An siRNA targeting Col2a1, a delivery system thereof and application thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a siRNA targeting Col2a1, a delivery system thereof and application, wherein the siRNA has a sense strand and an antisense strand, the nucleotide sequence of the sense strand comprises a modified or unmodified nucleotide sequence as shown in SEQ ID NO:1, the nucleotide sequence of the antisense strand comprises a modified or unmodified nucleotide sequence as shown in SEQ ID NO:2, and the delivery system contains the siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof. The siRNA targeting Col2a1 can specifically block the Col2a1 signal of local tissues, block the pathological communication with downstream cells, and then restore the vascular network, improve the local immune microenvironment, and promote the healing of soft tissues and bone tissues of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Col2a1-targeting siRNA, its delivery system and application. The Col2a1-targeting siRNA can specifically block Col2a1 signaling in local tissues, block its pathological communication with downstream cells, thereby restoring the vascular network, improving the local immune microenvironment, and promoting the healing of soft tissue and bone tissue in bisphosphonate-associated osteonecrosis of the jaw (BRONJ). Background Technology

[0002] Bisphosphonate-associated osteonecrosis of the jaw (BRONJ) is one of the most serious complications of anti-bone resorption therapy (such as bisphosphonates used to treat osteoporosis and bone metastases in cancer). Clinically, BRONJ presents as poorly healing exposure and necrosis of the jawbone, often accompanied by pain, infection, and soft tissue damage, and may even progress to pathological fractures and long-term jaw dysfunction. Currently, there is no effective cure for BRONJ in clinical practice, and conventional treatments (such as antibacterial mouthwash, systemic antibiotics, necrotic bone resection, and soft tissue closure) have an extremely high recurrence rate, mainly due to the irreversible failure of local vascular repair function.

[0003] Although impaired angiogenesis is considered a key mechanism in the pathogenesis of BRONJ, the molecular basis of endothelial cell dysfunction and how it disrupts the local microenvironment remain incompletely understood. Recent research confirms that bisphosphonates not only directly cause severe degeneration of the vascular network, but more importantly, in the BRONJ microenvironment, pathological endothelial cells abnormally upregulate their intracellular Col2a1 gene, regulating macrophages and fibroblasts through the Col2a1-Cd44 / Sdc4 signaling axis, leading to extracellular matrix disturbance and exacerbated inflammation in the mandibular microenvironment.

[0004] Against this backdrop, the development of a nucleic acid drug that can specifically silence local Col2a1 expression holds promise for blocking abnormal intercellular communication at its source, restoring endothelial function, rescuing the pathological phenotype of BRONJ, and providing a novel targeted therapy strategy for this refractory disease. Summary of the Invention

[0005] To address the shortcomings of the prior art, the first objective of this invention is to provide a Col2a1-targeting siRNA and its pharmaceutically acceptable formulation. This siRNA can specifically silence Col2a1 gene expression in target cells, thereby disrupting the Col2a1-Cd44 / Sdc4 pathological signaling axis, inhibiting abnormal extracellular matrix remodeling and local inflammation, and clearing obstacles to vascularization and bone regeneration in the jawbone microenvironment.

[0006] The second objective of this invention is the application of the aforementioned Col2a1-targeting siRNA in the preparation of drugs for treating bisphosphonate-associated osteonecrosis of the jaw (BRONJ), promoting jawbone angiogenesis, lymphangiogenesis, and / or tissue repair. This strategy breaks through the limitations of simply "promoting angiogenesis" in previous approaches, and by regulating the three-dimensional microenvironment network of "endothelium-osteogenesis-immunity," it provides an optimized solution for clinical intervention of BRONJ and other diseases related to local microenvironment imbalance.

[0007] A third objective of this invention is to provide a delivery system comprising the aforementioned siRNA targeting Col2a1, enabling efficient, in-situ drug release.

[0008] To achieve the above objectives, the present invention employs a technical solution consisting of the following technical measures.

[0009] In one aspect, the present invention provides a siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof, the siRNA having a sense strand and an antisense strand, the nucleotide sequence of the sense strand being a modified nucleotide sequence as shown in SEQ ID NO: 1; and the nucleotide sequence of the antisense strand being a modified nucleotide sequence as shown in SEQ ID NO: 2;

[0010] The modifications include methoxy modification, thiosclerosis modification, and cholesterol modification of nucleotides;

[0011] The methoxy modification is performed along the 5' to 3' direction, including methoxy modification (2'-O-methyl modification) at the 2' position of the nucleotide sugar ring at positions 1, 2, 3, 6, 8, 9, 11, 12, 15, 16, 17, 18, 22, and 23 on the sense strand; and along the 5' to 3' direction, including methoxy modification (2'-O-methyl modification) at the 2' position of the nucleotide sugar ring at positions 1, 2, 3, 8, 9, 12, 15, 17, 18, 22, and 23 on the antisense strand.

[0012] The thiophosphate backbone modification is performed in the direction from the 5' end to the 3' end, including between the 1st to 3rd nucleotides and between the 21st to 23rd nucleotides of the sense strand, with adjacent nucleotides linked by thiophosphate bonds; and in the direction from the 5' end to the 3' end, including between the 1st to 3rd nucleotides and between the 21st to 23rd nucleotides of the antisense strand, with adjacent nucleotides linked by thiophosphate bonds.

[0013] The cholesterol modification is performed along the 5' to 3' direction, including cholesterol modification at the 3' position of the nucleotide sugar ring at position 23 of the positive strand.

[0014] In this document, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt for the Col2a1-targeting siRNA provided by this invention. Generally, "pharmaceutically acceptable salt" as used in this invention refers to a salt that retains the biological activity targeting Col2a1 and is suitable for administration to humans or animals, and the method for preparing the salt for the siRNA is known in the art, typically involving reacting the siRNA with a pharmaceutically acceptable base or acid.

[0015] In one of the technical solutions, the modified sense strand nucleotide sequence is as follows:

[0016] 5'-mC mC mUGGmCAmUmUGmCmUGGmCmUmUmUAAA mU mU-3'-Cholesteryl;

[0017] The modified antisense nucleotide sequence is as follows:

[0018] 5'-mU mU mUAAAGmCmCAGmCAAmUGmCmCAGG mU mU-3';

[0019] Where mC and mU represent nucleotides that have undergone 2'-O-methyl modification;

[0020] This indicates that adjacent nucleotides are linked by a phosphate thioester bond;

[0021] 3'-Cholesteryl indicates that the 3' end is modified with cholesterol.

[0022] In one of the technical solutions, the modification further includes dangling deoxyribonucleotide dTdT at the 3' end of the sense strand and / or antisense strand; or, adding ribonucleotide UU at the 3' end of the sense strand and / or antisense strand.

[0023] On the other hand, the present invention provides a delivery system comprising the above-mentioned siRNA targeting Col2a1, which contains the above-mentioned siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof.

[0024] In one of the technical solutions, the delivery system further includes a delivery vector, wherein the siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof is loaded in the delivery vector, or mixed, compounded or conjugated with the delivery vector.

[0025] Furthermore, the delivery carrier preferably comprises hydrogels, liposomes, nanoparticles, or exosomes.

[0026] To better illustrate the present invention and provide a preferred embodiment for reference, the delivery carrier is a photocrosslinked methacrylamide gelatin (GelMA) hydrogel. Specifically, the preparation method of the photocrosslinked methacrylamide gelatin hydrogel loaded with the above-mentioned siRNA includes the following steps:

[0027] (1) Dissolve the photoinitiator LAP in phosphate buffer (PBS buffer, 0.01 M phosphate) to prepare a 0.25 w / v% initiator standard solution;

[0028] (2) Dissolve the methacrylamide gelatin in the initiator standard solution obtained in step (1) at a ratio of 5~15 w / v% and mix them evenly to obtain a precursor solution;

[0029] (3) Dissolve 100 μg of siRNA targeting Col2a1 in 0.2 mL of precursor solution to obtain a precursor solution loaded with siRNA;

[0030] (4) Irradiate the precursor solution loaded with siRNA under a 405 nm light source for 30 s to obtain photocrosslinked methacrylamide gel hydrogel loaded with siRNA.

[0031] In another aspect, the present invention also relates to the use of the above-mentioned Col2a1-targeting siRNA or a pharmaceutically acceptable salt thereof, and a delivery system containing the above-mentioned Col2a1-targeting siRNA, in the preparation of a drug for treating bisphosphonate-associated osteonecrosis of the jaw (BRONJ).

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] 1. This invention provides a siRNA that targets Col2a1. This siRNA directly intervenes in Col2a1, a core hub molecule in the BRONJ pathological process. Compared with traditional broad-spectrum anti-inflammatory or angiogenic drugs, it has higher specificity and blocks pathological endothelial-matrix communication at the source.

[0034] 2. This invention provides a delivery system containing the above-mentioned siRNA targeting Col2a1. Animal in vivo experiments have confirmed that local application of hydrogel loaded with this siRNA can not only effectively restore the morphology and function of blood vessels, but also reverse the pathological state of stromal cells and immune cells, promote osteoogenesis, and relieve inflammation, thereby achieving a comprehensive remodeling of the local microenvironment.

[0035] 3. In the mouse BRONJ tooth extraction defect model, it was demonstrated that the immediate in situ application of GelMA hydrogel loaded with siRNA after tooth extraction can significantly accelerate soft tissue healing and promote bone regeneration in the extraction socket, successfully rescuing the BRONJ phenotype.

[0036] 4. Combined with a photocrosslinkable GelMA hydrogel delivery system, it can achieve seamless filling and in-situ continuous release within the extraction socket, which is easy to operate and has great potential for clinical translation. Attached Figure Description

[0037] Figure 1 The flowchart and histological identification comparison of the mouse model of osteonecrosis of the jaw in a specific embodiment of the present invention are shown. In particular, Figure (A) is the flowchart of the mouse model of osteonecrosis of the jaw model; (B) is a comparison of representative Micro-CT images and histological evaluation of the control group and the osteonecrosis group at 1 day and 2 weeks after tooth extraction. The white scale bar is 500 micrometers, the black scale bar is 50 micrometers, and the arrows indicate continuous empty bone lacunae.

[0038] Figure 2 The present invention provides a schematic diagram of the experimental process and a comparison of gross healing assessment of the isotopic contralateral treatment model using siRNA-loaded hydrogel in a specific embodiment. Figure (A) is a schematic diagram of the experimental process of the isotopic contralateral treatment model using siRNA-loaded hydrogel; Figure (B) is a photograph documenting the placement of the precursor solution during tooth extraction and a comparison of soft / hard tissue healing assessment images two weeks post-operation.

[0039] Figure 3 A summary of the evaluation and comparison of the effects of siRNA-loaded hydrogels on bone regeneration and osteogenic formation in extraction sockets in specific embodiments of the present invention. Figure (A) shows representative three-dimensional reconstructed images of extraction sockets in the NC siRNA-loaded hydrogel group and the siRNA-loaded hydrogel group at 2 weeks post-operation using Micro-CT; Figure (B) shows representative images of HE staining, Masson staining, and osteopontin (OPN) immunohistochemical staining of tissue sections from the two extraction sockets; the white scale bar is 500 micrometers, the green scale bar is 100 micrometers, and the black scale bar is 50 micrometers.

[0040] Figure 4 This invention presents a summary chart comparing the evaluation of the angiogenesis-osteogenic coupling effect of siRNA-loaded hydrogels on tooth extraction sockets in specific embodiments. Figure (A) shows representative immunofluorescence staining images of CD31-positive vessels in the extraction sockets of the NC siRNA-loaded hydrogel group and the siRNA-loaded hydrogel group; Figure (B) shows representative dual immunofluorescence staining images of Emcn-positive vessels and surrounding BMP2-positive osteoblasts in the extraction sockets of the two groups; the black scale bar is 50 micrometers.

[0041] Figure 5A summary chart comparing the effects of siRNA-loaded hydrogels on alleviating local inflammation in extraction sockets according to specific embodiments of the present invention. Figure (A) shows representative immunofluorescence staining images of CD68-positive macrophage infiltration in the extraction sockets of the NC siRNA-loaded hydrogel group and the siRNA-loaded hydrogel group; Figure (B) shows representative immunohistochemical staining images of the inflammatory factors IL-1β and TNF-α in the two groups; the black scale bar is 50 micrometers, and the blue scale bar is 20 micrometers. Detailed Implementation

[0042] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. Although it is believed that those skilled in the art will fully understand the following terms, the following definitions are set forth to help illustrate the subject matter disclosed in the present invention.

[0043] In one aspect, the present invention provides a siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof, the siRNA having a sense strand and an antisense strand, the nucleotide sequence of the sense strand being a modified nucleotide sequence as shown in SEQ ID NO: 1; and the nucleotide sequence of the antisense strand being a modified nucleotide sequence as shown in SEQ ID NO: 2;

[0044] The modifications include methoxy modification, thiosclerosis modification, and cholesterol modification of nucleotides;

[0045] The methoxy modification is performed along the 5' to 3' direction, including methoxy modification (2'-O-methyl modification) at the 2' position of the nucleotide sugar ring at positions 1, 2, 3, 6, 8, 9, 11, 12, 15, 16, 17, 18, 22, and 23 on the sense strand; and along the 5' to 3' direction, including methoxy modification (2'-O-methyl modification) at the 2' position of the nucleotide sugar ring at positions 1, 2, 3, 8, 9, 12, 15, 17, 18, 22, and 23 on the antisense strand.

[0046] The thiophosphate backbone modification is performed in the direction from the 5' end to the 3' end, including between the 1st to 3rd nucleotides and between the 21st to 23rd nucleotides of the sense strand, with adjacent nucleotides linked by thiophosphate bonds; and in the direction from the 5' end to the 3' end, including between the 1st to 3rd nucleotides and between the 21st to 23rd nucleotides of the antisense strand, with adjacent nucleotides linked by thiophosphate bonds.

[0047] The cholesterol modification is performed along the 5' to 3' direction, including cholesterol modification at the 3' position of the nucleotide sugar ring at position 23 of the positive strand.

[0048] In this document, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt for the Col2a1-targeting siRNA provided by this invention. Generally, "pharmaceutically acceptable salt" as used in this invention refers to a salt that retains the biological activity targeting Col2a1 and is suitable for administration to humans or animals, and the method for preparing the salt for the siRNA is known in the art, typically involving reacting the siRNA with a pharmaceutically acceptable base or acid.

[0049] In one embodiment, the modified sense strand nucleotide sequence is as follows:

[0050] 5'-mC mC mUGGmCAmUmUGmCmUGGmCmUmUmUAAA mU mU-3'-Cholesteryl;

[0051] The modified antisense nucleotide sequence is as follows:

[0052] 5'-mU mU mUAAAGmCmCAGmCAAmUGmCmCAGG mU mU-3';

[0053] Where mC and mU represent nucleotides that have undergone 2'-O-methyl modification;

[0054] This indicates that adjacent nucleotides are linked by a phosphate thioester bond;

[0055] 3'-Cholesteryl indicates that the 3' end is modified with cholesterol.

[0056] In one embodiment, the modification further includes dangling deoxyribonucleotide dTdT at the 3' end of the sense strand and / or antisense strand; or, adding ribonucleotide UU at the 3' end of the sense strand and / or antisense strand.

[0057] On the other hand, the present invention provides a delivery system comprising the above-mentioned siRNA targeting Col2a1, which contains the above-mentioned siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof.

[0058] In one embodiment, the delivery system further includes a delivery vector in which the siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof is loaded, mixed, compounded, or conjugated with the delivery vector.

[0059] Furthermore, the delivery carrier preferably comprises hydrogels, liposomes, nanoparticles, or exosomes.

[0060] To better illustrate the present invention and provide a preferred embodiment for reference, the delivery carrier is a photocrosslinked methacrylamide gelatin (GelMA) hydrogel. Specifically, the preparation method of the photocrosslinked methacrylamide gelatin hydrogel loaded with the above-mentioned siRNA includes the following steps:

[0061] (1) Dissolve the photoinitiator LAP in phosphate buffer (PBS buffer, 0.01 M phosphate) to prepare a 0.25 w / v% initiator standard solution;

[0062] (2) Dissolve the methacrylamide gelatin in the initiator standard solution obtained in step (1) at a ratio of 5~15 w / v% and mix them evenly to obtain a precursor solution;

[0063] (3) Dissolve 100 μg of siRNA targeting Col2a1 in 0.2 mL of precursor solution to obtain a precursor solution loaded with siRNA;

[0064] (4) Irradiate the precursor solution loaded with siRNA under a 405 nm light source for 30 s to obtain photocrosslinked methacrylamide gel hydrogel loaded with siRNA.

[0065] In another aspect, the present invention also relates to the use of the above-mentioned Col2a1-targeting siRNA or a pharmaceutically acceptable salt thereof, and a delivery system containing the above-mentioned Col2a1-targeting siRNA, in the preparation of a drug for treating bisphosphonate-associated osteonecrosis of the jaw (BRONJ).

[0066] The present application will be further explained in detail below with reference to embodiments. However, those skilled in the art should understand that these embodiments are provided for illustrative purposes only and are not intended to limit the present application.

[0067] Example

[0068] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of this application. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products. This application should not be construed as being limited to the specific embodiments described.

[0069] 1. Main experimental materials and instruments

[0070] Laboratory animals: C57BL / 6J mice (female, 8-12 weeks old, with suitable body length / weight, purchased from Chengdu Dashuo Laboratory Animal Co., Ltd.).

[0071] Zoledronic acid (ZA, purchased from MCE, catalog number HY-13777).

[0072] Hydrogel raw materials (methacrylamide gelatin, GelMA, EFL-DexMA-500K).

[0073] Main instruments: High-resolution Micro-CT scanner (SCANCO μCT50 / 100), rotating confocal fluorescence microscope (Olympus IXplore IX83 SpinSR), all-slide digital scanner (Olympus SLIDEVIEW VS200), cryostat (Leica CM1950), etc.

[0074] Example 1

[0075] In this embodiment, the siRNA targeting Col2a1 has a modified sense strand and a modified antisense strand. It was synthesized by Huzhou Hippo Biotechnology Co., Ltd. The synthesized product was purified by high performance liquid chromatography (HPLC), and its molecular weight was confirmed to be correct by mass spectrometry (MS). The purity was greater than 90%, and the solvent was DEPC water.

[0076] The modified sense strand nucleotide sequence is as follows:

[0077] 5'-mC mC mUGGmCAmUmUGmCmUGGmCmUmUmUAAA mU mU-3'-Cholesteryl;

[0078] The modified antisense nucleotide sequence is as follows:

[0079] 5'-mU mU mUAAAGmCmCAGmCAAmUGmCmCAGG mU mU-3'.

[0080] Example 2

[0081] This embodiment utilizes the Col2a1-targeting siRNA from Example 1 to prepare a Col2a1-targeting siRNA delivery system. The delivery carrier is a photocrosslinked methacrylamide gelatin (GelMA) hydrogel, and its preparation method is as follows:

[0082] (1) Dissolve the photoinitiator LAP in phosphate buffer (PBS buffer, 0.01 M phosphate) to prepare a 0.25 w / v% initiator standard solution;

[0083] (2) Dissolve the methacrylamide gelatin in the initiator standard solution obtained in step (1) at a ratio of 10 w / v% and mix well to obtain a precursor solution;

[0084] (3) Dissolve 100 μg of siRNA targeting Col2a1 in 0.2 mL of precursor solution to obtain a precursor solution loaded with siRNA, and keep it for later use.

[0085] Comparative Example 1

[0086] Comparative Example 1 served as a blank negative control for Example 1, using a disordered siRNA (NC siRNA) that did not target any coding gene and had the same GC content as Example 1, provided by Huzhou Hippo Biotechnology Co., Ltd.

[0087] The modified blank negative control sense strand nucleotide sequence is as follows:

[0088] 5'-mU mU mCmUmCmCGAAmCGmUGmUmCAmCGmU mU mU-3'-Cholesteryl;

[0089] The modified blank negative control antisense strand nucleotide sequence is as follows:

[0090] 5'-A mC GmUGAmCAmCGmUmUmCGGAGAA mU mU-3'.

[0091] Comparative Example 2

[0092] Comparative Example 2 is a delivery system loaded with the disordered siRNA (NC siRNA) prepared using the disordered siRNA from Comparative Example 1. The preparation method is the same as in Example 2. Finally, a precursor solution loaded with NC siRNA was prepared and stored for later use.

[0093] To test the drug's relevance, a mouse model of osteonecrosis of the jaw needs to be established and identified. The specific methods are as follows:

[0094] C57BL / 6J mice were intraperitoneally injected with zoledronic acid (dose: 200 μg / kg, 3 times a week) for 8 consecutive weeks (day 1 to day 56) to induce microvascular damage and necrosis in the jawbone, serving as the jawbone necrosis group. The control group was injected with an equal volume of physiological saline.

[0095] On day 42 of drug administration, the bilateral mandibular first molars of the mice were extracted under aseptic conditions (see the model establishment procedure). Figure 1 (A) Mice were sacrificed on day 1 and day 14 (day 56) after tooth extraction. Mandibular bone tissue was collected, fixed with 4% paraformaldehyde, and then subjected to Micro-CT scan to assess bone resorption and necrosis. Subsequently, the tissue was decalcified, embedded, and sectioned with 0.5 M EDTA solution, and then stained with HE and Masson staining, respectively.

[0096] like Figure 1 As shown in Figure B, Micro-CT images taken 1 day and 2 weeks after tooth extraction show that in the control group, the extraction sockets gradually filled with new bone; while in the osteonecrosis group, the extraction sockets showed obvious bone resorption, sequestrum formation, and bone defects. Histological results (HE and Masson staining) showed ( Figure 1 (B): Two weeks after surgery, continuous hollow bone depressions were observed in the necrotic bone area of ​​the jawbone necrosis group (as shown by the arrows in the figure), accompanied by microvascular collapse and disordered collagen fiber arrangement, confirming the successful establishment of a mouse model of jawbone necrosis.

[0097] Test 1: Establishment and Gross Healing Assessment of an Isologous Heterologous Therapy Model Loaded with siRNA Hydrogel

[0098] On day 42 of drug administration, the bilateral mandibular first molars of the mice were extracted under aseptic conditions using the method described above.

[0099] An isotropic, contralateral controlled experiment was conducted: During tooth extraction, 60 μg of the precursor solution loaded with NC siRNA (as obtained in Comparative Example 2) was immediately placed in the fresh extraction socket on the left side of the mouse, and 60 μg of the precursor solution loaded with siRNA (as obtained in Example 2) was placed in the extraction socket on the right side. In-situ UV crosslinking and curing were then performed (irradiated at 405 nm for 30 s), resulting in NC siRNA-loaded hydrogels and siRNA-loaded hydrogels, respectively. The experimental procedure is illustrated below. Figure 2As shown in (A), the implantation process is recorded in photographs as follows. Figure 2 (B) As shown in the left figure.

[0100] Two weeks after the operation, the mice were euthanized, and the bilateral mandibles were harvested to observe and evaluate the soft tissue coverage of the extraction sockets and the healing of the hard tissue wounds.

[0101] like Figure 2 As shown in (B), two weeks after tooth extraction, the gingival soft tissue wound on the side loaded with NC siRNA hydrogel (left side) had not healed, with exposed necrotic bone and wound surface, and significantly poor bone formation in the lower part; in contrast, the soft tissue on the side loaded with siRNA hydrogel (right side) had completely healed, the extraction socket was filled with a large amount of new tissue, and bone formation was good. This indicates that the siRNA-loaded hydrogel, which targets and inhibits the abnormal expression of Col2a1 in endothelial cells, has a significant ability to promote the healing of soft and hard tissue wounds in extraction sockets of the jawbone necrosis.

[0102] Test 2: Evaluation of the effects of siRNA-loaded hydrogels on bone regeneration and osteogenic activity in tooth extraction sockets

[0103] Two weeks post-surgery, mandibular bone samples were taken from both sides of the test specimen and subjected to Micro-CT scanning and 3D reconstruction. After decalcification and embedding, the samples were sectioned and stained with HE, Masson's trichrome, and immunohistochemically (IHC) for osteopontin (OPN, a key marker of osteogenic bone).

[0104] like Figure 3 (A) Micro-CT three-dimensional reconstruction results show that compared with the NC siRNA-loaded hydrogel group, the amount of new bone filling in the extraction socket of the siRNA-loaded hydrogel group is significantly increased.

[0105] like Figure 3 (B) Histological and immunohistochemical results showed that HE and Masson staining confirmed the formation of a large number of mature mineralized bone trabeculae in the extraction socket of the siRNA-loaded hydrogel group, and the necrotic area was significantly reduced. OPN immunohistochemical staining showed that the OPN positive expression area and expression intensity in the siRNA-loaded hydrogel group were significantly higher than those in the NC siRNA-loaded hydrogel group, indicating that the siRNA-loaded hydrogel can significantly induce osteogenic differentiation and bone regeneration in the extraction socket.

[0106] Test 3: Evaluation of the effect of siRNA-loaded hydrogel on angiogenesis-osteoogenesis coupling in tooth extraction sockets

[0107] Frozen or paraffin sections of mandibular bone tissue from both groups were taken 2 weeks post-surgery in Test 1. Immunofluorescence (IF) staining was used to detect the expression of the endothelial cell marker CD31 and to assess the local microvascular morphology and formation.

[0108] We further evaluated the coupling effect between angiogenesis and osteoogenesis in the microcirculation by performing dual immunofluorescence staining on H-type vascular markers endomucin (Emcn) and bone morphogenetic protein 2 (BMP2, an osteoblast marker).

[0109] like Figure 4 As shown in (A), the CD31-positive vascular structure in the extraction socket of the siRNA-loaded hydrogel group was intact, and the number of vessels and the density of luminal vessels were higher than those of the severely sparse NC siRNA-loaded hydrogel group.

[0110] like Figure 4 (B) The results of dual immunofluorescence showed that the siRNA-loaded hydrogel group not only promoted the proliferation of a large number of Emcn-positive blood vessels, but also accumulated a large number of BMP2-positive osteoblasts around the Emcn-positive blood vessels, forming a good "angiogenic-osteogenic" coupled microenvironment, indicating that blocking the Col2a1 paracrine axis can synergistically accelerate jawbone repair by remodeling the local microvascular network.

[0111] Test 4: Evaluation of the effect of siRNA-loaded hydrogel on alleviating local inflammation in tooth extraction sockets

[0112] Two weeks after the surgery, mandibular tissue sections were taken from both groups in Test 1, and the degree of infiltration of the pan-macrophage marker CD68 was detected by immunofluorescence (IF) staining.

[0113] Immunohistochemical staining (IHC) was used to detect the expression levels of major pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6).

[0114] like Figure 5 (A) Immunofluorescence results showed that a large number of CD68-positive macrophages were observed to infiltrate the extraction socket in the NC siRNA-loaded hydrogel group, indicating a strong immune imbalance and inflammatory state; while the number of CD68-positive cells was significantly reduced in the siRNA-loaded hydrogel group.

[0115] like Figure 5 (B) Immunohistochemical results showed that the positive expression of pro-inflammatory factors IL-1β, TNF-α and IL-6 in the siRNA-loaded hydrogel group was significantly lower than that in the NC siRNA-loaded hydrogel group.

[0116] The above results indicate that siRNA-loaded hydrogels can effectively block pro-inflammatory communication between endothelial cells and macrophages, inhibit excessive inflammatory response in the extraction socket of osteonecrosis of the jaw, and reshape the anti-inflammatory microenvironment, thereby providing favorable conditions for jawbone regeneration.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof, characterized in that: The siRNA has a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is a modified nucleotide sequence as shown in SEQ ID NO: 1; and the nucleotide sequence of the antisense strand is a modified nucleotide sequence as shown in SEQ ID NO:

2. The modifications include methoxy modification, thiosclerosis modification, and cholesterol modification of nucleotides; The methoxy modification is performed at the 2' position of the nucleotide sugar ring at positions 1, 2, 3, 6, 8, 9, 11, 12, 15, 16, 17, 18, 22, and 23 on the sense strand, in a direction from the 5' end to the 3' end; and at the 2' position of the nucleotide sugar ring at positions 1, 2, 3, 8, 9, 12, 15, 17, 18, 22, and 23 on the antisense strand, in a direction from the 5' end to the 3' end. The thiophosphate backbone modification is performed in the direction from the 5' end to the 3' end, including between the 1st to 3rd nucleotides and between the 21st to 23rd nucleotides of the sense strand, with adjacent nucleotides linked by thiophosphate bonds; and in the direction from the 5' end to the 3' end, including between the 1st to 3rd nucleotides and between the 21st to 23rd nucleotides of the antisense strand, with adjacent nucleotides linked by thiophosphate bonds. The cholesterol modification is performed along the 5' to 3' direction, including cholesterol modification at the 3' position of the nucleotide sugar ring at position 23 of the positive strand.

2. The siRNA targeting Col2a1 according to claim 1, characterized in that: The modification also includes dangling deoxyribonucleotide dTdT at the 3' end of the sense strand and / or antisense strand; or, adding ribonucleotide UU at the 3' end of the sense strand and / or antisense strand.

3. A delivery system, characterized in that: The delivery system contains the siRNA targeting Col2a1 as described in claim 1, or a pharmaceutically acceptable salt thereof.

4. The delivery system according to claim 3, characterized in that: The delivery system further includes a delivery vector in which the siRNA targeting Col2a1 or a pharmaceutically acceptable salt thereof is loaded, mixed, compounded, or conjugated with the delivery vector.

5. The delivery system according to claim 4, characterized in that: The delivery carrier includes hydrogels, liposomes, nanoparticles, or exosomes.

6. The use of the siRNA targeting Col2a1 as described in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating bisphosphonate-associated osteonecrosis of the jaw.

Images