Preparation method and application of renal tubular targeting siRNA delivery system mediated by gamma-glutamyltransferase

Abstract

The application discloses a preparation method and application of a kidney tubule-targeted siRNA delivery system mediated by gamma-glutamyltransferase, and belongs to the technical field of siRNA delivery systems.A kind of kidney tubule-targeted siRNA delivery system mediated by gamma-glutamyltransferase according to the application, kidney tubule-targeted siRNA delivery system siRNA-GSH mediated by GGT is prepared by biorthogonal reaction, and glutathione (GSH) which can be recognized and transported by kidney tubule area gamma-glutamyltransferase GGT is chemically coupled with methoxyl siRNA with a maleimide group.The application discloses a brand-new kidney tubule GGT-based targeted siRNA delivery system, which is expected to be used in the field of gene therapy of non-druggable kidney-related disease targets such as diabetic nephropathy and renal fibrosis, and provides a feasible scheme for solving the targeted siRNA treatment of kidney-related diseases.

Description

Technical Field

[0001] This invention relates to the field of siRNA delivery systems, and in particular to the preparation method and application of a renal tubular targeted siRNA delivery system mediated by γ-glutamyltransferase. Background Technology

[0002] The kidneys are vital metabolic organs, and kidney-related diseases significantly impact health and quality of life, especially for those requiring long-term medication or exhibiting irreversible progression (such as diabetic nephropathy and renal fibrosis), for which currently, no consistently effective drugs exist. Furthermore, the kidneys, as crucial metabolic organs, are also important intervention targets for some metabolic diseases (such as diabetes and hypertension). Small interfering ribonucleic acid (siRNA) is a promising nucleic acid drug that specifically degrades mRNA within target organ tissues and cells through base complementarity, thereby downregulating the expression levels of related proteins. However, siRNA lacks specific targeting capabilities in vivo and is easily degraded and rendered ineffective by nucleases. Therefore, efficient and specific in vivo delivery of siRNA drugs is crucial for their clinical application. Currently, chemical modification is a common method for improving siRNA drugs. Chemically modified siRNA (such as 2'-methoxy substitution or thiophosphate substitution) can significantly enhance its resistance to nuclease degradation. Meanwhile, by using chemical or physical methods to form nucleic acid complexes with siRNA, functional fragments (proteins, peptides, sugars, etc.) can be formed, which can greatly improve the problems of low tissue targeting, poor plasma pharmacokinetics, and difficulty in crossing cell barriers that are inherent to siRNA drugs. Furthermore, currently approved clinical siRNA drugs are all chemically modified nucleic acid complexes with functional fragments, indicating that constructing chemically modified siRNA delivery systems with superior performance is an important direction for the development of siRNA drugs.

[0003] Gamma-glutamyl transferase (GGT) is an important protein involved in the transport of glutathione (GSH) in the body, and it is highly expressed on the cell membranes of tissues with absorption and secretion functions. The kidney, as a vital metabolic organ, plays a crucial role in GSH reabsorption. The brush border of proximal renal tubular epithelial cells is the main distribution site of GGT in the kidney and also the site with the highest GGT content in the body. Under physiological conditions, approximately 90% of the filtered GSH in the primary urine (about 250 mg daily) is reabsorbed via GGT-mediated processes. Furthermore, GGT in the kidney can recognize and mediate the reabsorption of more than 80% of γ-glutamylated short peptides in the primary urine; this process is essential for maintaining the body's amino acid pool and antioxidant reserves. Therefore, GGT-mediated renal GSH reabsorption is a potential kidney-targeted delivery pathway and holds promise for kidney-specific siRNA delivery.

[0004] Currently, some studies have attempted to achieve targeted delivery of siRNA drugs to kidney tissue by constructing multifunctional nanodelivery systems (viral vectors, extracellular vesicles, liposomes, and polymer carriers, etc.). However, due to the size limitation of the basement membrane barrier of the kidney glomerulus (<6nm) and the high non-specific accumulation of most nanocarriers in organs such as the liver, the delivery efficiency of kidney-targeted siRNA delivery systems is low. There are no nanodelivery systems that directly deliver drugs to kidney microstructures (such as the microvilli region of the proximal renal tubule) in clinical trials.

[0005] Meanwhile, although only chemically modified siRNA complex delivery systems have passed clinical trials, all six siRNA drugs are liver-targeted delivery systems. Among them, five are chemically modified siRNAs conjugated with N-acetylgalactosamine (GalNAC) ligands that target hepatocytes. No siRNA drugs targeting kidney tissue have passed clinical trials. Therefore, finding a safe and effective kidney-targeted siRNA delivery technology remains a significant unresolved challenge. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing and applying a renal tubule-targeting siRNA delivery system mediated by γ-glutamyltransferase, in order to solve the problems of low delivery efficiency of renal-targeting siRNA delivery systems and the lack of nanodelivery systems that can directly deliver drugs to the renal microstructure.

[0007] To achieve the above objectives, on the one hand, the present invention provides a renal tubule-targeting siRNA delivery system mediated by γ-glutamyltransferase. The siRNA-GSH delivery system mediated by GGT is prepared by chemically coupling glutathione GSH, which can be recognized and transported by γ-glutamyltransferase GGT in the renal tubule region, with methoxy siRNA containing maleimide group through a bioorthogonal reaction.

[0008] The sequence of methoxysiRNA with a maleimide group is as follows:

[0009] The positive chain is 5'-Mal-mGmCmAmGmCmUmCmUmUmCmGmAmUmUmAmCmAmUmUdTdT-3', with the core nucleotide sequence shown in SEQ ID NO.1;

[0010] Antisense strand: 5'-mAmAmUmGmUmAmAmUmCmGmAmAmGmAmGmCmUmGmCdTdT-3', core nucleotide sequence as shown in SEQ ID NO.2;

[0011] Where m represents the 2'-methoxy modification of the corresponding nucleotide.

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned γ-glutamyltransferase-mediated renal tubular targeted siRNA delivery system, comprising the following steps:

[0013] S1. Dissolve reduced GSH in DEPC water to prepare a 1 mg / mL GSH reaction stock solution:

[0014] S2. Take the GSH reaction mother liquor prepared in S1, add DEPC water, add 0.1M NaOH solution to adjust the pH to 6.7-7.4, transfer to a volumetric flask and make up to volume to obtain a GSH reaction solution with a concentration of 0.1 mg / mL;

[0015] S3. Mix the methoxy siRNA solution with maleimide group and GSH reaction solution at a molar ratio of 1:10, vortex at 600 rpm and 4℃ for 3 h. After the reaction is completed, the reaction solution is ultrafiltered 3 times and centrifuged to obtain siRNA-GSH solution.

[0016] On the other hand, the present invention provides the application of the above-mentioned γ-glutamyltransferase-mediated renal tubular targeted siRNA delivery system in the preparation of drugs for treating kidney-related diseases, including diabetic nephropathy and renal fibrosis; the delivery system can specifically target proximal renal tubular epithelial cells and silence the expression of the target gene, which is sodium-glucose cotransporter 2.

[0017] Therefore, the preparation method and application of the γ-glutamyltransferase-mediated renal tubular targeted siRNA delivery system of the present invention have the following beneficial effects:

[0018] (1) The present invention uses maleimide-thiol click reaction to couple the GGT substrate glutathione (GSH) with chemically modified siRNA to prepare a renal tubular targeted siRNA delivery system (siRNA-GSH) mediated by renal GGT. The preparation method is simple and only requires one click reaction to obtain the product.

[0019] (2) Compared with unmodified naked siRNA, methoxy chemical modification can enhance the in vivo resistance to nuclease degradation of siRNA, so that siRNA can stably maintain its structure and function during in vivo circulation.

[0020] (3) siRNA-GSH modified with GSH as a GGT targeting ligand can effectively cross the glomerular basement membrane barrier and be recognized by GGT on the renal tubular epithelial cell membrane and transported into the proximal renal tubular cells, targeting and silencing the expression of target genes and proteins in the renal tubules.

[0021] (4) A single low-dose tail vein injection of this invention can target and silence the expression of sodium-glucose cotransporter 2 (Sglt-2) in renal tubular epithelial cells, with the silencing efficiency reaching its peak at 48 h. Compared with unmodified GSH meo-siRNA, the siRNA-GSH group can increase urinary glucose levels by more than 3 times in a single time period, and the cumulative urinary glucose level can reach more than 4 times in 72 h. In the GSH-depleted animal model, the siRNA-GSH group can increase urinary glucose levels by more than 6 times in a single time period, with the largest increase of 10 times in urinary glucose levels during the 24 to 48 h period. This indicates that GSH ligands can effectively improve the delivery efficiency of siRNA into renal tubular epithelial cells.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is the siRNA-GSH standard curve;

[0025] Figure 2HPLC analysis of siRNA-GSH and meo-siRNA;

[0026] Figure 3 This is Example 2 of the present invention, which is a live real-time fluorescence imaging test.

[0027] Figure 4 This is Test Example 2 of the present invention: 24-hour in vitro organ imaging analysis, where A is the 24-hour in vitro organ imaging and B is the fluorescence intensity of the in vitro organ after 24 hours.

[0028] Figure 5 Fluorescence imaging of kidney tissue sections for test case 3;

[0029] Figure 6 This study analyzed the antiserum degradation ability of siRNA-GSH; where A represents the gel imaging band; and B represents the fluorescence grayscale value of the gel imaging band.

[0030] Figure 7 The changes in Sglt-2 protein levels after transfection with the target sequence are shown; where A is the Western blot band and B is the protein expression level of Sglt-2.

[0031] Figure 8 Example 6 was used to analyze the degree of inhibition of the function of the target protein in cells by flow cytometry. Among them, A is the distribution of fluorescence intensity of 2-NBDG uptake by cells in different treatment groups detected by flow cytometry; B is the statistical graph of 2-NBDG uptake fluorescence intensity in cells in different treatment groups.

[0032] Figure 9 Analysis of urinary glucose levels at different time points for meo-siRNA and siRNA-GSH in normal and GSH-depleted mouse models;

[0033] Figure 10 Analysis of cumulative urinary glucose levels in the first 72 hours for meo-siRNA and siRNA-GSH in normal and GSH-depleted mouse models. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0037] Example 1

[0038] A method for preparing a GGT-mediated renal tubule-targeting siRNA delivery system (siRNA-GSH) includes the following steps:

[0039] S1. Preparation of glutathione (GSH) stock solution: Dissolve 10 mg of reduced GSH completely in a small beaker containing 5 mL of DEPC water, transfer to a 10 mL volumetric flask and make up to volume to prepare a 1 mg / mL GSH stock solution.

[0040] S2. Preparation of GSH reaction solution: Use a pipette to transfer 1 mL of GSH stock solution to a small beaker containing 4 mL of DEPC water. Then, add an appropriate amount of NaOH solution (0.1 M) to adjust the pH to 6.7. Finally, transfer the above solution to a 10 mL volumetric flask and make up to volume to prepare a 0.1 mg / mL GSH reaction solution.

[0041] Preparation and purification of S3 and siRNA-GSH:

[0042] Dissolve aliquots of lyophilized methoxysiRNA (meo-siRNA-Mal) powder with maleimide groups (2 OD, 5 nmol, 66 µg) in 100 µL of DEPC water, then add 153.4 µL of 0.1 mg / mL GSH reaction solution (10 eq, 50 nmol, 15.34 µg), and vortex at 600 rpm and 4 °C for 3 h. After the reaction is complete, transfer the reaction solution to a 500 µL 10 kDa ultrafiltration centrifuge tube, centrifuge (7250 rpm, 5000 g) for 15 min, repeat 3 times to obtain the siRNA-GSH solution.

[0043] The meo-siRNA-Mal sequence is:

[0044] Chain of Justice:

[0045] 5'-Mal-mGmCmAmGmCmUmCmUmUmCmGmAmUmUmAmCmAmUmUdTdT-3', the core nucleotide sequence is shown in SEQ ID NO.1;

[0046] antisense chain:

[0047] 5'-mAmAmUmGmUmAmAmUmCmGmAmAmGmAmGmCmUmGmCdTdT-3', the core nucleotide sequence is shown in SEQ ID NO.1;

[0048] Where m represents the 2'-methoxy modification of the corresponding nucleotide.

[0049] Test Example 1

[0050] The product siRNA-GSH was analyzed using an amino normal phase column (TOSOH, TSK-GEL Amide-80, 3µm; No. 0021867, 4.6mm ID×15cm).

[0051] The analytical conditions were as follows: Mobile phase A was 0.1 M triethylamine acetate buffer solution (pH 6.0); mobile phase B was acetonitrile; VA:VB = 4:1; flow rate was 0.8 mL / min, and isobaric elution was used. The fluorescence excitation wavelength was 360 nm, and the detection wavelength was 590 nm.

[0052] The sample pretreatment steps are as follows: The prepared siRNA-GSH and unmodified meo-siRNA were subjected to agarose gel electrophoresis for quantitative analysis. 2.5 nmol (1 OD, 33 µg) of meo-siRNA was dissolved in 250 µL of DEPC water, and then 10 µL was diluted to 100 µL to prepare a 1 µM meo-siRNA standard solution. 10 µL of each siRNA-GSH stock solution was diluted to 200 µL to prepare siRNA-GSH dilution buffers. Subsequently, 1, 2, 3, 4, and 5 µL of the meo-siRNA standard solution were diluted to 10 µL to obtain nucleic acid standard curve solutions. Similarly, 5 µL of each siRNA-GSH dilution buffer was diluted to 10 µL to prepare the siRNA-GSH quantitative analysis solution. Add 2µL of 5×loading buffer to the diluted standard solution and quantitative analysis solution, and perform electrophoresis at 120V for 10 min in a 2% agarose gel. Then, image and analyze the fluorescence intensity in the gel imaging system (Bio-Rad).

[0053] Quantitative analysis of siRNA-GSH standard curve, such as Figure 1 As shown, quantitative analysis revealed that the siRNA content per well of the siRNA-GSH sample was 2.59 pmol, which translates to a total product amount of 2.07 nmol, with a yield of 41.55%.

[0054] A HPLC test solution was prepared by diluting 50 pmol of siRNA-GSH and meo-siRNA stock solutions with DEPC water to 99 µL, followed by the addition of 1 µL of 100× SerRed (Servicebio) nucleic acid dye. SerRed dye does not exhibit fluorescence excitation in the absence of nucleic acid base pairs or under incomplete nucleic acid chains; however, when the dye is embedded within complete base pairs, SerRed emits strong 590 nm emission light under 360 nm excitation light. Based on this SerRed luminescence principle, the sample was analyzed.

[0055] Sample analysis results are as follows Figure 2 As shown, the results indicate that the elution time of unmodified meo-siRNA is around 7 min, while that of siRNA-GSH is around 5 min. This is because GSH cross-linking reduces the interaction between the siRNA backbone and the amino groups on the HPLC column, thus advancing the elution time of siRNA-GSH compared to the unmodified form. Integral area under the curve analysis shows that the purity of the siRNA-GSH stock solution after ultrafiltration purification is above 95%. This demonstrates that the method in Example 1 can successfully prepare the ligand-conjugated drug siRNA-GSH.

[0056] Test Example 2

[0057] The biodistribution of the GSH-modified fluorescent dye Cy7 was evaluated to verify its GGT-mediated targeted transport process. Cy7-labeled Cy7-GSH was prepared using the bioorthogonal reaction method described in Example 1. Cy7 and Cy7-GSH were injected via tail vein at concentrations with the same UV absorption intensity as Cy7, and their distribution in ICR mice and in vitro tissue distribution 24 hours after perfusion were observed. Furthermore, the renal GGT activity was inhibited and the in vivo GSH content was reduced (thus decreasing the concentration of GSH filtered into the renal tubules) by tail vein injection of the GGT-specific inhibitor GGsTop and intraperitoneal injection of diethyl maleate (DEM), respectively, thus verifying the correlation between GSH renal targeted delivery function and renal GGT activity.

[0058] Results of live real-time fluorescence imaging are as follows Figure 3 As shown, the results indicated that Cy7, after tail vein injection, can be metabolized by the kidneys, with its fluorescence intensity peaking at 30 minutes and being completely excreted from the body within 2 hours. In contrast, Cy7-GSH exhibited a significantly prolonged metabolic time in vivo, with its renal fluorescence signal persisting up to 24 hours, demonstrating a clear renal targeting phenomenon. In mice pre-injected with the GGT-specific inhibitor GGsTop (5 mg / kg), the renal accumulation of Cy7-GSH was significantly reduced and almost completely cleared after 2 hours.

[0059] 24-hour ex vivo organ imaging analysis results are as follows Figure 4As shown in the figures, A represents the imaging of isolated organs after 24 hours, and B represents the fluorescence intensity of isolated organs after 24 hours. The results show that under the same exposure conditions, no fluorescence accumulation of Cy7 was observed in any organ, indicating that it had been completely metabolized and cleared. However, Cy7-GSH exhibited a significant kidney-specific accumulation phenomenon. Simultaneously, the fluorescence of isolated kidneys treated with GGsTop showed a significant decrease, indicating that inhibiting GGT activity in kidney tissue reduces Cy7-GSH accumulation in the kidney. Conversely, the fluorescence of isolated kidneys treated with DEM showed a significant increase, indicating that reducing the concentration of free GSH in the renal tubules enhances Cy7-GSH accumulation in the kidney. In summary, in vivo fluorescence imaging analysis demonstrates that GSH has the ability to act as a kidney-targeted delivery ligand, and that renal GGT plays a crucial role in GSH ligand-mediated kidney targeting function.

[0060] Test Example 3

[0061] Kidney tissue from mice injected with Cy7 and Cy7-GSH 24 hours prior to test example 2 was frozen sectioned and analyzed under a microscope. The results are as follows: Figure 5 As shown in the figure, no fluorescence signal was observed in the kidney tissue of the Cy7 group mice, while the kidney tissue of the Cy7-GSH group mice showed significant fluorescence intensity, mainly distributed in the renal cortex region. This indicates that the targeted transport process of GGT in the kidney tissue mainly occurs in the proximal tubule region, which is also the site of highest GGT expression in vivo. No fluorescence signal was observed in the glomeruli, distal tubules, and vascular tissue structures, indicating that Cy7-GSH has been completely metabolized and cleared from the circulatory system, and there is no reabsorption and refiltration of Cy7-GSH. Therefore, the fluorescence in the proximal tubule region is emitted by Cy7-GSH within the epithelial cells, which proves that Cy7-GSH undergoes a significant intracellular targeted transport process in the proximal tubule region, verifying the function of GSH as a targeted ligand in the renal tubule region.

[0062] Test Example 4

[0063] Take 5 µL of the meo-siRNA standard solution and siRNA-GSH dilution buffer from Test Example 1 and dilute to 9 µL. Add 1 µL of fresh mouse serum and incubate at 37 °C for 0, 5, 30, and 60 min, respectively. Then add 2 µL of 5× loading buffer and perform electrophoresis at 120 V on a 2% agarose gel for 30 min. Imaging and fluorescence intensity analysis are then performed using a gel imaging system (Bio-Rad). The results are as follows: Figure 6 As shown in Part A of the diagram.

[0064] The siRNA-GSH was evaluated using the fluorescence grayscale values ​​of the gel imaging bands, and the results are as follows: Figure 6As shown in Part B, the results indicate that unmethoxylated siRNA was degraded by more than 95% by serum nucleases within 5 minutes, while methoxylated siRNA resisted nuclease degradation and maintained more than 75% nucleic acid integrity within 60 minutes. In contrast, due to the interaction between the siRNA-GSH ligand and serum, the bands were more dispersed than those of Meo-siRNA. However, grayscale analysis showed that the siRNA ligand-conjugated drug still possessed the ability to resist nuclease degradation and maintained more than 75% of the nucleic acid undegraded.

[0065] Test Example 5

[0066] The protein expression level of Sglt-2 was detected by Western blotting (WB). The following is a detailed procedure for cell seeding, administration, and WB:

[0067] (1) TCMK-1 cell seeding plate:

[0068] After washing the cultured cells twice with PBS, trypsin was added and the cells were digested at 37°C for 5 min. Then, complete culture medium was added to stop the digestion. The cell suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended and counted. The cell suspension was diluted to an appropriate concentration and then injected at 5 × 10⁻⁶ cells / mL. 4 Inoculate one cell per well into a 24-well plate, incubate for 24 hours, and then administer the drug.

[0069] (2) siRNA drug administration:

[0070] The candidate siRNA sequences were pretreated according to the instructions of the commercial reagent Lipo 3000, and then injected into serum-free medium at a concentration of 75 pmol / well for 6 h. After injection, the medium was replaced with complete medium and cultured for another 42 h, followed by Western blotting.

[0071] The specific steps for using WB are as follows:

[0072] ① Protein extraction: Discard the culture medium, wash the cells with PBS solution, add 100µL RIPA cell lysis buffer to each well, scrape the cells and transfer them to EP tubes, lyse on ice for 30 min, centrifuge at 12000 rpm and 4℃ for 10 min, collect the supernatant, measure the protein concentration with the BCA kit, add 5× protein loading buffer and heat at 95℃ for 10 min to denature the protein, centrifuge at 13000 rpm for 3 min and collect the supernatant.

[0073] ② Electrophoresis: Prepare a 10% polyacrylamide gel for electrophoresis, assemble the electrophoresis apparatus, load the protein samples in sequence, set the voltage to 80V for the stacking gel, and change the voltage to 120V after the samples enter the separating gel. After electrophoresis, remove the glass plate, remove the gel, and rinse it with water.

[0074] ③ Transfer: Activate the PVDF membrane by immersing it in anhydrous methanol for 2 minutes, then immerse it together with two sheets of filter paper in transfer buffer. Open the safety cover of the semi-dry transfer tank, remove the cathode device, and place the moistened filter paper, PVDF membrane, gel, and another moistened filter paper sequentially on the anode plate. Remove air bubbles with a roller, cover the cathode, and then close the safety cover of the device. Turn on the power and transfer at a constant current of 400mA for 20 minutes. After the transfer is complete, disassemble the transfer device and remove the PVDF membrane.

[0075] ④ Sealing: Immerse the PVDF membrane in 5% skim milk powder and seal it on a decolorizing shaker for 2 hours. After sealing, wash it 3 times with TBST solution for 1 minute each time.

[0076] ⑤ Incubate with primary antibody: Dilute the primary antibodies for Sglt-2 and GAPDH with TBST solution according to the instructions. Immerse the PVDF membrane in the primary antibody solution and shake overnight at 4°C. Recover the primary antibody solution and wash the PVDF membrane three times with TBST solution, 5 min each time.

[0077] ⑥ Secondary antibody incubation: Dilute the secondary antibody with TBST solution according to the instructions, immerse the PVDF membrane in the secondary antibody solution, and incubate on a shaker for 1 hour. Recover the secondary antibody solution, and wash the PVDF membrane three times with TBST solution, 5 minutes each time.

[0078] ⑦ Development: Place the PVDF membrane flat on plastic wrap. Mix liquids A and B from the ultrasensitive ECL chemiluminescence kit in a 1:1 ratio and evenly drop the mixture onto the PVDF membrane, ensuring the membrane is fully covered. Cover the PVDF membrane with plastic wrap and incubate for 1 minute. Then, develop the membrane using a developing apparatus.

[0079] The results of the WB experiment are as follows: Figure 7 As shown, A represents the Western blot (WB) band, and B represents the protein expression level of Sglt-2. Compared with the random sequence group and the normal cell group, the cellular protein level decreased by 50% after transfection with the target sequence siRNA, indicating that this sequence can effectively reduce the expression level of Sglt-2 within 48 hours.

[0080] Test Example 6

[0081] After administration of siRNA and culturing for 48 hours, the cells were incubated with a glucose fluorescent analog (2-NBDG) and then analyzed by flow cytometry. The specific experimental steps are as follows:

[0082] The siRNA administration procedure was the same as that in Test Example 5. Cells were incubated for 40 min in 100 µM 2-NBDG serum-free low-glucose medium, followed by washing three times with PBS. Trypsin was then added to prepare a cell suspension. The supernatant was discarded, and the cells were resuspended in PBS before flow cytometry analysis.

[0083] Flow cytometry analysis results as follows Figure 8 As shown, the intracellular fluorescence intensity was 5 × 10⁻⁶ in both the untransfected and randomly sequence-transfected groups after 2-NBDG administration. 3 The fluorescence intensity of cells transfected with the target sequence siRNA was approximately 25 times that of normal cells without 2-NBDG administration; while the fluorescence intensity of cells transfected with the target sequence siRNA was approximately 8 × 10⁻⁶. 2 The fluorescence level decreased by more than 80% compared to the random sequence group, indicating that the function of Sglt-2 protein was significantly inhibited. The target sequence can reduce the expression level of Sglt-2 protein while inhibiting its ability to take up glucose.

[0084] Test Example 7

[0085] In normal mice, glucose within the renal tubule lumen is transported to the renal tubular epithelial cells via the Sglt-2 transporter and then released back into the bloodstream, resulting in virtually undetectable glucose in mouse urine. However, when Sglt-2 expression in proximal renal tubular epithelial cells is targeted and silenced by siRNA, glucose reabsorption within the renal tubule lumen is inhibited, leading to the presence of glucose in mouse urine. Therefore, changes in urinary glucose levels can be used to evaluate the targeted silencing efficiency of siRNA-GSH on Sglt-2 in renal tubular cells. The specific experimental steps are as follows:

[0086] The siRNA-GSH stock solution, after quantitative analysis in Example 1, was diluted and added to 0.1 eq of 10×PBS. The solution was administered via tail vein injection to each ICR mouse at a dose of 1 nmol nucleic acid equivalent. Urine samples were collected at each time point and analyzed for glucose content using a biochemical analyzer. Simultaneously, a short-term plasma GSH depletion animal model was established by administering diethyl maleate (DEM) at a dose of 1 mg / kg. The same dose of siRNA-GSH was then administered via tail vein injection, and urine samples were collected at each time point and analyzed for glucose content using a biochemical analyzer.

[0087] Glucose content in mouse urine at different time points as follows Figure 9 As shown, unmodified methoxysiRNA, after tail vein injection, showed no or very low glucose levels in urine, indicating that although meo-siRNA has excellent anti-ribozyme capabilities, its renal targeting silencing efficiency is low.

[0088] In contrast, after tail vein injection of siRNA-GSH at the same dose, the amount of glucose in mice showed differences after 12 hours, and the difference increased significantly to more than three times during the 24-48 hour period. This indicates that GSH ligand modification significantly improved the targeted silencing efficiency of meo-siRNA on the kidney.

[0089] Furthermore, the siRNA-GSH group showed a significant increase in urinary glucose in a short-term plasma GSH depletion mouse model, with the maximum increase being more than 10 times that of the meo-siRNA group. This indicates that a low GSH environment can further enhance the uptake of siRNA-GSH by renal GGT, thereby improving the renal targeting ability and gene silencing effect of siRNA-GSH. In contrast, there was no statistically significant difference in urinary glucose between the meo-siRNA group and the normal mouse model in the GSH depletion mouse model, indicating that a low GSH environment does not promote the production of non-GGT-targeted meo-siRNA.

[0090] Analysis of the cumulative urinary glucose levels over the first 72 hours yielded the following results: Figure 10 As shown in the figure. The results showed that within 72 hours, the average cumulative urinary glucose level in the meo-siRNA group was only 5 µmol, while the average cumulative urinary glucose level in the siRNA-GSH group was 22 µmol, which was 4 times that of the meo-siRNA group. In the GSH exhaustion model, the average cumulative urinary glucose level in the siRNA-GSH group was significantly increased to 44 µmol, which was twice that of the normal mouse model.

[0091] In summary, siRNA-GSH exhibits significantly enhanced renal-targeted gene silencing ability compared to meo-siRNA, suggesting that the strategy of conjugating siRNA to GGT substrates can serve as a novel renal-targeted siRNA delivery system.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kidney tubule targeting siRNA delivery system mediated by gamma-glutamyl transferase, characterized by: A siRNA delivery system targeting renal tubules mediated by gamma glutamyltransferase GGT, siRNA-GSH is prepared by bio-orthogonal reaction of glutathione GSH, which can be recognized and transported by gamma glutamyltransferase GGT in the renal tubule region, and methoxy siRNA with a maleimide group; The methoxy siRNA sequence with a maleimide group is as follows: Positive strand: 5'-Mal-mGmCmAmGmCmUmCmUmUmCmGmAmUmUmAmCmAmUmUdTdT-3', the core nucleotide sequence is shown as SEQ ID NO. 1; Negative strand: 5'-mAmAmUmGmUmAmAmUmCmGmAmAmGmAmGmCmUmGmCdTdT-3', the core nucleotide sequence is shown as SEQ ID NO. 2; Wherein, m represents 2'-methoxy modification of the corresponding nucleotide.

2. A method for preparing the tubular targeting siRNA delivery system mediated by γ-glutamyltransferase according to claim 1, characterized by, The method comprises the following steps: S1, dissolve reduced GSH in DEPC water to prepare a GSH reaction mother liquor with a concentration of 1 mg / mL; S2, take the GSH reaction mother liquor prepared in S1, add DEPC water, drop 0.1M NaOH solution to adjust pH to 6.7-7.4, transfer to a volumetric flask and dilute to volume to obtain a GSH reaction solution with a concentration of 0.1 mg / mL; S3, mix the methoxy siRNA solution with a maleimide group and the GSH reaction solution according to a molar ratio of 1:10, vortex at 600 rpm and 4℃ for 3h, after the reaction is completed, the reaction solution is ultrafiltrated and centrifuged for 3 times to obtain the siRNA-GSH solution.

3. Use of the kidney targeting siRNA delivery system mediated by γ-glutamyltransferase according to claim 1 for the preparation of a medicament for the treatment of a kidney related disease, characterized in that: The kidney-related diseases include diabetic nephropathy and renal fibrosis; The delivery system can specifically target proximal tubular epithelial cells of the kidney, silence the expression of target genes, and the target gene is sodium-glucose cotransporter 2.

Images