Particle-bound lipid particle, method of preparation, kit, and combination composition

Abstract

To provide a technique for delivering an active substance to the surface of a target cell.SOLUTION: According to one embodiment, a particle-bound lipid particle includes a biodegradable lipid particle, a particle, and active substances, wherein the particle is bound to an outer surface of the lipid particle, and the active substances are bound to a surface of the particle.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] SUMMARY OF THE INVENTION Embodiments of the present invention relate to particle-associated lipid particles, methods of making, kits and combination compositions. [Background technology]

[0002] Lipid particles are known as a means of delivering desired active substances, such as therapeutic genes and drugs, into cells. Lipid particles encapsulate the active substance to be delivered within a lipid membrane capsule. Lipid particles generally release the active substance into target cells via pathways such as membrane fusion and endocytosis.

[0003] Meanwhile, TRAIL receptors have been attracting attention as molecular targets for cancer therapy. TRAIL (TNF-related apoptosis-inducing ligand) is a cytokine involved in tumor immune surveillance. In recent years, it has been revealed that the expression level of Lewis sugar chains attached to lipids on cancer cells is key to promoting cancer cell death mediated by TRAIL receptors. In such treatments that utilize pathways to deliver active substances to the cell membrane surface, it is necessary to retain the active substance on the cell membrane surface, which is difficult to achieve with conventional lipid particle forms that deliver active substances inside cells. Summary of the Invention [Problem to be solved by the invention]

[0004] The problem to be solved by the present invention is to provide a technique for delivering active substances to the surface of target cells. [Means for solving the problem]

[0005] The particle-bound lipid particles according to the embodiment include a biodegradable lipid particle, a particle, and an active substance. The particle is bound to the outer surface of the biodegradable lipid particle. The active substance is bound to the surface of the particle. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 is a schematic diagram showing an example of a particle-bound lipid particle according to a first embodiment. [Figure 2] FIG. 1 is a schematic diagram showing an example of a particle-bound lipid particle according to a first embodiment. [Figure 3] FIG. 1 is a schematic diagram illustrating the concept of an example of a manufacturing method according to a second embodiment. [Figure 4] FIG. 4 is a schematic diagram showing an example of a manufacturing method according to a second embodiment. [Figure 5] FIG. 1 is a schematic diagram illustrating an example according to an embodiment. [Figure 6] 1A and 1B are schematic diagrams showing a comparative example (a) and an example (b) used in the experiment of Example 1. [Figure 7] An image showing the experimental results. [Figure 8] An image showing the experimental results. [Figure 9] An image showing the experimental results. [Figure 10] Schematic diagram showing a comparative example (a) and an example (b) used in the experiment of Example 2. FIG. [Figure 11] An image showing the experimental results. [Figure 12] An image showing the experimental results. DETAILED DESCRIPTION OF THE INVENTION

[0007] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially identical components are denoted by the same reference numerals, and some of their descriptions may be omitted. The drawings are schematic, and the relationship between the thickness of each component and the planar dimensions, the thickness ratio of each component, etc. may differ from the actual ones.

[0008] As used herein, the term "lipid particle" refers to a particle whose main component is lipid. For example, the term "lipid particle" as used herein includes commonly known forms such as lipid nanoparticles (LNPs), liposomes, and microemulsions.

[0009] (First embodiment) A particle-bound lipid particle according to an embodiment will be described with reference to FIG. 1 . The particle-bound lipid particle 10 includes a biodegradable lipid particle 11, an active substance 12, and a particle 14. The particle 14 is bound to the outer surface of the lipid particle 11. The active substance 12 is bound to the surface of the particle 14. Regarding the binding between the lipid particle 11 and the particle 14, for example, the particle 14 may be bound directly to a portion of the lipid constituting the lipid particle 11, or may be bound to the lipid particle 11 via a linker 13 contained in the lipid particle 11. For example, the linker 13 may be a known linker structure that is bound to and / or extends from a functional group of any lipid constituting the lipid particle 11. For example, the linker 13 may be a portion of any lipid constituting the biodegradable lipid particle 11, such as a portion of a PEG-modified lipid or a portion of a lipid having a functional group capable of binding a ligand. For example, the linker 13 may be a PEG-modified group having a thiol group at its terminus. FIG. 1 shows an example of a lipid bound to a modifying group, in which the lipid is bound to cholesterol. However, the linker 13 is not limited to this. As will be described in detail later, the lipids constituting the lipid particle 11 may contain one or more types of PEG-modified lipids, lipids having functional groups capable of binding ligands, etc., and when the lipid particle 11 is formed, a portion of the lipid or the modifying group may be used as the linker 13. Examples of functional groups capable of binding ligands include, but are not limited to, active ester groups, thiol groups, amino groups, maleimide groups, and carboxy groups. For example, the linker 13 may be contained in the lipid material or the formed lipid particle 11 at a linker density of 0.1 to 5%. By using the linker 13 and the particles 14, the particles 14 can be stably bound to the lipid particle 11 and positioned outside the lipid particle 11.

[0010] For example, the bonding mode between the linker 13 and the particle 14 may be determined depending on the material of the particle and / or the structure of the linker 13. As described above, the example in FIG. 1 shows an example in which a thiol group is present at the outer end of the linker 13. However, this is not limiting, and functional groups such as amino groups, active ester groups, maleimide groups, and carboxy groups may also be used. Meanwhile, the particle 14 may be composed of a material capable of bonding with or having affinity for the linker 13. For example, the particle 14 may be composed of a material capable of bonding with or having affinity for the thiol group and capable of bonding with the desired active substance 12. Such particles 14 may be selected from the group consisting of, for example, gold, silver, iron oxide, titanium oxide, zinc oxide, silica, lipid particles, polymer particles, and combinations of any two or more thereof, or may be particles primarily composed of these materials. Furthermore, the particle surface may be physically treated. Such surface treatment may be, for example, hydrophilization, which may facilitate bonding with the linker and / or immobilization or binding of the radioactive substance. The particles may be cell surface-retaining microparticles. In other words, for example, they have a diameter that does not pass through the cell membrane. Figure 2 shows an image of cell surface-retaining microparticle-bound lipid particles when applied to cells. The particles do not pass through the cell membrane 21 of the cell 20 and enter the cell 22, and therefore remain on the outside 23 of the cell membrane. For example, the particles have the property of remaining on the cell membrane for a certain period of time. Such a property may depend, for example, on the size of the particle. It is desirable that the particles are of a size that does not penetrate the cell membrane or that allows minimal penetration of the cell membrane. The certain period of time may be the time required for the active substance on the particle to act, such as 10 minutes, 1 hour, 1 day, or 1 week.

[0011] For example, the particle diameter may be 1 nm to 10 μm. More preferably, it is in the range of 10 nm to 5 μm, and even more preferably, 100 nm to 5 μm. For some cells, if the particle size is too small, uptake by endocytosis or the like may be promoted, making retention on the cell membrane difficult. Therefore, it is preferable to select an appropriate particle size each time. For example, one or more active substances 12 may be bound, attached, or immobilized on the surface of such particles 14. In the example of FIG. 1, three active substances 12 are immobilized on the surface of particles 14, but this is not limited to this. Furthermore, the active substance 12 bound to one lipid particle 11 may be one type or a combination of two or more types. In other words, the active substance 12 bound to one lipid particle 11 may consist of one type of component, or may consist of multiple different types of components. Alternatively, different types of active substances 12 may be selected and combined among multiple cell surface-bound particle-bound lipid particles 11 used in a single system containing the target cells to be targeted. Furthermore, for example, although the form of the active substance 12 is depicted in Figure 1 as being typically circular or spherical, it may also be a molecule represented as a chain or string as shown in Figure 2, and in reality, it is not limited to these shapes and may be any form depending on the substance.

[0012] The active substance 12 may be a synthetic substance or a natural substance. Examples of the active substance 12 include active substances that target cell surfaces and / or molecules present on cell surfaces, structures on cell surfaces, etc. Examples of such substances include active substances that have an effect on specific receptors, channels, surface structures, etc., or substances that have an effect on target cells via them, and include, for example, substances with any pharmacological action, receptor antagonists, receptor inhibitors, receptor agonists, anticancer drugs, etc.

[0013] Here, the term "target cell" refers to a cell on which an active substance is to act. For example, the target cell may be a cell or a cell-like structure having a phospholipid bilayer, such as a cell having a cell membrane. For example, the target cell may be an animal-derived cell or a bacterial-derived cell. The target cell may also be, for example, a cancer cell, a proliferative cell, or a cell affected by any other disease or injury. Examples of cancer cells include metastatic cancer, blood cancer such as leukemia, ovarian cancer, thyroid cancer, pheochromocytoma, multiple myeloma, melanoma, glioma, leukemia, prostate cancer, breast cancer, and ovarian cancer. Alternatively, for example, at the laboratory level, normal cells may be used as target cells. For example, the target cell may be selected depending on the type of active substance used, the type or characteristics of the biodegradable lipid particles, the subject to be treated, and / or the desires of the practitioner. Application of the particle-bound lipid particles to target cells can be, for example, clinical administration, laboratory administration, in vivo administration, in vitro administration, systemic administration, local administration, or any suitable route, such as intravascular administration, intraperitoneal administration, organ or intraorgan administration, etc.

[0014] It is desirable that the biodegradable lipid particles 11 have target cell targeting properties. Here, "target cell targeting properties" means, for example, having an appropriate affinity for target cells. Here, "appropriate affinity" means having a higher affinity for target cells under normal and / or typical contact conditions with the target cells than a similar delivery carrier of a general design, and / or having a higher affinity for target cells than the affinity for cells other than the target cells. Target cell targeting, i.e., appropriate affinity, is achieved by adjusting the lipid composition of the biodegradable lipid particles.

[0015] The biodegradable lipid particles 11 may have a lipid composition that exhibits the desired target cell targeting. The biodegradable lipid particles 11 are spherical or nearly spherical lipid particles formed by a lipid membrane, in other words, hollow lipid particles. They may be lipid membrane particles, such as lipid bilayer membrane particles, that encapsulate an aqueous solution core. Any known lipid particle may be used as the lipid particle. For example, the lipid composition forming the lipid particle may contain a first lipid (FFT-10) of formula (I) and / or a second lipid (FFT-20) of formula (II) as its constituent components. These lipids are biodegradable lipids. By adjusting the lipid composition of the biodegradable lipid particles using these lipids, appropriate affinity can be achieved. [ka]

[0016] Biodegradable lipid particles, i.e., lipid particles, may contain additional lipids in addition to the first and second lipids described above. Among the lipid molecular materials constituting the lipid particles, a fraction consisting of the first and second lipids will be referred to as the "first fraction" below. Furthermore, a fraction consisting of lipid molecular materials other than the first lipid or the second lipid will be referred to as the "second fraction" below. The lipids contained in the second fraction will also be collectively referred to as the "third lipid" below.

[0017] The terms "first fraction" and "second fraction" refer to the composition of the constituent components of the lipid particle, but do not refer to the physical location of the lipids contained therein. For example, the constituent components of the first fraction and the second fraction do not necessarily have to be individually grouped within the lipid particle; the lipids contained in the first fraction and the lipids contained in the second fraction may exist as a mixture. The proportion of the first fraction relative to the total lipid material constituting the lipid particle may be 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or less, 40% or less, 30% or less, 20% or less, for example, 10% to 50%, or 15% to 45%, etc.

[0018] In other words, the total content of FFT-10 and / or FFT-20, as a proportion of the lipid particles, may be, for example, in the range of 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 15% to 50%, 15% to 45%, 15% to 40%, 10% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, or 20% to 25%. The maximum content of FFT-10 and FFT-20 in the lipid particles may be, for example, an amount that allows the lipid particles to form lipid particles. The blending ratio of the second lipid in the first fraction may be 0% or more to 100%, for example, 15% to 75%, 20% to 60%, 24% to 50%, etc. Similarly, the blending ratio of the first lipid in the first fraction may be 0% or more to 100%, for example, 15% to 75%, 20% to 60%, 24% to 50%, etc. Here, percentages are expressed in mol / mol% unless otherwise specified.

[0019] The particle size and cell permeability of the lipid particles may vary depending on the blending ratio of the first lipid and the second lipid in the first fraction. For example, the larger the amount of the second lipid, the larger the particle size of the lipid particles. The average particle size of the lipid particles can be changed depending on the application. For example, it may be adjusted to about 20 nm to about 300 nm. For example, it may be about 50 nm to about 100 nm.

[0020] The type of third lipid contained in the second fraction of the lipid particles is not limited, and the second fraction may contain, for example, a base lipid. For example, a lipid that is a main component of a biological membrane can be used as the base lipid. The base lipid may be a phospholipid or a sphingolipid, such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, or cerebroside, or a combination thereof.

[0021] For example, as a base lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-Dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphochlorin (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphochlorin (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol, Alternatively, it is preferable to use any combination of these. As the base lipid, it is particularly preferable to use a cationic lipid or a neutral lipid, and the acid dissociation constant of the lipid particles can be adjusted by the content thereof. It is preferable to use DOTAP as the cationic lipid, and it is preferable to use DOPE as the neutral lipid.

[0022] The proportion of cationic lipids such as FFT10, FFT20, and DOTAP in the total lipid particle is preferably about 10% to about 50% in order to adjust the appropriate affinity for target cells. The proportion of cationic lipids in the total lipid particle may be, for example, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 15% to 50%, 15% to 45%, 15% to 40%, 10% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, or 20% to 25%. Furthermore, the lipid composition can be adjusted to obtain appropriate affinity by, for example, changing or providing a gradient in the component ratio of the cationic lipid contained in the lipid particles depending on the type and state of the target cells, thereby designing a lipid composition that exhibits appropriate affinity for the target cells. For example, the component ratio of the cationic lipid can be adjusted to provide appropriate affinity for target cells in a specific state.

[0023] It is also preferred that the second fraction contains a lipid that prevents aggregation of lipid particles. For example, the lipid that prevents aggregation can further contain a PEG-modified lipid, such as polyethylene glycol (PEG) dimyristoylglycerol (DMG-PEG), a polyamide oligomer derived from omega-amino(oligoethylene glycol)alkanoic acid monomer (U.S. Pat. No. 6,320,017), or monosialoganglioside. The second fraction can further contain lipids such as a lipid with relatively low toxicity for adjusting toxicity; a lipid with a functional group that allows a ligand to be bound to the lipid particle; or a lipid that prevents leakage of encapsulated substances such as sterols, for example, cholesterol. In particular, it is preferred to contain cholesterol.

[0024] The type and composition of lipids used in the second fraction may be appropriately selected taking into consideration the acid dissociation constant (pKa) or particle size of the lipid particles of interest, the type of active agent contained therein, or stability in cells.

[0025] One or more of the above lipids may be selected as desired as the lipid for linker 13. For example, linker 13 may be formed by modifying the hydrophobic end of a lipid such as DMG, DSPE, cholesterol, or DOPE with PEGylation or the like.

[0026] The linker 13 is preferably contained in a molar fraction of 0.01% to 1.0% of the constituent molecules of the biodegradable lipid particle (e.g., the first lipid, second lipid, and third lipid described above, i.e., including the lipids of the first and second fractions). In other words, if the amount of lipid used in the lipid particle is 1 mole, the amount of lipid of the linker 13 contained therein can be 0.0001 mole to 0.01 mole. More preferably, the linker is contained in a molar fraction of 0.3% of the constituent molecules of the biodegradable lipid particle.

[0027] For example, additional components may be contained within the biodegradable lipid particles. The additional components may be, for example, additional active substances, substances with other pharmacological activity, or nucleic acid constructs encoding genes. Furthermore, additional components may be incorporated as needed. Examples of additional components include pH adjusters, osmotic pressure adjusters, gene activators, etc. pH adjusters include organic acids such as citric acid and their salts. Osmotic pressure adjusters include sugars or amino acids. Here, the gene activator may be any substance that promotes or supports the activity of a gene when the additional active agent is a gene. Alternatively, for example, the biodegradable lipid particles may incorporate a labeling substance that makes the particle-bound lipid particles detectable or visualizeable. For example, such a labeling substance may be a radioactive substance, a fluorescent substance, a dye, a chemiluminescent substance, etc. For example, the above additional substances and / or active agents may be one type or a combination of two or more types. Alternatively, such additional substances and / or active agents may consist of one type of component or multiple different types of components.

[0028] As described above, particle-bound lipid particles according to the embodiments include biodegradable lipid particles, particles, and an active substance. The particles are bound to the outer surface of the biodegradable lipid particles. The active substance is bound to the surface of the particles. The particle-bound lipid particles remain on the cell surface, facilitating the action of the active agent on the cell surface. Conventionally, lipid particles have been used as systems for delivering a target substance to the interior of a cell by incorporating the target substance within the lipid particles. This embodiment is a novel delivery system based on a groundbreaking and innovative idea discovered by the inventor that by deliberately binding a target substance to the surface of such lipid particles, the substance can be delivered to the cell surface. By binding an active moiety to the surface of the particle bound to the outer surface of the biodegradable lipid particles, the retention of such particle-bound lipid particles on the target cell surface can be further promoted. The above-described embodiments make it possible to provide a novel technology for delivering an active substance as an active ingredient to a target cell. It is also expected that side effects resulting from the delivery system will be reduced.

[0029] (Second embodiment) An example of a method for producing particle-bound lipid particles 10 with an active substance according to the second embodiment will be described with reference to Figures 3 and 4. First, lipid material with a desired lipid composition is used to form lipid particles 11, for example, by the above-mentioned known method (Figure 3(a)). At least a portion of the lipid material used at this time is desirably modified, for example, so that the lipid particles 11 contain a desired linker 13. Immobilizable particles 14 are added to the end of the linker 13, and the mixture is stirred and incubated (Figure 3(a)). An active substance 12 is then added, stirred, and incubated (Figure 3(b)). This results in particle-bound lipid particles 10 with an active substance (Figure 3(c)). Incubation here may be performed, for example, by leaving the mixture at a constant temperature. These steps can be rephrased as follows: That is, the method for producing cell surface-retaining microparticle-bound lipid particles 10 includes preparing biodegradable lipid particles 11 (FIG. 3(a) and FIG. 4(S31)), mixing the biodegradable lipid particles 11 with an active substance 12 (FIG. 3(b) and FIG. 4(S32)), incubating the resulting mixture (FIG. 3(b) and FIG. 4(S33)), and obtaining particle-bound lipid particles 10 (FIG. 3(c) and FIG. 4(S34)). Here, the microparticles 14 may be bound to the outer surface of the biodegradable lipid particles 11 (not shown) prior to mixing the biodegradable lipid particles 11 with the active substance 12 (FIG. 3(b)), or may be bound or immobilized to the active substance 12 (not shown) prior to this mixing (FIG. 3(b)).

[0030] The biodegradable lipid particles 11 may be prepared, for example, by forming lipid particles using desired materials by the Bangham method, organic solvent extraction, surfactant removal, freeze-thaw method, or the like. For example, lipid particles can be formed by preparing a lipid mixture obtained by incorporating the materials for the biodegradable lipid particles in an organic solvent such as alcohol in a desired ratio, and an aqueous buffer solution, adding the aqueous buffer solution to the lipid mixture, and stirring the resulting mixture to suspend it. Lipid particles obtained in this manner are an example of biodegradable lipid particles 11. For example, if additional active agents or additional components are to be encapsulated in the lipid particles 11, this may be achieved by incorporating the components to be encapsulated in the aqueous buffer solution.

[0031] For example, incubation conditions can be selected according to the properties of the active substance used and within pharmaceutically acceptable conditions. For example, incubation can be carried out at a temperature of about 4°C to about 37°C for about 10 minutes to about 1 hour. Alternatively, incubation can be carried out by leaving the mixture at room temperature.

[0032] Obtaining particle-bound lipid particles 10 containing an active substance may simply involve forming the desired particle-bound lipid particles 10. If desired, additional steps may be included. For example, the method may further include isolating the formed particle-bound lipid particles 10, or may further include washing the obtained particle-bound lipid particles 10.

[0033] This method for producing particle-bound lipid particles 10 can provide a new technology for delivering an active substance as an active ingredient to the cell membrane surface of target cells. Target cell-directed lipid particles can be more easily obtained as a means for delivering an active substance to target cells.

[0034] (Third embodiment) The particle-bound lipid particles according to the first embodiment described above may be provided in a state ready for immediate use on desired target cells, or may be provided as a particle-bound lipid particle preparation kit in the form of materials so that the user of the particle-bound lipid particles can adjust the particle-bound lipid particle at the time of use. In this case, as shown in Fig. 5, the kit may include, for example, biodegradable lipid particles 11 or biodegradable lipid particle materials (not shown) configured to be directed to desired target cells, and an active substance 12 (Fig. 5(a)). Alternatively, a portion of the configuration of biodegradable lipid particles 11, for example, particles 14, may be provided in a form independent of the lipid particles (Fig. 5(b)).

[0035] By providing the particle-bound lipid particle as a production kit, it becomes easy to handle each component or constituent in an appropriate environment.

[0036] (Fourth embodiment) The particle-bound lipid particles according to the first embodiment, e.g., cell surface-retaining microparticle-bound lipid particles, and the particle-bound lipid particle preparation kit according to the second embodiment, e.g., cell surface-retaining microparticle-bound lipid particle preparation kit, may be provided as a composition ready for immediate use on desired target cells, e.g., a pharmaceutical composition, or as a combined composition prepared just before use by a user of the particle-bound lipid particles, e.g., cell surface-retaining microparticle-bound lipid particles. For example, when provided as a combined composition, as shown in FIG. 5, the combined composition may comprise, for example, a first composition comprising biodegradable lipid particles 11 configured to target desired target cells to which microparticles 14 are attached on the exterior, and a second composition comprising an active substance 12 (FIG. 5(a)). Alternatively, the combined composition may comprise a first composition comprising base lipid particles 11, a second composition comprising active substance 12, and a third composition comprising microparticles 14 (FIG. 5(b)). Alternatively, lipid materials (not shown) for constituting the biodegradable lipid particles may be provided as a composition to be combined with the biodegradable lipid particles 11 instead of the biodegradable lipid particles. The compositions and combined compositions contain particle-bound lipid particles, for example, cell surface-attached microparticle-bound lipid particles, and may further have known components and / or compositions as desired. For example, the components and / or compositions may be selected so as to be supplied physically and / or chemically stable, so as to be pharmaceutically and / or medically stable, or so as to meet necessary and sufficient physical, chemical, and / or pharmaceutical and / or medical requirements.

[0037] The composition or combination composition may be a pharmaceutical composition. For example, the composition or combination composition contains particle-bound lipid particles or particle-bound lipid particle materials in a pharmaceutically acceptable state and / or as a component for delivering an active substance as an active ingredient to the cell membrane surface of target cells. The composition may be used in clinical or non-clinical fields. Such a composition or combination composition may contain appropriate additives, such as stabilizers, pH adjusters, buffers, viscosity adjusters, excipients, etc., depending on the desired method of use and / or administration route, the target cells used, or the subject to be administered. For example, when provided as a pharmaceutical composition to be administered to a subject, the components contained therein are selected and designed within a pharmaceutically acceptable range.

[0038] Such a composition or a combination composition can provide a novel technique for delivering an active substance as an active ingredient to the cell membrane surface of a target cell, and is also expected to reduce side effects resulting from the delivery system.

[0039] [example] Hereinafter, as an example of particle-bound lipid particles according to the embodiment, a drug delivery system mimicking cell surface-retaining microparticle-bound lipid particles was constructed, and an experimental example was described in which its target cell directivity was investigated.

[0040] Experiment 1. Preparation of target cell-directed lipid particles Biodegradable lipid particles were prepared, and streptavidin beads were immobilized on the outer surface of the lipids that comprised the lipid particles. Specifically, as shown in Figure 6(b), cell surface-retention microparticle-bound lipid particles 50 comprised biodegradable lipid particles 11, linkers 13 (DSPE-PEG2000-Biotin in this lipid particle) formed by modifying a portion of the lipids that comprised the lipid particles 11, and beads 61 (φ=3 μm, manufactured by Bang Biosciences) with streptavidin attached to the surface as microparticles bound to the linker 13 (Figure 6(b)). The target cell targeting of these lipid particles was tested. The fluorescent substance used was Rhodamine-PE (manufactured by Avanti Biosciences). Resin beads 61 without lipid particles attached were prepared as microparticles (Figure 6(a)).

[0041] The biodegradable lipid particle materials were prepared using FFT-20, DOPE, DOTAP, cholesterol, DSPE-PEG2000-Biotin, and Rhodamine-PE in molar ratios of 31.7:4.5:9.0:51.4:3.4:0.1. These materials were dissolved in ethanol to obtain a lipid solution. The lipid solution and the 10 mM HEPES (pH 7.3) solution were mixed using a microflow chip and a syringe pump. The mixed solution was further diluted 10-fold with 10 mM HEPES (pH 7.3) and then concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, Merck) to obtain biodegradable lipid particles 11 equipped with linkers 13. Streptavidin beads were then added in a buffer solution to obtain particle-bound lipid particles 50 of Example 1.

[0042] Experiment 2. Binding of biotin to streptavidin beads A streptavidin bead suspension (Comparative Example 1) was prepared in a microtube, to which a buffer solution was added as a control, followed by centrifugation. An image obtained by photographing the precipitate is shown in Figure 7(a). Particle-bound lipid particles 50 (Example 1) prepared in Experiment 1, suspended in buffer solution, were added to a microtube, and an image obtained by photographing the precipitate after centrifugation is shown in Figure 7(b). Since the color of the streptavidin beads in Figure 7(b) was the same as that of rhodamine, it was thought that the lipid particles and avidin beads were bound to each other.

[0043] Experiment 3: Observation under a microscope The streptavidin beads of Comparative Example 1 and the particle-bound lipid particles 50 of Example 1 were each added to a culture dish and observed under a microscope in bright field and fluorescent field, respectively. In bright field, both Comparative Example 1 and Example 1 were observed to be dispersed (FIGS. 8(a-1) and (b-1)). However, in fluorescent field, only the fluorescent substance contained in the particle-bound lipid particles 50 of Example 1 was observed (FIGS. 8(a-2) and (b-2)).

[0044] Experiment 4. Examination of target cell specificity against cancer cells The particle-bound lipid particles 50 of Example 1 were suspended in a buffer solution (composition: HEPES solution, pH 7.4, containing 200 mM glucose) and added to GUVs (giant unilamellar vesicles) modeling normal cells and cancer cells, respectively. After incubation at 37°C for 10 minutes, the GUVs modeling cancer cells were observed under a fluorescence microscope. The normal cell GUVs were composed of a lipid composition containing only DOPC, while the cancer cell GUVs were composed of a lipid composition containing DOPC:DOPS:DOPE in an 8:1:1 ratio, mimicking the lipid compositions of normal cells and cancer cells. Figure 9 shows the results of mixing GUVs and lipid particles. As a result, in the normal cell GUVs, the particle-bound lipid particles 50 and the normal cell GUVs were observed to be in separate states (Figure 9(a)). In contrast, the particle-bound lipid particles 50 were observed bound to the surface of the cancer cell GUVs (Figure 9(b)).

[0045] [Example 2] Next, we will explain an experimental example in which we prepared biodegradable lipid particles containing linkers extending from their ends, and then prepared microparticle-bound lipid particles by combining the same biodegradable lipid particles with 1.9 nm microparticles, and examined the effects of these particles on cellular uptake.

[0046] Experiment 5. Preparation of microparticle-bound lipid particles The microparticle-bound lipid particle of Example 2 differs from the microparticle-bound lipid particle of Example 1 in the type of cell surface-retaining microparticle. Specifically, as shown in Figure 10(b), the microparticle-bound lipid particle 70 comprises a biodegradable lipid particle 11, a nucleic acid 82 encapsulated in the lipid particle 11, a linker 13 formed by modifying a portion of the lipid constituting the lipid particle 11, and a gold nanoparticle 81 (manufactured by Nanoprobes, Inc., diameter 1.9 nm) as a nanoparticle bound to the linker 13 (Figure 10(b)). A comparative example of the microparticle-bound lipid particle of Figure 10(b) is a biodegradable lipid particle 11 that does not contain a gold nanoparticle or a linker (Figure 10(a)).

[0047] The method for producing the microparticle-bound lipid particles of Example 2 is described below. First, lipid particles of the following four types of compositions (referred to as (A), (B), (C), and (D)) were produced as biodegradable lipid particles 11. The material for biodegradable lipid particles 11 of composition (A) was a lipid solution obtained by mixing FFT-10, DOTAP, cholesterol, DMG-PEG2000, and DMG-PEG2000-Thiol in ethanol at molar fractions of 31.7:26.6:9.0:38.0:2.2:0.3. The material for biodegradable lipid particles 11 of composition (C) was a lipid solution obtained by mixing FFT-20, DOPE, DOTAP, cholesterol, DMG-PEG2000, and DMG-PEG2000-Thiol in ethanol at molar fractions of 31.7:4.5:9.0:51.4:3.1:0.3. That is, the lipid particles of composition (A) and the lipid particles of composition (C) contain DMG-PEG2000 and DMG-PEG2000-Thiol in a molar ratio of 9:1.

[0048] The material for biodegradable lipid particles 11 of composition (B) is a lipid solution obtained by mixing FFT-10, DOPE, cholesterol, and DMG-PEG2000 in ethanol in a molar ratio of 31.7:26.6:9.0:38.0:2.5, respectively. The material for biodegradable lipid particles 11 of composition (D) is a lipid solution obtained by mixing FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG2000 in ethanol in a molar ratio of 31.7:4.5:9.0:51.4:3.4, respectively. As described above, compositions (A) and (B) are based on the same lipid composition, but composition (B) does not contain DMG-PEG2000-Thiol, i.e., a linker. The lipid particles of compositions (C) and (D) are based on the same lipid composition, but composition (D) does not contain a linker.

[0049] Next, 1 mg / ml GFP-mRNA (TriLink BioTechnologies, approximately 1000 bp) was mixed with 10 mM HEPES (pH 7.3) solution at a volume ratio of 1:9 to obtain a nucleic acid solution. This nucleic acid solution was mixed with each of the four lipid solutions at a 1:1 ratio using a microflow chip and a syringe pump to obtain four mixed solutions. In other words, the nucleic acid solution used was the same for all four types of biodegradable lipid particles 11.

[0050] Of the four mixed solutions, the mixed solutions containing biodegradable lipid particles of compositions (A) and (C) were diluted 4-fold with 10 mM HEPES (pH 7.3), and then gold nanoparticles (NanoPartzs, Inc., diameter 1.8 nm) were added in the buffer solution and reacted at room temperature for 30 minutes. On the other hand, the mixed solutions containing biodegradable lipid particles of compositions (B) and (D) were diluted 10-fold with 10 mM HEPES (pH 7.3), but were not reacted with the gold nanoparticles. Each mixed solution was then concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, Merck) to obtain microparticle-bound lipid particles 70 from the mixed solution of compositions (A) and (C), and biodegradable lipid particles 11 containing nucleic acid 82 from the mixed solution of compositions (B) and (D).

[0051] Experiment 6. Introduction of lipid particles into cancer cell lines A total of four types of lipid particles, namely, microparticle-bound lipid particles obtained from the mixed solution of composition (A) and composition (C), and biodegradable lipid particles obtained from the mixed solution of composition (B) and composition (D), were introduced into breast cancer cell line MDA-MB231 or human hepatoma-derived cell line Huh7. Specifically, MDA-MB231 was introduced into a 96-well plate at 2 × 10 5 The cells were seeded at 1 × 10 cells per well of a 96-well plate, and then each lipid particle solution containing 200 ng of encapsulated nucleic acid was added dropwise. 5 The cells were seeded with the number of cells, and then each lipid particle solution containing a total of 100 ng of encapsulated nucleic acid was added dropwise.

[0052] Fluorescence micrographs taken 24 hours after transfection are shown in Figure 11. In both the comparative example (i.e., compositions (B) and (D)) and the working example (i.e., compositions (A) and (C)), the same level of GFP fluorescence was observed from the breast cancer cell lines, and no differences in morphology were observed.

[0053] The GFP fluorescence intensity measured using a plate reader is shown in Figure 12. The fluorescence intensity values for each lipid particle composition (A), (B), (C), and (D) were calculated by subtracting the fluorescence intensity value of cells not transfected with any lipid particles (i.e., background) from the fluorescence brightness of cells transfected with each lipid particle (n = 3, error bars indicate standard error). The results in Figure 12 indicate that lipid particles with compositions (A) and (B) exhibited similar Huh7 targeting, while lipid particles with compositions (C) and (D) exhibited similar MDA-MB231 targeting. This indicates that the target cell targeting of lipid particles is independent of the presence or absence of cell surface-retaining microparticles such as gold nanoparticles. In other words, the target cell targeting of lipid particles is maintained even when cell surface-retaining microparticles are bound to them. This also suggests that microparticles can be delivered to desired cells regardless of the type of ionized lipid, such as FFT-10 or FFT-20.

[0054] These results suggest that by utilizing lipid particles with target cell specificity, cell surface-retaining microparticle-bound lipid particles can be provided as novel particle-bound lipid particles for delivering active agents to target cells.

[0055] Further exemplary embodiments are described below. [1] A particle-bound lipid particle comprising a biodegradable lipid particle, a particle bound to the outer surface of the lipid particle, and an active substance bound to the surface of the particle. [2] The particle-bound lipid particle according to [1], wherein the biodegradable lipid particle includes a linker extending from an end of a portion of the constituent lipid, and the particle is immobilized to the linker. [3] The particle-bound lipid particle according to [1] or [2], wherein the active substance interacts with a cell surface or a structure present on a cell surface to exhibit an active effect. [4] The particle-bound lipid particle according to any one of [1] to [3], wherein the biodegradable lipid particle has a lipid composition that exhibits target cell-directedness. [5] The particle-bound lipid particle according to any one of [1] to [4], wherein the particle has the property of remaining on a cell membrane for a certain period of time. [6] The lipid particle according to any one of [2] to [5], wherein the linker is contained in a molar fraction of 0.01% to 1% of the constituent molecules of the biodegradable lipid particle containing the constituent lipid. [7] The particle-bound lipid particle according to any one of [1] to [6], wherein the particle is selected from the group consisting of gold, silver, iron oxide, titanium oxide, zinc oxide, silica, lipid particles, polymer particles, or combinations thereof. [8] The particle-bound lipid particle according to any one of [1] to [7], wherein the particle has a diameter of 1 nm to 10 μm. [9] The particle-bound lipid particles according to any one of [1] to [8], wherein the biodegradable lipid particles encapsulate an additional active substance or additional component within the lipid particles.

[10] A method for producing the particle-bound lipid particle according to [1], comprising: providing said biodegradable lipid particles; mixing the biodegradable lipid particles with the particles; The active substance is mixed with the resulting mixture to obtain particle-bound lipid particles. 1. A method of making a particle-associated lipid particle comprising:

[11] The method according to [1], wherein the biodegradable lipid particles contain a linker extending from an end of a portion of the constituent lipids, and the particles are immobilized to the linker.

[12] A particle-bound lipid particle production kit comprising biodegradable lipid particles, nanoparticles bound or capable of being bound to the outer surface of the lipid particles, and an active substance.

[13] The particle-bound lipid particle production kit according to

[12] , wherein the biodegradable lipid particle includes a linker extending from an end of a portion of the constituent lipid, and the particle can be immobilized to the linker.

[14] A combination composition comprising a first composition comprising biodegradable lipid particles and particles bound to the lipids of the lipid particles, and a second composition comprising an active agent.

[15] The combination composition according to

[14] , wherein the biodegradable lipid particles contain a linker extending from an end of a portion of the constituent lipids, and the particles are immobilized to the linker.

[0056] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0057] 10...particle-bound lipid particle, 11...lipid particle (lipid particle), 12...active substance, 13...linker, 14...immobilization particle (particle), 40a, 40b...particle-bound lipid particle preparation kit (particle-bound lipid particle composition or combination composition), 61...streptavidin beads, 50...particle-bound lipid particle, 52...fluorescent substance

Claims

1. A particle-bound lipid particle comprising a biodegradable lipid particle, a particle bound to the outer surface of the lipid particle, and an active agent bound to the surface of the particle.

2. The particle-bound lipid particle according to claim 1, wherein the biodegradable lipid particle comprises a linker extending from an end of a portion of the constituent lipid, and the particle is immobilized to the linker.

3. The particle-bound lipid particle according to claim 1 , wherein the active substance interacts with a cell surface or a structure present on a cell surface to exhibit an active effect.

4. The particle-bound lipid particle of claim 1 , wherein the biodegradable lipid particle has a lipid composition that exhibits target cell targeting.

5. The particle-bound lipid particle according to claim 1, wherein the particle has the property of remaining on a cell membrane for a certain period of time.

6. The lipid particle according to claim 2, wherein the linker is contained in a molar fraction of 0.01% to 1% of the constituent molecules of the biodegradable lipid particle containing the constituent lipids.

7. The particle-bound lipid particle of claim 1 , wherein the particle is selected from the group consisting of gold, silver, iron oxide, titanium oxide, zinc oxide, silica, lipid particles, polymer particles, or combinations thereof.

8. 2. The particle-bound lipid particle of claim 1, wherein the particle has a diameter of 1 nm to 10 μm.

9. The particle-bound lipid particle of claim 1 , wherein the biodegradable lipid particle encapsulates an additional active substance or additional component within the lipid particle.

10. 10. A method of making the particle-associated lipid particles of claim 1, comprising: providing said biodegradable lipid particles; mixing the biodegradable lipid particles with the particles; The active substance is mixed with the resulting mixture to obtain particle-bound lipid particles.

1. A method of making a particle-associated lipid particle comprising:

11. A particle-bound lipid particle production kit comprising biodegradable lipid particles, nanoparticles bound or capable of being bound to the outer surface of the lipid particles, and an active substance.

12. The particle-bound lipid particle production kit according to claim 11, wherein the biodegradable lipid particle comprises a linker extending from an end of a portion of the constituent lipid, and the particle can be immobilized to the linker.

13. A combination composition comprising a first composition comprising biodegradable lipid particles and particles associated with the lipids of said lipid particles, and a second composition comprising an active agent.

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