DESC:Priority Claim:

[0001] This application claims priority from the provisional application numbered 202041050881 filed with Indian Patent Office, Chennai on 23rd November 2020 entitled “A process for preparation of liposomal powder with bioactive molecules”, the entirety of which is expressly incorporated herein by reference
Preamble to the Description
[0002] The following specification describes the invention and the manner in which it is to be performed:
DESCRIPTION OF THE INVENTION
Technical field of the invention
[0003] The present invention relates to a process for preparation of formulation of liposomal powder for encapsulation of bio active molecules with nano fibers for drug delivery. More particularly, the invention discloses a process of preparation of the liposomal powder encapsulating the bio active molecules by nano fiber weaving technology. The invention also discloses a formulation of liposomal powder.

Background of the invention
[0004] Liposome is a spherical vesicle having at least one lipid bilayer. Liposomes are used as a vehicle for administration of nutrients, biomolecules and pharmaceutical drugs. Liposomes are small, closed vesicles made up of lipids, especially phospholipids. Lipids generally have a hydrophobic "tail" and a hydrophilic "end" and which when mixed with water, tend to join toward a common center while the hydrophobic tails become oriented outwardly to interface with the water. The lipids form a monomolecular layer or arrange themselves in more than one layer of lipids to form a "liposome" having an inner space and an outer surface. Liposomes are composed of more than one layer.

[0005] Liposomes are generally used as delivery agent to deliver active constituents, biomolecules, drug molecules etc. Liposomes are promising systems for drug delivery due to their size in addition to the hydrophobic and hydrophilic character and biocompatibility.

[0006] Liposomes are of different types such as the multilamellar vesicle, the small unilamellar liposome vesicle, the large unilamellar vesicle and the cochleate vesicle. Multilamellar vesicles are made up of several lamellar phase lipid bilayers. The small unilamellar liposome vesicles are made up of one lipid bilayer.

[0007] The liposome is made up of aqueous solution core surrounded by a hydrophobic membrane in the form of a lipid bilayer and the hydrophilic solutes dissolved in the core, which cannot easily pass through the lipid bilayer. Hydrophobic chemicals associate with the bilayer. As a result, liposomes are loaded with hydrophobic and hydrophilic molecules.

[0008] The lipid bilayer fuse with other bilayers to deliver the molecules to a site of action such as the cell membrane thus delivering the liposome contents.

[0009] The size of the liposome varies from 0.025 µm to 2.5 µm vesicles. Moreover, liposomes have one or bilayer membranes. The vesicle size is an acute parameter in determining the circulation half-life of liposomes and both size and number of bilayers affect the amount of drug encapsulation in the liposomes.

[0010] The biomolecules are encapsulated into the liposomes to retain the activity of the biomolecule as well as for efficient delivery. Although the encapsulation of biologically active materials in liposomes has significant potential for delivering such materials to targeted sights in the human body, the production of encapsulated materials on a commercially feasible scale is not technically feasible.

[0011] The US Application No. US20060280691A1 titled “Spray freeze dried liposomal ciprofloxacin powder aerosol drug delivery” discloses a powder for inhalatory aerosol delivery. The powder is obtained by spray freeze dried liposome particles with a biologically active agent such as antibiotic encapsulated within a phospholipid. The invention also discloses a method of producing a powder for inhalatory aerosol delivery and the method comprises the steps of mixing a biologically active agent with a phospholipid to form a liquid liposome suspension and spray freeze drying the liposome suspension to form particles of powder. The formulation resulted in effective sustained release of the active ingredient from the liposome. Further, the formulation also exhibited aerodynamic and dissolution properties. However, the liposomal phospholipids are associated with electrostatic interactions and the phospholipids spontaneously encapsulate a particle of suitable charge in an ionic solution.

[0012] The US Application No. US5567433A titled “Liposome preparation and material encapsulation method” discloses a method of preparation of liposomes for encapsulation and delivery of a wide variety of biologically active materials. The invention provides liposomes and a production method which is simple, feasible and inexpensive for the large-scale commercial manufacturing of liposomes and encapsulated materials. The method involves the formation of liposome dispersion in the absence of any organic solvent or detergent followed by one or several cycles of freezing and thawing the liposomes and finally dehydration of the liposome dispersion to form a lipid powder. When desired, the lipid powder is hydrated in the presence of the biologically active material whereby the material is encapsulated in reconstituted liposomes. The method also includes combining the liposome dispersion with a bulking agent prior to the dehydration and formation of the lipid powder to facilitate the handling of the lipid powder as well as its rapid dispersal upon hydration. However, the formulation is associated with short span of life after delivery.
[0013] The Japanese Application No. JPS5782311A titled “Production of liposome preparation” discloses a phospholipid composition wherein the phospholipid is dispersed in an aqueous medium, freeze-dried and dispersed in an aqueous medium containing an active ingredient. The phospholipid is selected from yolk, soybean or any other animal or plant tissues and the aqueous medium selected from sodium chloride or a saccharide. The active ingredient is selected from the group iltiazem, glutathione, vitamins and the enzyme preparation such as L-asparginase. The formulation is not associated with controlled release of the active ingredient and involves high doses of active ingredient.
[0014] Even though the composition of liposomes, methods of encapsulation of active ingredients in liposomes using spray drying freeze technology along with and the potential delivery of liposomes in target delivery sites are available in the prior arts, the production of liposomes on commercial scale is not feasible. The use of solvents may be expensive, and few solvents may unfavorably interact with the active ingredients and use of multiple steps is not feasible to achieve a good yield. Hence, there is a requirement for a process of preparation of formulation of liposomal powder for encapsulation of bio active molecules which is feasible on commercial scale.
Summary of the invention
[0015] The present invention overcomes the drawbacks of the existing prior arts by converting liposomal dispersion to liposomal powder. Also, the formulation comprises a bioactive molecule, which is encapsulated in the liposome along with turmeric nanofibers.
[0016] The present invention discloses a formulation of liposomal powder. The formulation comprises phospholipids at a concentration range of 10%-25%, turmeric nanofibers at a concentration range of 1%-10%, modified food starch or gum Arabic at a concentration range of 40%-80% and a bioactive molecule at a concentration range of 20%-40%.
[0017] The present invention relates to process of preparation of liposomal powder with bioactive molecules. The process begins with dissolving phospholipid in Millipore water followed by addition of bioactive molecule. The turmeric nanofibers and modified starch/gum arabic are added to the mixture and stirred to form a liposomal emulsion. The liposomal emulsion is subjected to spray drying in order to obtain liposomal powder.
[0018] The bioactive molecule used in the invention is selected from a group comprising curcuminoids, withanolides, ascorbic acid, lutein and Docosahexaenoic Acid (DHA).
[0019] The turmeric nanofibers form the core of the liposome. When the nanofiber is dispersed in water, it forms an unorganized network. When the liposomal dispersion is subjected to spray drying, organized structure is formed by the nanofiber.
[0020] The modified food starch or gum Arabic are added to the formulation as an encapsulating agent to facilitate the formation of liposomal powder.
[0021] The liposomal powder has the particle size range of 50-400 nm depending on the type of bioactive molecule used ensuring delivery at the target site.
[0022] The dehydration of the liposomal dispersion and reconstitution of the powder form is advantageous in improving the encapsulation efficiency of the bioactive molecule and shows improved stability. The liposomal powder aids in controlled delivery of the bioactive molecules.
Brief description of drawings
[0023] FIG 1 tabulates the composition of the liposomal powder.
[0024] FIG 2 illustrates a process for preparation of the liposomal powder.
[0025] FIG 3 illustrates in-vitro drug release studies of native curcumin and liposomal powder.
[0026] FIG 4 illustrates in-vitro drug release studies of ß-Caryophyllene and liposomal powder.
[0027] FIG 5 illustrates the pharmacokinetic activity of ascorbic acid in the liposomal powder.
Detailed description of the invention
[0001] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definition is provided for specific terms, which is used in the following written description.
[0028] The term “Encapsulation” refers to an action of enclosing a particle in a capsule.
[0029] The invention discloses a spray dried formulation of liposomal powder for encapsulation of bioactive molecules with nanofibers for drug delivery.
[0030] FIG 1 tabulates the composition of the liposomal powder. The formulation of the present invention comprises phospholipids at a concentration range of 10%-25%, turmeric nanofibers at a concentration range of 1%-10%, modified food starch/ gum Arabic at a concentration range of 40%-80% and a bioactive molecule at a concentration range of 20%-40%.
[0031] The composition of the formulation is the liposomal powder. The formulation comprises a bioactive molecule encapsulated in the liposome with well-arranged nanofibers. The liposomes are made up of phospholipids and the nanofibers used are turmeric nanofibers. The bioactive molecules are selected from a group comprising curcuminoids, withanolides, ascorbic acid, Docosahexaenoic Acid (DHA), lutein and resveratrol. The nanofiber which is in the core of the liposome forms an un-organized network when dispersed in water and modified food starch or gum Arabic are added to facilitate encapsulation. Upon spray drying, water is evaporated, modified food starch or gum Arabic facilitate the formation of liposomes and the nano fiber forms an organized network and maintains the liposomal structure. The combination of liposome with nanofiber protects the bioactive molecule and allows the delivery of the bioactive molecule at the target site.
[0032] The particle size of the liposomal powder varies with the type of the biomolecule used and the particle size of the liposomal power is usually in the range of the 50- 400 nanometers allowing for convenient delivery at the target site.
[0033] The invention also discloses a process for preparation of the liposomal powder with nanofibers encapsulated with bioactive molecule. The process comprises the initial step of preparing the liposome dispersion with the bioactive molecule and nanofibers. The liposome dispersion is subjected to high pressure homogenization and spray drying to obtain the formulation in the powder form.
[0034] FIG 2 illustrates the process for preparation of the liposomal powder. The process (200) of preparation of liposomal powder starts with a step (201) of dissolving a phospholipid in Millipore water at a temperature of 800C. At step (202), turmeric nanofibers and modified food starch or gum Arabic are added to the stirred mixture. At step (203), bioactive molecule is added to the phospholipid mixture with constant stirring which is continued for 30 minutes. At step (204), the mixture is homogenized for 30 minutes at the temperature of 800C to form a liposomal emulsion. At step (205), the liposomal emulsion is subjected to one or more cycles of spray drying to obtain the liposomal powder.
[0035] The bioactive molecule used in the invention is selected from a group comprising curcuminoids, withanolides, ascorbic acid, lutein and DHA. The nanofibers selected are turmeric nanofibers, which form the structural organization that retains the bioactive molecule firmly during encapsulation and also allows the release of bioactive molecule in a systematic manner. The modified food starch or gum Arabic is selected to act as an encapsulating agent that facilitate encapsulation of the bioactive to form liposomes. The liposomal powder prepared accordingly to the process of the present invention is analyzed for release of curcumin and ß-Caryophyllene.
[0036] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner
Example 1: Analysis of in-vitro drug release studies of curcumin from liposomal powder.
[0037] The in-vitro drug release studies were performed for native curcumin and liposomal powder carrying curcumin at two different pH of 5.5 and 7.4 at 370 C. The negative charge of the phospholipids in the liposomal powder is helpful for the uptake and good for sustainable drug release at pathological sites.
[0038] FIG 3 illustrates in-vitro drug release studies of native curcumin and liposomal powder carrying curcumin. The study was performed at pH 5.5 and 7.4 at 370 C. Curcumin was released from the liposomal powder by a fast initial rupture followed by a sustained release. The amount of curcumin released was 46.2% at pH 5.5 and 40.4% at pH 7.4.
Example 2: Analysis of in-vitro drug release studies of ß-Caryophyllene from liposomal powder
[0039] The in-vitro drug release studies were performed for ß-Caryophyllene and the liposomal powder carrying ß-Caryophyllene at pH of 6.8 at 370 C. ß-Caryophyllene and the liposomal powder exhibited a two-phase release pattern by a quicker release in the primary phase and later a sustained release.
[0040] FIG 4 illustrates in-vitro drug release studies of ß-Caryophyllene and liposomal powder ß-Caryophyllene. The study was performed at pH 6.8 at 370 C. The results indicated that 81% of fast primary rupture until 12 hours and later sustained release of liposomal power was observed.
[0041] The liposomal powder prepared accordingly to the process of the present invention is analyzed for pharmacokinetic properties.
Example 3: Analysis of pharmacokinetic properties of the liposomal powder
[0042] Various ADMET (Adsorption, Digestion, Metabolism, Excretion and Toxicity) parameters were evaluated for the liposomal powder carrying ascorbic acid.
[0043] FIG 5 illustrates the pharmacokinetic activity of ascorbic acid in the liposomal powder. Ascorbic acid has gastrointestinal absorption of 39.154%. The ascorbic acid present in the liposomal powder is found to be non-toxic and non-carcinogenic.
[0044] The unique process of the present invention includes the combined steps of preparing the liposomal dispersion and subjecting the liposomal dispersion to the spray drying to obtain the final product in the powder form. The powder form allows for feasible storage to retain the bioavailable parameters of the bioactive molecule and is reconstituted with the suitable material.
[0045] The method involving the dehydration of the liposomal dispersion and later reconstitution of the powder form is also advantageous in improving the encapsulation efficiency of the bioactive molecule. The liposomal powder of the invention shows improved stability and is useful for controlled delivery of the bioactive molecules.
[0046] The use of liposomes in powdered form increases storage stability of the liposomal powder. The stability shown by liposomal powder determines the range of application such as in nutraceutical, food and beverage industries where the liposomal powder is used. The present invention does not exhibit any transformation in physical form and color when stored at different temperatures over a period of time. Also, the concentration of the bioactive molecules does not decrease drastically when stored over a period of time.
[0047] The liposomal powder of the present invention overcomes the drawback of the liposome in dispersion and is useful in food and drug delivery products. The conversion of liposomal liquids to powder form extends the usage of the product in multiple formats. Further, liposomal powders ensure the delivery of bioactive molecules into the blood stream in a sustained manner and the presence of the structural organization of the nanofibers allows the sustained release of the bioactive molecules without degradation thus retaining the biological activity of the bioactive molecules. The pharmacokinetic studies depict the liposomal powder to be non-toxic and non-carcinogenic.

,CLAIMS:1. A process for preparation of liposomal powder with bioactive molecules, the process comprising the steps of:
a) dissolving 10%-25% phospholipid in Millipore water at a temperature of 800 °C;
b) adding 1%-10% turmeric nanofibers to the stirred mixture;
c) adding 40%-80% modified food starch/gum arabic to the stirred mixture;
d) adding 20%-40% bioactive molecule to the phospholipid mixture with constant stirring and continuing the stirring for 30 minutes;
e) homogenizing the mixture for 30 minutes at the temperature of 800C to form a liposomal emulsion; and
f) subjecting the liposomal emulsion to spray drying to obtain the liposomal powder.

2. The process as claimed in claim 1, wherein said bioactive molecule is selected from a group comprising curcuminoids, withanolides, ascorbic acid, lutein and Docosahexaenoic Acid (DHA).

3. A formulation of liposomal powder with bioactive molecule, the formulation comprising:
a) a phospholipid at a concentration range between 10% -25%;
b) a turmeric nanofiber at a concentration range between 1%-10%;
c) a modified food starch/gum Arabic at a concentration range between 40%-80%; and
d) a bioactive molecule at a concentration range between 20%-40%.

4. The formulation as claimed in claim 3, wherein the liposomal powder delivers bioactive molecule into the blood stream in a sustained manner.

5. The formulation as claimed in claim 3, wherein the liposomal powder exhibits particle size in the range of 50-400 nm.

6. The formulation as claimed in claim 3, wherein the liposomal powder exhibits extended storage stability.

7. The formulation as claimed in claim 3, wherein the liposomal powder exhibits improved encapsulation efficiency.