Lipid particle for radiation therapy, manufacturing method, combination composition, and kit

Abstract

To provide a technique for delivering a radioactive substance as an active ingredient to a target cell.SOLUTION: According to one arrangement, a lipid particle for radiation therapy includes a biodegradable lipid particle, and radioactive substances as an active ingredient. The radioactive substances are bound to the biodegradable lipid particle and located outside the biodegradable lipid particle.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] Radioactive materials are widely used in medical treatment and diagnosis. For example, targeted isotope therapy is a method of treating targeted cells using radioactive materials. Traditionally, artificial radioactive materials, i.e., artificial radionuclides, have generally been produced by nuclear reactors such as research reactors. In recent years, there has been a global shortage of radioactive materials produced by research reactors, and instead, treatments using radioactive materials produced by accelerators have attracted attention. Summary of the Invention [Problem to be solved by the invention]

[0003] The problem to be solved by the present invention is to provide a technique for delivering a radioactive substance as an active ingredient to a target cell. [Means for solving the problem]

[0004] A radiotherapeutic lipid particle according to an embodiment comprises a biodegradable lipid particle and a radioactive substance as an active ingredient, the radioactive substance being bound to the lipid particle and located outside the lipid particle. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 is a schematic diagram showing an example of a lipid particle for radiotherapy according to a first embodiment. [Figure 2] FIG. 1 is a schematic diagram illustrating the concept of an example of a manufacturing method according to a second embodiment. [Figure 3] FIG. 4 is a schematic diagram showing an example of a manufacturing method according to a second embodiment. [Figure 4] FIG. 10 is a schematic diagram showing an example of a combination composition according to the third embodiment. [Figure 5]Schematic diagram showing a comparative example (a) and an example (b) used in the experiment. [Figure 6] An image showing the experimental results. [Figure 7] An image showing the experimental results. [Figure 8] An image showing the experimental results. [Figure 9] Schematic diagram showing a comparative example (a) and an example (b) used in the experiment. [Figure 10] An image showing the experimental results. [Figure 11] An image showing the experimental results. [Figure 12] An image showing the experimental results. [Figure 13] An image showing the experimental results. [Figure 14] An image showing the experimental results. DETAILED DESCRIPTION OF THE INVENTION

[0006] 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.

[0007] 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 also includes forms commonly known as lipid nanoparticles (LNPs), liposomes, microemulsions, and the like.

[0008] (First embodiment) A radiotherapeutic lipid particle according to an embodiment will be described with reference to FIG. 1. The radiotherapeutic lipid particle 10 includes a biodegradable lipid particle 11 and a radioactive substance 12 bound to the lipid particle 11 and located outside the lipid particle. The lipid particle 11 and the radioactive substance 12 may be bound, for example, directly to a portion of the lipid constituting the lipid particle 11, or may be bound via an intermediate portion 15 of the lipid particle 11, such as a linking unit 15. The linking unit 15 includes, for example, a linker 13 and a nanoparticle 14 bound to the linker 13. 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 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. FIG. 1 shows an example of the linker 13, which is a PEG-modified lipid having a thiol group at its terminal, such as a portion of cholesterol. However, the linker 13 is not limited to this. As will be described in detail later, by including one or more types of PEG-modified lipids, lipids having functional groups capable of binding ligands, etc. as the lipids constituting the lipid particle 11, a portion of these lipids can be used as the linker 13 when the lipid particle 11 is formed. Examples of functional groups capable of binding ligands include, but are not limited to, thiol groups, amino groups, maleimide groups, and carboxy groups. For example, the linker 13 can be included in the lipid material or the formed lipid particle 11 at a molar ratio of 0.01% to 1% as a linker density. In other words, if the amount of lipid used in the lipid particle is 1 mole, the amount of lipid in the linker 13 included therein can be 0.0001 mole to 0.01 mole. By using the linker 13 and the nanoparticles 14, the radioactive substance 12 can be stably bound to the lipid particle 11 and positioned on the outside thereof.

[0009] For example, the linker 13 is bonded to a nanoparticle 14 capable of binding or having affinity for the radioactive substance to be used. 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. In this case, the nanoparticle 14 may be composed of a material capable of binding or having affinity for the thiol group and capable of binding or having affinity for the radioactive substance 12. Such nanoparticles 14 may be made of, for example, Au, Ag, SiO2, Si, glass, resin, etc., or may be nano-sized particles primarily composed of these materials. The surface of the nanoparticle may also be specifically functionalized. Such functionalization may include, for example, active ester group modification, maleimide activation, thiol group modification, amino group modification, carboxy group modification, methyl group modification, avidin modification, biotin modification, etc. This may facilitate binding to the linker and / or immobilization or binding of the radioactive substance. The diameter ratio of the biodegradable lipid particles to the nanoparticles may be, for example, 1:1 to 100:1, 10:1 to 100:1, 50:1 to 100:1, etc. For example, one or more radioactive substances 12 may be bound, attached, or immobilized to the surface of such nanoparticles 14. In the example of FIG. 1, three radioactive substances 12 are immobilized on the surface of the nanoparticles 14, but this is not limiting. Furthermore, the radioactive substance 12 bound to one lipid particle 11 may be one type or a combination of two or more types. In other words, the radioactive 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 radioactive substances 12 may be selected and combined among multiple radiotherapeutic lipid particles used for a single system containing target cells to be treated. The particle diameter may be 1 nm to 1 μm, for example, 1 nm to 100 nm, for example, 1 nm to 10 nm, etc.

[0010] The radioactive material 12 may be an artificial radioactive material, i.e., an artificial radionuclide, or may be a natural radioactive material, i.e., a natural radionuclide. For example, examples of radioactive materials include, for example: 211 At, 213 Bi, 225Alpha-emitting nuclides such as Ac, 89 Sr, 186 Re, 192 Ir, 67 Cu, 177 Lu, 64 Cu, 86 Y, 124 β-ray nuclides such as I, 67 Ga, 111 In, 114m In, 169 They may be, for example, radioactive materials produced by an accelerator. For example, they may be alpha-ray nuclides, alpha emitters, etc. For example, an example of an alpha emitter is astatine ( 211 At), radium ( 223 Ra), actinium ( 225 For example, an embodiment using astatine will be described later as a third embodiment.

[0011] Here, the term "target cell" refers to a cell to be acted upon by a radioactive substance. For example, the target cell may be a cancer cell, a proliferating cell, or a cell affected by any other disease or damaged cell. 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. For example, the target cell may be selected depending on the type of radioactive substance used, the type or characteristics of the biodegradable lipid particle, the subject to be treated, and / or the desires of the practitioner. The application of the radiotherapeutic lipid particle to the target cell may be, for example, clinical administration, laboratory administration, in vivo administration, in vitro administration, systemic administration, local administration, or any other appropriate route, such as intravascular administration, intraperitoneal administration, or intraorgan administration.

[0012] The biodegradable lipid particles 11 can have target cell tropism. Here, "target cell tropism" means, for example, having an appropriate affinity for target cells. Here, "appropriate affinity" means, under normal and / or typical contact conditions with the target cells, a higher affinity than a similar delivery carrier of a general design, and / or a higher affinity for the target cells than the affinity for cells other than the target cells. Target cell tropism, i.e., appropriate affinity, is achieved by adjusting the lipid composition of the biodegradable lipid particles.

[0013] The biodegradable lipid particles 11 may have a lipid composition that exhibits desired target cell targeting. The lipid particles 11 are spherical or nearly spherical lipid particles formed by a lipid membrane, in other words, hollow lipid particles. In other words, the biodegradable lipid particles are 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]

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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 hydrophilic end of a lipid such as DMG, DSPE, cholesterol, or DOPE with PEGylation or the like.

[0024] For example, an additional active agent may be contained within the biodegradable lipid particles. The additional active agent may be, for example, an additional radioactive substance, a component having other pharmacological activity, or a nucleic acid construct encoding a gene. Furthermore, additional components may be encapsulated as necessary. Examples of the additional component include a pH adjuster, an osmotic pressure adjuster, a gene activator, etc. The pH adjuster may be, for example, an organic acid such as citric acid and its salt. The osmotic pressure adjuster may be, for example, a sugar or an amino acid. Here, the gene activator may be any substance that promotes or supports the activity of the additional active agent when the additional active agent is a gene. Alternatively, for example, the biodegradable lipid particles may encapsulate a labeling substance that makes the radiotherapeutic lipid particles detectable or visualizeable. For example, such a labeling substance may be an additional 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 may consist of multiple components of different types.

[0025] Such lipid particles for radiotherapy can provide a novel technique for delivering radioactive materials as active ingredients to target cells, and are also expected to reduce side effects caused by the delivery system.

[0026] (Second embodiment) An example of a method for producing lipid particles 10 for radiotherapy according to the second embodiment will be described with reference to Figures 2 and 3. First, lipid particles 11 are formed using a lipid material having a desired lipid composition, for example, by the above-mentioned method known per se (Figure 2(a)). At least a portion of the lipid material used at this time is modified, for example, so that the lipid particles 11 contain a desired linker 13. Nanoparticles 14 that can be immobilized at the end of the linker 13 are added thereto, and the mixture is stirred and incubated (Figure 2(a)). Further, a radioactive substance 12 is added, stirred, and incubated (Figure 2(b)). This results in the production of lipid particles 10 for radiotherapy (Figure 2(c)). Here, incubation 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 lipid particles 10 for radiotherapy includes preparing biodegradable lipid particles 11 (Figure 2(a), Figure 3(S31)), mixing the biodegradable lipid particles 11 with radionuclides 12 (Figure 2(b), Figure 3(S32)), incubating the resulting mixture (Figure 2(b), Figure 3(S33)), and obtaining lipid particles 10 for radiotherapy (Figure 2(c), Figure 3(S34)).

[0027] 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.

[0028] For example, incubation conditions can be selected according to the properties of the radioactive 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.

[0029] Obtaining the radiotherapeutic lipid particles 10 may be achieved by forming the desired radiotherapeutic lipid particles 10. If desired, additional steps may be included. For example, the method may further include isolating the formed radiotherapeutic lipid particles 10, or may further include washing the obtained radiotherapeutic lipid particles 10.

[0030] This method for producing the radiotherapeutic lipid particles 10 can provide a new technology for delivering a radioactive substance as an active ingredient to target cells. Target cell-directed lipid particles can be more easily obtained as a means for delivering a radioactive substance to target cells.

[0031] (Third embodiment) The radiotherapeutic lipid particles according to the third embodiment contain astatine ( 211 The radiotherapeutic lipid particle 10 may be similar to the lipid particle for radiotherapy according to the first embodiment, except that it contains astatine (Asp, At). A specific configuration will be described using FIG. 1. The lipid particle for radiotherapy 10 contains a biodegradable lipid particle 11 and astatine (Asp, At) as a radioactive substance 12 bound to the lipid particle 11 and located outside the lipid particle. The binding between the lipid particle 11 and the astatine 12 is achieved by a linking unit 15. The linking unit 15 includes, for example, a linker 13 and a nanoparticle 14 bound to the linker 13. For example, the linker 13 may be a lipid that is PEG-modified and has a terminal thiol group, such as DSPE and / or cholesterol. The nanoparticle 14 may be, for example, an Au nanoparticle. The binding mode between astatine and Au is as shown in the following formula 1 and Tables 1 and 2.

number

[0032] Astatine is an alpha emitter and can have a strong therapeutic effect in small amounts. For example, astatine ( 211 Lipid particles 10 for radiotherapy containing At) as an active ingredient can be used to select, for example, metastatic cancer as a target cell. By using biodegradable lipid particles 11 with target cell specificity, it is possible to suppress the side effects of astatine on normal cells. Furthermore, since astatine has a short half-life of approximately 7 hours, when lipid particles for radiotherapy containing astatine as an active ingredient are used for treatment, it is desirable to bind the lipid particles 11 and astatine 12 immediately before administration to a patient. In this case, astatine and biodegradable lipid particles 11 may be provided as a combination composition for radiotherapy, as described below.

[0033] Astatine may be produced by any known method. For example, the production method includes three processes: nuclear transmutation, separation and recovery, and synthesis. Specifically, the production method may be carried out as follows: First, in the nuclear transmutation, astatine is accelerated to about 28 MeV by an accelerator. 4 He 2+ By irradiating ions onto the bismuth target, 209 Bi(α,2 n) 211 By At reaction 211 Generate At.

[0034] The separation and recovery process is as follows: After irradiation, the target contains the raw material. 209 Produced by nuclear reaction with Bi 211 Since At is mixed in, the two are separated and only At is recovered. As an example of a separation method, a Bi target is placed in a quartz tube and heated to 850 °C in an electric furnace to remove the gas. 211At was passed through a fluororesin tube cooled to -100°C together with an oxygen stream. 211 At is solidified and collected on the inner wall of the tube, or a wet method in which it is dissolved in an acid or the like may be used.

[0035] The recovery form may vary depending on the separation method. For example, the recovered material may be finally collected in a quartz tube or a filter. 211 The At can be washed with a washing solution and collected in solution. The collected solution is then combined with biodegradable lipid particles designed for the cancer being treated by a synthesis device. 211 Radiotherapeutic lipid particles may be prepared by chemically binding At.

[0036] For information on the production method of astatine, please refer to the following literature, for example, "Development of a mass production method for the artificial element astatine - Accelerating the development of cancer treatment drugs using alpha rays" (https: / / www.riken.jp / press / 2023 / 20230831_3 / index.html) and Shigeki Watanabe et al., "Separation of At-211 by dry and wet methods," 15th Radiopharmaceutical and Imaging Diagnostic Agents Research Meeting (2015).

[0037] Although not limited thereto, the astatine-containing radiotherapeutic lipid particles may be administered to target cells by, for example, intravenous administration, thereby allowing delivery to lesions such as cancer metastases.

[0038] Such lipid particles for radiotherapy containing astatine as an active ingredient can provide a novel technology for delivering radioactive substances as active ingredients to target cells. It is also expected that side effects resulting from the delivery system will be reduced. Alpha emitters such as astatine can achieve strong therapeutic effects with small amounts. Furthermore, by using lipid particles with target cell specificity, it is possible to efficiently deliver the substance to target cells while preventing the effects on normal cells that cause side effects during treatment. Furthermore, by changing the lipid composition of the lipid particles, it is relatively easy to adjust the specificity for targeting desired tumor cells. Conventionally, the selection of antibodies or the development of new antibodies for each disease was required, limiting the scope of application. However, the lipid particles for radiotherapy containing astatine according to the embodiments are expected to be easier to design and have a wider range of applications than conventional methods.

[0039] (Fourth embodiment) The radiotherapeutic lipid particles according to the first and third embodiments described above may be provided in a state where they can be immediately used on desired target cells, or may be provided as a kit for preparing radiotherapeutic lipid particles in the form of materials so that the user of the radiotherapeutic lipid particles can adjust them at the time of use. In this case, as shown in FIG. 4, the kit may include, for example, biodegradable lipid particles 11 or a biodegradable lipid particle material (not shown) configured to be directed to desired target cells, and a radioactive substance 12 (FIG. 4(a)). Alternatively, a portion of the configuration of the biodegradable lipid particles 11, for example, nanoparticles 14, may be provided in a form independent of the lipid particles (FIG. 4(b)). For example, the kit for preparing radiotherapeutic lipid particles may include, independently, biodegradable lipid particles for radiotherapeutic lipid particles and a radioactive substance as an active ingredient. For example, being provided independently means that the first component and the second component are stored in different containers, and delivered to the user together in a container such as a single box, or the first component and the second component may be delivered to the user separately from different suppliers.

[0040] By providing the lipid particles for radiotherapy as a production kit, it becomes easy to handle each component or constituent in an appropriate environment.

[0041] (Fifth embodiment) The radiotherapeutic lipid particles according to the first and third embodiments and the radiotherapeutic lipid particle preparation kit according to the fourth embodiment may be provided as a composition ready for immediate use on desired target cells, such as a pharmaceutical composition, or as a combined composition, such as a combined pharmaceutical composition, prepared immediately before use by a user of the above-mentioned radiotherapeutic lipid particles. For example, when provided as a combined composition, as shown in FIG. 4, the combined composition may comprise, for example, a first composition containing biodegradable lipid particles 11 or a biodegradable lipid particle material (not shown) configured to be directed to desired target cells, and a second composition containing a radioactive substance 12 (FIG. 4(a)). Alternatively, the combined composition may comprise a first composition containing base lipid particles 11, a second composition containing the radioactive substance 12, and a third composition containing a portion of the linking moiety 15, which is part of the configuration of the biodegradable lipid particles 11, such as nanoparticles 14 (FIG. 4(b)). The compositions and combination compositions contain particle-bound lipid particles, e.g., cell surface-resident microparticle-bound lipid particles, and may further contain 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 satisfy physically and / or chemically and / or pharmacologically and / or medically necessary and sufficient conditions.

[0042] The composition or combination composition may be a pharmaceutical composition. For example, the composition or combination composition may contain radiotherapeutic lipid particles or radiotherapeutic lipid particle materials in a pharmaceutically acceptable state and / or as an ingredient for delivering a radioactive substance as an active ingredient to 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 to be included are selected and designed within a pharmaceutically acceptable range. For example, a combination composition containing radiotherapeutic lipid particles may be a combination composition comprising a first composition containing biodegradable lipid particles for the radiotherapeutic lipid particles and a second composition containing a radioactive substance as an active ingredient to be bound to the lipid particles. The biodegradable lipid particles may further include a linker extending from the end of a portion of the constituent lipids thereof, and nanoparticles immobilized on the linker for immobilizing the radioactive substance. The first composition and the second composition may be contained in a first container and a second container, respectively, and further contained in a third container, and provided. Alternatively, the first composition and the second composition may be contained in a first container and a second container, respectively, and provided in an independent state. In this case, for example, they may be provided to the user by different manufacturers or providers.

[0043] Such a composition or combination composition can provide a novel technique for delivering a radioactive substance as an active ingredient to target cells, and is also expected to reduce side effects resulting from the delivery system.

[0044] [Example 1] Hereinafter, a drug delivery system that mimics lipid particles for radiotherapy according to an embodiment was constructed, and experimental examples in which its target cell directivity was investigated will be described.

[0045] Experiment 1. Preparation of target cell-directed lipid particles Biodegradable lipid particles were prepared, and fluorescent lipids were used instead of radioactive materials on the exterior of the lipids that make up these lipid particles. Specifically, as shown in Figure 5(b), a lipid particle model 50 for radiotherapy comprises a biodegradable lipid particle 11, a linker 13 formed by modifying a portion of the lipid that makes up the lipid particle 11, and a bead 61 with streptavidin attached to its surface as a nanoparticle bound to the linker 13 (Figure 5(b)). This model was tested for target cell targeting. The fluorescent substance used was Rhodamine-PE (Avanti). Resin beads 61 (φ=3 μm, Bang) were prepared for comparison (Figure 5(a)).

[0046] 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, respectively. These materials were dissolved in ethanol to obtain a lipid solution. The lipid solution and 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, manufactured by Merck) to obtain biodegradable lipid particles 11 equipped with linkers 13. Streptavidin beads were then added to the buffer solution to obtain the radiotherapy lipid particle model 50 of Example 1.

[0047] Experiment 2. Visualization of gold nanoparticle binding to lipid particles 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 6(a). A radiotherapy lipid particle model 50 (Example 1) prepared in Experiment 1 suspended in buffer solution was added to a microtube, and an image obtained by photographing the precipitate after centrifugation is shown in Figure 6(b). The color of the streptavidin beads was the light pink color of rhodamine-PE, suggesting that the lipid particles and avidin beads were bound to each other.

[0048] Experiment 3: Observation under a microscope The streptavidin beads of Comparative Example 1 and the lipid particle model 50 for radiotherapy of Example 1 were each added to a culture dish, and the results of observation under a microscope in bright field and fluorescent field are shown in Figure 7. In bright field, both Comparative Example 1 and Example 1 were observed to be in a dispersed state. However, in fluorescent field, only the fluorescent substance contained in the lipid particle model 50 for radiotherapy of Example 1 was observed.

[0049] Experiment 4: Examination of target cell tropism against normal and cancer cell models The lipid particle model 50 for radiotherapy of Example 1 was suspended in a buffer solution (composition: HEPES solution, pH 7.4, with 200 mM glucose dissolved), and added to GUVs (giant unilamellar vesicles) modeling normal cells and cancer cells, respectively. After incubation at 37°C for 10 minutes, the samples were observed under a fluorescence microscope. The GUVs for normal cells were composed of a lipid composition containing only DOPC, while the GUVs for cancer cells were composed of a lipid composition containing DOPC:DOPS:DOPE in a ratio of 8:1:1. The results of mixing the GUVs and lipid particles are shown in Figure 8. As a result, in the GUVs for normal cells, the lipid particle model 50 for radiotherapy and the cells were observed to be independent of each other (Figure 8(a)). In contrast, the lipid particle model 50 for radiotherapy was observed to be bound to the surface of the GUVs for cancer cells (Figure 8(b)).

[0050] [Example 2] Next, we will explain an experimental example in which biodegradable lipid particles containing gold nanoparticles for immobilizing radioactive substances and linkers extending from the ends were prepared, and the effect of attaching gold nanoparticles on cellular uptake was examined.

[0051] Experiment 5. Preparation of Gold Nanoparticle-Conjugated Lipid Particles We prepared biodegradable lipid particles with gold nanoparticles attached. Specifically, as shown in Figure 9(b), biodegradable lipid particles 70 comprise biodegradable lipid particles 11, nucleic acids 82 encapsulated in the lipid particles 11, linkers 13 formed by modifying a portion of the lipids that make up the lipid particles 11, and gold nanoparticles 81 as nanoparticles attached to the linkers 13 (Figure 9(b)). For comparison, we prepared biodegradable lipid particles 71 that did not contain gold nanoparticles or linkers (Figure 9(a)).

[0052] Experiments were conducted with two compositions (hereafter referred to as B and C). The materials for biodegradable lipid particles 70 (B) were prepared as follows: FFT-20, DOPE, DOTAP, cholesterol, DMG-PEG2000, and DMG-PEG2000-Thiol in molar ratios of 31.7:4.5:9.0:51.4:3.1:0.3. The materials for biodegradable lipid particles 70 (C) were prepared as follows: FFT-20, DOPE, DOTAP, cholesterol, DMG-PEG2000, and DMG-PEG2000-Thiol in molar ratios of 31.7:4.5:9.0:51.4:2.1:1.3. Specifically, lipid particle B contained DMG-PEG2000 and DMG-PEG2000-Thiol at a molar ratio of 9:1, while lipid particle C contained DMG-PEG2000 and DMG-PEG2000-Thiol at a molar ratio of 7:3. These materials were dissolved in ethanol to obtain a lipid solution. A nucleic acid solution was obtained by mixing 1 mg / ml GFP-mRNA (TriLink BioTechnologies, approximately 1000 bp) with 10 mM HEPES (pH 7.3) solution at a volume ratio of 1:9. The same nucleic acid solution was used for all lipid particles. The lipid solution and the nucleic acid solution were mixed at a 1:1 ratio using a microflow chip and syringe pump. The mixture was then diluted 4-fold with 10 mM HEPES (pH 7.3). Gold nanoparticles (Nanoprobes, Inc., diameter 1.9 nm) were then added to the buffer solution and allowed to react for 30 minutes at room temperature. The mixture was concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain biodegradable lipid particles 70 of Example 2.

[0053] The materials for the biodegradable lipid particles 71(A) of Comparative Example 2 were prepared as follows: FFT-20, DOPE, DOTAP, cholesterol, DMG-PEG2000, and DMG-PEG2000-Thiol in a molar ratio of 31.7:4.5:9.0:51.4:3.1:0.3. The lipid solution and the nucleic acid solution were mixed using a microflow chip and a syringe pump. The mixed solution was diluted 10-fold with 10 mM HEPES (pH 7.3) and then concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain biodegradable lipid particles 71 encapsulating nucleic acid 82.

[0054] Experiment 6: Observation under an electron microscope Biodegradable lipid particles 71 (A) of Comparative Example 2 and biodegradable lipid particles 70 (C) of Example 2, both of which encapsulate nucleic acid 82, were negatively stained using an aqueous uranyl acetate solution, and the results of their observation are shown in Figure 10. Dispersed lipid particles were observed in both Comparative Example 2 and Example 2. In addition, black dots representing gold nanoparticles were observed on the outside of biodegradable lipid particles 70 in Example 2, suggesting that gold nanoparticles 81 were successfully bound to the surface of lipid particles 11 via linkers 13.

[0055] Experiment 7. Introduction of lipid particles into breast cancer cell lines 2 x 10 cells in a 96-well plate 5The nucleic acid 82 was encapsulated in lipid particles 71 (A) of Comparative Example 2 and biodegradable lipid particles 70 (B) and (C) of Example 2, both of which were introduced into the breast cancer cell line MCF-7. After 24 hours of culture, the cells were subjected to fluorescence observation under a microscope. The results are shown in Figure 11(a) . Samples containing only the cell line to which no lipid particles were added are shown as "no liposome." Each sample contained 200 ng of lipid particles containing nucleic acid. In both Comparative Example 2 and Example 2, similar levels of GFP fluorescence were observed from the breast cancer cell lines, and the cells exhibited almost identical morphologies. The GFP fluorescence intensity of MCF-7 cells measured using a plate reader is also shown in Figure 11(b) . The fluorescence intensity values of lipid particles A, B, and C were calculated by subtracting the fluorescence intensity value of no liposomes from the fluorescence brightness of cells transfected with each lipid particle. No significant differences in fluorescence were observed between cells transfected with lipid particles A, B, or C. This suggests that the gold nanoparticle-conjugated lipid particles have the same targeting ability as biodegradable lipid particles 71 that do not contain gold nanoparticles or linkers, but have no difference in toxicity.

[0056] Experiment 8. Introduction of lipid particles into hepatocellular carcinoma cell lines Figure 12 shows the results of introducing biodegradable lipid particles 71 (A) of Comparative Example 2, biodegradable lipid particles 70 (B) of Example 2, and biodegradable lipid particles 70 (C) of Example 2, each containing nucleic acid 82, into the human hepatoma-derived cell line Huh-7. All conditions except for the cell type were the same as in Experiment 6. As in Experiment 6, no significant differences were observed in the experimental results using fluorescent photographs and a plate reader. This suggests that, with the same composition, the trends in targeting and toxicity are similar even when the cell type is changed.

[0057] Experiment 9. Verification of cell targeting of biodegradable lipid particles with different lipid compositions Biodegradable lipid particles with lipid compositions A and B, as well as two other lipid compositions (hereafter referred to as D and E) different from those used in Experiment 8, were used to verify whether cell targeting was maintained regardless of the type of ionized lipid in their components. First, in addition to biodegradable lipid particles 70(B) and 71(A) prepared in Experiment 8, four types of lipid particles were prepared: biodegradable lipid particles 70(D) and 71(E), each containing nucleic acid 82. Biodegradable lipid particles 71(D) were prepared as a lipid solution containing FFT-10, DOTAP, cholesterol, DMG-PEG2000, and DMG-PEG2000-Thiol in molar ratios of 31.7:26.6:9.0:38.0:2.2:0.3. Gold nanoparticles were reacted under the same conditions as in Experiment 5 to produce biodegradable lipid particles 71(D). The material for biodegradable lipid particles 71(E) is a lipid solution containing FFT-10, DOTAP, cholesterol, and DMG-PEG2000 in the molar ratios of 31.7:26.6:9.0:38.0:2.5. Each of the four types of lipid particles was introduced into the breast cancer cell line MDA-MB231 or the human hepatoma cell line Huh7 to verify cell targeting. MDA-MB231 cells were plated at 2 × 10 5 The cells were seeded at 1 × 10 cells / well in a 96-well plate, and each lipid particle containing 200 ng of nucleic acid was transfected. 5 The cells were seeded at a cell number of 100 ng, and each lipid particle containing 100 ng of nucleic acid was then introduced.

[0058] Figure 13 shows fluorescence micrographs of each cell type transfected with each lipid particle 2 hours after transfection. Observations were performed using a 20x objective lens. Figure 14 also shows the GFP fluorescence intensity measured using a plate reader. Each fluorescence intensity value was calculated using a plate reader by subtracting the fluorescence intensity of cells not transfected with lipid particles (i.e., background fluorescence intensity) from the fluorescence intensity of cells transfected with each lipid particle (n = 3, error bars indicate standard error). Figures 13 and 14 show that compositions (D) and (E) both exhibited Huh7 targeting, while compositions (B) and (A) both exhibited MDA-MB231 targeting. This demonstrates that varying the lipid composition can confer lipid particles targeting different cell types. Furthermore, it was shown that the target cell targeting of lipid particles is independent of the presence or absence of nanoparticles, such as gold nanoparticles; in other words, the target cell targeting of lipid particles is maintained even when nanoparticles are bound to them. In other words, it is possible to achieve both surface modification of nanoparticles and cell delivery properties regardless of the type of ionized lipid, such as FFT-10 or FFT-20.

[0059] These results suggest that the use of lipid particles with target cell specificity can provide novel lipid particles for radiotherapy, which can deliver radioactive substances as active agents to target cells.

[0060] Further exemplary embodiments are described below. [1] Lipid particles for radiotherapy, comprising biodegradable lipid particles and a radioactive substance as an active ingredient bound to the lipid particles and located outside the lipid particles. [2] The lipid particle according to [1], wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid, and a nanoparticle immobilized on the linker for immobilizing a radioactive substance. [3] The lipid particles according to [1] or [2], wherein the biodegradable lipid particles have a lipid composition that exhibits target cell specificity. [4] The lipid particles according to any one of [1] to [3], wherein the lipid composition of the biodegradable lipid particles contains 10% to 50% of a cationic lipid. [5] The lipid particle according to any one of [1] to [4], wherein the lipid composition of the biodegradable lipid particle contains FFT10 and FFT20 in an amount of 10% to 50% based on the total amount of lipid. [6] The lipid particles according to any one of [1] to [5], wherein the biodegradable lipid particles further contain a labeling substance that makes the lipid particles for radiotherapy detectable. [7] The lipid particle according to any one of [2] to [6], wherein the linker is contained in the biodegradable lipid particle at a specific linker density. [8] The lipid particles according to any one of [2] to [7], wherein the main component of the nanoparticles is Au, Ag, SiO2, Si, glass, or resin. [9] The lipid particle according to any one of [2] to [8], wherein the surface of the nanoparticle is functionalized.

[10] The lipid particles according to any one of [2] to [9], wherein the diameter ratio of the biodegradable lipid particles to the nanoparticles is 1:1 to 100:1.

[11] The lipid particle according to any one of [1] to

[10] , wherein the radioactive substance is an alpha emitter.

[12] The lipid particle according to any one of [1] to

[11] , wherein the radioactive substance is astatine.

[13] Providing the biodegradable lipid particles; mixing the biodegradable lipid particles with the radioactive material; incubating the resulting mixture; and Obtaining lipid particles for radiotherapy A method for producing the lipid particle according to any one of [1] to

[12] , comprising:

[14] The method according to

[13] , wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid, and a nanoparticle immobilized on the linker for immobilizing the radioactive substance.

[15] The method according to

[13] or

[14] , wherein the radioactive substance is an alpha emitter.

[16] The method according to any one of

[13] to

[15] , wherein the radioactive substance is astatine.

[17] A combination composition comprising a first composition containing biodegradable lipid particles for radiotherapy, and a second composition containing a radioactive substance as an active ingredient to be bound to the lipid particles.

[18] The combination composition according to

[17] , wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid, and a nanoparticle immobilized to the linker for immobilizing the radioactive substance.

[19] The combination composition according to

[17] or

[18] , wherein the radioactive substance is an alpha emitter.

[20] The combination composition according to any one of

[17] to

[19] , wherein the radioactive substance is astatine.

[21] A kit for producing lipid particles for radiotherapy, which independently comprises biodegradable lipid particles for radiotherapy and a radioactive substance as an active ingredient.

[22] The kit according to

[21] , wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid, and a nanoparticle immobilized to the linker for immobilizing the radioactive substance.

[23] The kit according to

[21] or

[22] , wherein the radioactive substance is an alpha emitter.

[24] The kit according to any one of

[21] to

[23] , wherein the radioactive substance is astatine.

[0061] 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]

[0062] 10...lipid particle for radiotherapy, 11...lipid particle (lipid particle), 12...radioactive substance, 13...linker, 14...particle for fixation (e.g., gold nanoparticle), 15...linking unit, 40a, 40b...lipid particle preparation kit for radiotherapy (lipid particle composition for radiotherapy or combination composition), 61...streptavidin beads, 50...lipid particle model for radiotherapy, 52...fluorescent substance, 70...biodegradable lipid particle, 71...biodegradable lipid particle (linker and gold nanoparticle not bound), 81...gold nanoparticle, 82...nucleic acid

Claims

1. A lipid particle for radiotherapy comprising a biodegradable lipid particle and a radioactive substance as an active ingredient, the radioactive substance being bound to the lipid particle and located outside the lipid particle.

2. The lipid particle of claim 1, further comprising a linker extending from an end of a portion of the constituent lipid of the biodegradable lipid particle, and a nanoparticle immobilized on the linker for immobilizing the radioactive substance.

3. The lipid particle according to claim 1, wherein the biodegradable lipid particle has a lipid composition that exhibits target cell targeting.

4. 2. The lipid particle according to claim 1, wherein the lipid composition of the biodegradable lipid particle comprises 10% to 50% of a cationic lipid.

5. The lipid particle according to claim 1, wherein the lipid composition of the biodegradable lipid particle contains FFT10 and FFT20 in an amount of 10% to 50% based on the total amount of lipid.

6. The lipid particle according to claim 1, wherein the biodegradable lipid particle further contains a labeling substance that makes the radiotherapeutic lipid particle detectable.

7. The lipid particle according to claim 2, wherein the linker is contained in the biodegradable lipid particle at a molar ratio of 0.01 to 1% among the constituent molecules of the lipid particle.

8. The main components of the nanoparticles are Au, Ag, and SiO ２ 3. The lipid particle according to claim 2, which is made of silicon, glass, or resin.

9. The lipid particle of claim 2, wherein the surface of the nanoparticle is functionalized.

10. 3. The lipid particle according to claim 2, wherein the diameter ratio of the biodegradable lipid particle to the nanoparticle is 1:1 to 100:

1.

11. The lipid particle of claim 1, wherein the radioactive substance is an alpha emitter.

12. The lipid particle according to claim 1, wherein the radioactive substance is astatine.

13. providing said biodegradable lipid particles; mixing the biodegradable lipid particles with the radioactive material; incubating the resulting mixture; and Obtaining lipid particles for radiotherapy 2. A method for producing the lipid particle of claim 1, comprising:

14. The method according to claim 13, wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid thereof, and a nanoparticle immobilized on the linker for immobilizing the radioactive substance.

15. 14. The method of claim 13, wherein the radioactive material is an alpha emitter.

16. 14. The method of claim 13, wherein the radioactive substance is astatine.

17. A combination composition comprising a first composition containing biodegradable lipid particles for radiotherapy, and a second composition containing a radioactive substance as an active ingredient to be bound to the lipid particles.

18. The combination composition according to claim 17, wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid thereof, and a nanoparticle immobilized to the linker for immobilizing the radioactive substance.

19. 18. The combination composition of claim 17, wherein the radioactive material is an alpha emitter.

20. 18. The combination composition of claim 17, wherein the radioactive substance is astatine.

21. A kit for producing lipid particles for radiotherapy, which independently comprises biodegradable lipid particles for use in radiotherapy and a radioactive substance as an active ingredient.

22. The kit according to claim 21, wherein the biodegradable lipid particle further comprises a linker extending from an end of a portion of the constituent lipid thereof, and a nanoparticle immobilized on the linker for immobilizing the radioactive substance.

23. 22. The kit of claim 21, wherein the radioactive material is an alpha emitter.

24. 22. The kit of claim 21, wherein the radioactive substance is astatine.

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