Application of macrophage membrane coated SPHK1 particles in uveitis
By preparing SPHK1 nanoparticles coated with macrophage membranes, the problem of toxic side effects in the treatment of uveitis was solved, achieving highly efficient targeted therapy for uveitis and reducing retinal damage and inflammation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-03-24
AI Technical Summary
Existing medications for treating uveitis have toxic side effects, and there is a need to seek safe and effective prevention/treatment methods.
SPHK1 nanoparticles were prepared by using macrophage membrane-coated SPHK1 particles, loading TBG promoter-driven SPHK1 expression plasmids with PLGA as nanocarriers, and coating the surface of the plasmids with lipopolysaccharide-stimulated macrophage membranes to achieve targeted gene intervention.
It enhances the targeting function of uveitis lesions, reduces retinal damage and inflammatory response, prevents damage to the retinal barrier, and has good biocompatibility.
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Figure CN121714535A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of new drug development technology, and in particular to the application of macrophage membrane-coated SPHK1 particles in uveitis. Background Technology
[0002] Uveitis is a group of inflammatory diseases affecting the iris, ciliary body, choroid, retina, and blood vessels. Its clinical manifestations are diverse, including ciliary or mixed conjunctival injection, posterior synechiae, fundus exudates, retinal edema, and hemorrhage. This disease is characterized by its tendency to recur, especially in cases of weakened immune function. If not treated promptly and properly, it often leads to serious complications and is one of the leading causes of visual impairment and blindness. Common complications include cataracts, glaucoma, retinal detachment, macular edema, and corneal diseases (such as band keratosis and edema).
[0003] Current treatment methods focus on rapidly controlling inflammation, primarily relying on topical or systemic corticosteroids, supplemented by immunosuppressants, antibiotics, or antiviral drugs depending on the cause. However, long-term use can produce significant toxic side effects. Therefore, finding safe and effective clinical drugs for the prevention and treatment of uveitis is a crucial challenge that the medical community urgently needs to address. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of macrophage membrane-coated SPHK1 particles in uveitis.
[0005] SPHK1 is a key enzyme catalyzing the production of the signaling lipid S1P, playing an "accelerator" role in inflammatory and immune responses. It amplifies inflammatory responses by promoting the release of large amounts of pro-inflammatory factors from macrophages and neutrophils; simultaneously, the S1P it generates directs lymphocytes from lymphoid organs into the circulatory system, regulating their migration and function. In recent years, nanoparticle-based therapies, prevention, and detection methods have significantly impacted disease management. With the abundance of available nanoparticles, the design of application-specific nanocarriers has become increasingly common. However, once inside the body, nanoparticles encounter a highly complex environment adept at recognizing and eliminating foreign elements. For example, blood contains various protein- and cellular components, and contact with any of them can rapidly affect performance.
[0006] This invention provides macrophage membrane-coated SPHK1 nanoparticles. Using polylactic-coated glycolic acid copolymer (PLGA) as a nanocarrier, an SPHK1 expression plasmid driven by the TBG promoter is loaded to prepare PLGA-mSPHK1 nanoparticles. Then, macrophage membranes extracted after lipopolysaccharide stimulation are coated on the surface of PLGA-mSPHK1 nanoparticles to prepare macrophage membrane-coated nanoparticles LPS-MM / PLGA-mSPHK1, which have both cell-targeting and tissue-targeting properties.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A macrophage membrane-coated SPHK1-loaded nanoparticle (LPS-MM / PLGA-mSPHK1) was prepared by using polylactic-coated glycolic acid copolymer (PLGA) as a nanocarrier and loading an SPHK1 expression plasmid driven by the TBG promoter. Then, macrophage membranes extracted after lipopolysaccharide stimulation were coated on the surface of the PLGA-mSPHK1 nanoparticles to obtain the final product.
[0008] The sequence of the specific promoter is shown in SEQ ID NO.1: GGGGCTGGAAGCTACCTTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCTACAGAACCTATTAGAAAGGATCACCCAGCCTCTGCTTTTGTACAACTTTCCCTTAAAAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAACTTTTTCCTGCTGCCTCTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCAGCATGGACTT AAACCCCTCCAGCTCTGACAATCCTCTTTCTCTTTTGTTTTACATGAAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTTCAAACCTTATCATTTTTTGCTTTGTTCCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTTAATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGAAAGAT.
[0009] Preferably, the SPHK1 sequence is a mouse SPHK1 nucleic acid sequence, as shown in SEQ ID NO. 2:
[0010] Preferred method: The preparation method of the PLGA-mSPHK1 nanoparticles is as follows: PLGA is dissolved in dichloromethane, SPHK1 expression plasmid and polyethyleneimine (PEI) are added, wherein the SPHK1 expression plasmid and PEI are in a transfection ratio of plasmid:PEI = 1:2-3; then the mixture is added to a 1% polyvinyl alcohol (PVA) aqueous solution, sonicated in an ice bath for 3-8 min, stirred with a magnetic stirrer to evaporate the organic solvent for 3-5 h, then dialyzed in ultrapure water for 10-15 h, and passed through a 200 nm polycarbonate membrane to obtain PLGA-mSPHK1 nanoparticles.
[0011] Preferred method: The extraction method of the lipopolysaccharide-stimulated macrophage membrane is as follows: Macrophages are stimulated with 100 ng / ml LPS, and the macrophages are polarized to M1 type. The polarized macrophages are then subjected to a hypotonic solution gradient to obtain cell lysate, which is extracted by differential centrifugation and probe sonication. The hypotonic solution gradient is specifically: the cell suspension is added to a 0.25X buffer solution containing a protease inhibitor, lysed on ice for 2 h, and then sonicated for 5 min to fully lyse the cells. The cell lysate is collected, centrifuged at 1000 rpm and 4℃ for 5 min, and the supernatant is carefully collected. Then, the cell fragments and organelles are removed by centrifugation at 14000 rpm and 4℃ for 10 min, and the precipitate is collected to obtain the macrophage membrane. The obtained macrophage membrane is resuspended in ultrapure water and then extruded through a 400 nm polycarbonate membrane using a micromembrane extruder, and repeatedly extruded 10-20 times to obtain the macrophage membrane.
[0012] The application of the macrophage membrane-coated SPHK1 nanoparticles (LPS-MM / PLGA-mSPHK1) in the preparation of products for the prevention or treatment of uveitis.
[0013] The beneficial effects of this invention are as follows: 1. In this invention, polylactic acid-glycolic acid copolymer (PLGA) is used as a nanocarrier to load SPHK1 expression plasmid driven by TBG promoter, and then coated with lipopolysaccharide-stimulated macrophage membrane to prepare LPS-MM / PLGA-mSPHK1.
[0014] 2. This invention can recruit LPS-MM / PLGA-mSPHK1 to retinal lesion sites, thereby increasing the accumulation of LPS-MM / PLGA-mSPHK1 at the lesion sites, enhancing the gene intervention effect, and achieving highly efficient targeting function of autoimmune uveitis lesion sites. It can not only reduce retinal damage and inflammatory response and prevent retinal barrier damage, but also has good biosafety. Attached Figure Description
[0015] Figure 1SPHK1 is highly expressed in patients with experimental autoimmune uveitis (EAU). Figure 1 A represents the SPHK1 representation in the GEO database, specifically in Control and EAU. Figure 1 B is the SPHK1 expression differential analysis in the GEO database. Figure 2 Expression map of plasmid TBG-mSPHK1; Figure 3 Characterization diagrams of PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1 nanoparticles (among others) Figure 3 A is an LPS-MM / PLGA-mSPHK1 transmission electron microscope; Figure 3 B represents the zeta potential of PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1. Figure 3 C represents the LPS-MM / PLGA-mSPHK1 particle size; Figure 3 D represents PLGA-mSPHK1, LPS-MM / PLGA-mSPHK1, macrophage membrane, lipopolysaccharide-stimulated macrophage membrane, and macrophage Coomassie Brilliant Blue. Figure 4 Stability testing of PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1 nanoparticles (among others) Figure 4 A represents the particle size and PDI of PLGA-mSPHK1 over 7 days; Figure 4 B represents the 7-day particle size and PDI of LPS-MM / PLGA-mSPHK1. Figure 5 This is a diagram showing the results of cellular uptake experiments of PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1 nanoparticles; Figure 6 The image shows the results of LPS-MM / PLGA-mSPHK1 nanoparticles reducing anterior segment inflammation in EAU mice (wherein) Figure 6 A is the result of the mouse anterior segment; Figure 6 B is a statistical chart of the anterior segment clinical score; Figure 7 The image shows the results of LPS-MM / PLGA-mSPHK1 nanoparticles alleviating fundus inflammation in EAU mice (wherein) Figure 7 A is a fundus image of a mouse; Figure 7 B is a statistical chart of fundus clinical scoring; Figure 8 This is an image of the optical coherence tomography (OCT) results of LPS-MM / PLGA-mSPHK1 nanoparticles reducing EAU mice (wherein) Figure 8 A is the mouse OCT result image; Figure 8B is the OCT clinical score statistical chart; Figure 9 The image shows the results of LPS-MM / PLGA-mSPHK1 nanoparticles reducing retinal vascular leakage in EAU mice (wherein) Figure 9 A shows the results of Evans blue staining in mice; Figure 9 B is the Evans Blue clinical score statistical chart. Detailed Implementation
[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0017] All raw materials and reagents used in this invention are commercially available products. Unless otherwise specified, all parts mentioned in this invention are parts by weight, and all percentages are mass percentages. Example 1: SPHK1 is highly expressed in patients with experimental autoimmune uveitis (EAU). Based on the GEO database (GSE7850), intraocular imaging was performed on healthy individuals and patients with experimental autoimmune uveitis. SPHK1 expression analysis revealed that SPHK1 expression was increased in the eyes of patients with experimental autoimmune uveitis (e.g., Figure 1 (As shown).
[0018] Example 2: Construction of plasmid TBG-mSPHK1.
[0019] The expression map of plasmid TBG-mSPHK1 is as follows: Figure 2 As shown, the specific promoter sequence in plasmid TBG-mSPHK1 is shown in SEQ ID NO.1: GGGGCTGGAA GCTACCTTTG ACATCATTTC CTCTGCGAAT GCATGTATAA TTTCTACAGAACCTATTAGA AAGGATCACC CAGCCTCTGC TTTTGTACAA CTTTCCCTTA AAAAACTGCC AATTCCACTGCTGTTTGGCC CAATAGTGAG AACTTTTTCC TGCTGCCTCT TGGTGCTTTT GCCTATGGCC CCTATTCTGCCTGCTGAAGA CACTCTTGCC AGCATGGACT TAAACCCCTC CAGCTCTGAC AATCCTCTTT CTCTTTTGTTTTACATGAAG GGTCTGGCAG CCAAAGCAAT CACTCAAAGT TCAAACCTTA TCATTTTTTG CTTTGTTCCTCTTGGCCTTG GTTTTGTACA TCAGCTTTGA AAATACCATC CCAGGGTTAA TGCTGGGGTT AATTTATAACTAAGAGTGCT CTAGTTTTGC AATACAGGAC ATGCTATAAA AATGGAAAGA T The nucleic acid sequence of mouse SPHK1 is shown in SEQ ID NO.2: ATGGAACCA GTAGAATGCC CTCGAGGACT GCTCCCACGG CCATGCAGAG TGCTGGTGCTGCTGAACCCC CAGGGTGGCA AGGGCAAGGC TCTGCAGCTC TTCCAGAGCC GTGTGCAGCC CTTCCTGGAGGAGGCAGAGA TAACCTTTAA ACTGATACTC ACCGAACGGA AGAACCATGC CAGGGAGCTG GTGTGTGCAGAGGAGTTGGG TCACTGGGAC GCCCTGGCAG TCATGTCCGG TGATGGTCTG ATGCATGAGG TGGTGAATGGGCTAATGGAA CGGCCAGACT GGGAGACTGC CATCCAGAAA CCCCTGTGTA GCCTCCCTGG AGGCTCCGGCAATGCGCTGG CAGCTTCTGT GAACCACTAT GCTGGGTACG AGCAGGTGAC TAATGAAGAC CTGCTCATCAACTGCACACT GCTGTTGTC CGCCGGCGCC TTGTACCCAT GAACCTGCTGTCCCGACA CTGCTTCTGGCTGCGGCTC TATTCTGTGC TCAGTCTGTC CTGGGGCTTT GTTGCTGACG TGGACCTCGA GAGTGAGAAGTACAGGCGCT TGGGGGAGAT TCGTTTCACA GTGGGCACCT TCTTTCGCCT AGCAAGCCTG CGCATCTACCAAGGCCAACT GGCCTACCTT CCTGTAGGAA CTGTGGCCTC TAAGAGACCC GCCTCTACAC TGGTGCAGAAGGGCCCCGTC GACACACACC TTGTTCCTCT GGAGGAGCCA GTGCCTTCTC ATTGGACTGT GGTACCAGAACAGGACTTCG TCCTGGTGCT GGTGCTGCTA CACACCCACC TGAGCTCCGA GCTGTTTTGCA GCACCCATGGGCCGCTGTGA GGCTGGTGTT ATGCATCTGT TCTACGTACG TGCGGGGTG TCAAGGGCTG CGCTGCTGCGCCTCTTCCTG GCCATGCAGAAGGGCAAGCA TATGGAACTT GACTGTCCAT ACCTGGGTTCA TGTGCCCGTGGTTGCTTTCC GCCTGGAGCC CAGGAGCCAG AGGGGCGTGT TTTCTGTGGA TGGAGAGCTG ATGGTATGTGAAGCTGTGCA GGGCCAAGTG CACCCAAACT ACCTTTGGAT GGTCTGTGGC AGCAGAGATG CCCCATCCGGCCGGGACTCC CGGCGGGGGC CACCTCCAGA AGAACCATAA Example 3: Characterization of the preparation of PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1 nanoparticles.
[0020] (1) The extraction method of lipopolysaccharide-stimulated macrophage membrane is as follows: macrophages are stimulated with 100 ng / ml LPS and polarized to M1 type. The polarized macrophages are then subjected to a hypotonic solution gradient to obtain cell lysate, which is extracted by differential centrifugation and probe sonication. The hypotonic solution gradient is specifically: the cell suspension is added to a 0.25X buffer solution containing protease inhibitor, lysed on ice for 2 h, and then sonicated for 5 min to fully lyse the cells. The cell lysate is collected, centrifuged at 1000 rpm and 4℃ for 5 min, and the supernatant is carefully collected. Then, the cell debris and organelles are removed by centrifugation at 14000 rpm and 4℃ for 10 min, and the precipitate is collected to obtain the macrophage membrane. The obtained macrophage membrane is resuspended in ultrapure water and then extruded through a 400 nm polycarbonate membrane using a micro membrane extruder 10-20 times to obtain the macrophage membrane, which is used for subsequent coating of PLGA nanoparticles.
[0021] (2) Preparation of PLGA-mSPHK1 nanoparticles: Weigh 10 mg of PLGA, add 1 ml of dichloromethane, and sonicate for 60 s to fully dissolve the PLGA. Then add 1 mg of SPHK1 expression plasmid and 2 ml of PEI (calculated according to the transfection ratio plasmid:PEI = 1:2), and mix well. Slowly add the above solvent dropwise to 3 ml of 1% polyvinyl alcohol (PVA) aqueous solution, sonicate in an ice bath for 5 min, with 5 s intervals. Stir with a magnetic stirrer to evaporate the organic solvent for 4 h, then dialyze in ultrapure water for 12 h, and pass through a 200 nm polycarbonate membrane to obtain PLGA-mSPHK1 nanoparticles.
[0022] (3) The prepared PLGA-mSPHK1 nanoparticles were thoroughly mixed with the macrophage membrane stimulated by the extracted lipopolysaccharide at a ratio of 1:10 and ultrasonicated in a water bath for 10 min. The nanoparticles were then passed through a 400 nm polycarbonate membrane and then through a 200 nm polycarbonate membrane using a micro membrane extruder to obtain LPS-MM / PLGA-mSPHK1 nanoparticles.
[0023] (4) The LPS-MM / PLGA-mSPHK1 nanoparticles were diluted 10 times with PBS, and their microstructure was observed using a transmission electron microscope. The results are as follows: Figure 3 As shown in Figure A, LPS-MM / PLGA-mSPHK1 nanoparticles exhibit a distinct "core-shell" structure.
[0024] (5) PLGA-mSPHK1 nanoparticles and LPS-MM / PLGA-mSPHK1 nanoparticles were diluted 20-fold with PBS, and their particle size was determined using a Malvern laser particle size analyzer; they were also diluted 200-fold with ddH2O, and their zeta potential was determined using a Malvern laser particle size analyzer. The results are as follows: Figure 3 As shown in Figure B: The potential of LPS-MM / PLGA-mSPHK1 nanoparticles is lower than that of PLGA-mSPHK1 nanoparticles. The results are as follows... Figure 3 As shown in C, the particle size of LPS-MM / PLGA-mSPHK1 nanoparticles is between 200nm and 300nm.
[0025] (6) Macrophages, macrophage membranes, lipopolysaccharide-stimulated macrophage membranes, PLGA-mSPHK1 nanoparticles, and LPS-MM / PLGA-mSPHK1 nanoparticles were extracted with protein lysis buffer, and then characterized by DS-PAGE gel electrophoresis and classic Coomassie brilliant blue staining. The results are as follows: Figure 3 As shown in D.
[0026] Example 4: Stability testing of PLGA-mSPHK1 nanoparticles and LPS-MM / PLGA-mSPHK1 nanoparticles.
[0027] The particle size and dispersion index (PDI) of PLGA-mSPHK1 nanoparticles and LPS-MM / PLGA-mSPHK1 nanoparticles were measured continuously for 7 days. The results are as follows. Figure 4 As shown, both PLGA-mSPHK1 nanoparticles and LPS-MM / PLGA-mSPHK1 nanoparticles are relatively stable, with particle size and PDI value fluctuating within a small range.
[0028] Example 5: Cellular uptake experiment of PLGA-mSPHK1 nanoparticles and LPS-MM / PLGA-mSPHK1 nanoparticles.
[0029] (1) AML-12 cells were plated and divided into three groups (Blank group, PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1). After adhesion, intervention was performed.
[0030] (2) After cell adhesion, 100 μL of PLGA-mSPHK1 and LPS-MM / PLGA-mSPHK1 nanoparticles were added at the 12h time point.
[0031] (3) After the intervention time is over, discard the supernatant and fix the PFA for 10 minutes.
[0032] (4) Discard the supernatant, wash once with PBS, and stain in DIO staining solution in an incubator for 20 min.
[0033] (5) Discard the supernatant and wash once with PBS, then stain with DAPI for 10 min.
[0034] (6) Discard the supernatant, wash once with PBS, mount with fluorescent mounting medium, and then perform laser confocal imaging. Results are as follows: Figure 5 As shown, LPS-MM / PLGA-mSPHK1 nanoparticles have a higher uptake rate in AML-12 cells than PLGA-mSPHK1 nanoparticles.
[0035] Example 6: Establishment and treatment of experimental autoimmune uveitis (EAU) Experimental autoimmune uveitis (EAU) is a standardized laboratory animal model induced by immunological methods that reproduces the key immunopathogenic mechanisms and pathological features of human uveitis. This model exhibits a high degree of similarity to the human disease in terms of clinical manifestations and histopathology, and is a recognized experimental system for studying the pathogenesis of uveitis, assessing the degree of inflammation, and validating drug treatment regimens.
[0036] (1) Eight-week-old female C57BL / 6J mice (purchased from the Animal Experiment Center of Chongqing Medical University) were selected and anesthetized with sodium pentobarbital. Wild-type mice (WT mice) were divided into three groups: Blank group, EAU group and EAU+LPS-MM / PLGA-mSPHK1 group.
[0037] (2) Mice in the EAU+LPS-MM / PLGA-mSPHK1 group were injected with LPS-MM / PLGA-mSPHK1 nanoparticles via the tail vein.
[0038] (3) One week after the nanoparticle injection, EAU modeling was performed on mice in the EAU group and the EAU+LPS-MM / PLGA-mSPHK1 group. IRBP was dissolved in DMSO and PBS, and Mycobacterium tuberculosis was dissolved in CFA. The mixture was emulsified with an equal volume of IRBP and CFA for 1 hour. 200 μL of IRBP-CFA mixture was injected subcutaneously into each mouse, followed by 200 μL of PTX injected intraperitoneally.
[0039] (4) Two weeks after modeling, the inflammation of the mouse eyes was observed by taking pictures of the anterior segment, fundus, and OCT.
[0040] Example 7: Clinical scoring of anterior segment inflammation in the EAU model Two weeks after immunization, the degree of anterior segment inflammation was examined using a slit lamp and clinically scored using a double-blind method. The clinical score for anterior segment inflammation was based on the severity of five signs: corneal opacity, conjunctival or ciliary injection, mixed injection, anterior chamber inflammatory cells, and posterior synechiae, rated from 0 to 5 points. Results were as follows: Figure 6 As shown, compared with the EAU group, the EAU+LPS-MM / PLGA-mSPHK1 group can significantly improve inflammation such as anterior segment congestion in mice.
[0041] Example 8: Clinical scoring of fundus inflammation in the EAU model Two weeks after immunization, the degree of fundus inflammation was observed by fundus photography and clinically scored using a double-blind method. The clinical score for fundus retinal inflammation was based on the severity of five signs: patchy soft exudates, linear vasculitis, macular edema, retinal hemorrhage, and retinal detachment, rated from 0 to 5. Results are as follows: Figure 7 As shown, compared with the EAU group, the EAU+LPS-MM / PLGA-mSPHK1 group significantly improved fundus inflammation in mice.
[0042] Example 9: Clinical scoring of EAU model optical coherence tomography (OCT) Two weeks after immunization, fundus images were acquired using optical coherence tomography (OCT) to assess the degree of retinal inflammation. A double-blind clinical scoring method was used to assess the OCT images, based on four main morphological features: degree of inflammatory cell infiltration, retinal fold formation, focal retinal detachment, and granulomatous lesions. Each sign was scored from 0 to 4 points according to its severity, and the final total score served as a quantitative indicator of the degree of inflammation. Results are as follows: Figure 8 As shown, compared with the EAU group, the EAU+LPS-MM / PLGA-mSPHK1 group significantly improved fundus inflammation in mice.
[0043] Example 10: Evans Blue Staining of Retinal Patches Two weeks after modeling, the tail veins of mice were fully dilated by wiping them with alcohol swabs. After fixing the tail, 100 μl of 2% Evans Blue (Sigma, USA) was injected into the tail vein. Upon successful injection, the mice's hands and feet rapidly turned blue. Two hours later, the mice were euthanized by sodium pentobarbital anesthesia, and the eyeballs were removed and fixed in 4% paraformaldehyde for 3 hours. Under a microscope, a circular incision was made 1-2 mm medially along the limbus, and the anterior segment tissues such as the cornea, iris, and vitreous humor were removed. The retina was radially incised in four quadrants, bluntly dissected, and laid flat on a clean glass slide, facing upwards towards the vitreous humor. Residual vitreous fibers were removed, and the slide was mounted with glycerin. The retinal leakage was observed under a fluorescence microscope. The results are as follows: Figure 9 As shown, compared with the EAU group, the EAU+LPS-MM / PLGA-mSPHK1 group can significantly improve retinal vascular leakage in mice.
[0044] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.