Description:FIELD OF THE INVENTION
The present invention relates to liposomal formulations for co-encapsulating water-soluble vitamins and water-insoluble compounds. More specifically, the present invention relates to a novel liposomal composition designed to enhance the stability, absorption, and bioavailability of the formulation, thereby providing an improved method for their delivery and utilization in various health applications.
BACKGROUND OF THE INVENTION
Vitamin C, also known as ascorbic acid, and plant sterols, are eminent and widely used for their diverse physiological benefits. Vitamin C is useful in collagen synthesis, immune function, and antioxidant defence, plant sterols are known for their cholesterol-lowering properties and cardiovascular benefits. Both compounds face challenges relating to bioavailability at the therapeutic/effective dose, and stability (both in vitro and in the gastrointestinal environment). and dissolution when administered individually.
Plant sterols have poor solubility and thus has poor dissolution rate affecting its bioavailability. Vitamin C, however, is highly soluble hydrophilic molecule which is absorbed primarily through active transport. Ascorbic acid (ASC), and its oxidized form, dehydroascorbic acid (DHA), exist in a dynamic equilibrium in the body. At physiological pH, ascorbic acid predominantly exists in its ionized form, ascorbate (ASC), which is the more stable and prevalent form in biological systems. Due to their hydrophilic nature, both ASC and DHA have difficulty crossing biological membranes, such as the cell membrane. Therefore, they primarily rely on specific transporter molecules that are anchored in the cell membrane to facilitate their movement in and out of cells. These transporters, such as the sodium-dependent vitamin C transporters (SVCTs), are essential for maintaining adequate intracellular levels of vitamin C. So at the clinical dose of higher the 500mg the bioavailability is fairly reduced
The effective delivery of these nutrients to target tissues presents challenges, particularly for water-soluble vitamins and water-insoluble lipids. Traditional delivery systems often fail to adequately protect these compounds from degradation in the gastrointestinal tract or to facilitate their dissolution into the body. A liposomal delivery system addresses these challenges efficiently. Liposomal formulations have emerged as promising carriers for bioactive compounds due to their unique structure and properties. Liposomes, lipid-based vesicles, offer advantages such as improved stability, enhanced bioavailability, and targeted delivery to specific tissues. Numerous studies have demonstrated the suitability of liposomal delivery systems for encapsulating individual vitamins or phytochemicals. Liposomes have the potential to encapsulate both hydrophilic molecules (in the core) and hydrophobic molecules in the bilayer. Utilizing this property, we have co-encapsulated the vitamin C (and other water-soluble ingredients) and plant sterols (and other lipid-soluble ingredients) in a single liposomal system to harness a synergistic effect.
US2020069599A1 discloses features of novel lipids and compositions involving the same. Lipid nanoparticles include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Lipid nanoparticles further including therapeutics and/or prophylactics such as RNA are useful in the delivery of therapeutics and/or prophylactics to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression.
US10064954B2 discloses an invention comprises compounds, methods of making, and methods of using. A group of polymer-cyclodextrin-lipid conjugates having a centre backbone and three or four appended functional groups are disclosed, wherein one of the hydrophilic components is cyclodextrin. The compounds may have a backbone with three or four appended functional groups: one or two lipophilic compounds including sterols or “fat-soluble” vitamins or fatty acids, one or two hydrophilic polymers, and one cyclodextrin. Specific functional groups may be selected for specific applications in formulating pharmaceuticals, cosmetics, nutraceuticals, and the like. Typical coupling reactions of the conjugates may involve one or more combinations or in series of alkylation including N-alkylation or O-alkylation, etherification, and esterification processes. A variety of linkers between the centre backbone and functional groups may also be selected to modify the carriers or centre backbones for the coupling reactions and optimize performance of the conjugates.
While nanocarriers encapsulated within liposomes and lipid-based formulations hold great promise for drug delivery, they are subject to several limitations that hinder their widespread application. These limitations include low drug loading capacity, premature drug leakage, susceptibility to degradation, and insufficient targeting efficiency. Addressing these challenges is crucial to unlock the full potential of these innovative drug delivery platforms and improve their therapeutic efficacy.
The present invention addresses the aforementioned challenges by providing a novel liposomal formulation for encapsulating water-soluble vitamins and water-insoluble lipids. The formulation comprises liposomes composed of lipid bilayers encapsulating an aqueous core containing the desired nutrients. The liposomal composition is carefully optimized to ensure stability, compatibility, and efficient encapsulation of the water-soluble vitamins and water-insoluble lipids.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a co-encapsulation formulation for water-soluble vitamins (more specifically vitamin C) and water-insoluble lipids (more specifically beta sitosterol) in a single liposomal delivery.
Another objective of the present invention is to provide an enhanced stability formulation of water-soluble vitamins and water-insoluble lipids within liposomes to protect these compounds from degradation, oxidation, or other forms of deterioration.
Yet another objective of the present invention is to improve bioavailability by facilitating their transport across biological membranes and bypassing barriers to absorption in the gastrointestinal tract.
Yet another objective of the present invention is to provide targeted delivery as liposomes can be designed to target specific tissues or organs, allowing for precise delivery.
Yet another objective of the present invention is to provide versatility and convenience and instead of having to take multiple supplements or formulations separately, individuals can benefit from a single, comprehensive product.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided hereinbelow, in which various embodiments of the disclosed invention are illustrated by way of example.

SUMMARY OF THE INVENTION
The present invention relates to a liposomal formulation for co-encapsulating a water-soluble vitamin (specifically vitamin C and analogue) and a water-insoluble lipid (specifically plant sterols and analogue). Vitamin C has a concentration range from 10% w/w to 60% w/w. In the preferred embodiment, the sterol analogues have a concentration range from 0.5% w/w to 6.0% w/w. The phospholipid has a concentration range from 40.0% w/w to 65.0% w/w. The complexing agent has a concentration range from 1.0% w/w to 10.0% w/w. The spray drying aid has a concentration range from 5.0% w/w to 30.0% w/w. The liposomal formulation includes a lyophilization aid. Herein, the encapsulation efficiency of the liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid ranges from 20% to 85%. Herein, liposomes encapsulating β-sitosterol with vitamin -C is in range the of 100 nm to 2000 nm. Herein, the liposomal formulation shows the sustained dissolution profile for both hydrophilic and hydrophobic molecules that are expected to enhance the bioavailability of the liposomal formulation. Herein the liposomal formulation is a controlled and sustained release dosage over at least a 12-hour period. Herein, the dosage form for liposomal co-encapsulation includes but are not limited to tablet, capsule, gummy, mouth strip, lozenges, juice, cheese, and yogurt. Herein, the capsule, gummy, mouth strips and juice dosage forms of the liposomal formulation deliver at least 40-1000mg of ascorbic acid efficiently.
In the embodiment, a method for preparing the liposomal formulation is by spray drying and lyophilization, where the formulation is prepared in the required dosage form, and the formulation goes through the lyophilization cycle optimization to pre-freeze and then conduct primary and secondary drying to remove residue moisture.
The main advantage of the present invention is that it provides a co-encapsulation formulation for water-soluble vitamins and water-insoluble lipids in a single liposomal delivery.
Another advantage of the present invention is that it provides an enhanced stability formulation of water-soluble vitamins and water-insoluble lipids within liposomes to protect these compounds from degradation, oxidation, or other forms of deterioration.
Yet another advantage of the present invention is that it improves bioavailability by facilitating their transport across biological membranes and bypassing barriers to absorption in the gastrointestinal tract.
Yet another advantage of the present invention is that it provides targeted delivery as liposomes can be designed to target specific tissues or organs, allowing for precise delivery.
Yet another advantage of the present invention is that it provides versatility and convenience and instead of having to take multiple supplements or formulations separately, individuals can benefit from a single, comprehensive product.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.

BRIEF DESCRIPTION OF DRAWINGS
The accompanying drawings are incorporated in and constitute a part of this specification to provide a better understanding of the invention. The drawings illustrate one embodiment of the invention and together with the description, serves to explain the principles of the invention.
Fig. 1 is a graphical representation of the difference in the permeability of Vitamin C plain and Vitamin C liposomal system.
Fig. 2 is a graphical representation of the difference in the permeability of beta-sitosterol plain and beta-sitosterol liposomal system.
Fig. 3 is graphical representation of particle size of spray dried liposomal vitamin C and with β-sitosterol dispersed in aqueous media.
Fig. 4 is graphical representation of Zeta potential of spray dried liposomal vitamin C and with β-sitosterol dispersed in aqueous media.
Fig. 5 shows Scanning Electron Microscopic image of spray dried liposomal vitamin C with β-sitosterol.

DETAILED DESCRIPTION OF THE INVENTION
Definition
The term “a” or “an”, as used herein, is defined as one. The term “plurality”, as used herein, is defined as two as or more than one. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language).
The term “comprising” is not intended to limit the present invention with such terminology rather is used in a wider sense. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of”. The term “comprising” may be used interchangeably with the terms “having” or “containing”.
Reference in this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms, throughout the document means that a specific feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places, this specification throughout are not necessarily all referring to the same embodiment. Furthermore, the specific features, structures, or characteristics are combined in any suitable manner in one or more embodiments without limitation.
The term “or” as used herein is to be interpreted as inclusive or meaning any one or more combinations. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in mutually exclusive, inherently.
As used herein, the term "one or more" generally refers to, but is not limited to, singular as well as the plural form of the term.
The present invention relates to a liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid. The liposomal formulation includes vitamin C, plant sterols and analogs, a phospholipid, a complexing agent, a spray drying aid, and a lyophilization. The vitamin C having a concentration range from 10% w/w to 60% w/w. The plant sterols and analogs has a concentration range from 0.5% w/w to 6.0% w/w. The plant sterols and analogs includes but is not limited to beta-sitosterol(phytosterol), cholesterol, ergosterol, stigmasterol, campesterol and bile acids. The phospholipid has a concentration range from 40.0% w/w to 65.0% w/w. The phospholipid includes but is not limited to phosphatidylcholine (PC), phosphatidylethanolamine (PE), Phosphatidylserine (PS), phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin, Sphingomyelin and soya/sunflower lecithin. The complexing agent has a concentration range from 1.0% w/w to 10.0% w/w. The complexing agent includes but is not limited to α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. The spray drying aid has a concentration range from 5.0% w/w to 30.0% w/w. The spray drying aid includes but is not limited to silicon dioxide(silica), calcium silicate, magnesium stearate, maltodextrin, modified starch, gum arabic, cellulose derivatives, lecithin, talc, zein, lactose, polyvinylpyrrolidone, sodium aluminium silicate, microcrystalline cellulose, hydrocolloids, polyethylene glycol (PEG), cyclodextrin, trehalose, sorbitol, and dextrin. The lyophilization aid ranges from 1.0% w/w-10.0% w/w. The lyophilization aid includes but is not limited to trehalose, mannitol, sucrose, lactose, citrate, phosphate, albumin gelatin, ascorbic acid, glutathione, tween 80 and polysorbate. Herein the encapsulation efficiency of the liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid range from 20% to 85%. Herein, liposomes encapsulating β-sitosterol with vitamin -C is in range the of 100 nm to 2000 nm. Herein the liposomal formulation enhances the dissolution and bioavailability of liposomal formulation as apparent from ex-vivo permeability studies. Herein the liposomal formulation shows a controlled and sustained release dosage over at least a 12-hour period. Herein, the dosage form for liposomal co-encapsulation is selected from tablet, capsule, gummy, mouth strip, lozenges, juice, cheese, and yogurt. Herein, the capsule, gummy, mouth strips and juice dosage forms of the liposomal formulation deliver at least 40-1000mg of ascorbic acid efficiently.
In the embodiment, a method for preparing the liposomal formulation is by single step liquid liposomal dispersion followed by spray drying or lyophilization, lyophilization cycle optimization was done by pre-freeze and then conducting primary and secondary drying to remove residue moisture.

In the embodiment, a method is for preparing the liposomal formulation. The method includes:
water having a temperature of not more than 10℃ is added to a homogenizer;
vitamin C is added to the homogenizer and vacuum is applied;
the homogenizer is activated till a solution is obtained;
the phospholipid, the plant sterols and analogs, the complexing agent and spray drying aid are added to the homogenizer;
vacuum is applied to the homogenizer keeping the temperature of not more than 10℃;
the homogenizer is activated till a homogenized solution is obtained;
pressurized nitrogen is applied in the homogenizer to release vacuum;
probe sonication is performed over the solution and the solution is transferred for spray drying.
Herein, in the spray drying method, controlled rate of the solution is pumped through a rotary or nozzle atomizers into fine droplets; the fine droplets are transferred into the drying chamber to evaporate the liquid content and result in the formation of solid particles or powder substances; the solid particles or powder substances are removed from the drying chamber with the help of a cyclone separator and cooled.
Herein, in the lyophilization cycle optimization, the homogenized liposomal dispersion is pre-frozen to a specified temperature; the pre-frozen containers are primarily dried in a lyophilized chamber to sublime the ice into vapours; subsequently secondary drying is conducted over the container to remove moisture residues till the moisture in the container reaches a pre-determined level; after the end of lyophilization cycle, the final product is packaged and stored.
Herein, the dissolution of liposomal vitamin C with plant sterols and analogs release is at least 80% in the capsule dosage.
The present invention relates to a liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid. The liposomal formulation includes one or more vitamin C, one or more plant sterols and analogs, one or more phospholipids, one or more complexing agents, one or more spray drying aids, and one or more lyophilization’s. The one or more vitamin C having a concentration range from 10% w/w to 60% w/w. The one or more plant sterols and analogs having a concentration range from 0.5% w/w to 6.0% w/w. In the embodiment the one or more plant sterols and analogs includes but are not limited to beta-sitosterol(phytosterol), cholesterol, ergosterol, stigmasterol, campesterol and bile acids. The one or more phospholipids has a concentration range from 40.0% w/w to 65.0% w/w. In the embodiment the one or more phospholipids includes but are not limited to phosphatidylcholine (PC), phosphatidylethanolamine (PE), Phosphatidylserine (PS), phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin, Sphingomyelin and soya/sunflower lecithin. The one or more complexing agents has a concentration range from 1.0% w/w to 10.0% w/w. The one or more complexing agents includes but are not limited to α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. The one or more spray drying aids has a concentration range from 5.0% w/w to 30.0% w/w. The one or more spray drying aids includes but are not limited to silicon dioxide(silica), calcium silicate, magnesium stearate, maltodextrin, modified starch, gum arabic, cellulose derivatives, lecithin, talc, zein, lactose, polyvinylpyrrolidone, sodium aluminium silicate, microcrystalline cellulose, hydrocolloids, polyethylene glycol (PEG), cyclodextrin, trehalose, sorbitol, and dextrin. The one or more lyophilization aids ranges from 1.0% w/w-10.0% w/w. The one or more lyophilization aids includes but are not limited to trehalose, mannitol, sucrose, lactose, citrate, phosphate, albumin gelatin, ascorbic acid, glutathione, tween 80 and polysorbate 20. Herein the encapsulation efficiency of the liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid range from 20% to 85%. Herein, liposomes encapsulating β-sitosterol with vitamin -C is in range the of 100 nm to 2000 nm. Herein the liposomal formulation shows sustained release the dissolution of Vitamin C and the plant sterol, thus enhancing the bioavailability. Herein the liposomal formulation is a controlled and sustained release dosage over at least a 12-hour period. Herein, the dosage form for liposomal co-encapsulation includes tablet, capsule, gummy, mouth strip, lozenges, juice, cheese, and yogurt. Herein, the capsule, gummy, mouth strips and juice dosage forms of the liposomal formulation deliver at least 40-1000mg of ascorbic acid efficiently.
In the embodiment, a method for preparing the liposomal formulation is preparing homogenous liquid liposomal dispersion followed by spray drying and lyophilization, where the formulation is prepared in the required dosage form, and the formulation goes through the lyophilization cycle optimization to pre-freeze and then conduct primary and secondary drying to remove residue moisture.

In the embodiment, a method is for preparing the liposomal formulation. The method includes:
water having a temperature of not more than 10℃ is added to a homogenizer;
vitamin C is added to the homogenizer and vacuum is applied;
the homogenizer is activated till a solution is obtained;
the phospholipid, the plant sterols and analogs, the complexing agent and spray drying aid are added to the homogenizer;
vacuum is applied to the homogenizer at a temperature of not more than 10℃;
the homogenizer is activated till a homogenized solution is obtained;
pressurized nitrogen is applied in the homogenizer to release vacuum;
probe sonication is performed over the solution and the solution is transferred for spray drying.
Herein, in the spray drying method, controlled rate of the solution is pumped through a rotary or nozzle atomizers into fine droplets; the fine droplets are transferred into the drying chamber to evaporate the liquid content and result in the formation of solid particles or powder substances; the solid particles or powder substances are removed from the drying chamber with the help of a cyclone separator and cooled.
Herein, in the lyophilization cycle optimization, the liquid liposomal dispersion is pre-frozen to a specified temperature; the pre-frozen containers are primarily dried in a lyophilized chamber to sublime the ice into vapours; subsequently secondary drying is conducted over the container to remove moisture residues till the moisture in the container reaches a pre-determined level;
after the end of lyophilization cycle, the final product is packaged and stored.

Herein, the dissolution of liposomal vitamin C with plant sterols and analogs release is at least 80% in the capsule dosage.

Example 1: 400 mg/g Ascorbic acid and 40mg/g β-sitosterol containing liposomal preparation
A 400 mg/g Ascorbic acid containing liposomal vitamin c with β-sitosterol powder using maltodextrin powder as spray drying agent. Scanning electron microscopic image of spray dried liposomal vitamin c with β-sitosterol is shown in figure 7. , Claims:1. A liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid, the liposomal formulation comprising of:
a vitamin C having a concentration range from 10% w/w to 60% w/w;
a plant sterols and analogs having a concentration range from 0.5% w/w to 6.0% w/w;
an at least one phospholipid having a concentration range from 40.0% w/w to 65.0% w/w;
an at least one complexing agent having a concentration range from 1.0% w/w to 10.0% w/w;
an at least one spray drying aid having a concentration range from 5.0% w/w to 30.0% w/w; and
an at least one lyophilization aid range from 1.0% w/w-10.0% w/w;
characterized in that, the dual loading encapsulation efficiency of the nano liposomal formulation for co-encapsulating a water-soluble vitamin and a water-insoluble lipid ranges from 20% to 85%;
characterized in that, liposomes encapsulating β-sitosterol with vitamin -C is in range the of 100 nm to 2000 nm;
characterized in that, the liposomal formulation shows sustained release the dissolution of the Vitamin C and the plant sterol and analogs, thus enhancing the bioavailability and permeation of water soluble vitamin and water insoluble lipid;
characterized in that, the liposomal formulation shows a controlled and sustained release dosage over at least a 12-hour period;

wherein, the dosage form for liposomal co-encapsulation is selected from tablet, capsule, gummy, mouth strip, lozenges, juice, cheese, and yogurt;

wherein, a method for preparing the liposomal formulation is by single step liquid liposomal dispersion followed by spray drying or lyophilization, where the formulation is prepared in the required dosage form, and the formulation goes through the lyophilization cycle optimization was done by pre-freeze and then conducting primary and secondary drying to remove residue moisture;

wherein, β-sitosterol also function as cholesterol to increase permeability and rigidity.

2. The liposomal formulation as claimed in claim 1, wherein, the plant sterols and analogs is selected from beta-sitosterol(phytosterol), cholesterol, ergosterol, stigmasterol, campesterol and bile acids.

3. The liposomal formulation as claimed in claim 1, wherein, the at least one phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), Phosphatidylserine (PS), phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin, Sphingomyelin and soya/sunflower lecithin.

4. The liposomal formulation as claimed in claim 1, wherein, the at least one complexing agent is selected from α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

5. The liposomal formulation as claimed in claim 1, wherein, the at least spray drying aid is selected from silicon dioxide(silica), calcium silicate, magnesium stearate, maltodextrin, modified starch, gum arabic, cellulose derivatives, lecithin, talc, zein, lactose, polyvinylpyrrolidone, sodium aluminium silicate, microcrystalline cellulose, hydrocolloids, polyethylene glycol (PEG), cyclodextrin, trehalose, sorbitol, and dextrin.

6. The liposomal formulation as claimed in claim 1, wherein, the at least one lyophilization aid is selected from trehalose, mannitol, sucrose, lactose, citrate, phosphate, albumin gelatin, ascorbic acid, glutathione, tween 80 and polysorbate 20.

7. The liposomal formulation as claimed in claim 1, wherein, a method is for preparing the liposomal formulation, the method comprising:
water having a temperature of not more than 10℃ is added to a homogenizer;
vitamin C is added to the homogenizer and vacuum is applied;
the homogenizer is activated till a solution is obtained;
the at least one phospholipid, the plant sterols, and analogs, the at least one complexing agent and the at least one spray drying aid are added to the homogenizer;
vacuum is applied to the homogenizer keeping the temperature of not more than 10℃;
the homogenizer is activated till a homogenized solution is obtained;
pressurized nitrogen is applied in the homogenizer to release vacuum;
probe sonication is performed over the solution and the solution is transferred for spray drying;
wherein, in the spray drying method, controlled rate of the solution is pumped through a rotary or nozzle atomizers into fine droplets; the fine droplets are transferred into the drying chamber to evaporate the liquid content and result in the formation of solid particles or powder substances; the solid particles or powder substances are removed from the drying chamber with the help of a cyclone separator and cooled;
wherein, in the lyophilization cycle optimization, the homogenized liposomal dispersion is pre-frozen to a specified temperature; the pre-frozen containers are primarily dried in a lyophilized chamber to sublime the ice into vapours; subsequently secondary drying is conducted over the container to remove moisture residues till the moisture in the container reaches a pre-determined level;
after the end of lyophilization cycle, the final product is packaged and stored.

8. The liposomal formulation as claimed in claim 1, wherein, the dissolution of liposomal vitamin C with plant sterols and analogs release is at least 80% in the capsule dosage.

9. The liposomal formulation as claimed in claim 1, wherein, the capsule, gummy, mouth strips and juice dosage forms of the liposomal formulation deliver at least 40-1000mg of ascorbic acid efficiently.