A method for preparation of highly bioavailable and stable dry liposomes

Abstract

The present invention discloses a method of preparation of dry liposomes using nano-milling, homogenization followed by spray drying to enhance the bioavailability and stability of the liposomes. The method of preparation of dry liposomes comprises the steps of dissolving gum Arabic in water and nano milling the mixture, dissolving phospholipids in equal parts in water and mixing with gum Arabic, dissolving bioactive molecule in water and mixing with phospholipid mixture and gum Arabic mixture to obtain liposomal complex and nano milling the mixture. The nano milled liposome is subjected to homogenization under high pressure and the process is repeated at least three times. Finally, the homogenized liposome is dried by subjecting to spray drying to obtain a stabilized dry liposome which exhibits improved stability, solubility, absorption and hence bioavailability.

Description

TITLE OF THE INVENTIONA method for preparation of highly bioavailable and stable dry liposomesPriority Claim

[0001] This application claims priority from the provisional application numbered 202341053557 filed with the Indian Patent Office, Chennai on 10thAugust 2023 post-dated to 10thSeptember 2023 entitled “A method for preparation of highly bioavailable and stable dry liposomes ”, the entirety of which is expressly incorporated herein by reference.Preamble to the description

[0002] The following specification particularly describes the invention and the manner in which it is to be performed:Description of the inventionTechnical field of the invention

[0003] The present invention relates to a method of preparation of dry liposomes. More particularly, the invention relates to a method of preparation of dry liposomes using nano-milling, homogenization to enhance the bioavailability and stability of the liposomes.Background of the invention

[0004] Liposomes are small artificial vesicles of spherical shape that are formed from cholesterol and natural non-toxic phospholipids. 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 active molecule encapsulation in the liposomes. Based on the structure, liposomes are classified into four categoriesbased on size and number of bilayers namely small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), multilamellar vesicle (MLV), and multivesicular vesicles (MW).

[0005] Liposomes are the most popular and promising approach to drug delivery systems compared to the numerous drug carriers created to increase the bioavailability and therapeutic efficacy of bioactives. Liposomes are associated with improved stability, drug delivery, and bioavailability of both hydrophilic and hydrophobic compounds, which are encapsulated or entrapped into various products.

[0006] Liposomal products are mostly available in various forms such as liquid or suspension form, as phospholipids are stabilized through a water-in-oil emulsion as a vehicle-like compartment for encapsulation. Although, liposomes exhibit poor stability because of the random nature of the bilayer collapsible of phospholipids in the presence of water, typically leading to non-uniform development in both size and shape.

[0007] However, in recent times, liposomes are also utilized for many active molecules in dietary and nutraceutical industry. Liposomes have one or bilayer membranes. The amphipathic nature of the liposomes has added advantage as the hydrophilic part is mainly phosphoric acid bound to a water-soluble molecule whereas the hydrophobic part consists of two fatty acid chains with 10-24 carbon atoms and 0-6 double bonds in each chain. They form lamellar sheets when dispersed in aqueous medium by aligning themselves in such a way that the polar head group faces outwards the aqueous region while fatty acid groups face each other forming a spherical, vesicle like structures called as liposomes.

[0008] Liposomes are composed of naturally derived phospholipids with mixed lipid chains, such as egg phosphatidylethanolamine, or of pure surfactant components such as dioleoyl phosphatidylethanolamine (DOP). It is common for liposomes to also contain a core aqueous solution that is trapped by one or more bilayers. The phospholipid bilayers of a liposome originate from natural sources, which are biologically inert, immunogenic and exhibit a lower inherent toxicity.

[0009] The use of liposomes is associated with low stability in aqueous dispersions. Liposomes are associated with chemical and physical degradation eventually resulting in reduced efficacy owing to decreased quality of the formulation.

[0010] In order to overcome this, a suitable liposomal formulation is achieved by choosing an adequate method of preparation. The selection of phospholipids, head group and chain length, as well as the ratio of liposomes components are crucial features to determine safety, stability, and efficiency of liposome.

[0011] Nano milling is the process by which the particle size of an the molecule is reduced in a liquid vehicle typically aqueous through grinding using polymeric or ceramic media. Nano milling is a proven, commercially validated process for formulating poorly water-soluble molecules. The key is the tremendous increase in surface area which directly correlates to an increase in dissolution rate.

[0012] Nano milling is a “top-down” approach of converting large, coarse particles into smaller, finer particles. The process entails the application of mechanical energy to physically break down crystalline structures of molecules. The main technique used for nano milling the molecule is high energy media milling. This technique relies on milling media — 0.2-1 pm beads that are made from ceramics or highly crosslinked polystyrene. The beads shear and collide with the particles during the milling process, reducing their particle size.

[0013] During nano milling, the molecule is suspended in a solution containing water and at least one stabilizer to prevent reaggregation of particles over time. The nano milling process generates an intermediate consisting of nanoparticles suspended in an aqueous vehicle. This is often referred to as nanoparticulate suspension or nanosuspension. Nanosuspensions have been converted into a wide range of dosage forms, including oral liquids, capsules, tablets, films, injectables, aerosols, and more.

[0014] Homogenization refers to any of several processes used to make a mixture of two mutually non-soluble liquids the same throughout. This is achieved byturning one of the liquids into a state consisting of extremely small particles distributed uniformly throughout the other liquid.

[0015] The Patent Application No. US10722508B2 entitled “Liposomes useful for drug delivery ' discloses liposome compositions containing substituted ammonium and polyanion, and optionally with a desired therapeutic or imaging entity. The invention also provides methods of making the liposome.

[0016] The Patent Application No. EP3138558B1 entitled “Liposome composition and production method therefore" discloses a method for preparation of liposomes. The method of adsorbing and retaining a drug onto a lipid membrane of a liposome, release of the drug to the outside of the liposome becomes difficult due to strong interactions such as hydrophobic interactions and electrostatic interactions. Therefore, the configuration of the liposome composition after production can be maintained, whereby it is easy to secure long-term preservation stability.

[0017] The Patent Application No. US4830858A entitled “Spray-drying method for preparing liposomes and products produced thereby’' discloses a method for preparing a stable liposome precursor in the form of a mixture of spray-dried liposomal components including biologically active compounds which may be stored dry and reconstituted with water to form a liposomal preparation immediately prior to use. The dry liposome precursor is also provided.

[0018] In spite of various advantages of the use of liposomes as drug delivery systems, the liposomes are associated with few challenges. One of the drawbacks of liposomes is the rapid degradation due to reticuloendothelial system (RES) and inability to achieve sustained drug delivery over a prolonged period of time. In addition, liposome-based delivery systems are known to possess limitations such as instability, short half-life, and rapid clearance.

[0019] Hence, there is a need for a method for preparation of dried liposomes that are stable and in dried form thus enhancing the stability and thus bioavailability of the encapsulated active molecules.Summary of the invention

[0020] The present invention overcomes the drawbacks of existing methods to produce liposomes. The present invention discloses a method of preparation of highly bioavailable and stable dry liposomes of bioactive molecules.

[0021] The present invention discloses a method of preparation of stable dry liposomes comprising steps of dissolving gum Arabic in water and nano milling the mixture, dissolving phospholipids in equal parts in water and mixing with gum Arabic, dissolving bioactive molecule in water and mixing with phospholipid mixture and gum Arabic mixture to obtain liposomal complex and nano milling the mixture. The nano milled liposome is subjected to homogenization under high pressure and the process is repeated at least three times.

[0022] The homogenized liposomal mixture is dried by subjecting to spray drying to obtain a stabilized dry liposome, which exhibits improved physical and chemical stability and bioavailability. The method disclosed is energy and cost effective also the dried liposomes hence obtained is safe, stable and therapeutically effective.Brief description of the drawings

[0023] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.

[0024] FIGURE 1 illustrates a flow chart for a method of preparation of the dried liposomes according to an embodiment of the invention.

[0025] FIGURE 2 illustrates a flow chart for a method of preparation of the dried Vitamin C liposomes according to another embodiment of the invention.

[0026] FIGURE 3 tabulates the total average pharmacokinetic variables for dried liposomal vitamin C and conventional vitamin C.

[0027] FIGURE 4 illustrates the mean concentrations of dried liposomal vitamin C and conventional vitamin C.

[0028] FIGURE 5 illustrates the Dynamic Light Scattering (DLS) potential of the dried liposomal vitamin C at durations of 0 months and 6 months.

[0029] FIGURE 6 illustrates the zeta potential of the dried liposomal vitamin C at durations of 0 months and 6 months.Detailed Description of the Invention

[0030] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0031] The term ''Liposomes ' refers to a small artificial vesicle, spherical in shape, having at least one lipid bilayer.

[0032] The term “Bioavailability” refers to the extent a substance or drug becomes completely available to its intended biological destination.

[0033] The present invention discloses a method of preparation of dry liposomes using nano-milling, homogenization followed by spray drying to enhance the bioavailability and stability of the liposomes.

[0034] The present invention is specific to the method of preparation of powdered form of liposomes using nano-milling process subsequently high-pressure homogenization, followed by removing the water through a spray drying process. The method results in the formation of dried liposomes that exhibit increased stability, solubility, absorption and hence bioavailability of the dried liposomes making it a suitable for drug delivery system for wide range of active agents.

[0035] FIG 1 illustrates a flow chart for a method of preparation of the dried liposomes according to another embodiment of the invention. The method (100) of preparation of liposomes with the bioactive molecule starts with a step (101) of dissolving gum Arabic in water at a temperature of 80°C. The mixture is subjected to nano milling process to obtain flexible gum Arabic colloidal nano spheres. The mixture subjected to nano milling undergoes particle size reduction wherein the chamber capacity is 80ml with a bead size of 0.5mm and 3.7 kg / m3density perbeads. The rotation conditions incorporated are 6000rpm for a duration of 10 minutes. The process of nano milling aims at particle size reduction for enhancing the solubility, absorption and hence bioavailability of the bioactive molecule.

[0036] At step (102), one half of phospholipids dissolved in water at a temperature of 80°C is incorporated into the nano sphere mixture. Nano milling aids in improved solubility, absorption and bioavailability. Phospholipids further facilitate effective delivery of the bioactive molecule and enhance the overall stability of the liposome.

[0037] At step (103), resultant nano sphere mixture is further subjected to nano milling process to obtain gum Arabic - phospholipid complex. The mixture subjected to nano milling undergoes particle size reduction wherein the chamber capacity is 80ml with a bead size of 0.5mm and 3.7 kg / m3density per beads. The rotation conditions incorporated are 6000rpm for a duration of 10 minutes. The high energy mechanical forces involved during the nano milling process enables to achieve particle size reduction hence enhancing the stability and dispersibility of the gum Arabic -phospholipid complex.

[0038] At step (104), remaining half of the phospholipids is subjected to dissolution in water at a temperature of 80°C. The dissolved mixture is further subjected to nano-milling process to facilitate particle size reduction. The mixture subjected to nano milling undergoes particle size reduction wherein the chamber capacity is 80ml with a bead size of 0.5mm and 3.7 kg / m3density per beads. The rotation conditions incorporated are 6000rpm for a duration of 10 minutes.

[0039] At step (105), bioactive molecule is dissolved in water at a dissolution rate of 25:75. The dissolved bioactive molecule is further subjected to mixing with the phospholipid mixture to obtain bioactive molecule- phospholipid complex. The mixture is further subjected to the process of nano milling. The mixture subjected to nano milling undergoes particle size reduction wherein the chamber capacity is 80ml with a bead size of 0.5mm and 3.7 kg / m3density per beads. The rotation conditions incorporated are 6000rpm for a duration of 10 minutes.

[0040] The bioactive molecule is selected from curcuminoids, nicotinamide mononucleotide (NMN), quercetin, resveratrol, hyaluronic acid, coenzyme Q10 (CoQlO), vitamin C, vitamin B 12, pyrroloquinoline quinone (PQQ), palmitoylethanolamide (PEA), ferrous bisglycinate, magnesium bisglycinate, vitamin D3, vitamin K2.

[0041] At step (106), the obtained bioactive molecule- phospholipid complex and gum Arabic- phospholipid mixture is subjected to mixing to obtain a liposomal mixture. The obtained liposomal mixture is subjected to nano milling process. The mixture subjected to nano milling undergoes particle size reduction wherein the chamber capacity is 80ml with a bead size of 0.5mm and 3.7 kg / m3density per beads. The rotation conditions incorporated are 6000rpm for a duration of 10 minutes. The integration of bioactive molecule- phospholipid complex and gum Arabic- phospholipid mixture occurs while the liposomes are in a liquid state, ensuring the encapsulation of bioactive molecule, flexible advanced colloidal spheres, and water within the liposomal structure.

[0042] At step (107), the nano milled liposome mixture is subjected to homogenization under high pressure and the process is repeated at least three times. The process of homogenization is carried out at a pressure of 300 Bar for a duration of 30 minutes under controlled temperature range of 25° C to 30° C. Upon subjecting the nano milled mixture to homogenization, the particle size of the mixture is reduced hence increasing the surface area. The increase in surface area facilitates even dispersion and easier dispersion of the liposome mixture. Hence, the bioavailability and efficacy of the liposome mixture is enhanced. The texture and taste are further improved. Additionally, the liposome mixture exhibits improved stability.

[0043] At step (108), the homogenized liposome mixture is dried by subjecting to spray drying to obtain a stabilized dry liposomal powder comprising the bioactive molecule. The inlet temperature of the spray dryer is 170° C and the outlet temperature is 70° C. Spray drying of the liposomal mixture enhances the practical surface area of the molecules. The encapsulation and product efficiency are furtherimproved by spray drying. Also, the control of shape, size and morphology is achieved. Having obtained dried powder, ease of transportation, accessibility and cost and energy efficiency is feasible. Physical and chemical stability of the dried liposomal powder is achieved.

[0044] The dried liposomes exhibit improved physical and chemical stability, extend half-life, delays the degradation as a result of spray drying. Further, liposomes prepared by said method are stable, results in controlled release and enhances the bioavailability of the encapsulated active molecules. Since the liposomes are subjected to spray drying, the moisture content is removed thereby preventing phospholipid hydrolysis. Also, as the method results in obtaining a dried liposome, the movement of molecules is reduced hence facilitating ease of storage. The stable and dried liposomes of the present invention are useful for encapsulation of various ranges of active molecules.

[0045] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.Example 1: A method of preparation Vitamin C dry liposomes and aalysis of bioavailability and stability and

[0046] The present invention discloses a method of preparation of highly bioavailable and stable dry liposomes. The embodiment further describes the method of preparation and the analysis of bioavailability and stability considering vitamin C as the bioactive molecule. FIG 2 illustrates a flow chart for a method of preparation of the dried vitamin C liposomes according to another embodiment of the invention. The method (200) of preparation of vitamin C liposomes with a step (201) of Dissolving gum Arabic in water at a temperature of 80°C to obtain a mixture and subjecting the mixture to nano milling process for particle size reduction. At step (202), one half of lecithin is dissolved in water at a temperature of 80°C is added to the gum Arabic mixture. At step (203), the resultant mixture is subjected to nano milling process. The nano milling chamber capacity is 80ml witha bead size of 0.5mm and 3.7 kg / m3density per bead. The rotation conditions incorporated are 6000rpm for a duration of 10 minutes.

[0047] At step (204), remaining lecithin is dissolved in water at a temperature of 80°C and the dissolved mixture is subjected to nano-milling process. At step (205), vitamin C is dissolved in water at a ratio of 1:3 and further mixing the lecithin mixture to obtain vitamin C-lecithin liposomal complex. The vitamin C-lecithin liposomal complex is further subjected to nano milling process for particle size reduction. At step (206), the obtained vitamin C-lecithin and gum Arabic-lecithin mixture is subjected to mixing and subjecting the obtained liposomal mixture to nano milling process for particle size reduction. At step (207), the nano milled vitamin C liposome mixture is subjected to homogenization under high pressure and the process is repeated at least three times. The process of homogenization is carried out at a pressure of 300 Bar for a duration of 30 minutes under controlled temperature range of 25° C to 30° C. At step (208), the homogenized formulation is dried by subjecting to spray drying to obtain a stabilized dry liposomal vitamin C. The inlet temperature of the spray dryer is 170° C and the outlet temperature is 70° C.

[0048] The obtained dried vitamin C liposomes exhibit enhanced solubility and bioavailability. The oral bioavailability of the powder was thus evaluated. The samples considered were dried liposomal vitamin C of 700-800 mg capsule comprising vitamin C at a concentration of 500mg per capsule and a conventional vitamin C capsule at the same concentration. Oral bioavailability was conducted on double blind, randomized, single dose, two -treatment, two-way crossover, in healthy adult human participants examined under fasting conditions. The sample size of the study was 20 comprising 10 participants randomized to each of the two study arms at a 1: 1 ratio and received dosing as per randomization. During the second period of study, the participants were interchanged for the crossover study after the washout period of 7 days. The subjects were fasted for at least 10 hours before dose administration. During morning hours, a single oral dose of 700mg to 800mg of either test product (T) or reference product (R) administered orally, along with 250 ml water. 8 blood samples were collected including the pre-dose samplingin each period. After the collection of blood samples, the samples were placed in a thermo-insulated box containing wet ice. The samples were transferred the box to the sample processing room where the blood samples were centrifuged within 30 minutes at a speed of 4000 ± 50 rpm for a duration of 10 minutes at a temperature range of 2 °C to 8 °C to separate the plasma.

[0049] The analysis of vitamin C was performed by adding one millimolar citric acid buffer to the plasma samples to precipitate out the proteins and centrifuged and filtered through 0.22 micron and the aliquot inject to the UPLC system. Vitamin C content in the plasma was measured by ultra performance liquid chromatography (UPLC) C18 column, 5 pm, with a volume of 4.6 x 250 mm. The mobile phase was 0.1 % acetic acid and methanol in the ratio of (90: 10). The flow was isocratic at a rate of 1 ml / min. at 32 °C. The eluate was detected using an Agilent VWD detector set at 254 nm.

[0050] Further, the effects of the dried liposomal vitamin C were compared with conventional vitamin C. FIG 3 tabulate the total average pharmacokinetic variables for dried liposomal vitamin C and conventional vitamin C. The maximum plasma vitamin C (Cmax) and the AUC for the dried liposomal vitamin C were approximately 2.76 pg / mL and 88.66 pg.h / mL, respectively. In comparison, these values for the conventional vitamin C were 0.82 pg / mL and 25.97 pg.h / mL, respectively. The time to reach the maximum plasma concentration (Tmax) is 2 h for both liposomal vitamin C and vitamin C, which indicates that the release patterns of vitamin C in both the groups are the same.

[0051] FIG 4 illustrates the mean concentrations of dried liposomal vitamin C and conventional vitamin C. The absorption of the dried liposomal vitamin C was 3.41- fold greater than the vitamin C in terms of absorption (AUCo-t) and 3.37-fold greater in terms of the rate of absorption (Cmax). AUCo ® is defined as the area under the plasma concentration versus time curve from time zero to infinity. Where AUCo- □o = AUCo-t + Ct / Kei, Ct is the last measurable concentration and Keiis the terminal elimination rate constant AUCo ® was 191.37 pg.h / mL and 55.34 pg.h / mL for dried liposomal vitamin C group and conventional vitamin C group, respectively. t2isdefined as the elimination or terminal half-life will be calculated as 0.693 / Kel. t2was 51.05 hours and 47.73 hours for liposomal vitamin C group and conventional vitamin C group, respectively. The present invention discloses a method of preparation to obtain dried liposomal vitamin C which significantly exhibits enhanced absorption.

[0052] The dried liposomal vitamin C was further subjected to stability analysis. An accelerated stability study was performed as per the guidelines of the International Conference on Harmonization (ICH) of technical requirements for the registration of pharmaceuticals for human use. Dried liposomal vitamin C at a weight of 10 g sample packets of samples were packed in polyethylene kit and kept in air-tight high-density polypropylene (HDPE) bottles and incubated at temperatures of 40°C and 75% relative humidity for a duration of six months in a stability chamber. The samples were retrieved after time intervals of 0,1, 2, 3 and 6 months and further subjected to stability analysis for parameters including color and appearance, active content, moisture content, and microbial parameters.

[0053] The liposomal stability evaluation was performed by retrieving the samples after the first and sixth month and analyzing the liposomal stability by Dynamic Light Scattering (DLS) and zeta potential measurements. FIG 5 illustrates the Dynamic Light Scattering (DLS) potential of the dried liposomal vitamin C at durations of 0 months and 6 months. FIG 6 illustrates the zeta potential of the dried liposomal vitamin C at durations of 0 months and 6 months. The accelerated stability study of the dried liposomal vitamin C indicates the product is stable for a duration of 24 months. The Zeta potential and DLS values of 0thand 6thmonth respectively showed no significant variations there by confirming that liposomes remain stable, and the morphological parameters remain intact.

[0054] The present invention discloses a method of preparation of dried liposomes with improved bioavailability and stability. The method disclosed overcomes the drawbacks of conventional liposomes including chemical instability such as oxidation of phospholipid layer, increased bilayer permeability, precipitation, leakage of liposomal components, physical instability such as degradation byheating and freezing. The method comprises steps of optimized nano-milling, homogenization followed by spray drying to enhance the bioavailability and stability of the liposomes. The method disclosed is energy and cost effective also the dried liposomes hence obtained is safe, stable and therapeutically effective.

Claims

Claims:We Claim:

1. A method of preparation dry liposomes, the method comprising steps of: a) subjecting gum Arabic to dissolution in water at a temperature of 80°C to obtain a mixture subjecting the mixture to nano milling process to obtain flexible gum Arabic colloidal nano spheres (101); b) adding one half of phospholipids dissolved in water at a temperature of 80°C to the nano sphere mixture (102); c) subjecting the resultant nano sphere mixture to particle size reduction by nano milling process to obtain gum Arabic - phospholipid complex (103); d) subjecting remaining half of the phospholipids to dissolution in water at a temperature of 80°C and subjecting the dissolved mixture to nano-milling process for particle size reduction (104); e) subjecting the bioactive molecule to dissolution in water and further mixing the phospholipid mixture to obtain bioactive moleculephospholipid complex and subjecting the mixture to nano-milling process for particle size reduction (105); f) subjecting the obtained bioactive molecule- phospholipid complex and gum Arabic- phospholipid mixture to mixing and subjecting the obtained liposomal mixture to nano milling process for particle size reduction (106); g) homogenizing the nano milled liposome mixture at high pressure for three times (107); and h) subjecting the homogenized liposome mixture to spray drying to obtain dried liposomes (108).

2. The method as claimed in claim 1, wherein the bioactive molecules is selected from group of curcuminoids, nicotinamide mononucleotide (NMN), quercetin, resveratrol, hyaluronic acid, coenzyme Q10 (CoQlO), vitamin C, vitamin B12, pyrroloquinoline quinone (PQQ), palmitoylethanolamide (PEA), ferrous bisglycinate, magnesium bisglycinate, vitamin D3, vitamin K2.

3. The method as claimed in claim 1 , wherein the phospholipid used is lecithin.

4. The method as claimed in claim 1, wherein the nano milling process for particle size reduction is carried out at a chamber capacity of 80ml, bead size of 0.5mm, density per beads 3.7 kg / m3rotating at 6000rpm for a duration of 10 minutes.

5. The method as claimed in claim 1, wherein the bioactive molecule is dissolved in water at a dissolution rate of 25:75.

6. The method as claimed in claim 1, wherein the homogenization of the liposome mixture is carried out at high pressure conditions at 300 Bar for a duration of 30 minutes under controlled temperature range of 25° C to 30° C.

7. The method as claimed in claim 1, wherein the spray drying to obtain dried liposomes is carried at inlet temperature of the spray dryer is 170° C and the outlet temperature is 70° C.

8. The method as claimed in claim 1, wherein the obtained dried liposomes comprising bioactive molecule are physically and chemically stable due to optimized particle size reduction and the process of drying.

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