a cellular membrane (referred to as trans-membrane transport). The present invention also relates to a process for preparing such vessels and, further, to processes for preparing compositions and preparations containing such vessels. According to a first aspect thereof, the present invention provides for a lipid transport vessel, the vessel comprising a biologically active substance making up the vessel core Which biologically active substance is, at least partially, coated with fatty acids said fatty acids functioning, in use, to facilitate trans-membrane transport of the biologically active substance.

Description

TRANSPORT VESSELS FOR FACILITATING TRANS-MEMBRANE TRANSPORT

TECHNICAL FIELD The present invention relates to transport vessels for facilitating the transport of a biologically active substance across a cellular membrane (referred to as trans-membrane transport). The present invention also relates to a process for preparing such vessels and, further, to processes for preparing compositions and preparations containing such vessels.

BACKGROUND TO THE INVENTION The cell membrane of a cell, which is also known as the plasma membrane or the cytoplasmic membrane, is a biological membrane which functions to separate the interior of the cell (the intracellular environment) from the outside environment (the extracellular environment) surrounding the cell. The basic function of the cell membrane is to protect the cell from its surroundings and to control the movement of substances into and out of the cell, which the membrane does by virtue of the fact that it is selectively permeable to ions and organic molecules. Cell membranes are also involved in a variety of cellular processes, such as, cell adhesion, ion conductivity, cell signalling. These membranes also serve as the attachment surface for several extracellular structures, for example the glycocalyx. Although the abovementioned processes are also essential for the functioning of a cell, the present patent specification focuses particularly on trans-membrane transport and the features of the cell membrane which are pertinent to such transport.

As mentioned above, the cell membrane is selectively permeable and regulates which substances enter and leave the cell, thereby facilitating the transport of materials required for survival. Transport of substances into and out of the cell can be either by way of a passive process (which does not require the input of cellular energy) or an active process (which does require the input of cellular energy).

With respect to passive membrane transport; some small molecules and ions (for example, carbon dioxide and oxygen) move across the cell membrane directly, via osmosis and diffusion. Certainnutrients, such as sugars and amino acids diffuse into and out of the cell via protein channels such as aquaporins and permeases.

Turning to active membrane transport mechanisms; certain molecules are absorbed by cells through the process of endocytosis. In this process, the cell membrane invaginates around the substance to be transported. The captured substance is engulfed and enters the cell on the intracellular side of the cellular membrane as a vesicle. Exocytosis is the reverse process to endocytosis and is utilized to allow certain substances and bi-products of metabolism to exit the cell.

Membrane transport is directly related to the structure of the cell membrane. The cell membrane is comprised of a lipid bilayer which is formed through the process of self-assembly. The cell membrane consists of three classes of amphipathic lipids: phospholipids, glycolipids and sterols, as well as carbohydrate and protein components.

The amphipathic phospholipids which are most abundant, in the majority of cells, assemble so that the hydrophilic “head” regions of the phospholipids are orientated towards the intracellular and extracellular spaces which have contact with polar fluids whilst the hydrophobic “tail” regions orientate inwards such that they are isolated from the polar fluids thereby resulting in the formation of the lipid bilayer through the advent of such non-covalent hydrophobic interactions between the “tail” regions. The lipid bilayer is continuous and spherical and forces such as van der Waals forces, electrostatic hydrogen bonds and non-covalent interactions all contribute to the formation of this bilayer.

The structure of the lipid bilayer is such that it is generally impermeable to polar molecules and hence generally prevents polar solutes, such as, amino acids, nucleic acids, carbohydrates, proteins and ions, from diffusing across the membrane. However, the lipid bilayer is structured so as to allow for the passive diffusion of hydrophobic molecules across it. In this respect, the cell membrane is considered to be lipophilic (permeable to lipids) and hence lipophobic molecules (generally the polar hydrophilic molecules) are unable to directly penetrate the cell membrane. For the purposes of this specification, lipophobic refers to substances which are not soluble in lipids or in non-polar solvents, which is contrasted with lipophilic substances which are soluble in lipids and/or non-polar solvents.

Biologically active molecules, such as vitamins, minerals and pharmaceutical compositions for the treatment of various conditions must be delivered to the relevant body cells in order to have an effect. Trans-membrane transport mechanisms are therefore highly relevant with respect to the delivery of such compounds and compositions and hence mechanisms to facilitate such trans-membrane transport have been developed in the art. Three commonly known manners in which to facilitate the trans-membrane transport of biologically active substances include the use of liposomes, the use of pheroids and the use of nanoparticle technology. A liposome is a spherical vesicle which is composed of a lipid bilayer which is artificially prepared through the disruption of biological membranes utilizing processes such as sonication, for example. As regards the delivery of biologically active substances using liposomes, the liposome encapsulates the substance to be delivered such that the molecules are encapsulated within a lipid bilayer. The lipid bilayer of the liposome is able to fuse with other lipid bilayers, such as the cell membrane, and to diffuse there through thus enabling the delivery of the substance into the cell. Generally speaking the substances for delivery which are encapsulated within the liposome are hydrophilic substances which are unable to diffuse through the cell membrane due to their polar nature. However, it will be appreciated by those skilled in the art that liposomes can be used to transport both hydrophobic substances, hydrophilic substances and charged molecules (which given their charged nature, are generally speaking hydrophilic in nature).

As stated above, liposome mediated delivery is typically by means of diffusion. However, liposomes can be made within a particular size range so that they become targets for macrophage phagocytosis. Such liposomes are then digested within the macrophage’s phagosome which results in the release of the biologically active substances contained within the liposome. Furthermore, liposomes can be made and subsequently decorated with opsonins and ligands which activate endocytosis in specific cell types, which endocytosis will be responsible for the uptake of the liposome into the cell and will facilitate the delivery of the biologically active substances to such cell. It will be appreciated by those skilled in the art that liposomes can be utilized to deliver a wide variety of biologically active substances to cells, including: carbohydrates, vitamins, amino acids, drugs, dietary and nutritional supplements and DNA (for the purposes of transfection), to name but a few examples.

As can be appreciated, liposomes can be customized to facilitate the delivery of certain biologically active substances and liposome structure can be customized to facilitate easier trans-membrane transport. In this respect, and by way of example, specific reference is made to certain types of liposomes: e Elastic Liposomes: These are liposomes that have specific ratios of phosphatidyl choline, cholesterol and cetyl pyridium within their structure, together with the biologically active substances as this composition ensures elasticity of the liposomes which facilitates trans- membrane transport.

e Ethosomes: Ethosomes are liposomes embodying high concentrations of ethanol which is a permeation enhancer. These liposomes which have a softer structure than standard liposomes and show enhanced delivery to skin cells and hence are utilized for this purpose. + Niosomes: Niosomes are comprised of non-ionic amphiphilic lipids in lamellar phase and such liposomes also have specific delivery properties.

Typically, liposomes encapsulate both the biologically active substances and the medium within which they are suspended. This can lead to decreased dosage for delivery which can be disadvantageous. Therefore it would be advantageous to provide for an alternative and improved transport vessel for trans-membrane transport wherein suspending media is not included in the transport vessel.

Furthermore, liposomes encapsulate the hydrophilic (lipophobic biologically active substance) thereby facilitating delivery across the lipophilic cell membrane rather than allowing for a modification of the lipophilicity of the active substances which means that in liposomes the volume to mass ratio of the vesicle relative to the biologically active substance is also higher. This also results in decreased active substance delivery which is disadvantageous.

Turning now to consider the use of Pheroids™ and Pheroid™ technology in trans-membrane transport; the so-called pheroid comprises an organic carbon backbone composed of unsaturated fatty acids with side-chain interactions that result in self-emulsifying characteristics. These vesicles are utilized to entrap hydrophilic, hydrophobic and amphiphilic compounds. Furthermore, these vesicles can be manipulated as to loading ability, mechanical resistance, permeability, size and solubility. The vesiclesare able to cross the cell membrane thereby allowing for the trans-membrane transport of the encapsulated compounds and the subsequent release thereof within the cell. Pheroids™ are manufactured by sparging nitrous oxide through an aqueous environment containing a low concentration of vitamin F. 5 Pheroids™ are typically utilized in trans-membrane delivery as adjuvants to enhance vaccine efficacy, in the treatment of infectious diseases, in instances where a reduction of immune recognition is required for the purposes of drug delivery (which also allows for the delivery of reduced dosage) and to minimize cytotoxicity and membrane damage.

Pheroids™, like liposomes, require a high vesicle component to active substance ratio, due to the incorporation of non-active suspending media with the active component, which tends to lead to less active substances being incorporated therein and consequently less active substances being delivered in a single vesicle. This leads to a higher dosage requirement which is disadvantageous.

Therefore, as stated above, it would be advantageous to provide an improved transport vessel which optimizes the vesicle to active substance ratio and which allows for less of the active substance to be delivered in order to obtain an effective dosage.

Both pheroid™ and liposome technology are reliant on the encapsulation of the active substance, meaning that the entire active substance must be encapsulated within the vesicle before trans- membrane transport can take place. As such, partial treatment of the lipophobic active substance is not possible which can also be disadvantageous.

Nanoparticles are small objects that behave as whole units with respect to their transport and properties. Typically, such particles are internalized by cells by way of endocytosis which facilitates the delivery of the biologically active substances making up such particle to the cell. However, nanoparticles containing lipid ligands can be delivered to cells directly. Typically, nanoparticles are utilized in systems wherein the controlled release of a drug is required.

OBJECT OF THE INVENTION It is an object of the present invention to provide an improved transport vessel for facilitating trans- membrane transport of biologically active substances across cell membranes which at least in part obviates the disadvantages associated with the technology of the prior art. It is a further object of the present invention to provide a method for manufacturing said improved transport vessels.

It is a further object of the present invention to provide for medicaments and preparations containing the transport vessels of the invention, these medicaments and preparations indicated for use in the treatment of diseases in mammalian subjects.

SUMMARY OF THE INVENTION According to a first aspect thereof, the present invention provides for a lipid transport vessel, the vessel comprising a biologically active substance making up the vessel core which biologically active substance is, at least partially, coated with fatty acids said fatty acids functioning, in use, to facilitate trans-membrane transport of the biologically active substance.

The general structure of the lipid transport vessel, as described and identified herein, is characterised in accordance with the appended microscopy data.

In an embodiment of the invention, the fatty acids may be polyunsaturated free fatty acids.

The polyunsaturated free fatty acids may include linoleic acid or alpha-linolenic acid. In an alternative embodiment, the polyunsaturated free fatty acid may comprise Vitamin F.

Vitamin F is commonly used as a collective term for essential fatty acids (EFAs) of oleic acid 21wt%, linoleic acid 34wt%, and linolenic acid 30wt%.

The fatty acids may either partially coat or fully coat the core. The coating accounts for the lipid transport vessel's ability to transport the biologically active substances contained in the core across a cellular membrane.

The trans-membrane transport is facilitated primarily by a diffusion process. However, it is envisaged that trans-membrane transport by the lipid transport vessels of the present invention may be facilitated by an active trans-membrane transport process, such as, endocytotic and phagocytotic processes.

The lipid transport vessel may be utilised to facilitate the transport of biologically active substances across a variety of cellular membranes, including but not limited to: oral membranes, gastric membranes, intestinal mucosa, nasal mucosa, alveolar mucosa, the stratum corneum, the epidermis of the skin and the conjunctiva of the eye.

The core of the lipid transport vessel is anhydrous and is generally a lipophobic substance but it is envisaged that the core may be a lipophilic substance. In particular, the core may be understood to be a substance possessing lipid characteristics but which does not move over membranes easily due to its size or polarity. Examples of such substances would include cholesterol-derived compounds such as steroids. In addition hereto, linoleic acids are known in the art to increase the transport of steroids over membranes.

In the instance that the core is a lipophobic core, it is envisaged that the biologically active substances comprising the core may include zwitterions, charged molecules, molecules having surface hydroxyl groups, polar molecules or a combination of the aforementioned. The biologically active substances contained within the core may include: amino acids (such as I-glutamine), pharmaceutical drugs (such as acetylsalicylic acid), biological metabolites (such as l-carnitine), vitamins (such as vitamin C), minerals (such as magnesium oxide) and other nutritional agents (such as nutrients and/or nutraceuticals). However, it will be appreciated by those skilled in the art that the core could contain a vast number of biologically active substances and that, as such, this disclosure is by no means limiting with respect to the content of the core.

The polyunsaturated free fatty acids associate with the lipophobic vessel core through a non-covalent interaction wherein pi-electrons from the double bonds in the polyunsaturated free fatty acids contribute to such association.

This results in the charges in the lipophobic core becoming dispersed which leads to decreased polarity of the core and increased lipophilicity which allows the lipid transport vessel to cross the cellular membrane.

According to a second aspect thereof, the present invention provides a method of manufacturing a lipid transport vessel, the method comprising mixing an anhydrous biologically active substance with at least one polyunsaturated free fatty acid composition in a ratio of 3.75:1 biologically active substance: polyunsaturated free fatty acid.

The biologically active substance comprises the core of the lipid transport vessel and is generally comprised of a lipophobic chemical substance.

However, it will be appreciated by those skilled in the art that a lipophilic biologically active chemical substance may also comprise the core of the lipid transport vessel.

The polyunsaturated free fatty acid may be selected from the group comprising: linoleic acid, alpha- linolenic acid, Vitamin F or a combination of the aforementioned.

In the instance that the core is comprised of a lipophobic chemical substance it is envisaged that it may include zwitterions, charged molecules, molecules having surface hydroxyl groups, polar molecules or a combination of the aforementioned.

The core may include: amino acids, such as I-glutamine, pharmaceutical drugs, such as acetylsalicylic acid, biological metabolites, such as |-carnitine, vitamins, such as vitamin C, minerals, such as magnesium oxide and other nutritional agents, such as nutrients and/or nutraceuticals.

However, it will be appreciated by those skilled in the art that the core could contain a vast number of lipophobic chemical substances and that, as such, this disclosure is by no means limiting with respect to the content of the core.

In an exemplary embodiment of the present invention, the lipid transport vessel manufactured in accordance with the second aspect of the invention may be incorporated into a secondary lipophilicchemical substance which secondary lipophilic chemical substance may subsequently be included in preparations for epidermal application.

The secondary lipophilic chemical substance may be selected from the group consisting of lanolin, petroleum jelly, waxes, mineral oil, natural oils and processed oils.

It will be appreciated by those skilled in the art that a variety of secondary lipophilic substances could be utilised for this purpose and hence the scope of the invention is by no means limited by the above provided list of secondary lipophilic substances.

The secondary lipophilic chemical substance provides an environment where the lipid transport vessel is stable.

Secondary substances should exclude substances such as silica (Aerosol™) which is used to thicken oils.

In this regard, silica will interfere with the lipid component (polyunsaturated fatty acids) of the transport vessel due to its hydrophilic nature.

In fact, silica would compete with the hydrophilic core due to its very hydrophilic nature.

Silica is usually used to dehydrate substances but it may also be used to thicken oils.

According to a third aspect thereof, the present invention provides for the use of a lipid transport vessel, as described above, in a method of facilitating trans-membrane transport.

Primarily, the lipid transport vessel is utilised to facilitate transport of lipophobic substances across cell membranes.

However, it will be appreciated by those skilled in the art that the vessel may be utilised to transport substances which are not strictly lipophobic.

The lipid transport vessel comprises a core area containing the biologically active lipophobic chemical substance which is required to be transported into the cell; this core area is coated, in the manner described above, by polyunsaturated free fatty acids.

The coating of polyunsaturated free fatty acids increases the lipophilicity of the core thereby allowing for the diffusion of core across the lipophilic cell membrane.

It will be appreciated by those skilled in the art that active trans-membrane transport processes may also play a role in such transport.

The method of facilitating trans-membrane transport may be utilised in delivery systems for the delivery of a wide range of biologically active compounds, such as, pharmaceuticals and nutritional supplements.

According to a fourth aspect thereof, the present invention provides for the use of the lipid transport vessel described herein in the manufacture of a medicament for the treatment of mammalian diseases. It will be appreciated by those skilled in the art that the disease or diseases indicated for treatment will be wholly dependent on the selection of the biologically active substance incorporated within the lipid transport vessel, the active substance being indicated for the treatment of one or more diseases. Treatment in this instance shall refer to prophylactic treatment, alleviating treatment and/or curative intervention and shall in no way be limited by the nature and/or the scope of the benefit provided.

Administration of the medicament and/or lipid transport vessel of the present invention can be done in any acceptable manner known in the medicinal arts. Specific non-limiting examples of administration methods which may be used in accordance with the embodiments of the invention include: topical administration, injection, intravenous administration, rectal administration, transdermal administration, ophthalmic administration, lymphatic administration and nasal administration. Further, non-limiting examples of administration methods which may be used in accordance with embodiments of the present invention include oral application by means of a soft (preferably) or hard gelatine capsule. In an embodiment, said gelatine capsule may be coated with an enteric coating in order to ensure that the contents are released in the duodenum (the first part of the small intestine). In accordance with a fifth aspect thereof, the present invention provides a preparation comprising a lipid transport vessel, the vessel containing a biologically active substance, making up the vessel core, the vessel core being at least partially coated with fatty acids which increase the lipophilicity of the core and thereby facilitate its trans-membrane transport and a secondary lipophilic chemical substance within which the vessel is suspended, the secondary lipophilic substance functioning, in use, as a suspending medium for the vessel for preparations intended for epidermal application.

The secondary lipophilic chemical substance may be selected from the group consisting of lanolin, petroleum jelly, waxes, mineral oil, natural oils and processes oils. It will be appreciated by those skilled in the art that a variety of secondary lipophilic substances could be utilised for this purpose and hence the scope of the invention is by no means limited by the above provided list of secondary lipophilic substances.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: is a light micrograph of magnesium oxide (MgO) crystals (a biologically active substance) which have been treated with vitamin F which appears as a stained oily substance in the micrographs; Figure 2: is a light micrograph of Asprin crystals (a biologically active substance) which have been treated with vitamin F which appears as a stained oily substance in the micrographs; Figure 3: is a light micrograph of L-glutamine crystals (a biologically active substance) which have been treated with vitamin F which appears as a stained oily substance in the micrographs; Figure 4: is a fluorescence micrograph of MgO crystals (a biologically active substance) which have been treated with vitamin F which fluoresces as shown in the micrographs; Figure 5: is a fluorescence micrograph of Asprin crystals (a biologically active substance) which have been treated with vitamin F which fluoresces as shown in the micrographs; Figure 6: is a fluorescence micrograph of L-glutamine crystals (a biologically active substance) which have been treated with vitamin F which fluoresces as shown in the micrographs; Figure 7: is a graph depicting the nuclear magnetic resonance (NMR) spectrum of a preparation prepared in accordance with the fifth aspect of the invention, said preparation includinga lipid transport vessel having an L-glutamine core and a lanolin secondary lipophilic chemical substance; Figure 8: is a further graph depicting the nuclear magnetic resonance (NMR) spectrum of the preparation detailed with reference to Figure 7; Figure 9: is a graph depicting the nuclear magnetic resonance spectrum (NMR) of the lanolin component of the preparation detailed in Figure 7; Figure 10: is a graph depicting the nuclear magnetic resonance spectrum (NMR) of the vitamin F component of the preparation detailed in Figure 7; Figure 11: is a graph depicting the nuclear magnetic resonance spectrum (NMR) of the L- glutamine component of the preparation detailed in Figure 7; Figure 12: 12A depicts a light micrograph and the corresponding fluorescent image of PSA with vitamin F is shown in 12B; Figure 13: 13A depicts a light micrograph and the corresponding fluorescent image of aspirin with vitamin F is shown in 13B;

Figure 14: 14A depicts a light micrograph and the corresponding fluorescent image of aspirin with citriole 100 oil (where no vitamin F is added) is shown in Figure 14B; Figure 15: 15A depicts a light micrograph and the corresponding fluorescent image of aspirin with vitamin F in citriole 100 oil is shown in Figure 15B; Figure 16: 16A depicts a light micrograph and the corresponding fluorescent image of citrimis, CAS and vitamin F is shown in Figure 16B; Figure 17: 17A depicts a light micrograph and the corresponding fluorescent image of citriole, CAS and vitamin F is shown in Figure 17B;

Figure 18: 18A depicts a light micrograph and the corresponding fluorescent image of CAS without vitamin F is shown in Figure 18B; and Figure 19: 19A depicts a light micrograph and the corresponding fluorescent image of CAS with vitamin F is shown in Figure 19B. The presently disclosed subject matter will now be described more fully hereinafter. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

DESCRIPTION OF THE INVENTION Example: Microscopic examination of compound mixtures To ascertain the gross structure of the lipid transport vessel of the present invention, a number of microscopic studies were undertaken. As such, the following crystalline compounds were examined using light and fluorescence microscopy: e Vitamin F blended with Magnesium oxide (MgO) in accordance with the method described hereunder; e Vitamin F blended with Asprin in accordance with the method described hereunder; and e Vitamin F blended with L-glutamine in accordance with the method described hereunder.

As briefly set out in the summary of the present invention herein above, the Inventors are of the opinion that vitamin F binds to the various biologically active substances via non-covalent interactions and more particularly via cohesion or capillary forces. The Nile Red stain was utilised as a probe to determine the presence of fatty acids given that it produces a red fluorescent signature in the presence of fatty acids at approximately 600 nm. When utilised, the Nile Red is dissolved in 100% acetone.

Light microscopy The light microscopic observations, based on transmitted illumination, indicate the presence of crystalline compounds stained by Nile Red. Such compounds show differently hued red diffractions.

The acetone utilised in the preparation of the Nile Red stain extracted some of the fatty base component (i.e. the vitamin F), which led to a background fluorescent signal on the mounting slide and coverslip when excitation illumination of 540 nm was utilised. The treated crystals are embedded in a fat-like base and upon examination post staining it is clear that this base appears to cover the surface of the crystals, the base appearing red due to the adherence of the stain. The results of such microscopic studies are shown in Figures 1, 2 and 3 appended hereto. Figure 1 depicts MgO crystals, Figure 2 shows the Asprin crystals and Figure 3 depicts the L-glutamine crystals.

As is evident in Figures 1 to 3, the shape and the size of the crystals does not have an effect on the adherence of the fatty acid compound such that the fatty acid compound can be seen to adhere to all crystal shapes and sizes.

Fluorescence microscopy Fluorescence microscopy was conducted utilising the same compositions as those utilised for light microscopy. The resultant micrographs are presented in Figures 4 to 6, wherein Figure 4 corresponds to the MgO crystals. Figure 5 corresponds to the Asprin crystals and Figure 6 corresponds to L- glutamine crystals.

For the purposes of fluorescence excitation, illumination of 540 nm was used and this showed distinct red fluorescence at ca. 600nm which corresponded to the fatty acid being bound to the crystals. The presence of such red fluorescence was clearly indicative of strong adherence of the stained fatty acid (in this instance, vitamin F) to the surface of the crystal. Notably MgO, Asprin and L-glutamine showed no differential binding activity with respect to the binding of vitamin F and vitamin F appears to be strongly associated with the various crystals.

Analysis of microscopy results The micrographs do not show the exact bonding mechanism between the crystals and the vitamin F (oily base). However, the micrographs clearly indicate a distinct association between the oily base and the crystals.

The lipid transport vessel As specified in the first aspect thereof, the invention provides for a lipid transport vessel. This lipid transport vessel has a core area and a coating and the vessel functions in use to facilitate the transport of lipophobic biologically active substances across cell membranes, which are primarily lipophilic in nature.

In the context of the present patent specification, lipophobic substances shall be considered to be hydrophilic and lipophilic substances shall be considered to be hydrophobic. In this instance, hydrophobicity is the physical property of a molecule which is repelled by water. Such molecules are typically non-polar, neutral molecules which therefore tend to be soluble in other non-polar solvents.

Hydrophilicity is the physical property of a molecule which tends to be attracted to water and dissolved therein. A hydrophilic molecule or portion of a molecule is one that is typically charge-polarized (a polar molecule) and capable of hydrogen bonding, enabling it to dissolve more readily in water than in oil or other non-polar solvents.

The core area contains the biologically active substance, which is typically hydrophobic (lipophilic). In this respect the transport vessel's core volume consists only of the biologically active substance and does not contain any suspending media (i.e. the core is anhydrous). The fact that the core volume only contains the biologically active substance is significant, in that, this structure ensures that only the biologically active substance is delivered through a cell membrane thereby resulting in a higher dosage delivery which is beneficial and distinct from the delivery systems of the prior art which are described here above. It will be appreciated by those skilled in the art that the vessel's core may contain biologically active substances which are hydrophilic (lipophobic) or at least partially hydrophilic or lipophilic molecules with hydroxy groups on their surface. In an further embodiment of the invention,

the vessel's core may contain polar molecules or a combination thereof such as steroids and cholesterol derived molecules. The vessel core is typically anhydrous, as stated above, and at least partially coated with fatty acids; these fatty acids functioning, in use, to facilitate the trans-membrane transport of the biologically active substance.

The biologically active substances in the core area may include zwitterions, charged molecules, molecules having surface hydroxyl groups, polar molecules or a combination of the aforementioned (steroids would fall in the latter two groups). The biologically active substances contained within the core may include: amino acids, such as I-glutamine, pharmaceutical drugs, such as acetylsalicylic acid, biological metabolites, such as |-carnitine, vitamins, such as vitamin C, minerals, such as magnesium oxide and other nutritional agents, such as nutrients and/or nutraceuticals. Specific exemplary embodiments in this respect are discussed hereunder in the section dealing with compositions. It will be appreciated by those skilled in the art that the core could contain a vast number of biologically active substances and that, as such, this disclosure is by no means limiting with respect to the content of the core. Referring specifically to the fatty acid coating portion of the lipid transport vessel; the free fatty acids are polyunsaturated free fatty acids. It is envisaged that polyunsaturated free fatty acids such as linoleic acid, alpha-linolenic acid or vitamin F may be utilised as the coating portion.

Linoleic acid Linoleic acid is a polyunsaturated omega-6 fatty acid which is a colorless liquid at room temperature. Furthermore, it is an essential fatty acid which cannot be directly synthesized in the body from other food components and therefore must form part of the diet. Chemically, linoleic acid is a carboxylic acid with an 18-carbon chain and two cis double bonds; with the first double bond located at the sixth carbon from the methyl end. The structure of linoleic acid is given hereunder:

JA PN HO" + 9 12 Alpha(a)-Linolenic Acid a-Linolenic acid is an omega-3 fatty acid and organic compound found in seeds, nuts (notably walnuts), and many common vegetable oils. It is also an essential fatty acid.

Chemically, a-Linolenic acid is a carboxylic acid with an 18-carbon chain and three cis double bonds. The first double bond is located at the third carbon from the methyl end of the fatty acid chain, known as the n end. Thus, o-linolenic acid is a polyunsaturated n-3 (omega-3) fatty acid. The structure of a- Linolenic acid is given hereunder: . 9 6 3 1 w HO 1 9 12 15 18 Vitamin F Vitamin F is commonly used as a collective term for essential fatty acids (EFAs) of oleic acid 21wt% linoleic acid 34wt% and linolenic acid 30wt%. These fatty acids may either partially coat or fully coat the core. In a preferred embodiment the polyunsaturated free fatty acids partially coat the core area. The polyunsaturated free fatty acids associate with the lipophobic vessel core through a non-covalent interaction wherein pi-electrons from the double bonds in the polyunsaturated free fatty acids contribute to such association. This results in the charges in the lipophobic core becoming dispersed which leads to decreased polarity of the coreand increased lipophilicity which allows the lipid transport vessel to cross the cellular membrane. This trans-membrane transport is facilitated primarily by a diffusion process.

It will be appreciated by those skilled in the art that the above described lipid transport vessel may be used to facilitate the transport of biologically active substances across a variety of cellular membranes, including but not limited to: oral membranes, gastric membranes, intestinal mucosa, nasal mucosa, alveolar mucosa, the stratum corneum, the epidermis of the skin and the conjunctiva of the eye. The membranes that are targeted in this regard will depend on the type of biologically active substance being delivered and the indicated treatment regime. For example, topical preparations containing the lipid transport vessel of the invention may contain biologically active substances indicated for the treatment of skin conditions. In this respect, the lipid transport vessel will function to deliver the biologically active ingredient to the epidermis of a patient in need of treatment.

The method of manufacturing the lipid transport vessel The second aspect of the present invention provides for a method of manufacturing the lipid transport vessel described here above In accordance with this method an anhydrous biologically active substance is slowly mixed, using a planetary mixer, with at least one polyunsaturated free fatty acid composition in a ratio of 3.75:1 biologically active substance: polyunsaturated free fatty acid.

Typically, the polyunsaturated free fatty acid is vitamin F, although it will be appreciated by those skilled in the art that other types of polyunsaturated free fatty acids, such as, linoleic acid and/or alpha- linolenic acid may also be utilised.

When preparing the same, the anhydrous biologically active substance is mixed with the polyunsaturated free fatty acids until none of the polyunsaturated free fatty acids are freely present in the mixture. It is thus clear that the anhydrous biologically active substance will form the core area of the lipid transport vessel and the polyunsaturated free fatty acid or acids will form the coating of the lipid transport vessel. Accordingly, mixing, as described above facilitates the formation of the non- covalent interactions between the core area and the coating, which association is described in greater detail above.

It is envisaged that the lipid transport vessel manufactured in accordance with the above described method may be incorporated into a secondary lipophilic chemical substance which secondary lipophilic chemical substance may subsequently be included in preparations for epidermal application. Specific compositions that may be manufactured in this respect are discussed hereunder. Silicon dioxide (SiOz) has dehydrating capabilities and it is possible for it to compete with the anhydrous core of the lipid transport vessel. This could potentially result in the occurrence that vitamin F would rather associate with silicon dioxide than with the anhydrous core.

Lipid transport vessel for use in the transport of biologically active substances The lipid transport vessel of the present invention shows applicability for use in methods of facilitating trans-membrane transport. Primarily this method applies to the transport of lipophobic biologically active substances across cell membranes. However, it will be appreciated by those skilled in the art that the vessel may be utilised to transport substances which are not strictly lipophobic. The abovementioned trans-membrane transport is facilitated by the increased lipophilicity of the biologically active substance which is brought about via the coating process described above. This coating of the core area by polyunsaturated free fatty acids increases the lipophilicity of the core area thereby allowing for the diffusion across the lipophilic cell membrane. It will be appreciated by those skilled in the art that active trans-membrane transport processes may also play a role in such transport. This method of facilitating trans-membrane transport may be utilised in delivery systems for the delivery of a wide range of biologically active compounds, such as, pharmaceuticals and nutritional supplements. Medicaments The lipid transport vessel of the invention may be utilised in the manufacture of a medicament for the treatment of mammalian diseases. It will be appreciated by those skilled in the art that the disease ordiseases indicated for treatment will be wholly dependent on the selection of the biologically active substance incorporated within the lipid transport vessel, the active substance being indicated for the treatment of one or more diseases. Specific compositions shall be considered hereunder in the section dealing with compositions.

Treatment in this instance shall refer to prophylactic treatment, alleviating treatment and/or curative intervention and shall in no way be limited by the nature and/or the scope of the benefit provided. Administration of the medicament and/or lipid transport vessel of the present invention can be done in any acceptable manner known in the medicinal arts. Specific non-limiting examples of administration methods which may be used in accordance with the embodiments of the invention include: topical administration, injection, intravenous administration, rectal administration, transdermal administration, ophthalmic administration, lymphatic administration and nasal administration. Further, non-limiting examples of administration methods which may be used in accordance with embodiments of the present invention include oral application by means of a soft gelatine capsule. In an embodiment, said gelatine capsule may be coated with an enteric coating in order to ensure that the contents are released in the duodenum (the first part of the small intestine). Preparations The lipid transport vessel of the invention may be incorporated into a secondary lipophilic substance which functions, in use, as a suspending medium for the vessel in preparations intended for epidermal application. In this way, the secondary lipophilic chemical substance, which comprises the largest portion of the final product, may be selected from the group comprising: lanolin, petroleum jelly, waxes, mineral oil, natural oils and processes oils. It will be appreciated by those skilled in the art that a variety of secondary lipophilic substances could be utilised for this purpose and hence the scope of the invention is by no means limited by the above provided list of secondary lipophilic substances. Specific exemplary embodiments of preparations prepared in accordance with the invention include a preparation comprising a lipid vessel containing a biologically active core of L-glutamine and a coating of vitamin F suspended in lanolin as the secondary lipophilic chemical substance.

Specific reference in this regard is had to Figures 7 and 8 which are graphs depicting the nuclear magnetic resonance spectrum of the said preparation. The nuclear magnetic resonance (NMR) spectra of the secondary lipophilic chemical substance (the lanolin), vitamin F and L-glutamine are shown in Figures 9 to 11, respectively. Such NMR data was obtained in a previous study and is included herein for the sake of completeness. Other specific exemplary embodiments of preparations and medicaments (as alluded to above) may include a lipid transport vessel containing a biologically active substance selected from the group consisting of acetyl salicylic acid, MgO, dry L-carnitine and vitamin C which biologically active substances have been coated with vitamin F in accordance with the method of manufacturing the lipid transport vessel detailed here above. Example: Light and fluorescence microscopy The following compound mixtures were examined using light and fluorescence microscopy: Sample 12 PSA and vitamin F (PSA = propionyl salicylic acid); Sample 13 Aspirin and vitamin F; Sample 14 Aspirin and citriole 100 (no vitamin F has been added); Sample 15 Aspirin and citriole 100 and vitamin F; Sample 16 Citrimis, CAS and vitamin F (CAS = copper 2 aspirinate); Sample 17 Citriole 100, CAS and vitamin F; Sample 18 CAS (no vitamin F has been added); and Sample 19 CAS and vitamin F.

The binding of vitamin F or oils to the different crystals was tested using the stain Nile Red (NR) as a probe to determine fatty acids or oil present on the surface of the crystals, thus showing red fluorescence. Stained or unstained crystalline structures did not show fluorescence in the absence of either vitamin F or the oils citriole or citrimis.

Observations

1. Light microscopy (Figurers 12A to Figures 19A) Light microscope observations using transmitted white light indicates, primarily, the presence of crystalline components stained by Nile Red (dissolved in 100% acetone) showing some red diffractions. The crystals, either PSA, aspirin or CAS are embedded in the oil or vitamin F base such that the oil or vitamin F covered the surfaces of all the crystals.

The observations of vitamin F and oil, as bonded to the crystals, are illustrated in Figures 12A to 19A. The aspirin crystals are stained well with the red stain also in the visible light region (excluding the normal UV-light excitation required for fluorescent emission of the Nile Red). The CAS crystal compound has a slight inherent blue tint and did not stain red as some of the PSA or aspirin crystals. However, suspended in the oily compounds that are closely bonded to the surface, all of the crystals showed a distinct red fluorescence.

2. Fluorescence microscopy (Figures 12B to 19B) The samples as listed above were also illuminated by the required 540nm excitation for NR to show distinct red fluorescence at ca. 640nm of the oily compounds as bonded to the different crystals. This clearly indicates the strong adherence and interaction of the oily compounds to the surface of the crystals. The corresponding fluorescent microscopic images of a specific area, are respectively shown in Figures 12B to 19B.

3. Discussion Looking at Figure 12, where the sample contains PSA and vitamin F, it can be seen that the crystals are covered by a stained oily substance showing an intense fluorescence of vitamin F.

Turning to Figure 13 where the samples contain aspirin and vitamin F, the crystals are covered by a stained oily substance and have a less intense fluorescence.

As can be seen in Figure 14 where the samples contain aspirin with citriole 100 oil (no vitamin F added), the crystals adhere to and are covered by the stained oily substance.

In Figure 15, the samples contained aspirin with vitamin F in citriole 100 oil and the crystals showed an intense fluorescence of vitamin F.

Looking at Figure 16 where the samples contain citrimis, CAS and vitamin F, it can be observed that the crystals are covered by and show an intense fluorescence of an oily substance.

Turning to Figure 17 where the samples contain citriole, CAS and vitamin F, the crystals can be seen to show an intense fluorescence of an oily substance.

Figure 18 shows the samples (containing CAS without vitamin F) whereby no fluorescence occurred due to lack of an oily compound (green hue was 540 nm excitation). Lastly, Figure 19 (where the samples contained CAS with vitamin F) shows the crystals covered by vitamin F having an intense Nile Red fluorescence.

Conclusions

From the foregoing, it is submitted that microscopic examinations cannot show specific bonding mechanisms between the oily base (e.g. vitamin F) and the crystals.

The oily base is distinctly associated and linked to crystals; either PSA, aspirin or the CAS compound mixture.

Nile Red was used as a selective probe for fats fluorescing at a red wavelength (840nm). In this regard, no fluorescence occurred in the absence of the either vitamin F or the oils.

In addition hereto, and ascribed to high external oily content, internal bonding could not be detected by fluorescence.

It will be appreciated by persons skilled in the art that the current description is by no means an exhaustive account of all embodiments falling within the spirit of the invention as described herein and that, as such, the invention should not be limited to the the examples contained in the description.

Claims

25. CONCLUSIONS

A lipid transport vessel, the vessel comprising a biologically active substance forming the vascular core, which biologically active substance is coated, at least in part, with polyunsaturated free fatty acids by mixing an anhydrous biologically active substance with the polyunsaturated free fatty acids in a ratio of 3.75: 1 biologically active substance: polyunsaturated free fatty acid, said polyunsaturated fatty acids, in use, functioning to transport the biologically active substance through the membrane by diffusion, endocytic and phagocytic processes.

The lipid transport vessel of claim 1, wherein the polyunsaturated free fatty acids comprise linoleic acid or alpha linolenic acid.

The lipid transport vessel of claim 1, wherein the polyunsaturated free fatty acids comprise vitamin F.

Lipid transport vessel according to any one of claims 1-3, wherein the polyunsaturated free fatty acids partially coat or completely coat the core.

The lipid transport vessel of claim 1, wherein trans-membrane transport through the lipid transport vessels is facilitated by a diffusion process.

The lipid transport vessel of claim 1, wherein trans-membrane transport through the lipid transport vessels is facilitated by an active trans-membrane transport process, such as endocytic and phagocytic processes.

A method of making a lipid transport vessel, the method comprising mixing an anhydrous biologically active substance with at least one polyunsaturated free fatty acid in a ratio of 3.75: 1 biologically active: polyunsaturated free fatty acid.

The method of claim 7, wherein the biologically active substance comprises the core of the lipid transport vessel and consists of a lipophobic chemical.

The method of claim 7 or 8, wherein the polyunsaturated free fatty acid is selected from the group comprising: linoleic acid, alpha linolenic acid, vitamin F or a combination of the above.

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The method of claim 9, wherein the polyunsaturated free fatty acid is vitamin F.

Use of a lipid transport vessel according to any of the preceding claims, in a method of transporting the biologically active substance through the membrane by diffusion, endocytic and phagocytic processes.

The use of a lipid transport vessel according to claim 11, wherein the lipid transport vessel is used to transport lipophobic substances through cell membranes by diffusion, endocytic and phagocytic processes.

The use of a lipid transport vessel according to claim 11, wherein the lipid transport vessel is used to transport substances that are not lipophobic through cell membranes by diffusion, endocytic and phagocytic processes.

Use of the lipid transport vessel according to any of the preceding claims in the manufacture of a medicament for the treatment of mammalian diseases.

A composition comprising a lipid transport vessel, the vessel containing a biologically active substance forming the vascular core, the vascular core being at least partially coated with polyunsaturated free fatty acids by mixing an anhydrous biologically active substance with the polyunsaturated free fatty acids in a ratio of 3.75: 1 biologically active substance: polyunsaturated free fatty acid, which increases core lipophilicity and thereby transports the biologically active substance through the membrane by diffusion, endocytic and phagocytic processes and a secondary lipophilic chemical containing the vessel suspended, the secondary lipophilic substance functions, in use, as a vessel suspending medium for preparations intended for epidermal administration.

The formulation of claim 15, wherein the secondary lipophilic chemical is selected from the group consisting of lanolin, petroleum jelly, waxes, mineral oil, natural oils, and process oils.