Nanoparticle and pharmaceutical composition

Abstract

The present invention provides a nanoparticle with which it is possible, safely, efficiently, and stably over a long period of time, to introduce siRNA used for RNA interference into an intervertebral disk cell. This nanoparticle is used for prevention or treatment of intervertebral disk degeneration, and has a biodegradable hydrogel and a ribonucleic acid molecule that is retained by a particle of the biodegradable hydrogel and that selectively inhibits an activity of a mammalian target of rapamycin (mTOR) complex.

Description

Nanoparticles and pharmaceutical compositions

[0001] The present invention relates to nanoparticles and pharmaceutical compositions.

[0002] Low back pain is the most common illness in Japan. Furthermore, in the United States, low back pain is a serious problem that causes 1% of the workforce to be unable to work, resulting in an annual economic loss of $100 billion. One of the main causes of low back pain is spinal disc degeneration, which can lead to conditions such as disc herniation and spinal stenosis. Particularly in the elderly, spinal disc degeneration (hereinafter simply referred to as "disc degeneration") is often accompanied by lower limb pain, numbness, paralysis, and bladder and rectal disorders. Elderly people suffering from spinal disc degeneration often end up bedridden or in need of care.

[0003] The current main treatment for intervertebral disc degeneration is surgical resection. However, surgical resection has issues such as a high reoperation rate and poor outcomes associated with tissue destruction and loss of function. Meanwhile, research is also being conducted on drugs and other treatments for intervertebral disc degeneration without surgical resection (Patent Documents 1 to 4, etc.).

[0004] Autophagy, a mechanism for the breakdown and recycling of intracellular waste products and abnormal organelles, is important for maintaining cellular homeostasis. Mammalian target of rapamycin (mTOR) plays a central role in the signaling pathway that inhibits autophagy. Regulation of autophagy and the mTOR signaling pathway has been shown to be important for maintaining the homeostasis of intervertebral disc nucleus pulposus cells (Non-Patent Documents 1-3).

[0005] One known method for introducing drugs into cells is to use biodegradable hydrogel particles as a carrier (Patent Document 5). The biodegradable hydrogel particles are taken up by living cells themselves, and then gradually release the drug as they are degraded within the cells. Patent Document 5 describes that when biodegradable hydrogel particles were loaded with magnetic material and introduced into mouse hepatocytes, the ratio of the amount of magnetic material remaining seven days after introduction to the amount of magnetic material one day after introduction was 0.5 to 0.6.

[0006] International Publication No. 2017 / 130756 International Publication No. 2019 / 151444 Japanese Patent Application Laid-Open No. 2021-178860 Patent Publication No. 2022-551002 International Publication No. 2019 / 088292

[0007] Ito et al., “Selective interference of mTORC1 / RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction,” Osteoarthritis Cartilage, 2017, 25(12), p. 2134-2146, Kakiuchi et al., “Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction", Osteoarthritis Cartilage, 2019, 27(6), p.965-976, Yurube et al., "Autophagy and mTOR signaling during intervertebral disc aging and degeneration", JOR Spine, 2020, 3(1), e1082

[0008] As described in Patent Documents 1 to 4, methods of treating intervertebral disc degeneration by introducing drugs or the like have been investigated. However, the safety and effectiveness of these methods have not been guaranteed, and they have not yet been put to practical use. One of the reasons for this is thought to be the unique characteristics of the intervertebral disc.

[0009] The intervertebral disc is an avascular tissue with a central nucleus pulposus surrounded by a surrounding annulus fibrosus and cartilaginous endplates. This makes it susceptible to nutritional deficiencies due to aging and smoking, which are considered to be the primary cause of disc degeneration. Furthermore, the avascular nature of the intervertebral disc makes it one of the earliest tissues to degenerate in the human body, with early degeneration particularly evident in the nucleus pulposus. In humans, after the age of 11–16, nucleus pulposus cells lose their phenotype and are replaced by chondrocyte-like cells. This leads to increased histological degeneration and cell death. Maintaining homeostasis in the intervertebral disc is difficult, making it unsuitable for repair and regeneration. Therefore, strategies that take into account the unique internal environment are crucial for cell-biological treatment of disc degeneration.

[0010] In recent years, technologies for introducing specific genes or drugs into cells have attracted attention in the fields of regenerative medicine and gene therapy. In particular, gene expression suppression technologies such as RNA interference (RNAi) are expected to be useful in the treatment and prevention of diseases by suppressing the function of specific genes. Gene expression suppression technologies that have minimal impact on homeostasis and are less likely to cause long-term cytotoxicity are particularly useful for intervertebral discs, which have the characteristics described above. However, in order to put gene expression suppression technologies into practical use, a method for efficiently and safely introducing genes into cells is required.

[0011] Viral vectors and lipofection have been widely used as conventional gene transfer methods, but these methods have several issues. Viral vectors have high transfer efficiency, but they carry the risk of immune reactions and genetic recombination. Lipofection is safer than viral vectors, but has low transfer efficiency and still poses problems of cytotoxicity.

[0012] It is thought that hydrogel particles such as those described in Patent Document 5 can be used to safely introduce genes into intervertebral disc cells. However, according to the findings of the present inventors, not all cells take up hydrogel particles; some cells take up hydrogel particles while others do not. Furthermore, it is thought that intervertebral disc cells, which are avascular tissues with low metabolic activity, have low uptake efficiency of hydrogel particles.

[0013] Furthermore, since the puncture performed during local administration of drugs can cause degeneration of the intervertebral disc, it is desirable to use a method that can reduce the number of drug administrations to the patient when treating and preventing intervertebral disc degeneration. Patent Document 5 describes that after the administration of hydrogel particles containing a drug (contrast agent), the amount of contrast agent was reduced by half within about one week. However, when treating and preventing intervertebral disc degeneration, it is desirable for the gene to remain in the cells for a longer period of time.

[0014] Given this background, there is a need for the development of safer and more efficient gene transfer methods. In particular, considering the characteristics of the intervertebral disc described above, it is desirable to develop a minimally invasive method that allows stable gene transfer over a long period of time.

[0015] As described above, an object of the present invention is to provide a safe and efficient gene transfer method, thereby bringing about great progress in the fields of regenerative medicine and gene therapy.

[0016] The present invention was made based on the above findings, and aims to provide nanoparticles that can safely, efficiently, and stably introduce ribonucleic acid molecules that selectively knock out target genes, such as siRNA used in RNA interference, into intervertebral disc cells over a long period of time, and a pharmaceutical composition containing the nanoparticles.

[0017] The above-mentioned problems are solved by the nanoparticles and pharmaceutical compositions of [1] to

[10] below. [1] Nanoparticles for use in preventing or treating intervertebral disc degeneration, comprising: a biodegradable hydrogel; and a ribonucleic acid molecule retained in the biodegradable hydrogel particles, the ribonucleic acid molecule selectively inhibiting the activity of a mammalian target of rapamycin (mTOR) complex. [2] The nanoparticles of [1], wherein the ribonucleic acid molecule is an siRNA that selectively inhibits the activity of mTOR complex 1. [3] The nanoparticles of [1] or [2], wherein the ribonucleic acid molecule is an siRNA that selectively inhibits the expression of regular-associated protein of mTOR (RAPTOR). [4] The nanoparticles of any of [1] to [3], wherein the amount of the ribonucleic acid molecule is 1 pmol to 100 pmol per 1 μg of hydrogel. [5] The nanoparticles according to any one of [1] to [4], wherein the biodegradable hydrogel is cationized gelatin. [6] The nanoparticles according to any one of [1] to [5], wherein an aqueous solution containing the nanoparticles at a concentration of 60 pmol / μl, calculated as ribonucleic acid molecules, is administered to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat, and four weeks later, the rate of ribonucleic acid-positive cells in the administration area is 80% or more. [7] The nanoparticles according to any one of [1] to [6], wherein an aqueous solution containing the nanoparticles at a concentration of 60 pmol / μl, calculated as ribonucleic acid molecules, is administered to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat, and four weeks later, the rate of ribonucleic acid-positive cells in the administration area is 80% or more. [8] A pharmaceutical composition for use in preventing or treating intervertebral disc degeneration, comprising the nanoparticles according to any one of [1] to [7]. [9] The pharmaceutical composition according to [8], wherein the administration interval is 2 weeks or more.

[10] The pharmaceutical composition according to [8] or [9], wherein the amount of the nanoparticles converted into the amount of the ribonucleic acid molecules is 10 pmol to 400 nmol per ml of the pharmaceutical composition.

[0018] The present invention provides nanoparticles that can safely, efficiently, and stably introduce ribonucleic acid molecules that selectively knock out target genes, such as siRNA used in RNA interference, into intervertebral disc cells over a long period of time, and a pharmaceutical composition containing the nanoparticles.

[0019] Figure 1 shows a pathway diagram illustrating how mTOR is involved in mRNA translation, protein synthesis, cell division and proliferation, and autophagy. Ｒａｐ Figure 2B is a graph showing cell viability after lipofection at different concentrations. Figure 3A is a graph showing cell viability after lipofection with cGNS. Ｒａｐ－Ｆｌ Figure 3B shows a fluorescence image of the cGNS siRNA at a concentration of 400 pmol / ml. Ｒａｐ－Ｆｌ 3C is a fluorescent image of intervertebral disc nucleus pulposus cells after cell division after 4 days when the siRNA concentration was set at 400 pmol / ml using cGNS. Ｒａｐ－Ｆｌ 4A is a graph showing the siRNA-positive cell rate after 1 day, 4 days, 7 days, 10 days, and 14 days when the siRNA concentration was 400 pmol / ml using cGNS. Ｒａｐ－Ｆｌ The siRNA concentration was set at 400 pmol / ml, and cGNS Ｒａｐ－Ｆｌ Figure 4B is a graph showing the siRNA-positive cell rate after 1 day, 4 days, and 7 days in each test shown in Figure 4A. Figure 5A shows the cGNS-positive cell rate after 1 day, 4 days, and 7 days in each test shown in Figure 4A. Ｒａｐ Figure 5B shows the bands representing RAPTOR (150 kDa) and α-tubulin (49 kDa) obtained by Western blot for the group transfected with 60 pmol / ml siRNA (60 pmol / ml siRNA) and the control group transfected by lipofection (60 pmol / ml siRNA). Ｒａｐ5B is a graph showing the expression level of RAPTOR (relative to the expression level of α-tubulin) after 1 day, 2 days, 3 days, 5 days, and 7 days for a group transfected with siRNA (60 pmol / ml). FIG. 5C is a graph showing the expression level of RAPTOR (relative to the expression level of α-tubulin) after 1 day, 2 days, 3 days, 5 days, and 7 days for a group transfected by lipofection (60 pmol / ml). FIG. 5D is a graph showing both FIG. 5B and FIG. 5C. FIG. 6 shows the expression level of RAPTOR (relative to the expression level of α-tubulin) after 1 day, 2 days, 3 days, 5 days, and 7 days for a group transfected by lipofection (60 pmol / ml). Ｒａｐ The Western blot analysis of the group transfected with 400 pmol / ml siRNA shows bands representing RAPTOR (150 kDa), LC3-I (16 kDa), LC3-II (14 kDa), p62 / SQSTM1 (62 kDa), and α-tubulin (49 kDa). Ｒａｐ－Ｆｌ Fluorescence images taken 7 and 28 days after administration using siRNA (60 pmol / μl) and 7 and 28 days after administration using lipofection (60 pmol / μl). Ｒａｐ－Ｆｌ Figure 9A shows fluorescence images taken 7, 28, 56, and 84 days after administration of cGNS fluorescently labeled with Cy5 (cGNS-FI) to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat. Figure 9B shows a graph showing the total fluorescence intensity (ratio to the fluorescence intensity immediately after administration) immediately, 1 day, 4 days, 7 days, 14 days, 28 days, 56 days, and 84 days after administration of cGNS fluorescently labeled with Cy5 (cGNS-FI) to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat. Figure 10 is a schematic diagram showing the experimental protocol for Animal Experiment 3. FIG. 11A shows the cGNS in vivo in rat intervertebral disc tissue. Ｒａｐ , cGNSｃｔｒｌ , and PBS were administered, and 7 days after administration, a static compression force (1.3 MPa) was applied for 24 hours to the compression group (loaded). Figure 11B shows X-ray images taken immediately after static compression, and 7, 28, 56, and 84 days after static compression. Ｒａｐ , cGNS ｃｔｒｌ 12A and 12B are graphs showing the normalized disc height calculated using the disc height index when a static compression force (1.3 MPa) was applied for 24 hours after 7 days of administration to the compression group (loaded) in which cGNS and PBS were administered. Ｒａｐ , cGNS ｃｔｒｌ , and PBS were administered, and a static compression force (1.3 MPa) was applied for 24 hours only to the compression group (loaded) 7 days after administration. Safranin O / Fast green staining images of NP (nucleus pulposus), AF (annulus fibrosus), and CEP (cartilage endplate) were obtained immediately after static compression, 28 days later, 56 days later, and 84 days later. Figure 12B shows the effect of cGNS on rat intervertebral disc tissue in vivo. Ｒａｐ , cGNS ｃｔｒｌ 13A and 13B are graphs showing the degeneration score evaluated using the Orthopaedic Research Society spine section histopathological grading score when a static compressive force (1.3 MPa) was applied for 24 hours 7 days after administration to the compression group (loaded) alone. Ｒａｐ and cGNS ｃｔｒｌ 13B shows fluorescent images obtained by multi-fluorescence staining 28 days after static compression when a static compression force (1.3 MPa) was applied to the compression group (loaded) 7 days after administration of the drug or no drug, respectively. Ｒａｐ and cGNS ｃｔｒｌ13C is a graph showing the rate of RAPTOR positive cells when a static compressive force (1.3 MPa) was applied for 24 hours 7 days after administration to the compression group (compression (+)) alone, or without administering any drug. Ｒａｐ and cGNS ｃｔｒｌ 13D is a graph showing the rate of TUNEL-positive cells when a static compressive force (1.3 MPa) was applied for 24 hours 7 days after administration to the compression group (compression (+)) alone, or without administration of any drug. Ｒａｐ and cGNS ｃｔｒｌ 14A is a graph showing the percentage of p16 / INK4A positive cells when a static compressive force (1.3 MPa) was applied for 24 hours 7 days after administration to the compression group (compression (+)) alone, or without administration of any drug. Ｒａｐ－Ｆｌ , and cGNS ｃｔｒｌ Normal tissue (-), cGNS (without drug administration), and sham compression (+) were administered to the compression group (loaded) and the compression group (compression (+)) 7 days after the administration. ｃｔｒｌ (loaded), and cGNS Rａｐ Figure 14B shows the immunohistochemical staining (DAB staining) of the cGNS in the rat intervertebral disc tissue in vivo. Ｒａｐ－Ｆｌ and cGNS ｃｔｒｌ The cGNS obtained 28 days after static compression when a static compression force (1.3 MPa) was applied for 24 hours 7 days after administration to the compression group (compression (+)) alone, or without administration of any drug. ｃｔｒｌ (compression(+)), and cGNS Rａｐ Figure 14C is a graph showing the ratio of DIPEN production rate adjusted by cell number in the rat intervertebral disc tissue in vivo (compression (+)) to normal tissue. Ｒａｐ－Ｆｌ and cGNS ｃｔｒｌThe cGNS obtained 28 days after static compression when a static compression force (1.3 MPa) was applied for 24 hours 7 days after administration with or without drug administration. ｃｔｒｌ (compression(+)), and cGNS Rａｐ 1 is a graph showing the ratio of ARGSV production rate in (compression(+)) to normal tissue, adjusted by cell number.

[0020] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0021] One embodiment of the present invention relates to nanoparticles for use in the prevention or treatment of intervertebral disc degeneration, comprising a biodegradable hydrogel and a ribonucleic acid molecule that selectively inhibits the activity of the mammalian target of rapamycin (mTOR) complex by RNA interference, the ribonucleic acid molecule being retained on the biodegradable hydrogel particle.

[0022] (Biodegradable Hydrogel) Nanoparticles include biodegradable hydrogels. Biodegradable hydrogels are materials that form nanoparticles and hold ribonucleic acid molecules inside the nanoparticles. Furthermore, nanoparticles formed from biodegradable hydrogels are taken up by certain living cells and gradually decompose within the living cells. Therefore, nanoparticles formed from biodegradable hydrogels can serve as carriers for delivering ribonucleic acid molecules to intervertebral disc cells and can gradually release the ribonucleic acid molecules within the cells.

[0023] A hydrogel is a material that can form a gel using water as a solvent, and typically contains a hydrophilic polymer that forms a network structure and water trapped in the network structure. The hydrogel used in this embodiment is a biodegradable hydrogel, which is a material that is hydrolyzed within cells or decomposed by enzymes or lysosomes secreted within cells.

[0024] Examples of hydrogels include polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, agarose, carboxymethylcellulose (CMC), starch, and pectin; proteins such as gelatin, collagen, fibrin, and albumin; polyamino acids such as poly-γ-glutamic acid, poly-L-lysine, and polyarginine; and synthetic polymers such as acrylamide, silicone, polyvinyl alcohol, polyethylene oxide, and polyvinylpyrrolidone.

[0025] Among these, polysaccharides, proteins, and polyamino acids are preferred from the viewpoint of facilitating their uptake into living cells themselves and suppressing damage to living cells during uptake. Furthermore, from the viewpoint of cellular uptake and ease of acquisition or production, polysaccharides and proteins are more preferred, and gelatin is even more preferred.

[0026] Gelatin is a protein that contains 300 or more glycine residues out of 1,000 amino acid residues when analyzed with an amino acid analyzer, and also contains both alanine and proline. Any known gelatin can be used, as long as it can be formed into particles. It can be obtained by denaturing collagen derived from bovine bone, bovine hide, pig skin, pig tendon, fish scales, fish meat, etc. Gelatin has long been used for food and medical purposes, and poses little harm to the human body when ingested. Furthermore, since gelatin disperses and disappears in vivo, it does not need to be removed from the body. Gelatin may contain components other than gelatin as long as they can be taken up into cells when granulated. It is preferable that the amounts of components other than gelatin contained in nanoparticles as biodegradable hydrogels are within a range that poses negligible harm to the human body when ingested. Furthermore, it is preferable that the components other than gelatin are substances that do not accumulate in the body and are easily excreted.

[0027] From the viewpoint of facilitating the formation of hydrogel particles, the weight-average molecular weight of gelatin is preferably 1,000 or more and 100,000 or less. The weight-average molecular weight can be a value measured in accordance with, for example, the PAGI Method, 10th Edition (2006).

[0028] Gelatin may be crosslinked. The crosslinking may be performed by a crosslinking agent or may be self-crosslinking without the use of a crosslinking agent.

[0029] The crosslinking agent may be, for example, a compound having multiple functional groups capable of forming chemical bonds with hydroxyl groups, carboxyl groups, amino groups, thiol groups, imidazole groups, etc. Examples of such crosslinking agents include glutaraldehyde, water-soluble carbodiimides including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate (CMC), compounds having two or more epoxy groups including ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, and glycerol polyglycidyl ether, and propylene oxide. Of these, glutaraldehyde and EDC are preferred, with glutaraldehyde being more preferred, from the viewpoint of enhancing reactivity.

[0030] Examples of the self-crosslinking include crosslinking by application of heat or irradiation with electron beams or ultraviolet rays.

[0031] The nanoparticles preferably contain at least two hydrogels (a first hydrogel and a second hydrogel) that differ in degree of crosslinking or composition. The first hydrogel and the second hydrogel may be selected from the hydrogels described above, and may be a combination of hydrogels with different degrees of crosslinking or a combination of hydrogels with different compositions.

[0032] The difference in the composition of the hydrogel refers to the difference in the ratio of functional groups, etc., detected by, for example, TEM-EDS measurement, i.e., the ratio of S atoms, N atoms, O atoms, C atoms, and H atoms. By preparing a cross-sectional sample of a hydrogel particle and performing TEM-EDS measurement, the difference in the ratio of S atoms, N atoms, O atoms, C atoms, etc. can be detected. In this specification, the difference in the composition of the hydrogel means that the content of at least one of S atoms, N atoms, O atoms, C atoms, and H atoms that constitute the hydrogel differs by 2% or more.

[0033] Differences in the degree of crosslinking of hydrogels refer to differences in density detected by, for example, TEM-EELS measurement. These differences in density can be confirmed as differences in TEM-EELS loss spectra. In this specification, differences in the degree of crosslinking mean that the signal intensity of any energy band in the TEM-EELS loss spectrum differs by 2% or more.

[0034] The combination of hydrogels with different crosslinking degrees or compositions is preferably a combination of hydrogels with different intracellular degradability. Combining two or more types of hydrogels with different intracellular degradability facilitates controlled release of the encapsulated substance. The intracellular degradability of the hydrogel can be evaluated by preparing hydrogel particles carrying a labeling substance, introducing the particles into cells, and then measuring the particle decomposition rate using the labeling substance as an indicator.

[0035] In this case, the nanoparticle preferably includes an internal domain made of a first hydrogel and an external matrix made of a second hydrogel that encapsulates the internal domain.

[0036] The specific combination of hydrogels can be selected to achieve the desired release behavior. Particularly from the viewpoint of facilitating the sustained release of ribonucleic acid molecules from hydrogel particles, it is preferable to use a hydrogel that is less susceptible to degradation as the first hydrogel constituting the internal domain and a hydrogel that is more susceptible to degradation as the second hydrogel constituting the external matrix. For example, a hydrogel with a higher degree of crosslinking may be used as the first hydrogel and a hydrogel with a lower degree of crosslinking may be used as the second hydrogel. When hydrogel particles having such a structure are taken up by living cells, the easily degradable external matrix composed of the second hydrogel is first degraded from the outside by enzymes present in the cytoplasm (e.g., collagenase if the hydrogel is gelatin). As a result, the microparticles that were in the less susceptible internal domain composed of the first hydrogel are released into the cytoplasm, starting with those located closest to the outside of the external matrix, followed by the microparticles that were in the internal domain.

[0037] The nanoparticle may include two or more internal domains, including an internal domain formed by a first hydrogel and an internal domain formed by a third hydrogel that differs from the first hydrogel in degree of crosslinking or composition.

[0038] From the viewpoint of further enhancing biocompatibility, it is preferable that at least one of the first hydrogel and the second hydrogel contains gelatin. Furthermore, in order to facilitate the uptake of the hydrogel particles by the activity of the cells themselves, it is more preferable that the second hydrogel constituting the external matrix that forms the outer layer of the particles contains gelatin, and it is even more preferable that both the first hydrogel and the second hydrogel contain gelatin.

[0039] (Ribonucleic acid molecule) The nanoparticles contain a ribonucleic acid molecule that selectively inhibits the activity of the mammalian target of rapamycin (mTOR) complex. The ribonucleic acid molecule may be any small RNA that inhibits the activity of the mTOR complex, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA). The ribonucleic acid molecule, which is a small RNA, preferably has 15 to 100 bases, more preferably 20 to 70 bases, and even more preferably 30 to 50 bases.

[0040] These ribonucleic acid molecules may be fluorescently labeled, etc. By labeling the ribonucleic acid molecule, it is possible to confirm whether the ribonucleic acid molecule has been sufficiently introduced into cells after administration to the intervertebral disc.

[0041] Figure 1 shows a pathway diagram illustrating the involvement of mTOR in apoptosis, mRNA translation, protein synthesis, cell division and proliferation, senescence, extracellular matrix degradation, and autophagy. mTOR, a serine / threonine kinase, forms two protein complexes: mTOR complex 1 (mTORC1), which is a complex with the regulatory-associated protein of mTOR (RAPTOR), and mTOR complex 2 (mTORC2), which is a complex with the rapamycin-insensitive companion of mTOR (RICTOR). When cells receive nutrients, growth factors, and energy, these signals are transmitted to the enzyme PI3K. PI3K activates the serine / threonine kinase Akt directly or via mTORC2. Akt then suppresses apoptosis. Akt also regulates the activity of the downstream signaling pathway, mTORC1. mTORC1 promotes cell proliferation and protein synthesis through the kinase p70 / S6K, while also inducing senescence and extracellular matrix degradation. mTORC1 also suppresses autophagy. Thus, the mTOR complex is involved in apoptosis, senescence, extracellular matrix degradation, autophagy, and other processes. Therefore, inhibiting the activity of the mTOR complex and reducing these functions can promote the maintenance of homeostasis in intervertebral disc cells and potentially treat or prevent intervertebral disc degeneration.

[0042] The ribonucleic acid molecule may inhibit the activity of either mTORC1 or mTORC2. According to the findings of the present inventors, activating autophagy in intervertebral disc cells and improving the cells' own cleaning and healing abilities through autophagy facilitates the maintenance of homeostasis in intervertebral disc cells, and can more effectively suppress cell death, aging, and extracellular matrix degradation in intervertebral disc cells. Therefore, the ribonucleic acid molecule preferably inhibits the activity of mTORC1, which suppresses autophagy, and particularly preferably inhibits the expression of RAPTOR. Ribonucleic acid molecules that inhibit these activities or expressions may be produced by known methods or obtained from commercial sources.

[0043] These ribonucleic acid molecules typically remain in the cytoplasm and disappear after several passages, restoring normal gene function. Therefore, the gene knockdown effect is temporary and is unlikely to have a permanent effect on cellular activity. On the other hand, since ribonucleic acid molecules inhibit mTOR activity as long as they remain in the cytoplasm, they can be expected to have a longer-lasting effect than drug or protein administration, and the number of administrations to patients can be reduced. Since punctures performed during local administration of drugs, etc. can sometimes cause degeneration of the intervertebral disc, reducing the number of administrations to patients can also suppress the progression of intervertebral disc degeneration caused by punctures, etc. during administration.

[0044] The molecular weight of the ribonucleic acid in the nanoparticles is preferably 1 pmol to 100 pmol, more preferably 5 pmol to 70 pmol, and even more preferably 10 pmol to 50 pmol, per 1 μg of hydrogel.

[0045] (Nanoparticles) Nanoparticles include particles formed from the hydrogel and the ribonucleic acid molecules held in the hydrogel.

[0046] The ribonucleic acid molecule may be retained on the surface of the nanoparticle or may be retained inside the nanoparticle. When the nanoparticle has a multi-layer structure having an internal domain and an external matrix, the ribonucleic acid molecule may be contained in either the internal domain or the external matrix, or in both the internal domain and the external matrix. When the ribonucleic acid molecule is contained in the internal domain, the ribonucleic acid molecule is released over a longer period of time, so that the therapeutic or preventive effect on intervertebral disc degeneration due to the inhibition of mTOR activity is exerted over a longer period of time. When the ribonucleic acid molecule is contained in the external matrix, the ribonucleic acid molecule is released immediately after uptake into cells, so that the therapeutic or preventive effect on intervertebral disc degeneration is exerted early after administration.

[0047] The average particle diameter of the nanoparticles is preferably 100 nm or more and 1000 nm or less. When the average particle diameter of the nanoparticles is 1000 nm or less, they are easily taken up into cells by the cell's own activity. In order to take up a large number of nanoparticles into cells in a shorter time, the average particle diameter of the nanoparticles is more preferably 800 nm or less. On the other hand, nanoparticles with an average particle diameter of 100 nm or more are more likely to carry ribonucleic acid molecules within the particles, and the amount of ribonucleic acid molecules that can be accommodated can be increased. Furthermore, the larger the average particle diameter of the nanoparticles within the range of 1000 nm or less, the more easily they are taken up into cells by the cell's own activity. From the above perspective, the average particle diameter of the nanoparticles is preferably 150 nm or more. The average particle diameter of the nanoparticles can be the apparent particle diameter of the nanoparticles measured by dynamic light scattering.

[0048] The nanoparticles may contain drugs or probes other than the above-mentioned ribonucleic acid molecules.

[0049] Examples of the above-mentioned drugs include pharmaceutical proteins, plasmids, aptamers, antisense nucleic acids, ribozymes, nucleic acid molecules used for pharmaceutical purposes (excluding the above-mentioned ribonucleic acid molecules that selectively inhibit the activity of the mTOR complex), including tRNA, snRNA, siRNA, shRNA, miRNA, ncRNA, and condensed DNA, as well as antigens used for pharmaceutical purposes. Examples of the above-mentioned probes include fluorescent probes and molecular beacons.

[0050] The amount of water contained in the nanoparticles is not particularly limited, but after the swelling treatment, it is preferably 1% by mass or more and 99% by mass or less, more preferably 10% by mass or more and 90% by mass or less, and even more preferably 15% by mass or more and 80% by mass or less, relative to the total mass of the nanoparticles.

[0051] In this specification, nanoparticles after swelling treatment refer to nanoparticles obtained by immersing dry nanoparticles in water at 40°C under atmospheric pressure for 60 minutes, and in this specification, nanoparticles in a dry state refer to nanoparticles after being left to stand in the air at 80°C for 24 hours.

[0052] It is preferable that the surface of nanoparticles be positively charged. This is because, since the surface of a cell is negatively charged, if the total surface charge of nanoparticles is positive, nanoparticles can easily approach cells and be easily taken up by cells. Furthermore, if the total surface charge of nanoparticles is positive, after they are taken up by cells via endocytosis, the release of nanoparticles from endosomes into the cytoplasm due to the proton sponge effect is likely to be promoted.

[0053] More specifically, the zeta potential of the nanoparticles in a solution having a pH of 7.4 is preferably greater than 0 mV and less than or equal to 30 mV. When the zeta potential is greater than 0 mV, the nanoparticles are easily taken up. When the zeta potential is less than or equal to 30 mV, the nanoparticles are sufficiently flexible. From the above viewpoint, the zeta potential is more preferably 2 mV or more and 30 mV or less, and even more preferably 4 mV or more and 15 mV or less.

[0054] The zeta potential can be a value measured using a known zeta potential measuring device (for example, a measuring device using the formula described in Hiroyuki Mori and Yoshio Okamoto: Flotation, 27, 117, 1-124 (1980)).

[0055] To achieve the above state, the hydrogel is preferably cationized. The cationization of the hydrogel can be achieved by crosslinking or PEG-NH ２ This can be achieved by reducing the amount of carboxyl or aldehyde groups in the molecule through surface modification with cations such as cationization with glycine or capping with glycine. The greater the degree of cationization, the easier the nanoparticles are to be taken up, resulting in a greater therapeutic or preventive effect.

[0056] (Comparison with other methods) In this embodiment, particles formed from a biodegradable hydrogel are used as a carrier to introduce ribonucleic acid molecules into intervertebral disc cells. In this method, the cells themselves take up the nanoparticles, and the biodegradable hydrogel is degraded within the cells, gradually releasing the ribonucleic acid molecules. All materials contained in the nanoparticles are highly biocompatible and disappear within the cells after inhibiting only the activity of the target protein. Therefore, problems such as immune reactions and genetic recombination, which are issues with viral vector methods, are unlikely to occur.

[0057] Furthermore, because nanoparticles are made of highly biocompatible materials, cytotoxicity is less of a problem, unlike lipofection. For example, in the examples described below, when the lipofection method was used, a significant decrease in cell viability was observed when the ribonucleic acid molecule concentration (around the cells) in the culture medium was 80 pmol / ml. However, when using the nanoparticles of this embodiment, no significant decrease in cell viability was observed even when the ribonucleic acid molecule concentration was 400 pmol / ml (see Figures 2A and 2B). Therefore, by using the nanoparticles of this embodiment, it is possible to introduce a larger amount of ribonucleic acid molecules into cells, and a higher therapeutic or preventive effect can be expected. Note that the optimal dosage may vary depending on the animal species and administration method. For example, when administered to a living body such as a human, a higher concentration is usually administered to enhance the therapeutic and preventive effect.

[0058] Furthermore, while the lipofection method has the problem of low transfection efficiency, the use of nanoparticles can dramatically increase the rate of transfection of ribonucleic acid molecules into cells. For example, in the Examples described below, the rate of transfection of ribonucleic acid molecules into cells using nanoparticles was 100% after one day, which is significantly higher than that of lipofection (see Figures 4A and 4B).

[0059] According to the findings of the present inventors, not all types of cells uniformly take up hydrogel particles, and some cells may not take up hydrogel particles at all. It is conceivable that intervertebral disc cells, which have low metabolic activity, would have a low uptake efficiency of hydrogel particles, but the high uptake rate demonstrated, contrary to this expectation, is a surprising result. Although the reason for this is unclear, it is thought that intervertebral disc cells, located in a closed environment in vivo, are less susceptible to external influences, resulting in a high uptake rate.

[0060] Furthermore, nanoparticles can release ribonucleic acid molecules in cells over a long period of time because they slowly release the ribonucleic acid molecules. For example, in the Examples described below, the amount of ribonucleic acid molecules remaining in cells decreased over time when using lipofection, but when using nanoparticles, the percentage of cells in which the ribonucleic acid molecules remained for a long period of time after administration was very high, and the ribonucleic acid molecules remained in daughter cells after cell division (see Figures 3A to 3C, 4A and 4B).

[0061] According to the inventors' new findings, the sustained-release effect observed when biodegradable hydrogel particles are introduced into intervertebral disc cells is significantly longer than when biodegradable hydrogel particles are introduced into other cells. For example, in the Examples described below, when biodegradable hydrogel particles (gelatin particles) were administered to the nucleus pulposus region of the caudal intervertebral disc of 12-week-old male Sprague-Dawley rats, the presence of ribonucleic acid and biodegradable hydrogel was confirmed even after 8 weeks (56 days) and 12 weeks (84 days) (see Figures 8, 9A, and 9B). This is thought to be due to the fact that the intervertebral disc is an avascular tissue and intervertebral disc cells are located in a closed environment, resulting in relatively low metabolic activity.

[0062] In other words, by using nanoparticles to deliver ribonucleic acid molecules to intervertebral disc cells, it is possible to deliver a large amount of ribonucleic acid molecules compared to lipofection and even achieve sustained release over a very long period of time. This is thought to enable a high therapeutic or preventive effect to be achieved while reducing the number of administrations of ribonucleic acid molecules for treating or preventing intervertebral disc degeneration. Because punctures performed during local administration can cause degeneration of the intervertebral disc, it is preferable to administer ribonucleic acid molecules as few times as possible.

[0063] Furthermore, as will be shown in the Examples below, animal experiments have also shown that nanoparticles can be used to introduce and slowly release ribonucleic acid molecules into intervertebral disc cells (see Figures 7 and 8). These results demonstrate that methods using nanoparticles have a high potential for the treatment or prevention of intervertebral disc degeneration by non-surgical means, which has been difficult to achieve until now.

[0064] For example, in the Examples described below, an aqueous solution containing the nanoparticles was administered to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat at a concentration of 60 pmol / μl, calculated as ribonucleic acid molecules. After one week (7 days) and four weeks (28 days), the ribonucleic acid-positive cell rate in the administration area was 100%. While the ribonucleic acid-positive cell rate may vary somewhat depending on the molecular weight of the ribonucleic acid contained in the nanoparticles and the size of the nanoparticles, after one week, it can be 60% or higher, preferably 80% or higher, and more preferably 90% or higher. Similarly, after four weeks, it can be 60% or higher, preferably 80% or higher, and more preferably 90% or higher.

[0065] In the examples described below, an aqueous solution containing the nanoparticles was administered to the nucleus pulposus region of the caudal intervertebral disc of 12-week-old male Sprague-Dawley rats at a concentration of 60 pmol / μl, calculated as ribonucleic acid molecules. The siRNA-positive cell rates were 82.0% (56 days) and 32.5% after 8 weeks (56 days) and 12 weeks (84 days), respectively (see Figure 8). While the ribonucleic acid-positive cell rate may vary somewhat depending on the molecular weight of the ribonucleic acid contained in the nanoparticles and the size of the nanoparticles, after 8 weeks, the rate can be between 40% and 100%, preferably between 60% and 90%, and more preferably between 70% and 90%. Similarly, after 12 weeks, the rate can be between 10% and 100%, preferably between 20% and 60%, and more preferably between 30% and 50%.

[0066] As a result, in this embodiment, the effects of reducing apoptosis, senescence, extracellular matrix degradation, autophagy, and other activities are maintained for a longer period than previously expected (see Figures 12A, 12B, 13A, 13B, 13C, 13D, 14A, and 14B). This promotes the maintenance of homeostasis in intervertebral disc cells, and provides the effect of treating or preventing intervertebral disc degeneration for a long period of time.

[0067] Therefore, the introduction of ribonucleic acid molecules using biodegradable hydrogel nanoparticles could be a very promising method for treating or preventing intervertebral disc degeneration. To date, no local gene transfer technology into living intervertebral discs has been proven safe and effective and has been put to practical use. Biodegradable hydrogel nanoparticles enable minimally invasive, long-lasting, and stable gene transfer. This could potentially lead to applications in many other areas and diseases, including degenerative diseases such as osteoarthritis and inflammatory diseases such as rheumatoid arthritis, and could have a significant impact on medical applications.

[0068] Furthermore, the introduction of ribonucleic acid molecules in this embodiment is based on an improvement in the internal environment, making it less likely that adjacent intervertebral disc disorders or lumbar spondylolysis will occur after spinal fusion surgery. This makes it possible to preserve intervertebral disc function, which will be beneficial for many young people, athletes, and postoperative patients, and is expected to contribute to the medical economy by reducing the number of expensive spinal surgeries.

[0069] Furthermore, gene therapy using ribonucleic acid molecules targets cells that are naturally present in the body, eliminating the need for foreign substances. Furthermore, it has the advantage of a longer duration of action compared to other drugs with a long half-life. Because of its long duration of action, it is possible to reduce the frequency of punctures and prevent tissue damage and degeneration caused by punctures. Furthermore, the mechanism of action of other drugs is often unclear, which can lead to unwanted interactions and unexpected effects. In particular, there are many unknowns about the metabolic mechanisms that affect the intervertebral disc. In contrast, the introduction of ribonucleic acid molecules that knock out only the target gene has a limited effect and is less likely to produce unexpected effects.

[0070] Pharmaceutical Compositions The nanoparticles described above can be included in pharmaceutical compositions for the treatment or prevention of intervertebral disc degeneration.

[0071] The dosage form of the pharmaceutical composition is not particularly limited, and may be a solid dosage form or a liquid dosage form.The dosage form of the pharmaceutical composition may be an oral dosage form such as a tablet, capsule, powder, granule, liquid, syrup, and sublingual, or a parenteral dosage form such as an injection for subcutaneous, intramuscular, intra-articular, and intravenous administration, a suppository for rectal administration, a patch, an ointment, a cream, a lotion, and an inhalant for intranasal administration.Among these, a liquid dosage form that can be easily administered locally to the intervertebral disc is preferred, and an injection is more preferred.

[0072] In addition to the nanoparticles, the pharmaceutical composition may contain additives such as a solvent such as water as a carrier, a solubilizing agent, a suspending agent, an emulsifying agent, a stabilizer, a preservative, an isotonic agent, a viscosity agent, a pH adjuster, a buffer, an antioxidant, and a base for intra-articular administration.

[0073] The pharmaceutical composition may be prepared as a kit in which the nanoparticles, carrier, and additives (or a combination of multiple of these) are sealed in separate containers, and are mixed at the time of use to form the pharmaceutical composition for administration.

[0074] The pharmaceutical composition can be produced by a known method according to the dosage form, etc. The nanoparticles described above can be used in producing the pharmaceutical composition by these methods.

[0075] The method of administration of the pharmaceutical composition is not limited, and may be oral or parenteral administration, but is preferably administered parenterally to the intervertebral disc. For example, the affected area may be exposed by a known surgical technique, or the pharmaceutical composition may be applied to the nucleus pulposus site using a syringe and a filling device under a microscope or endoscope.

[0076] The amount of nanoparticles in the liquid pharmaceutical composition, converted into the amount of the ribonucleic acid molecule, is preferably 10 pmol to 400 nmol per ml of the pharmaceutical composition, more preferably 100 pmol to 300 nmol, even more preferably 1 nmol to 200 nmol, and particularly preferably 10 nmol to 100 nmol. Because nanoparticles have low toxicity, the concentration of the ribonucleic acid molecule can be increased, thereby further improving the therapeutic or preventive effect per administration.

[0077] The dosage of the pharmaceutical composition is not particularly limited and may be determined depending on the therapeutic or preventive effect. For example, if the pharmaceutical composition is in liquid form, 0.01 ml to 10 ml can be administered to the affected area per dose, preferably 0.1 ml to 5 ml, and more preferably 0.2 ml to 3 ml.

[0078] The administration interval of the pharmaceutical composition is not particularly limited and may be determined depending on the therapeutic or preventive effect, but may be, for example, one day or more, preferably one week or more, more preferably two weeks or more, even more preferably four weeks or more, and particularly preferably eight weeks or more. Because the nanoparticles slowly release ribonucleic acid molecules within cells over a long period of time, the administration interval can be extended, which can suppress the onset of intervertebral disc degeneration due to puncture or the like during administration and can also improve the patient's quality of life (QOL).

[0079] Methods of Treatment or Prevention The nanoparticles described above can be used in methods of treatment or prevention of intervertebral disc degeneration.

[0080] The specific method for the treatment or prevention is not particularly limited, and a pharmaceutical composition containing the nanoparticles in a solid or liquid form may be administered to a patient or a human or non-human vertebrate for whom prevention is desired. The pharmaceutical compositions described above can be widely used as the pharmaceutical composition containing the nanoparticles.

[0081] The method of administration of the pharmaceutical composition is not limited, and may be oral or parenteral administration, but is preferably administered parenterally to the intervertebral disc. For example, the affected area may be exposed by a known surgical technique, or the pharmaceutical composition may be applied to the nucleus pulposus site using a syringe and a filling device under a microscope or endoscope.

[0082] The amount of nanoparticles in the liquid pharmaceutical composition, converted into the amount of the ribonucleic acid molecule, is preferably 10 pmol to 400 nmol per ml of the pharmaceutical composition, more preferably 100 pmol to 300 nmol, even more preferably 1 nmol to 200 nmol, and particularly preferably 10 nmol to 100 nmol. Because nanoparticles have low toxicity, the concentration of the ribonucleic acid molecule can be increased, thereby further improving the therapeutic or preventive effect per administration.

[0083] The dosage of the pharmaceutical composition is not particularly limited and may be determined depending on the therapeutic or preventive effect. For example, if the pharmaceutical composition is in liquid form, 0.01 ml to 10 ml can be administered to the affected area per dose, preferably 0.1 ml to 5 ml, and more preferably 0.2 ml to 3 ml.

[0084] The administration interval of the pharmaceutical composition is not particularly limited and may be determined depending on the therapeutic or preventive effect, but may be, for example, one day or more, preferably one week or more, more preferably two weeks or more, even more preferably four weeks or more, and particularly preferably eight weeks or more. Because the nanoparticles slowly release ribonucleic acid molecules within cells over a long period of time, the administration interval can be extended, which can suppress the onset of intervertebral disc degeneration due to puncture or the like during administration and can also improve the patient's quality of life (QOL).

[0085] As used herein, treatment or prevention includes treatment, prevention, suppression of recurrence, reduction, suppression, improvement, elimination, reduction in incidence rate, delay in onset, suppression of progression, reduction in severity, reduction in recurrence rate, delay in recurrence, alleviation of clinical symptoms, and the like.

[0086] Specific examples of the present invention will be described below together with comparative examples, but the present invention is not limited to these.

[0087] The following cell and animal experiments were conducted under the review and approval of the Kobe University Animal Experiment Committee.

[0088] Data values ​​were expressed as mean ± standard deviation (ratio scale following normal distribution, interval scale) or median [range] (ordinal scale, ratio scale not following normal distribution) based on a frequency distribution table. For continuous variables, repeated measures analysis of variance or multi-way analysis of variance was used, with the Tukey-Kramer test performed as a post-hoc test. For ordinal scale variables, the Kruskal-Wallis test was used, with the DUNN test added post-hoc. In all tests, significance was assessed using p<0.0500 as the criterion.

[0089] 1. Preparation of Materials The following materials were prepared: Gelatin prepared by acidic treatment of pigskin was supplied by Nitta Gelatin, Inc. (Osaka, Japan). Glutaraldehyde (GA, 25 wt% aqueous solution), glycine, concentrated hydrochloric acid, acetone, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and penicillin / streptomycin were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) solution was purchased from Dojindo Laboratories, Inc. (Kumamoto, Japan). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Spermine and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Inc. (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM) and Opti minimal essential medium (Opti-MEM) were purchased from GIBCO Life Technologies Co. (Carlsbad, CA). Lipofectamine® RNAiMAX transfection reagent, Invivofectamine® 3.0 Reagent, and Silencer® Select Raptor siRNA (Assay ID: s143003), including Alexa Fluor-555-labeled siRNA, were purchased from Thermo Fisher Scientific (Waltham, MA). Silencer Select Negative Control No. 1 siRNA (4390843, sequence not published), used as a negative control, was purchased from Thermo Fisher Scientific (Waltham, MA).The sense strand of siRNA has the sequence shown in SEQ ID NO: 1, the antisense strand of siRNA has the sequence shown in SEQ ID NO: 2, and the Alexa Fluor-555-labeled siRNA has the sequence shown in SEQ ID NO: 3. Cy5 (53020) used to fluorescently label cationized gelatin was purchased from Lumiprobe Corporation (Huntvale, MD). Antibodies used in Western blotting were purchased from Cell Signaling Technology (Danvers, MA), RAPTOR (#2983), LC3 (#3868), and α-Tubulin (#2144), p62 / SQSTM1 (ab56416) from Abcam (Cambridge, UK), and anti-rabbit secondary antibody (NA934) and anti-mouse secondary antibody (NA931) from GE Healthcare (Chicago, IL).

[0090] Antibodies used for immunohistochemical staining were COL2A1 (sc-518017), Aggrecan (sc-33695), and CDKN2A / p16 (sc-1661) from Santa Cruz Biotechnology (Dallas, TX). ARGSV (1042003) and DIPEN (1042001) were purchased from MD Bio Products (Zurich, Switzerland). Safranin O / Fast green staining was performed using safranin-O (S-0145) from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fast green (10720) was purchased from Chroma Gesellschaft Schmidt (Stuttgart, Germany). Anti-mouse secondary antibody (414341) and DAB substrate set (425011) for DAB staining were purchased from Nichirei Biosciences Corporation (Tokyo, Japan). A TUNEL staining kit (MK500) for detecting apoptotic cell death was purchased from Takara Bio Inc. (Shiga, Japan).

[0091] 2. Preparation of Nanoparticles: 2.0 g of gelatin with an isoelectric point of 9.0 and a weight-average molecular weight of 99,000 Da was dissolved in 50 ml of double-distilled water. A polyamine compound (spermine) was added to the aqueous solution at a molar ratio of 50:1 relative to the carboxyl groups of the gelatin, and the polyamine compound was chemically introduced into the gelatin. Then, 11 M concentrated hydrochloric acid was added to the aqueous solution to adjust the pH of the gelatin solution to 5.3. Next, EDC was added at a molar ratio of 3:1 relative to the carboxyl groups of the gelatin, and the mixture was reacted at 40°C for 18 hours. The mixture was then dialyzed against distilled water for 72 hours, and the resulting dialyzed solution was freeze-dried to obtain cationized gelatin.

[0092] The amino group introduction rate of the prepared cationized gelatin, measured by the TNBS method, was 42.6 mol%. Subsequently, 1.25 ml of the cationized gelatin solution (50 mg / ml) was weighed and heated to 40°C, and 5 ml of acetone was added dropwise to form a coacervate. 20 ml of GA was added to the formed coacervate, and chemical crosslinking was carried out at 40°C for 6 hours. Next, 3 ml of glycine solution (100 mM) was added to inactivate unreacted aldehyde groups. The solution was then stirred at room temperature for 12 hours to evaporate unreacted acetone. The cGNS was then collected by centrifugation at 16,000 G for 30 minutes at 25°C and resuspended in distilled water. This process was repeated three times to obtain the final cGNS for use.

[0093] 1 μg of cGNS was mixed with 20 pmol of Raptor siRNA, stirred at room temperature for 15 minutes, and then centrifuged at 11,000 G for 15 minutes at 25° C. to obtain cGNS incorporating Raptor siRNA (cGNS Ｒａｐ ) was prepared.

[0094] 500 μl of cGNS adjusted to 5 mg / ml was prepared, and 400 μl of 0.25 M sodium bicarbonate (NaHCO3) was added to this to create a 2.5 mg cGNS / 900 μl solution. The amount of FITC required was 2.5 g × 10- 3The calculation was: / 10000 (moles of gelatin) x 4 x 389.38 (molecular weight of FITC) = 389.38 μg. FITC was dissolved in DMSO to prepare a 389.38 μg / 100 μl FITC solution. The cGNS containing sodium bicarbonate was mixed with the FITC solution to prepare a mixture, which was then reacted at 25 ° C, 300 rpm, and protected from light for 3 hours. The mixture was then centrifuged at 25 ° C, 10,000 G for 15 minutes, and the supernatant was collected and redispersed in distilled water to prepare fluorescently labeled cGNS with FITC.

[0095] cGNS except that Raptor siRNA fluorescently labeled with Alexa Fluor-555 and cGNS fluorescently labeled with FITC were used. Ｒａｐ In the same manner as in the preparation of Example 1, Raptor siRNA and gelatin were used to prepare fluorescently labeled cGNS (cGNS Ｒａｐ－Ｆｌ ) was prepared.

[0096] (Method for preparing cGNS fluorescently labeled with Cy5) 500 μl of cGNS prepared at 5 mg / ml was prepared, and 400 μl of 0.25 M sodium bicarbonate (NaHCO3) was added to this to prepare a solution of 2.5 mg of cGNS / 900 μl. The amount of Cy5 required was 2.5 g × 10 -3 The calculation was: / 10000 (moles of gelatin) x 4 x 792.96 (molecular weight of Cy5) = 792.96 μg. Cy5 was dissolved in DMSO to prepare a Cy5 solution of 792.96 μg / 100 μl. The cGNS with added sodium bicarbonate was mixed with the Cy5 solution to prepare a mixture, which was then reacted at 25°C, 300 rpm for 3 hours in the dark. After that, the mixture was centrifuged at 25°C, 10,000G for 15 minutes to collect the supernatant, which was then redispersed in distilled water to prepare the cGNS fluorescently labeled with Cy5 (cGNS Ｃｙ５ ) was prepared.

[0097] The apparent average particle size of cGNS was determined by dynamic light scattering at 37 ° C. using a DLS-7000 (manufactured by Otsuka Electronics Co., Ltd.), and was found to be 245.4 ± 17.0 nm. The PdI (polydispersity index) of the average particle size was 0.096 ± 0.0084, and particles with a uniform particle size were obtained. The zeta potential of cGNS in a solution at pH 7.4 was determined by electrophoretic light scattering using a DLS-8000 (manufactured by Otsuka Electronics Co., Ltd.), and was found to be 8.90 ± 0.07 mV.

[0098] cGNS Ｒａｐ The apparent average particle size of cGNS was determined by dynamic light scattering at 37°C using DLS-7000 manufactured by Otsuka Electronics Co., Ltd., and was found to be 303.4±2.0 nm. The PdI (polydispersity index) of the average particle size was 0.074±0.0080, indicating that particles with a uniform particle size were obtained. Furthermore, the average particle size of cGNS in a solution with a pH of 7.4 was 303.4±2.0 nm. Ｒａｐ The zeta potential of the polymer was determined by electrophoretic light scattering using DLS-8000 manufactured by Otsuka Electronics Co., Ltd., and was found to be 4.32±0.40 mV.

[0099] The apparent average particle size of cGNS fluorescently labeled with FITC was determined by dynamic light scattering at 37°C using DLS-7000 manufactured by Otsuka Electronics Co., Ltd., and was found to be 286.8±5.9 nm. The PdI (polydispersity index) of the average particle size was 0.170±0.0100, indicating that particles with a uniform particle size were obtained. Furthermore, the cGNS in a solution with a pH of 7.4 Ｒａｐ The zeta potential of the polymer was determined by electrophoretic light scattering using DLS-8000 manufactured by Otsuka Electronics Co., Ltd., and was found to be 4.20±0.30 mV.

[0100] The apparent average particle size of cGNS fluorescently labeled with Cy5 was determined by dynamic light scattering at 37°C using DLS-7000 manufactured by Otsuka Electronics Co., Ltd., and was found to be 270.0±4.4 nm. The PdI (polydispersity index) of the average particle size was 0.074±0.0080, indicating that particles with a uniform particle size were obtained. Furthermore, in a solution with a pH of 7.4, Ｃｙ５The zeta potential of the polymer was determined by electrophoretic light scattering using DLS-8000 manufactured by Otsuka Electronics Co., Ltd., and was found to be 4.87±1.29 mV.

[0101] 3. Preparation for Lipofection Lipofectamine® RNAiMAX was used for the lipofection method, and 3.3 pmol of Raptor siRNA was mixed with 1 μl of the reagent according to the manual, followed by stirring at room temperature for 5 minutes.

[0102] 4. Experiments and Evaluation The following experiments were carried out using the prepared materials.

[0103] [Cell Experiment 1: Cell Viability] Primary intervertebral disc nucleus pulposus cells collected from the caudal intervertebral disc of 12-week-old male Sprague-Dawley rats were used for the cell experiment. The intervertebral disc nucleus pulposus cells were cultured in DMEM medium containing 10 vol% FBS and 1 vol% penicillin-streptomycin at 37°C and 5% CO ２ After the cell occupancy reached 70% to 80%, the medium was changed to Opti-MEM.

[0104] Then, various concentrations of cGNS Ｒａｐ was added to the medium, and transfection was carried out for 3 hours.

[0105] As a control group, transfection was carried out under the same conditions using lipofectamine lipofection with varying concentrations of siRNA.

[0106] After siRNA was introduced by each method, cell viability was measured by WST-8 assay. Specifically, WST-8 reagent was introduced into each medium immediately after transfection, and 3 hours later, the absorbance of each medium at a wavelength of 450 nm was measured at 37°C. The ratio of the absorbance measured in each medium introduced with siRNA to the absorbance measured in a control medium not introduced with siRNA was calculated and used as the cell viability of each medium.

[0107] Figure 2A shows different concentrations of cGNS. Ｒａｐ2A and 2B are graphs showing cell viability after lipofection at different concentrations. The horizontal axis of each graph shows the amount of siRNA introduced (pmol of siRNA per ml of medium), and the vertical axis shows cell viability (%).

[0108] As shown in Figure 2A, cGNS Ｒａｐ When siRNA was introduced by this method, the cell survival rate was higher even when a higher concentration of siRNA was introduced than by lipofection. Ｒａｐ When 400 pmol (20 μg cGNS) / ml of siRNA was introduced by the lipofection method, the cell viability was comparable to that when 60 pmol / ml of siRNA was introduced by the lipofection method. Ｒａｐ It was found that when siRNA was introduced by this method, it was possible to administer 6.7 times higher concentrations of siRNA than by lipofection without toxicity.

[0109] [Cell Experiment 2: Intracellular distribution and duration of siRNA] cGNS Ｒａｐ Instead of cGNS Ｒａｐ－Ｆｌ Transfection into intervertebral disc nucleus pulposus cells was carried out in the same manner as in Experiment 1, except that siRNA was used at a concentration of 400 pmol / ml or 60 pmol / ml.

[0110] As a control group, transfection was carried out by lipofection in the same manner as in Experiment 1, except that the siRNA concentration was 60 pmol / ml.

[0111] After transfection using each method, FITC fluorescence images (distribution of cGNS) and Alexa Fluor-555 fluorescence images (distribution of siRNA) were obtained 1, 4, 7, 10, and 14 days later. To examine cell distribution, fluorescence images were also obtained after adding 4',6-diamidino-2-phenylindole (DAPI) to stain the nuclei. The siRNA-positive cell rate was also measured at each time point. Five ROIs (regions of interest) were randomly set using Image J, and the ratio of siRNA-positive cells (cells with fluorescent dye localized in the cytoplasm) to the total number of cells within each ROI was calculated. The average value of the five ROIs was used to calculate the positive cell rate.

[0112] Figure 3A shows the cGNS Ｒａｐ－Ｆｌ The images are fluorescence images taken using a FITC fluorescent image (cGNS distribution), an Alexa Fluor-555 fluorescent image (siRNA distribution), a DAPI fluorescent image (cell distribution), and a composite image of these. The upper panel shows a fluorescence image taken one day after transfection, and the lower panel shows a fluorescence image taken 14 days after transfection.

[0113] Figure 3B shows the cGNS Ｒａｐ－Ｆｌ Fluorescence images of intervertebral disc nucleus pulposus cells after cell division after 4 days were captured using a fluorescent imager (FITC) at an siRNA concentration of 400 pmol / ml. From left to right, the images show a fluorescent image of FITC (distribution of cGNS), a fluorescent image of Alexa Fluor-555 (distribution of siRNA), a fluorescent image of DAPI (distribution of cells), and a composite image of these.

[0114] Figure 3C shows the cGNS Ｒａｐ－Ｆｌ 3C is a graph showing the siRNA-positive cell rate after 1 day, 4 days, 7 days, 10 days, and 14 days when 400 pmol / ml siRNA was used. The horizontal axis of FIG. 3C shows the number of days since transfection, and the vertical axis shows the siRNA-positive cell rate (%).

[0115] Figure 4A shows fluorescence images taken 1 day, 4 days, and 7 days after transfection using each method. From left to right, the images show an Alexa Fluor-555 fluorescence image (distribution of siRNA), a DAPI fluorescence image (distribution of cells), and a composite image of these. The upper column shows cGNS. Ｒａｐ－Ｆｌ The center column shows the fluorescence image when the siRNA concentration was 400 pmol / ml using cGNS. Ｒａｐ－Ｆｌ The bottom panel shows a fluorescent image obtained by adjusting the siRNA concentration to 60 pmol / ml using the lipofection method, and the bottom panel shows a fluorescent image obtained by adjusting the siRNA concentration to 60 pmol / ml using the lipofection method.

[0116] Figure 4B is a graph showing the siRNA-positive cell rates after 1 day, 4 days, and 7 days for each test shown in Figure 4A. The horizontal axis of Figure 4B represents the number of days since transfection, and the vertical axis represents the siRNA-positive cell rate (%).

[0117] As shown in Figure 3A, cGNS and siRNA were uniformly colocalized in the cytoplasm. Furthermore, as shown in Figures 3A and 3C, the siRNA-positive cell rate was 100.0% ± 0.0% one day after transfection. Although the siRNA-positive cell rate decreased over time, it remained at 52.4 ± 7.3% even after 14 days of subculture.

[0118] As is clear from FIG. 3B, cGNS and siRNA were uniformly co-localized in the cytoplasm even in daughter cells after cell division.

[0119] As is clear from Figures 4A and 4B, when cGNS was used, the rate of siRNA-positive cells was higher than that by the lipofection method at all time points (p = 0.0080 to 0.0490).

[0120] [Cell Experiment 3: RAPTOR Knockdown Efficiency] The same procedure as in Experiment 1 was used to knock down cGNS. Ｒａｐ The siRNA was transfected into intervertebral disc nucleus pulposus cells by lipofection at a concentration of 60 pmol / ml.

[0121] After transfection using each method, the expression levels of RAPTOR and α-tubulin as a loading control were measured by Western blotting 1, 2, 3, 5, and 7 days later.

[0122] Figure 5A shows the cGNS Ｒａｐ The figures show bands representing RAPTOR (150 kDa) and α-tubulin (49 kDa) obtained by Western blotting for a group transfected using cGNS and a control group transfected by lipofection. From the left, the figures show bands for a control group that was not transfected, and bands obtained 1 day, 2 days, 3 days, 5 days, and 7 days after transfection. The upper column shows bands representing cGNS. Ｒａｐ The bottom panel shows bands representing RAPTOR and α-tubulin for the group transfected using the ELISA kit, and the bottom panel shows bands representing RAPTOR and α-tubulin for the group transfected by lipofection.

[0123] Figure 5B shows the cGNS Ｒａｐ 5B is a graph showing the expression levels of RAPTOR (relative to the expression level of α-tubulin) after 1 day, 2 days, 3 days, 5 days, and 7 days for a group transfected using α-tubulin. FIG. 5C is a graph showing the expression levels of RAPTOR (relative to the expression level of α-tubulin) after 1 day, 2 days, 3 days, 5 days, and 7 days for a group transfected by lipofection. FIG. 5D is a graph showing both FIG. 5B and FIG. 5C. The horizontal axis of FIG. 5B to FIG. 5D represents the number of days since transfection, and the vertical axis represents the expression level of RAPTOR.

[0124] As is clear from Figures 5A to 5D, cGNS ＲａｐUsing cGNS, the highest knockdown efficiency of RAPTOR protein was observed at 2 days after transfection (53.9% ± 5.1%, p = 0.0003), and significant knockdown of RAPTOR protein expression was confirmed up to 7 days after transfection (64.7% ± 19.8%, p = 0.044). On the other hand, lipofection showed the highest knockdown efficiency at 1 day after transfection (59.2% ± 22.3%, p = 0.0010), with significant knockdown disappearing by 2 days. Comparing knockdown efficiencies between the two groups at each evaluation time point, cGNS significantly increased knockdown efficiency at 2 days (p = 0.0483), 3 days (p = 0.0065), 5 days (p < 0.0001), and 7 days (p = 0.0015). Ｒａｐ A significantly higher knockdown efficiency was confirmed in the group transfected with

[0125] [Cell Experiment 4: Enhancement of autophagy] Using the same procedure as in Experiment 1, Ｒａｐ The siRNA concentration was 400 pmol / ml.

[0126] After transfection, the expression levels of RAPTOR, autophagy indicators LC3-I, LC3-II, and p62 / SQSTM1, and α-tubulin as a loading control were measured by Western blotting 1, 2, 3, 5, and 7 days later.

[0127] Figure 6 shows bands obtained by Western blotting, representing RAPTOR (150 kDa), LC3-I (16 kDa), LC3-II (14 kDa), p62 / SQSTM1 (62 kDa), and α-tubulin (49 kDa). From left to right, the bands are those of a non-transfected control, and those of 1, 2, 3, 5, and 7 days after transfection.

[0128] As is clear from FIG. ＲａｐRNA interference using α-glucan β-glucan β-glucan induced an increase in LC3-II and a decrease in p62 / SQSTM1 due to inhibition of mTOR complex 1 via suppression of RAPTOR protein expression. Furthermore, enhanced autophagy was observed.

[0129] [Animal Experiment 1: siRNA-Positive Cell Rate] cGNS was implanted into the nucleus pulposus region of the caudal intervertebral disc of 12-week-old male Sprague-Dawley rats using a 33G needle under X-ray fluoroscopy. Ｒａｐ－Ｆｌ 2 μl of an aqueous solution containing Raptor siRNA (at a concentration of 60 pmol / μL) was administered. For comparison, Raptor siRNA was administered locally using Invivofectamine® 3.0, a lipofection-based siRNA transfection reagent for living organisms. Frozen sections were prepared 7 and 28 days after administration, and the efficiency of siRNA intracellular introduction was evaluated as the siRNA-positive cell rate, and a comparison was made between the two groups. In addition, cGNS Ｒａｐ－Ｆｌ For the group into which siRNA was administered, frozen sections were prepared 56 and 84 days after administration, and the intracellular introduction efficiency of siRNA was evaluated as the siRNA-positive cell rate, and the time course of the siRNA-positive cell rate was examined.

[0130] Figure 7 shows fluorescence images taken from each group 7 and 28 days after administration. Ｒａｐ－Ｆｌ The images show fluorescence images taken 7 and 28 days after administration using the lipofection method, as well as fluorescence images taken 7 and 28 days after administration using the lipofection method. The upper image shows a fluorescence image of the whole disc, and the lower image shows an enlarged image of the area enclosed by the dotted line. From the top, the images show a fluorescence image of Alexa Fluor-555 (distribution of siRNA), a fluorescence image of DAPI (distribution of cells), and a composite image of these.

[0131] cGNS Ｒａｐ－Ｆｌ After administration of siRNA, the siRNA-positive cell rates were 100.0% ± 0.0% at 7 and 28 days, respectively. In contrast, after administration by lipofection, the siRNA-positive cell rates were 63.0% ± 2.0% and 26.6% ± 5.2% at 7 and 28 days, respectively (both p<0.001).

[0132] From these results, cGNS Ｒａｐ By using the method, a significantly higher and longer-lasting efficiency of Raptor siRNA delivery into living cells was observed.

[0133] Figure 8 shows the cGNS at 7, 28, 56 and 84 days after administration. Ｒａｐ－Ｆｌ From the left, the fluorescent images are from the group using cGNS. Ｒａｐ－Ｆｌ The figures show fluorescence images taken 7, 28, 56, and 84 days after administration when using Alexa Fluor-555 (Figure 7 shows the same trend images taken 7 and 28 days after administration for reference). The upper image shows a fluorescence image of the whole disc, and the lower image shows an enlarged image of the dotted line area. From top to bottom, the images show an Alexa Fluor-555 fluorescence image (distribution of siRNA), a DAPI fluorescence image (distribution of cells), and a composite image of these.

[0134] cGNS Ｒａｐ After administration, the siRNA-positive cell rates at 56 and 84 days were 82.0% ± 14.0% and 32.5% ± 16.7%, respectively. Although the siRNA-positive cell rate decreased over time, siRNA persisted in the cells for 84 days.

[0135] [Animal Experiment 2: Degradability of cGNS in vivo] Cy5-fluorescently labeled cGNS (cGNS-FI) was administered to the nucleus pulposus region of the caudal intervertebral disc of 12-week-old male Sprague-Dawley rats, and quantitative evaluation was performed over time up to 84 days using an In Vivo Imaging System.

[0136] Figure 9A shows fluorescence images taken immediately after administration and 1, 7, 14, 28, and 84 days after administration. Figure 9B is a graph showing the total fluorescence intensity (ratio to the fluorescence intensity immediately after administration) immediately after administration and 1, 4, 7, 14, 28, 56, and 84 days after administration. Although the fluorescent area shrank over time due to the degradation of the gelatin nanoparticles, even after 84 days, 26.1% ± 13.5% of the fluorescence remained compared to immediately after administration (p < 0.0001).

[0137] [Animal Experiment 3: Intervertebral Disc Protective Effect] FIG. 10 is a conceptual diagram showing the experimental protocol of Animal Experiment 3.

[0138] As a degeneration model, we selected a caudal intervertebral disc static compression model in 12-week-old male Sprague-Dawley rats. cGNS was inserted into the coccygeal (Co) intervertebral disc at Co8 / 9 and Co11 / 12. Ｒａｐ , Co9 / 10, and Co12 / 13 were transfected with negative control siRNA (cGNS ｃｔｒｌ PBS was administered to Co13 / 14 (2 μl each, 120 pmol of siRNA). Seven days after administration, Co8 / 9 and Co9 / 10 in the compression group were subjected to static compression (1.3 MPa) for 24 hours. After compression was released (8 days after administration in the uncompressed group), radiological evaluations, histological evaluations (Safranin O / Fast green staining), multifluorescent staining (RAPTOR, p16 / INK4A, TUNEL), and immunohistochemical staining (COL2A1, Aggrecan, DIPEN, an MMP-derived Aggrecan degradation product, and ARGSV, an ADAMTS-derived Aggrecan degradation product) were performed over time.

[0139] Figure 11A shows X-ray images taken immediately after static compression and 7, 28, 56, and 84 days later. Figure 11B shows a graph of normalized disc height calculated using the disc height index (Masuda et al., "A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration," Spine (Phila Pa 1976), 30(1):5-14, 2005). Figure 11B shows the cGNS of the compression group. Ｒａｐ The site where the cGNS was administered Ｒａｐ(loaded)), cGNS of the non-compressed group Ｒａｐ The site where the cGNS was administered Ｒａｐ (unloaded)), cGNS of the compression group ｃｔｒｌ The site where the cGNS was administered ｃｔｒｌ (loaded)), cGNS of the non-compressed group ｃｔｒｌ The site where the cGNS was administered ｃｔｒｌ 1 shows the normalized disc height for the site of the non-compressed group where PBS was administered (PBS (unloaded)), and the site of the non-compressed group where PBS was administered (PBS (unloaded)).

[0140] At 28 days, 56 days, and 84 days after the degeneration caused by compression loading, ｃｔｒｌ cGNS compared with the administered disc Ｒａｐ Significant maintenance of disc height was observed in the treated discs (after 28 days: 78.9% ± 6.1% vs. 67.2% ± 10.4%; p = 0.0468, after 56 days: 80.1% ± 10.2% vs. 61.1% ± 4.5%; p = 0.0002, after 84 days: 77.7% ± 4.3% vs. 52.1% ± 9.2%; p < 0.0001, all cGNS). Ｒａｐ Against cGNS ｃｔｒｌ ).

[0141] Figure 12A shows Safranin O / Fast green stained images of the NP (nucleus pulposus), AF (annulus fibrosus), and CEP (cartilage endplate) obtained immediately, 28 days, 56 days, and 84 days after static compression. Figure 12B shows a graph showing the degeneration score (severity) assessed using the Orthopaedic Research Society spine section histopathological grading score (Lai et al., "Development of a standardized histopathology scoring system for intervertebral disc degeneration in rat models: An initiative of the ORS spine section," JOR Spine, 4(2):e1150, 2021). Figures 12A and 12B also show the cGNS, as in Figure 11B. Ｒａｐ (loaded), cGNS Ｒａｐ(unloaded), cGNS ｃｔｒｌ (loaded), cGNS ｃｔｒｌ The staining images and degeneration scores for PBS (unloaded), and PBS (unloaded) are shown.

[0142] 84 days after degeneration was induced by compression loading, ｃｔｒｌ cGNS compared with the administered disc Ｒａｐ Treated discs had significantly lower degeneration scores (3.5 [2 to 5] points vs. 8.0 [7 to 12] points; p=0.0142).

[0143] Fig. 13A shows a fluorescent image obtained by multiple fluorescent staining 28 days after static compression. Fig. 13A shows normal tissue (indicated by (-) in Fig. 13A) to which no drug administration by puncture or degeneration induction by compression load was performed, as well as a cGNS tissue similar to that in Fig. 11B. ｃｔｒｌ (unloaded), cGNS Ｒａｐ (unloaded), cGNS ｃｔｒｌ (loaded), and cGNS Ｒａｐ 13B, 13C, and 13D are graphs showing the percentages of RAPTOR-, TUNEL-, and p16 / INK4A-positive cells, respectively.

[0144] Multiplex immunofluorescence staining after 28 days showed that cGNS was significantly increased in both the group with and without degeneration induced by compression load. ｃｔｒｌ cGNS for administered intervertebral discs Ｒａｐ The administration of acetaminophen suppressed the expression of RAPTOR in the intervertebral discs (42.0% ± 8.4% vs. 73.7% ± 14.0%; p = 0.0155, 39.4% ± 12.9% vs. 68.1% ± 11.6%; p = 0.0287, respectively). ｃｔｒｌ cGNS for administered intervertebral discs ＲａｐA decrease in the rate of TUNEL-positive cells was observed in the treated discs, demonstrating an inhibitory effect on apoptotic cell death (16.5% ± 2.5% vs. 26.8% ± 7.0%; p = 0.0061). ｃｔｒｌ cGNS for administered intervertebral discs Ｒａｐ A decrease in the rate of p16 / INK4A-positive cells was observed in the treated intervertebral discs, and the inhibitory effect on senescence cell aging was also confirmed (24.0% ± 5.4% vs. 52.0% ± 7.6%; p = 0.0005).

[0145] Fig. 14A shows an immunohistochemical staining (DAB staining) image obtained 28 days after static compression. Fig. 14A shows normal tissue (-) to which no drug administration by puncture or degeneration induction by compression load was performed, cGNS ｃｔｒｌ (loaded), and cGNS Ｒａｐ 13B, 13C, and 13D are stained images of COL2A1 (an indicator of the cell matrix), Aggrecan, DIPEN, and ARGSV, respectively. 14B and 14C are stained images of cGNS. ｃｔｒｌ (loaded), and cGNS Ｒａｐ 1 is a graph showing the ratio of DIPEN and ARGSV production rates adjusted by cell number in the cGNS (loaded) relative to normal tissue. ｃｔｒｌ cGNS for administered intervertebral discs Ｒａｐ The expression of ARGSV, an aggrecan degradation product by ADAMTS, was reduced in the administered intervertebral discs (163.0%±62.8% vs. 381.9%±74.1%; p=0.0010), demonstrating an inhibitory effect on the catabolism of the extracellular matrix.

[0146] In these animal experiments, cGNS was not detected in the nucleus pulposus of the living intervertebral disc. ＲａｐIt was confirmed that local administration of cGNS resulted in the uptake of siRNA into cells for a longer period than lipofection. IVIS also confirmed that cGNS remained in the intervertebral disc for a long period after local administration. This is thought to be due to the biocompatibility of cGNS, which suppressed local inflammation and prevented rapid degradation. Furthermore, experiments using a rat caudal intervertebral disc degeneration model showed that cGNS Ｒａｐ The degeneration inhibitory effect of cGNS was confirmed. Ｒａｐ It was observed that administration of the compound inhibited the decrease in radiographic disc height and the progression of histological degenerative findings, and it was also confirmed that it contributed to the suppression of apoptotic cell death, senescence (cell senescence), and extracellular matrix degradation.

[0147] The siRNA-mediated target gene expression suppression observed in these animal studies lasted for 84 days, significantly longer than the 3-4 weeks typically observed in living cells. Furthermore, a significant inhibitory effect on intervertebral disc degeneration was observed, sufficient to withstand mechanical load. The unprecedented efficacy and extended treatment period are likely due in large part to the unique characteristics of spinal disc tissue, the selection of a gene therapy strategy targeting the PI3K / Akt / mTOR signaling pathway, particularly Raptor, and the synergistic effects of improvements and optimization of gene expression suppression technologies, including cGNS.

[0148] This application claims priority from Japanese Patent Application No. 2024-147560, filed August 29, 2024. The matters set forth in the specification, claims and drawings of said application are incorporated herein by reference.

[0149] According to the present invention, there are provided nanoparticles that can be effectively used for the treatment and prevention of intervertebral disc degeneration, and pharmaceutical compositions containing the same.

[0150] The technology of this invention is also useful for many young people, athletes, and postoperative patients who wish to preserve intervertebral disc function, such as those with adjacent segment disorders after spinal fusion surgery or discs affected by lumbar spondylolysis. This is expected to reduce the number of expensive spinal surgeries and contribute to medical economics.

Claims

1. Nanoparticles for use in preventing or treating intervertebral disc degeneration, comprising: a biodegradable hydrogel; and a ribonucleic acid molecule that selectively inhibits the activity of a mammalian target of rapamycin (mTOR) complex, the ribonucleic acid molecule being retained in the biodegradable hydrogel.

2. The nanoparticle according to claim 1, wherein the ribonucleic acid molecule is an siRNA that selectively inhibits the activity of mTOR complex 1.

3. The nanoparticles according to claim 1, wherein the ribonucleic acid molecule is an siRNA that selectively inhibits the expression of a regular-associated protein of mTOR (RAPTOR).

4. The nanoparticles according to claim 1, wherein the amount of the ribonucleic acid molecule is 1 pmol to 100 pmol per 1 μg of hydrogel.

5. The nanoparticles according to claim 1, wherein the biodegradable hydrogel is cationized gelatin.

6. The nanoparticles according to claim 1, wherein an aqueous solution containing the nanoparticles at a concentration of 60 pmol / μl, calculated as ribonucleic acid molecules, is administered to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat, and four weeks later, the rate of ribonucleic acid-positive cells in the administered region is 80% or more.

7. The nanoparticles according to claim 1, wherein an aqueous solution containing the nanoparticles at a concentration of 60 pmol / μl, calculated as ribonucleic acid molecules, is administered to the nucleus pulposus region of the caudal intervertebral disc of a 12-week-old male Sprague-Dawley rat, and 12 weeks later, the rate of ribonucleic acid-positive cells in the administered region is 20% or more.

8. A pharmaceutical composition for use in preventing or treating intervertebral disc degeneration, comprising the nanoparticles according to any one of claims 1 to 7.

9. The pharmaceutical composition according to claim 8, wherein the administration interval is 2 weeks or longer.

10. The pharmaceutical composition according to claim 8, wherein the amount of the nanoparticles converted into the amount of the ribonucleic acid molecules is 10 pmol or more and 400 nmol or less per ml of the pharmaceutical composition.

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