Lipid nanoparticles and method for producing same

Abstract

The present invention relates to a method for producing lipid nanoparticles, the method comprising: a step for dissolving a hyaluronic acid derivative in an aqueous solvent to form an aqueous phase; a step for dissolving a phospholipid in an organic solvent to form an oil phase; and a step in which the oil phase is dropped into the aqueous phase and dispersed. The hyaluronic acid derivative is represented by general formula (1). [In general formula (1), R1, R2, R3, R4, and R5 each independently represent a hydroxyl group or a group represented by-O-CH2-CH (OH)-CH2-OR6. And R6 represents a linear or branched alkyl group or alkenyl group. And n represents an integer of 1-2500 (inclusive). In the formula, R1, R2, R3, R4 and R5 are not all hydroxyl groups. ]. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to lipid nanoparticles and a method for producing the same. Background Art

[0002] Lipid nanoparticles are nanoparticles with a layer formed by a lipid membrane in the outer shell. Lipid nanoparticles usually have a core that can dissolve poorly water-soluble substances inside the layer formed by the lipid membrane. Lipid nanoparticles can encapsulate poorly water-soluble drugs to dissolve them, and can encapsulate nucleic acids (such as mRNA, siRNA) that are easily decomposed in the body to prevent their decomposition, so they can be used as drug carriers for drug delivery systems. For example, lipid nanoparticles containing mRNA of the spike protein of COVID-19 are used as COVID-19 vaccines.

[0003] In order to selectively deliver lipid nanoparticles to specific cells, the surface of lipid nanoparticles is also modified. For example, a technology is disclosed in patent documentation 1, in order to improve the uptake efficiency to the cells (cancer cells etc.) with CD44, lipid nanoparticles are made to contain hyaluronic acid derivatives, which are formed by directly or indirectly connecting the neutral lipids with the hydrophobic groups of straight-chain shapes and the hydroxyl of the N-acetylglucosamine (acetyl glucosamine) as the reducing sugar at the end of hyaluronic acid. In addition, for example, a technology is disclosed in patent documentation 2, in order to regulate or promote the uptake of lipid nanoparticles to target cells and the surface of lipid nanoparticles coated with glycosaminoglycans such as hyaluronic acid.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2019-189574

[0007] Patent Document 2: Japanese Patent Application No. 2020-530778 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] Lipid nanoparticles are usually composed of phospholipids, cholesterol, and lipids modified with polyethylene glycol (PEG). In addition, nucleic acid-encapsulating lipid nanoparticles usually contain cationic lipids that can complex with negatively charged nucleic acid molecules (such as mRNA molecules).

[0010] PEGylated lipids are important components that inhibit the increase in particle size of lipid nanoparticles due to aggregation and agglomeration, and help improve the stability of lipid nanoparticles in formulations and in the blood. However, it has been suggested that PEGylated lipids may cause allergic reactions, and the possibility of causing local allergic reactions (such as the Moderna arm) or more serious systemic allergic reactions cannot be ruled out. Therefore, there is a need to increase the selection of lipid nanoparticles as drug carriers, such as lipid nanoparticles composed of alternative materials.

[0011] Although Patent Documents 1 and 2 disclose using hyaluronic acid to improve the uptake of lipid nanoparticles into target cells, they do not disclose improving the stability of lipid nanoparticles.

[0012] The object of the present invention is to provide novel lipid nanoparticles having excellent emulsifying power and emulsification stability and a method for producing the same.

[0013] Solutions for solving problems

[0014] The inventors have found that although the emulsifying power (ability to generate smaller particles) and emulsification stability (ability to suppress the increase of particle size during storage) of the specific hyaluronic acid derivative itself described later are extremely low, by combining with phospholipids, the emulsifying power (ability to generate smaller particles) and emulsification stability (ability to suppress the increase of particle size during storage) are significantly improved. It is also found that the lipid nanoparticles of the present invention described later can efficiently encapsulate oil, drugs, etc. The present invention is completed based on this new insight.

[0015] That is, the present invention relates to, for example, the following technical aspects.

[0016] [1] A lipid nanoparticle comprising a hyaluronic acid derivative and a phospholipid,

[0017] The above-mentioned hyaluronic acid derivative is represented by the following general formula (1).

[0018]

[0019] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer greater than 1 and less than 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is not included. ].

[0020] [2] The lipid nanoparticle according to [1], wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, and a mixture of two or more thereof.

[0021] [3] The lipid nanoparticle according to [1] or [2], wherein the R6 is a linear alkyl group having 8 to 22 carbon atoms.

[0022] [4] The lipid nanoparticle according to any one of [1] to [3], wherein the modification rate of the hyaluronic acid derivative is 0.1% to 50%.

[0023] [5] The lipid nanoparticle according to any one of [1] to [4], further comprising a substance enclosed in an outer shell composed of at least the hyaluronic acid derivative and the phospholipid.

[0024] [6] The lipid nanoparticle according to [5], wherein the substance is a substance with poor water solubility.

[0025] [7] The lipid nanoparticle according to [5] or [6], wherein the substance is selected from the group consisting of a poorly water-soluble drug, a complex of a nucleic acid and a cationic lipid, an oil, and a mixture of two or more thereof.

[0026] [8] A method for producing lipid nanoparticles, comprising:

[0027] A step of dissolving a hyaluronic acid derivative in an aqueous solvent to form an aqueous phase;

[0028] A step of dissolving phospholipids in an organic solvent to form an oil phase; and

[0029] The oil phase is added dropwise to the water phase to disperse the oil phase.

[0030] The above-mentioned hyaluronic acid derivative is represented by the following general formula (1).

[0031]

[0032] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer greater than 1 and less than 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is not included. ].

[0033] [9] The production method according to [8], further comprising the step of subjecting the dispersion obtained in the dispersing step to a high-pressure emulsification treatment.

[0034] Effects of the Invention

[0035] According to the present invention, novel lipid nanoparticles and methods for making the same having excellent emulsifying power and emulsification stability can be provided. Lipid nanoparticles of the present invention are excellent in emulsifying power and emulsification stability by using specific hyaluronic acid derivatives and phospholipids in combination. In addition, lipid nanoparticles of the present invention can be efficiently enclosed in inner materials such as oil and medicine, so that they can be used as drug carriers of drug delivery systems without using PEG-modified lipids that demonstrate the possibility of causing allergic reactions. DETAILED DESCRIPTION

[0036] The following describes in detail the mode for carrying out the present invention. However, the present invention is not limited to the following embodiment.

[0037] <Features of the Invention>

[0038] (Lipid Nanoparticles)

[0039] The present invention is characterized by providing a lipid nanoparticle comprising a hyaluronic acid derivative and a phospholipid, wherein the hyaluronic acid derivative is represented by the following general formula (1).

[0040]

[0041] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups is excluded.]

[0042] (States of phospholipids and hyaluronic acid derivatives in lipid nanoparticles)

[0043] The hyaluronic acid derivative contained in the lipid nanoparticles of the present invention is covered with a lipid membrane layer formed of phospholipids (and, if necessary, shell (lipid membrane layer) constituents other than the hyaluronic acid derivative, such as lipids other than phospholipids).

[0044] More specifically, since the portion of the group represented by -O-CH2-CH(OH)-CH2-OR6 in the hyaluronic acid derivative (particularly the R6 portion) is adsorbed on the lipid membrane layer serving as the outer shell, it does not form a covalent bond with the components of the outer shell (lipid membrane layer) other than the hyaluronic acid derivative such as phospholipids and lipids other than phospholipids, and is contained in the lipid nanoparticles.

[0045] (Particle size of lipid nanoparticles and changes in particle size after storage)

[0046] The particle size of the lipid nanoparticles is greater than 0 nm and less than 1000 nm. In the present invention, "particle size" refers to the average particle size measured by dynamic light scattering. The particle size can be measured by a particle size measuring device using dynamic light scattering (e.g., Zetasizer Nano, manufactured by Malvern Panalytical).

[0047] From the perspective of excellent emulsifying power, the particle size of the lipid nanoparticles of the present invention can specifically be greater than 0 nm and less than 900 nm, greater than 10 nm and less than 800 nm, greater than 15 nm and less than 700 nm, greater than 20 nm and less than 600 nm, or greater than 25 nm and less than 500 nm.

[0048] In addition, the lipid nanoparticles of the present invention have excellent emulsification stability. Specifically, the average particle size immediately after preparation and the average particle size after storage at 40°C for 1 week are within 80 nm, can be within 70 nm, and can further be within 60 nm, within 50 nm, within 40 nm, and within 35 nm. It should be noted that the average particle size of the lipid nanoparticles of the present invention after storage is within the above range.

[0049] (Method for producing lipid nanoparticles)

[0050] The present invention is characterized in that it provides a method for producing lipid nanoparticles, which comprises: a step of dissolving a hyaluronic acid derivative in an aqueous solvent to form an aqueous phase; a step of dissolving a phospholipid in an organic solvent to form an oil phase; and a step of dropping the oil phase into the aqueous phase and dispersing it, wherein the hyaluronic acid derivative is represented by the following general formula (1).

[0051]

[0052] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups is excluded.]

[0053] <Hyaluronic acid derivatives>

[0054] The hyaluronic acid derivative used in the lipid nanoparticles of the present embodiment is a derivative represented by the following general formula (1).

[0055]

[0056] The hyaluronic acid derivative of the present embodiment has a structure in which a part or all of the hydroxyl groups are substituted by a group represented by -O-CH2-CH(OH)-CH2-OR6, wherein the hydroxyl groups refer to the hydroxyl groups bonded to the carbon atoms at positions 4 and 6 of N-acetylglucosamine constituting hyaluronic acid, the hydroxyl groups bonded to the carbon atoms at positions 2 and 3 of glucuronic acid constituting hyaluronic acid, and the hydroxyl group in the carboxyl group bonded to the carbon atom at position 5. It should be noted that, in the group represented by -O-CH2-CH(OH)-CH2-OR6, the oxygen atom recorded on the far left is bonded to the carbon atom constituting hyaluronic acid.

[0057] In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. It should be noted that when there are multiple R1s (when n is 2 or more), the R1s contained in different repeating units may be the same or different. Similarly, when there are multiple R2s (when n is 2 or more), the R2s contained in different repeating units may be the same or different. When there are multiple R3s (when n is 2 or more), the R3s contained in different repeating units may be the same or different. When there are multiple R4s (when n is 2 or more), the R4s contained in different repeating units may be the same or different. When there are multiple R5s (when n is 2 or more), the R5s contained in different repeating units may be the same or different.

[0058] Examples of the linear or branched alkyl group represented by R6 include a linear or branched alkyl group having 8 to 22 carbon atoms, a linear or branched alkyl group having 9 to 21 carbon atoms, a linear or branched alkyl group having 10 to 20 carbon atoms, a linear or branched alkyl group having 11 to 19 carbon atoms, and a linear or branched alkyl group having 12 to 18 carbon atoms.

[0059] More specific examples of the linear or branched alkyl group represented by R6 include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 2-methylbutyl, 1-methylbutyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, n-heptyl, n-octyl, isooctyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl (myristyl), n-hexadecyl (palmityl), n-octadecyl (stearyl), and n-eicosyl.

[0060] Examples of the straight-chain or branched alkenyl group represented by R6 include a straight-chain or branched alkenyl group having 8 to 22 carbon atoms, a straight-chain or branched alkenyl group having 9 to 21 carbon atoms, a straight-chain or branched alkenyl group having 10 to 20 carbon atoms, a straight-chain or branched alkenyl group having 11 to 19 carbon atoms, and a straight-chain or branched alkenyl group having 12 to 18 carbon atoms.

[0061] More specific examples of the straight-chain or branched alkenyl group represented by R6 include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl and oleyl.

[0062] As the linear or branched alkyl or alkenyl group represented by R6, a linear or branched alkyl group is preferred, and a linear alkyl group is more preferred, from the viewpoint of safety to a living body.

[0063] In the general formula (1), n ​​represents an integer of 1 to 2500. For example, n may be 2 to 2500, 4 to 2450, 6 to 2400, 8 to 2350, 10 to 2300, 12 to 2250, 14 to 2200, 16 to 2150, 17 to 2100, 18 to 2050, or 19 to 2000.

[0064] In general formula (1), when all of R1, R2, R3, R4 and R5 are hydroxyl groups, hyaluronic acid is obtained. Therefore, the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups is excluded from general formula (1).

[0065] (Modification rate of hyaluronic acid derivatives)

[0066] The modification rate of the hyaluronic acid derivative of the present embodiment can be, for example, greater than 0.1% and less than 50%. In this specification, the modification rate of the hyaluronic acid derivative is a value expressing the number of groups represented by -O-CH2-CH(OH)-CH2-OR6 contained in one structural unit of hyaluronic acid as a percentage. Here, one structural unit of hyaluronic acid refers to one structural unit formed by a disaccharide of glucuronic acid and N-acetylglucosamine. The number of groups represented by -O-CH2-CH(OH)-CH2-OR6 contained in one structural unit of hyaluronic acid can be, for example, expressed by 1 It was identified by H-NMR spectroscopy analysis.

[0067] From the perspective of improving emulsion stability, the modification rate of the hyaluronic acid derivative of this embodiment can be, for example, 0.5% to 45%, 1% to 40%, 2% to 30%, 3% to 20%, or 4% to 16%.

[0068] (Method for producing hyaluronic acid derivative)

[0069] The hyaluronic acid derivative of this embodiment can be prepared, for example, by reacting hyaluronic acid or its salt with a compound represented by the following general formula (2) (hereinafter also referred to as "Compound 1"). It should be noted that, in order to improve reactivity, the raw material hyaluronic acid or its salt can be replaced with an alkylammonium salt and then reacted with Compound 1.

[0070]

[0071] [R6 in the general formula (2) has the same meaning as R6 in the general formula (1).]

[0072] The raw material hyaluronic acid or its salt can be extracted from biological tissues such as animals (e.g., cockscombs, umbilical cords, skin, joint fluid), and can be obtained by culturing microorganisms, animal cells or plant cells (e.g., fermentation methods using bacteria of the genus Streptococcus), chemical or enzyme-synthesized substances, etc. There is no particular limitation on the salt of hyaluronic acid, but it is preferably a food or pharmaceutically acceptable salt, and for example, sodium salts, potassium salts, calcium salts, zinc salts, magnesium salts, ammonium salts, alkylammonium salts, etc. can be cited.

[0073] The average molecular weight of the raw material hyaluronic acid or its salt can be, for example, 411 to 1,020,000, but from the perspective of the ease of adsorption of lipid nanoparticles to the shell, improving the emulsification power and emulsification stability, it can be 1,000 to 1,000,000, 2,000 to 800,000, 4,000 to 700,000, 5,000 to 600,000, 6,000 to 500,000, 7,000 to 400,000, or 8,000 to 300,000.

[0074] In this specification, the average molecular weight of hyaluronic acid or a salt thereof is a value measured by the following method.

[0075] The following solution was used as the sample solution: about 0.05 g of hyaluronic acid and / or its salt (this product) was accurately weighed and dissolved in a 0.2 mol / L sodium chloride solution to accurately prepare a 100 mL solution; and 8 mL, 12 mL and 16 mL of the solution were accurately weighed, and a 0.2 mol / L sodium chloride solution was added thereto respectively to accurately prepare a 20 mL solution. For these sample solutions and 0.2 mol / L sodium chloride solution, the specific viscosity (Formula (A)) was measured at 30.0 ± 0.1 ° C according to the viscosity determination method (Method 1, capillary viscometer method) of the general test method of the Japanese Pharmacopoeia (15th edition revision), and the reduced viscosity (Formula (B)) at each concentration was calculated. The vertical axis is the reduced viscosity, and the concentration of this product relative to the converted dry product (g / 100 mL) is used as the horizontal axis to draw a graph, and the intrinsic viscosity is obtained from the intersection of the straight line connecting each point and the vertical axis. The intrinsic viscosity obtained here is substituted into the Laurent formula (formula (C)) to calculate the average molecular weight (M) (reference: TC Laurent, M. Ryan, A. Pietruszkiewicz,: BBA, 42, 476-485 (1960)).

[0076] (Formula A): Specific viscosity = {the number of seconds required for the sample solution to flow down) / (the number of seconds required for the 0.2 mol / L sodium chloride solution to flow down)} - 1

[0077] (Formula B): Reduced viscosity (dL / g) = specific viscosity / (concentration of the product relative to the converted dry product g / 100mL))

[0078] (Formula C): Intrinsic viscosity (dL / g) = 3.6 × 10 -4 M 0.78

[0079] (Content of hyaluronic acid derivatives)

[0080] The content of the hyaluronic acid derivative in the lipid nanoparticles of the present embodiment only needs to be a sufficient amount to cover the lipid membrane layer and improve the emulsification power and emulsification stability. For example, based on the total amount of lipid nanoparticles, it can be greater than 1 mass % and less than 90 mass %, or greater than 1.5 mass % and less than 85 mass %, greater than 2 mass % and less than 80 mass %, greater than 2.5 mass % and less than 70 mass %, greater than 3 mass % and less than 60 mass %, greater than 3.5 mass % and less than 50 mass %, greater than 4 mass % and less than 45 mass %, or greater than 5 mass % and less than 40 mass %.

[0081] <Phospholipids>

[0082] The lipid nanoparticles of the present embodiment include phospholipids as constituent components. As phospholipids, for example, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and sphingophospholipids such as nerve sheath myelin can be listed. One phospholipid can be used alone, or two or more can be used in combination.

[0083] As the phospholipid, for example, phospholipids derived from animal or plant materials such as egg yolk, soybean, or rapeseed can be used without particular limitation.

[0084] The content of phospholipids in the lipid nanoparticles of the present embodiment only needs to be a sufficient amount to form the lipid membrane layer. For example, based on the total amount of lipid nanoparticles, it can be greater than 1 mass % and less than 90 mass %, or greater than 2 mass % and less than 85 mass %, greater than 3 mass % and less than 80 mass %, greater than 4 mass % and less than 70 mass %, greater than 5 mass % and less than 60 mass %, greater than 7 mass % and less than 50 mass %, greater than 8 mass % and less than 40 mass %, greater than 9 mass % and less than 30 mass %, or greater than 10 mass % and less than 20 mass %.

[0085] From the perspective of improving emulsification power and emulsion stability, the content ratio of the hyaluronic acid derivative to the phospholipid in the lipid nanoparticles of the present embodiment (the content of the hyaluronic acid derivative (mass %): the content of the phospholipid (mass %)) can be, for example, in the range of 55:1 to 1:25, or in the range of 50:1 to 1:20, or in the range of 45:1 to 1:15. It should be noted that in the case of containing factors such as salts that make the emulsion state unstable as described later, the content ratio of the hyaluronic acid derivative can be higher in order to improve the emulsion stability.

[0086] <Contained material>

[0087] The lipid nanoparticles of the present embodiment have a core inside the shell consisting of at least hyaluronic acid derivatives and phospholipids, and can contain substances (included substances) in the core. The shell consisting of at least hyaluronic acid derivatives and phospholipids is composed of a lipid membrane layer, which is formed by the part of the group (particularly the part of R6) shown in the -O-CH2-CH (OH) -CH2-OR6 in the hyaluronic acid derivative and the fatty acid part in the phospholipid. In the case where the external phase of the lipid nanoparticles is composed of a carrier based on an aqueous solvent, the hydrophilic part of the hyaluronic acid derivative and the phospholipid is configured on the surface of the shell, and the inside of the shell becomes a hydrophobic environment, and a water-soluble substance can be appropriately included. In addition, in the case where the external phase of the lipid nanoparticles is composed of a carrier based on an organic solvent, the hydrophobic part of the hyaluronic acid derivative and the phospholipid is configured on the surface of the shell, and the inside of the shell becomes a hydrophilic environment, and a water-soluble substance can be appropriately included.

[0088] The type of the contained substance is not particularly limited, and may be any of a poorly water-soluble substance and a water-soluble substance. A poorly water-soluble substance refers to a substance with low solubility in water, specifically, a substance described in the 16th revised edition of the Japanese Pharmacopoeia as "slightly soluble" (the amount of solvent required to dissolve 1 g or 1 mL of solute is 30 mL or more and less than 100 mL), "slightly soluble" (the amount of solvent is 100 mL or more and less than 1000 mL), "very slightly soluble" (the amount of solvent is 1000 mL or more and less than 10000 mL), or "almost insoluble" (the amount of solvent is 10000 mL or more).

[0089] The poorly water-soluble substance may be a substance having physiological activity (poorly water-soluble drug). Examples of the poorly water-soluble substance include docetaxel, paclitaxel, capecitabine, oxaliplatin, gefitinib, doxorubicin, irinotecan, gemcitabine, pemetrexed, temozolomide, imatinib, vinorelbine, letrozole, teniposide, etoposide, podophyllotoxin, camptothecin, 10-hydroxycamptothecin, 9-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, irinotecan, vinblastine , vincristine, vindesine, vinflunine, vinpocetine, norcantharidin, silybin, propofol, florfenicol, mitiglinide, artemisinin, dihydroartemisinin, sirolimus, ibuprofen, nitrendipine, nicardipine, nimodipine, rebamipide, gliclazide, cisapride, felodipine, glibenclamide, acyclovir, oleanolic acid, scutellarin, ferulic acid, paracetamol, palmitoyl rhizoxin), Penclomedine, Tamoxifen, Vinblastine, Valproic acid, Tacrolimus, Cyclosporine A, Amphotericin B, Ketoconazole, Domperidone, Sulpiride, Fenofibrate, Bezafibrate, Azithromycin, Itraconazole, Miconazole, Brimonidine, Latanoprost, Silibinin, Erythromycin, Roxithromycin, Rifaximin, Cisapride, Cyclosporine, Diclofenac, Felodipine, Ibuprofen, Indomethacin, Acemetacin, Nicardi The water-soluble substances include phenoxyethanol, hydroxybenzoic acid, hydroxycinnamic acid, hydroxybenzoic ...

[0090] The water-soluble substance can be a substance with physiological activity (water-soluble drug). As a water-soluble substance, for example, nucleic acid, peptide, low molecular weight compound, etc. can be listed. As a nucleic acid, for example, DNA, mRNA, siRNA, microRNA, aptamer, ribozyme, etc. can be listed. It should be noted that nucleic acid can be used as a compound with cationic lipids as a poorly water-soluble substance, and is included in the lipid nanoparticles of the present embodiment. The water-soluble substance can be used alone or in combination of two or more.

[0091] When the lipid nanoparticles of the present embodiment contain an encapsulated substance, the encapsulated substance can be efficiently encapsulated in the lipid nanoparticles. The amount of the encapsulated substance contained in the lipid nanoparticles of the present embodiment (in the case of a water-soluble substance, it is expressed by the encapsulation rate; in the case of a poorly water-soluble substance, it is expressed by the content of the encapsulated substance in the lipid nanoparticles) can be 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, or 90% by mass or more after being stored at 40° C. for one week, based on the initial amount of the encapsulated substance.

[0092] The initial amount of the contained substance (content of contained substance) in the lipid nanoparticles of the present embodiment can be, for example, 1% by mass or more and 95% by mass or less, 1.1% by mass or more and 94% by mass or less, 1.2% by mass or more and 93% by mass or less, 1.3% by mass or more and 92% by mass or less, 1.4% by mass or more and 91.5% by mass or less, 1.5% by mass or more and 91% by mass or less, 1.6% by mass or more and 90.9% by mass or less, 2% by mass or more and 90% by mass or less, 2.5% by mass or more and 80% by mass or less, 3% by mass or more and 70% by mass or less, 3.5% by mass or more and 60% by mass or less, 4% by mass or more and 50% by mass or less, 4.5% by mass or more and 40% by mass or less, 5% by mass or more and 30% by mass or less, or 5.5% by mass or more and 20% by mass or less.

[0093] <Carrier>

[0094] The lipid nanoparticles of the present embodiment are usually prepared into a suspension containing the lipid nanoparticles of the present embodiment and a carrier. The suspension can also be said to be a composition containing lipid nanoparticles (composition containing lipid nanoparticles). The carrier constitutes the external phase of the lipid nanoparticles. As a carrier, for example, an aqueous solvent (such as water, buffer) used when forming an aqueous phase, an organic solvent used when forming an oil phase, etc. can be listed. As a buffer, for example, phosphate buffered saline (PBS), acetate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, MES buffer, HEPES buffer, etc. can be listed. As water, for example, distilled water, ordinary water, purified water, sterile purified water, water for injection, and distilled water for injection defined in the 18th edition of the revised Japanese Pharmacopoeia can be listed.

[0095] The content of the carrier in the lipid nanoparticle-containing composition of the present embodiment may be, for example, 0% by mass or more and less than 100% by mass based on the total amount of the lipid nanoparticle-containing composition.

[0096] <Other ingredients>

[0097] The composition containing lipid nanoparticles of this embodiment may further contain other food or pharmaceutically acceptable ingredients within the scope of not impairing the effects of the present invention. Other ingredients include, for example, lipids other than phospholipids, oils, organic solvents, sugars, salts, pH adjusters, buffers, etc.

[0098] (Lipids other than phospholipids)

[0099] As lipids other than phospholipids, for example, sterol lipids such as cholesterol and cholesterol derivatives (such as esters of cholesterol and fatty acids), cationic lipids such as oleylamine and 3,5-bis(dodecyloxy)benzylamine, etc. can be listed. Sterol lipids, for example, help to form the structure (lipid membrane layer) of lipid nanoparticles. For example, when a negatively charged substance such as nucleic acid is used as an inclusion substance, the cationic lipid forms a complex with the substance and is used to help the inclusion substance dissolve.

[0100] (grease)

[0101] The fat is used, for example, to help dissolve the contained substance. Examples of the fat include vegetable oils such as soybean oil, sesame oil, rapeseed oil, safflower oil, olive oil, castor oil, corn oil, cottonseed oil, rice bran oil, sunflower seed oil, grape seed oil, and wheat germ oil, medium chain triglycerides (MCT), and long chain triglycerides (LCT).

[0102] (carbohydrate)

[0103] Sugars are used, for example, to adjust osmotic pressure. Examples of sugars include sorbitol, xylitol, mannitol, glucose, trehalose, maltose, sucrose, raffinose, lactose, and dextran.

[0104] (Salts)

[0105] Salts are used, for example, to increase the inclusion rate of the inclusion substance (e.g., nucleic acid) or to adjust the osmotic pressure. As salts, for example, sodium chloride, potassium chloride, etc. can be cited. Salts are substances that cause the emulsification stability of lipid nanoparticles to decrease, but the lipid nanoparticles of the present embodiment have excellent emulsification power and emulsification stability, so even if salts are contained, emulsification stability can be maintained. Specifically, the content of salts (e.g., sodium chloride) in the lipid nanoparticles of the present embodiment, for example, based on the total amount of lipid nanoparticles, can be 0% by mass or more and 1500% by mass or less, 0% by mass or more and 1400% by mass or less, 0% by mass or more and 1300% by mass or less, 0% by mass or more and 1200% by mass or less, 0% by mass or more and 1156% by mass or less. It should be noted that the content of salts mentioned here also includes the amount of salts present in the external phase.

[0106] (pH adjuster)

[0107] Examples of the pH adjuster include strong acids such as hydrochloric acid and strong bases such as sodium hydroxide.

[0108] (Buffer)

[0109] Examples of the buffer include a phosphate buffer, a boric acid buffer, and a Tris buffer.

[0110] <Stabilizer>

[0111] The lipid nanoparticles of the present embodiment are excellent in emulsification stability due to the combination of hyaluronic acid derivatives and phospholipids. In addition, hyaluronic acid derivatives and phospholipids are excellent in the safety of organisms, and the possibility of causing allergic reactions is small. Therefore, the lipid nanoparticles of the present embodiment may be substantially free of stabilizers that may cause allergic reactions. As stabilizers that may cause allergic reactions, for example, polyethylene glycol (including free forms and forms combined with lipids, etc.) etc. can be listed. "Substantially free of stabilizers that may cause allergic reactions" means that, based on the total amount of lipid nanoparticles, the content of stabilizers that may cause allergic reactions is below 5 mass %, and may also be below 4 mass %, below 3 mass %, below 2 mass %, below 1 mass %, below 0 mass %.

[0112] <Lipid Nanoparticles>

[0113] (form)

[0114] The form of the composition containing lipid nanoparticles of this embodiment is not particularly limited and can be prepared in any form according to the intended use. Specifically, for example, it can be in the form of a suspension containing a carrier, the suspension itself, or a powder obtained by freeze-drying after removing the carrier.

[0115] (use)

[0116] The lipid nanoparticles of this embodiment can be used as, for example, a drug carrier that contains a physiologically active substance inside the shell (lipid membrane layer). Specifically, the lipid nanoparticles of this embodiment can be used as injections, eye drops, fat emulsions, etc.

[0117] (Giving form)

[0118] The lipid nanoparticles of this embodiment can be administered by injection, and can also be administered by spraying, dipping, rinsing, coating with a pad or a ball roller, brushing, dripping, aerosol spraying, or pump spraying.

[0119] (Given to)

[0120] The subject to which the lipid nanoparticles of the present embodiment are administered may be, for example, a mammal such as a human.

[0121] (Dosage)

[0122] The administration amount, administration timing, and administration period of the lipid nanoparticles of the present embodiment can be set according to the type of the physiologically active substance used, the type of the administration subject, and the like.

[0123] <Method for producing lipid nanoparticles>

[0124] The method for producing lipid nanoparticles of the present embodiment comprises: a step of dissolving a hyaluronic acid derivative in an aqueous solvent to form an aqueous phase (aqueous phase forming step); a step of dissolving a phospholipid in an organic solvent to form an oil phase (oil phase forming step); and a step of dropping the oil phase into the aqueous phase and dispersing it (dispersion step). In addition to the above steps, the method for producing lipid nanoparticles of the present embodiment may further comprise a step of high-pressure emulsification treatment of the dispersion obtained in the dispersion step (fine emulsification step).

[0125] (Water Phase Formation Step)

[0126] In the aqueous phase forming step, the hyaluronic acid derivative is dissolved in an aqueous solvent to form an aqueous phase. In addition, it is preferred that a water-soluble component other than the hyaluronic acid derivative is dissolved in an aqueous solvent together with the hyaluronic acid derivative in the aqueous phase forming step.

[0127] Examples of aqueous solvents include water and buffer solutions, and examples of buffer solutions include phosphate buffered saline (PBS), acetate buffer, citrate buffer, borate buffer, tartaric acid buffer, Tris buffer, MES buffer, and HEPES buffer.

[0128] The method for dissolving the hyaluronic acid derivative and, if necessary, the water-soluble component other than the hyaluronic acid derivative in the aqueous solvent is not particularly limited, and the hyaluronic acid derivative may be dissolved, for example, by stirring, etc. Alternatively, the hyaluronic acid derivative may be dissolved by heating to 40 to 80° C., if necessary.

[0129] (Oil Phase Forming Step)

[0130] In the oil phase forming step, the phospholipid is dissolved in the organic solvent to form the oil phase. In addition, it is preferred that a water-insoluble component other than the phospholipid is dissolved in the organic solvent together with the phospholipid in the oil phase forming step.

[0131] Examples of the organic solvent include ethanol, dimethylformamide (DMF), propylene glycol, benzene, acetone, dimethylformamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone, tetrahydrofuran, and chloroform.

[0132] The method for dissolving the phospholipid and, if necessary, the water-insoluble component other than the phospholipid in the organic solvent is not particularly limited, and the phospholipid may be dissolved, for example, by stirring, etc. Alternatively, the phospholipid may be dissolved by heating to 40 to 80° C. as necessary.

[0133] In the manufacturing method of this embodiment, the order of implementing the water phase forming step and the oil phase forming step is arbitrary. For example, the water phase forming step can be implemented first, the oil phase forming step can be implemented first, or the water phase forming step and the oil phase forming step can be implemented simultaneously.

[0134] (Dispersion process)

[0135] In the dispersion step, the oil phase is added dropwise to the aqueous phase and dispersed. By performing the dispersion step, the lipid nanoparticles of the present embodiment are obtained. The dispersion of the oil phase can be performed, for example, by slowly dropping the oil phase into the aqueous phase at a constant speed, then heating at 5000 to 15000 rpm for 1 to 30 minutes at 50 to 70° C. and stirring. The particle size of the lipid nanoparticles obtained in the dispersion step is usually in the range of 1 nm to 500 nm.

[0136] It should be noted that, by implementing the above-mentioned dispersion process, the hydrophilic part of the hyaluronic acid derivative and the phospholipid is arranged on the surface of the shell, and the inside of the shell becomes a hydrophobic environment, and a substance with poor water solubility can be appropriately included. If it is desired to make the inside of the shell a hydrophilic environment, for example, the water phase can be added dropwise to the oil phase in the dispersion process and dispersed.

[0137] (Emulsification process)

[0138] In the fine emulsification step, the dispersion obtained in the dispersion step is subjected to high-pressure emulsification treatment. The lipid nanoparticles of the present embodiment can also be obtained by implementing the fine emulsification step. The high-pressure emulsification treatment can be implemented, for example, using a high-pressure emulsifier. The high-pressure emulsification treatment can be implemented, for example, by passing the liquid through 3 to 30 times at a pressure of 50 to 200 MPa under a temperature condition of 50 to 70°C. As high-pressure emulsifiers, chamber-type high-pressure homogenizers such as Microfluidizer (Microfluidics Corp.), Nanomizer (Yoshida Machinery Co., Ltd.), and STAR BURST (SUGINO MACHINE), Gaulin-type homogenizer (APV), Lannier-type homogenizer (Lannier), high-pressure homogenizer (Niro Soavi), homogenizer (Sanwa Machinery Co., Ltd.), high-pressure homogenizer (Izumi Food Machinery Co., Ltd.), and ultra-high-pressure homogenizer (IKA) can be listed. Homogenizing valve-type high-pressure homogenizers. The particle size of the lipid nanoparticles obtained in the fine emulsification step is usually in the range of 1 nm to 250 nm.

[0139] (Other processes)

[0140] The method for producing lipid nanoparticles according to the present embodiment may further include a sterilization step and a pH adjustment step in addition to the above-mentioned steps, if necessary.

[0141] (Sterilization process)

[0142] In the sterilization step, the lipid nanoparticles obtained in the dispersion step or the refined emulsification step are sterilized. The sterilization of the lipid nanoparticles can be carried out according to conventional methods. Specifically, for example, the lipid nanoparticles can be sterilized by passing through a membrane filter (e.g., a nylon syringe filter) having a pore size of 0.2 to 0.22 μm.

[0143] (pH adjustment process)

[0144] In the pH adjustment process, a pH adjusting agent is mixed to adjust the pH of the lipid nanoparticles. The pH adjustment process can be implemented as needed and is not a necessary process. It is preferred to implement the pH adjustment process before the sterilization process. Specifically, for example, the pH adjustment process can be implemented by further adding a pH adjusting agent in the aqueous phase formation process, further adding a pH adjusting agent in the oil phase formation process, further adding a pH adjusting agent in the dispersion process, and further adding a pH adjusting agent in the refined emulsification process. The amount of the pH adjusting agent added can be set so that the pH of the lipid nanoparticles finally obtained is within the desired range.

[0145] Example

[0146] Hereinafter, the present invention will be described in more detail based on Examples etc. However, the present invention is not limited to the following Examples.

[0147] The corresponding relationship between the components and abbreviations used in the Examples is as follows.

[0148] MCT: COCONARD RK (trade name), manufactured by Kao Corporation (caprylic acid triglyceride)

[0149] PC: PC-98T (trade name), manufactured by Kewpie Co., Ltd. (egg yolk lecithin purified to a phosphatidylcholine content of 98% or more)

[0150] PBS: Phosphate buffered saline (pH 7.4), manufactured by Nacalai Tesque Co., Ltd.

[0151] CH: Cholesterol, manufactured by Tokyo Chemical Industry Co., Ltd. (cholesterol)

[0152] CHE: Cholesterol Oleate, manufactured by Tokyo Chemical Industry Co., Ltd. (cholesterol oleate)

[0153] OA: Oleic acid, manufactured by Tokyo Chemical Industry Co., Ltd.

[0154] CsA: Cyclosporine A, manufactured by LKT Laboratories

[0155] Tac: Tacrolimus hydrate, manufactured by Tokyo Chemical Industry Co., Ltd.

[0156] PL: PL-100M (trade name), Kewpie Co., Ltd. (egg yolk lecithin containing 80% phosphatidylcholine and 20% phosphatidylethanolamine)

[0157] LCT: Japanese Pharmacopoeia soybean oil, manufactured by Kao Corporation (long-chain triglyceride with fatty acids mainly containing 18 carbon atoms)

[0158] RD: Rebapate, manufactured by Tokyo Chemical Industry Co., Ltd.

[0159] NP: Nimodipine, manufactured by Tokyo Chemical Industry Co., Ltd.

[0160] [Test Example 1: Production of hyaluronic acid derivatives, and evaluation of emulsifying power and emulsification stability]

[0161] <Manufacturing of Hyaluronic Acid Derivatives>

[0162] The hyaluronic acid derivative was prepared according to the method described in Example 1 of Japanese Patent No. 4845071. Specifically, 5.0 g of hyaluronic acid (average molecular weight 8000, manufactured by Kewpie Co., Ltd.) was dissolved in 500 mL of water in a 1 L beaker, and a 40 w / v% tetrabutylammonium hydroxide aqueous solution was added while stirring to adjust the pH to 7.2. After pH adjustment, freeze drying was performed to obtain 10.2 g of tetrabutylammonium salt of hyaluronic acid. 1.0 g of tetrabutylammonium salt of hyaluronic acid, C 200,000 and 10.2 g of tetrabutylammonium hydroxide were added to a 30 mL sample bottle. 12～13 2.0 g of alkyl glycidyl ether (reaction reagent) (manufactured by Yokkaichi Synthetic Co., Ltd.) and 10 mL of dimethylformamide (DMF) were reacted in a water bath at 80°C for 8 hours while stirring. After the reaction was completed, 10 mL of 12.5 w / v% sodium chloride aqueous solution was added, and the pH was adjusted to 1.0 with 8 w / v% hydrochloric acid. Then, 50 mL of ethanol was slowly added while stirring to precipitate hyaluronic acid. Then, the pH was adjusted to 7.0 with 25 w / v% sodium hydroxide, the precipitate was recovered by filtration, and washed 3 times with 50 mL of 80 v / v% ethanol aqueous solution. The obtained precipitate was vacuum dried at 60°C to obtain 0.48 g of a hyaluronic acid derivative represented by the following general formula (1).

[0163]

[0164] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6.]

[0165] In the hyaluronic acid derivative produced in Test Example 1, R6 is an alkyl group having 12 or 13 carbon atoms, and n is an integer of 1-50.

[0166] <Manufacturing of lipid nanoparticles>

[0167] Lipid nanoparticles encapsulating MCT as a poorly water-soluble substance were prepared by the following method.

[0168] PC and MCT are dissolved in ethanol in the mode of becoming 0.1 mass % and 0.5 mass % respectively, thereby obtain ethanol solution.For the solution that 0.5mg hyaluronic acid derivative is dissolved in 4.5mL PBS, vigorously stir, drip 0.5mL ethanol solution simultaneously, prepare the lipid nanoparticle (embodiment 1) of inner MCT.In addition, except that ethanol solution does not include PC, prepare the lipid nanoparticle (comparative example 1) of inner MCT according to the method same as embodiment 1.

[0169] <Evaluation of Particle Size>

[0170] The particle size of the lipid nanoparticles of Example 1 and Comparative Example 1 was measured just after preparation and after storage at 40°C for 1 week. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 1. It should be noted that in this embodiment, the phospholipid content, salt content, content of the inner substance, and the content of the hyaluronic acid derivative are values ​​calculated according to the formula, and are based on the total amount of lipid nanoparticles. Here, PBS contains 0.9% by mass of sodium chloride.

[0171] [Table 1]

[0172]

[0173] The results of lipid nanoparticles prepared without using PC (Comparative Example 1) show that the emulsifying power (ability to generate smaller particles) and emulsification stability (ability to inhibit particle size increase during storage) of the hyaluronic acid derivative represented by general formula (1) itself are extremely low.

[0174] [Test Example 2: Evaluation of the emulsification-assisting effect of hyaluronic acid derivatives (pH 7.4)]

[0175] <Manufacturing of Hyaluronic Acid Derivatives>

[0176] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0177] <Manufacturing of lipid nanoparticles>

[0178] Lipid nanoparticles encapsulating MCT as a poorly water-soluble substance were prepared by the following method.

[0179] PC, CH and MCT are dissolved in ethanol in the mode of becoming 0.1 mass %, 0.2 mass % and 0.5 mass %, respectively, thus obtain ethanol solution.For the solution that hyaluronic acid derivative 5mg is dissolved in 4.5mL PBS, vigorously stir, drip 0.5mL ethanol solution simultaneously, prepare the lipid nanoparticle (embodiment 2) of inner bag MCT.In addition, except that hyaluronic acid derivative is not included in PBS, prepare the lipid nanoparticle (comparative example 2) of inner bag MCT according to the method same as embodiment 2.

[0180] <Evaluation of Particle Size>

[0181] The particle sizes of the lipid nanoparticles of Example 2 and Comparative Example 2 were measured immediately after preparation and after storage at 40° C. for 1 week. The particle sizes were measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 2.

[0182] [Table 2]

[0183]

[0184] It can be seen that by adding the hyaluronic acid derivative represented by the general formula (1) to lipid nanoparticles mainly composed of phospholipids, the emulsifying power (the ability to generate smaller particles) and the emulsification stability (the ability to inhibit the increase in particle size during storage) are significantly improved. In other words, it can be seen that the hyaluronic acid derivative represented by the general formula (1) has an excellent effect of assisting emulsification.

[0185] [Test Example 3: Evaluation of the emulsification-assisting effect of hyaluronic acid derivatives (pH 4.0)]

[0186] <Manufacturing of Hyaluronic Acid Derivatives>

[0187] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0188] <Manufacturing of lipid nanoparticles>

[0189] Lipid nanoparticles encapsulating MCT as a poorly water-soluble substance were prepared by the following method.

[0190] PC, CH and MCT are dissolved in ethanol in the mode of becoming 0.125 mass %, 0.25 mass % and 0.625 mass % respectively, so as to obtain ethanol solution.For the solution that hyaluronic acid derivative 5mg is dissolved in 100mM acetate buffer (pH4.0) 5mL is vigorously stirred, 0.5mL ethanol solution is added dropwise simultaneously, the lipid nanoparticle (embodiment 3) of inner MCT is prepared.In addition, except that hyaluronic acid derivative is not included in 100mM acetate buffer, the lipid nanoparticle (comparative example 3) of inner MCT is prepared according to the method same as embodiment 3.

[0191] <Evaluation of Particle Size>

[0192] The particle size of the lipid nanoparticles of Example 3 and Comparative Example 3 was measured immediately after preparation and after storage at room temperature for 1 day. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 3. Here, 100 mM acetic acid buffer contains 0.18 mass % of sodium acetate.

[0193] [Table 3]

[0194]

[0195] It can be seen that, similar to Test Example 2, even when the pH is different, by adding the hyaluronic acid derivative represented by the general formula (1) to lipid nanoparticles mainly composed of phospholipids, the emulsifying power (the ability to generate smaller particles) and the emulsification stability (the ability to inhibit the increase in particle size during storage) are significantly improved.

[0196] [Test Example 4: Evaluation of the emulsification-assisting effect (freeze-thaw resistance) of hyaluronic acid derivatives]

[0197] <Manufacturing of Hyaluronic Acid Derivatives>

[0198] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0199] <Manufacturing of lipid nanoparticles>

[0200] Lipid nanoparticles encapsulating MCT as a poorly water-soluble substance were prepared by the following method.

[0201] PC, CH and MCT are dissolved in ethanol in the mode of becoming 0.125 mass %, 0.25 mass % and 0.625 mass %, respectively, thus obtain ethanol solution.For the solution that hyaluronic acid derivative 5mg is dissolved in 5mL PBS, vigorously stir, drip 0.5mL ethanol solution simultaneously, prepare the lipid nanoparticle (embodiment 4) of inner bag MCT.In addition, except that hyaluronic acid derivative is not included in PBS, prepare the lipid nanoparticle (comparative example 4) of inner bag MCT according to the method same as embodiment 4.

[0202] <Evaluation of Particle Size>

[0203] The lipid nanoparticles of Example 4 and Comparative Example 4 were freeze-thawed. That is, each lipid nanoparticle was cooled to -40°C to freeze rapidly and then thawed at room temperature. Next, the particle size of the lipid nanoparticles of Example 4 and Comparative Example 4 was measured just after preparation and after freeze-thaw. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 4.

[0204] [Table 4]

[0205]

[0206] It is known that by adding the hyaluronic acid derivative represented by the general formula (1) to lipid nanoparticles based on phospholipids, the emulsification stability during freeze-thaw (the ability to inhibit the increase in particle size during storage) is significantly improved. It should be noted that easily decomposable pharmaceutical products are usually stored frozen, but lipid nanoparticles based on phospholipids are difficult to store frozen. The lipid nanoparticles of the present invention can also be stored frozen.

[0207] [Test Example 5: Evaluation of Modified Examples of Hyaluronic Acid Derivatives]

[0208] <Manufacturing of Hyaluronic Acid Derivatives>

[0209] (Hyaluronic acid derivative A)

[0210] A hyaluronic acid derivative A was prepared in the same manner as in Test Example 1. The average molecular weight of hyaluronic acid, a raw material of the hyaluronic acid derivative A, was 8000, and R6 in the general formula (1) was an alkyl group having 12 or 13 carbon atoms.

[0211] (Hyaluronic acid derivative B)

[0212] Using C 18 Alkyl glycidyl ether replaces C 12～13 Hyaluronic acid derivative B was prepared in the same manner as in Test Example 1 except that alkyl glycidyl ether was used. The average molecular weight of hyaluronic acid as a raw material of hyaluronic acid derivative B was 8000, and R6 in general formula (1) was an alkyl group having 18 carbon atoms.

[0213] (Hyaluronic acid derivative C)

[0214] Hyaluronic acid derivative C was obtained in the same manner as in Test Example 1 except that high molecular weight hyaluronic acid (average molecular weight 100,000, manufactured by Kewpie Co., Ltd.) was used instead of hyaluronic acid (average molecular weight 8,000, manufactured by Kewpie Co., Ltd.). The average molecular weight of hyaluronic acid as a raw material of hyaluronic acid derivative C was 100,000, and R6 in general formula (1) was an alkyl group having 12 or 13 carbon atoms.

[0215] <Manufacturing of lipid nanoparticles>

[0216] Lipid nanoparticles encapsulating MCT as a poorly water-soluble substance were prepared by the following method.

[0217] PC, CH, and MCT were dissolved in ethanol to give 0.1 mass %, 0.2 mass %, and 0.5 mass %, respectively, to obtain ethanol solutions.

[0218] For the solution that hyaluronic acid derivative A5mg is dissolved in 4.5mL PBS, vigorous stirring is carried out, and 0.5mL ethanol solution is added dropwise simultaneously, and the lipid nanoparticle (embodiment 7) of MCT is prepared. In addition, hyaluronic acid (average molecular weight 8000, Kewby Co., Ltd. system) 2.5mg and hyaluronic acid derivative A2.5mg are used to replace hyaluronic acid derivative A5mg, in addition, the lipid nanoparticle (embodiment 6) of MCT is prepared according to the same method as Example 7. Further, hyaluronic acid (average molecular weight 8000, Kewby Co., Ltd. system) 4mg and hyaluronic acid derivative A1mg are used to replace hyaluronic acid derivative A5mg, in addition, the lipid nanoparticle (embodiment 5) of MCT is prepared according to the same method as Example 7. Further in addition, hyaluronic acid (average molecular weight 8000, Kewby Co., Ltd. system) 5mg is used to replace hyaluronic acid derivative A5mg, in addition, the lipid nanoparticle (comparative example 5) of MCT is prepared according to the same method as Example 7. Comparative Example 5, Example 5, Example 6 and Example 7 correspond to the case where the modification rate of hyaluronic acid derivative A is changed to 0%, 1%, 2.5% and 5%, respectively. It should be noted that the modification rates of hyaluronic acid derivative A, hyaluronic acid derivative B and hyaluronic acid derivative C are modified by 1 The content of MgCl2O3 was determined by H-NMR spectral analysis.

[0219] To a solution prepared by dissolving 5 mg of hyaluronic acid derivative B or 5 mg of hyaluronic acid derivative C in 4.5 mL of PBS, 0.5 mL of ethanol solution was added dropwise while vigorously stirring to prepare lipid nanoparticles encapsulating MCT (Examples 8 and 9).

[0220] <Evaluation of Particle Size>

[0221] The particle sizes of the lipid nanoparticles of Comparative Example 5 and Examples 5 to 9 were measured immediately after preparation and after storage at room temperature for 1 day. The particle sizes were measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 5.

[0222] [Table 5]

[0223]

[0224] As shown in Table 5, even when the average molecular weight of the raw material hyaluronic acid, the number of carbon atoms of the alkyl group represented by R6 in the general formula (1), or the modification rate are changed, the emulsifying power (the ability to generate smaller particles) and the emulsification stability (the ability to inhibit the increase in particle size during storage) are significantly improved by adding the hyaluronic acid derivative represented by the general formula (1) to lipid nanoparticles mainly composed of phospholipids.

[0225] [Test Example 6: Evaluation of Variations of Enclosed Substances]

[0226] <Manufacturing of Hyaluronic Acid Derivatives>

[0227] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0228] <Manufacturing of lipid nanoparticles>

[0229] Lipid nanoparticles containing poorly water-soluble substances (MCT, CHE or OA) were prepared by the following method.

[0230] PC, CH and poorly water-soluble substances (MCT) are dissolved in ethanol in the mode of becoming 0.1 mass %, 0.2 mass % and 0.5 mass %, respectively, so as to obtain ethanol solution. For the solution of hyaluronic acid derivative 5mg being dissolved in 4.5mL PBS, vigorous stirring is carried out, 0.5mL ethanol solution is added dropwise simultaneously, the lipid nanoparticles (embodiment 10) of the substance (MCT) of inner bag poor water solubility are prepared. PC and poorly water-soluble substances (CHE or OA) are dissolved in ethanol in the mode of becoming 0.1 mass % and 0.5 mass %, respectively, so as to obtain ethanol solution. For the solution of hyaluronic acid derivative 5mg being dissolved in 4.5mL PBS, vigorous stirring is carried out, 0.5mL ethanol solution is added dropwise simultaneously, the lipid nanoparticles (embodiments 11 and 12) of the substance (CHE or OA) of inner bag poor water solubility are prepared.

[0231] <Evaluation of Particle Size>

[0232] The particle sizes of the lipid nanoparticles of Examples 10 to 12 were measured immediately after preparation and after storage at 40° C. for 1 week. The particle sizes were measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 6.

[0233] [Table 6]

[0234]

[0235] As shown in Table 6, the lipid nanoparticles of the present invention can dissolve various poorly water-soluble substances (MCT, CHE or OA), and at this time have excellent emulsification power (the ability to generate smaller particles) and emulsification stability (the ability to inhibit the increase in particle size during storage).

[0236] [Test Example 7: Evaluation of Variations of Enclosed Substances]

[0237] <Manufacturing of Hyaluronic Acid Derivatives>

[0238] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0239] <Manufacturing of lipid nanoparticles>

[0240] Lipid nanoparticles containing nucleic acid-lipid complexes were prepared by the following method.

[0241] PC, CH and oleylamine were dissolved in ethanol in a manner of 0.25 mass %, 0.5 mass % and 1.25 mass % respectively to obtain an ethanol solution. A solution of 1 mg of salmon testis-derived deoxyribonucleic acid and 1.2 mg of a hyaluronic acid derivative dissolved in 9 mL of 100 mM acetic acid buffer (pH 1.5) was vigorously stirred, and 1 mL of the ethanol solution was added dropwise to prepare lipid nanoparticles containing nucleic acid-lipid complexes (Example 13).

[0242] <Evaluation of Particle Size and Nucleic Acid Encapsulation Rate>

[0243] The particle size and nucleic acid inclusion rate of the lipid nanoparticles of Example 13 were measured just after preparation. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The nucleic acid inclusion rate was measured by the following method. The prepared lipid nanoparticles were passed through an ultrafiltration membrane (trade name: Vivaspin 20, manufactured by Sartorius, molecular weight cutoff: 100KDa), and the filtered external aqueous phase was used as a measurement sample. Using a spectrophotometer (manufactured by Shimadzu Corporation), the measurement sample was measured together with a quantitative DNA solution prepared to 0.1 mg / mL to 0.001 mg / mL, and the absorption wavelength at 260 nm was measured to quantify the DNA concentration. The nucleic acid inclusion rate is calculated using the following formula. Here, the total DNA concentration is a concentration corresponding to the total amount of deoxyribonucleic acid derived from the salmon testis used, which is 0.1 mg / mL.

[0244] Nucleic acid inclusion rate = (total DNA concentration - external water phase DNA concentration) / total DNA concentration

[0245] The results are shown in Table 7.

[0246] [Table 7]

[0247]

[0248] As shown in Table 7, the lipid nanoparticles of the present invention can efficiently dissolve nucleic acid lipid complexes (complexes of oleylamine as a cationic lipid and nucleic acids) (nucleic acid inclusion rate 70.3%), and also have excellent emulsification power (ability to generate smaller particles).

[0249] [Test Example 8: Evaluation of Variations of Enclosed Substances]

[0250] <Manufacturing of Hyaluronic Acid Derivatives>

[0251] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0252] <Manufacturing of lipid nanoparticles>

[0253] CsA-encapsulated lipid nanoparticles were prepared by the following method.

[0254] PC and CsA are dissolved in ethanol in the mode of becoming 10 mass % and 1 mass %, respectively, thereby obtain ethanol solution.For the solution that hyaluronic acid derivative 250mg is dissolved in 4.5mL PBS, vigorously stir, drip 0.5mL ethanol solution simultaneously, prepare the lipid nanoparticle (embodiment 14) of inner bag CsA.In addition, except that PBS does not comprise hyaluronic acid derivative, prepare the lipid nanoparticle (comparative example 6) of inner bag CsA according to the method same as embodiment 14.

[0255] <Evaluation of Particle Size and Appearance>

[0256] The particle size and appearance of the lipid nanoparticles of Example 14 and Comparative Example 6 were measured and observed immediately after preparation. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The appearance was observed visually. The results are shown in Table 8.

[0257] [Table 8]

[0258]

[0259] As shown in Table 8, the lipid nanoparticles of the present invention can dissolve various poorly water-soluble substances (CsA in this test example), and the emulsifying power (ability to generate smaller particles) is excellent. In addition, it can be confirmed that the hyaluronic acid derivative can be blended without any problem even at 5% by mass.

[0260] [Test Example 9: Evaluation of Variations of Enclosed Substances]

[0261] <Manufacturing of Hyaluronic Acid Derivatives>

[0262] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0263] <Manufacturing of lipid nanoparticles>

[0264] Lipid nanoparticles containing a poorly water-soluble substance (CsA or Tac) were prepared by the following method.

[0265] PC, a substance with poor water solubility (CsA or Tac) and ethanol are mixed and heated to 60°C to form an oil phase. A hyaluronic acid derivative is dissolved in PBS to form an aqueous phase. The aqueous phase heated to 60°C is stirred, and the oil phase is added dropwise for 5 minutes for coarse emulsification. The coarse emulsion is treated 10 times with a high-pressure emulsifier (Microfluidizer, manufactured by Microfluidics Corp.) at 150 MPa to prepare lipid nanoparticles containing a substance with poor water solubility (CsA or Tac) (Examples 15 to 17). It should be noted that since the contained substance has poor water solubility, it can be said that the contained substance that is not present in the external phase but present in the suspension can be dissolved in the lipid nanoparticles.

[0266] <Evaluation of Particle Size, Appearance, and Content of Inclusion Material in Suspension>

[0267] The particle size, appearance and content of the contained substance in the suspension of the poorly water-soluble substance (CsA or Tac) of the lipid nanoparticles of Examples 15 to 17 were measured immediately after preparation and after storage at 40°C for 1 week. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The appearance was observed visually. The content of contained substances in the suspension was measured using high performance liquid chromatography (manufactured by Nihon Waters). Specifically, the chromatographic column used was Waters Eclipse XDB-C18 (4.6×150mm, 5μm), the mobile phase was acetonitrile: water: trifluoroacetic acid = 75:25:0.2, the detection method was UV (210nm), the flow rate during CsA determination was 1.2mL / min, the temperature was 60°C, and the flow rate during Tac determination was 1.0mL / min, the temperature was set to 40°C, and the contents of CsA and Tac in the suspension were quantified. The results are shown in Table 9.

[0268] [Table 9]

[0269]

[0270] As shown in Table 9, the lipid nanoparticles of the present invention can dissolve various poorly water-soluble substances (CsA or Tac), and at this time have excellent emulsification power (ability to generate smaller particles) and emulsion stability (ability to inhibit particle size increase during storage).

[0271] [Test Example 10: Evaluation of variations of phospholipids and encapsulated substances]

[0272] <Manufacturing of Hyaluronic Acid Derivatives>

[0273] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0274] <Manufacturing of lipid nanoparticles>

[0275] Lipid nanoparticles containing a poorly water-soluble substance (LCT) were prepared by the following method.

[0276] PL and LCT are mixed and heated to 60°C to form an oil phase. A hyaluronic acid derivative is dissolved in PBS to form an aqueous phase. The aqueous phase heated to 60°C is stirred and the oil phase is added dropwise for coarse emulsification for 5 minutes. The coarse emulsion is treated 5 times with a high-pressure emulsifier (Microfluidizer, manufactured by Microfluidics Corp.) at 150 MPa to prepare lipid nanoparticles containing LCT (Examples 18 to 19). In addition, lipid nanoparticles containing LCT are prepared in the same manner as in Examples 18 and 19, except that PL is not included in the oil phase (Comparative Example 7). Furthermore, lipid nanoparticles containing LCT are prepared in the same manner as in Examples 18 and 19, except that the hyaluronic acid derivative is not included in the aqueous phase (Comparative Example 8).

[0277] <Evaluation of Particle Size>

[0278] 10 mL of the suspension of lipid nanoparticles prepared in Comparative Examples 7 to 8 and Examples 18 to 19 was weighed into a vial, sealed after adding 90 mg of sodium chloride and stored at 40°C. The particle size was measured just after preparation and after being stored at 40°C for 1 week. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 10.

[0279] [Table 10]

[0280]

[0281] When a poorly water-soluble substance (LCT) is dissolved using a phospholipid monomer (Comparative Example 8) or a hyaluronic acid derivative monomer (Comparative Example 7), the emulsification stability is extremely low. However, by using a combination of phospholipids and hyaluronic acid derivatives, it is confirmed that the emulsification stability (the ability to inhibit the increase in particle size during storage) is significantly improved (Examples 18 and 19).

[0282] [Test Example 11: Evaluation of Variations of Enclosed Substances]

[0283] <Manufacturing of Hyaluronic Acid Derivatives>

[0284] A hyaluronic acid derivative was prepared in the same manner as in Test Example 1.

[0285] <Manufacturing of lipid nanoparticles>

[0286] Lipid nanoparticles containing poorly water-soluble substances (MCT and RD, or MCT and NP) were prepared by the following method.

[0287] PL, MCT, a drug (RD or NP) as a poorly water-soluble substance and an organic solvent (ethanol or dimethylformamide (DMF)) are mixed and heated to 60°C to form an oil phase. A hyaluronic acid derivative is dissolved in pure water or in a solution formed by adding 1 mol / L sodium hydroxide aqueous solution to pure water to form an aqueous phase. The aqueous phase heated to 60°C is stirred while the oil phase is dropped and coarsely emulsified for 5 minutes. The coarse emulsion is treated 5 times at 150 MPa with a high-pressure emulsifier (Microfluidizer, manufactured by Microfluidics Corp.) to prepare lipid nanoparticles containing poorly water-soluble substances (MCT and RD, or MCT and NP) (Examples 20 to 22).

[0288] <Evaluation of Particle Size>

[0289] The particle sizes of the lipid nanoparticles of Examples 20 to 22 were measured immediately after preparation. The particle sizes were measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 11. Here, the 1 mol / L sodium hydroxide aqueous solution contained 0.4 mass % of sodium hydroxide.

[0290] [Table 11]

[0291]

[0292] As shown in Table 11, it can be seen that the lipid nanoparticles of the present invention can dissolve various poorly water-soluble substances (MCT and RD, or MCT and NP), and in this case, the emulsifying power (ability to generate smaller particles) is excellent.

[0293] [Test Example 12: Evaluation of Modified Examples of Hyaluronic Acid Derivatives]

[0294] <Manufacturing of Hyaluronic Acid Derivatives>

[0295] A hyaluronic acid derivative with a high modification rate was obtained by the same method as the hyaluronic acid derivative A of Test Example 5 except that the reaction time in the 80°C water bath was extended to more than 8 hours. 1 Determined by H-NMR spectral analysis.

[0296] <Manufacturing of lipid nanoparticles>

[0297] The LCT-encapsulated lipid nanoparticles were prepared by the following method.

[0298] PL and LCT were mixed and heated to 60°C to form an oil phase. A hyaluronic acid derivative was dissolved (suspended) in PBS to form an aqueous phase. The aqueous phase heated to 60°C was stirred while the oil phase was added dropwise for 5 minutes for rough emulsification. The rough emulsion was treated 5 times with a high-pressure emulsifier (Microfluidizer, manufactured by Microfluidics Corp.) at 150 MPa to prepare lipid nanoparticles containing LCT (Example 23).

[0299] <Evaluation of Particle Size>

[0300] The particle size of the lipid nanoparticles of Example 23 was measured immediately after preparation and after storage at 40° C. for 1 week. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical). The results are shown in Table 12.

[0301] [Table 12]

[0302]

[0303] As shown in Table 12, the lipid nanoparticles of the present invention are found to be excellent in emulsifying power (ability to generate smaller particles) and emulsification stability (ability to suppress increase in particle size during storage) even when the modification ratio of the hyaluronic acid derivative is changed.

Claims

1. A method for producing lipid nanoparticles, comprising: A step of dissolving a hyaluronic acid derivative in an aqueous solvent to form an aqueous phase; A step of dissolving phospholipids in an organic solvent to form an oil phase; and The oil phase is added dropwise to the water phase to disperse the oil phase. The hyaluronic acid derivative is represented by the following general formula (1): In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6, R6 represents a linear or branched alkyl or alkenyl group, and n represents an integer of 1 to 2500, wherein: The case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded. 2 . The production method according to claim 1 , further comprising a step of subjecting the dispersion obtained in the dispersing step to a high-pressure emulsification treatment.

3. The manufacturing method according to claim 1 or 2, wherein: The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, and a mixture of two or more thereof.

4. The production method according to any one of claims 1 to 3, wherein The R6 is a linear alkyl group having 8 to 22 carbon atoms.

5. The production method according to any one of claims 1 to 4, wherein The modification rate of the hyaluronic acid derivative is 0.1% or more and 50% or less.

6. The production method according to any one of claims 1 to 5, wherein The lipid nanoparticles further contain a substance enclosed in an outer shell composed of at least the hyaluronic acid derivative and the phospholipid.

7. The manufacturing method according to claim 6, wherein: The substance is a poorly water-soluble substance.

8. The manufacturing method according to claim 6 or 7, wherein: The substance is selected from the group consisting of poorly water-soluble drugs, complexes of nucleic acids and cationic lipids, oils, and mixtures of two or more thereof.

9. A lipid nanoparticle comprising a hyaluronic acid derivative and a phospholipid, The hyaluronic acid derivative is represented by the following general formula (1): In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6, R6 represents a linear or branched alkyl or alkenyl group, and n represents an integer of 1 to 2500, wherein: The case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.

10. The lipid nanoparticle according to claim 9, wherein The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, and a mixture of two or more thereof.

11. The lipid nanoparticle according to claim 9 or 10, wherein The above-mentioned R6 is a linear alkyl group having 8 to 22 carbon atoms.

12. The lipid nanoparticle according to any one of claims 9 to 11, wherein The modification rate of the hyaluronic acid derivative is 0.1% or more and 50% or less. 13 . The lipid nanoparticle according to claim 9 , further comprising a substance enclosed in an outer shell composed of at least the hyaluronic acid derivative and the phospholipid.

14. The lipid nanoparticle according to claim 13, wherein The substance is a poorly water-soluble substance.

15. The lipid nanoparticle according to claim 13 or 14, wherein The substance is selected from the group consisting of poorly water-soluble drugs, complexes of nucleic acids and cationic lipids, oils, and mixtures of two or more thereof.

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