KR102475540B1 - Method and apparatus for manufacturing drug-loaded lipid nanoparticles - Google Patents

Abstract

The present invention relates to a method and a device for manufacturing lipid nanoparticles containing a drug capable of manufacturing a large quantity of lipid nanoparticles with easy delivery of a low-polar drug by dissolving a low-polar phospholipid compound unlike a microfluidic method using an only polar organic solvent of alcohols which can be mixed with water when manufacturing the lipid nanoparticles. In addition, in a case of the lipid nanoparticles manufactured by the manufacturing method and the manufacturing device of the present invention, the drug can be delivered with high stability by including antibodies which are specifically bonded to receptors or antigens of a target cell surface and selectively targeting specific cells in a body.

Description

Method and apparatus for manufacturing drug-loaded lipid nanoparticles {Method and apparatus for manufacturing drug-loaded lipid nanoparticles}

The present invention relates to a method and apparatus for preparing drug-loaded lipid nanoparticles.

Although the development of new active ingredients is important in the pharmaceutical industry, the development of drug delivery systems for how well such active ingredients can be delivered to the body is also a very important technical field. The development of such drug delivery systems has been very active in recent years, and such drug delivery systems have largely been developed into implants, microparticles, and nanoparticles. However, implants and microparticles are easy to use for various sustained-release parenteral administration of drugs, but their size is too large to be used in release-type injections, limiting the development of intravascular injection formulations. Therefore, in recent years, nanotechnology has been in the limelight to improve drug delivery systems and increase efficiency.

Lipid-based drug delivery (LBDD) systems are advanced technologies for delivering various bioactive molecules, such as small molecule inhibitors and vaccine components, to target cells and tissues. There are various advantages over drug delivery methods, including drug stability and bioavailability. Lipid Nanoparticle (LNP) technology, one of the LBDD systems, uses various biocompatible lipids for the purpose of preventing the drug from being degraded by intravascular enzymes before it is delivered to cells and takes effect when injected into the body. With the delivery technology created, researchers have continuously studied lipid nanoparticles as a delivery vehicle for oligonucleotide-based therapeutics.

On the other hand, as a method for preparing drug-containing lipid nanoparticles, a method using a microfluidics method using a flow path is known. For example, it has been reported that lipid nanoparticles with a diameter of about 30 nm can be produced with good reproducibility by using a microchannel with a built-in three-dimensional micromixer capable of achieving instantaneous mixing of two liquids (Non-Patent Document 0001). In addition, a flow path structure using a conventional three-dimensional mixer is obtained by using a simple two-dimensional flow path structure in which baffles (baffle plates) of a certain width are arranged alternately on both sides with respect to the flow path width in a micro-sized flow path through which the raw material solution flows. It has also been reported that a nano-sized lipid particle formation system with higher particle size controllability can be formed (Patent Document 0001). These nanoparticle preparation methods have recently been mainly adopted for the preparation of lipid nanoparticles loaded with fat-soluble drugs or nucleic acids such as siRNA (short interfering RNA) or mRNA. For example, as lipid nanoparticles serving as carriers for efficiently delivering nucleic acids such as siRNA into target cells, lipid nanoparticles containing pH-sensitive, ionizable cationic lipids as constituent lipids have been reported. (Patent Document 0002). The present inventors have disclosed a method for preparing a nano-liposome-microbubble conjugate in which a drug for hair loss treatment is encapsulated by ultrasonic treatment after adding a drug solution for treating hair loss to a lipid film composition (Patent Document 0003).

On the other hand, in the case of the above microfluidic method, basically only water-miscible organic solvents such as alcohols are used, and devices applying the method do not dissolve in alcohols, so low-polar drugs or low-polarity organic solvents must be used. In the case of using a phospholipid compound, layer separation occurs and is not mixed, so it is not suitable for forming drug-loaded lipid nanoparticles. In addition, the existing microfluidic manufacturing apparatus has a problem in that it is difficult to prepare an antibody-bound lipid nanoparticle because it is difficult to add a device for binding an antibody capable of recognizing an antigen to the surface of the lipid nanoparticle.

Accordingly, as a result of repeated experiments, the present inventors have produced lipid nanoparticles and antibody attachment with a manufacturing apparatus using a thin film hydration method rather than a microfluidic method to prepare lipid nanoparticles composed of a low polarity organic compound or a low polarity phospholipid compound. By developing a process that performs sequentially, the present invention has been completed.

Japanese Patent Registration No. 6942376 (registration date: 2021.09.10) Republic of Korea Patent Publication No. 10-2020-0018782 (published date: 2020.02.20) Republic of Korea Patent Registration No. 10-1918250 (registration date: 2018.11.07)

Leung et al., Journal of Physical Chemistry C Nanomater Interfaces, 2012, vol.116(34), p.18440-18450.

An object of the present invention is to provide a method and apparatus for preparing drug-loaded lipid nanoparticles capable of selectively delivering low-irritability drugs to target cells.

In order to achieve the above purpose,

The present invention includes (a) preparing a lipid solution containing an organic solvent and a lipid in a lipid solution supply unit;

(b) introducing the lipid solution into a reaction chamber;

(c) removing the organic solvent from the lipid solution of step (b);

(d) preparing an aqueous solution containing water, a buffer and a drug in an aqueous solution supply unit;

(e) introducing the aqueous solution into the reaction chamber and dispersing it using a stirrer to form a dispersion containing lipid nanoparticles;

In addition, the present invention provides a lipid solution supply unit in which a lipid solution containing an organic solvent and lipid is stored;

An aqueous solution supply unit in which an aqueous solution containing water, a buffer and a drug is stored;

a reaction chamber connected to the lipid solution supply unit and the aqueous solution supply unit, provided with a thermostat for setting a temperature in the space and a stirrer for stirring the solution; and

It provides a drug-loaded lipid nanoparticle manufacturing apparatus comprising a; receiving part connected to the reaction chamber and accommodating the dispersion containing the lipid nanoparticles generated in the reaction chamber.

When lipid nanoparticles are prepared using the method and apparatus for preparing lipid nanoparticles of the present invention, unlike the microfluidic method in which only polar organic solvents such as alcohols that are miscible with water are used, non-polar or low-polarity organic solvents are used. By dissolving the low-polarity phospholipid compound through the, it is possible to prepare a large amount of lipid nanoparticles that are easy to deliver low-polarity drugs.

In addition, in the case of the lipid nanoparticles prepared by the manufacturing method and manufacturing apparatus of the present invention, including antibodies capable of specifically binding to receptors or antigens on the surface of target cells, they selectively target only specific cells in the body and have high drug stability. can deliver.

1 is a schematic diagram of an apparatus for producing lipid nanoparticles according to an embodiment of the present invention.

Hereinafter, a method and apparatus for preparing drug-loaded lipid nanoparticles according to the present invention will be described in detail.

The terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to explain their invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

The present invention includes (a) preparing a lipid solution containing an organic solvent and a lipid in a lipid solution supply unit;

(b) introducing the lipid solution into a reaction chamber;

(c) removing the organic solvent from the lipid solution of step (b);

(d) preparing an aqueous solution containing water, a buffer and a drug in an aqueous solution supply unit;

(e) introducing the aqueous solution into the reaction chamber and dispersing it using a stirrer to form a dispersion containing lipid nanoparticles;

Hereinafter, the drug-loaded lipid nanoparticle manufacturing method of the present invention will be described step by step in detail.

In the method for preparing drug-loaded lipid nanoparticles of the present invention, step (a) is a step of preparing a lipid solution containing an organic solvent and a lipid in the lipid solution supply unit.

The lipid solution supply unit may store a lipid solution. The lipid solution may include an organic solvent and a lipid.

The type of the organic solvent is not particularly limited, and any one can be used as long as it can dissolve the lipid used in the preparation of the lipid nanoparticles of the present invention. The organic solvent may preferably be a non-polar or low-polarity organic solvent, and benzene, butanol, butyl acetate, carbon tetrachloride, chloroform, cyclohexane, dichloroethane, dichloromethane, diethyl ether, diisopropyl ether , selected from the group consisting of ethyl acetate, heptane, hexane, isooctane, methyl ethyl ketone (MEK), methyl t-butyl ether (MTBE), pentane, toluene, trichlorethylene, xylene, and mixed solvents thereof It can be.

The lipid may be selected from the group consisting of ionizable lipids, phospholipids, cholesterol, PEG-modified lipids, and combinations thereof.

The ionizable lipid is an ionizable compound having properties similar to lipids and allows the drug to be encapsulated in lipid nanoparticles with high efficiency through electrostatic interactions with drugs (eg, anionic drugs and / or nucleic acids) role can be fulfilled. The ionizable lipid may be protonated (positively charged) at a pH below the pKa of the cationic lipid and substantially neutral at a pH above the pKa. In one example, the lipid nanoparticle may include a protonated ionizable lipid and/or a neutral ionizable lipid.

As the phospholipid, any phospholipid capable of promoting fusion of the lipid nanoparticles may be used without limitation.

The cholesterol imparts rigidity in terms of shape to the lipid filling in the lipid nanoparticles, and serves to improve the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles. Also, as a cholesterol derivative, it may have cationic properties.

The PEG-modified lipid refers to a conjugated form of lipid and PEG, and refers to a lipid in which a polyethylene glycol (PEG) polymer, a hydrophilic polymer, is bound to one end. The PEG-modified lipid contributes to particle stability in serum of the nanoparticles within the lipid nanoparticles and serves to prevent aggregation between lipid nanoparticles. In addition, the PEG-modified lipid protects the nucleic acid from degradation enzymes during in vivo delivery of the nucleic acid, thereby enhancing the stability of the nucleic acid in the body and increasing the half-life of the drug encapsulated in the lipid nanoparticle.

In the PEG-modified lipid, the PEG can be directly conjugated to the lipid or linked to the lipid through a linker moiety. Any linker moiety suitable for linking PEG to a lipid may be used, including, for example, ester-free linker moieties and ester-containing linker moieties. The ester-free linker moiety is an amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O- ), urea (-NHC (O) NH-), disulfide (-S-S-), ether (-O-), succinyl (- (O) CCH CH C (O)-), succinimidyl (-NHC (O )CHCHC(O)NH-), ethers, disulfides, as well as combinations thereof (e.g., linkers containing both carbamate linker moieties and amido linker moieties). The ester-containing linker moiety may be, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and these Combinations of, but not limited to.

The lipid in the PEG-modified lipid may be used without limitation as long as it is a lipid capable of binding to polyethylene glycol, and phospholipid and/or cholesterol, which are other components of the lipid nanoparticle, may also be used. Specifically, lipids in PEG-modified lipids include ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine , DSPC), distearoylphosphatidylethanolamine (DSPE), or cholesterol, but is not limited thereto.

PEG in the PEG-modified lipid is a hydrophilic polymer and has the ability to inhibit the adsorption of plasma proteins, thereby increasing the circulation time of lipid nanoparticles in the body and preventing aggregation between nanoparticles. In addition, PEG-modified lipids can exhibit a stealth function in vivo to prevent degradation of nanoparticles.

The PEG may be one in which a functional group is bound to a side not bound to a lipid (functionalized PEG). At this time, the usable functional group is one or more selected from the group consisting of succinyl, carboxylic acid, maleimide, amine, biotin, cyanur, and folate. can

The PEG refers to conventional polyethylene glycol, and in the present invention, POZ [poly(2-oxazoline)], which can replace it, can be used.

In the method for preparing drug-loaded lipid nanoparticles of the present invention, step (b) is a step of introducing the lipid solution into a reaction chamber.

Specifically, the lipid solution may be continuously supplied from the lipid solution supply unit to the reaction chamber through a flow path connecting the lipid solution supply unit and the reaction chamber. A raw material supply controller is formed in the flow path, so that the amount of the lipid solution required by the reaction chamber can be appropriately controlled and supplied.

In the method for preparing drug-loaded lipid nanoparticles of the present invention, step (c) is a step of removing the organic solvent from the lipid solution of step (b).

The removing of the organic solvent may include maintaining the inside of the reaction chamber in a vacuum state using a vacuum pump and evaporating the organic solvent in the lipid solution introduced into the reaction chamber. In this case, only the organic solvent may be evaporated by setting the temperature in the reaction chamber to a high temperature using a thermostat provided in the reaction chamber. In this way, by evaporating and removing the organic solvent, the remaining lipid components other than the organic solvent in the lipid solution form a film on the inner wall and bottom of the reaction chamber.

In the drug-loaded lipid nanoparticle manufacturing method of the present invention, step (d) is a step of preparing an aqueous solution containing water, a buffer and a drug in the aqueous solution supply unit.

The aqueous solution supply unit may store an aqueous solution including water, a buffer, and a drug. The aqueous solution may further include an injection. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like.

The buffer may be any buffer suitable for maintaining a pH suitable for administration into the body, and preferably, the pH of the buffer may be 5.0 to 8.0.

The drug may be selected from the group consisting of ionic drugs, nucleic acid drugs, protein drugs, and combinations thereof. The type of the drug is not particularly limited, and examples thereof include antipsychotic drugs such as dementia treatment agents, Parkinson's disease treatment agents, anticancer agents, anti-anxiety agents, antidepressants, tranquilizers, and antipsychotic agents; cardiovascular agents such as antihypertensive agents, antihypertensive agents, antihypertensive agents, antithrombotic agents, vasodilators, and antiarrhythmic agents; epilepsy medication; gastrointestinal agents such as anti-ulcer agents; rheumatic drugs; antispasmodic; tuberculosis treatment; muscle relaxants; osteoporosis medication; erectile dysfunction treatment; styptic; hormones such as sex hormones; diabetes medication; Antibiotic; antifungal agents; antiviral agents; antipyretic analgesic anti-inflammatory agent; autonomic nervous modulators; corticosteroids; diuretic; antidiuretic; painkiller; anesthetic; antihistamines; antiprotozoal agents; anti-anemic agents; anti-asthmatics; antispasmodic; antidote; antimigraine drugs; antiemetic; antiparkinsonian agents; antiepileptic drugs; antiplatelet agents; antitussive expectorants; bronchodilators; cardiac; immunomodulator; and mixtures thereof.

In the drug-loaded lipid nanoparticle manufacturing method of the present invention, step (e) is a step of introducing the aqueous solution into a reaction chamber and dispersing it using a stirrer to form a dispersion containing lipid nanoparticles.

In step (e), the lipid component present in the form of a film on the inner wall and bottom of the reaction chamber is dispersed in the aqueous solution through an agitator, thereby forming an O/W emulsion type dispersion. Any agitator may be used as long as it is for forming a dispersion, but it is preferable to use an ultrasonic agitator. Specifically, when the dispersion is formed using the ultrasonic stirrer, the uniformity of the lipid nanoparticles in the dispersion is improved and the encapsulation efficiency is also improved, whereas in the case of physical stirring, the uniformity of the lipid nanoparticles is relatively lowered , Agitation may be inappropriate in the process of freezing/thawing the dispersion as follows. Therefore, it is preferable to use an ultrasonic stirrer in order to mass-produce lipid nanoparticle dispersions with excellent uniformity and encapsulation efficiency.

In the step (e), freezing/thawing of the dispersion liquid and melting the frozen dispersion liquid may be repeated 1 to 12 times. Specifically, the process of freezing the dispersion liquid by exposing it to liquid nitrogen for 5 minutes and then exposing it to hot water again to melt it may be repeated. At this time, by repeating the steps of freezing and thawing the lipid film composition, a lipid nanoparticle dispersion of a more uniform size can be formed, the drug encapsulation efficiency of lipid nanoparticles can be increased, and the mass production efficiency of lipid nanoparticles can be further improved. can improve However, if the number exceeds 12 times, the encapsulation efficiency and mass production yield of lipid nanoparticles may rather decrease, so less than 12 times is preferable.

In the step (e), after the freezing/thawing procedure, the dispersion may be reacted at a temperature of 0 to 10° C. for 1 to 10 hours. If the temperature is out of the range, the dispersion stability of the lipid nanoparticles may deteriorate, and if the reaction time is less than 1 hour or exceeds 10 hours, the reaction time is not sufficient to properly prepare the lipid nanoparticles. Otherwise, the reaction time is too long, and the problem of low efficiency may occur.

The lipid nanoparticles may have a particle size of 10 to 2,000 nm. When the size of the lipid nanoparticle is less than 10 nm, it may be difficult for the drug to be encapsulated in the lipid nanoparticle, and stability may be lowered when injected into the body, which is undesirable. In addition, even if it exceeds 2,000 nm, stability may be lowered when the composition containing the lipid nanoparticles is injected into the body, which is not preferable.

The drug-loaded lipid nanoparticle manufacturing method of the present invention may further include, after step (e), preparing an antibody solution containing an antibody capable of recognizing an antigen in an antibody solution supply unit (f).

The antibody is an antibody that can specifically bind to a receptor or antigen on the cell surface, and can improve the delivery efficiency of a substance into the nucleus of a target cell. For example, polyclonal antibodies or monoclonal antibodies against biological components such as cell membrane protein receptors that are specifically expressed in target tissues or organs are preferably disposed on the surface of lipid nanoparticles.

Generating such antibodies can be readily prepared using techniques well known in the art. The polyclonal antibody can be obtained from serum collected after injecting a protein specifically expressed on the surface of a target cell into an animal. As the animal, any animal host such as goat, rabbit, or pig may be used. The monoclonal antibody, as is widely known in the art to which the present invention pertains, is prepared using a hybridoma method (Kohler G. and Milstein C.) or a phage antibody library (Clackson et al.; Marks et al.) technology. It can be manufactured by In order to perform the hybridoma method, cells of an immunologically suitable host animal such as a mouse and cancer or myeloma cell lines may be used. Thereafter, after fusing these two types of cells by a method using polyethylene glycol or the like, that is, a method widely known in the art to which the present invention pertains, the antibody-producing cells can be proliferated by a standard tissue culture method. Then, after obtaining a uniform cell population by subcloning by the limited dilution technique, a hybridoma capable of producing an antibody specific to one protein specifically expressed on the target cell surface is a standard technology. Depending on the method, it can be mass-cultured in vitro or in vivo. In the phage antibody library method, an antibody gene for one protein specifically expressed on the surface of a target cell is obtained, expressed in the form of a fusion protein on the surface of a phage, and an antibody library is prepared in vitro, It can be performed by isolating and preparing a monoclonal antibody that binds to one type of protein specifically expressed on the target cell surface from the library. Antibodies prepared by the above methods can be separated by methods such as electrophoresis, dialysis, ion exchange chromatography, and affinity chromatography.

The antibody may include a functional fragment of the antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') ₂ , F(ab) ₂ , Fv, and the like.

The antibody is 1,4-bis-maleimidobutane, 1,11-bis-maleimidotetraethylene glycol, 1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride, succinimidyl-4- [N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]] and its sulfonated salt (sulfo-SMCC), succimidyl 6-[3-(2-pyridyldithio)- fionamido] hexanoate] and its sulfonated salt (sulfo-SPDP), m-maleimidobenzoyl-N-hydrosisuccinimide ester and its sulfonated salt (sulfo-MBS), and succimidyl [4-(p- Maleimidophenyl) butylate] and one or more crosslinking agents selected from the group consisting of sulfonated salts thereof (sulfo-SMPB) may be used as a linker to be bonded. At this time, the linker is characterized in that it connects the phospholipid containing the amine group of the lipid nanoparticle and the antibody.

In addition, the drug-loaded lipid nanoparticle manufacturing method of the present invention, after the step (f), introduces the antibody solution into the reaction chamber, and reacts the dispersion with the antibody solution to form antibody-linked lipid nanoparticles. A step (g) of forming a dispersion may be further included.

The lipid nanoparticles contain a lipid derivative capable of reacting with a mercapto group in polyclonal antibodies, monoclonal antibodies and functional fragments of the antibody molecules, for example, DSPE-PEG(2000)-amine, etc. , the antibody can be bound to the membrane surface of the lipid nanoparticle.

In the step (g), a crosslinking agent may be further included in the reaction chamber. Specifically, the cross-linking agent may be mixed for 1 to 5 hours in a reaction chamber in which the lipid nanoparticle dispersion is present, and then the antibody solution may be added and mixed for 1 to 5 hours. In this case, the lipid nanoparticle, the crosslinking agent and the antibody may be combined in a weight ratio of 10:1:1 to 30:5:1.

In the step (g), the dispersion and the antibody solution may be reacted at a temperature of 0 to 10 ° C for 5 to 20 hours. If the temperature is out of the range, the dispersion stability of the antibody-bound lipid nanoparticles may deteriorate, and if the reaction time is less than 1 hour or exceeds 10 hours, the reaction time is not sufficient to lipid nanoparticles. The antibody may not bind properly, or the reaction time may be too long, resulting in low efficiency.

In addition, the drug-loaded lipid nanoparticle manufacturing method of the present invention may further include the step (h) of removing the lipid nanoparticles to which the antibody is not bound from the dispersion formed in the step (g) after the step (g). can

Specifically, the dispersion containing the antibody-coupled lipid nanoparticles is moved to a purification unit using a rotary pump, and then antibody-coupled lipid nanoparticles and antibody-unbound lipid nanoparticles are separated through a column purification device. can do. At this time, the lipid nanoparticle to which the antibody is not bound is removed.

In addition, the drug-loaded lipid nanoparticle manufacturing method of the present invention, after the step (h), recovers the dispersion from which the lipid nanoparticles to which the antibody is not bound in the step (h) is removed, and then passes through a sterilization filter. It may further include step (i).

The sterile filter may have a pore size of 0.25 or 0.4 μm, preferably, the sterile filter has a pore size of 0.25 μm.

In addition, the present invention provides a drug-loaded lipid nanoparticle preparation device.

Hereinafter, a schematic diagram of a lipid nanoparticle production apparatus according to an embodiment of the present invention of FIG. 1 will be described.

Referring to FIG. 1 , the lipid nanoparticle manufacturing apparatus 100 includes a lipid solution supply unit 110, an aqueous solution supply unit 120, a reaction chamber 200, and a receiving unit 300.

The lipid solution supply unit 110 may store a lipid solution containing an organic solvent and lipid. Since the lipid solution is the same as described above, description thereof is omitted.

The aqueous solution supply unit 120 may store an aqueous solution including water, a buffer and a drug. Since the aqueous solution is as described above, description thereof is omitted.

The reaction chamber 200 may receive the lipid solution, the aqueous solution, and the antibody solution from the lipid solution supply unit 110 and the aqueous solution supply unit 120 . A lipid nanoparticle dispersion may be formed while sequentially receiving the lipid solution and the aqueous solution.

The lipid solution supply unit 110 and the reaction chamber 200 are connected by a first connecting passage FL1, so that the lipid solution is continuously supplied from the lipid solution supply unit 110 to the reaction chamber 200. can Similarly, the aqueous solution supply unit 120 and the reaction chamber 200 are connected by a second connection passage FL2, so that the aqueous solution can be continuously supplied from the aqueous solution supply unit 120 to the reaction chamber 200. have.

A flow control unit 250 is formed in the first connection path FL1 and the second connection path FL2 to appropriately control and supply the amount of the lipid solution and the aqueous solution required by the reaction chamber 200. can The flow control unit 250 may be any material capable of controlling the flow of one or more solutions supplied to the reaction chamber, and for example, the flow control unit 250 may be a solenoid valve.

The reaction chamber 200 is connected to a thermostat 210 for setting the temperature in the space within the chamber, an agitator 220 for agitating the dispersion, and the reaction chamber 200, and nitrogen gas is supplied to the reaction chamber 200. It may include a nitrogen gas supply unit 230 for supplying and a vacuum pump 240 for discharging residual gas in the reaction chamber 200 .

Specifically, in the reaction chamber 200, in a state in which residual gas is removed from the reaction chamber through the vacuum pump 240 and maintained in a vacuum, the lipid solution supply unit 110 by the first connection passage FL1 The lipid solution supplied from may be removed by heating the thermostat 210 above the boiling point of the organic solvent to vaporize the organic solvent. At this time, the remaining lipid components except for the organic solvent form a thin film on the inner wall and bottom surface of the reaction chamber 200 in the form of a film. After removing the organic solvent, nitrogen gas is sufficiently supplied into the reaction chamber 200 through the nitrogen gas supply unit 230, and then the aqueous solution is supplied from the aqueous solution supply unit 120 through the second connection passage FL2. In addition, a dispersion may be formed by ultrasonically stirring the lipid component and the aqueous solution using the stirrer 220. At this time, for uniformity of the size of the dispersed lipid nanoparticles, the dispersion may be frozen using the thermostat 210, and freezing/thawing of melting the frozen dispersion may be repeated 1 to 12 times. By repeating the freezing/thawing process, a dispersion having a uniform size of the lipid nanoparticles may be formed.

In addition, the lipid nanoparticle manufacturing apparatus 100 may further include an antibody solution supply unit 130 connected to the reaction chamber 200 .

The antibody solution supply unit 130 may store an antibody solution containing an antibody capable of recognizing an antigen. Since the antibody solution is as described above, description thereof is omitted.

The antibody solution supply unit 130 and the reaction chamber 200 are connected by a third connection path FL3, so that the antibody solution is continuously supplied from the antibody solution supply unit 130 to the reaction chamber 200. can Similarly, a flow control unit 250 is formed in the third connection passage FL3 so that the amount of the antibody solution required by the reaction chamber 200 can be appropriately adjusted and supplied.

Specifically, the antibody solution stored in the antibody solution supply unit 130 is supplied to the reaction chamber 200 in which a dispersion containing homogenized lipid nanoparticles is formed, and the lipid nanoparticles and the antibody react, so that the drug is loaded and the antibody A lipid nanoparticle dispersion to which is bound can be prepared.

The accommodating part 300 may receive and accommodate the dispersion liquid formed from the reaction chamber 200 . The accommodating part 300 and the reaction chamber 200 may be connected by a fourth connection passage FL4. A first dispersion liquid supply control unit (not shown) and a first rotary pump 310 are formed in the fourth connection passage FL4 to selectively supply the dispersion liquid to the accommodating unit 300 as needed.

The lipid nanoparticle manufacturing apparatus 100 may further include a purification unit 400 .

The purification unit 400 is connected to the accommodation unit 300, receives and receives the dispersion stored in the accommodation unit 300, and in the dispersion, the lipid nanoparticles to which the antibody is not bound and the lipid to which the antibody is bound Nanoparticles can be separated. The purification unit 400 may remove the lipid nanoparticles to which the antibody is not bound, and recover the lipid nanoparticles to which the antibody is bound. The purification part 400 and the accommodating part 300 may be connected by a fifth connection passage FL5. A second dispersion liquid supply controller (not shown) and a second rotary pump 410 are formed in the fifth connection passage FL5 to selectively supply the dispersion liquid to the purifier 400 as needed.

The lipid nanoparticle manufacturing apparatus 100 may further include a sterilization filter 500 and a final receiving unit 600. The method of sterilizing the lipid nanoparticles in the sterilization filter 500 is not particularly limited, and sterilization may be performed by passing the lipid nanoparticle dispersion through a syringe filter having a pore size of 0.25 or 0.4 μm. . The aseptic dispersion liquid passing through the sterilization filter 500 may be supplied and accommodated in the final receiving unit 600 . The final receiving unit 600 may be washed by recovering antibody-coupled lipid nanoparticles from the sterile dispersion. A method for recovering, washing, and drying the antibody-bound lipid nanoparticles is not particularly limited, and may be recovered using a method such as filtration or centrifugation, followed by washing with water. Through this, it is possible to remove remaining solvents and surfactants. The washing step may be typically performed using water, and the washing step may be repeated several times. The final receiving unit 600 may dry the washed lipid nanoparticles to obtain lipid nanoparticle powder. After filtering and washing, the obtained lipid nanoparticles may be dried using a conventional drying method to finally obtain a dried lipid nanoparticle powder. A method of drying the lipid nanoparticles is not particularly limited. The drying method may be performed using freeze drying, vacuum drying or reduced pressure drying.

The lipid nanoparticle preparation apparatus 100 may further include a controller (not shown) commonly used in the art to automate the lipid nanoparticle preparation. Regarding the control unit, any control system for automatically producing lipid nanoparticles is not particularly limited.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following preparation examples and examples.

Preparation of antibody-conjugated lipid nanoparticles

Lipid nanoparticles were prepared using a thin film hydration method. Briefly, helper lipid : cholesterol : ionized lipid : DSPE-PEG(2000)-amine (molar ratio 0.1 : 0.25 : 0.56 : 0.1) was dissolved in 1 mL of chloroform and the solvent was evaporated to form a film in the reaction chamber. Helper lipids used herein refer to zwitterionic lipid compounds such as lecithin, HSPC, DSPC, DPPC, and DOPE, and the ionized lipids include NGS-NTA (Ni) or DOTAP, DLin-KC2-DMA, DLin-MC3-DMA, and the like. Means an ionic lipid compound. The film was hydrated with a complex (drug of interest) in which 383 nM of each purified gene, protein, drug, etc. was dissolved in 1 mL phosphate buffered saline (PBS, pH 7.2). After the freeze-thaw procedure, the lipid nanoparticle solution was incubated with sulfo-SMCC ^* for 2 hours at 4 °C.

*(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)

Unreacted substances were removed by dialysis, and thiolated antibodies were bound to the lipid nanoparticles at 4° C. for 16 hours. The resulting antibody-conjugated lipid nanoparticle solution was filtered through a 0.25 μm syringe filter and then dialyzed through a 1,000 kDa membrane at 4° C. for 16 hours.

Characterization of lipid nanoparticles

The prepared lipid nanoparticle composition was evaluated for size and stability by performing dynamic light scattering (DLS) analysis and zeta potential measurement after 200 nm filtration and 1,000 kDa dialysis. The zeta potential of the lipid nanoparticles was measured using a Zetasizer Nano ZS (Malvern Instruments).

As a result of DLS measurement, it was confirmed that the lipid nanoparticles had a nanometer size of 141.0 ± 1.28 nm. In addition, as a result of measuring the zeta potential, it was confirmed that the surface charge of the nano-lipid particles exhibited a negative charge of -6.58 ± 0.10 mV, thereby confirming that nano-lipid particles having excellent dispersibility and stability were well prepared by the preparation method of the present invention.

Although the present invention has been described through specific details and limited examples as described above, this is only provided to help the overall understanding of the present invention, the present invention is not limited to the above examples, and the field to which the present invention belongs Those skilled in the art can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention should not be limited to the described embodiments, and it will be said that not only the claims to be described later, but also all modifications equivalent or equivalent to these claims belong to the scope of the present invention. .

100: lipid nanoparticle manufacturing device
110: lipid solution supply unit
120: aqueous solution supply unit
130: antibody solution supply unit
200: reaction chamber
210: thermostat
220: agitator
230: nitrogen gas supply unit
240: vacuum pump
250: flow control unit
300: receiving part
310: first rotary pump
400: purification unit
410: second rotary pump
500: sterile filter
600: final receiving part
FL1: first connecting path
FL2: Second link
FL3: 3rd link
FL4: 4th link
FL5: 5th link
FL6: 6th link

Claims (15)

delete delete delete delete delete delete delete delete delete delete A lipid solution supply unit in which a lipid solution containing an organic solvent and lipid is stored;
an aqueous solution supply unit in which an aqueous solution containing water, a buffer and a drug is stored;
A reaction chamber connected to the lipid solution supply unit and the aqueous solution supply unit, provided with a thermostat for setting the temperature in the space to enable freezing/thawing and a stirrer for stirring the solution;
an accommodating unit connected to the reaction chamber and accommodating a dispersion containing the lipid nanoparticles generated in the reaction chamber; and
An antibody solution supply unit connected to the reaction chamber and storing an antibody solution containing an antibody capable of recognizing an antigen;
The organic solvent is benzene, butanol, butyl acetate, carbon tetrachloride, chloroform, cyclohexane, dichloroethane, dichloromethane, diethyl ether, diisopropyl ether, ethyl acetate, heptane, hexane, isooctane, methyl It is selected from the group consisting of ethyl ketone (MEK), methyl t-butyl ether (MTBE), pentane, toluene, trichlorethylene, xylene, and mixed solvents thereof,
The lipid is selected from the group consisting of ionizable lipids, phospholipids, cholesterol, PEG-modified lipids, and combinations thereof;
The drug is characterized in that selected from the group consisting of ionic drugs, nucleic acid drugs, protein drugs and combinations thereof, the drug-loaded lipid nanoparticle manufacturing device.
delete According to claim 11,
The manufacturing apparatus includes a nitrogen gas supply unit connected to the reaction chamber and supplying nitrogen gas to the reaction chamber; and
A vacuum pump for discharging the residual gas in the reaction chamber; Characterized in that it further comprises, the drug-carrying lipid nanoparticle manufacturing apparatus.
According to claim 11,
The manufacturing apparatus is connected to the receiving part, the purification unit for removing the lipid nanoparticles to which the antibody is not bound in the dispersion; characterized in that it further comprises, the drug-loaded lipid nanoparticle manufacturing apparatus.
According to claim 14,
The manufacturing apparatus is connected to the purification unit, a sterilization filter for filtering the dispersion; characterized in that it further comprises, the drug-loaded lipid nanoparticle manufacturing apparatus.

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