A nucleic acid drug delivery system, and a method of preparing and using the same

Abstract

The application discloses a nucleic acid drug delivery system and a preparation method and application thereof. The nucleic acid drug delivery system comprises a photocrosslinked hydrogel and a cationic lipid nanoparticle, the cationic lipid nanoparticle is loaded in the photocrosslinked hydrogel, the cationic lipid nanoparticle encapsulates siRNA for inhibiting REST gene expression, and a nucleotide sequence of the siRNA comprises sequences shown in SEQ ID No. 1 and SEQ ID No. 2. After the nucleic acid drug delivery system is implanted in local tissues of spinal cord injury, the cationic lipid nanoparticle encapsulating siRNA is released slowly, REST gene is continuously silenced to promote axon regeneration, the photocrosslinked hydrogel plays a ROS scavenging role, in addition, the photocrosslinked hydrogel can promote M2 polarization of macrophages to regulate an immune microenvironment, so that nerve repair is synergistically promoted at a molecular and cellular level, and the "immune regulation-axonal regeneration" double-track parallelism is realized, and therefore, the nucleic acid drug delivery system has a wide application prospect in the treatment of spinal cord injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nucleic acid drug delivery system, its preparation method, and its uses. Background Technology

[0002] Spinal cord injury (SCI) is a common and severe type of central nervous system injury, characterized by loss of neurological function and severe disability. Currently, there are no effective repair and treatment methods available clinically. The pathological process of SCI is generally divided into two stages: primary injury and secondary injury. Secondary injury includes inflammatory responses, apoptosis, blood-spinal cord barrier disruption, oxidative stress, and abnormal gene regulation. In secondary injury following spinal cord injury, excessive ROS production significantly promotes apoptosis by triggering harmful cascade reactions, including membrane lipid peroxidation, endoplasmic reticulum stress, and intracellular calcium imbalance. The large accumulation of reactive oxygen species (ROS) is considered a key factor in inducing cell death and exacerbating tissue damage. Therefore, scavenging ROS is crucial for improving the inflammatory microenvironment and thus promoting the treatment of spinal cord injury.

[0003] Neuroinflammation plays a crucial role in the pathogenesis of spinal cord injury, ultimately leading to neuronal apoptosis and scar formation, thus hindering the repair process after spinal cord injury. Macrophages play a key role in the inflammatory response following spinal cord injury. After spinal cord injury, M1 macrophages exacerbate the inflammatory cascade by recruiting additional inflammatory cells and generating reactive oxygen species (ROS). M2 macrophages have anti-inflammatory effects, promote axonal regeneration, and facilitate functional recovery. Studies have shown that macrophages can switch between M1 and M2 phenotypes under specific stimuli. Therefore, promoting the phenotypic shift of macrophages from M1 to M2 has become a key therapeutic strategy for modulating the immune microenvironment in the treatment of spinal cord injury.

[0004] Limited neuronal axonal regeneration capacity after spinal cord injury (SCI) is one of the core challenges to neurological function recovery. Recent studies have found that the transcriptional repressor REST (RE1-Silencing Transcription factor) is significantly upregulated after spinal cord injury (SCI). Its abnormal expression inhibits the transcription of neural repair-related genes, hindering axonal regeneration and thus limiting neurological function recovery. REST (repressor element-1-silencing transcription factor), also known as NRSF, is a neuron-restrictive silence factor that binds to neuron-restrictive silence elements (NRSEs) in DNA, inhibiting the transcription of neuron-specific genes containing NRSE elements in non-neurons. Recent research confirms that REST is an upstream repressor of pro-regeneration gene programs related to central nervous system axonal regeneration and is an important regulator of axonal regeneration in the central nervous system; inhibiting REST leads to increased neural regeneration. Therefore, regulating REST gene expression is a potential therapeutic approach to promote axonal regeneration after spinal cord injury.

[0005] Nucleic acid drugs have emerged as a potential treatment for various diseases. These drugs include antisense oligonucleotides (ASO), small interfering RNA (siRNA), messenger RNA (mRNA), aptamers, and microRNA (miRNA) mimics / inhibitors. Among them, small interfering RNA (siRNA), as a key effector molecule in RNA interference (RNAi) technology, induces specific degradation of target gene mRNA in vivo through double-stranded RNA (dsRNA), thereby causing gene silencing at different levels and demonstrating significant therapeutic potential. Compared to small molecule drugs, siRNA offers advantages such as abundant target sites, low resistance to drug resistance, long-lasting effects, and ease of synthesis and production. Compared to antibodies, which face challenges such as high cost, potential systemic toxicity, and the development of anti-antibody agents, siRNA offers longer-lasting effects and greater safety. Furthermore, siRNA drugs have a higher probability of successful drug development and are more time-saving and convenient in research and production, leading to increased favor from pharmaceutical companies in recent years. Small interfering RNA (siRNA) can specifically silence the REST gene, thereby relieving its inhibition of genes related to neural regeneration, promoting axonal regeneration and neuronal survival, and achieving effective treatment, showing promising application prospects. However, siRNA suffers from poor stability, easy degradation, low delivery efficiency, and potential non-specific effects when used in vivo, severely limiting its clinical translation. Therefore, achieving efficient, stable, and targeted delivery of siRNA is a key technical challenge that urgently needs to be addressed in this field.

[0006] Lipid nanoparticles (LNPs) have been widely used in nucleic acid drug and vaccine development due to their excellent biocompatibility and nucleic acid delivery capabilities. LNPs can effectively encapsulate and protect siRNA, preventing its degradation by nucleases in vivo, and achieve targeted delivery through surface modification. Therefore, loading REST siRNA into LNPs holds promise for significantly improving its delivery efficiency and therapeutic efficacy in spinal cord injury treatment.

[0007] Furthermore, hydrogels, as a three-dimensional polymer network material, possess excellent tissue compatibility and controllable release characteristics, enabling them to form a scaffold within the spinal cord injury cavity and provide a platform for sustained local drug release. Introducing ROS-scavenging components into the hydrogel can not only alleviate oxidative stress in the injury microenvironment but also provide favorable conditions for nerve repair.

[0008] In summary, current technologies for treating spinal cord injuries still suffer from limited efficacy, short-lasting drug effects, and a lack of comprehensive multi-faceted intervention. Therefore, there is an urgent need for a comprehensive treatment strategy that can simultaneously achieve ROS clearance and REST gene silencing to improve neurological function repair after spinal cord injury. Summary of the Invention

[0009] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a nucleic acid drug delivery system, its preparation method and uses.

[0010] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0011] A first aspect of the present invention protects a nucleic acid drug delivery system comprising a photocrosslinked hydrogel and cationic lipid nanoparticles, wherein the cationic lipid nanoparticles are loaded in the photocrosslinked hydrogel and the cationic lipid nanoparticles are encapsulated with siRNA that inhibits the expression of the REST gene, wherein the nucleotide sequence of the siRNA comprises the sequences shown in SEQ ID No. 1 and SEQ ID No. 2.

[0012] Another aspect of the present invention protects a method for preparing the nucleic acid drug delivery system described above, comprising the following steps:

[0013] 1) Obtain cationic lipid nanoparticles encapsulated with siRNA;

[0014] 2) The cationic lipid nanoparticles are dispersed in a photocrosslinked hydrogel to obtain the nucleic acid drug delivery system.

[0015] Another aspect of the present invention protects a pharmaceutical composition comprising the nucleic acid drug delivery system as described above.

[0016] Another aspect of the present invention protects the use of the nucleic acid drug delivery system as described above or the pharmaceutical composition as described above in the preparation of products for treating spinal cord injury.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1) The nucleic acid drug delivery system of the present invention comprises a photocrosslinked hydrogel and cationic lipid nanoparticles encapsulating siRNA (LNP-siRNA REST). The photocrosslinked hydrogel has good biocompatibility and provides a stable support structure for the entire nucleic acid drug delivery system. It not only protects the cationic lipid nanoparticles from interference and damage from the external environment, but also achieves sustained release of siRNA through its own properties. At the same time, the lipid bilayer structure of the cationic lipid nanoparticles can efficiently encapsulate siRNA, preventing it from being degraded by nucleases during in vivo transport, and ensuring that the siRNA can accurately and completely reach the target cells.

[0019] 2) The nucleic acid drug delivery system of the present invention is composed of cationic lipid nanoparticles encapsulating siRNA and photocrosslinked hydrogel, and has a three-dimensional porous network structure hydrogel morphology with large specific surface area and good swelling performance.

[0020] 3) After the nucleic acid drug delivery system of the present invention is implanted into the local tissue of spinal cord injury, the nucleic acid drug delivery system slowly releases LNP-siRNA REST, continuously silencing the REST gene. At the same time, the photocrosslinked hydrogel plays a role in ROS scavenging. In addition, it can also promote the polarization of macrophage M2, thereby synergistically promoting nerve repair at the molecular and cellular levels, thus promoting the repair of spinal cord injury. Attached Figure Description

[0021] Figure 1 The image shown is a WB test result of three groups of siRNA silencing REST proteins in Example 1 of the present invention.

[0022] Figure 2A The image shown is a transmission electron microscope image of the cationic lipid nanoparticle LNP-siRNA REST in Example 2 of the present invention.

[0023] Figure 2B The NTA data for LNP-siRNA REST of cationic lipid nanoparticles in Example 2 of the present invention are shown.

[0024] Figure 2C This illustrates the inhibitory effect of cationic lipid nanoparticles LNP-siRNA REST on REST in nerve cells in Example 2 of the present invention.

[0025] Figure 3A The image shown is a transmission electron microscope image of the NXCGA@LNP-siRNA REST nucleic acid drug delivery system in Example 3 of the present invention.

[0026] Figure 3B The graph shows the swelling rate results of the NXCGA@LNP-siRNA REST nucleic acid drug delivery system in Example 3 of the present invention.

[0027] Figure 3C The results shown are from a drug release performance study of the NXCGA@LNP-siRNA REST nucleic acid drug delivery system in Example 3 of this invention.

[0028] Figure 4A The image shown is a graph illustrating the results of H2O2 removal using the NXCGA@LNP-siRNA REST nucleic acid drug delivery system in Example 4 of this invention.

[0029] Figure 4B The image shown is a graph illustrating the results of ROS removal using the NXCGA@LNP-siRNA REST nucleic acid drug delivery system in Example 4 of this invention.

[0030] Figure 4CThe image shown is a graph illustrating the results of the NXCGA@LNP-siRNA REST nucleic acid drug delivery system in Example 4 of this invention in scavenging DPPH and PITO free radicals.

[0031] Figure 5A The diagram shows the results of in vitro inhibition of the REST gene in various groups in Example 5 of the present invention.

[0032] Figure 5B The diagram shows the results of dorsal root ganglion cell axon growth in each group in Example 5 of the present invention.

[0033] Figure 5C The image shows the expression results of GAP43 and Tuj1, proteins related to nerve regeneration, in each group in Example 5 of the present invention.

[0034] Figure 6A The image shows the BBB scores of rats after 6 weeks of administration in each group in Example 6 of this invention.

[0035] Figure 6B The images shown are rat spinal cord specimens from each group of rats after administration at 6 weeks, as well as HE staining and LFB staining images, in Example 6 of this invention.

[0036] Figure 6C The figure shown is a graph illustrating the effect of drug administration on the inhibition of 4-HNE in rats in Example 6 of the present invention.

[0037] Figure 6D The figure shown is a graph illustrating the effect of drug administration on the inhibition of 8-OHdG in rats in Example 6 of the present invention.

[0038] Figure 6E The figure shows the effect of drug administration on neuronal markers (NF200) and glial cell markers (GFAP) in each group of rats in Example 6 of the present invention.

[0039] Figure 6F The figure shown is a graph of the expression levels of marker proteins GAP43 and Tuj1 in rats after administration in each group in Example 6 of the present invention.

[0040] Figure 7A The image shown is a result of Western blot detection of macrophage polarization-related phenotypic markers in Example 7 of the present invention.

[0041] Figure 7B The image shown is a quantitative analysis diagram of the expression of the macrophage M1 marker iNOS detected by Western blot in Example 7 of the present invention.

[0042] Figure 7CThe image shown is a quantitative analysis diagram of the expression of Arg1, a macrophage M2 marker, detected by Western blot in Example 7 of the present invention.

[0043] Figure 8A The figure shown is a diagram of the CCK-8 cell proliferation assay results in Example 8 of the present invention.

[0044] Figure 8B The image shown is an HE staining image of the major organs of rats in each group 42 days after spinal cord injury surgery in Example 8 of the present invention. Detailed Implementation

[0045] In secondary spinal cord injury, excessive reactive oxygen species (ROS) significantly promote neuronal apoptosis by triggering harmful cascade reactions such as membrane lipid peroxidation, endoplasmic reticulum stress, and intracellular calcium imbalance. Limited neuronal axonal regeneration capacity is a core challenge to neuronal function recovery. To address this need, this invention provides a nucleic acid drug delivery system, its preparation method, and its applications.

[0046] A first aspect of the present invention protects a nucleic acid drug delivery system comprising a photocrosslinked hydrogel and cationic lipid nanoparticles, wherein the cationic lipid nanoparticles are loaded in the photocrosslinked hydrogel and the cationic lipid nanoparticles are encapsulated with siRNA that inhibits the expression of the REST gene, wherein the nucleotide sequence of the siRNA comprises the sequences shown in SEQ ID No. 1 and SEQ ID No. 2.

[0047] While siRNA possesses high specificity, it is susceptible to degradation by nucleases. This invention addresses this issue by encapsulating siRNA within cationic lipid nanoparticles (LNPs). The lipid bilayer structure protects the siRNA, effectively resisting enzymatic degradation, and leverages the inherent membrane fusion properties of LNPs for efficient delivery to target cells. Furthermore, the siRNA is loaded into a photocrosslinked hydrogel, and its release is sustained and controlled through the slow degradation of the hydrogel network. Simultaneously, the photocrosslinked hydrogel serves as a temporary tissue scaffold, filling cavities in spinal cord injuries and providing physical guidance and mechanical support for axonal migration and elongation. Moreover, the photocrosslinked hydrogel exhibits strong antioxidant properties, eliminating excessive reactive oxygen species (ROS) released from spinal cord injuries. This promotes nerve regeneration, enhances oxidative stress resistance, promotes axonal regeneration, and regulates the macrophage immune microenvironment, thereby comprehensively improving spinal cord injury.

[0048] GAGCGUGUCUACAAGUGUA(SEQ ID No.1)

[0049] UACACUUGUAGACACGCUC(SEQ ID No.2)

[0050] In some embodiments, the raw materials for the cationic lipid nanoparticles include phospholipids, cationic lipids, cholesterol, and long-cycle materials.

[0051] In some embodiments, the phospholipid is selected from dioleoylphosphatidylethanolamine (DOPE).

[0052] In some embodiments, the cationic lipid is selected from dioleoylphosphatidylcholine-dimethacrylate (Dlin-MC3-DMA).

[0053] In some embodiments, the cholesterol is selected from one or both of cholesterol monosuccinate and DC-cholesterol.

[0054] In some embodiments, the long-cycle material is selected from polyethylene glycol (PEG) and its derivatives, wherein the PEG derivative is selected from one or more of polyethylene glycol-vitamin E succinate (TPGS), polyethylene glycol-cholesterol (CHEMS-PEG), polyethylene glycol-modified distearylphosphatidylethanolamine (DSPE-PEG), polyethylene glycol-modified dimyristoylphosphatidylethanolamine (DMPE-PEG), polyethylene glycol-modified dipalmitoylphosphatidylethanolamine (DPPE-PEG), and polyethylene glycol-modified distearylmethylpropanediol (DMG-PEG).

[0055] In some specific embodiments, the phospholipids in the raw materials of the cationic lipid nanoparticles are selected from DOPE, the cholesterol is selected from DC-cholesterol, the cationic lipids are selected from Dlin-MC3-DMA, and the long-cycle material is selected from DMG-PEG 2000.

[0056] In some specific embodiments, the mass ratio of the phospholipids, cationic lipids, cholesterol, and long-cycle materials is (1-6):1:(1-10):1. In one specific embodiment, it is 2.3:1:4.7:1.2.

[0057] In some embodiments, the mass ratio of the cationic lipid nanoparticles to the photocrosslinked hydrogel is (20-30):1, or it can be (20-28):1, or it can be (25-30):1, or it can be 20:1, 22:1, 25:1, 28:1, or 30:1.

[0058] In some embodiments, based on the molar number of nitrogen atoms in the cationic lipid nanoparticles and the molar number of phosphorus atoms in the siRNA, the N / P molar ratio of the cationic lipid nanoparticles to the siRNA is (2-8):1, or it can be (2-5):1, or it can be (4-8):1, or it can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1.

[0059] In some specific embodiments, an aqueous phase of siRNA is obtained, and an ethanol phase of cationic lipid nanoparticles is obtained. The cationic lipid nanoparticles encapsulating siRNA are then obtained by an organic phase infusion method. The organic infusion method refers to adding the ethanol phase of the cationic lipid nanoparticles to the aqueous phase of the siRNA; the volume ratio of the ethanol phase of the cationic lipid nanoparticles to the aqueous phase of the siRNA is 1:3; the concentration of the siRNA is 0.1 mg / mL based on the total volume of the aqueous phase of the siRNA; and the concentration of the cationic lipid nanoparticles is 0.1 mg / mL based on the total volume of the ethanol phase of the cationic lipid nanoparticles.

[0060] In some embodiments, the raw materials of the photocrosslinked hydrogel include one or more of acryloylglycine, xanthan gum, carbomer, methacrylamide hyaluronic acid, methacrylamide gelatin, photoinitiator, and antioxidant. The carbomer is selected from one or more of carbomer 940, carbomer 941, and carbomer 934. Preferably, it is carbomer 940. Carbomer 940 is a polymer copolymerized by crosslinking acrylic monomers with crosslinking agents such as allyl sucrose or pentaerythritol. Its unique three-dimensional network structure gives it better thickening ability and suspension stability compared to linear polyacrylic acid polymers.

[0061] In some embodiments, the photoinitiator is selected from 2-hydroxy-2-methylphenylacetone, i.e., Irgacure1173, which is a liquid photoinitiator.

[0062] In some embodiments, the antioxidant is selected from one or more of gallic acid, quercetin, catechin, tea polyphenols, epigallocatechin, and tannic acid. In one specific embodiment, it is gallic acid.

[0063] In some specific embodiments, the raw materials of the photocrosslinked hydrogel include acryloylglycine, xanthan gum, carbomer, photoinitiator, and antioxidant.

[0064] In some more specific embodiments, the mass ratio of acryloylglycine, xanthan gum, and carbomer is (10-30):(1-6):1, or it can be (10-22):(1-6):1, or it can be (20-30):(1-6):1, or it can be 10:(1-6):1, 15:(1-6):1, 20:(1-6):1, 25:(1-6):1, or 30:(1-6):1. In one specific embodiment, it is 20:3:1.

[0065] In some more specific embodiments, the volume-to-mass ratio of the photoinitiator to acryloylglycine is 1 μL:(0.01-10) g, or it can be 1 μL:(0.01-5) g, or 1 μL:(4-8) g, or 1 μL:(6-10) g, or 1 μL:0.01 g, 1 μL:0.05 g, 1 μL:0.1 g, 1 μL:0.5 g, 1 μL:2 g, 1 μL:3 g, 1 μL:5 g, 1 μL:7 g, 1 μL:8 g, or 1 μL:10 g. In one specific embodiment, it is 10 μL:2 g.

[0066] In some more specific embodiments, the mass ratio of the antioxidant to acryloylglycine is (0.01-1):1, or it can be (0.01-1):1, (0.5-1):1, (0.3-1):1, or 0.01:1, 0.05:1, 0.1:1, or 0.5:1. In one specific embodiment, it is 0.2g:2g.

[0067] In some embodiments, the photocrosslinked hydrogel can be crosslinked under ultraviolet irradiation of 280 nm-320 nm.

[0068] In some specific embodiments, acryloylglycine, xanthan gum, and carbomer are mixed in water for 12 hours; then a photoinitiator is added and mixed for 2 hours to obtain a precursor solution; the precursor solution is crosslinked by ultraviolet light irradiation for 25 minutes; then the crosslinked product is mixed with gallic acid to obtain the photocrosslinked hydrogel.

[0069] Another aspect of the present invention protects a method for preparing the nucleic acid drug delivery system as described above, comprising the following steps:

[0070] 1) Obtain cationic lipid nanoparticles encapsulated with siRNA;

[0071] 2) The cationic lipid nanoparticles are dispersed in a photocrosslinked hydrogel to obtain the nucleic acid drug delivery system.

[0072] In some embodiments, the preparation method of the siRNA-encapsulated cationic lipid nanoparticles is as follows: lipid nanoparticle raw materials dissolved in ethanol and siRNA in an aqueous phase are mixed at a controlled flow rate in a microfluidic device; the liquid is collected at the outlet of the microfluidic channel and purified by dialysis to obtain the siRNA-encapsulated liposomes. The flow rate ratio of the ethanol phase to the aqueous phase is 1:1-5.

[0073] In some embodiments, the photocrosslinked hydrogel is prepared by mixing acryloylglycine (NAGA), xanthan gum (XG), carbomer 940 (CBP940) with water, then adding a photoinitiator to obtain a precursor solution; transferring the precursor solution to a 3D printer for printing and crosslinking under ultraviolet light irradiation; then adding an antioxidant gallic acid solution to obtain the photocrosslinked hydrogel (NXCGA). The applicant prepared NXC without adding an antioxidant to the photocrosslinked hydrogel, and loaded it with cationic lipid nanoparticles without any siRNA encapsulation (NXC@LNP) and cationic lipid nanoparticles encapsulated with siRNA (NXC@LNP-siRNA REST). It was found that these nanoparticles effectively scavenge H2O2, ROS, DPPH, and PTIO free radicals; while the antioxidant properties of NXCGA@LNP-siRNA REST, NXCGA, and NXCGA@LNP-siRNA scramble were improved after adding an antioxidant. After co-incubation with dorsal root ganglion neurons in vitro, the expression levels of GAP43 and Tuj1 and axons of NXCGA@LNP-siRNA REST were significantly better than those of NXC@LNP-siRNA REST.

[0074] In some embodiments, the photocrosslinked hydrogel and the cationic lipid nanoparticles are mixed and incubated to obtain the nucleic acid drug delivery system.

[0075] In some embodiments, the incubation is carried out at room temperature for 0.5-2 hours, or for 0.5 hours, 0.8 hours, 1 hour, 1.5 hours, or 2 hours.

[0076] Another aspect of the present invention protects a pharmaceutical composition comprising a nucleic acid drug delivery system as described above and pharmaceutically acceptable excipients.

[0077] In the pharmaceutical compositions of this invention, the pharmaceutically acceptable excipients are selected from one or more of carriers, diluents, binders, lubricants, and wetting agents. Specific examples of substances that can serve as pharmaceutically acceptable carriers, diluents, binders, lubricants, and wetting agents include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize nucleic acid drug delivery systems or to improve their activity or bioavailability.

[0078] The pharmaceutical compositions of this invention are one or more of the following: patches, solutions, injections, sprays, nasal drops, aerosols, powders, tablets, capsules, and granules. All of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0079] The pharmaceutical composition of the present invention can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. The pharmaceutical composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0080] Another aspect of the present invention protects the use of the nucleic acid drug delivery system as described above or the pharmaceutical composition as described above in the preparation of products for treating spinal cord injury.

[0081] In some embodiments, the product has at least one of the following functions: 1) promoting nerve regeneration; 2) resisting oxidative stress; 3) promoting axon regeneration; 4) regulating the macrophage immune microenvironment.

[0082] In some embodiments, promoting neurogenesis refers to upregulating the expression level of the neuronal marker NF200 while reducing the expression level of the astrocyte activation marker GFAP.

[0083] In some embodiments, the antioxidant stress refers to the scavenging of reactive oxygen species (ROS) and free radicals, including H2O2 and ROS, and DPPH and PTIO free radicals. In vitro cell experiments have shown that the nucleic acid drug delivery system effectively scavenges H2O2, ROS, DPPH free radicals, and PTIO free radicals. Furthermore, in vivo experiments have revealed that it can reduce the levels of 4-HNE, a lipid peroxidation metabolite, and 8-OHdG, a DNA oxidative damage marker, in spinal cord tissue.

[0084] In some embodiments, promoting axonal regeneration refers to promoting axonal length extension and upregulating the expression of GAP43 and Tuj1. In vitro cell experiments have shown that the nucleic acid drug delivery system can specifically act on nerve cells, inhibiting the expression of REST; co-culturing with dorsal root ganglion cells promotes significant axonal elongation and significantly upregulates the expression of axonal growth cone marker proteins GAP43 and Tuj1. Further in vivo experiments have shown that the nucleic acid drug delivery system can upregulate the expression of marker proteins GAP43 and Tuj1 in the spinal cord.

[0085] In some embodiments, the regulation of the macrophage immune microenvironment refers to inhibiting macrophage polarization towards M2 and inducing it towards M1. Through in vitro cell experiments, this invention has found that the nucleic acid drug delivery system can effectively reduce LPS-induced iNOS upregulation and enhance Arg-1 expression, indicating that it can inhibit macrophage polarization towards M2 and induce it towards M1.

[0086] The present invention further provides a method for treating or preventing spinal cord injury, comprising: administering an effective amount of the nucleic acid drug delivery system or the pharmaceutical composition described above to a subject in need. The method may also be in vitro or non-therapeutic.

[0087] The target organism is a mammal, including but not limited to humans, primates, livestock, pets, laboratory test animals, or captured wild animals. Primates are preferred. Humans are the most preferred target organism. The target organism can be a patient with spinal cord injury or an individual seeking to prevent spinal cord injury. The nucleic acid drug delivery system or the drug composition can be administered to the target organism before, during, or after spinal cord injury treatment.

[0088] In summary, the nucleic acid drug delivery system of the present invention has good mechanical properties and biocompatibility. The prepared cylindrical hydrogel can macroscopically simulate natural spinal cord tissue, which can promote nerve regeneration. It can also effectively reduce reactive oxygen species bursting in the center of spinal cord injury, thereby reducing nerve cell death. In addition, no pathological changes occurred in multiple organs after administration, demonstrating extremely high safety. It has no adverse effects on cell proliferation, thus exhibiting extremely high safety.

[0089] The purpose of this invention is to provide a nucleic acid drug delivery system that combines cationic lipid nanoparticles (LNPs) loaded with REST gene small interfering RNA (siRNA) with a hydrogel possessing reactive oxygen species (ROS) scavenging function, for the treatment of spinal cord injury. This nucleic acid drug delivery system can deliver cationic lipid nanoparticles encapsulated with siRNA, achieving sustained-release therapy for spinal cord injury. Through this system, effective ROS scavenging is achieved, alleviating oxidative stress in secondary spinal cord injury, improving the pathological microenvironment after spinal cord injury, and simultaneously silencing the REST gene through RNA interference, relieving the inhibition of genes related to nerve repair, thereby promoting the recovery of nerve function and overcoming the shortcomings of existing treatments, such as insufficient efficacy, short duration of action, and lack of comprehensive intervention.

[0090] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0091] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0092] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention may be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, provided that one of those skilled in the art possesses the prior art and that this invention is described.

[0093] In the following embodiments of this application, N-acryloylglycine was purchased from Zhengzhou Alpha Chemical Co., Ltd., with a molecular weight of 128.13 and CAS number 2479-62-1; xanthan gum was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a molecular weight of 241.11496; and carbomer 940 was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a molecular weight of 280.747.

[0094] Example 1: Design of siRNA

[0095] Targeting the REST gene (ID: 5978), the REST gene sequence was selected from GenBank, and three sets of specific small interfering RNAs (siRNAs) were designed according to the design principles of RNAi design. Simultaneously, a non-specific siRNA (Scramble) was designed. The corresponding siRNA powders were produced by Shanghai Sangon Biotech Co., Ltd. The sequences of the three siRNAs are as follows:

[0096] siRNA REST3 group:

[0097] GAGCGUGUCUACAAGUGUA(SEQ ID No.1)

[0098] UACACUUGUAGACACGCUC(SEQ ID No.2)

[0099] siRNA REST2 group:

[0100] CGACCUCUCGAAAGCUGAA (SEQ ID No.3)

[0101] UUCAGCUUUCGAGAGGUCG(SEQ ID No.4)

[0102] siRNA REST1 group:

[0103] GAAGUGUACAGCUCGAAUA (SEQ ID No.5)

[0104] UAUUCGAGCUGUACACUUC (SEQ ID No.6)

[0105] Scramble group:

[0106] UUCUCCGAACGUGUCACGUTT(SEQ ID No.7)

[0107] ACGUGACACGUUCGGAGAATT (SEQ ID No.8)

[0108] In addition, a control group (CON group) was set up, which did not receive any treatment.

[0109] PC12 nerve cells were prepared at a concentration of 1×10⁻⁶. 6 Inoculate one well per cell into a 6-well plate, and begin transfection the following day.

[0110] (1) Dilution of siRNA: Carefully check the label on the EP tube containing the siRNA powder to confirm the nmol amount of RNA, and dilute it to a 20 μM stock solution. Then take four sterile EP tubes and add 125 μL of Opti-MEM™ I serum-reduced medium (Gibco, catalog number 31985070) to each tube. Then add 7.5 μL of the three designed siRNA groups and Scramble stock solution respectively, and gently pipette to mix.

[0111] (2) Diluting Liposome 3000: Take four more sterile EP tubes and add 125 μL of Opti-MEM™ I serum-reduced medium to each tube. Then add 5 μL of Lipofectamine™ 3000 reagent (Thermo Fisher Scientific, catalog number L3000015) to each tube and gently pipette to mix, thus obtaining the diluted Liposome 3000 solution.

[0112] (3) Add all of the diluted Liposome3000 solution from step (2) to the diluted siRNA solution from step (1) and gently mix by pipetting or by vortexing briefly for 1 minute to obtain a mixture.

[0113] (4) Let the mixture from step (3) stand at room temperature for 10-15 minutes to allow the liposome-siRNA complex to form.

[0114] (5) Remove the P12 cells from the 6-well plate, aspirate the old culture medium from the wells, and replace it with 1250 μL of fresh serum-free DMEM medium (Gibco). Add 250 μL of the liposome-siRNA complex from step (4) dropwise and evenly to the corresponding cell wells. Gently shake the cell plate back and forth and side to side to distribute the cells evenly. Place the cell plate back in a 37°C, 5% CO2 incubator for further culture. After 6-8 hours of transfection, replace the medium with complete culture medium (Gibco). Continue culturing. Complete culture medium refers to DMEM medium containing 10% FBS.

[0115] (6) Cell proteins were extracted 48-72 h after transfection and the expression of the target protein was detected by Western Blot.

[0116] The results of Western Blot (WB) analysis are shown below. Figure 1 β-tubulin was used as an internal reference gene to calculate the expression level of REST protein.

[0117] from Figure 1As shown in A and 1B, compared with the control group (CON group) and the negative control group (Lipo3000+siRNA Scramble), the expression level of REST protein in the siRNA treatment groups (siRNA REST1, siRNA REST2, siRNA REST3) was significantly reduced. Among them, compared with the control group (CON group), siRNA REST3 could significantly silence the expression level of REST (p<0.01). Subsequent experiments were conducted using siRNA REST3.

[0118] Example 2: Preparation and characterization of cationic lipid nanoparticles encapsulating siRNA

[0119] In Example 2, the siRNA REST3 designed in Example 1 was encapsulated in cationic lipid nanoparticles to prepare cationic lipid nanoparticles encapsulated with siRNA, which were then characterized. The characteristics included the following:

[0120] 2.1 Preparation of cationic lipid nanoparticles encapsulating siRNA

[0121] 2.1.1 Obtaining the lipid organic phase

[0122] Four raw materials were weighed: dioleoylphosphatidylcholine-dimethacrylate (Dlin-MC3-DMA), dioleoylphosphatidylethanolamine (DOPE), cholesterol (DC-cholesterol), and DMG-PEG2000 (distearyl methyl propylene glycol-polyethylene glycol 2000). They were dissolved in anhydrous ethanol to obtain 10 mg / mL Dlin-MC3-DMA stock solution, 5 mg / mL DOPE stock solution, 10 mg / mL cholesterol stock solution, and 10 mg / mL DMG-PEG2000 stock solution. Then, 12 μL of Dlin-MC3-DMA stock solution, 56 μL of DOPE stock solution, 56 μL of cholesterol stock solution, and 14 μL of DMG-PEG2000 stock solution were respectively taken and made up to 333 μL with ethanol to obtain the lipid organic phase.

[0123] 2.1.2 Obtaining the siRNA REST aqueous phase

[0124] Based on the ratio of the molar number of nitrogen atoms (N) in the cationic lipid to the molar number of phosphorus atoms (P) in the siRNA, with an N / P ratio of 6:1, the amount of REST siRNA in the aqueous phase in step 2.1.2 is determined. Mass = molar amount × average molecular weight of nucleotides (330 Da)

[0125] The siRNA REST (also known as siRNA REST3) from Example 1 was added to DEPC water to prepare the siRNA REST stock solution. Then, the siRNA REST stock solution was diluted to 0.1 mg / mL with sodium citrate buffer (50 nM, pH=4.5) to obtain the siRNA REST aqueous phase.

[0126] 2.1.3 LNP-siRNA REST Preparation

[0127] The lipid organic phase obtained in step 2.1.1 was added dropwise to the siRNA REST aqueous phase obtained in step 2.1.2 (V organic phase:V aqueous phase = 1:3) using the organic phase injection method. The mixture was then vortexed, allowed to stand, and assembled to form cationic lipid nanoparticles encapsulated with siRNA (labeled LNP-siRNA REST). The specific steps are as follows:

[0128] The organic phase was injected into the aqueous phase using a microfluidic chip (Hangzhou Tingke Biotechnology Co., Ltd., NSR-E20). Specifically, 333 μL of the lipid organic phase obtained in step 2.1.1 was drawn into a syringe, and 1 mL of the siRNA REST aqueous phase obtained in step 2.1.2 was drawn into another syringe. The flow rate ratio of the organic phase to the aqueous phase was set to 1:3, and the total flow rate was set to 12 mL / min. The organic phase and the aqueous phase were injected into the microfluidic chip at the same time. Inside the chip, the organic phase and the aqueous phase met and mixed rapidly in a specific mixing region to form cationic lipid nanoparticles. The cationic lipid nanoparticles encapsulating siRNA (abbreviated as LNP-siRNA REST) ​​were collected from the chip outlet.

[0129] Meanwhile, nonspecific siRNA (Scramble) was prepared using the same method as siRNA REST3 to obtain cationic lipid nanoparticles (LNP-siRNA Scramble) encapsulating nonspecific siRNA.

[0130] 2.2 Characterization

[0131] 2.2.1 Transmission electron microscopy characterization

[0132] The LNP-siRNA REST obtained in step 2.1.3 was imaged using transmission electron microscopy. The results are shown in [Figure 1]. Figure 2A .

[0133] from Figure 2A It can be seen that LNP-siRNA REST particles are approximately spherical or near-spherical, with a relatively uniform distribution and no obvious aggregation. The typical core-shell structure can be clearly observed, and the particle surface is relatively smooth with clear boundaries.

[0134] 2.2.2 Particle Size Distribution

[0135] The particle size distribution of the LNP-siRNA REST obtained in step 2.1.3 was detected using NTA, and the results are shown in [Figure 1]. Figure 2B .

[0136] from Figure 2B It is known that the average particle size of LNP-siRNA REST is around 200 nm.

[0137] 2.3 Western blot analysis of the effect of LNP-siRNA REST on REST expression in nerve cells

[0138] The effect of LNP-siRNA REST prepared in step 2.1.3 on REST expression in nerve cells was detected by Western blotting.

[0139] PC12 neurons were seeded in 6-well plates at a seeding rate of 220,000 cells per well and divided into 3 groups:

[0140] CON group: PC12 neurons were supplemented with only complete culture medium. Complete culture medium refers to DMEM containing 10% serum.

[0141] LNP-siRNA Scramble group: Add complete culture medium containing 100 nM of the LNP-siRNA Scramble prepared in step 2.1.3.

[0142] LNP-siRNA REST group: Add complete culture medium containing 100 nM of LNP-siRNA REST prepared in step 2.1.3.

[0143] The cells were incubated at 37°C for 48 hours before Western blot analysis.

[0144] Test results are shown Figure 2C .

[0145] from Figure 2C It is known that LNP-siRNA REST can be well transfected into P12 nerve cells and then taken up, significantly downregulating REST expression.

[0146] Example 3: Preparation and Release Performance Study of Nucleic Acid Drug Delivery System

[0147] In Example 3, the cationic lipid nanoparticles (LNP-siRNA REST) ​​obtained in Example 2 were prepared into a hydrogel-like nucleic acid drug delivery system together with other raw materials, and their release performance was investigated. This included the following:

[0148] 3.1 Preparation of nucleic acid drug delivery system

[0149] 3.1.1 Construction of the hydrogel (NXCGA)

[0150] 1) Mix water, acryloylglycine (NAGA), xanthan gum (XG), and carbomer 940 (CBP940) in the following ratio of 10 mL: 2 g: 0.3 g: 0.1 g. Stir continuously at room temperature using a magnetic stirrer for 12 hours until a homogeneous viscous solution is formed.

[0151] 2) Then, in step 1), add photoinitiator Irgacure 1173 (10 μL) and continue stirring for 2 hours under light-protected conditions to ensure thorough mixing and obtain the precursor solution.

[0152] 3) The precursor solution obtained in step 2) is transferred to the pneumatic extrusion barrel of the 3D printer for printing, and crosslinked by irradiation with 300nm ultraviolet light for 25 minutes to obtain NXC.

[0153] 4) Prepare a 10 w / v% concentration of gallic acid (GA) aqueous solution, and soak 2-3 mL of GA aqueous solution and the cross-linked product NXC from step 3) at room temperature for 12 h to form NXCGA hydrogel.

[0154] The resulting NXCGA hydrogel was stored at 4°C for testing.

[0155] 3.1.2 Preparation of the NXCGA@LNP-siRNA REST nucleic acid drug delivery system

[0156] The 20 mg NXCGA hydrogel prepared in step 3.1.1 and the 1 mg LNP-siRNAREST prepared in Example 2 were incubated at room temperature for 1 h to prepare a nucleic acid drug delivery system in the form of a hydrogel (abbreviated as NXCGA@LNP-siRNAREST), which is a cylinder with a diameter of 1 mm and a height of 2 mm, and the amount of siRNA loaded is 50 nmol.

[0157] Meanwhile, nonspecific siRNA (Scramble) was used to prepare a hydrogel loaded with cationic lipid nanoparticles (NXCGA@LNP-siRNA Scramble) using the same method as siRNA REST3.

[0158] In addition, the NXC obtained in step 3.1.1 was not mixed with gallic acid, and the rest was prepared in the same way as siRNA REST3 to obtain lipid nanoparticles (NXC@LNP) encapsulating siRNA REST3, and further prepared into a hydrogel loaded with cationic lipid nanoparticles (NXC@LNP-siRNA REST).

[0159] In addition, the NXC obtained in step 3.1.1 was not mixed with gallic acid, and instead loaded with cationic lipid nanoparticles without siRNA to obtain NXC@LNP.

[0160] NXCGA@LNP-siRNA REST, NXCGA@LNP-siRNA Scramble, NXC@LNP-siRNA REST, NXCGA hydrogel, and NXC@LNP are all cylindrical hydrogels with a diameter of 1 mm and a height of 2 mm; among them, the amount of siRNA loaded in NXCGA@LNP-siRNA REST, NXCGA@LNP-siRNA Scramble, and NXC@LNP-siRNA REST is 50 nmol.

[0161] 3.2 Characterization of Nucleic Acid Drug Delivery Systems

[0162] 3.2.1 Microscopic morphology characterization

[0163] The sample from step 3.1.2 was continuously freeze-dried at -80℃ for 36 hours to remove moisture, resulting in NXCGA@LNP-siRNAREST freeze-dried powder. The cross-section of the NXCGA@LNP-siRNAREST freeze-dried powder was sputter-coated with gold and attached to the sample stage, and then imaged and observed on a field emission scanning electron microscope.

[0164] 3.2.2 Characterization of swelling properties

[0165] The swelling degree of NXCGA@LNP-siRNA REST at room temperature was measured, and the swelling ratio (%) was calculated. The specific steps are as follows:

[0166] Seven groups were set up. In each group, 2g of the hydrogel-like NXCGA@LNP-siRNA REST prepared in step 3.1.2 was immersed in 1mL of deionized water. The seven groups were immersed for 2, 4, 6, 8, 10, 12, 24, 48, 72, 86, and 120 hours, respectively, and then the samples were removed. The swelling weight (W) of the NXCGA@LNP-siRNA REST was recorded. S The expanded NXCGA@LNP-siRNA REST was then freeze-dried at -80℃ for 36 h, and the dry weight (W) of NXCGA@LNP-siRNA REST was recorded. d The expansion rate can be defined as:

[0167]

[0168] from Figures 3A-3BIt can be seen that NXCGA@LNP-siRNA REST exhibits a typical three-dimensional porous network structure, and the swelling rate also increases with time, indicating that the NXCGA@LNP-siRNA REST obtained in this invention has the advantage of good swelling performance.

[0169] 3.2 Study on the release performance of nucleic acid drug delivery systems

[0170] First, siRNA REST was labeled with Cy5.5 (the Cy5.5 labeling of siRNA REST was performed by Shanghai Sangon Biotech Co., Ltd.). Then, NXCGA@LNP-siRNA REST was prepared according to step 3.1.2 above. This REST was placed in an EP tube, and 100 µL of PBS buffer was added. 50 µL of supernatant was collected daily, and 50 µL of fresh PBS buffer was added daily. The experiment lasted for 10 days. The concentration of siRNA in the collected supernatant was determined using a fluorescence spectrophotometer (Eppendorf, Hamburg, Germany). All experimental consumables were RNase-free, and the PBS buffer used was autoclaved to ensure RNase-free contamination. Daily release curves over time were calculated and plotted. Results are shown below. Figure 3C The daily release amount is referenced in reference 1 (Sun J et al. M2 Macrophage Membrane-Mediated Biomimetic-Nanoparticle Carrying COX-siRNA Targeted Delivery for Prevention of Tendon Adhesions by Inhibiting Inflammation. Small. 2023;19(33):e2300326. doi:10.1002 / smll.202300326).

[0171] from Figure 3C It can be seen that the release rate gradually increases over time and can be sustained for about 7 days.

[0172] Example 4 In vitro antioxidant test

[0173] In Example 4, the in vitro antioxidant properties of the nucleic acid drug delivery system were investigated. These included the following:

[0174] 4.1 H2O2 Removal Experiment

[0175] After each group of samples was prepared, it was immersed in an aqueous solution containing 100 μM H2O2 and incubated for 1 hour. The concentration of H2O2 in the (Beyotime) aqueous solution was then detected using a hydrogen peroxide assay kit.

[0176] Divided into 5 groups, the specific grouping is as follows:

[0177] NXC@LNP group: The NXC@LNP prepared in step 3.1.2 was incubated in an aqueous solution of H2O2 for 1 h.

[0178] NXC@LNP-siRNA REST group: The NXC@LNP-siRNA REST prepared in step 3.1.2 was incubated in an aqueous solution of H2O2 for 1 h.

[0179] NXCGA group: The hydrogel (NXCGA) prepared in step 3.1.1 was incubated in an aqueous solution of H2O2 for 1 h.

[0180] NXCGA@LNP-siRNA Scramble group: The NXCGA@LNP-siRNAScramble prepared in step 3.1.2 was incubated in an aqueous solution of H2O2 for 1 h.

[0181] NXCGA@LNP-siRNA REST group: The NXCGA@LNP-siRNA RES prepared in step 3.1.2 was incubated in an aqueous solution of H2O2 for 1 h.

[0182] See results Figure 4A .

[0183] from Figure 4A It can be seen that the concentration of H2O2 in the NXCGA group, NXCGA@LNP-siRNA Scramble group, and NXCGA@LNP-siRNA REST group was significantly reduced, indicating that NXCGA has a good antioxidant capacity.

[0184] 4.2 ROS Removal Experiment

[0185] PC12 cells were seeded into 6-well plates at a seeding density of 200,000 cells / well. Each group (same as step 4.1) was co-cultured with PC12 cells for 24 h. Then, the culture medium was replaced with medium containing 100 μM H2O2 and cultured for another 24 h. The ROS assay kit purchased from Beyotime was then used for detection, and each group was observed by fluorescence microscopy.

[0186] See results Figure 4B .

[0187] from Figure 4B It can be seen that, compared with the NXC group and the NXC@LNP-siRNA REST group, the ROS level in PC12 cells cultured in the NXCGA group, NXCGA@LNP-siRNA REST group, and NXCGA@LNP-siRNA scramble group was significantly reduced.

[0188] 4.3 Free radical scavenging experiment

[0189] To evaluate the antioxidant activity of each group, DPPH and PTIO free radical scavenging experiments were performed, as described in reference 2 (Cite This: ACS Appl. Polym. Mater. 2025, 7, 457−466).

[0190] See results Figure 4C .

[0191] from Figure 4C It can be seen that, compared with the NXC group and the NXC@LNP-siRNA REST group, the NXCGA group, the NXCGA@LNP-siRNA REST group, and the NXCGA@LNP-siRNA scramble group have DPPH and PTIO scavenging rates of over 75%, indicating excellent antioxidant capacity.

[0192] Example 5: Study on the ability of in vitro axonal regeneration promotion

[0193] In Example 5, the nucleic acid drug delivery system was tested in vitro with dorsal root ganglion (DRG) neurons to investigate the effect of the nucleic acid drug delivery system on axonal regeneration capacity. The DRG neurons were isolated and cultured according to Reference 3 (Reference 3: Lei M et al. Cell-cell and cell-matrix adhesion regulated by Piezo1 is critical for stiffness-dependent DRG neuron aggregation. Cell Rep.2023;42(12):113522. doi:10.1016 / j.celrep.2023.113522).

[0194] In this embodiment, the siRNA loading in each drug group was 0.5 nmol, and the preparation methods for the others were the same. The difference was that when preparing the cationic lipid nanoparticles encapsulating siRNA, the siRNA REST stock solution was diluted to 0.1 mg / mL with sodium citrate buffer (50 nM, pH=4.5) to obtain 100 μL of siRNA REST aqueous phase and 33 μL of lipid organic phase. In other embodiments, the siRNA loading was 50 nmol.

[0195] In this embodiment, the cells were divided into 5 groups, with each group of cells seeded at a rate of 10,000-20,000 cells per well in a 12-well plate:

[0196] PBS group: Obtained by incubating PBS and dorsal root ganglion neurons at room temperature for 48 hours.

[0197] NXCGA group: The hydrogel (NXCGA) prepared in step 3.1.1 and dorsal root ganglion neurons were incubated at room temperature for 48 h.

[0198] NXCGA@LNP-siRNA Scramble group: NXCGA@LNP-siRNA Scramble with siRNA loading of 0.5 nmol was prepared according to step 3.1.2, and then incubated with dorsal root ganglion nerve cells at room temperature for 48 h.

[0199] NXCGA@LNP-siRNA REST group: The NXCGA@LNP-siRNA REST nucleic acid drug delivery system with an siRNA loading of 0.5 nmol was prepared according to step 3.1.2, and then incubated with dorsal root ganglion nerve cells at room temperature for 48 h.

[0200] NXC@LNP group: NXC@LNPs and dorsal root ganglion neurons prepared in step 3.1.2 were obtained by incubating at room temperature for 48 hours.

[0201] NXC@LNP-siRNA REST group: NXC@LNP-siRNA REST with an siRNA loading of 0.5 nmol was prepared according to step 3.1.2, and then incubated with dorsal root ganglion nerve cells at room temperature for 48 h.

[0202] 5.1. In vitro inhibition of the REST gene

[0203] After culturing for 48 hours, cell proteins were extracted. Cell protein extraction and sample preparation specifically included the following:

[0204] ① Cell protein extraction: Add cell lysis buffer and vortex on an instrument for thorough lysis. Repeat the procedure of vortexing for 2 minutes and then incubating on ice for 10 minutes, 4 times.

[0205] ② Centrifuge at 13000 g for 15 min at 4℃, then transfer the supernatant to a new 1.5 mL EP tube, discard the cell pellet, and obtain the sample.

[0206] ③ Take 10 μL for protein concentration determination, add 1 / 3 volume of loading buffer to the remaining sample, vortex to mix, boil in a water bath for 10 min, and then cool on ice. Store the prepared sample frozen at -20℃.

[0207] The expression of REST in the sample was detected by Western blot. The specific experimental steps are as follows.

[0208] from Figure 5A It is known that the NXCGA@LNP-siRNA REST group and the NXC@LNP-siRNA REST group, nucleic acid drug delivery systems can release LNP-siRNA REST, which acts on nerve cells and inhibits the expression of REST in nerve cells.

[0209] 5.2 Effects on the axons of dorsal root ganglion neurons

[0210] After co-culturing each group with dorsal root ganglion (DRG) neurons for 48 h, fixation was performed with 4% formaldehyde, followed by washing with PBS. Blocking with 5% bovine serum albumin (BSA) for at least 30 min was then performed, followed by removal of excess liquid and overnight incubation at 4°C with primary antibody (Tuj1 antibody, 1:200 dilution). The primary antibody was recovered, and the cells were washed with PBS. Subsequent steps were performed in the dark, with incubation at room temperature for 45 min with the corresponding fluorescent antibody (1:200 dilution). Gentle washing with PBS was performed. Nuclear staining was performed at room temperature for 5 min, followed by DAPI (1:200 dilution), and washing with PBS. Slides were mounted and photographed using a laser confocal microscope.

[0211] See results Figure 5B .

[0212] from Figure 5B It can be seen that knocking down REST expression in both the NXCGA@LNP-siRNA REST group and the NXC@LNP-siRNA REST group significantly increased the axonal growth of DRG neurons.

[0213] 5.3 Effects on the expression levels of GAP43 and Tuj1 in dorsal root ganglion cells

[0214] Each group was co-cultured with DRG neurons for 48 h, then relevant proteins were extracted, and the levels of GAP43 and Tuj1 were detected by Western blotting.

[0215] See results Figure 5C .

[0216] from Figure 5C It can be seen that, after inhibiting REST expression, the expression of axon growth-related proteins GAP43 and Tuj1 was significantly upregulated in both the NXCGA@LNP-siRNA REST group and the NXC@LNP-siRNA REST group.

[0217] Western blot experimental procedures

[0218] I. Cell Protein Extraction and Sample Preparation

[0219] ① Cell protein extraction: Add cell lysis buffer and vortex to fully lyse the cells. Repeat the process of vortexing for 2 minutes followed by incubation on ice for 10 minutes, four times. ② Centrifuge at 13000 g for 15 minutes at 4℃. Transfer the supernatant to a new 1.5 mL EP tube, discard the cell pellet, and obtain the sample. ③ Use 10 μL for protein concentration determination. Add 1 / 3 volume of loading buffer to the remaining sample, vortex to mix, boil in a water bath for 10 minutes, and then cool on ice. Store the prepared sample at -20℃.

[0220] II. Specific Steps

[0221] (1) Prepare SDS-PAGE separating gels of appropriate concentrations according to the molecular weight of the proteins. Prepare commonly used reagents in advance according to the corresponding formulas: electrophoresis buffer, transfer buffer, TBST, 5% skim milk, etc.

[0222] (2) Take out the sample to be tested and add it. After adding the sample, set the electrophoresis parameters: 80 V for 30 min, 100 V for 1 h. Stop electrophoresis after all the target bands have separated.

[0223] (3) Cut a polyvinylidene fluoride (PVDF) membrane of appropriate size, activate the membrane with methanol, place the SDS-PAGE gel on the membrane, and finally place it in the transfer clamp. Transfer the membrane for 2 hours in constant current mode of 350mA.

[0224] (4) After the transfer is completed, the PVDF membrane is placed in 5% skim milk and sealed on a shaker at room temperature for 2 hours.

[0225] (5) After blocking, wash three times with TBST for 5 min each time. Then, cut the PVDF membrane into strips according to the molecular weight of the target protein. Dilute the primary antibody with the primary antibody dilution buffer to prepare the primary antibody working solution. Incubate the strips with the corresponding primary antibody working solution at 4°C overnight. The next day, remove the strips, recover the primary antibody, and wash four times with 1×TBST for 5 min each time.

[0226] (6) Incubation of secondary antibody: Dilute HRP-labeled secondary antibody with TBST at a ratio of 1:3000 and incubate at room temperature on a shaker for 1 hour.

[0227] (7) Wash 4 times with 1×TBST, 5 min each time.

[0228] (8) Development and quantification: Prepare the ECL chemiluminescence working solution and use it immediately. Add it evenly to the PVDF membrane and use an exposure instrument to develop the protein expression on the PVDF membrane.

[0229] Example 6 In vivo test

[0230] This Example 6 presents an in vivo trial of a nucleic acid drug delivery system in a spinal cord injury hemisection model. The trial includes the following:

[0231] 6.1 Construction of a spinal cord injury hemisection model

[0232] Thirty-six SD female rats aged 6-8 weeks and weighing 200-220g were divided into 6 groups of 6 rats each.

[0233] After weighing and anesthetizing, the rats were placed in a prone position on the operating table. The fur on the rat's back was prepared. After disinfection with povidone-iodine, a midline dorsal incision was made, separating the skin and muscle to fully expose the spine. After accurate positioning, a T9-T11 laminectomy was performed, with the movements as gentle as possible to avoid spinal cord damage. The laminectomy was removed to fully expose the spinal cord. The T9-T11 segments were exposed, and a wedge-shaped incision was made close to the left side of the median spinal vein, creating a defect of approximately 2 mm in the left hemispinal cord. Different groups of hydrogel nucleic acid drug delivery systems (each group of 1 mm diameter, 2 mm high cylindrical hydrogels cut into semi-cylinders) were placed in the defect. After thorough hemostasis, the wound was sutured layer by layer. Artificial urination was assisted three times daily until the rats regained spontaneous urination function.

[0234] There are a total of 6 groups:

[0235] Sham group: sham surgery group, only the muscles were separated, the spinal cord was exposed but no damage was done, and then sutured.

[0236] SCI group: Spinal cord injury group, T9-T11 laminectomy was performed after separation and muscle removal.

[0237] NXCGA group: NXCGA hydrogel prepared in step 3.1.1 was implanted in rats with a spinal cord injury hemisection model.

[0238] NXCGA@LNP-siRNA Scramble group: NXCGA@LNP-siRNA Scramble prepared in step 3.1.2 was implanted into rats with a spinal cord injury hemisection model.

[0239] NXCGA@LNP-siRNA REST group: NXCGA@LNP-siRNA REST prepared in step 3.1.2 was implanted in rats with a spinal cord injury hemisection model.

[0240] 6.2 Exercise Scoring

[0241] A spinal cord injury hemisection model was established in SD rats, and the hydrogel samples prepared in step 3.1.2 were implanted into the corresponding groups. The rats were divided into different groups, and their Basso-Beattie-Bresnahan (BBB) ​​scores were measured and compared on days 1, 7, 14, 21, 28, 35, and 42 after surgery.

[0242] Score Scoring Details 0 No visible hindlimb movement 1 Slight movement of one or two joints, usually the hip and / or knee joints. 2 One joint has extensive range of motion, or one joint has extensive range of motion while the other joint has slight range of motion. 3 Both joints have wide range of motion 4 All three joints of the hind limbs can move slightly. 5 Two joints have slight movement, while the third joint has a wide range of motion. 6 Two joints have wide range of motion, and the third joint has slight range of motion. 7 All three joints of the hind limbs can move extensively. 8 The paw pads can touch the ground when not under load. 9 Sometimes the claw's palm bears weight and supports movement, or the claw's back bears weight and moves; sometimes the claw's palm bears weight and moves without support. 10 Occasionally, weight-bearing movement is observed on the palmar surface; there is no coordinated movement of the forelimbs or hindlimbs. 11 Palmar weight-bearing movement is frequently observed, but there is no coordinated movement of the forelimbs or hindlimbs. 12 Weight-bearing movement on the palm is frequently observed, with occasional coordinated movements of the forelimbs and hindlimbs. 13 Commonly seen in palm-weight-bearing movements, and can also be seen in coordinated movements of the forelimbs and hindlimbs. 14 There is sustained palmar weight-bearing movement and coordinated forelimb and hindlimb movements; or there is common palmar movement, sustained coordinated forelimb and hindlimb movements, and occasional dorsal claw movement. 15 Sustained palmar movement and sustained coordinated forelimb and hindlimb movements; little or no gripping during forelimb forward movement; the active claw is parallel to the body upon initial contact. 16 The gait shows continuous palmar movement and continuous coordinated forelimb and hindlimb movements. The forelimbs often grip the ground during forward movement. At initial contact, the active claw is parallel to the body, and rotates after weight transfer. 17 The gait shows continuous palmar movement and continuous coordinated forelimb and hindlimb movements; the forelimbs frequently grip the ground during forward movement; the active claw is positioned parallel to the body during initial contact and after weight transfer. 18 The gait exhibits continuous palmar movement and continuous coordinated forelimb and hindlimb movements, with sustained claw gripping during forelimb forward movement; upon initial contact, the active claw is positioned parallel to the body, rotating after weight transfer. 19 The gait exhibits continuous palmar movement and coordinated forelimb and hindlimb movements, with sustained claw gripping during forelimb forward movement; the active claws remain parallel to the body during initial contact and after weight transfer. The tail is sometimes or always drooping. 20 Sustained palmar movement, consistent and coordinated gait, continuous gripping of the toes, with the active paws positioned parallel to the body upon initial contact and after weight transfer, unstable torso, and a continuously held tail. 21 Continuous palmar movement, coordinated gait, sustained gripping of the toes, active paws always parallel to the body throughout the activity, stable torso, and tail always held high.

[0243] See the rating results Figure 6A .

[0244] from Figure 6A The BBB scores showed that on postoperative day 1, rats in the Sham group could move normally, scoring 21 points, while rats in the other four groups dragged their hind limbs behind them, exhibiting bilateral hind limb paralysis, indicating a successful establishment of the spinal cord injury model. Furthermore, with the passage of time, spinal cord function gradually recovered in the SCI, NXCGA, NXCGA@LNP-siRNA scramble, and NXCGA@LNP-siRNA REST groups, with scores gradually increasing. Notably, at 6 weeks postoperatively, the score in the NXCGA@LNP-siRNA REST treatment group was significantly higher than that in the SCI group, with a statistically significant difference.

[0245] 6.3. HE and LFB staining of spinal cord tissue

[0246] Forty-two days after spinal cord injury surgery, SD rats were anesthetized, fixed in a supine position, and the chest was opened to expose the heart for cardiac puncture. Simultaneously, the right atrial appendage was cut open. Pre-cooled 0.9% saline solution (4°C) was rapidly perfused into the left ventricle. When the tissue turned white and the fluid flowing from the right atrial appendage became clear, 100 mL of pre-cooled 4% paraformaldehyde (PFA) (4°C) was injected. Immediately after PFA injection, the rats exhibited limb twitching and tail shaking, indicating successful perfusion. Perfusion continued until the rats were completely rigid. After perfusion, spinal cord tissue was extracted, with the sampling area 1 cm above and below the injury site. The extracted spinal cord tissue was immersed in 4% PFA for 48 hours, then embedded in paraffin, sectioned, and stained using hematoxylin-eosin (HE) and Laucker's Fast Blue (LFB).

[0247] Hematoxylin-eosin (HE) staining procedure:

[0248] Paraffin sections were placed on stainless steel slides and baked in a 65°C oven for 8 hours. They were then sequentially immersed in xylene for 15 min, xylene for 15 min, anhydrous ethanol for 2 min, anhydrous ethanol for 2 min, 95% ethanol for 2 min, 85% ethanol for 2 min, 75% ethanol for 2 min, ddH2O for 2 min, and histochemical PBS for 3 min. This step was dewaxing to water. Following this, they were gently washed three times in ddH2O for 2 min each time. Hematoxylin staining was performed for 5 min, followed by rinsing the dye with running water. 1% hydrochloric acid-ethanol was added for 20 s, followed by washing with ddH2O for 2 min. 1% ammonia solution was used for blueing for 20 s, followed by microscopic observation and washing with ddH2O for 2 min. 0.5% eosin staining was performed for 2 min, followed by washing with ddH2O for 30 s. The sections were then dehydrated using the reverse dewaxing to water procedure, mounted with neutral resin, scanned using a slide scanner, and the observations were recorded.

[0249] LFB staining steps:

[0250] (1) Place the paraffin sections on a stainless steel glass slide rack and bake them in an oven at 65°C for 8 hours. Then, soak them in xylene for 15 minutes, xylene for 15 minutes, anhydrous ethanol for 2 minutes, anhydrous ethanol for 2 minutes, 95% ethanol for 2 minutes, 85% ethanol for 2 minutes, 75% ethanol for 2 minutes, ddH2O for 2 minutes, and histochemical PBS for 3 minutes. This step is to dewax them to water.

[0251] (2) Soak the slices in 0.1% LFB solution and seal them in an oven at 60℃ for 15 hours.

[0252] (3) The next day, take out the slices, soak them in 95% alcohol for 4 min, then wash them twice in double-distilled water for 3 min each time. Then, use 0.05% lithium carbonate aqueous solution for 15 s to separate the colors, and continue to separate the colors in 70% alcohol for 10 s. Repeat step 2 and this step until the boundary between gray matter and white matter is clear when observed under a microscope.

[0253] (4) Final dehydration: Dehydration is carried out in reverse order of the dewaxing to water step.

[0254] (5) Mounting and observation: Spin dry the slides and then scan them with a slide scanner to obtain the staining results.

[0255] See results Figure 6B .

[0256] from Figure 6BHE staining results showed that the tissue structure in the Sham group was intact and neatly arranged; while in the other four groups, round or irregular cavities of varying sizes were observed at the site of spinal cord injury, surrounded by residual nerve tissue. Furthermore, the NXCGA@LNP-siRNA REST group exhibited significantly better spinal cord structural integrity than the SCI group, improving the degree of structural disorder, and the NXCGA@LNP-siRNA REST group had the smallest cavity area.

[0257] Myelin is a membrane that surrounds the axon of a nerve cell. It is a multi-layered lipid bilayer structure formed by the plasma membrane of the nerve cell spirally winding along the axon. LFB can bind to the myelin sheath, thus staining it. The myelin sheath appears bright blue, which can reveal the morphology, structure, and pathological changes of the myelin sheath, allowing observation of its integrity, degree of necrosis, and repair status.

[0258] from Figure 6B As can be seen from the LFB staining results, the myelin sheath structure in the SCI group was severely damaged and lost. After NXCGA@LNP-siRNA REST treatment, the myelin sheath condition improved, and the myelin sheath structure was more intact. The more intact myelin sheath structure indicates that the insulating layer of nerve fibers was not significantly damaged, which helps to maintain the stable transmission of nerve signals.

[0259] The combined results indicate that NXCGA@LNP-siRNA REST can play a repair role and improve the pathological condition of the spinal cord after SCI.

[0260] 6.4. Enhance antioxidant capacity

[0261] Seven days after spinal cord injury surgery, spinal cord tissue was collected from rats in each group, sectioned, and subjected to immunofluorescence staining. The specific steps are as follows:

[0262] Paraffin sections were placed on stainless steel slides and baked in a 65°C oven for 8 hours. They were then sequentially immersed in xylene for 15 min, xylene for 15 min, anhydrous ethanol for 2 min, anhydrous ethanol for 2 min, 95% ethanol for 2 min, 85% ethanol for 2 min, 75% ethanol for 2 min, ddH2O for 2 min, and histochemical PBS for 3 min. This step dewaxing to water. The sections were then immersed in 3% hydrogen peroxide at room temperature for 30 min to inactivate endogenous peroxidase in the spinal cord tissue. The sections were washed with histochemical PBS, immersed in citrate buffer, and then boiled in an egg cooker for 30 min. This step was for antigen heat retrieval. Cool, wash, and block with 5% bovine serum albumin (BSA) for at least 30 min. Then, remove excess liquid and incubate overnight at 4°C with primary antibodies (NF (1:200), GFAP (1:200), 4-hydroxynonenal (4-HNE) (1:200 dilution), and 8-hydroxy-2-deoxyguanosine (8-OHdG) (1:200 dilution). Recover the primary antibodies and wash with histochemical PBS. All subsequent steps are performed in the dark, incubating with the corresponding fluorescent antibodies at room temperature for 45 min (1:200 dilution). Gently wash with histochemical PBS. Stain the nuclei at room temperature for 5 min, then stain with DAPI (1:200 dilution), and wash with histochemical PBS. Mount the slide and capture images using a laser confocal microscope.

[0263] See results Figure 6C and 6D .

[0264] 4-Hydroxynonenal (4-HNE) is a metabolite of lipid peroxidation that induces cellular dysfunction and even apoptosis. 8-Hydroxydeoxyguanosine (8-OHdG), on the other hand, is a specific marker of DNA oxidative damage, and its level can quantitatively reflect the degree of DNA oxidative damage. After spinal cord injury (SCI), the microenvironment of the damaged area undergoes severe oxidative stress, manifested as excessive ROS production and an imbalance in the antioxidant defense system. At this time, the expression levels of 4-HNE and 8-OHdG in spinal cord tissue are significantly upregulated. Elevated levels of both not only directly reflect the severity of oxidative damage but can also further exacerbate neuronal death, glial cell activation, and inflammatory responses by activating downstream signaling pathways (such as NF-κB and MAPK), forming a vicious cycle that ultimately hinders the recovery of neuronal function.

[0265] from Figure 6C and 6DThe expression levels of 4-HNE and 8-OHdG were very high in the SCI group. However, after treatment with the NXCGA group, the NXCGA@LNP-siRNA scramble group, and the NXCGA@LNP-siRNA REST group, the expression of 4-HNE and 8-OHdG was significantly reduced, indicating that the NXCGA group and the NXCGA@LNP-siRNA REST group can effectively inhibit the oxidative stress environment after SCI in vivo.

[0266] 6.5. Promotes nerve regeneration

[0267] Forty-two days after spinal cord injury surgery, spinal cord tissue was collected from rats in each group, sections were prepared, and immunofluorescence staining was performed. Neuronal marker (NF200) and glial cell marker (GFAP) were selected for immunofluorescence staining to observe the fluorescence expression of the sections.

[0268] Neurofilaments (NF) are primarily found within neuronal axons under normal physiological conditions. Following spinal cord injury (SCI), with degenerative necrosis of neuronal axons, NF within the axons undergoes depolymerization and degradation, resulting in a significant decrease in its content, which is then replaced by cystic cavities or scar tissue forming in the injury area. Increased expression of NF200 (neurofilament heavy chain, 200 kDa subunit) is a direct marker of enhanced axonal regeneration capacity, indicating better recovery of spinal cord nerve function.

[0269] Glial fibrillary acidic protein (GFAP) is mainly found in astrocytes. After spinal cord injury (SCI), the injury microenvironment triggers reactive gliosis in astrocytes, manifested as cell proliferation, hypertrophy, and significantly enhanced GFAP synthesis and expression. High levels of GFAP expression are closely related to excessive glial scar formation, severely hindering axonal regeneration and functional connectivity reconstruction. Decreased GFAP expression helps inhibit glial scar formation, suppress reactive gliosis, and promote neurogenesis.

[0270] See results Figure 6E .

[0271] from Figure 6E It is known that in vivo, NXCGA@LNP-siRNA REST can promote the increase of NF200 level, reduce the level of GFAP, and play a role in promoting nerve regeneration.

[0272] 6.6 Western blot detection of spinal cord proteins

[0273] Proteins were extracted from rat spinal cord 42 days after surgery, and Western blot analysis was performed on the marker proteins GAP43 and Tuj1.

[0274] Growth-associated protein 43 (GAP43) is a marker of neuronal differentiation, and increased expression reflects enhanced axonal regeneration capacity. Neuron-specific β-tubulin III (Tuj1) is a marker of neuronal differentiation and maturation; elevated expression levels indicate that neurons have entered the terminal differentiation stage, further supporting the active state of axonal growth.

[0275] See results Figure 6F .

[0276] from Figure 6F It can be seen that the NXCGA@LNP-siRNA REST group can upregulate the expression of the marker proteins GAP43 and Tuj1.

[0277] Example 7: Study on the in vitro regulation of the macrophage immune microenvironment

[0278] RAW246.7 macrophages were cultured using standard methods, and 10 cells were cultured per well at a ratio of (2-5) × 10⁶ cells / well. 5 Cells were seeded into appropriate 12-well culture plates; the next day, except for the LPS group, the other groups were placed in the corresponding hydrogel samples and co-cultured with the cells for 48 hours. The groups are as follows:

[0279] Control group: RAW246.7 macrophages were added to normal culture and incubated at room temperature for 48 hours.

[0280] LPS group: RAW246.7 macrophages were added to LPS (100 ng / mL) and incubated at room temperature for 48 h.

[0281] NXC@LNP group: The NXC@LNP and RAW246.7 macrophages prepared in step 3.1.2 were incubated at room temperature for 48 h.

[0282] NXC@LNP-siRNA REST group: The NXC@LNP and RAW246.7 macrophages prepared in step 3.1.2 were incubated at room temperature for 48 h.

[0283] NXCGA group: The NXCGA cells and RAW246.7 macrophages prepared in step 3.1.1 were incubated at room temperature for 48 h.

[0284] NXCGA@LNP-siRNA Scramble group: The NXCGA@LNP-siRNA Scramble prepared in step 3.1.2 and RAW246.7 macrophages were incubated at room temperature for 48 h.

[0285] NXCGA@LNP-siRNA REST group: The nucleic acid drug delivery system NXCGA@LNP-siRNA REST prepared in step 3.1.2 and macrophages RAW246.7 were incubated at room temperature for 48 h.

[0286] Macrophages are immune cells and can be classified into M1 and M2 types. Of these two phenotypes, M1 macrophages initiate pro-inflammatory cytokine responses, not only directly engulfing tumor cells but also inhibiting tumor development by participating in efficient antigen presentation and promoting tumor-killing T helper type 1 cell responses. Conversely, M2 macrophages inhibit the initiation of pro-inflammatory cytokine responses.

[0287] After culture, cellular proteins were extracted from each group, and the expression levels of intracellular markers such as the M2 marker Arg1 and the M1 marker iNOS were detected by Western blotting. The results are shown in [Figure 1]. Figures 7A-7C .

[0288] Figures 7A-7C It is evident that NXCGA@LNP-siRNA REST effectively reduces LPS-induced iNOS upregulation and enhances Arg-1 expression. This indicates that NXCGA@LNP-siRNA REST can inhibit M1 macrophage polarization and induce M2 macrophage polarization.

[0289] Example 8: Safety Assessment

[0290] 8.1 Cell proliferation assay

[0291] The NXC hydrogel and NXCGA hydrogel prepared in step 3.1.1 were co-cultured with PC12 cells for 1, 3, and 5 days, respectively, and CCK8 assays were performed. The results are shown in the figure. Figure 8A .

[0292] A control group (CON) and blank wells were also set up. The CON group consisted of cells seeded in culture medium, while the blank wells contained cells seeded only in culture medium.

[0293] Pre-culture at 37℃ for 4 hours to allow PC12 cells to adhere to the culture vessel. After adhesion, PC12 cells were harvested and cultured at 5 × 10⁶ cells per well. 3 Cells were seeded in 96-well plates and co-cultured with hydrogel. Incubation was performed in a CO2 incubator for 1, 3, and 5 days. The medium was changed every two days. At each time point, 100 μL of 10% CCK-8 solution was added to each well of the plate using a pipette, and the plate was incubated for another 4 hours. Absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using a formula, and statistical analysis of the absorbance values ​​was performed using statistical software to generate cell proliferation and cell viability graphs.

[0294] Cell viability (%) = (As - Ab) / (Ac - Ac) × 100%

[0295] Where As is the absorbance of the composite scaffold pores in the experimental group, Ac is the absorbance of the control pores, and Ab is the absorbance of the blank.

[0296] from Figure 8A It is evident that the nucleic acid drug delivery system of this application has no adverse effect on cell proliferation.

[0297] 8.2. Organ HE staining

[0298] Forty-two days after spinal cord injury surgery, major organs, including heart, liver, spleen, intestine, and kidney, were harvested from rats in each group. Sections were prepared and stained with hematoxylin and eosin (HE) to assess the safety of hydrogel therapy. Results are shown below. Figure 8B .

[0299] from Figure 8B It can be seen that there were no obvious pathological changes in any of the organs, indicating that it has good safety.

[0300] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A nucleic acid drug delivery system, characterized in that, The invention comprises a photocrosslinked hydrogel and cationic lipid nanoparticles, wherein the cationic lipid nanoparticles are loaded in the photocrosslinked hydrogel and encapsulated with siRNA that inhibits the expression of the REST gene, and the nucleotide sequence of the siRNA is shown in SEQ ID No. 1 and SEQ ID No.

2.

2. The nucleic acid drug delivery system as described in claim 1, characterized in that, The mass ratio of the cationic lipid nanoparticles to the photocrosslinked hydrogel is (20-30):

1.

3. The nucleic acid drug delivery system as described in claim 1, characterized in that, Based on the molar number of nitrogen atoms in the cationic lipid nanoparticles and the molar number of phosphorus atoms in the siRNA, the N / P molar ratio of the cationic lipid nanoparticles and siRNA is (2-8):

1.

4. The nucleic acid drug delivery system as described in claim 2, characterized in that, The raw materials of the photocrosslinked hydrogel include acryloylglycine, xanthan gum, carbomer, photoinitiator, and antioxidant.

5. The nucleic acid drug delivery system as described in claim 3, characterized in that, The raw materials for the cationic lipid nanoparticles include phospholipids, cationic lipids, cholesterol, and long-cycle materials.

6. The nucleic acid drug delivery system as described in claim 4, characterized in that, The photoinitiator is selected from 2-hydroxy-2-methylphenylacetone.

7. The nucleic acid drug delivery system as described in claim 4, characterized in that, The antioxidant is selected from one or more of gallic acid, quercetin, catechin, tea polyphenols, epigallocatechin, and tannic acid.

8. The nucleic acid drug delivery system as described in claim 4, characterized in that, The mass ratio of acryloylglycine, xanthan gum and carbomer is (10-30):(1-6):

1.

9. The nucleic acid drug delivery system as described in claim 4, characterized in that, The volume-to-mass ratio of the photoinitiator to acryloylglycine is 1 μL:(0.01-10)g.

10. The nucleic acid drug delivery system as described in claim 4, characterized in that, The mass ratio between the antioxidant and acryloylglycine is (0.01~1):

1.

11. The nucleic acid drug delivery system as described in claim 5, characterized in that, The phospholipid is selected from dioleoylphosphatidylethanolamine.

12. The nucleic acid drug delivery system as described in claim 5, characterized in that, The cationic lipid is selected from dioleoylphosphatidylcholine-dimethacrylate.

13. The nucleic acid drug delivery system as described in claim 5, characterized in that, The cholesterol is selected from one or both of cholesterol monosuccinate and DC-cholesterol.

14. The nucleic acid drug delivery system as described in claim 5, characterized in that, The long-cycle material is selected from polyethylene glycol and its derivatives, wherein the polyethylene glycol derivative is selected from one or more of polyethylene glycol-vitamin E succinate, polyethylene glycol-cholesterol, polyethylene glycol-modified distearylphosphatidylethanolamine, polyethylene glycol-modified dimyristoylphosphatidylethanolamine, polyethylene glycol-modified dipalmitoylphosphatidylethanolamine, and polyethylene glycol-modified distearylmethylpropylene glycol.

15. The nucleic acid drug delivery system as described in claim 5, characterized in that, The mass ratio of the phospholipids, cationic lipids, cholesterol and long-cycle materials is (1-6):1:(1-10):

1.

16. The nucleic acid drug delivery system as described in claim 1, characterized in that, The photocrosslinked hydrogel can be crosslinked under ultraviolet irradiation of 280 nm-320 nm.

17. A method for preparing a nucleic acid drug delivery system according to any one of claims 1-16, characterized in that, Includes the following steps: 1) Obtain cationic lipid nanoparticles encapsulated with siRNA; 2) The cationic lipid nanoparticles are dispersed in a photocrosslinked hydrogel to obtain the nucleic acid drug delivery system.

18. A pharmaceutical composition, characterized in that, Including the nucleic acid drug delivery system as described in any one of claims 1-16.

19. Use of the nucleic acid drug delivery system of any one of claims 1-16 or the pharmaceutical composition of claim 18 in the preparation of a product for treating spinal cord injury.

20. The use as described in claim 19, characterized in that, The product has at least one of the following functions: 1) promoting nerve regeneration; 2) resisting oxidative stress; 3) promoting axon regeneration; 4) regulating the macrophage immune microenvironment.

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