CircRNA nano delivery system capable of reversing nucleus pulposus progenitor cell senescence and preparation method and application of circRNA nano delivery system

Abstract

The invention discloses a circRNA nano delivery system capable of reversing nucleus pulposus progenitor cell senescence and a preparation method and application of the circRNA nano delivery system. The circRNA nano delivery system comprises: a) Klotho circRNA, and b) a liposome, and the liposome comprises a vascular peptide vasculotide, an ionizable lipid molecule, a phospholipid, a PEG lipid, and a steroid. The invention also provides application of the circRNA nano delivery system capable of reversing nucleus pulposus progenitor cell senescence in preparation of drugs for treating intervertebral disc degeneration. The invention provides liposome particles (Vas-Klotho-LNP) which have targeted NPPCs and wrap circRNA, Klotho circRNA is efficiently introduced into the NPPCs through Tie2 agonist peptide, so that rejuvenation of the NPPCs is promoted, ECM synthesis is promoted, and regeneration and repair of intervertebral discs are synergistically promoted in vivo and in vitro.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a circRNA nano delivery system capable of reversing aging of nucleus pulposus progenitor cells, and a preparation method and application thereof. Background Art

[0002] Intervertebral Disc Degeneration (IVDD) is a chronic degenerative disease that leads to progressive destruction of the intervertebral disc and has a significant impact on the quality of life. Currently, there is no approved effective therapy to prevent the progression of IVDD, and clinical treatment mainly adopts symptomatic relief and ultimate spinal fusion. With age, the regenerative potential of endogenous stem cells / progenitor cells in the intervertebral disc decreases, and their ability to differentiate into functional cells also weakens, which is closely related to the degeneration of intervertebral disc structure and health. Therefore, restoring the regenerative potential of endogenous intervertebral disc stem cells / progenitor cells may contribute to the structural and functional remodeling of IVDD, but effective strategies have not yet been formed.

[0003] Nucleus pulposus progenitor cells (NPPCs) are potential seed cells for cell therapy and tissue engineering in the field of intervertebral disc repair and regeneration. They form extracellular matrix (ECM) by secreting type II collagen and proteoglycans, and play a key role in maintaining intervertebral disc homeostasis. The proportion of NPPCs gradually decreases with the increase of IVDD grade, indicating that ECM metabolism and NP cell differentiation ability are weakened in degenerated intervertebral discs. In addition, the regenerative ability of NPPCs changes over time during degeneration and aging, and the cells enter a senescent state. Therefore, it is urgent to restore their regenerative ability through the reactivation of endogenous NPPCs and rebuild the youthful structure and functional characteristics of the intervertebral disc.

[0004] Klotho is an anti-aging single-transmembrane protein whose decreased levels are associated with cellular aging and stem cell dysfunction. Klotho consists of a large extracellular domain (130 kDa), a transmembrane domain, and a short intracellular domain consisting of 10 amino acids. In humans, Klotho gene polymorphisms are associated with age-related bone loss, spinal disease, osteocalcin levels, and bone mineral density.

[0005] Vasculotide is a Tie2 agonist peptide with a polyethylene glycol cluster structure that mimics the effects of Ang-1. In different models of pneumococcal pneumonia, vasculotide can promote pulmonary endothelial stability and reduce pulmonary permeability (Birgitt Gutbier, Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia).

[0006] In the field of intervertebral disc degeneration treatment, no therapeutic method using Klotho on nucleus pulposus progenitor cells (NPPCs) has been published. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a circRNA nano-delivery system with the ability to reverse the aging of nucleus pulposus progenitor cells, as well as a preparation method and application thereof. The constructed liposome particles encapsulating circRNA targeting NPPCs efficiently introduce Klotho circRNA into NPPCs through Tie2 agonist peptide, thereby promoting their rejuvenation and ECM synthesis, and synergistically promoting intervertebral disc regeneration and repair in vivo and in vitro.

[0008] In order to achieve the above technical objectives, the technical solution implemented by the present invention is:

[0009] In one aspect, the present invention provides a circRNA nano-delivery system capable of reversing the aging of nucleus pulposus progenitor cells, comprising:

[0010] a) Klotho circRNA, and

[0011] b) Liposomes, wherein the liposomes contain vasculotide, ionizable lipid molecules, phospholipids, PEG lipids, and steroids.

[0012] In the embodiment of the present invention, the ionizable lipid molecule is named T12, and the structural formula is:

[0013]

[0014] Preferably, the phospholipid is selected from dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), distearoylphosphatidylethanolamine (DP ... distearoylphosphatidylethanolamine (DPPEA), distearoylphosphatidylcholine (DPPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), distearoylphosphatidylethanolamine (DPPEA), distearoylphosphatidylcholine (DPPEA), distearoylphosphatidylethanolamine (DPPEA), -phosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-trans-phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and a combination of one or more of their derivatives and isomers.

[0015] In the embodiment of the present invention, the steroid is cholesterol.

[0016] In an embodiment of the present invention, the PEG lipid is PEG-DMG.

[0017] Preferably, the circRNA nano delivery system comprises DSPE-PEG-Vas, T12, PEG-DMG, DOPE and cholesterol in a molar ratio of 2:40:3:10:45.

[0018] On the other hand, the present invention also provides a method for preparing the circRNA nano delivery system, comprising the following steps:

[0019] Step 1, preparation of DSPE-PEG-Vas: DSPE-PEG-MAL solution and vasculotide solution were mixed and stirred to prepare DSPE-PEG-Vas.

[0020] Step 2, accurately weigh DSPE-PEG-Vas, T12, PEG-DMG, DOPE and cholesterol and dissolve them in solvents to prepare lipid stock solutions, convert the molar mass of each component into volume, and take the prescribed volume of the lipid stock solutions and mix them thoroughly to obtain a lipid organic phase; use enzyme-free sodium hydrogen phosphate-citrate buffer pH = 5.0 to dissolve α-Klotho-circRNA to obtain an α-Klotho-circRNA aqueous phase;

[0021] Step 3: The lipid organic phase and the α-Klotho-circRNA aqueous phase were injected into the microfluidic system for mixing. Then, the mixture was dialyzed in RNase-free PBS pH 7.4 to remove the solvent and concentrated by ultrafiltration to obtain circRNA-loaded LNP, namely Vas-Klotho@LNP.

[0022] Furthermore, in step 1, the molar ratio of DSPE-PEG-MAL to vasculotide is 1:1.

[0023] Furthermore, in step 1, the DSPE-PEG-MAL solution, the vasculotide solution and the solvent in step 2 are organic solvents, which may be methanol, ethanol, etc., preferably anhydrous ethanol.

[0024] On the other hand, the present invention also provides the use of the circRNA nano delivery system in the preparation of drugs for treating intervertebral disc degeneration.

[0025] The drug may be in an oral dosage form or an injectable dosage form, and may be administered to a desired subject (such as a human or non-human mammal) by oral administration, injection, or the like.

[0026] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, granules, etc. Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. The present invention provides a Klotho circular RNA (circRNA) nanoparticle that uses TEK receptor tyrosine kinase (Tie2) as an active target receptor because it is highly expressed in NPPCs and has limited expression in other cells.

[0028] 2. The liposome particles (Vas-Klotho@LNP) encapsulating circRNA targeting NPPCs constructed by the present invention efficiently introduced Klotho circRNA into NPPCs through Tie2 agonist peptide, and Vas-Klotho@LNP showed excellent ability in NPPC internalization, lysosomal escape and successful Klotho transfection.

[0029] 3. The circRNA-encapsulated liposome particles provided by the present invention delivered the Klotho gene via Vas-Klotho@LNP, significantly enhancing the ability of NPPCs to differentiate into nucleus pulposus-like cells; at the same time, the expression of FGF23 was significantly downregulated, and CDK4, MMP10, MMP12, MMP13, ADAMTS5 and ADAMTS7 were downregulated, indicating that it effectively inhibited the aging of NPPCs, promoted the rejuvenation of NPPCs and promoted ECM synthesis, and synergistically promoted the regeneration and repair of intervertebral discs in vitro and in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 A circRNA nano-delivery system with the ability to reverse the aging of nucleus pulposus progenitor cells is provided in Example 1, wherein a is the molecular structure formula of the new ionizable lipid T12; b is a schematic diagram of the microfluidic mixing construction of circRNA-LNP, which is composed of ionizable lipids, cholesterol, DOPE and PEG lipids.

[0032] Figure 2 The test results of a circRNA nanodelivery system for reversing the aging of nucleus pulposus progenitor cells provided in Example 1, wherein a is the particle size distribution; the data are expressed as mean ± standard deviation, n = 3; b is the potential distribution of the LNP nanoformulation; c is the transmission electron microscopy (TEM) image analysis of LNP; d is the gel retardation experiment of LNP.

[0033] Figure 3 The immunofluorescence images in Example 2 show the uptake of Vas-Klotho@LNP and Klotho@LNP in NPPCs.

[0034] Figure 4 The performance evaluation results of Vas-Klotho@LNP provided in Example 2, wherein a is the flow cytometry results and quantitative analysis of the uptake of Vas-Klotho@LNP and Klotho@LNP in NPPCs; b is the flow cytometry results and quantitative analysis of the transfection efficiency of Vas-Klotho@LNP and Klotho@LNP in NPPCs; c is a confocal microscopy image showing cells expressing green fluorescence in NPPCs after successful transfection of Klotho, Vas-Klotho@LNP and Klotho@LNP.

[0035] Figure 5 The confocal microscopy images in Example 2 show the lysosomal escape phenomenon that occurs in cells after co-incubation with Vas-Klotho@LNP.

[0036] Figure 6 The performance evaluation results of Vas-Klotho@LNP provided in Example 2, wherein a is the transcriptome sequencing volcano plot result of differentially expressed genes in NPPCs after co-incubation with Vas-Klotho@LNP; b is the KEGG pathway analysis result, highlighting 10 significantly regulated pathways.

[0037] Figure 7 This is a heat map of differentially expressed genes in NPPCs after co-incubation with Vas-Klotho@LNP in Example 2.

[0038] Figure 8 The immunofluorescence method in Example 2 was used to quantitatively analyze the expression of ACAN and MMP2 proteins after NPPCs were co-incubated with Klotho, Vas-Klotho@LNP and Klotho@LNP for 3 days.

[0039] Fig. 9 The confocal microscopy images in Example 2 show the expression of ACAN protein after NPPCs were co-incubated with Klotho, Vas-Klotho@LNP and Klotho@LNP for 3 days.

[0040] Fig.10 This is a validation diagram of the Klotho circRNA circularization interface. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] Studies have found that supplementing exogenous Klotho can reduce the secretion of age-related senescent secretory phenotype (SASP), restore degenerative nucleus pulposus tissue, and slow down IVDD. By supplementing Klotho, it is possible to restore aged NPPCs to a young level of bioenergetic state and enhance the functional regeneration of aged intervertebral discs. The present invention selects TEK receptor tyrosine kinase (Tie2) as an active target receptor because it is highly expressed in NPPCs and its expression in other cells is limited. The liposome particles (NT-LNPs) encapsulated with circRNA targeting NPPCs constructed by the present invention efficiently introduce Klotho circRNA into NPPCs through Tie2 agonist peptide, thereby promoting its rejuvenation and promoting ECM synthesis, and synergistically promoting intervertebral disc regeneration and repair in vivo and in vitro.

[0043] "PEG-lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include PEG-DMG and the like.

[0044] Phospholipids include dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), ... Acyl-phosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-trans-phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and their derivatives and isomers.

[0045] Example 1, a method for preparing a circRNA nano-delivery system capable of reversing aging of nucleus pulposus progenitor cells

[0046] Preparation of DSPE-PEG-Vas: An anhydrous ethanol solution of DSPE-PEG-MAL and an anhydrous ethanol solution of vasculotide were mixed at a molar ratio of 1:1 and stirred to prepare DSPE-PEG-Vas.

[0047] DSPE-PEG-Vas, PEG-DMG, DOPE and cholesterol (molar ratio = 2:40:3:10:45) were accurately weighed and dissolved in anhydrous ethanol to prepare a lipid stock solution with a concentration of 5 mg / mL. At the same time, T12 was accurately weighed and dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 10 mg / mL; according to the molar mass of each lipid component converted to the added volume, the above lipid stock solutions of the prescribed volume were taken and mixed thoroughly to obtain the lipid ethanol phase. α-Klotho-circRNA was diluted to 1 mg / mL using enzyme-free sodium hydrogen phosphate-citrate buffer (pH = 5.0) to obtain the α-Klotho-circRNA aqueous phase.

[0048] α-Klotho-circRNA was prepared by cyclization of Mus musculus klotho (Kl) mRNA. The CDS sequence of klotho mRNA is shown in the sequence listing SEQ ID NO.1, and its NCBI reference sequence number is NM_013823.2. The cyclization interface verification is shown in Fig.10 shown.

[0049] The lipid ethanol phase and the α-Klotho-circRNA aqueous phase were injected into the microfluidic system (INano) at a mass ratio of 10:1 and a flow rate of 1:3. TM L, Micro&Nano (Shanghai) Biologics Co. Ltd.), using a microfluidic chip to quickly mix the two. The mixture was then dialyzed in RNase-free PBS (pH 7.4) to remove ethanol and concentrated by ultrafiltration to obtain LNPs loaded with circRNA (Vas-Klotho@LNP). The size distribution and zeta potential of LNPs were measured by dynamic light scattering (DLS) using a Malvern instrument. All LNPs were stored at 4°C and used within three days after preparation.

[0050] In the present invention, a novel ionizable lipid molecule (T12) was designed ( Figure 1 a). Subsequently, the liposome component (Vas-Klotho@LNP) for delivering Klotho-circRNA was prepared using a microfluidic system ( Figure 1 b). The particle size and potential of LNP were measured by dynamic light scattering (DLS). Compared with Klotho@LNP without vasculotide (Vas) (LNP in the figure, ordinary lipid particles, a mixture of DSPE-PEG, T12, PEG-DMG, DOPE and cholesterol (molar ratio = 2:40:3:10:45)), the particle size of Vas-Klotho@LNP (Vas-LNP in the figure) after binding with vasculotide (Vas) increased slightly, while the potential remained close to zero ( Figure 2 a and 2b). Figure 2 As shown in c, transmission electron microscopy (TEM) clearly observed the spherical morphology of circRNA-loaded LNP (Vas-Klotho@LNP).

[0051] In order to evaluate the protective ability of lipid nanoparticles against RNase, circRNA was incorporated into each lipid nanoparticle. The operation method is as follows: 0.8% agarose gel preparation: accurately weigh 0.28mg agarose and dissolve it in 35mL ultrapure water, and place it in a microwave oven and heat it to boil three times. Then, add 3.5μL nucleic acid dye to the above gel solution and mix it, then pour it into the gel tray and insert the comb to wait for room temperature to solidify. A sample containing 400ng of mRNA was added to each well (the CDS sequence of the mRNA sequence is shown in the sequence table SEQ ID NO.1), and four groups were set up, namely naked Fluc mRNA solution (Free mRNA), mRNA-LNPs (Vas-LNP), mRNA-LNPs incubated with 10ng / mL RNase at room temperature for 15min (Vas-LNP+RNase), and mRNA-LNPs incubated with 1% triton at 37℃ for 15min (Vas-LNP+Triton X-100), to verify the protective effect of LNP on mRNA. Mix well with 5 μL RNA loading buffer (2×), add enzyme-free water to unify the volume to 10 μL, and heat at 65°C for 10 minutes. Use a micropipette to add the samples to the gel wells. Perform electrophoresis at 120V constant voltage for 30-40 minutes, and observe the position of mRNA bands with the help of a gel imager. The integrity of the RNA fragments was evaluated on the first day, and it was observed that the circRNA in the LNP was well preserved and resistant to degradation by ribonucleases (RNases) ( Figure 2 d).

[0052] Example 2, Performance Evaluation of Vas-Klotho@LNP

[0053] To evaluate the cellular uptake of circRNA nanoformulations (Vas-Klotho@LNP), Cy5-labeled circRNA was concentrated to form Cy5-circRNA LNPs. NPPCs were exposed to each formulation, and the uptake of circRNA-LNPs was examined qualitatively and quantitatively. Cells were initially treated with Klotho@LNP and Vas-Klotho@LNP. After 4 h of incubation, the cells were washed three times with ice-cold PBS and analyzed using confocal microscopy (Opera Phenix, PerkinElmer, USA) and Gallios flow cytometer (Beckman). For confocal microscopy, cell nuclei were stained with DAPI. Confocal microscopy analysis confirmed that Klotho circular RNA (circRNA) can be efficiently delivered to nucleus pulposus progenitor cells (NPPCs). Notably, Vas-modified LNPs showed significant efficiency in delivering Klotho circular RNA ( Figure 3Flow cytometry analysis showed that Vas-modified LNPs were more efficiently internalized into cells in NPPCs ( Figure 4 a). This result highlights the significant improvement in internalization efficiency after Vas modification of LNPs (Vas-Klotho@LNP).

[0054] To evaluate the effect of LNP-mediated gene delivery on Klotho expression in NPPCs, the cells were comprehensively analyzed. In in vitro gene transfection assays, NPPCs were treated with free circKlotho, Klotho@LNP, and Vas-Klotho@LNP, respectively. After 48 h of incubation, the proportion of EGFP-positive cells was quantified using a confocal microscope (Opera Phenix, PerkinElmer, USA) and a Gallios flow cytometer (Beckman). Flow cytometry data showed that 43.88±4.81% of NPPCs in the Vas-Klotho@LNP group showed obvious green fluorescence positive staining, while the proportion of positive cells in the Klotho@LNP group was relatively low, only 14.00±2.81% ( Figure 4 b). In addition, confocal fluorescence imaging further emphasized this difference, with the number of positively stained NPPCs in the NT-KLNP group being significantly higher than that in the Klotho@LNP group and the Klotho group ( Figure 4 c) These results collectively demonstrated that Vas-Klotho@LNP delivery successfully induced Klotho gene expression in NPPCs.

[0055] To evaluate the endosomal escape of circRNA nanoformulations, Cy5-labeled circRNA was concentrated to produce Cy5-circRNA LNPs. Lysosomes / endosomes were stained with Lysotracker Green for 1 hour and then washed three times. Subsequently, the nuclei were stained with DAPI for 1 minute. Imaging was performed using a high-content screening system. Subsequently, the temporal dynamic distribution of Cy5-labeled Klotho circular RNA in NPPCs was monitored by confocal real-time microscopy. Interestingly, after prolonged incubation time, Cy5-Klotho circular RNA was widely distributed in the cytoplasm, indicating that the LNP successfully escaped from the lysosome and entered the cytoplasm ( Figure 5 ). These observations clearly demonstrated that Vas-Klotho@LNP exhibited superior capabilities in NPPC internalization, lysosomal escape, and successful transfection of Klotho.

[0056] The present invention further performed RNA sequencing analysis on aged NPPCs co-cultured with Vas-Klotho@LNP. RNA sequencing was used to analyze the number of differentially expressed genes after successful Vas-Klotho@LNP transfection, the fold change of genes related to cell rejuvenation during aging, and the enrichment of up-regulated / down-regulated genes. Pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, a heat map was generated to visualize the results. The transcriptome sequencing results showed that compared with untreated NPPCs, aged NPPCs co-cultured with Vas-Klotho@LNP showed 816 genes upregulated and 839 genes downregulated, indicating that Klotho gene delivery caused significant differential gene expression ( Figure 6 a). Among them, genes related to extracellular matrix (ECM) synthesis (SOX9 and ACAN) were significantly upregulated, while genes related to ECM decomposition (MMP10 and MMP13) were significantly downregulated. This indicates that the delivery of Klotho gene via Vas-Klotho@LNP significantly enhanced the ability of NPPCs to differentiate into nucleus pulposus-like cells.

[0057] Figure 6 b shows five upregulated and five downregulated pathways. Pathways associated with rejuvenation (such as cAMP, apelin, insulin secretion, and Wnt signaling pathways) were significantly upregulated. In contrast, the IL17 signaling pathway and pathways associated with inflammation and aging (such as the regulation of TRP channels by inflammatory mediators, calcium signaling pathways, RIG-I-like receptor signaling pathways, and chemokine signaling pathways) were significantly downregulated. Heat map results show changes in the expression of key genes. The results showed that after the delivery of Klotho, genes associated with aging were downregulated, while genes associated with rejuvenation were upregulated. After the delivery of Klotho, silent information regulator 6 (SIRT6) was significantly upregulated, forming a protein complex with NRF2 and RNA polymerase II, promoting the binding of NRF2 to antioxidant response elements (AREs), and activating the transcription of antioxidant genes including heme oxygenase 1 (HO-1). This protein complex plays an important role in regulating mitochondrial energy conversion, thereby directly controlling the differentiation of stem cells. The increase of FGF23 is associated with aging, while the delivery of Klotho significantly downregulated the expression of FGF23, indicating its anti-aging effect. In addition, the downregulation of CDK4, MMP10, MMP12, MMP13, ADAMTS5, and ADAMTS7 indicated that the delivery of Klotho effectively inhibited the aging of NPPCs ( Figure 7 ).

[0058] After the successful delivery of Klotho via Vas-Klotho@LNP, it was observed that ACAN protein expression was significantly upregulated, while MMP2 protein expression was significantly downregulated. These results indicate that Klotho was successfully transcribed in cells, successfully achieving the rejuvenation of NPPCs, and further inducing their differentiation into nucleus pulposus cells, bringing hope for nucleus pulposus regeneration and repair ( Figure 8 , 9 ).

[0059] Example 3, a drug for treating intervertebral disc degeneration

[0060] The circRNA-loaded LNP (Vas-Klotho@LNP) provided in Example 1 is used as an active ingredient to prepare a drug for treating intervertebral disc degeneration. The drug can be in an oral dosage form or an injectable dosage form and can be administered to a desired subject (such as a human or non-human mammal) by oral administration, injection, or the like.

[0061] The medicament may further comprise excipients.

[0062] The oral dosage forms include tablets, capsules, films, granules and the like.

[0063] The injection contains Vas-Klotho@LNP, which is injected into the intervertebral disc of aging IVDD patients through a syringe to treat intervertebral disc degeneration.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A circRNA nano-delivery system capable of reversing aging of nucleus pulposus progenitor cells, characterized in that: Include: a) Klotho circRNA, and b) Liposomes, wherein the liposomes contain vasculotide, ionizable lipid molecules, phospholipids, PEG lipids, and steroids.

2. The circRNA nano-delivery system for reversing aging of nucleus pulposus progenitor cells according to claim 1, characterized in that: The ionizable lipid molecule is named T12, and its structural formula is:

3. The circRNA nano-delivery system for reversing aging of nucleus pulposus progenitor cells according to claim 1, characterized in that: The phospholipid is selected from dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, palmitoyloleoyl-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, distearoyl-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, 18-1-trans-phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine, and a combination of one or more of their derivatives and isomers.

4. The circRNA nano-delivery system for reversing aging of nucleus pulposus progenitor cells according to claim 1, characterized in that: The steroid is cholesterol.

5. The circRNA nano-delivery system for reversing aging of nucleus pulposus progenitor cells according to claim 1, characterized in that: The PEG lipid is PEG-DMG.

6. The circRNA nano-delivery system for reversing aging of nucleus pulposus progenitor cells according to claim 1, characterized in that: The circRNA nano delivery system comprises DSPE-PEG-Vas, T12, PEG-DMG, DOPE and cholesterol, with a molar ratio of 2:40:3:10:

45.

7. The method for preparing the circRNA nano-delivery system for reversing aging of nucleus pulposus progenitor cells according to claim 1, characterized in that: The steps include: Step 1, preparation of DSPE-PEG-Vas: mixing DSPE-PEG-MAL solution with vasculotide solution, stirring, to prepare DSPE-PEG-Vas; Step 2, accurately weigh DSPE-PEG-Vas, T12, PEG-DMG, DOPE and cholesterol and dissolve them in solvents to prepare lipid stock solutions, convert the molar mass of each component into volume, and take the prescribed volume of the lipid stock solutions and mix them thoroughly to obtain a lipid organic phase; use enzyme-free sodium hydrogen phosphate-citrate buffer pH = 5.0 to dissolve α-Klotho-circRNA to obtain an α-Klotho-circRNA aqueous phase; Step 3: The lipid organic phase and the α-Klotho-circRNA aqueous phase were injected into the microfluidic system for mixing. Then, the mixture was dialyzed in RNase-free PBS pH 7.4 to remove the solvent and concentrated by ultrafiltration to obtain circRNA-loaded LNP, namely Vas-Klotho@LNP.

8. The preparation method of the circRNA nano delivery system capable of reversing the aging of nucleus pulposus progenitor cells according to claim 7, characterized in that: In step 1, the molar ratio of DSPE-PEG-MAL to vasculotide is 1:

1.

9. The preparation method of the circRNA nano delivery system capable of reversing the aging of nucleus pulposus progenitor cells according to claim 8, characterized in that: In step 1, the DSPE-PEG-MAL solution, the vasculotide solution and the solvent in step 2 are organic solvents, preferably anhydrous ethanol.

10. Use of the circRNA nano-delivery system capable of reversing the aging of nucleus pulposus progenitor cells according to claim 1 in the preparation of a drug for treating intervertebral disc degeneration.

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