JP2018016642A - Manufacturing method of lipid particles, and nucleic acid delivery carrier having lipid particles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of lipid particles which is capable of holding nucleic acid or the like at a high inclusion ratio, and a nucleic acid delivery carrier having lipid particles.SOLUTION: A manufacturing method of lipid particles including nucleic acid includes processes (1) to (4) below: (1) a process of heating oil phase containing phospholipid, alcohol and ester; (2) a process of mixing water phase containing nucleic acid and the oil phase prepared in process (1); (3) a process of cooling liquid mixture containing the oil phase and the water phase obtained in process (2) to crystallize lipid particles; and (4) a process of removing alcohol and ester from the liquid mixture containing the oil phase and the water phase obtained in process (3).SELECTED DRAWING: None

Description

  The present invention relates to a method for producing lipid particles and uses of lipid particles, and preferably relates to a method for producing lipid particles useful for delivering nucleic acids into cells and uses of lipid particles.

  Nucleic acid drugs have a clear mechanism of action against diseases, have few side effects, and are described as next-generation drugs. For example, a nucleic acid drug using RNA interference (RNAi) can cause degradation of mRNA of a target gene present in a cell and inhibit target gene expression. As a result, it is possible to reduce or treat a disease symptom caused by abnormal expression of a specific gene or gene group. In such nucleic acid pharmaceuticals utilizing RNA interference, for example, nucleic acids such as siRNA are used. In order to express functions of these nucleic acids, it is necessary to deliver the nucleic acids into cells.

  In general, a carrier (vector) is used as a method for effectively delivering a nucleic acid into a cell. Since the nucleic acid is anionic, cationic liposomes using a cationic lipid as a carrier (vector) (Non-patent Document 1), amphoteric liposomes having both cationic and anionic (Patent Documents 1 and 2), etc. Is being studied.

The Bangham method is known as the most common method for producing liposomes. The bangham method is to dissolve phospholipids in an organic solvent such as chloroform in a container, then evaporate the organic solvent to create a lipid thin film on the inner surface of the container, and then add water to the thin film to swell the thin film. In this method, liposomes are obtained by shaking the container.
In addition, methods such as an organic solvent extraction method, a surfactant removal method, and a freeze-thaw method are known.

Japanese Unexamined Patent Publication No. 2011-21026 JP 2005-517739

Gene Therapy, Vol. 6, p271, 1999

Inclusion rate is an important index for evaluating a carrier (vector). The encapsulation rate is an index indicating what percentage of the hydrophilic substance added to retain in the inner aqueous phase when the carrier (vector) is produced is retained in the inner aqueous phase.
Conventional methods for producing carriers (vectors) have not been sufficient to obtain carriers (vectors) that can retain nucleic acids at a high encapsulation rate, for example.

  An object of the present invention is to provide a method for producing lipid particles capable of retaining nucleic acids and the like at a high encapsulation rate, and to provide a nucleic acid delivery carrier having lipid particles.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have (1) a step of heating an oil phase containing phospholipid, alcohol and ester, (2) an aqueous phase containing nucleic acid, and step (1). A step of mixing the oil phase prepared in step (3), a step of cooling the mixed solution containing the oil phase and the aqueous phase obtained in step (2) to crystallize lipid particles, and (4) step (3). And the step of removing alcohol and ester from the mixed solution containing the oil phase and the aqueous phase obtained in step 1 above, and found that the production method of lipid particles that solves the above problems can be provided, and to complete the present invention It came.

（式中、Ｒ^１およびＲ^２は、同一または異なって、炭素数１０〜２２のアルキル基、炭素数１０〜２２のアルキルオキシアルキレン基、炭素数１０〜２２のアルカノイルオキシアルキレン基および炭素数１０〜２２のアルキルオキシカルボニルアルキレン基から選択される置換基である）で表される化合物である、［４］に記載の脂質粒子の製造法。
［６］ 工程（１）において、リン脂質、アルコールおよびエステルを含む油相を４０〜７０℃で加熱する、［１］〜［５］のいずれか１つに記載の脂質粒子の製造法。
［７］ 工程（３）において、油相および水相を含む混合液を１０〜３０℃で冷却する、［１］〜［６］のいずれか１つに記載の脂質粒子の製造法。
［８］ ［１］〜［７］のいずれか１つに記載の製造法によって得られる脂質粒子を有する核酸送達キャリア。 That is, the means for solving the problems are as follows.
[1] Steps (1) to (4) below:
(1) a step of heating an oil phase containing phospholipid, alcohol and ester; (2) a step of mixing an aqueous phase containing nucleic acid and an oil phase prepared in step (1); (3) in step (2) A step of cooling the liquid mixture containing the obtained oil phase and water phase to crystallize lipid particles; and (4) removing alcohol and ester from the liquid mixture containing the oil phase and water phase obtained in step (3). A process for producing lipid particles encapsulating nucleic acids.
[2] The method for producing lipid particles according to [1], wherein the alcohol is an alcohol having 1 to 6 carbon atoms.
[3] The method for producing lipid particles according to [1] or [2], wherein the ester is an acetate ester.
[4] The production of lipid particles according to any one of [1] to [3], wherein in step (1), the oil phase further comprises a compound having at least one amino group and at least one imidazolyl group. Law.
[5] A compound having at least one amino group and at least one imidazolyl group is represented by the general formula (1):

(In the formula, R ¹ and R ² are the same or different and each represents an alkyl group having 10 to 22 carbon atoms, an alkyloxyalkylene group having 10 to 22 carbon atoms, an alkanoyloxyalkylene group having 10 to 22 carbon atoms, and 10 carbon atoms. The method for producing lipid particles according to [4], which is a compound represented by the formula (1) to a compound selected from ˜22 alkyloxycarbonylalkylene groups.
[6] The method for producing lipid particles according to any one of [1] to [5], wherein in step (1), the oil phase containing phospholipid, alcohol and ester is heated at 40 to 70 ° C.
[7] The method for producing lipid particles according to any one of [1] to [6], wherein in the step (3), the liquid mixture containing the oil phase and the aqueous phase is cooled at 10 to 30 ° C.
[8] A nucleic acid delivery carrier having lipid particles obtained by the production method according to any one of [1] to [7].

  According to the method for producing lipid particles of the present invention, it is possible to provide a method for producing lipid particles capable of retaining nucleic acids and the like with a high encapsulation rate, and a nucleic acid delivery carrier having lipid particles.

It is a figure of ¹ H-NMR of compound A in a synthesis example. It is a figure of MS spectrum of compound A in a synthesis example.

  Hereinafter, the present invention will be described in detail.

  In the present specification, “to” indicates a range including numerical values described before and after that as a minimum value and a maximum value, respectively.

(1) Method for Producing Lipid Particles The lipid particles of the present invention are produced by the following steps (1) to (4):
(1) heating an oil phase containing phospholipid, alcohol and ester;
(2) A step of mixing the aqueous phase containing nucleic acid and the oil phase prepared in step (1);
(3) a step of cooling the liquid mixture containing the oil phase and the water phase obtained in step (2) (hereinafter sometimes referred to as an oil phase-water phase mixture) to crystallize lipid particles; and (4 ) A method for producing lipid particles encapsulating nucleic acid (hereinafter referred to as the production method of the present invention), which comprises the step of removing alcohol and ester from the mixed solution containing the oil phase and aqueous phase obtained in step (3). Is).

The production method of the present invention is a method for forming particles using coacervation. Coacervation separates into two phases: a colloid-rich liquid phase and a colloid-poor liquid phase when other substances are added to the hydrocolloid, diluted with a poor solvent, or when the pH is changed. It is a phenomenon that does.
In the production method of the present invention, an oil phase containing phospholipid, alcohol and ester is heated, mixed with an aqueous phase containing nucleic acid, and the obtained oil phase, and then cooled over time, a kind of core cell. By causing self-organization so that a phospholipid encapsulates the nucleic acid, a liposome that holds the nucleic acid at a high encapsulation rate can be obtained.

[Step (1)]
The production method of the present invention includes step (1) an oil phase containing phospholipid, alcohol and ester (an oil phase means an oily component contained in a composition obtained by mixing phospholipid, alcohol and ester) ).

  The phospholipid used in the present invention is not particularly limited. Examples include hydrogenated phospholipids in which unsaturated carbon chains of lipids are saturated with hydrogen, and synthetic phospholipids obtained by modifying natural phospholipids by synthesis. These may be used alone or in combination of two or more. Good.

  The total amount of phospholipid in the present invention is preferably 10: 1 to 5000: 1, more preferably 100: 1 to 1000: 1, in terms of molar ratio to the nucleic acid.

  Although it does not specifically limit as alcohol used by this invention, It is preferable to use a C1-C6 alcohol. Specific examples include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and 3-methyl-1-butanol. As alcohol used by this invention, it is more preferable to use ethanol from a polar viewpoint.

  Although it does not specifically limit as ester used by this invention, It is preferable to use an acetate ester. Specific examples include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, and isobutyl acetate. As ester used by this invention, it is more preferable to use ethyl acetate from a polar or lipophilic viewpoint.

  In the present invention, the ratio of ester to alcohol is preferably 90:10 to 10:90, more preferably 80:20 to 30:70, and 70:30 to 40:60 in volume ratio. Is more preferable.

  In the production method of the present invention, in the step (1), it is preferable to further include a compound having at least one amino group and at least one imidazolyl group (preferably as a lipid).

  In the present invention, the compounding amount when using a compound having at least one amino group and at least one imidazolyl group is preferably 10 mol% to 70 mol%, and preferably 15 mol% to 60 mol% with respect to the total amount of lipid. More preferably, it is more preferably 20 mol% to 50 mok%.

式中、Ｒ^１およびＲ^２は、同一または異なって、炭素数１０〜２２のアルキル基、炭素数１０〜２２のアルキルオキシアルキレン基、炭素数１０〜２２のアルカノイルオキシアルキレン基および炭素数１０〜２２のアルキルオキシカルボニルアルキレン基から選択される置換基である。 Although it does not specifically limit as a compound which has at least 1 amino group and at least 1 imidazolyl group used by this invention, For example, it is preferable to use the compound represented by General formula (1).

In the formula, R ¹ and R ² are the same or different and each represents an alkyl group having 10 to 22 carbon atoms, an alkyloxyalkylene group having 10 to 22 carbon atoms, an alkanoyloxyalkylene group having 10 to 22 carbon atoms, and 10 to 10 carbon atoms. A substituent selected from 22 alkyloxycarbonylalkylene groups.

R ¹ and R ² are the same or different and are more preferably an alkyl group having 10 to 22 carbon atoms, and more preferably an alkyl group having 14 to 20 carbon atoms. Moreover, you may have a double bond in the alkyl group.

  The compound having at least one amino group and at least one imidazolyl group represented by the general formula (1) used in the production method of the present invention is not particularly limited. For example, the compound can be synthesized by the following method. it can.

In the formula, PG represents a protecting group, and X represents a leaving group constituting an active ester. R ¹ and R ² are the same as described above.

  That is, an active ester of histidine (A) protected with an appropriate protecting group is reacted with an amine derivative (B) in the presence of a base to obtain a compound (C), and then the general formula is obtained by an appropriate deprotection method. The compound represented by (1) can be synthesized.

  Here, as a protecting group that can be used in the active ester (A) of histidine, for example, W.I. W. Greene et al., Protective Groups in Organic Synthesis 4th edition, pp. 255-265, 2007, John Wiley & Sons, INC.) And the like. Specifically, preferred examples include Boc group (tert-butoxycarbonyl group), Z group (benzyloxycarbonyl group) and the like.

  Examples of the active ester that can be used include phenyl ester, trifluorophenyl ester, pentaphenyl ester, and hydroxysuccinimide ester, and hydroxysuccinimide ester is preferable from the viewpoint of raw material availability or stability.

  Examples of the base that can be used include inorganic bases and organic bases. Examples of the inorganic base include sodium hydrogen carbonate and sodium carbonate. Examples of the organic base include triethylamine and diisopropylamine. The base to be used is preferably an appropriate base depending on the protecting group of the active ester (A) of histidine used in the reaction.

  Although it does not specifically limit as a solvent which can be used, A general organic solvent can be used. Specifically, ether solvents, ester solvents, amide solvents, and halogen solvents can be used. Preferred examples include ether solvents such as tetrahydrofuran and halogen solvents such as dichloromethane and chloroform.

  Examples of deprotection reactions that can be used include W.S. W. Greene et al., Protective Groups in Organic Synthesis 4th edition, pp. 255-265, 2007, John Wiley & Sons, INC.).

  As a compound having at least one amino group and at least one imidazolyl group used in the production method of the present invention, a compound represented by the formula (2) (2-amino-N, N-dihexadecyl-3- (1H— Preference is given to using imidazol-5-yl) propanamide) [also referred to herein as compound A].

  In the production method of the present invention, by using the compound represented by the formula (2), a nucleic acid can be retained with a high encapsulation rate and excellent in the release property of the nucleic acid in the target cell. be able to.

In the step (1) of the present invention, the temperature at which the oil phase containing phospholipid, alcohol and ester is heated is preferably 40 to 70 ° C, more preferably 45 to 65 ° C, and more preferably 50 to More preferably, it is 60 degreeC.
The heating time is not particularly limited as long as it can be confirmed that the temperature of the entire liquid is uniformly desired.

[Other ingredients]
In the oil phase of the present invention, in addition to phospholipids, alcohols and esters, other components can be included as necessary within a range not impairing the effects of the present invention.

(Sterol)
The production method of the present invention may contain sterol in the oil phase. In the present invention, by including sterol in the oil phase, the membrane fluidity can be lowered and the effect of stabilizing lipid particles can be obtained.
Although it does not specifically limit as sterol, Cholesterol, phytosterol (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether. Cholesteryl-4′-hydroxybutyl ether can be raised. Among these, cholesterol is preferable.
In the present invention, the sterol content is preferably 10 mol% to 60 mol%, more preferably 20 mol% to 55 mol%, and even more preferably 25 mol% to 50 mol%, based on the total lipid amount. .

(Lipid with polyethylene glycol chain)
The production method of the present invention may include a lipid having a polyethylene glycol chain (hereinafter referred to as “PEG chain”) in the oil phase. In the present invention, by including a lipid having a PEG chain in the oil phase, the effect of stabilizing the dispersion of lipid particles can be obtained.
The lipid having a PEG chain is not particularly limited, and examples thereof include PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, and ceramide PEG derivatives. Among these, PEG-modified phosphoethanolamine is preferable.
The weight average molecular weight of the PEG chain is preferably 500 to 5000, and more preferably 750 to 2000.
The PEG chain may be branched and may have a substituent such as a hydroxymethyl group.

  In the present invention, the amount of the lipid having a PEG chain is preferably 0.5 mol% to 12 mol%, more preferably 2 mol% to 10 mol%, more preferably 4 mol% to 8 mol% with respect to the total lipid amount. % Is more preferable.

[Step (2)]
The production method of the present invention includes a step (2): a step of mixing an aqueous phase containing a nucleic acid and an oil phase prepared in step (1).

[Nucleic acid]
The nucleic acid used in the present invention includes any known form of nucleic acid. Specific examples of the nucleic acid include general RNA, DNA, and derivatives thereof, which may be single-stranded DNA or RNA, double-stranded DNA or RNA, DNA- It may be an RNA hybrid. Specific examples of the nucleic acid that can be used in the present invention include antisense DNA, antisense RNA, DNA enzyme, ribozyme, siRNA, shRNA, miRNA, aiRNA, piRNA, decoy nucleic acid, and aptamer. As the nucleic acid used in the present invention, siRNA, miRNA, aiRNA, antisense DNA, and antisense RNA are preferably used.

The nucleic acid used in the present invention is not limited to the natural type, and in order to improve stability in vivo such as nuclease resistance, at least a part of the sugar or phosphate backbone constituting the nucleotide is included. The non-natural type may be modified.
Non-natural nucleic acids with modified sugar moieties include 2′-O-methyl RNA, 2′-O- (2-methoxy) ethyl RNA, 2′-deoxy-2′-fluoroarabino nucleic acid, and cross-linked type A nucleic acid (LNA / BNA) etc. are mentioned. Peptide nucleic acid (PNA) in which the sugar moiety is replaced with a peptide, morpholino nucleic acid in which morpholino is replaced, and the like can also be given as examples of non-natural nucleic acids.
Examples of the non-natural nucleic acid in which the phosphate backbone is modified include phosphorothioate and phosphorodithioate.

  The aqueous phase (the aqueous phase means an aqueous component) in the production method of the present invention can be obtained, for example, by dissolving nucleic acid in an aqueous component such as water.

  In the step (2) of the present invention, the aqueous phase and the oil phase obtained in the step (1) are mixed. The ratio (mass ratio) of mixing the water phase and the oil phase is preferably 3.0: 1.0 to 1.0: 1.0, and 1.6: 1.0 to 1.1: 1.0. More preferred.

The temperature at the time of mixing the water phase and the oil phase is preferably 40 to 70 ° C, more preferably 45 to 65 ° C, and further preferably 50 to 60 ° C.
The mixing time is not particularly limited as long as it can be confirmed that the entire liquid is uniform. The heating time is not particularly limited as long as it can be confirmed that the temperature of the entire liquid is uniformly desired.

[Step (3)]
The production method of the present invention includes a step (3): a step of cooling the oil phase-water phase mixture obtained in step (2) to crystallize lipid particles.

In the step (3) of the present invention, the cooling condition of the oil phase-water phase mixture is preferably 10 to 30 ° C, more preferably 15 to 25 ° C.
The cooling time is not particularly limited, but the cooling rate is preferably −3 ° C./min or less.

[Step (4)]
The production method of the present invention includes a step (4): a step of removing alcohol and ester from the oil phase-water phase mixture obtained in step (3).

  In the step (4) of the present invention, the method for removing alcohol and ester from the oil phase-water phase mixed solution is not particularly limited, and can be removed by a general method.

[Other processes]
The lipid particles obtained by the production method of the present invention can be sized as necessary. In sizing, the particle diameter can be reduced by ultrasonically treating a liquid (suspension) containing lipid particles by either in-tank ultrasonic treatment or probe ultrasonic treatment.
Moreover, the lipid particle obtained by the manufacturing method of this invention can be concentrated as needed. Various known methods can be employed for the concentration, and examples thereof include a concentration method using an ultrafiltration membrane.

(2) About lipid particle In this invention, a lipid particle means the particle | grains comprised from a lipid, and is not specifically limited. Lipid particles of the present invention include liposomes having lamellar structures that are closed vesicles composed of lipid bilayers. As the liposome, structures such as multi-liposome (MLV), small unilamellar liposome (SUV), and giant unilamellar liposome are known, but are not particularly limited. The lipid particles of the present invention also include particles that do not have a lipid bilayer structure (lamellar structure) like the above-mentioned liposome and that have a structure filled with constituent components inside the particle.

  The form of lipid formation can be confirmed by electron microscope observation or structural analysis using X-rays. For example, by a method using Cryo transmission electron microscope observation (CryoTEM method), lipid particles like lipids have a lipid bilayer structure (lamella structure), a structure having an inner water layer, or lipid particles like liposomes. It does not have a lipid bilayer structure (lamella structure) and inner water layer, and has a core with high electron density inside the particle, so it has a structure packed with components such as lipids, etc. Can be confirmed. The presence or absence of lipid bilayer structure (lamella structure) can also be confirmed by lipid X-ray scattering (SAXS) measurement.

  Although the particle diameter of the lipid particle of the present invention is not particularly limited, it is preferably 10 to 1000 nm, more preferably 50 to 500 nm, and further preferably 75 to 350 nm. The particle diameter of the lipid particles can be measured by a general method (for example, dynamic light scattering method, laser diffraction method, etc.).

(3) Utilization of lipid particles As an example of lipid particles obtained by the production method of the present invention, a nucleic acid or the like (for example, a gene or the like) may be introduced into a cell by introducing the lipid particle into the cell in vitro. it can. Moreover, when the nucleic acid obtained by the manufacturing method of this invention contains the nucleic acid which has a pharmaceutical use, a lipid particle can be administered to a biological body as a nucleic acid pharmaceutical.

When the lipid particles obtained by the production method of the present invention are used as a nucleic acid drug, the lipid particles of the present invention are used alone or with a pharmaceutically acceptable administration medium (for example, physiological saline or phosphate buffer). It can be mixed and administered to a living body.
The concentration of the lipid particles in the mixture with the pharmaceutically acceptable carrier is not particularly limited, and can generally be 0.05% by mass to 90% by mass. Moreover, the nucleic acid medicine containing the lipid particles of the present invention may contain other pharmaceutically acceptable additive substances such as a pH adjusting buffer and an osmotic pressure adjusting agent.

  The administration route when administering the nucleic acid drug containing the lipid particles of the present invention in vivo is not particularly limited, and can be administered by any method. Examples of the administration method include oral administration and parenteral administration (intra-articular administration, intravenous administration, intraperitoneal administration, intramuscular administration, etc.). The nucleic acid medicament containing the lipid particles of the present invention can also be administered by direct injection at the disease site.

  The dosage form of the lipid particles of the present invention is not particularly limited, but when oral administration is performed, the lipid particles of the present invention are combined with an appropriate excipient in combination with tablets, troches, capsules, pills, It can be used in the form of a suspension, syrup and the like. In addition, preparations suitable for parenteral administration include antioxidants, buffers, bacteriostats, and isotonic sterile injections, suspending agents, solubilizing agents, thickening agents, stabilizers or preservatives. Additives such as can be included as appropriate.

(4) Nucleic acid delivery carrier The lipid particles obtained by the production method of the present invention are very useful as a nucleic acid delivery carrier because they can hold nucleic acids at a high encapsulation rate. The nucleic acid delivery carrier of the present invention can introduce a nucleic acid or the like into a cell by mixing the obtained lipid particles with a nucleic acid or the like and transfecting the cell in vitro. The nucleic acid delivery carrier of the present invention is also useful as a nucleic acid delivery carrier in nucleic acid medicine.

The present invention is specifically described by the following examples, but the scope of the present invention is not limited to the following examples.
In the present invention, the cholesterol is Dishman's cholesterol HP, DPPC (dipalmitoylphosphatidylcholine) is NOF COATSOME MC6060, DSPE-PEG (polyethylene glycol-modified phosphoethanolamine, PEG chain molecular weight: 2000). NOF used SUNBRIGHT DSPE-020CN, and lecithin used NOF COATSOME NC-50.

[Synthesis Example: Synthesis of Compound Represented by Formula (2) (hereinafter, Compound A)]
First Step: Add 230 g of dihexadecylamine and 5.52 g of triethylamine to 230 mL of tetrahydrofuran, add 24.6 g of Boc-His (1-Boc) -OSU with stirring, stir at room temperature for 1 hour, and at 50 ° C. for 5 hours. Stir. Thereafter, tetrahydrofuran was distilled off under reduced pressure, and 450 mL of chloroform and 200 mL of water were added to the reaction product. The organic layer was separated, washed successively with saturated aqueous sodium hydrogen carbonate solution, 10% aqueous citric acid solution and saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 5/1 to 3/1) to obtain 24 g of an oily product.

Second Step 21.7 g of the protector obtained in the first step was added little by little to 35 mL of trifluoroacetic acid and stirred at room temperature for 24 hours. Thereafter, the mixture was gradually added to 600 mL of an aqueous solution containing 40 g of saturated sodium bicarbonate, and stirred for 1 hour. Chloroform 500 mL was added to the resulting reaction solution, the organic layer was separated, washed successively with a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (chloroform / methanol = 10/1) to obtain 11.6 g of colorless solid compound A. The compound was identified by NMR and MS.

[Example 1]
(Coacervation method)
Preparation of oil phase L-α-dipalmitoylphosphatidylcholine, lecithin, cholesterol, N- (carbonyl-methoxypolyethyleneglycol 2000) -1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (hereinafter DSPE- PEG) is weighed out to give a molar ratio of 61/15/20/4, 80 mg, 20 mg, 14 mg, and 20 mg, respectively, and 0.3 mL of ethanol and 0.7 mL of ethyl acetate are added and dissolved. Obtained.
Preparation of nucleic acid-retaining lipid particles To the oil phase obtained in the above step, 0.25 mL of a nucleic acid aqueous solution in which 5 mg of siRNA described below is dissolved in 0.263 mL of sterilized water and 1.0 mL of sterilized water are added and heated at 55 ° C. for 10 minutes. did. Thereafter, the mixture was allowed to cool at room temperature with stirring. Subsequently, the mixture was dialyzed with a 100 mM histidine solution at room temperature to remove the ethanol / ethyl acetate mixed solution. The obtained solution was sized by passing it through a 0.4 μm filter using an extruder (Mini Extruder manufactured by Avanti Polar Lipids) to obtain lipid particles retaining nucleic acids.

[Example 2]
(Coacervation method)
Preparation of 200 mM histidine solution 15.5 g of L-histidine was weighed and dissolved in 500 mL of sterile water. 200 mM HCl was added so that the pH was 7 to obtain a 200 mM histidine solution.
Preparation of oil phase L-α-dipalmitoylphosphatidylcholine, compound A, cholesterol, DSPE-PEG were weighed out to give a molar ratio of 26/26/44/4, respectively 37 mg, 30 mg, 33 mg and 20 mg, and ethanol was added. 0.3 mL and 0.7 mL of ethyl acetate were added and dissolved to obtain an oil phase.
Preparation of Nucleic Acid-Retaining Lipid Particles To the oil phase obtained in the above step, 0.25 mL of a nucleic acid aqueous solution in which 5 mg of siRNA described below was dissolved in 0.263 mL of sterile water, 0.625 mL of 200 mM histidine solution, and 0.375 mL of sterile water were added. And heated at 55 ° C. for 10 minutes. Subsequently, the mixture was dialyzed with a 100 mM histidine solution at room temperature to remove the ethanol / ethyl acetate mixed solution. The obtained solution was sized by passing through a 0.4 μm filter using an extruder (Mini Extruder manufactured by Avanti Polar Lipids) to obtain lipid particles retaining nucleic acid.

[Example 3]
(Coacervation method)
In the preparation of the oil phase, L-α-dipalmitoylphosphatidylcholine, compound A, cholesterol, DSPE-PEG were weighed out to a molar ratio of 26/22/44/8, respectively 31 mg, 31 mg, 33 mg, and 44 mg. Except for the above, it was prepared in the same manner as in Example 2 to obtain lipid particles retaining nucleic acid.

Comparative Example 1
(Bangham method)
Preparation of oil phase L-α-dipalmitoylphosphatidylcholine, compound A, cholesterol, DSPE-PEG were weighed out to give a molar ratio of 26/26/44/4, respectively 37 mg, 30 mg, 33 mg, 20 mg, ethanol 0 .3 mL and 0.7 mL of ethyl acetate were added and dissolved to obtain an oil phase.
Formation of Lipid Membrane The oil phase obtained in the above step was placed in a recovery flask, and ethanol and ethyl acetate were distilled off with an evaporator to obtain a lipid membrane.
Preparation of Nucleic Acid-Retaining Lipid Particles To the above-mentioned eggplant flask, add 0.25 mL of a nucleic acid solution prepared by dissolving 5 mg of siRNA described below with 0.263 mL of sterile water, 0.625 mL of 200 mM histidine solution, and 0.375 mL of sterile water, and vortex mixer. And hydrated with stirring at 55 ° C. The obtained liquid was sized by passing through a 0.4 μm filter using an extruder (Mini Extruder manufactured by Avanti Polar Lipids) to obtain lipid particles retaining nucleic acid.

[Comparative Example 2]
(Coacervation method)
In the preparation of the oil phase, lipid particles retaining nucleic acids were prepared in the same manner as in Example 2 except that a mixed solution of 0.3 mL of ethanol and 0.7 mL of ethyl acetate was dissolved instead of 1 mL of ethanol.

[Comparative Example 3]
(Bangham method)
Preparation of oil phase L-α-dipalmitoylphosphatidylcholine, compound A, cholesterol, DSPE-PEG were weighed out to give a molar ratio of 61/15/20/4, respectively 80 mg, 20 mg, 14 mg, 20 mg, chloroform 1 0.0 mL was added and dissolved to obtain an oil phase.
Formation of Lipid Membrane The oil phase obtained in the above step was placed in a recovery flask, and ethanol and ethyl acetate were distilled off with an evaporator to obtain a lipid membrane.
Preparation of Nucleic Acid-Retaining Lipid Particles To the above-mentioned eggplant flask, 0.25 mL of a nucleic acid aqueous solution prepared by dissolving 5 mg of siRNA described below in 0.263 mL of sterilized water and 1.0 mL of sterilized water are added and stirred at 55 ° C. using a vortex mixer. While hydrated. The obtained liquid was sized by passing through a 0.4 μm filter using an extruder (Mini Extruder manufactured by Avanti Polar Lipids) to obtain lipid particles retaining nucleic acid.

The siRNA having the following sequence was used.
5′-GUUCAGACCACUUCAGCUU-3 ′ (sense chain) (SEQ ID NO: 1)
3′-CAAGUCUUGGUGAAGUCGAA-5 ′ (antisense chain) (SEQ ID NO: 2)

Measurement of Lipid Particle Size The particle size of the lipid particles was measured as the undiluted solution using a lipid particle dispersion using a zeta potential / particle size measurement system ELS-Z2 manufactured by Otsuka Electronics Co., Ltd.

Evaluation of siRNA encapsulation rate (quantification of total nucleic acid concentration)
To 0.02 mL of lipid particles holding nucleic acid, 0.01 mL of 3M ammonium acetate solution and 0.003 mL of glycogen were added, and then 0.5 mL of ethanol was added to dissolve the lipid and precipitate only the nucleic acid. . After standing at −20 ° C. for 2 hours, the mixture was centrifuged at 14000 × g and 4 ° C. for 15 minutes to remove the supernatant. After air-drying for 15 minutes or more, water was added to redissolve, and the total nucleic acid concentration was quantified by measuring the concentration using Nanodrop NF1000 (Thermo Fisher Scientific).

(Quantification of nucleic acid concentration in the external water phase)
Quantification was performed using Quant-iT RiboGreen RNA Assay Kit (Invitrogen) according to the low-range assay described in the manual & protocol. First, 20 × TE buffer contained in the above-described kit was diluted with water to obtain 1 × TE buffer. In order to quantify only the nucleic acid in the outer aqueous phase, 0.095 mL of 1 × TE buffer was added to 0.005 mL of lipid particles holding nucleic acid to prepare a 20-fold diluted solution. Subsequently, 1.99 mL of 1 × TE buffer was added to 0.1 mL of a 20-fold diluted solution to obtain a sample diluted 4000 times with TE buffer without destroying the lipid particles holding the nucleic acid.
By adding 1.791 mL of 1 × TE buffer to 0.009 mL of RiboGreen reagent stock solution, adjusting the 200-fold dilution, and then adding 1.98 mL of 1 × TE buffer to 0.22 mL of the 200-fold dilution, RiboGreen reagent diluted 2000 times was obtained.
Place 0.1 mL of a sample diluted 4000 times in a 96-well plate, then add 0.1 mL of RiboGreen reagent diluted 2000 times to the sample, and use a plate reader Infinit EF200 (TECAN) to detect fluorescence (excitation wavelength: 485 nm, The nucleic acid concentration in the outer aqueous phase was quantified by measuring the fluorescence wavelength (535 nm).

(Calculation of inclusion rate)
Using the quantification results of the total nucleic acid concentration and the concentration in the outer aqueous phase obtained in the above steps, the inclusion rate of the lipid particles holding the nucleic acids of Examples 1 to 3 and Comparative Examples 1 to 3 was determined according to the following formula. Calculated.
Inclusion rate (%) = (total nucleic acid concentration−nucleic acid concentration in the outer aqueous phase) ÷ total nucleic acid concentration × 100

  The results are shown in Table 1.

  As shown in Table 1, the lipid particles prepared by the coacervation method are compared with the conventional preparation method Bangham method without using a compound having at least one amino group and at least one imidazolyl group. It was found that the inclusion rate was unexpectedly improved. In addition, as in Examples 2 and 3, lipid particles retaining nucleic acids obtained by a coacervation method using a compound having at least one amino group and at least one imidazolyl group have a very high encapsulation rate. It was found to have

Claims (4)

The following steps (1) to (4):
(1) heating an oil phase containing phospholipid, alcohol and ester;
(2) A step of mixing the aqueous phase containing nucleic acid and the oil phase prepared in step (1);
(3) a step of cooling the liquid mixture containing the oil phase and the water phase obtained in step (2) to crystallize lipid particles; and (4) a mixture containing the oil phase and the water phase obtained in step (3). Removing alcohol and esters from the liquid,
A method for producing a nucleic acid-containing lipid particle comprising:
The alcohol is ethanol, the ester is ethyl acetate,
In step (1), the oil phase further comprises a compound having at least one amino group and at least one imidazolyl group,
A compound having at least one amino group and at least one imidazolyl group has the general formula (1):

(In the formula, R ¹ and R ² are the same or different and each represents an alkyl group having 10 to 22 carbon atoms, an alkyloxyalkylene group having 10 to 22 carbon atoms, an alkanoyloxyalkylene group having 10 to 22 carbon atoms, and 10 carbon atoms. Is a substituent selected from an alkyloxycarbonylalkylene group of ˜22),
A method for producing lipid particles. The manufacturing method of the lipid particle of Claim 1 which heats the oil phase containing a phospholipid, alcohol, and ester at 40-70 degreeC in a process (1). The method for producing lipid particles according to claim 1 or 2, wherein in step (3), the mixed liquid containing the oil phase and the aqueous phase is cooled at 10 to 30 ° C. In a process (2), it mixes so that a water phase and an oil phase may be 1.6: 1.0-1.1: 1.0 by mass ratio. Manufacturing method.

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