Spermidine polymer and its mediated gene delivery system, preparation method and application thereof

Abstract

The present application relates to the field of organic nanomaterials and the field of biological medicine, and specifically provides a spermidine polymer and a gene delivery system mediated by the spermidine polymer, a preparation method and application thereof. The spermidine polymer is formed by condensation of spermidine monomers and glyoxal as a crosslinking agent molecule, can be used as a gene carrier with antioxidant and mitochondrial autophagy activity to prepare a gene delivery system, and is used for nucleic acid encapsulation and transfection; the spermidine polymer and specific siRNA self-assemble to generate a gene delivery complex, which can transfect a target to effectively silence; a kidney-targeting type gene delivery system with a core-shell structure, which is assembled by the gene delivery complex loaded with specific siRNA of an acute kidney injury key target Arginase-2 and kidney-targeting peptide modified hyaluronic acid, can be targeted to tubular epithelial cells, can effectively improve acute kidney injury by synergistically enhancing mitochondrial autophagy and relieving oxidative stress, and has good biological safety.

Description

Technical Field

[0001] This invention relates to the fields of organic nanomaterials technology and biomedicine, specifically to spermidine polymers and their mediated gene delivery systems, their preparation methods and applications. Background Technology

[0002] Oxidative stress is a major physiological stimulus in the body, referring to a state of imbalance between oxidation and antioxidation (leaning towards oxidation), and is also an important factor in cell damage. Oxidative stress leads to high levels of reactive oxygen species (ROS) in cells, which can directly damage mitochondrial DNA (mtDNA). Accumulation of damaged mtDNA can lead to mitochondrial damage and dysfunction, thereby inducing related mitochondrial diseases. Currently, oxidative stress and impaired mitophagy are important pathological phenotypes in the damage of organs rich in mitochondria, such as the heart and kidneys. For example, acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function within a short period of time. It has a rapid onset and high mortality rate, with an incidence rate of approximately 10%–15% in hospitalized patients and over 50% in intensive care patients. AKI can cause renal microcirculatory disturbances, excessive production of reactive oxygen species and inflammatory factors. Common triggers include drug toxins, sepsis, and ischemia-reperfusion injury. Studies have shown that impaired mitophagy, oxidative stress damage, and increased apoptosis are among the key pathological mechanisms common to various types of AKI. The common way to treat oxidative stress caused by disease is to take antioxidants. However, since conventional antioxidant therapy does not target key pathogenic targets, the treatment effect still needs to be improved. At present, there is a lack of targeted, specific and effective prevention and treatment measures. Therefore, developing new targeted diagnosis and treatment strategies is an urgent technical problem to be solved.

[0003] Gene therapy targeting key targets is currently a hot topic in drug development. However, commonly used viral vectors pose the risk of immunogenicity, while liposomes lack specificity in targeted transfection, and these vector materials only support the transcellular transport of functional nucleic acids, without possessing beneficial biological activity for disease treatment. Therefore, developing and designing non-invasive, non-viral, and bioactive gene vectors for targeted delivery of genetic material to target organs is a pressing technical challenge.

[0004] In various animal models and human epidemiological studies, spermidine has been found to activate mitophagy, inhibit oxidative stress, and improve mitochondrial function. With three positive charges, it has a natural advantage in constructing gene vectors to deliver functional nucleic acids to regulate therapeutic targets. It has been used for DNA self-assembly and functionalization, as well as for the side-chain modification of polyphosphamides. However, gene vectors developed using this method suffer from drawbacks such as low content of active ingredients and difficulty in forming free active monomers. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides, on the one hand, a spermidine polymer and its mediated gene delivery system, its preparation method, and its application. The spermidine polymer can serve as a bioactive gene carrier, activating mitophagy and alleviating oxidative stress. On the other hand, this invention provides a kidney-targeted gene delivery system that, through synergistic activation of mitophagy, alleviation of oxidative stress, and apoptosis, can improve acute kidney injury.

[0006] The objective of this invention is achieved through the following technical solution: A spermidine polymer, characterized in that it is formed by the condensation of spermidine monomer and crosslinking agent molecules; wherein the crosslinking agent is glyoxal, and the molar ratio of spermidine to glyoxal is 1:1 to 1:5.

[0007] Furthermore, the molecular weight of the polymer is 9000 Da to 20000 Da.

[0008] The second objective of this invention is to protect a method for preparing a spermidine polymer, wherein spermidine is dissolved in an organic solvent to obtain a primary solution, and glyoxal solution is added to the primary solution under low temperature conditions and a protective gas atmosphere, and the mixture is stirred and heated to react, thereby obtaining a secondary solution. In the secondary solution, the molar ratio of spermidine to glyoxal is 1:1 to 1:1.5. The secondary solution is then filtered, dialyzed, and freeze-dried to obtain the spermidine polymer.

[0009] The spermidine and glyoxal undergo condensation based on a Schiff base reaction to generate a spermidine polymer with conjugated imine bonds, wherein the spermidine polymer is a polyspermineimide.

[0010] Furthermore, the organic solvent is one or more of dimethyl sulfoxide, ethylenediamine, ethanol, anhydrous methanol, or anhydrous ethanol.

[0011] Furthermore, the protective gas is one or more of nitrogen, carbon dioxide, helium, or argon.

[0012] Furthermore, the low temperature condition is 0℃~4℃; the reaction time for stirring and heating is 12~24h, and the reaction temperature is gradually increased from 0℃~4℃ to 25℃.

[0013] Furthermore, a molecular sieve is added to the first solution to filter and remove water from the secondary solution, and the resulting filtrate contains spermidine polymer.

[0014] Furthermore, the dialysis time is 24-26 hours, and the freeze-drying time is 12-24 hours.

[0015] A third objective of this invention is to protect a gene delivery complex, which is self-assembled from the spermidine polymer and siRNA; wherein the mass ratio of the spermidine polymer to siRNA is 10:1 to 30:1.

[0016] Furthermore, the mass ratio of the spermidine polymer to siRNA is 10:1.

[0017] The present invention also provides a method for preparing a gene delivery complex, characterized in that siRNA and spermidine polymer are dissolved separately in enzyme-free water, mixed in a mass ratio, and allowed to stand for self-assembly reaction to obtain the gene delivery system; wherein the mass ratio is 10:1 to 30:1, the self-assembly reaction temperature is 0℃ to 4℃, and the reaction time is 0.5 to 1 h.

[0018] Through experiments, the inventors discovered that polysemine imine, synthesized via Schiff base reaction using spermidine as the active monomer and glyoxal as the cross-linking agent, can serve as a gene carrier. It can self-assemble with functional siRNA into a nanostructure, which can act as a gene delivery complex to transfect and deliver gene material into cells. Moreover, after releasing functional siRNA, the gene carrier still retains biological activity, enhancing mitophagy and alleviating oxidative stress. Simultaneously, the released functional siRNA can play a targeted silencing role. The combination of the two further improves the damage to target organs, especially organs such as the heart and kidneys.

[0019] As a further application, a fourth objective of the present invention is to protect a kidney-targeting gene delivery system, the gene delivery system comprising a core structure and a shell structure; the core structure being the gene delivery complex assembled from a spermidine polymer and siRNA, wherein the siRNA is Arginase-2 siRNA (SEQ ID NO.1: GTATATTACTGAAGAAATA); the shell structure being a hyaluronic acid-kidney-targeting peptide complex; the mass ratio of the spermidine polymer, Arginase-2 siRNA, and hyaluronic acid-kidney-targeting peptide complex is 10:1:(1~2.5).

[0020] Furthermore, the mass ratio of the spermidine polymer, Arginase-2 siRNA, and hyaluronic acid-kidney-targeting peptide complex is 10:1:1.

[0021] Furthermore, the particle size of the gene delivery system is 280~320 nm, and the zeta potential is -2~0 mV.

[0022] Furthermore, the kidney-targeting peptide is CKKEEEKKEEEKKEEEK (SEQ ID NO.2).

[0023] This invention also provides a method for preparing a kidney-targeted gene delivery system, comprising the following steps: S1, spermidine polymer undergoes a self-assembly reaction with Arginase-2 siRNA to form a nuclear structure; S2, hyaluronic acid reacts and condenses with kidney-targeting peptides to form a hyaluronic acid-kidney-targeting peptide complex; S3, the hyaluronic acid-kidney-targeting peptide complex is mixed with the core structure and a core-shell structure is formed by electrostatic adsorption reaction to obtain a gene delivery system.

[0024] Through further targeted experiments, the inventors discovered that the gene delivery complex, which is self-assembled from spermidine polymer and functional siRNA, not only possesses transfection function but also adsorbs hyaluronic acid modified with kidney-targeting peptides to form a core-shell structured gene delivery system. Hyaluronic acid is a natural anionic polysaccharide with good biocompatibility and biodegradability. It is one of the specific ligands of the adhesion molecule CD44. CD44 is a group of transmembrane glycoproteins with complex functions and structures, widely distributed on the type I transport membrane of proteoglycan receptors, and extensively distributed on leukocytes, epithelial cells, and endothelial cells. Studies have shown that CD44 is expressed less in normal kidney tissue but increased in kidney-damaged cells. Therefore, selecting hyaluronic acid as an important component of the shell structure of the gene delivery system can enhance the targeting of the gene delivery system and its uptake rate by the targeted cells, thereby improving transfection efficiency. More preferably, the reaction and condensation of hyaluronic acid with kidney-targeting peptides to form a hyaluronic acid-kidney-targeting peptide complex, as the shell structure, further enhances the targeting of the kidney-targeted gene delivery system. Subsequent mechanistic studies revealed that when the gene delivery system is loaded with specific Arginase-2 siRNA, it can target renal tubular epithelial cells, synergistically enhancing mitophagy and alleviating oxidative stress through gene silencing, thereby improving acute kidney injury. Studies have shown that Arginase-2 plays a crucial role in mitochondrial dynamics and oxidative respiration, and its targeted therapeutic applications in ischemia-reperfusion or drug-induced AKI have also been reported.

[0025] Further, in step S2, the hyaluronic acid is maleimide-modified hyaluronic acid, and the mass ratio of hyaluronic acid to the kidney-targeting peptide is 1:1 to 1:5; the reaction temperature of the click chemistry reaction is 0℃ to 4℃, the reaction time is 12 to 24 h, and the reaction pH is 7 to 8.

[0026] Using maleimide-modified hyaluronic acid, under suitable pH conditions, the maleimide group readily undergoes Michael addition with the thiol group in the peptide to form a thioether bond, thus strengthening the close connection between the kidney-targeting peptide and hyaluronic acid.

[0027] Further, in step S3, the mass ratio of the spermidine polymer gene carrier, Arginase-2 siRNA, and hyaluronic acid-kidney-targeting peptide complex is 10:1:(1~2.5); the reaction temperature of the electrostatic adsorption reaction is 0℃~4℃, and the reaction time is 0.5h~1h. The mass ratio of the spermidine polymer gene carrier and Arginase-2 siRNA is fixed at 10:1. When the mass fraction of the kidney-targeting peptide complex is greater than 2.5, the particle size increases, which will affect the delivery of the gene delivery system.

[0028] A fifth objective of this invention is to protect the use of the spermidine polymer in the preparation of gene delivery systems, antioxidant or mitochondrial autophagy stimulant drugs.

[0029] A sixth objective of this invention is to protect the use of the gene delivery complex in the preparation of antioxidant or mitochondrial autophagy stimulant drugs.

[0030] The seventh objective of this invention is to protect the use of the kidney-targeting gene delivery system in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0031] The beneficial effects of this invention are as follows: (1) The spermidine polymer provided by the present invention can be used as a gene carrier. While delivering genes, it retains the biological activity of spermidine in promoting mitochondrial autophagy and anti-oxidation, providing a new strategy for anti-oxidation and other therapeutic methods.

[0032] (2) The spermidine polymer provided by the present invention can successfully condense siRNA as a gene carrier and has a highly efficient silencing effect on related targets, providing a potential pathway for gene therapy.

[0033] (3) The kidney-targeted gene delivery system provided by the present invention delivers functional nucleic acids to regulate key AKI targets. Due to the acid instability of the imine structure, it can dissociate spermine active monomers in the acidic environment of the endostomy, and synergistically exert biological activities to promote mitochondrial autophagy, anti-oxidation and anti-apoptosis, thus enhancing the prevention and treatment of AKI.

[0034] (4) The kidney-targeted gene delivery system provided by the present invention has a medium-scale particle size and kidney-targeting ligand modification, has high kidney selectivity, and is located in the main lesion of AKI—renal tubular epithelial cells, to complete the targeted delivery of functional nucleic acids at the lesion site, which is of great significance for the prevention and treatment of AKI.

[0035] (5) The kidney-targeted gene delivery system provided by the present invention has good biosafety: through toxicity test evaluation, after intravenous administration to normal C57BL6 / N mice for 24 hours, the main organs were taken for pathological sections, and serum samples were taken to detect liver and kidney function and blood routine, and no obvious toxic side effects were found.

[0036] Instruction manual illustrations Figure 1 This is a synthetic strategy for spermidine polymers (polyspermineimide).

[0037] Figure 2 The 1H NMR spectra of spermidine (A) and polyspermineimide (B) are shown.

[0038] Figure 3 The images show the infrared spectra of spermidine and polyspermineimide.

[0039] Figure 4 This is a size exclusion chromatogram of spermidine and polyspermineimide.

[0040] Figure 5 Agarose gel images showing the particle size and zeta potential of nanoparticles self-assembled at different polysemine imine / siRNA mass ratios (A) and the siRNA condensation level at different polysemine imine / siRNA mass ratios (B).

[0041] Figure 6 This is a schematic diagram of the level of mitochondrial autophagy in the HK-2 cell injury model under polysemine intervention.

[0042] Figure 7 This diagram illustrates the levels of various reactive nitrogen / oxygen in the HK-2 cell injury model and the HCM cell injury model under polysemine intervention.

[0043] Figure 8 This diagram illustrates the transfection efficiency detection of polyspermine imine gene vectors loaded with different target siRNAs.

[0044] Figure 9 This is a schematic diagram showing the serum creatinine and blood urea nitrogen levels in mice with cardiorenal syndrome after intervention with polysemine.

[0045] Figure 10 This is a schematic diagram showing the levels of myocardial enzyme indicators (creatine kinase, creatine kinase isoenzyme MB, and lactate dehydrogenase) in mice with cardiorenal syndrome after intervention with polysemine.

[0046] Figure 11 The infrared spectrum of maleimide-modified hyaluronic acid and kidney-targeting peptide-hyaluronic acid complex.

[0047] Figure 12 The diameter and zeta potential of nanoparticles were obtained by preparing renal-targeting peptide-hyaluronic acid complex / polysemine / Arginase-2 siRNA at different mass ratios.

[0048] Figure 13Transmission electron microscopy (TEM) images of a kidney-targeted gene delivery system were obtained when the mass ratio of kidney-targeting peptide-hyaluronic acid complex / Arginase-2 siRNA / polysemine imine gene vector was 1:10:1.

[0049] Figure 14 This is a schematic diagram of Arginase-2 protein expression levels in an HK-2 cell injury model induced by a kidney-targeted gene delivery system.

[0050] Figure 15 This is a schematic diagram of the level of mitochondrial autophagy in an HK-2 cell injury model induced by a kidney-targeted gene delivery system.

[0051] Figure 16 This diagram illustrates the levels of various reactive nitrogen / oxygen in an HK-2 cell injury model induced by a kidney-targeted gene delivery system.

[0052] Figure 17 This is a schematic diagram of the in vivo targeting of a kidney-targeted gene delivery system.

[0053] Figure 18 This diagram illustrates the in vivo safety of a kidney-targeted gene delivery system. Detailed Implementation

[0054] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description. Unless otherwise defined, all technical and scientific terms used in the embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the methods used are conventional methods in the art; the reagents used can be prepared commercially or by known and disclosed methods.

[0055] Example 1: Preparation and Characterization of Spermine Polymer Based on the Schiff base reaction, a spermidine polymer (polysemidineimide) was synthesized using spermidine as a monomer and glyoxal as a crosslinking agent. The product structure was characterized using 1H NMR and infrared spectroscopy, and the molecular weight was measured using gel size exclusion chromatography. The specific implementation method is as follows: Spermine (2.43 mmol) was dissolved in 18 ml of anhydrous ethanol to obtain a primary solution. Molecular sieves (4 Å) were added to the primary solution, and then 40% glyoxal solution (2.43 mmol) was slowly added under a nitrogen atmosphere and at 0 °C with simultaneous stirring. The mixture was gradually heated to room temperature (25 °C) while maintaining continuous stirring, and the reaction was carried out for 12 h to obtain a secondary solution. The synthetic strategy is as follows: Figure 1As shown; after the reaction is complete, the secondary solution is filtered, and the filtrate is transferred to a dialysis membrane (MWCO = 1000), dialyzed with double-distilled water for 24 hours, and then freeze-dried for 24 hours to obtain polysemine imine.

[0056] The 1H NMR characterization results of spermidine and polyspermineimide are as follows: Figure 2 As shown in (AB), Figure 2 In (A), the peak at 4.75 ppm is a water peak, and the peaks at 2.55, 2.46, 1.55, and 1.37 ppm are hydrogen atoms at positions 1-4 of spermidine; Figure 2 (B) In the spectrum of polysemine imine, the peaks at 8.38, 2.69 and 1.52 ppm are attributed to hydrogen atoms at positions 1-3, respectively.

[0057] The infrared spectral characterization results of spermidine and polyspermineimide are as follows: Figure 3 As shown, 1653 cm is visible. -1 Characteristic peaks of C=N bonds are formed at this location; Size exclusion chromatography characterization results of spermidine and polyspermineimide are as follows: Figure 4 As shown, the polymer molecular weight is 14329 Da, indicating the formation of the polymer polysemine imine.

[0058] The above results demonstrate the successful synthesis of polysemine imine.

[0059] Example 2: Preparation and Characterization of Gene Delivery Complex Furthermore, the polysemine synthesized in Example 1 was used as a gene carrier to form a gene delivery system with siRNA through self-polymerization. During the process, a Malvern nanoparticle size analyzer was used to detect the particle hydration size and potential, and agarose gel electrophoresis (2%, 1×TBE buffer, 0.5 μg / ml SYBR Gold, 100V, 45 min) was used to detect the nucleic acid loading efficiency. The specific implementation method is as follows: Polysemine imide and siRNA were dissolved separately in enzyme-free water to obtain polysemine imide solutions with a concentration of 1 mg / mL and siRNA solutions with a concentration of 1 mg / mL, respectively. Polysemine imide and siRNA were mixed at mass ratios of 5:1, 10:1, 20:1, and 30:1, and each mixture was vortexed for 30 seconds using a Vortex mixer and then incubated at 4°C for 0.5 h to allow the polysemine imide and siRNA to undergo a self-assembly reaction to form nanoparticles (gene delivery system). The particle size variation and nucleic acid loading efficiency of the nanoparticles are shown in the figure below. Figure 5As shown in (AB), when the mass ratio of polyspermine imide to siRNA is ≥10:1, the polyspermine imide gene vector completely encapsulates the siRNA, with a particle size of 280 nm and a zeta potential of +18 mV. Subsequent experiments used a mass ratio of polyspermine imide to siRNA of 10:1.

[0060] Example 3: Detection of the activity of the polyspermine imine gene vector via a cellular pathway This embodiment uses renal tubular epithelial cells (HK-2 cells) and human cardiomyocytes (HCM cells) as experimental materials. Normal HK-2 cells and HCM cells incubated with polysemine were treated using a modeling method for vancomycin-induced HK-2 cell damage and hydrogen peroxide-induced HCM cell damage. Fluorescent probes were used to detect mitophagy and oxidative stress levels. The specific implementation method is as follows: 1. Establish a vancomycin-induced HK-2 cell damage model. The specific procedures are as follows: (1) Vaccination of 1×10 6 ~5×10 6 One HK-2 cell was cultured in each well of a 6-well plate containing 2 mL of complete culture medium; (2) Prepare 1990 µL of complete culture medium containing 4 mM vancomycin, add it to the culture well, and gently shake well; (3) Add 10 µL of polysemine containing 1 mg / mL to the culture medium and shake gently. (4) Place the culture plate in a CO2 incubator at 37℃ and incubate for 48 h; Based on the inventors' previous work, the concentration of vancomycin added was determined to be 4 mM, and the cell culture time was 48 h.

[0061] Meanwhile, a blank control group (the vancomycin in step (2) and the polyspermine in step (3) were replaced with the same volume of complete culture medium, and other conditions remained unchanged), a vancomycin-free group (the polyspermine in step (3) was replaced with the same volume of complete culture medium, and other conditions remained unchanged), and a vancomycin-incubated group (the polyspermine in step (3) was replaced with the same concentration of spermidine, and other conditions remained unchanged).

[0062] 2. Establish a hydrogen peroxide-induced HCM cell damage model. The specific procedures are as follows: (1) Vaccination of 1×10 6 ~5×10 6 One HCM cell was cultured in a 6-well plate containing 2 mL of complete culture medium. (2) Prepare 1990 µL of complete culture medium containing 100 μM hydrogen peroxide, add it to the culture well, and gently shake to mix; (3) Add 10 µL of polysemine containing 1 mg / mL to the culture medium and shake gently. (4) Place the culture plate in a CO2 incubator at 37℃ and incubate for 4 hours; Based on the inventors' previous work, the concentration of added hydrogen peroxide was determined to be 100 μM, and the cell culture time was 4 h.

[0063] Meanwhile, a blank control group (the hydrogen peroxide in step (2) and the polyspermine in step (3) were replaced with the same volume of complete culture medium, and other conditions remained unchanged), a hydrogen peroxide group without intervention (the polyspermine in step (3) was replaced with the same volume of complete culture medium, and other conditions remained unchanged), and a hydrogen peroxide group with spermine incubation intervention (the polyspermine in step (3) was replaced with the same concentration of spermine, and other conditions remained unchanged).

[0064] 3. Mitochondrial autophagy level detection, the specific operation is as follows: The colocalization concentration and colocalization of mitochondria and lysosomes within cells were simultaneously detected using Mito-Tracker Deep Red and Lyso-Tracker green fluorescent probes. The Mito-Tracker Deep Red and Lyso-Tracker green fluorescent probe kits were purchased from Beyotime Biotechnology Co., Ltd. The specific procedures are as follows: (1) According to the method of the corresponding kit, dilute Mito-Tracker Deep Red and Lyso-Tracker green with serum-free culture medium at a ratio of 1:20000 respectively; (2) Remove the cell culture medium from the 6-well plates of the four experimental groups in the above vancomycin-induced HK-2 cell damage model, add 1 mL of diluted Mito-Tracker Deep Red and Lyso-Tracker green; incubate in a cell culture incubator at 37°C for 30 min, and wash the cells three times with PBS buffer (pH 7.4) to fully remove fluorescent probes that have not entered the cells; (3) Detection and analysis were performed using a laser confocal imaging system (Leica microscopy system). The Mito-Tracker Deep Red used an excitation wavelength of 644 nm and an emission wavelength of 665 nm, while the Lyso-Tracker green used an excitation wavelength of 504 nm and an emission wavelength of 511 nm.

[0065] Test results as follows Figure 6 As shown, Figure 6 The results showed that polysemine intervention could reverse the vancomycin-induced decrease in mitochondrial autophagy in HK-2 cells to some extent.

[0066] 4. Oxidative stress level detection, the specific operation is as follows: Using DCFH-DA (Shanghai Beyotime Biotechnology Co., Ltd.), Dihydroethidium (Shanghai Beyotime Biotechnology Co., Ltd.), ONOO- (Beijing Bio-Rad Laboratories Co., Ltd.), and Mito-SOX (Invitrogen Molecular Probes) TM The fluorescent probe can simultaneously detect intracellular reactive oxygen species (ROS) levels, superoxide anion levels, peroxynitrosoanion levels, and mitochondrial superoxide levels.

[0067] (1) According to the method of the corresponding kit, take a small amount of DCFH-DA, Dihydroethidium, ONOO- and Mito-SOX fluorescent probes respectively, and dilute them with serum-free culture medium at a ratio of 1:1000 to form working solutions of each active oxygen / nitrogen probe with a final concentration of 10 μM / L.

[0068] (2) Remove the cell culture medium from the 6-well plates of the 8 experimental groups in the vancomycin-induced HK-2 cell damage model and the hydrogen peroxide-induced HCM cell damage model, add 1 mL of diluted working solution of each reactive oxygen species / nitrogen probe; incubate in a cell culture incubator at 37℃ for 20 min, and wash the cells 3 times with PBS to fully remove fluorescent probes that have not entered the cells; (3) Detection and analysis were performed using the Operetta high-content imaging system (PerkinElmer). DCFH-DA used an excitation wavelength of 488 nm and an emission wavelength of 525 nm; Dihydroethidium used an excitation wavelength of 535 nm and an emission wavelength of 610 nm; Mito-SOX used an excitation wavelength of 510 nm and an emission wavelength of 580 nm.

[0069] Test results as follows Figure 7 As shown in (AB), Figure 7 The results showed that polysemine intervention could reverse the increase of various reactive nitrogen / oxygen ratios in HK-2 cell and cardiomyocyte injury models to a certain extent.

[0070] Example 4: Detection of transfection efficiency of polyspermine imine gene vector via cellular pathway The polysemine synthesized in Example 1 was used as a gene carrier and self-assembled with siRNAs specific for XDH, HELB, and HNRNPK to form gene delivery complexes. All siRNAs were purchased from Sangon Biotech (Shanghai) Co., Ltd., and their specific sequences are shown in Table 1. The preparation process can be referred to in Example 2. Each nanoparticle was co-incubated with normal HK-2 cells, and a negative control group was established. The specific implementation method is as follows: (1) Vaccination of 1×10 6 ~5×10 6 One HK-2 cell was cultured in each well of a 6-well plate containing 2 mL of complete culture medium; (2) Add 2 mL of nanoparticles containing 1 mg / mL to the culture medium and shake gently. (3) Place the culture plate in a CO2 incubator at 37℃ and incubate for 24 h; (4) RNA was extracted from HK-2 cells after incubation, and the expression of related mRMA was detected by Realtime PCR.

[0071] Test results as follows Figure 8 As shown, the results indicate that the intervention of nanoparticles assembled from XDH, HELB, and HNRNPK-specific siRNA sequences can significantly knock down the mRNA levels of the corresponding molecules in HK-2 cells.

[0072] Table 1 Sequences involved in Example 4 name Sequence (5'→3') XDH UUUCUGAAACAGUAAAGUUGCTT (SEQ ID NO.3) HELB UUCAUCACAAAUAGAAACGCGTT (SEQ ID NO.4) HNRNPK UCUGGGACUGAAACACUGGCATT (SEQ ID NO.5) Example 5: Investigating the in vivo therapeutic effect of polyspermineimide via animal routes. A mouse model of cardiorenal syndrome was established. Before modeling, mice were treated with or without polysemine. After 72 hours, major organs were harvested, and the extent of damage was assessed by HE staining. Orbital blood was collected to detect renal function and other blood biochemical indicators. The specific implementation method is as follows: (1) Establishment of a mouse model of cardiorenal syndrome: Male C57BL / 6N mice (6-8 weeks old) were injected intraperitoneally once a day with isoproterenol (50 mg / kg), while the control group was injected intraperitoneally with an equal volume of physiological saline for 3 days.

[0073] (2) Vector intervention: 1 hour before modeling, mice in the control group and model group were injected with saline via the tail vein, while mice in the treatment group were injected with 60 mg / kg polysemine via the tail vein.

[0074] (3) Kidney function test: 72 h later, the mice in the three groups (control group, model group and treatment group) were sacrificed, and the kidneys were taken. The tissue sections were 3 μm thick. HE staining and renal tubular injury score were used to evaluate the morphology and structure of the kidney tissue and renal tubules and the extent of damage. Orbital blood was taken and serum creatinine, blood urea nitrogen, creatine kinase, creatine kinase isoenzyme MB and lactate dehydrogenase levels were detected by ELISA kit.

[0075] Test results as follows Figure 9 (AB) Figure 10As shown in (AC), the results indicated that after intervention with polysemine imine, the elevations in serum creatinine (9A) and blood urea nitrogen (9B) in mice were significantly reversed, and renal function was significantly restored; the elevations in myocardial enzyme levels (10A) creatine kinase, (10B) creatine kinase isoenzyme MB, and (10C) lactate dehydrogenase were also significantly reversed, and cardiac function was significantly restored. These results demonstrate that polysemine imine has a protective effect against damage to multiple organs.

[0076] Example 6: Preparation and Characterization of a Kidney-Targeted Gene Delivery System Polysemine imide was used as a gene carrier and self-assembled with siRNA to form a gene delivery complex, serving as the core structure of the gene delivery system. Based on the Michael addition reaction, maleimide-modified hyaluronic acid was conjugated with a kidney-targeting peptide to form a hyaluronic acid-kidney-targeting peptide complex, which was then coated around the core structure via electrostatic adsorption, forming a core-shell structure. The product structure was characterized using infrared spectroscopy; the particle hydration size and potential were detected using a Malvern nanoparticle size analyzer; and the particle microstructure was observed using transmission electron microscopy. The specific implementation method is as follows: 1. Preparation and characterization of nuclear structures in kidney-targeted gene delivery systems The polysemine synthesized in Example 1 was used as a gene carrier and self-assembled with Arginase-2 siRNA to form nanoparticles (core structure). The mass ratio of the two was selected from the preferred ratio in Example 2 (10:1). The particle hydration size and potential were detected using a Malvern nanoparticle size analyzer; the nucleic acid loading efficiency was detected by agarose gel electrophoresis (2%, 1×TBE buffer, 0.5 μg / ml SYBR Gold, 100V, 45 min). The specific implementation method is as follows: Arginase-2 siRNA sequence (5'→3'): GTATATTACTGAAGAAATA (SEQ ID NO.1), purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0077] Polysemine imide and Arginase-2 siRNA were dissolved separately in enzyme-free water to obtain polysemine imide solution with a concentration of 1 mg / mL and Arginase-2 siRNA solution with a concentration of 1 mg / mL. Polysemine imide and Arginase-2 siRNA were mixed at a mass ratio of 10:1 and vortexed for 30 s using a Vortex vortex mixer. The mixtures were then allowed to stand at 4 °C for 0.5 h to obtain the gene delivery complex.

[0078] 2. Preparation and characterization of shell structures in gene delivery systems Based on the Michael addition reaction, maleimide-modified hyaluronic acid (HA-MAL, Xi'an Ruixi Biotechnology Co., Ltd.) is conjugated with kidney-targeting peptide (Shanghai Qiangyao Biotechnology Co., Ltd.) (SEQ ID NO.2: CKKEEEKKEEEKKEEEK) to form a hyaluronic acid-kidney-targeting peptide complex.

[0079] Maleimide-modified hyaluronic acid was dissolved in enzyme-free water at a concentration of 1 mg / mL; a kidney-targeting peptide was dissolved in enzyme-free water at a concentration of 1 mg / mL; the hyaluronic acid and kidney-targeting peptide were mixed at a mass ratio of 1:1 and vortexed for 30 s using a Vortex mixer. The pH of the mixture was adjusted to 7.5 using 0.01 M sodium carbonate, and the mixture was allowed to stand at 4 °C for 12 h. Subsequently, the mixture was lyophilized to obtain the hyaluronic acid-kidney-targeting peptide complex.

[0080] The infrared spectral characterization results of the hyaluronic acid-kidney-targeting peptide complex (HA-KTP) are as follows: Figure 11 As shown, in the infrared spectrum of the complex, the NH stretching vibration peak appears at 3325 cm⁻¹. -1 This was not found in maleimide-modified hyaluronic acid, indicating the addition of an NH structure. 2936 and 2868 cm⁻¹ -1 The peaks are attributed to the antisymmetric and symmetric stretching vibrations of methylene CH4, respectively, at 1682 cm⁻¹. -1 The peak is the C=O stretching vibration peak of amide I, at 1554 cm⁻¹. -1 The peak is the NH bending vibration peak of amide II, 1206 cm⁻¹. -1 The peaks are due to the CN stretching vibration; these are characteristic peaks of the targeting peptide. 1398 and 1312 cm⁻¹ -1 The peaks belong to the -COO antisymmetric stretching vibration and the symmetric stretching vibration, respectively, indicating that the carboxyl group in the original structure was not destroyed. These results demonstrate the successful synthesis of the hyaluronic acid-kidney-targeting peptide complex.

[0081] 3. Preparation and characterization of the final product gene delivery system The hyaluronic acid-kidney-targeting peptide complex was dissolved in enzyme-free water at a concentration of 1 mg / mL. Then, the hyaluronic acid-kidney-targeting peptide complex and the gene delivery complex (polyseminate-imine gene: Arginase-2 siRNA = 10:1) were mixed at a specific mass ratio (w: 10:1), vortexed for 30 seconds, and allowed to stand at 4°C for 0.5 hours to form the final core-shell nanostructure. Figure 12As shown, with the increase of the proportion of hyaluronic acid-renal-targeting peptide complex added, the particle diameter increases and the zeta potential gradually decreases, due to shell formation. When the mass ratio of hyaluronic acid-renal-targeting peptide complex, polysemine imine gene carrier, and Arginase-2 siRNA is fixed at 1:10:1, the potential reverses, forming mesoscale nanoparticles conforming to the renal transport principle. At this point, the particle size is approximately 300 nm, and the zeta potential is -2 mV. Figure 13 As shown, the spherical morphology of the final nanoparticles was observed using transmission electron microscopy.

[0082] Example 7: Detection of the activity of a gene delivery system loaded with Arginase-2 siRNA via a cellular pathway. This embodiment uses renal tubular epithelial cells (HK-2 cells) as experimental material. Normal HK-2 cells and those incubated with a gene delivery system loaded with Arginase-2 siRNA were treated using a vancomycin-induced HK-2 cell damage modeling method. Western blot was used to detect changes in Arginase-2 protein expression levels, and fluorescent probes were used to detect mitophagy and oxidative stress levels. The specific implementation method is as follows: 1. Establish a vancomycin-induced HK-2 cell damage model. The specific procedures are as follows: (1) Vaccination of 1×10 6 ~5×10 6 One HK-2 cell was cultured in each well of a 6-well plate containing 2 mL of complete culture medium; (2) Prepare 1990 µL of antibiotic-free complete culture medium containing 4 mM vancomycin, add it to the culture well, and gently shake well; (3) Add 10 µL of a gene delivery system containing 1 mg / mL Arginase-2 siRNA to the culture medium and shake gently. (4) Place the culture plate in a CO2 incubator at 37℃ and incubate for 24~96 h; Based on the inventors' previous work, the concentration of vancomycin added was determined to be 4 mM, and the cell culture time was 24-96 h.

[0083] Meanwhile, a blank control group (in which the vancomycin in step (2) and the gene delivery system in step (3) were replaced with the same volume of complete culture medium, and other conditions remained unchanged), a vancomycin-free group (in which the gene delivery system in step (3) was replaced with the same volume of complete culture medium, and other conditions remained unchanged), and a vancomycin-negative control group (in which the gene delivery system loaded with Arginase-2 siRNA in step (3) was replaced with a gene delivery system loaded with scramble siRNA of the same concentration and volume, and other conditions remained unchanged) were set up.

[0084] 2. Transfection level detection, the specific operation is as follows: Arginase-2 expression levels were detected using Western blot, and the results are as follows: Figure 14 As shown, the intervention of the gene delivery system loaded with Arginase-2 siRNA has a significant inhibitory effect on vancomycin-induced upregulation of Arginase-2 expression.

[0085] 3. Mitochondrial autophagy level detection, the specific operation is as follows: The colocalization concentration and colocalization of mitochondria and lysosomes within cells were simultaneously detected using Mito-Tracker Deep Red and Lyso-Tracker green fluorescent probes. The Mito-Tracker Deep Red and Lyso-Tracker green fluorescent probe kits were purchased from Beyotime Biotechnology Co., Ltd. The specific procedures are as follows: (1) According to the method of the corresponding kit, dilute Mito-Tracker Deep Red and Lyso-Tracker green with serum-free culture medium at a ratio of 1:20000 respectively; (2) Remove the cell culture medium from the 6-well plates of the four experimental groups in the above vancomycin-induced HK-2 cell damage model, add 1 mL of diluted Mito-Tracker Deep Red and Lyso-Tracker green; incubate in a cell culture incubator at 37°C for 30 min, and wash the cells three times with PBS buffer (pH 7.4) to fully remove fluorescent probes that have not entered the cells; (3) Detection and analysis were performed using a laser confocal imaging system (Leica microscopy system). The Mito-Tracker Deep Red used an excitation wavelength of 644 nm and an emission wavelength of 665 nm, while the Lyso-Tracker green used an excitation wavelength of 504 nm and an emission wavelength of 511 nm.

[0086] Test results as follows Figure 15 As shown, Figure 15 The results showed that intervention with a gene delivery system loaded with Arginase-2 siRNA could maximally reverse the vancomycin-induced decrease in mitochondrial autophagy in HK-2 cells.

[0087] 4. Oxidative stress level detection, the specific operation is as follows: Using DCFH-DA (Shanghai Beyotime Biotechnology Co., Ltd.), Dihydroethidium (Shanghai Beyotime Biotechnology Co., Ltd.), ONOO- (Beijing Bio-Rad Laboratories Co., Ltd.), and Mito-SOX (Invitrogen Molecular Probes) TM The fluorescent probe can simultaneously detect intracellular reactive oxygen species (ROS) levels, superoxide anion levels, peroxynitrosoanion levels, and mitochondrial superoxide levels.

[0088] (1) According to the method of the corresponding kit, take a small amount of DCFH-DA, Dihydroethidium, ONOO- and Mito-SOX fluorescent probes respectively, and dilute them with serum-free culture medium at a ratio of 1:1000 to form working solutions of each active oxygen / nitrogen probe with a final concentration of 10 μM / L.

[0089] (2) Remove the cell culture medium from the 6-well plate culture wells of the 4 experimental groups in the above vancomycin-induced HK-2 cell damage model, add 1 mL of diluted reactive oxygen / nitrogen probe working solution; incubate in a cell culture incubator at 37 ℃ for 20 min, and wash the cells 3 times with PBS buffer to fully remove fluorescent probes that have not entered the cells. (3) Detection and analysis were performed using the Operetta high-content imaging system (PerkinElmer). DCFH-DA used an excitation wavelength of 488 nm and an emission wavelength of 525 nm; Dihydroethidium used an excitation wavelength of 535 nm and an emission wavelength of 610 nm; Mito-SOX used an excitation wavelength of 510 nm and an emission wavelength of 580 nm.

[0090] Test results as follows Figure 16 As shown, the results indicate that intervention with a gene delivery system loaded with Arginase-2 siRNA can maximally reverse the dramatic increases in various reactive nitrogen / oxygen ratios in the vancomycin-induced HK-2 cell damage model.

[0091] Example 8: Studying the in vivo targeting of a kidney-targeted gene delivery system via animal pathways Wild-type mice were treated with different formulations, and the in vivo distribution patterns and ex vivo organ distribution of the formulations at different time points were observed using a small animal in vivo imaging system. The specific procedures are as follows: (1) Male C57BL / 6N mice (6-8 weeks old) were divided into three groups: free siRNA group, gene delivery system without targeted peptide modification (with hyaluronic acid as the shell structure), and gene delivery system with targeted peptide modification (siRNA in all groups was labeled with Cy-5 fluorescence).

[0092] (2) After treating wild-type mice with the above-mentioned preparations, the in vivo process and kidney distribution of the drug delivery system after intravenous administration were observed at 4 h, 12 h and 24 h using the IVIS® Lumina II bioluminescence in vivo imaging system. After 24 h, major organs (such as heart, liver, spleen, lung, kidney, brain, lymph and bone marrow, etc.) were taken for in vitro Cy-5 fluorescence intensity quantification to analyze the distribution of the drug delivery system in the major organs.

[0093] (3) Separate kidney tissue, cut tissue sections 3 μm thick, and observe the renal suborganization of the gene delivery system using immunofluorescence.

[0094] Cy-5 was tested using an excitation wavelength of 535 nm and an emission wavelength of 610 nm. The detection results are as follows: Figure 17 As shown in (AD), the results indicate that (A) the half-life of free siRNA in plasma is 0.18 h, while the half-life of the targeted peptide-modified gene delivery system in plasma is 8.06 h, showing that the gene delivery system has a longer blood circulation time; (BC) the targeted peptide-modified gene delivery system group has the highest kidney enrichment, and fluorescence intensity quantification shows that the kidney selectivity is more than 10 times that of the liver at 12 h; (D) immunofluorescence shows that the nanosystem is localized in the renal tubules.

[0095] Example 9: In vivo safety study of the kidney-targeted gene delivery system via animal route. Wild-type mice were treated with or without the gene delivery system. Major organs were harvested 24 hours later, and damage to these organs was assessed using HE staining. Orbital blood was collected to detect liver and kidney function and other blood biochemical indicators to evaluate systemic toxicity. The specific implementation method is as follows: (1) Male C57BL / 6N mice (6-8 weeks old) were divided into two groups: a control group and a targeted peptide modification gene delivery system group. Mice in the above groups were given saline or 60 mg / kg of gene delivery system via tail vein injection.

[0096] (2) After 24 hours, the heart, liver, spleen, lung and kidney were collected and the tissue sections were 3μm thick. HE staining and renal tubular injury score were used to evaluate the morphology and structure of the kidney tissue and renal tubules and the extent of damage. Orbital blood was collected and the serum creatinine, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels were detected by ELISA kit.

[0097] Test results as follows Figure 18As shown in (AF), the results indicate that after 24 h of treatment with the gene delivery system, no significant changes were found in (A) alanine aminotransferase, (B) aspartate aminotransferase, (C) alkaline phosphatase, (D) serum creatinine, and (E) blood urea nitrogen in mice; and (F) HE staining of the heart, liver, spleen, lungs, and kidneys showed no significant organ damage, indicating that the gene delivery system has good biosafety.

[0098] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0099] The above-described embodiments are merely examples of several implementations of the present invention, and should not be construed as limiting the scope of the present invention. The scope of the present invention is defined by the appended claims rather than the foregoing description. It can be applied to various fields suitable for the present invention. For those skilled in the art, other implementations and modifications can be easily implemented based on the content disclosed in this patent document. Therefore, all changes that do not depart from the equivalent implementations and modifications of the present invention and fall within the meaning and scope of the equivalent elements of the claims are within the protection scope of the present invention.

Claims

1. A kidney-targeted gene delivery system, characterized in that, The gene delivery system comprises a core structure and a shell structure; the core structure is a gene delivery complex, which is self-assembled from a spermidine polymer and siRNA; wherein the siRNA is Arginase-2 siRNA; the shell structure is a hyaluronic acid-kidney-targeting peptide complex; the mass ratio of the spermidine polymer, Arginase-2 siRNA, and hyaluronic acid-kidney-targeting peptide complex is 10:1:(1~2.5); wherein the spermidine polymer is formed by the condensation of spermidine monomers and cross-linking agent molecules; wherein the cross-linking agent is glyoxal, and the molar ratio of spermidine to glyoxal is 1:1~1:5; The hyaluronic acid-kidney-targeting peptide complex is formed by Michael addition of maleimide-modified hyaluronic acid with thiol groups in the kidney-targeting peptide.

2. The kidney-targeted gene delivery system according to claim 1, characterized in that, The molecular weight of the spermidine polymer is 9000 Da to 20000 Da.

3. The kidney-targeted gene delivery system according to claim 1, characterized in that, The preparation method of the spermidine polymer includes the following steps: dissolving spermidine in an organic solvent to obtain a primary solution; adding glyoxal solution to the primary solution under low temperature conditions and a protective gas atmosphere; stirring and heating to react and obtain a secondary solution; wherein the molar ratio of spermidine to glyoxal in the secondary solution is 1:1 to 1:1.5; filtering, dialyzing, and freeze-drying the secondary solution to obtain the spermidine polymer.

4. The kidney-targeted gene delivery system according to claim 3, characterized in that, The organic solvent is one or more of dimethyl sulfoxide, ethylenediamine, ethanol, and anhydrous methanol; the protective gas is one or more of nitrogen, carbon dioxide, helium, or argon.

5. The kidney-targeted gene delivery system according to claim 1, characterized in that, The mass ratio of the spermidine polymer, Arginase-2 siRNA, and hyaluronic acid-kidney-targeting peptide complex is 10:1:

1.

6. The kidney-targeted gene delivery system according to claim 1, characterized in that, The mass ratio of hyaluronic acid to kidney-targeting peptides is 1:1 to 1:

5.

7. The use of the kidney-targeted gene delivery system as described in any one of claims 1-6 in the preparation of drugs for the prevention and treatment of acute kidney injury.

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