Claims:We claim:
1. A process for synthesis of liposomes comprising of,
I. Preparing feed stoke comprising of lipid, cholestrol and drug molecule dissolved in the solvents; 5
said lipids are choline lipids – phosphatidyl choline, Egg PC, sphingomyelin, pH sensitive lipids phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, phosphatidylcholine, distearoyl phosphatidyl choline and 10 their PEGylated forms in the range of 0.1 - 5 % w/w;
said cholesterol is in the range of - 0.01-0.5 % w/w;
said drug molecules are hydrophilic or hydrophobic drug in 15 the range of (0.1-3%) w/w;
said solvents are group of chloroform, dichloromethane, acetone, hexane ether benzene, in the range of 95-99.9 % w/w; 20
II. Generating tuned sized Nano droplets of feed stock varying the power to volume ratio in the nanodroplet generating apparatus;
III. Encapsulating said drug molecule by introducing carrier gas; 25 flashing high temperature and pressurized steam over said Nano droplets and generating liposome;
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Wherein,
said carrier gas are nitrogen, argon, helium and injected in the range of 5SCCM – 200 SCCM; wherein the carrier gas flow rate adjusted within the said given range, less or more number of droplets are carried by reducing or increasing 5 the flow rates of the carrier gas respectively; thereby said range provides uniform sized nanodroplets in each batches in a continuous process;
the temperature of steam is in ranges from 50 - 130ºC and 10 steam pressures is in range of 0.5 bar to 5 bar at flow rates of 10mL/minute to 200mL/minute;
IV. Separating said liposomes and recovering solvent.
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2. A process for synthesis of liposomes, comprising of:
I. Preparing feed stoke comprising of lipid, cholesterol and drug molecule dissolved in the solvents;
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said lipids are choline lipids – phosphatidyl choline, Egg PC, sphingomyelin, pH sensitive lipids phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, phosphatidylcholine, distearoyl phosphatidyl choline and their PEGylated forms in the range of 0.1 - 5 % w/w; 25
said cholesterol is in the range of - 0.01-0.5 % w/w;
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said drug molecules are hydrophilic or hydrophobic drug in the range of (0.1-3%) w/w;
said solvents are group of chloroform, dichloromethane, acetone, hexane ether benzene, in the range of 95-99.9 % 5 w/w;
II. Generating tuned sized Nano droplets of feed stock varying the power to volume ratio in the nanodroplet generating apparatus;
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III. Encapsulating said drug molecule into the Nano droplets by flashing high temperature and pressurized steam over the Nano droplets and generating liposome;
Wherein, 15
the temperature of steam is in range of 50 - 130ºC and steam pressure is in range of 0.5 bar to 5 bar at flow rates of 10mL/minute to 200mL/minute; 1. wherein the variation of steam flow rate and pressure in said range 20 provides uniform sized liposomes in each batches in a continuous process;
IV. Condensing and Extruding said liposomes.
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3. A process for synthesis of liposomes as claimed in claim 1 and 2, wherein the present process reduces the process time and in turn increasing the throughput rate of the process.
4. A process for synthesis of liposomes as claimed in claim 1 and 2, 5 wherein the size of the synthesized liposomes is in the range of 40 to 400 nm.
Dated this 8th Day of May 2019
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________________________
GOPI J. TRIVEDI (Ms)
IN/PA 993 15
At Y. J. Trivedi & Co.
(Authorized Agent of the Applicant)
To,
The Controller of Patents,
Patent Office, 20
Mumbai. , Description:FORM – 2
THE PATENTS ACT, 1970
(39 of 1970)
5
COMPLETE SPECIFICATION
(See section 10; rule 13)
“PROCESS FOR SYNTHESIS OF LIPOSOMES"
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Ahmedabad University
The Statutory University established under the State Act,
recognized under UGC Act
Having address at 15 Commerce Six Roads, Navrangpura, Ahmedabad-380009, Gujarat, India
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The following specification particularly describes the nature of this invention and the manner in which it is to be performed:
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FIELD OF THE INVENTION
The present invention relates to a process for synthesis of liposomes. More particularly, the present invention relates to a process for continuous synthesis of tunable and monomodal size distributed liposomes along with encapsulation of drugs within said synthesized 5 liposomes; thereby saving process time and energy resulting in efficient process for continuously synthesizing liposomes.
BACKGROUND OF THE INVENTION Liposomes are microscopic lipid vesicles that are composed of a central 10 aqueous cavity surrounded by a lipid membrane formed by concentric bilayer(s) (lamellas). Liposomes are able to incorporate hydrophilic substances (in the aqueous interior) or hydrophobic substances (in the lipid membrane). Liposomes can be unilamellar vesicles (“UMV”), having a single lipid bilayer, or multilamellar vesicles (“MLV”), having a 15 series of lipid bilayers (also referred to as “oligolamellar vesicles”). Liposomes or lipid nanoparticles are used as one of the drug delivery vehicles for multiple pharmaceutical applications particularly for the delivery of hydrophobic drugs and for increasing their circulation and residence time within the body. They have gained a lot of commercial 20 interest given the fact that liposomal formulation of anti-cancer drugs have been commercialized and many more are under clinical trials. Many processes for the synthesis of the liposomes have been developed in past that generally relates to mixing of two phases i.e. 25 mixing of an aqueous phase and an organic phase.
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There are four classical methods of liposome manufacture. The difference between the various methods is the way in which lipids are drying down from organic solvents and then redisposed in aqueous media. These steps are performed individually or are mostly combined. 5 • A conventional method to prepare liposome is hydration of a Thin Lipid Film also known as the Bangham Method. This is the original method which was initially used for liposomes production. A mixture of phospholipid and cholesterol were dispersed in organic solvent. Then, the organic solvent was 10 removed by means of evaporation (using a Rotary Evaporator at reduced pressure). Finally, the dry lipidic film deposited on the flask wall was hydrated by adding an aqueous buffer solution under agitation at temperature above the lipid transition temperature. 15 • Another conventional technique to prepare liposomes is Reverse-Phase Evaporation (REV) Technique wherein, a lipidic film is prepared by evaporating organic solvent under reduced pressure. The system is purged with nitrogen and the lipids are 20 re-dissolved in a second organic phase which is usually constituted by diethyl ether and/or isopropyl ether. Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into this mixture. The organic solvent is subsequently removed, and the system is maintained 25 under continuous nitrogen. These vesicles have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles.
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Most importantly, a substantial fraction of the aqueous phase (up to 62% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. 5 • Yet another conventional technique used is Solvent (Ether or Ethanol) Injection Technique: The solvent injection methods involve the dissolution of the lipid into an organic phase (ethanol or ether), followed by the injection of the lipid solution into aqueous media, forming liposomes. The ethanol injection 10 method was first described in 1973. The main relevance of the ethanol injection method resides in the observation that a narrow distribution of small liposomes (under 100 nm) can be obtained by simply injecting an ethanolic lipid solution in water, in one step, without extrusion or sonication. The ether injection 15 method differs from the ethanol injection method since the ether is immiscible with the aqueous phase, which is also heated so that the solvent is removed from the liposomal product. The method involves injection of ether-lipid solutions into warmed aqueous phases above the boiling point of the ether. The ether 20 vaporizes upon contacting the aqueous phase, and the dispersed lipid forms primarily unilamellar liposomes. An advantage of the ether injection method compared to the ethanol injection method is the removal of the solvent from the product, enabling the process to be run for extended periods forming a concentrated 25 liposomal product with high entrapment efficiencies. • Furthermore Detergent Dialysis technique is used wherein Liposomes, in the size range of 40–180 nm, are formed when
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lipids are solubilized with detergent, yielding defined mixed micelles. As the detergent is subsequently removed by controlled dialysis, phospholipids form homogeneous unilamellar vesicles with usefully large encapsulated volume. Other methods have been already used for liposomes preparation such as: calcium 5 induced fusion, nanoprecipitation, and emulsion techniques. • In Yet another process 6 g of solvent L-a-phosphatidylcholine (Soy) was dispersed in 100 mL of water using a magnetic stirrer at 200 rpm for 10 minutes at ambient temperature. The dispersed liposome was passed through a Microfluidic 10 homogenizer at 15,000 psi. Three cycles of passing resulted in a liposome less than 100 nm in diameter. Trehalose was then added to the liposome to a final concentration of 10% (w/w). The resulting stable isotonic liposome was either used as liquid or lyophilized 15 However, these conventional techniques are batch processes that require large amounts of organic solvent, which are harmful both to the environment and to human health, requiring complete removal of residual organic solvent. Furthermore, conventional methods consist 20 of many steps for size homogenization and consume a large amount of energy which is unsuitable for the mass production of liposomes. Also, dispersed-phospholipids in aqueous buffer yields a population of multilamellar liposomes (MLVs) heterogeneous both in size and shape (1–5 µm diameter). The liposome size obtained is in micrometers. 25 Thus, it takes several passes through a filter to obtain a population of liposomes having the desired narrow particle-size distribution. The
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filtering procedure can thus be time consuming and add significantly to the cost of producing the liposomes. Thus, said conventional processes are very time-consuming process and are not scalable. It further limits the tunability of size of liposomes as the size of the liposomes is controlled by the flow regime mixing and 5 by confined micro channels of microfluidic system. (Added above) Said micro channels of microfluidic system needs to be changed for each varying size of the liposomes meaning thereby it requires to change the entire reactor each time the size being changed and hence is severely limited in providing tuneable sized liposomes. 10 PRIOR ART AND IT’s DISADVANTAGES: US patent numbered US 8591942 B2 relates to provide methods for preparing liposomes comprising docetaxel and uses thereof. In certain 15 embodiments, liposomes are prepared without using heat, organic solvents, proteins, and/or inorganic salts in the process. In certain embodiments, the liposomal preparations are used in the treatment of diseases or disorders. However, in the said prior arts the process for synthesis of liposomes 20 is not size selective and further uses the micro fluidic homogenization process, wherein the process of microfluidic homogenization is a very slow process as the liquid flow rates achieved are very low resulting in lower yield, batch process at very high pressures of 15000 Psi. Thus, the prior art fails to provide a continuous process for synthesis of 25 liposomes and also fails to provide an energy efficient process. It also consumes more time.
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Another US patent numbered US 5540936 A relates to provide a process for the production of liposomes by combining an organic phase and an aqueous at a volume ratio of less than 3:1; the process can be conducted under conditions which obtain a single-modal population distribution of liposomes encompassing a pre-selected 5 mean particle size. A novel intermediate product obtained during the process, which can itself be used for the topical delivery of a bioactive agent, is also provided. However, it often takes several passes through a filter to obtain a 10 population of liposomes having the desired narrow particle-size distribution. The filtering procedure can thus be time consuming and add significantly to the cost of producing the liposomes. DISADVANTAGES OF PRIOR ART: 15 The prior art suffers from all or at least any of the following disadvantages: 1. Most of them fails to provide a continuous process for the 20 synthesis of liposomes. 2. Most of the process relates to the batch process that involves several intermediate steps which further consumes more time for the production of liposomes, and thereby fails to provide time efficient process. 25 3. Most of them fails to provide liposomes of tunable and monomodal, and uniform size distribution as it has to pass through several filters to obtain desired size liposomes.
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4. Fail to provide uniform size of liposomes in a short period of time. 5. Most of them consumes higher energy in size distribution of liposomes and thereby fails to provide an energy efficient process. 5 6. Most of the processes do not recover the solvent due to effluent generation thus not efficient and cost effective 7. Most of them utilizes large amount of solvent that leads to high use of raw material thus not cost effective. 8. Most of them fails to produce higher yield of liposomes in short 10 time as the time taken to produce the liposomes is in hours and therefore are not substantially efficient. 9. Most of them uses the reverse phase evaporation technique which thereby reduces the efficiency of drug encapsulation within liposomes. 15 Thus, there is an unmet need to come up with the invention that obviates the problem of prior art. OBJECTS OF THE INVENTION: 20 The primary object of the present invention is to provide a process for synthesis of liposomes. Further object of the present invention is to provide a process for 25 synthesis of liposomes, wherein said process continuously synthesizes liposomes.
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Further object of the present invention is to provide a process for synthesis of liposomes, that provides tunable and monomodal size distributed liposomes. Further object of the present invention is to provide a process for 5 synthesis of liposomes, which facilitates encapsulation of drugs within the liposomes in the continuous process. Further object of the present invention is to provide a process for synthesis of liposomes that is energy efficient. 10 Further object of the present invention is to provide a process for synthesis of liposomes that consumes less time. Further object of the present invention is to provide a process for 15 synthesis of liposomes that provides liposomes of uniform size. Further object of the present invention is to provide a process for synthesis of liposomes that produces higher yield of liposomes. 20 Further object of the present invention is to provide a process for synthesis of liposomes that provides proper recovery of the solvent used. Further object of the present invention is to provide a process for 25 synthesis of liposomes that eliminates the generation of effluent.
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Further object of the present invention is to provide a process for synthesis of liposomes that obviates the problems of prior art. SUMMARY OF THE INVENTION 5 In order to meet the above-mentioned objectives, the embodiment of the present invention provides a continuous process for synthesis of liposomes. Said process utilizes existing apparatuses for synthesizing liposomes. Said apparatus mainly comprises of a mixing chamber, a nano droplet generator or a bubbler, a receiving chamber, a carrier gas 10 ejector, a steam ejector, a condenser, a layer separator such as a sonicator or an extruder. the process to synthesize liposomes comprises of the following steps: Step 1: Preparing feed stoke 15 Step 2: Generating nanodroplets Step 3: Encapsulating of drug into the liposome Step 4: Separating the liposome and the solvent
BRIEF DESCRIPTION OF DRAWINGS 20 Fig 1.: Shows stained uniform sized liposomes synthesized by the present process for the synthesis of liposomes analyzed under the Transmission Electron microscope (TEM) Fig 2.: Shows unstained uniform sized drug loaded liposomes synthesized by the present process for the synthesis of 25
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liposomes analyzed under the Transmission Electron microscope (TEM) DETAILED DESCRIPTION OF THE INVENTION: In the present invention the features, nature, and advantages of the disclosed subject matter will become apparent from the detailed 5 description set forth below.
The present invention provides a process for synthesis of liposomes. The present invention further provides a process for continuous synthesis of tunable and monomodal size distributed liposomes along with encapsulation of drugs within said synthesized liposomes; 10 thereby saving process time and energy resulting in efficient process for continuously synthesizing liposomes.
The present invention a process for synthesis of liposomes mainly comprises of the following:
The materials used in the said process for synthesis of liposomes 15 comprises of:
1) Lipids: Lipids include such as but not limited to choline lipids – phosphatidyl choline, sphingomyelin, pH sensitive lipids such as phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, others such as phosphatidylcholine, 20 distearoyl phosphatidyl choline and their PEGylated and other functionalized forms; wherein lipid is the major component of liposomes and is the main ingredient used for synthesis of liposomes.
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2) solvent: solvent that includes but does not limit to chloroform, dichloromethane, acetone, hexane ether benzene and alike; wherein a solvent is utilized to dissolve the lipids.
3) cholesterol, used for providing rigidity/strength to liposomes
4) Drug molecules: Drug molecules includes but does not limits to type 5 of anticancer, antimicrobial, antibacterial drugs and small molecules that are stable at a temperature of 130-150 degrees. Some of the drugs are encapsulated in liposomes to increase their bioavailability. Some of the drugs are 10 sparsely soluble in water and need to be given in an encapsulated form within a liposome
The present process for synthesis of liposomes comprises of the following steps;
1. Step 1: Preparing feed stoke 15
Preparing feed stoke by mixing of lipids that includes but does not limits to phosphatidyl cholines, Egg PC and alike in the range of 0.1-5 % w/w, Chloroform in the range of 95-99.9 % w/w, Cholesterol in the range of - 0.01-0.5 % w/w and drug molecules in the range of (0.1-3%) w/w). 20
2. Step 2: Generating nanodroplets
Generating nano droplets, vapours, atomized solvent through either a Nano droplet generator (NDG), a bubbler or an ultrasonic horn
3. Step 3: Encapsulating of drug into the liposome 25
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Introducing carrier gas in the range of 5SCCM – 200 SCCM. The carrier gas is used to carry the droplets to the receiving chamber, where the droplets react with steam for encapsulation of the drug molecules along with lipids.
4. Step 4: Separating the liposome and the solvent 5
Soliciting or Extruding the droplets for the layer separation of liposomes and proper recovery of the solvent.
Said process for synthesis of liposomes utilizes existing apparatuses. The apparatuses used for the process for synthesis of liposomes comprises of a mixing chamber; a nanodroplet generator, a bubbler, 10 ultrasonic horn, an inert gas ejector, a carrier gas ejector, receiving chamber, a steam ejector; a separation column; a condenser; a layer separator; and a gas scrubber.
The present process for synthesis of liposomes is described in detail in the following steps; 15
Step I: Preparing feed stoke
In the process of synthesizing liposomes, said feed stoke is prepared by adding of lipid, and a drug molecule and is dissolved in the solvent that includes chloroform, dichloromethane, acetone, hexane ether benzene and alike. More preferably chloroform is used as a solvent; 20 wherein the lipid is the major component of liposomes and is the main ingredient used for synthesis of liposomes; solvent is utilized to dissolve the lipids and cholesterol to provide rigidity/strength to liposomes.
Preparation of feed stoke by mixing of lipids that includes but does not 25 limits to phosphatidyl choline, Egg PC and alike in the range of 0.1-5
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% w/w, Chloroform in the range of 95-99.9 % w/w, Cholesterol in the range of - 0.01-0.5 % w/w and drug molecules either hydrophilic or hydrophobic in the range of (0.1-3%) w/w. The above-mentioned values are the concentration of lipid, solvent and the drug which there by leads to various feed compositions of the feed stoke. Wherein said 5 solvent in the feed stoke may vary as water or as aqueous medium required to dissolve hydrophilic or hydrophobic drugs respectively. Said feed stoke is prepared in the mixing chamber.
Step II: Generating nanodroplets
In the next step, nanodroplets of said feed stoke is generated. For the 10 droplet generation, the lipid-solvent feed stoke is passed into the nanodroplet generator (NDG). The nano/micro droplets are generated by electro-magnetic (or mechanically produced) vibration energy produced by piezoelectric vibrations. This results in the formation of fine mist of nanodroplets each containing a feed stoke of the lipid and 15 the drug at the surface of the solvent. This step provides advantage of tunable and monomodal size of nano droplets which helps in achieving tunable and monomodal size distributed liposomes; which is achieved by adjusting the power to volume ratio; wherein the power to volume ratio is the ratio of the specific power supplied to the droplet generator 20 to the volume of solvent containing the lipid. The ratio is controlled by using a resistive control for controlling the power of droplet generator and varying it constantly with the change in the volume of solvent to keep the ratio constant. Thus, the power to volume ratio is varied by varying the amount of solvent in the feed stoke prepared according to 25 the power given to the nano droplet generator.
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Tunable size of nanodroplets is achieved by varying the power to volume ratio of the nanodroplet generator. Actual size of lipid particle inside the generated nanodroplet is smaller than the size of generated nanodroplets. The size of the lipid inside the nanodroplet and concentration of lipid in each nanodroplet depends upon the lipid-5 solvent feed stock composition. monomodal size of nanodroplets are obtained at a specific power to volume ratio set, size of nanodroplets is tuned (varied) by controlling the power to volume ratio;
Step III: Encapsulating of drug into the liposome
Said nano droplets containing of the lipid molecules of varied sizes 10 generated in the Nanodroplet Generator are further encapsulated with the drug molecule in the receiving chamber. For carrying the nano droplets containing of the lipid molecules of varied sizes generated from the nanodroplet generator to the receiving chamber, a carrier gas is injected in the nanodroplet generator. Wherein said carrier gas is a 15 reactive or non- reactive carrier gas that includes but does not limited to nitrogen, argon, helium and alike; most preferably argon into the solvent. The carrier gas is introduced in the range of 5SCCM – 200 SCCM; more preferably in the range of 50 SCCM - 70 SCCM in the Nanodroplet Generator. The gas flow rate is varied in the carrier gas 20 injector to tune (vary) the size of the nanodroplets to be carried. Depending upon the flow rate of the carrier gas adjusted, less or more number of droplets are carried by reducing or increasing the flow rates of the carrier gas respectively. This allows a variable amount of droplets to be mixed with speed depending upon the flow of the carrier 25 gas.
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Thus, specific size of nanodroplets are carried away by the flow adjusting the flow rate of the carrier gas. The size of the nanodroplets during a single step remains uniform.
The flow of carrier gas produces a negative draft that is a negative air pressure which is below the atmospheric pressure inside the 5 Nanodroplet Generator. Due to said negative air pressure lighter nanodroplets are carried away by negative draft through solvent. The adjustment of carrier gas flow rate gives size selected monomodal nanodroplets, that gets collected in the receiving chamber.
Also, the said flow rate of the carrier gas carrying the 10 nanodroplets/vapours of the solvent is varied as it allows more or less interaction with steam resulting in more/less heat to be transferred to the droplets respectively.
Further said nanodroplets containing the lipid molecules are flashed with high temperature and pressurized steam, for encapsulating the 15 drug molecules in the nanodroplets. High temperature pressurized steam is flashed on the nanodroplets in the receiving chamber; wherein the steam temperature is varying from 50 - 130ºC and steam pressures preferably ranging from 0.5 bar to 5 bar and flow rates of 10mL/minute to 200mL/minute; more preferably 1.5 bar and 20 70ml/minute.
The steam temperature and pressure is adjusted depending upon the concentration of solvent used and the stability of the drug molecule or protein to be encapsulated. The flow rates and the pressure of steam is adjusted within said given range above; wherein variation in the steam 25 flow rate and pressure gives varied size of liposomes specific to the
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batch cycle. The present process regulates the inflow of steam rate and its temperature and pressure thereby providing uniform sized liposomes in each batch in a continuous process. Depending upon the pressure of the steam the flow of the steam is adjusted. This, facilitates multiple ways to mix more or less (varying concentration) 5 droplets with different amount of steam allowing more or less heat to be transferred to the droplets accordingly.
As the steam flashes the solvent, the hydrophobic drug is encapsulated within the hydrophobic regions of the lipid while the hydrophilic drug is encapsulated within the hydrophilic region of the 10 liposome. Other molecules such as cholesterol tighten the liposome boundaries to prevent leakage. Thus, drug encapsulation is done in this step. Thus, it is a single step drug encapsulating process. At the end of this step drug encapsulated liposomes are obtained. The steam is ejected from the receiving chamber to the separation column and 15 the steam condenses in the separation column.
The steam ejected as abovementioned, acts as a solvent vaporiser due to the high temperature of the steam and it also creates a negative draft from the receiving chamber to the separation column by the flow of high temperature and pressurised steam. The solvent vapours 20 formed above is mixed with the steam and passes through a separation column due to the negative draft created; wherein the steam condenses in the separation column. Said liposomes in the vaporised solvent are transferred to the aqueous phase as the steam condenses in the separation column. There remains some 25 uncondensed steam along with non-condensable gases in the solvent in the separation column.
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In this step the drug molecule is efficiently encapsulated in said nanodroplets and liposomes are thus formed. Cholesterol tightens the boundaries of the liposome formed and ensures prevention of leakage in the liposomes. The flow rates and the pressure of steam is adjusted within said given range above; wherein variation in the steam flow rate 5 and pressure gives varied size of liposomes specific to the batch cycle. The present process regulates the inflow of steam flow rate and its temperature and pressure thereby providing uniform sized liposomes in each batch in a continuous process. Thus, this step provides stable, size selected, monomodal liposomes. 10
Step IV: Separating the liposome and the solvent
Said mixture of remaining uncondensed steam, solvent and non-condensable gas is passed through a condenser. The condenser has a below dew point temperature; wherein the below dew point temperature is the temperature at which the air cannot hold all the 15 vapour mixed with it. Here the below due point temperature is set between 45-75º C depending upon the flow rates of the steam and gas. The below due point temperature across the condenser allows complete recovery of solvent from the system. This condensed solvent and the liposomes are separated through a layer separator. Highly 20 dense solvent is collected at the bottom while the aqueous layer sits on top of the solvent. The recovered solvent is reused in the same process again. The gas generated during the process is collected in the gas scrubber.
Selected size liposomes may be obtained by attaching an extruder in 25 line with the layer separator. Extruding the liposomes through an
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extruder gives liposomes of selected size. Attaching an extruder is optional in the process.
The above described steps substantially reduces the processing time of synthesis of liposomes that is it takes only several minutes to synthesize the liposomes by adjusting the concentration of the solvent 5 , and reducing the use of water in the feed stock prepared as mentioned herein above; reducing the process time and in turn increasing the throughput rate of the process. . The reduction in time of preparation of liposomes is obtained as there are no independent steps requiring time such as solvent evaporation is not required as 10 high temperature and pressurised steam vapourises the solvent. Sonication and injecting aqueous medium is also eliminated which saves time. Moreover, High flow rates of carrier gas and steam, and continuous nature of process reduces the time required for synthesis of liposomes. The present process regulates the proportion of water in 15 the feedstock prepared thereby reducing the processing time in each batch in a continuous process.
Complete recovery of solvent is obtained in this step, which can be used in the same process again. Thus, it does not generate any effluent and reduces the consumption of solvent. Thus, saves cost of 20 raw materials.
In another embodiment of present invention, the feed stock for the preparation of liposomes is prepared using Step 1 as described herein above. Further process for the synthesis of liposomes is described 25 below:
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Step II: Generating nanodroplets
Generating of the Nano droplets from said feedstock in present embodiment comprises of;
i) Using an ultrasonic horn
ii) a pressurized jet of inert air 5
iii) passing the feed stoke through a bubbling stream of inert gas
Wherein;
• For generating nanodroplets through an ultrasonic horn, a continuous flow of the organic solvent directly enters a glass tube connected to an ultrasonic horn. The ultrasonic vibration 10 produces by the ultrasonic horn atomizes the solvent.; that is, it is broken into a fine mist of uniform nano sized droplets. Vibration of the solvent forms multiple nanodroplets by passing ultrasonic energy to the solvent which is continuously ejected out of the glass tube. 15
• For generating nano droplets by pressurised jet of inert air, the nanodroplets are formed as the pressurised inert gas and the feed stoke is prayed out from the nozzle into the receiving chamber.
• For generating of nanodroplets by a gas bubbler, said feed stoke 20 is carried with the bubbling stream of inert gas in the solvent. there is a negative draft created by the gas molecules bubbling through the solvent from the mixing chamber to the receiving chamber. The bubbling stream in the feed stoke creates nano droplets; wherein each droplet contains a lipid part inside the 25
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solvent particle. These nano droplets are then used for encapsulating the drug inside the nanodroplets formed.
Step III: Encapsulating of drug into the liposome
Further said nanodroplets containing the lipid molecules are flashed with high temperature and pressurized steam, which encapsulates the 5 drug molecules in the liposomes High temperature pressurized steam is flashed on the nanodroplets in the receiving chamber; wherein the steam temperature is varying from 50 - 130ºC and steam pressures preferably ranging from 0.5 bar to 5 bar and flow rates of 10ml/minute to 200ml/minute; more preferably 1.5 bar and 10 70ml/minute. The steam temperature and pressure are set depending upon the concentration of solvent used and the stability of the drug molecule or protein to be encapsulated.
The steam temperature and pressure is adjusted depending upon the concentration of solvent used and the stability of the drug molecule or 15 protein to be encapsulated. The flow rates and the pressure of steam is adjusted within said given range above; wherein variation in the steam flow rate and pressure gives varied size of liposomes specific to the batch cycle. The present process regulates the inflow of steam flow rate and its temperature and pressure thereby providing uniform sized 20 liposomes in each batch in a continuous process.
As the steam flashes over the solvent, the hydrophobic drug is encapsulated within the hydrophobic regions of the lipid while the hydrophilic drug is encapsulated within the hydrophilic region of the 25 liposome. Other molecules such as cholesterol tighten the liposome
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boundaries to prevent leakage. Thus, drug encapsulation is done in this step. Thus, it is a single step drug encapsulating process. At the end of this step drug encapsulated liposomes are obtained. The steam is ejected from the receiving chamber to the separation column and the steam condenses in the condenser. 5
In this step the drug molecule is efficiently encapsulated in said nanodroplets and liposomes are thus formed. Cholesterol tightens the boundaries of the liposome formed and ensures prevention of leakage in the liposomes. The flow rates and the pressure of steam is adjusted within said given range above; wherein variation in the steam flow rate 10 and pressure gives varied size of liposomes specific to the batch cycle. The present process regulates the inflow of steam flow rate and its temperature and pressure thereby providing uniform sized liposomes in each batch in a continuous process.
Step IV: Separating of liposome and solvent 15
The steam ejected as abovementioned, acts as a solvent vaporiser. A negative draft exists from the receiving chamber to the condenser by the flow of high temperature and pressurised steam. The high temperature of the steam flashes the solvent which is having low boiling point and vaporises the solvent in the receiving chamber. The 20 solvent vapours formed above is mixed with the steam and passes through condenser due to the negative draft created; wherein the stem condenses in the separation column. Said liposomes in the vaporised solvent are transferred to the aqueous phase as the steam condenses in the condenser. The condensed liposomes are collected in the 25 receiving chamber along with the solvent. Said liposomes then pass through a layer separator to sperate the solvent from the condensed
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liposomes. The solvent is separated and completely recovered, which is reused in the process again. The separated condensed liposomes are then collected in a receiving chamber. These separated liposomes are then size separated through an extruder.
Complete recovery of solvent is obtained in this step, which can be 5 used in the same process again. Thus, it does not generate any effluent and reduces the consumption of solvent. Thus, saves cost of raw materials.
Complete system is in continuous mode of operation in which the temperature, flow rate, pressure, power is set as per the range 10 provided. The synthesized liposomes are of uniform size ranging from 40 to 400 nm as per the change in parameter such as the gas flow rate, steam flow rate and its temperature and pressure.
The above described steps substantially reduces the processing time of synthesis of liposomes that is it takes only several to synthesize the 15 liposomes by adjusting the concentration of the solvent , and reducing the use of water in the feed stock prepared as mentioned herein above; reducing the process time and in turn increasing the throughput rate of the process. The reduction in time of preparation of liposomes is obtained as there are no independent steps requiring time such as 20 solvent evaporation is not required as high temperature and pressurised steam vaporises the solvent. Sonication and injecting aqueous medium is also eliminated which saves time. High flow rates of steam and continuous nature of process reduces the time required for synthesis of liposomes. The present process regulates the 25 proportion of water in the feedstock prepared thereby reducing the processing time in each batch in a continuous process.
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Having described what is considered the best from presently contemplated for embodying the present invention, various alterations, modifications, and/or alternative applications of the invention for any system will be promptly apparent to those skilled in the art. Therefore, it is to be understood that the present invention is not limited to the 5 practical aspects of the actual preferred embodiments hereby described and that any such modifications and variations must be considered as being within the scope and spirit of this invention, as described in the description of invention above.
The present invention (A process for synthesis of liposomes) and the 10 manner in which it is performed is described in detail below with working examples and is by the way of illustrations only. Therefore, these examples should not be constructed to limit the scope of the present invention as illustrated below.
EXAMPLE 1: 15
Said process for synthesis of liposomes is carried out by preparing a feedstock by taking a known amount of the lipid (egg PC) dissolved in a solvent (Chloroform) at a concentration of 10mg/ml. The above prepared solution is then placed in a piezoelectric droplet generator to generate micro/nanodroplets of solvent with lipids. A carrier gas argon 20 is injected inside the Nanodroplet Generator at a flow rate of 25SCCM which carries away the said nano droplets from the said droplet generator to the mixing chamber. The said nanodroplets are flashed with high temperature steam of 95º C temperature, 3 bar pressure and 70ml/minute flow rate. The said steam is condensed over a condenser. 25 The steam condenses transforming the solvent into multi-layered liposomes, which are collected with the condensed steam. The said
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liposomes are characterized by transmission electron microscopy and dynamic light scattering techniques.
EXAMPLE 2:
Said process for synthesis of liposomes is carried out by preparing a feedstock by using a known amount of the lipid (egg PC) dissolved in a 5 solvent (Chloroform) at a concentration of 10mg/ml and cholesterol is added to strengthen the lipid bilayers in the amount 1mg/ml. The above prepared solution is then placed in a piezoelectric droplet generator to generate micro/nanodroplets of solvent with lipid plus cholesterol. A carrier gas argon is injected inside the Nanodroplet 10 Generator at a flow rate of 25SCCM which carries away the said nano droplets from the said droplet generator to the mixing chamber. The said nanodroplets carrying lipid plus cholesterol are flashed with high temperature steam of 95º C temperature, 1 bar pressure and 70ml/minute flow rate. The said steam is condensed over a condenser. 15 The steam condenses transforming the solvent into multi-layered liposomes, which are collected with the condensed steam. The said liposomes are characterized by transmission electron microscopy and dynamic light scattering techniques. Flow rates of gas and steam are changed to obtain different sizes as given in table 1 . 20
Referring to Fig. 1, shows the Transmission Electron Microscope (TEM) analysis of the synthesized liposomes through the process of the present invention. The Transmission Electron Microscope (TEM) analysis shows the liposomes synthesized by the present process of synthesizing liposomes wherein the uniform sized liposomes range 25 from 40 – 400 nm.
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EXAMPLE 3:
Said process for synthesis of liposomes is carried out to encapsulate a hydrophobic drug such as quinine in a feed stock using a known amount of the lipid (egg PC) dissolved in a solvent (Chloroform) at a concentration of 10mg/ml and cholesterol is added to strengthen the 5 lipid bilayers in the amount 1mg/ml. The drug is pre-dissolved in the chloroform at concentration of 1mM. The above prepared solution is then placed in a piezoelectric droplet generator to generate micro/nanodroplets of solvent with lipid plus cholesterol plus the drug. A carrier gas argon is injected inside the Nanodroplet Generator 10 at a flow rate of 25SCCM which carries away the said nano droplets from the said droplet generator to the mixing chamber. The said nanodroplets carrying lipid plus cholesterol plus the said drug are flashed with high temperature steam of 95º C temperature, 1 bar pressure and 70ml/minute flow rate. The said steam is condensed 15 over a condenser. The steam condenses transforming the solvent into multi-layered liposomes, which are collected with the condensed steam. The said liposomes are characterized by transmission electron microscopy and dynamic light scattering techniques. Flow rates of gas and steam are changed to obtain different sizes as given in table 2. 20
Referring to Fig. 2, shows the Transmission Electron Microscope (TEM) analysis of the synthesized drug loaded liposomes through the process of the present invention. The Transmission Electron Microscope (TEM) analysis shows the liposomes synthesized by the present process of synthesizing liposomes wherein the uniform sized 25 drug loaded liposomes ranges from 40 to 400nm.
EXAMPLE 4:
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Said process for synthesis of liposomes is carried out to encapsulate a water-soluble drug, a feed stock of organic solvent and water is created that is aerosolized as droplets and dissolves the drug in the azeotropic mixture. The droplet formation and the flow rate are optimized for the process as shown in table 3 to reduce the overall time required for 5 evaporation of solvent. A known amount of the lipid (egg PC) dissolved in a solvent (Chloroform + water 90:10) at a concentration of 10mg/ml and cholesterol is added to strengthen the lipid bilayers in the amount 1mg/ml. To this mixture methotrexate, a water-soluble drug, is added at a concentration of 1 mM. The above prepared solution is then 10 placed in a piezoelectric droplet generator to generate micro/nanodroplets of solvent with lipid plus cholesterol plus the drug. A carrier gas argon is injected inside the Nanodroplet Generator at a flow rate of 25SCCM which carries away the said nano droplets from the said droplet generator to the mixing chamber. The said 15 nanodroplets carrying lipid plus cholesterol plus the said drug are flashed with high temperature steam of 95º C temperature, 1 bar pressure and 70ml/minute flow rate. The said steam is condensed over a condenser. The steam condenses transforming the solvent into multi-layered liposomes, which are collected with the condensed 20 steam. The said liposomes are characterized by transmission electron microscopy and dynamic light scattering techniques.
From all the experiments performed in examples 1-4, varying the different parameters such as the concentration of lipid in the solvent; the amount of drug and cholesterol; the energy supplied to 25 Nanodroplet Generator to generate solvent nanodroplets; carrier gas flow rate; steam temperature, pressure and flowrate; we observed that the process of synthesis of liposomes is a continuous and flexible
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process; wherein we obtained a variety of sized of liposomes by combination and varying multiple said parameters. There is no effluent generated, the solvent which is recovered in the end of the process can be reused again in the same process. Also, we observe that steam is used for phase transfer thus it provides a sterilized 5 environment making secondary sterilization of lipids redundant.
Results of steam flow rate and carrier gas flow rate affecting the size of liposomes
The steam flow rate and the carrier gas flow rates in the above 10 embodiments are combined together to generate liposomes which are monomodal in size, the steam pressure and flow rate and carrier gas pressure and flow rate can be varied, this variation varies the size of rhe synthesized liposomes. This is illustrated in the table below:
Feed Stoke Formulation
Steam pressure and flow rate
Carrier Gas pressure and flow rate
Size (Determined by dynamic light scattering techniques (DLS))
10mg/mL lipid in Chloroform
0.75 bar (50 mL/min)
3 psi (20 lit/min)
40-50 nm and 350 -380 nm
10mg/mL lipid in Chloroform
1.5 bar (75mL/min)
3 psi (10 lit/min)
110-125 nm
10mg/mL lipid in Chloroform
1.5 bar (50 mL/min)
10 psi (40 lit/min)
100-120nm
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10mg/mL lipid in Chloroform + 1mg/mL cholesterol
1.5 bar
70-80 mL/min
35 lit/min
80-100nm
Table 1
Table 1 shows the lipids, solvent cholesterol and drug amount is chosen as per the requirement, the carrier gas pressure and flow rate and the steam pressure and flow rate is also adjusted. This adjustments and variation in the amount of lipid, cholesterol and drug 5 into the solvent gives a range of liposomes which are monomodal in size, this variation and adjustment varies the size of the monomodal liposomes as it can be seen in size column of the table 1.
Experiment No.
Organic Solvent (Chloroform) Volume (µl)
Water volume (µl)
Carrier gas flow rate (lit/min)
Steam pressure (bar)
Time (min)
1
10000
-
25
No steam
2:45
2
9900
100
25
No steam
3:42
3
9900
100
25
3 bar, 70 ml/minute
3:15
4
9800
200
25
3 bar, 70 ml/minute
3:50
30
5
9700
300
25
3 bar, 70 ml/minute
3:47
6
9600
400
25
3 bar, 70 ml/minute
5:30
7
9500
500
25
3 bar, 70 ml/minute
6:30
8
9000
1000
25
3 bar, 70 ml/minute
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Table 2
Table 2 shows, the flow rate of carrier gas and the flow rate of the steam is kept constant; wherein the concentration of the feedstock is changed. High concentration of feed stoke with reduced amount of water, takes less process time to synthesize the liposomes This varies 5 the time to synthesize the liposomes. Further as shown the ratio of organic solvent volume to water volume affects the time of synthesis of a single run of 10mL of feed stoke in nanodroplet generator. As shown in the table the minimum and maximum volumes are maintained at the initial feed stoke input which further as described in the process 10 processes the feedstock input from the mixing chamber to the nanodroplet generator and continuously runs the process.
ADVANTAGES OF THE INVENTION
The present invention has many advantages over the prior art: 15
• The said invention provides a continuous process for synthesis of liposomes.
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• The present invention eliminates the batch wise processing, thus provides an energy efficient process for synthesis of liposomes.
• Provides a flexible process to vary various said parameters to obtain a tunable (varied) of sizes of liposomes.
• The process provides monomodal size distributed liposomes. 5
• The process provides higher yield of liposomes in less time. Hence time saving process with an increased throughput rate.
• The process provides single step efficient drug encapsulation.
• The solvent is flashed by the steam and thus there is no need to evaporate the solvent as in other processes. 10
• The solvent separated at the end of the process is reused in the process again in step I, minimizing the loss of solvent.
• Process eliminates the generation of effluent.
• the invention thus provides an economical process for synthesis of liposomes.