KR101402794B1 - The method of generating liposomes and vesosomes - Google Patents

Abstract

The present invention provides a method of making a single or multiple liposome or vesosome comprising the steps of: (a) preparing a water soluble and hydrophobic endoplasmic reticulum; (b) injecting the vesicles into microchannels to produce a reverse micelle precursor; (c) preparing a single or multiple lipid bilayer spherules by applying centrifugal force to the reverse micelle precursor. The preparation method of the present invention can be easily manufactured by preparing a reverse micelle precursor at a T-junction in a microchannel and automating single or multiple lipid bilayers using a rotating device. In addition, the manufacturing apparatus of the present invention comprises (i) a syringe pump; (Ii) a microfluidic device; And (iiic) a spinning device, which utilizes centrifugal force by a rotating device. Accordingly, the present invention provides a method for efficiently and easily producing a single or multiple lipid bilayer spherules at low cost, which can be used for various applications (e.g., therapeutic agents, diagnostic agents, etc.) through microencapsulation of drugs ). ≪ / RTI >

Description

The method of generating liposomes and vesosomes is a method for producing a lipid bilayer sphere and a lipid bilayer sphere,

The present invention relates to a process for the preparation of single or multiple lipid bilayers, the use of single or multiple lipid bilayers made therefrom, and an apparatus for the production of single or multiple lipid bilayers.

Liposome or vesosome is an empty sphere and it can be easily transferred into the cell by putting the materials desired by the experimenter or the user in the inside of the sphere, and the interest as a drug delivery system is increasing. Currently, in the industrial field, liposomes are widely used in the cosmetics industry for delivery of functional substances to skin cells and for encapsulation and transportation of various nutrients in the food industry. In the case of a microfluidic device, a test culture and culturing tendency such as experimental culture before a small-sized cell culture or a large-scale culture or a cell which can not be cultured in a large volume can be obtained by synthesizing nano- Is a state-of-the-art research technology. With this two technologies of the latest trends, the inventors of the present invention have developed a lipid bilayer that is a liposome within a single lipid bilayer. In contrast to conventional liposomes that capture only one lipid and move it to a desired position Liposomes containing different materials can be placed in one liposome to move and react with various substances at the same time. However, the production of the vesomosomes has been reported in a paper published in 2007 (Boyer, C., and Zasadzinski, JA, Multiple Lipid Compartments Slow Vesicle Contents Release in Lipases and Serum, ACS Nano. , 1 (3): 176-82 (2007)), and the method described in the above paper is very difficult. Only a skilled person who has made it for many years can make it and it takes about 2 days or more to make it. In the case of liposomes, which are relatively easy to make than beesomes, conventional techniques are difficult to make. Conventional liposome production technology is manufactured using expensive equipments such as a sonicator and a pore membrane, and this series of processes is a manufacturing method requiring a very skilled technique which is difficult for a beginner. In addition, the conventional method in which the size of the liposome is determined depending on the intensity of the sound wave crusher or the pore size of the microporous membrane is very inefficient. It is also a very difficult and difficult task to make the liposomes by wrapping them with other liposomes.

Accordingly, there is a need in the art for a process for producing a vesicosome that can be carried out more easily and easily.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a novel method for easily producing liposomes or vesomosomes. As a result, we have found that by crossing an aqueous phase and an organic solution phase in a microfluidic device to form reverse micelle precursors, The present invention has been accomplished based on the discovery that liposomes or vesicosomes can be easily prepared using the centrifugal force of a device.

Accordingly, an object of the present invention is to provide a method for producing a single or multiple liposome or vesosome.

Another object of the present invention is to provide a single or multiple lipid bilayers.

It is another object of the present invention to provide a composition for a cargo delivery vehicle.

It is another object of the present invention to provide an apparatus for producing single or multiple lipid bilayers.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method of producing a single or multiple liposome or vesosome comprising the steps of:

(a) preparing an aqueous endoplasmic reticulum and a hydrophobic endoplasmic reticulum;

(b) injecting the vesicles into microchannels to produce a reverse micelle precursor;

(c) preparing a single or multiple lipid bilayer spherules by applying centrifugal force to the reverse micelle precursor.

According to another aspect of the present invention, the present invention provides a single or multiple liposome or vesosome produced by the method described above.

According to another aspect of the present invention, there is provided a composition for a cargo delivery vehicle comprising the single or multiple lipid bilayer sphere described above as an active ingredient.

According to another aspect of the present invention, the present invention provides a method of making a syringe pump comprising: (a) a syringe pump; (b) a microfluidic device; And (c) a spinning device. ≪ IMAGE >

The present inventors have sought to develop a novel method for easily producing liposomes or vesomosomes. As a result, we have found that by crossing an aqueous phase and an organic solution phase in a microfluidic device to form reverse micelle precursors, device using the centrifugal force of liposome or vesomose.

The present invention provides a method for effectively and easily preparing a single or multiple lipid bilayer spheres which can be applied to various applications (e.g., therapeutic agents, diagnostic agents, etc.) through microencapsulation of the drug have.

Microencapsulation of drugs in biodegradable polymers or liposomes has been used to improve the pharmacokinetics of many drugs such as antibiotics and chemotherapeutic agents [J. A. Zasadzinski, Current Opinion in Solid State and Materials Science 2, 345 (1997); D. D. Lasic, D. Papahadjopoulos, Current Opinion in Solid State and Materials Science 1, 392 (1996); DD Lasic, Liposomes: From Physics to Applications (Elsevier, Amsterdam (1993); TM Allen, Current Opinion in Colloid and Interface Science 1, 645 Drug delivery vehicles are targeted either actively or passively to release drugs that are contained in a specific target location in the body. This targeted drug release is a measure of the effectiveness of the encapsulated drug For example, it is now known that unilamellar vesicles can inhibit the release of a large amount of unilamellar vesicles into the body to provide sustained release to a specific location in the body or targeted release. The drug that is released is used as a drug delivery carrier for the endoplasmic reticulum It is composed of solubility and the release is composed of a slow penetration through the membrane of the endoplasmic reticulum. Various modifications of the monomolecular endoplasmic reticulum membrane, such as increased stability, reduced permeability, insertion of the polymer into the bilayer membrane, For example, the modifications include modifications that polymerize or cross-link molecules in the bilayer.

Liposomes are always a closed, spherical, multilamellar endoplasmic reticulum capable of encapsulating a variety of drugs. Liposomes are the most studied endoplasmic reticulum to date and can be prepared to have many lipids and compositions that can modify stability, pharmacokinetics and biodistribution (T. M. Allen et al., Adv. Drug Deliv. Rev., 16, 267-284 (1995)). The lipid bilayer functions to encapsulate the drug and regulate its release rate. Typically, the liposomes comprise a polymer that is inserted into an endoplasmic reticulum membrane to protect the liposomes from macrophages that are to remove foreign material from the body. These polymers greatly increase the circulation time of the liposomes. In addition, liposomes can insert surface-specific binding agents to target the endoplasmic reticulum to specific target organs or cells.

Disadvantages of multi-membrane and monolayer liposomes as delivery systems are size. That is, the size of liposomes inhibits crossing with most normal membrane barriers and limits administration by an intravenous route. In addition, tissue selectivity of liposomes is limited to reticuloendothelial cells that recognize liposomes as foreign microparticles and concentrate liposomes in tissues such as liver and spleen. A further disadvantage of the liposomal system is that it relies on a single lipid membrane to control drug encapsulation, drug permeability and liposomal biocompatibility. It is known that it is very difficult to find a lipid membrane capable of effectively performing the above-mentioned functions.

Typically, beresomes are very small in size and can be manipulated by manipulating the lipid composition of the outer bilayer membrane or by mechanical processing (e. G., Evacuation of the vesicle through a filter of limited size) It is adjusted from micron to 5 micron. Basosomes can contain many water-soluble or lipid-soluble drugs or other solutes in the inner or outer capsule (i.e., the space between the endoplasmic reticulum and the outer layer bilayer membrane) or both. Thereafter, the above-mentioned drug or other water-soluble substance can progressively penetrate through the inner endocardial membrane and the outer layer double membrane.

The production of the above-mentioned beesome can be generally manufactured by a multi-step process. As a first step, interior containment units are made to load specific drugs or other agents to be carried. The second step is to prepare size-adjusted expanded aggregates without destruction of the endoplasmic reticulum membrane or contents when using aggregated endoplasmic reticulum. The third step is encapsulating the free or aggregated endoplasmic reticulum in the outer membrane. The above-described process for producing vesicosomes is not only complicated but also very difficult and inefficient.

According to the method of the present invention, the single or multiple liposome or vesosome of the present invention can be easily prepared as follows.

(a) preparing an aqueous endoplasmic reticulum and a hydrophobic endoplasmic reticulum.

Methods for preparing water-soluble and hydrophobic endoplates are well known in the art including, for example, detergent-dialysis, sonication, spontaneous vesicle preparations, reverse phase evaporation, etc., and all possible methods can be used for the production of an endoplasmic reticulum. That is, the various forms of the vesicle preparation are as follows: (a) a chemical preparation method of an endoplasmic reticulum such as reverse phase evaporation, surfactant-dialysis, pH jumping; (b) mechanical processing methods such as ultrasonication; And (c) a spontaneous vesicle preparation method that directly equilibrates the endoplasmic reticulum without special treatment [Jung, H. T. et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 1353]. The non-encapsulated biological agent can be removed by various dialysis techniques, ion exchange, chromatography, filtration or centrifugation at the stage of the vesicle production process described above. Also, the steps of preparing the inner endoplasmic reticulum and loading the drug are well known in the art (TM Allen et al., Advanced Drug Delivery Reviews, 16, 267-284 (1995); TM Allen, Current Opinion in Colloid and DD Lasic, Liposomes: From Physics to Applications, (Elsevier, Amsterdam (1993)); DD Lasic et al., Current Opinion in Solid State and Materials Science, 1, 392- 400 (1996)).

According to a preferred embodiment of the present invention, an organic solution suitable for the present invention is one selected from the group consisting of hexane, decane, n-decane, dodecane, squalene, cholesterol, tetradecane At least one organic solvent selected from the group consisting of hexadecane, nonane, cyclohexane, polyvinyl alcohol, lauric acid, acetic acid, formic acid, benzene, naphthalene, nitrobenzene, phenol and ethylene bromide, More preferably, the organic solution according to the invention comprises at least one organic solvent selected from the group consisting of Hexane, decane, n-decane, squalene and cholesterol do. Further, in order to produce a more rigid lipid bilayer, the mixing ratio of the organic solvent can be adjusted.

For example, when a mixture of decane and hexadecane is used in the preparation of a lipid organic solution, it is preferred to use a mixture having a weight ratio of decane: hexadecane of 1: 9-2: 8, and in the case of squalene and hexadecane It is preferable to use a mixture having a weight ratio of 1: 9-2: 8.

According to a preferred embodiment of the present invention, the lipids used in the preparation of lipid bilayer precursors as lipid bilayer precursors may be any lipids used in the manufacture of lipid bilayers, block copolymers including hydrophilic and hydrophobic (AB diblock copolymer, ABA triblock copolymer). For example, suitable lipids for the present invention are phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, cholesterol and sphingomyelin. More specifically, lipids suitable for the present invention include Asolectin, Diphytanoylphosphatidylcholine (DPhPC), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DHPC glycero-3-phosphocholine, DHPE (1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine), DMPC (1,2-dimyristoyl-sn- glycero-3-phosphocholine), DIODPC -DiODodecyl-sn-Glycero-3-Phosphocholine), DMPS (dimyristoyl phophatidylserine), DLPC (dimyristoyl phosphatidylglycerol, dilauryl phosphatidycholine), DMPE (1,2-dimyristoyl- glycero-3-phospho-rac- (1-glycerol)], Lyso PC (1-myristoyl-2-hydroxy-sn- glycero-3-phosphocholine), Lyso PE (1-oleoyl- glycero-3-phosphoethanolamine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dierucoyl- , DEPC (1,2-erucoyl-sn-glycero-3-phosphocholine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLOPC glycero-3-phosphocholine, DLPA-NA (1,2-dilauroyl-sn-glycero-3-phosphate sodium salt), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine ), DLPS-NA (1,2-dilauroyl-sn-glycero-3-phosphoserine sodium salt), DMPA-NA (1,2-dimyristoyl-sn- glycero- NA (1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt)), DOPA-NA (1,2-dioleoyl- glycero-3-phosphocholine, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPS-NA (1,2- dioleoyl-sn- glycero-3-phosphoserine , DPPA-NA (1,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt), DPPE (1,2-dipalmitoyl-sn-glycero 3-phosphoethanolamine), DPPS- -sn-glycero-3-phosphoserine (sodium salt), DSPA-NA (1,2-distearoyl-sn-glycero-3-phosphate phosphocholine, DSPE (1,2-diostearyl-sn-glycero-3-phosphoethanolamine), MSPC (1-myristoyl, 2-stearoyl-sn- glycero 3-phosphocholine), PMPC 2-oleoyl-sn-glycero-3-phosphoethanolamine, POPC (1-palmitoyl, 2-oleoyl-sn-glycero 3-phosphocholine), POPE (1-palmitoyl- 2-stearoyl-sn-glycero 3-phosphocholine, SMPC (1-stearoyl, 2-myristoyl-sn-glycero 3-phosphocholine), SOPC (1-stearoyl, 2-palmitoyl- glycero 3-phosphocholine, SPPC (1-stearoyl, 2-palmitoyl-sn-glycero 3-phosphocholine), PCDA (10,12-pentacosadiynoic acid), and PMOXA-PDMS- PMOXA (poly (methyloxazoline) -poly (dimethylsiloxane) but are not limited to, poly (methyloxazoline). At this time, the concentration of lipid in the lipid organic solution is not particularly limited, but is preferably 0.1-19 wt%, more preferably 1-3 wt%.

According to a preferred embodiment of the present invention, the hydrophobic endoplasmic reticulum of the present invention comprises at least one kind of hydrophobic endoplasmic reticulum and includes a target cargo of interest.

According to a preferred embodiment of the present invention, the objective carbohydrate that may be used in the present invention includes biological agents.

The term " biological agent " as used herein refers to a drug (e.g., a chemical substance, a nanoparticle, a peptide, a polypeptide, a nucleic acid, a carbohydrate, a quantum dots), a solute, a therapeutic agent, Contrast agent. The term " interior containment units " as used herein refers to structures having an interior space that may include agents such as therapeutic agents, diagnostic agents, cosmetics, or contrast agents. Typically, the structure is spherical, but is not limited thereto. For example, the containing unit is a lipid vesicle.

Suitable therapeutic agents that may be employed in the present invention include, but are not limited to, antibiotics, antifungal agents, chemotherapeutic agents, neovascular agents, anti-tuberculosis agents and antibodies. Wherein the antibiotic is selected from the group consisting of amikacin, kanamycin B, amphomycin, bacitracin, bicyclomycin, capreomycin, polyamicin E, cycloserine, chloramphenicol, dactinomycin, erythromycin, gentamicin, , Rifamycin, streptomycin, and tetramycin.

In addition, suitable therapeutic agents that can be used in the present invention may be drugs that function at neural junction sites, such as neurohumoral transmitters, cholinergic agonists, anticholinesterase agents, , Antimuscarinic drugs, catecholamines, sympathomimetic drugs, and adrenergic receptor antagonists.

The therapeutic agent may also be a drug that reduces inflammation, for example, a histamine antagonist, bradykinin, 5-hydroxytryptamine, lipid-derived autacoids, But are not limited to, methylxanthine, cromolyn sodium, analgesics, and antipyretics.

The therapeutic agent may be a drug that affects renal function and electrolyte metabolism, including, but not limited to, diuretic and organic compound tube transport inhibitors.

Furthermore, the therapeutic agent may be a drug that affects cardiovascular function. But are not limited to, for example, renin, angiotensin, organic nitrates, calcium channel blockers, beta-adrenergic antagonists, antihypertensives, digitalis and antiarrhythmics.

Suitable diagnostic agents that may be used in the present invention include, but are not limited to, radioactive labels, enzymes, chromophores, and fluorescers.

Suitable contrast agents for use in the present invention include, but are not limited to, radiation drugs. For example, the radiopharmaceuticals may include nuclides such as ²⁰¹ Tl, ^99m Tc, ¹³³ Xe, and the like, nucleocyte chelates, ¹¹ C-deoxy-D-glucose, ¹⁸ F-2-fluorodeoxy- Radiation-labeled metabolic agents such as D-glucose, [1- ¹¹ C] - and [ ¹²³ I] -beta-methyl fatty acid analogs, ¹³ N-ammonia and analogs thereof, ^99m Tc-tetracycline, ^99m Tc But are not limited to, infarct avid agents such as pyrophosphate, ²⁰³ Hg mercurial, ⁶⁷ Ga-citrate and the like, radiolabeled ligands, proteins, peptides and monoclonal antibodies no.

As described above, the cargo that may be used in the present invention may be labeled, and the label that may be used herein may include green fluorescence protein, radioactive isotopes (e.g., C ¹⁴ , I ¹²⁵ , P ³² , S ³⁵ ), chemicals (e.g., biotin), fluorescent substances (e.g., fluorescein isothiocyanate, rhodamine 6G, rhodamine B, TAMRA 6-carboxy-tetramethyl (4,6-diamidino-2-phenylindole) and Coumarin], luminescent materials, chemiluminescent materials and fluorescence resonance energy transfer (FRET) , But are not limited thereto.

(b) injecting the vesicles into a microchannel to produce a reverse micelle precursor.

In the present invention, when a liposome as a monomolecular vesicle is prepared, it is prepared by injecting it into an organic solution phase and an aqueous phase using a single syringe pump. That is, the liposome is emulsified as it is administered dropwise into the organic solution passage, and then is packaged while passing through the aqueous solution passage to produce a liposome (see FIG. 4).

Also, in the production of a multi-membrane vesicle (vesomes), the water-soluble and hydrophobic vesicles prepared as described above are injected into microchannels using different syringe pumps to produce a reverse micelle precursor at the junctions thereof 1 and 2).

According to a preferred embodiment of the present invention, the injection is carried out using two or more syringe pumps. The injection into the microchannel can regulate the flow rate of the flow rate through the control of the syringe pump, thereby producing a reverse micelle precursor of various sizes.

According to a preferred embodiment of the present invention, the flow rate of the microchannels is regulated through the adjustment of the strength of the syringe pump.

According to a preferred embodiment of the present invention, the syringe pump comprises an endoplasmic reticulum independent of each other.

According to a preferred embodiment of the invention, the microchannels have at least one point at which the tubes from the syringe pump intersect, and the point of intersection has various shapes (e.g., T-point or modified form thereof) .

According to a preferred embodiment of the present invention, the reverse micelle precursor is formed at an intersection of an aqueous phase and an organic solution phase in a microchannel, the size of which is proportional to the ratio of the flow rates of the aqueous solution and the organic solution .

Microfluidics have the following advantages through the development of miniaturized devices that move, mix, regulate and react fluid volumes in the micron range: (a) small amounts of reagents; (b) a more rapid and sensitive response due to the increased effectiveness of processes such as diffusion and mass transport; (c) simultaneous execution of multiple processes; And (d) cost savings. Most importantly, the fabrication of microfluidic devices is economical and allows integration of various modules to automate the analytical process (Duffy et al., Analytical Chemistry 70: 4974-4984 (1998); Jingdong et al., Analytical Chemistry 72: 1930-1933 (2000); and Martynova et al., Analytical Chemistry 69 (23): 4783-4789 (1997)). Microfluidic flow devices include various module units as an integral component of a micro-scale total analytical system (Manz et al., &Quot; Miniaturized total chemical analysis systems. &Quot; Transducers '89: Proceedings of the 5th International Conference on Solid-State Sensors and Actuators and Eurosensors III. Part 1, Montreux, Switzerland (Jun. 25-30, 1989); van den Berg et al., Proceedings of the International Symposium on Micromechanics and Human Science, pages 181 -184 (1994); and Dhawan et al., Analytical and Bioanlytical Chemistry, 373: 421-426 (2002)). Several strategies for mixing in microfluidic flow devices / systems can be implemented. Turbulence for large-scale mixing is not effective in most microfluidic flow devices / systems. Accordingly, other strategies for mixing have been developed, among which passive mixers are representative. The manual mixer uses only the geometry of the channel. For example, a manual mixer includes: (a) a manual mixer that generates a transverse flow using a robust arrangement of the herring-bone structures to increase the interfacial area between the mixed fluids Stroock et al., Science 295: 647-651 (2002)); (b) a manual mixer using a serpentine channel to stimulate a partially packed bed of chromatography columns (He et al., "A Picoliter Volume Mixer for Microfluidic Analytical Systems," Analytical Chemistry 73: 1942 (2001) ); And (c) a manual mixer using a T-junction mixer with a deep well structure (Johnson et al., "Rapid Microfluidic Mixing," Analytical Chemistry 74: 45 (2002)). According to a preferred embodiment of the present invention, the method of the present invention uses a manual mixer using a T-junction mixer.

(c) preparing a single or multiple lipid bilayer spherules by applying centrifugal force to the reverse micelle precursor.

The prepared reverse micelle precursor is made of a single lipid bilayer sphere (liposome) or a multiple lipid bilayer sphere (vesomosomes) under centrifugal force applied by a spinning device (see Figures 1 to 3).

According to a preferred embodiment of the present invention, the centrifugal forces important in the production of the single or multiple lipid bilayers of the present invention are controlled according to the ratio of the aqueous solution to the organic solution, and the size of the single or multiple lipid bilayers .

As used herein, the term " vesosome " is a structure formed by a variety of monomolecular or multimolar inclusion units (e. G., Vesicles) encapsulated within a unique outer layer bilayer structure.

Spinning devices that may be used in the present invention may be any device capable of imparting the centrifugal force necessary to form single or multiple bimorph spheres, including, for example, computer cooling fans, It is not.

The magnitude of the centrifugal force used in the method of the present invention can be variously determined according to the type and nature of the aqueous solution and the organic solution used, and the size of the single or multiple lipid bilayer spheres to be produced.

Compositions for the cargo delivery vehicle of the present invention include vesomes having an outer layer lipid bilayer for encapsulating various containment units (e.g., lipid vesicles) therein. The outer layer lipid bilayer may be produced from a variety of lipid compositions, and the bilayer may be multimodal or monomodal.

The inner containment units contained within the bilayer structure may be of uniform size or similar membrane or inner composition, and may also be of various sizes and / or other membranes or inner compositions. The membrane of the inner containment unit may be multi-membrane or mono-membrane. Also, the containment unit may be in a coherent or free form. The term " uniform size " as used herein refers to a very similar size, and does not necessarily mean an identical sized endoplasmic reticulum.

The composition of the present invention comprising a single or multiple lipid bilayer spheres as an active ingredient can be administered by a variety of methods including, for example, intramuscular injection, intravenous injection, oral administration, pulmonary adsorption, rectal Administration, subcutaneous injection, sublingual administration or topical administration.

The most effective modes of administration and dosage regimens for biological agents in the various containment units of the present invention will depend upon a variety of factors including the formulation method, the mode of administration, the age, weight, sex, pathology, food, time of administration, route of administration, And can be variously prescribed by factors such as sensitivity.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The invention relates to a process for the preparation of single or multiple lipid bilayers, the use of single or multiple lipid bilayers made therefrom, and an apparatus for the production of single or multiple lipid bilayers.

(b) The process of the present invention can be easily prepared by preparing a reverse micelle precursor at the T-junction in the microchannel and automating single or multiple lipid bilayers using a rotating device.

(c) The manufacturing apparatus of the present invention comprises (i) a syringe pump; (Ii) a microfluidic device; And (iiic) a spinning device, which utilizes centrifugal force by a rotating device.

(d) Accordingly, the present invention provides a method for effectively and easily producing a single or multiple lipid bilayer spherules at low cost, which can be used for various applications such as therapeutic agents, diagnostics, and the like through microencapsulation of drugs And the like).

1A is a schematic diagram of an automatic single or multiple lipid bilayer manufacturing system of the present invention.
Figure 1b is an actual photograph of a microfluidic device in an automatic single or multiple lipid bilayer manufacturing apparatus.
2 is a schematic diagram of a modification of a microfluidic device.
Figure 3 is a functional diagram of the rotating device, which is part of an automatic single or multiple lipid bilayer manufacturing device.
FIG. 4 is a schematic diagram illustrating a process in which a working reverse micelle precursor, which is part of an automatic single or multiple lipid bilayer manufacturing apparatus, forms a complete single or multiple lipid bilayer spherules.
5 is an actual photograph and a schematic diagram of a spinning device.
6 is a photomicrograph of a liposome which can be confirmed in Experimental Example 1. Fig.
Fig. 7 is a micrograph of a baclofen which can be confirmed by Experimental Example 2. Fig.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Structure and function of the invention

In the system used to accomplish the object of the present invention, a syringe pump (Harvard apparatus), a microfluidic device and a microfluidic device, which control the flow rate of the microfluidic device, And a spinning device which can be called a spinning device (Fig. 1A). In the case of a syringe pump, the organic solvent in which the lipid contained in the microfluidic device is dissolved and the solution in which the water-soluble substrate is melted flows at a constant rate. At this time, when the ratio of the flow rate of the organic solution to that of the aqueous solution is changed, it is possible to produce a single multi-lipid bilayer spheres of various sizes. In the case of a microfluidic device, a 'T' shaped channel (FIG. 1B) is present. An organic solution in which lipid is dissolved in one of the channels, and an aqueous solution in which a water soluble substrate is dissolved is flowed in the other channel. At this time, the syringe pump is pushed at a constant flow rate. At this time, the reverse micelle, which is a precursor of the lipid bilayer spheres, is produced by a method of cutting the solution from the aqueous solution channel by increasing the flow rate of the organic solution. In addition, a modified device such as that shown in FIG. 2 may be used instead of a T 'shaped channel to collect lipid bilayers having two substances or two or more different properties into a single lipid bilayer sphere. The generated reverse micelle is transferred to the next step, the rotating device. In the case of a rotating device, it is pre-filled with the same solution and the same organic solution as the above-mentioned aqueous solution. When the filled rotating device is rotated by a motor, the centrifugal force separates the two aqueous solutions and the organic solution, and a lipid mono layer is formed at the boundary between the aqueous solution and the organic solution. And a channel from the microfluidic device is connected to the center of the rotating device. The reverse micelle precursor formed in the microfluidic flow device is moved from the organic solution inside the rotating device to the aqueous solution by the centrifugal force. At this time, the reverse micelle is formed as a complete single or multiple lipid bilayer (liposome or vesosome) while passing through the lipid monolayer boundary inside the rotating device (FIGS. 3 and 4).

Example 1: Preparation of a lipid solution as a base material of the present invention

There are various lipid solutions used in the present invention. In particular, the type and concentration of the material in the lipid solution is an important factor in making a stable lipid bilayer sphere. Basically, Asolectin (Sigma), DPhPC (Avanti), DPPC (Avanti), etc. are added to solvents such as Hexane (Samchun), Decane (Junsei) and n-decane Of the solute. At this time, squalene or cholesterol is mixed at a certain ratio to make a more rigid lipid bilayer sphere not only of these solutes and solvents. Squalene (Sigma) or cholesterol (Sigma) is present between the lipid bilayers to make the lipid bilayer spheres more stable. In this experiment, the optimum ratio was found through several experiments. The ratio of lipid was optimum in the experiment in which asolectin and cholesterol were mixed at a ratio of 4: 1 and n-decane to squalene was mixed at a ratio of 9: 1. In addition, it was confirmed that the sample with the overall ratio of 20 mg / ml formed the most stable lipid bilayer sphere.

Example 2: Manufacture of rotating device

The manufacturing of a rotating device, which is a core device of the present invention, has been carried out. First of all, the rotating device that we want should have a device that indicates the relative centrifugal force called RCF (relative centrifugal force), or the RPM that can infer this relative centrifugal force. In addition, a device capable of smooth rotation is needed. There is a device called spin quota which is commercialized but it is expensive and bulky, and the experimenter used a cooling fan of a personal computer which is usually available in the general vicinity. The apparatus for rotation uses a cooling fan (Zalman) for a computer as described above and a cooling fan rotation rate control device that shows the number of revolutions of the cooling fan, and actually receives a reverse micelle precursor to form a single lipid bilayer or a multiple lipid bilayer Is made by drilling a hole in the center of the Petri dish (SPC) cover and attaching another cover to each other. At this time, the hole is provided with a conical auxiliary body to prevent the sample from protruding out. The device thus manufactured was attached to a fan to complete a rotation device (Fig. 5). When the aqueous solution and the lipid solution are put into the rotating device and rotated, the aqueous solution is pushed outward due to the specific gravity difference between the lipid solution and the aqueous solution, and the lipid solution is pushed inward. As a result, a single layer of lipid is formed between the aqueous solution and the lipid solution, which serves to complete the reverse micellar liposome (or vesicular) precursor completely with liposomes (or vesicosomes).

Example 3: Identification of an automatically formed lipid bilayer sphere (liposome or vesosome)

The method of identifying the liposomes or vesicosomes made is to optically measure the size of the liposomes by varying the concentration of the substances contained inside and outside the liposome.

In the case of the optical method, fluorescent lipid (NBD-PE; Avanti) was mixed with the liposome-forming lipophilic solution and the fluorescence was observed through a fluorescence microscope (Olympus, Japan) or a confocal microscope (Zeiss, Germany) It is a way to identify the sphere. At this time, in the case of berosomes, the liposomes in the liposomes and the lipid bilayers surrounding the liposomes were observed in different colors. The liposomes or vesicles observed in this manner were observed as green ring-shaped objects.

In the case of observing the change in size by varying the concentration of the substance, the osmotic pressure is generated by the difference in concentration between the inside and outside of the eye, thereby causing the solvent inside the liposome to escape out and the size of the liposome to be reduced. By confirming this, it can be confirmed that the lipid bilayer is single or multiple, and that the lipid is a liposome surrounded by a lipid membrane.

Experimental Example 1: Production of liposome was confirmed by using fluorescent lipid

0.05% NBD-PE (green fluorescing lipid) and 20% asolectin w / n-decane solution as a sample to be poured into the aqueous phase as the third distilled water and the oil phase, Prepared. Each solution was placed in a glass syringe and connected to a 'T' shaped microfluidic device. In the case of a rotating device, samples of components such as aqueous solution and oil-soluble solution flowing in a microfluidic device were put in an appropriate ratio. Thereafter, a tube was connected to the exit portion of the microfluidic device for collecting the liposome, and this tube was connected to the inlet of the rotating device. The microscope focuses on the 'T' shaped connection of the microfluidic device to ensure that the aqueous phase is well cut. Then, the fluid flow of the oil phase and the water-soluble layer was adjusted to a constant flow rate. At the same time, the rotating motor in the rotating device was operated to match the desired RCF value and operated. The liposomes were collected by adjusting the size of the cut by controlling the flow rate with a microscope. After collecting, the fat-soluble solution in the rotating device was removed (the fat-soluble solution could be removed easily), and the sample was taken from the remaining aqueous solution and observed with a fluorescence microscope or a confocal microscope. As a result, liposomes showed fluorescence unlike air bubbles and the like, and thus it was confirmed that liposomes were produced by observing samples having round fluorescence (FIG. 6).

EXPERIMENTAL EXAMPLE 2 Preparation and Identification of Vasomes Containing Different Fluorescent Colors in and out of the Fluorescent Lipid with Green Fluorescent Liquid Aqueous Solution of PCDA Liposome Containing Red Fluorescence

An aqueous solution of PCDA (Sigma) liposome was prepared as a sample to be poured into the aqueous channel. In this case, the aqueous solution of PCDA liposome is similar to the red lipid liposome. It is usually blue, and when it is subjected to impact, pH change, temperature change, etc., it becomes a red fluorescent substance . In the experiment, a red PCDA liposome aqueous solution was used. 0.05% NBD-PE, 20% asolectin w / n-decane solution was prepared as a sample to be flowed into the lipid-soluble channel. The procedure and the method of the remaining experiment are the same as the method described in Experimental Example 1. When the collected sample is observed using a fluorescence microscope or a confocal microscope, a red fluorescence dot is spread in a green circle, or a red (Fig. 7), which is a green fluorescent liposome contained in a red fluorescent liposome.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (19)

A method for producing a single or multiple lipid bilayer spherules comprising the steps of:
(a) preparing a reverse micelle precursor by injecting an aqueous solution and an organic solution into a microchannel using syringe pumps; And
(b) subjecting the reverse micelle precursor to centrifugal force to produce a single or multiple lipid bilayer sphere;
The organic solution of step (a) is a mixture of decane: hexadecane in a weight ratio of 1: 9-2: 8 or a mixture of squalene: hexadecane in a weight ratio of 1: 9-2: 8,
Wherein the microchannel has at least one intersection point of the tubes derived from the syringe pump and the flow rate of the microchannels is controlled by controlling the intensity of the syringe pump, And the flow rate of the organic solution, and the reverse micelle precursor is formed at an intersection of an aqueous phase and an organic solution phase, and the centrifugal force is adjusted according to the ratio of the aqueous solution to the organic solution , Wherein the adjustment of the centrifugal force determines the size of the single or multiple lipid bilayer spherules.
The method of claim 1, wherein the aqueous solution comprises one or more liposomes.
3. The method of claim 2, wherein the liposomes comprise a biological agent that is independent of each other.
4. The method of claim 3, wherein the biological agent is a chemical substance, a nanoparticle, a peptide, a polypeptide, a nucleic acid or a carbohydrate.
2. The method of claim 1, wherein the injection is performed using two or more syringe pumps.
6. The method of claim 5, wherein the syringe pump comprises liposomes independent of each other.
delete delete delete delete delete delete 6. A single or multiple lipid bilayer sphere produced by the method of any one of claims 1-6.
14. A composition for delivering a biological preparation comprising the single or multiple lipid bilayer spherules of claim 13 as an active ingredient.
15. The composition of claim 14, wherein the biological agent is a chemical substance, a nanoparticle, a peptide, a polypeptide, a nucleic acid or a carbohydrate.
(a) a syringe pump; (b) a microfluidic device; And (c) a spinning device;
The syringe pump is connected to one end of the microchannel of the microfluidic flow device, and the rotation device is connected to the other end of the microchannel of the microfluidic flow device. The centrifugal force is adjusted according to the ratio of the aqueous solution to the organic solution Characterized in that the organic solution is a mixture of decane: hexadecane in a weight ratio of 1: 9-2: 8 or a mixture of squalene: hexadecane in a weight ratio of 1: 9-2: 8 / RTI > a device for making a single or multiple lipid bilayer sphere.
17. The apparatus of claim 16, wherein the syringe pump comprises two or more syringe pumps.
17. The apparatus of claim 16, wherein the syringe pump comprises an endoplasmic reticulum independent of each other.
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