A nucleic acid delivery system targeting pancreatic beta cells and uses thereof

Abstract

The present application relates to the technical field of biological medicine and gene therapy, and particularly relates to a nucleic acid delivery system targeting pancreatic beta cells and application thereof. The nucleic acid delivery system comprises a nanocomplex formed by a cationic polymer modified with a targeting ligand and siRNA; the targeting ligand is a GLP-1 receptor targeting peptide; the cationic polymer is a PEI-PFA polymer; and the siRNA is siRNA targeting DYRK1A. The present application provides a novel pancreatic beta cell targeting delivery platform for gene therapy of type 2 diabetes. The platform realizes beta cell function regeneration by precisely regulating DYRK1A gene expression, and has targeting, safety and high efficiency, thereby providing a new strategy with broad clinical application prospect for diabetes treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and gene therapy, and in particular to a nucleic acid delivery system targeting pancreatic β cells and its application. Background Technology

[0002] Type 2 diabetes accounts for approximately 90% of all diabetes cases. The pathological essence of type 2 diabetes stems from two interrelated core defects: insulin resistance in peripheral tissues and a relative deficiency in insulin secretion due to insufficient compensation by pancreatic β-cells. Although various drugs, such as insulin sensitizers and insulin secretagogues, are widely used clinically, most existing therapies focus on controlling blood glucose levels and fail to fundamentally address the core issue of reduced β-cell numbers and dysfunction. Approximately 70% of diabetic patients fail to achieve ideal blood glucose control targets. Therefore, developing regenerative therapeutic strategies that can repair endogenous β-cell function and restore functional β-cell communities has become a cutting-edge direction in type 2 diabetes treatment research.

[0003] In recent years, dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) has attracted widespread attention as a potential target for promoting β-cell regeneration. DYRK1A is a widely expressed serine / threonine kinase in vivo that negatively regulates β-cell cycle progression through multiple mechanisms. On one hand, DYRK1A phosphorylates and activates T cell nuclear factor family transcription factors, promoting NFAT exocytosis and inactivation, thereby terminating the transcription of cell cycle-activating genes. On the other hand, DYRK1A can directly phosphorylate the Thr286 site of cyclin Cyclin D1, leading to Cyclin D1 degradation and cell cycle arrest. Recent studies have also found that DYRK1A kinase activity promotes the assembly of the DREAM complex, a key transcriptional repressor complex for maintaining β-cell quiescence; inhibition of DYRK1A promotes the conversion of the DREAM complex to the proliferative MMB complex, thereby driving β-cells into the cell cycle. Based on the above mechanisms, small molecule DYRK1A inhibitors (such as Harmine and Leucettinib-92) have been proven to induce the proliferation of human and rodent pancreatic β-cells in vitro and in vivo, and improve glycemic homeostasis in diabetic animal models. However, existing small molecule inhibitor strategies still face multiple technical bottlenecks: First, most DYRK1A inhibitors have significant off-target effects. For example, Harmine also potently inhibits monoamine oxidase MAO-A, which may cause adverse neuropsychiatric reactions. Second, there is a separation between the differentiation-proliferation-promoting effects of different DYRK1A inhibitors. Studies have found that although gene silencing DYRK1A can induce β-cell proliferation, it cannot upregulate the expression of key β-cell functional marker genes such as PDX1, MAFA, and NKX6.1, suggesting that simple pharmacological inhibition of DYRK1A is difficult to simultaneously achieve β-cell proliferation and functional maturation. Third, systemic administration of small molecule inhibitors lacks tissue specificity and poses a potential risk of extrapancreatic organ toxicity.

[0004] To overcome these limitations, combination therapy strategies have been proposed. Recent studies have shown that the combination of DYRK1A inhibitors and GLP-1 receptor agonists can achieve a 4-7 fold increase in β-cell clusters in immunodeficient mouse models of human islet transplantation and effectively reverse streptozotocin-induced hyperglycemia. This synergistic effect is partly mediated by the pro-islet hormone VGF, involving multiple mechanisms including β-cell proliferation, enhanced function, and improved survival. However, GLP-1 receptor agonists also face clinical translational challenges such as a short in vivo half-life, the need for frequent injections, and the risk of immunogenicity (38% of type 2 diabetes patients develop drug resistance antibodies after using exenatide).

[0005] Nanocarrier-based targeted delivery systems offer a new technological approach to addressing the aforementioned challenges. GLP-1 receptor-targeting peptides and their analogues (such as exenatide-4 and liraglutide) have been successfully conjugated to the surfaces of various nanocarriers, enabling pancreatic targeted imaging or drug delivery by specifically recognizing the highly expressed GLP-1 receptor on pancreatic β-cells. Studies have shown that exendin-4-modified hyaluronic acid-polycaprolactone nanocapsules can increase β-cell enrichment efficiency by approximately 400% in a non-obese diabetic severe combined immunodeficiency mouse model. A research team from the Czech Academy of Sciences also reported the use of magnetic nanoparticles modified with the GLP-1 analogue liraglutide for pancreatic β-cell magnetic resonance imaging, confirming the high efficiency and specificity of the GLP-1 receptor-targeting peptide-mediated active targeting strategy in vitro and in vivo. However, existing GLP-1 receptor-targeting nanosystems primarily focus on diagnostic imaging or small molecule drug delivery; their application to the targeted delivery of nucleic acid drugs (especially siRNA) to achieve precise silencing of key pathogenic genes on β-cells has not yet been systematically reported.

[0006] RNA interference (siRNA) technology, through the specific silencing of target gene expression using small interfering RNA (siRNA), has opened a new dimension for gene therapy in type 2 diabetes. Compared to small molecule inhibitors, siRNA exhibits higher target specificity, lower off-target risk, and can achieve gene silencing against "undruggable" targets. However, the clinical translation of siRNA has long been hampered by in vivo delivery bottlenecks: free siRNA is easily degraded by serum ribozymes, rapidly cleared by the kidneys, has poor cell membrane penetration, and lacks tissue targeting capabilities. While cationic polymers such as polyethyleneimine (PEI) can electrostatically complex siRNA and promote endosome escape, the excessively strong positive charge on the surface of unmodified PEI makes it prone to non-specific binding with blood components, triggering hemolytic reactions, complement activation, and reticuloendothelial system capture, severely limiting its in vivo application. Therefore, there is an urgent need to develop novel multifunctional nanodelivery platforms that combine targeted delivery capabilities, high gene silencing efficacy, and excellent biocompatibility to achieve a technological leap from "small molecule inhibition" to "precise gene silencing" in the DYRK1A targeted intervention strategy.

[0007] In summary, the existing technologies in the field of β-cell regeneration therapy for type 2 diabetes still have the following technical problems that urgently need to be solved: (1) Small molecule DYRK1A inhibitors have inherent defects such as off-target toxicity and separation of proliferative / differentiation effects; (2) Although GLP-1 receptor agonist combination therapy has significant efficacy, it lacks pancreatic targeting and has the risk of immunogenicity; (3) There is no effective pancreatic β-cell-specific delivery system for siRNA-based DYRK1A gene silencing strategies; (4) Existing cationic polymer nanocarriers have poor biocompatibility, high risk of hemolysis, and are difficult to meet the safety requirements for intravenous injection. Currently, there are no reports of organically combining GLP-1 receptor-targeting peptide active targeting modification, PEI-PFA cationic carrier, and DYRK1A-targeting siRNA to construct a nanodelivery system with targeting, safety, and efficient gene silencing function, and using it for gene therapy of type 2 diabetes. Summary of the Invention

[0008] The purpose of this invention is to provide a nucleic acid delivery system targeting pancreatic β cells and its application.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a nucleic acid delivery system targeting pancreatic β cells, comprising a nanocomposite formed by a cationic polymer modified with a targeting ligand and siRNA;

[0011] The targeting ligand is a GLP-1 receptor-targeting peptide;

[0012] The cationic polymer is a PEI-PFA polymer;

[0013] The siRNA is a siRNA that targets DYRK1A.

[0014] Preferably, in the PEI-PFA polymer, PEI and PFA are coupled through an acylation reaction.

[0015] Preferably, the GLP-1 receptor-targeting peptide is coupled to the PEI-PFA polymer via a cross-linking agent.

[0016] Preferably, the crosslinking agent is Sulfo-SMCC.

[0017] Preferably, the sequence of the siRNA targeting DYRK1A is shown in SEQ ID NO.2.

[0018] Preferably, the nanocomposite has a particle size of 50–100 nm and a zeta potential of +20–+60 mV; the targeted ligand-modified cationic polymer and siRNA form the nanocomposite through electrostatic self-assembly, wherein the N / P ratio is 5–10:1.

[0019] The present invention provides a pharmaceutical composition comprising the aforementioned nucleic acid delivery system targeting pancreatic β cells, and pharmaceutically acceptable excipients.

[0020] The present invention provides the use of the aforementioned nucleic acid delivery system targeting pancreatic β cells or the aforementioned pharmaceutical composition in the preparation of a medicament for treating type 2 diabetes.

[0021] Furthermore, the drug is administered via intravenous injection, and the delivery system enriches pancreatic islet β cells through active targeting mediated by GLP-1 receptor-targeting peptides. By silencing DYRK1A gene expression, it promotes β cell proliferation, inhibits apoptosis, and restores glucose-stimulated insulin secretion function, thereby improving metabolic abnormalities associated with type 2 diabetes.

[0022] Preferably, the treatment of type 2 diabetes includes at least one of the following: lowering blood glucose, improving glucose tolerance, improving insulin sensitivity, and promoting pancreatic β-cell proliferation.

[0023] This invention provides a method for preparing a PEI-PFA polymer modified with a GLP-1 receptor-targeting peptide, comprising the following steps:

[0024] (1) In the presence of a crosslinking agent, PEI and PFA are subjected to an acylation reaction to obtain a PEI-PFA polymer; the crosslinking agent is EDC and NHS;

[0025] (2) The PEI-PFA polymer was reacted with maleimide crosslinking agent to obtain Mal-PEI-PFA;

[0026] (3) The GLP-1 receptor-targeting peptide was reacted with Mal-PEI-PFA to obtain GLP-1-PEI-PFA.

[0027] Specifically, the synthesis method of the GLP-1-PEI-PFA polymer includes the following steps:

[0028] Synthesis of S1 and PEI-PFA: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and perfluorobutyric acid (HFA) were dissolved in 1 mL of methanol solution at a molar ratio of 3:3:1. After stirring for 1 h, PEI (mol ratio of HFA to 2 mL of methanol) was slowly added dropwise while stirring, until the final total volume was 3 mL. The stirring speed was maintained at 800 rpm throughout the process, and the reaction was allowed to proceed overnight for 12 h. The reaction solution was then transferred to a 3.5 kDa dialysis bag and dialyzed for 48 h, with the dialysate (ddH2O) replaced every 2 h. After dialysis, the solution was frozen at -80 ℃ for 3 h, and then freeze-dried using a vacuum freeze dryer for more than 24 h to obtain a transparent, viscous solid PEI-PFA.

[0029] Synthesis of S2 and Mal-PEI-PFA: 50 mg of PEI-PFA was dissolved in 2 mL of methanol solution, and 10.91 mg of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). The Sulfo-SMCC solution was slowly added dropwise to the PEI-PFA solution while stirring, and the reaction was allowed to proceed overnight for 16 h. The reaction solution was then transferred to a 3.5 kDa dialysis bag and dialyzed for 12 h, with the water changed every 2 h. After dialysis, the solution was frozen at -80 ℃ for 3 h, and then freeze-dried using a vacuum freeze dryer for 35-36 h to obtain a white, fluffy solid, Mal-PEI-PFA.

[0030] Synthesis of S3 and GLP-1-PEI-PFA: 30.923 mg of Mal-PEI-PFA was dissolved in 1 mL of ddH2O, and 10 mg of GLP-1 receptor-targeting peptide was dissolved in 1 mL of ddH2O. The GLP-1 solution was slowly added dropwise to the Mal-PEI-PFA solution, with a total volume of 2 mL. The mixture was stirred while being added, and the reaction was allowed to proceed overnight for 16 h. After the reaction was completed, the mixture was frozen at -80 ℃ for 3 h, and then freeze-dried in a vacuum freeze dryer for 30-33 h to obtain a white, fluffy solid GLP-1-PEI-PFA polymer.

[0031] Furthermore, this invention identifies the structures of Mal-PEI-PFA and GLP-1-PEI-PFA using 1H NMR spectroscopy, confirming that the GLP-1 receptor-targeting peptide is successfully linked to Mal-PEI-PFA. Full-wavelength scanning reveals that both GLP-1-PEI-PFA and GLP-1 exhibit characteristic absorption peaks at 280 nm, further confirming the successful linkage between the GLP-1 receptor-targeting peptide and Mal-PEI-PFA.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention presents the first design and synthesis of a GLP-1-PEI-PFA polymer, which, through modification with a GLP-1 receptor-targeting peptide, endows it with the ability to actively target pancreatic β-cells, specifically recognizing and binding to the highly expressed GLP-1 receptor on the surface of β-cells. Experimental results show that the GLP-1-PEI-PFA@siDYRK1A nanocomposite significantly enhanced the internalization efficiency of MIN6 cells in cellular uptake experiments. Clear Cy5 fluorescence signals were observed in the cytoplasmic region under laser confocal microscopy, and the cell nucleus diameter was significantly increased. This confirms that the active targeting mediated by the GLP-1 receptor-targeting peptide effectively improves the targeting recognition and entry capability of the nanodelivery system into pancreatic β-cells, laying a crucial foundation for subsequent efficient gene delivery.

[0034] The GLP-1-PEI-PFA polymer of this invention complexes DYRK1A-targeting siRNA via electrostatic interactions to form a GLP-1-PEI-PFA@siDYRK1A nanocomposite. This nanocomposite exhibits uniform particle size and moderate surface charge, effectively protecting siRNA from nuclease degradation, significantly reducing the risk of hemolysis, and efficiently delivering siRNA into the cytoplasm of pancreatic β-cells. In vitro gene silencing experiments showed that this nanocomposite could knock down DYRK1A mRNA levels by approximately 80% and significantly downregulate protein expression. Furthermore, Ki67 and EdU staining confirmed that it significantly promoted β-cell proliferation under both normal and glycolipid toxicity stress conditions, demonstrating excellent gene delivery efficiency and biological activity.

[0035] This invention provides a novel pancreatic β-cell-targeted delivery platform for gene therapy of type 2 diabetes. In vivo pharmacodynamic studies confirmed that the GLP-1-PEI-PFA@siDYRK1A nanocomposite significantly reduced random blood glucose, improved oral glucose tolerance and insulin sensitivity, increased serum insulin levels, and reduced glycated hemoglobin in a type 2 diabetic mouse model, without significantly affecting mouse body weight. This platform achieves β-cell functional regeneration through precise regulation of DYRK1A gene expression, combining targeting, safety, and high efficiency, providing a new strategy with broad clinical application prospects for diabetes treatment. Attached Figure Description

[0036] Figure 1 This is a synthetic route diagram for the polymer GLP-1-PEI-PFA.

[0037] Figure 2 The light absorption curves of GLP-1, Mal-PEI-PFA, and GLP-1-PEI-PFA in the wavelength range of 250-450 nm are shown.

[0038] Figure 3 The cytotoxicity of PEI, PEI-PFA, and GLP-1-PEI-PFA was detected by MTT assay. MIN6 cells were treated with five different concentrations of PEI, PEI-PFA, and GLP-1-PEI-PFA for 48 h, and their cell viability was then assessed.

[0039] Figure 4 The results of hemolysis experiments on PEI, PEI-PFA and GLP-1-PEI-PFA are shown. (a) is the result of the hemolysis experiment, and (b) is the hemolysis rate.

[0040] Figure 5 Agarose gel electrophoresis images of siRNA complexed under different N / P conditions are shown. (a) is the PEI-PFA group, (b) is the Mal-PEI-PFA group, and (c) is the GLP-1-PEI-PFA group.

[0041] Figure 6 The results of the potential and particle size experiments of the complexes are shown in (a) and (b) are the particle sizes of the polymers PEI-PFA, GLP-1-PEI-PFA, PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA.

[0042] Figure 7To evaluate the protective effect of PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA nanocomplexes on siRNA in a nuclease environment. Lanes 1-2: Free siRNA group. Lane 1 is the negative control without RNase A and SDS treatment; siRNA remains intact and a band is observed. Lane 2 is the control with RNase A and without SDS treatment; free siRNA is degraded by RNase A and no band is observed. Lanes 3-5: PEI-PFA@siRNA complex group. Lane 3 is the control without RNase A and without SDS treatment; the complex is intact, and siRNA is encapsulated by the vector and cannot migrate, resulting in no band. Lane 4 is the control without RNase A and with SDS treatment; SDS dissociates the complex, and the released siRNA is detectable, resulting in a band. Lane 5 is the control with RNase A and SDS treatment; the complex is first treated with RNase A and then dissociated with SDS to evaluate the protective ability of the PEI-PFA complex on siRNA, and a band is observed. Lanes 6-8: GLP-1-PEI-PFA@siRNA complex. Lane 6 was without RNase A and without SDS treatment; the complex was intact and showed no band. Lane 7 was without RNase A and with SDS treatment; the SDS dissociated the complex, and the released siRNA was detectable, showing a band. Lane 8 was with RNase A and with SDS treatment; the protective ability of the GLP-1-PEI-PFA complex against siRNA was evaluated, and a band was observed.

[0043] Figure 8 Transmission electron microscopy image of GLP-1-PEI-PFA@siDYRK1A.

[0044] Figure 9 The fluorescence intensity of Cy5 cells was analyzed by flow cytometry after incubation of GLP-1-PEI-PFA@siDYRK1A and MIN6 cells for 12 and 24 h.

[0045] Figure 10 The results are shown in the laser confocal microscopy observations. (a) The cell uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite and MIN6 cells after 12 h of incubation is observed by laser confocal microscopy. (b) Quantitative analysis of cell nucleus diameter in each group.

[0046] Figure 11 To analyze the expression level of the DYRK1A gene in MIN6 cells after incubation of GLP-1-PEI-PFA@siDYRK1A with MIN6 cells for 24 h using RT-qPCR.

[0047] Figure 12The results show the protein expression levels. (a) shows the expression level of DYRK1A protein in MIN6 cells after 24 h of incubation with GLP-1-PEI-PFA@siDYRK1A by Western Blot analysis, and (b) shows the quantitative analysis results. 1, 2, 3, 4, and 5 represent the blank control group (Blank), the free siRNA group (siDYRK1A), the intermediate vector group (PEI-PFA@siDYRK1A), the optimal vector group (GLP-1-PEI-PFA@siDYRK1A), and the positive control commercial liposome group (LNP@siDYRK1A), respectively.

[0048] Figure 13 The effect of GLP-1-PEI-PFA@siDYRK1A nanocomposite on MIN6 cell proliferation was detected using Ki67 staining. The left panel shows the Ki67 staining results after cells were treated with different treatment groups (siDYRK1A concentration of 100 nM) for 6 h, followed by 48 h of culture in DMEM low-glucose medium containing 10% FBS. The right panel shows the Ki67 staining results after cells were treated with different treatment groups (siDYRK1A concentration of 100 nM) for 6 h, followed by 48 h of culture in DMEM high-glucose, high-lipid medium containing 10% FBS, which is toxic to glycolipids. Proliferating cells were labeled with Ki67 staining (red), and cell nuclei were labeled with DAPI (blue). Cell proliferation was observed using confocal microscopy.

[0049] Figure 14 The image shows the effect of EdU staining on the proliferation of MIN6 cells. (a) is a representative image of EdU staining in each group of cells under normal culture conditions, (b) is a representative image of EdU staining in each group of cells under glycolipid toxic stimulation conditions, (c) is the quantitative analysis result of the EdU positive cell rate in each group under normal culture conditions, and (d) is the quantitative analysis result of the EdU positive cell rate in each group under glycolipid toxic stimulation conditions.

[0050] Figure 15 The results of the in vivo pharmacodynamic study of GLP-1-PEI-PFA@siDYRK1A nanocomposite in treating a mouse model of type 2 diabetes are as follows: (a) the curves showing the changes in body weight of mice in each group; (b) the curves showing the changes in blood glucose levels randomly; (c) the blood glucose curves of mice in each group after the intraperitoneal glucose tolerance test (IPGTT) following treatment; (d) the quantitative analysis of the area under the blood glucose curve (AUC) of the IPGTT following treatment; (e) the blood glucose curves of mice in each group after the insulin tolerance test (ITT) following treatment; (f) the quantitative analysis of the area under the blood glucose curve (AUC) of the ITT following treatment; (g) the results of the glycated hemoglobin test of mice in each group after treatment; and (h) the results of the insulin test of mice in each group. Detailed Implementation

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] The material information used in the following examples is shown in Table 1.

[0053] Table 1. Names and Manufacturers of Experimental Materials

[0054]

[0055] Example 1: Synthesis, characterization and biocompatibility of delivery carriers PEI-PFA and GLP-1-PEI-PFA polymer

[0056] 1 Experimental Methods

[0057] 1.1 Synthesis of PEI-PFA and GLP-1-PEI-PFA polymers

[0058] 1.1.1 Synthesis of PEI-PFA

[0059] (1) Dissolve EDC, NHS and HFA in methanol solution in a molar ratio of 3:3:1. That is, weigh 115 mg of EDC and 69 mg of NHS in 1.5 mL EP tube, dissolve them in 200 μL of methanol solution respectively, mix them, transfer the mixture to a clean brown glass bottle, add 26 μL of HFA to the mixture, and make up the system to 1 mL with methanol solution. The reaction is carried out in the dark for 1 h.

[0060] (2) Weigh 1 g of PEI (PEI to HFA molar ratio of 1:2) using a 4 mL EP tube and add 2 mL of methanol solution to dissolve it.

[0061] (3) Slowly add (2) to (1) while stirring. The final total volume is 3 mL. Stir at 800 rpm throughout the process and react overnight for 12 h.

[0062] (4) After the reaction is complete, the solution is transferred to a pre-treated dialysis bag and dialyzed for 12 h using ultrapure water as the dialysate. The dialysate is changed every 2 h during the reaction, and the reaction lasts for 48 h.

[0063] (5) After dialysis, transfer the reaction solution to a 50 mL centrifuge tube and freeze it at -80 ℃ for 3 h.

[0064] (6) After freezing, place the sample in a vacuum freeze dryer and freeze dry for more than 24 hours. After freeze drying, the sample is a transparent viscous solid. Weigh the obtained solid and store it at 4°C or use it for subsequent experiments.

[0065] 1.1.2 Synthesis of Mal-PEI-PFA

[0066] (1) Weigh 50 mg of PEI-PFA into a 4 mL EP tube, add 2 mL of methanol solution and sonicate to dissolve it.

[0067] (2) Weigh 10.91 mg of Sulfo-SMCC into a 4 mL EP tube, add 1 mL of DMSO and sonicate to dissolve it;

[0068] (3) Transfer (1) to a clean, transparent glass vial, slowly add (2) dropwise to (1) while stirring, and make up the total volume with methanol solution to 4 mL. Stir at 800 rpm throughout the process and react overnight for 16 h.

[0069] (4) After the reaction is complete, the solution is transferred to a pre-treated dialysis bag and dialyzed for 12 h using ultrapure water as the dialysate. The dialysate is changed every 2 h during the reaction, and the reaction lasts for 12 h.

[0070] (5) After dialysis, transfer the reaction solution to a 50 mL centrifuge tube and freeze it at -80 ℃ for 3 h.

[0071] (6) After freezing, the sample is placed in a vacuum freeze dryer and freeze-dried for 35-36 h. After freeze-drying, the sample is a white fluffy solid, which is the Mal-PEI-PFA polymer. The obtained solid is weighed and stored at -20 ℃ or used for subsequent experiments.

[0072] 1.1.3 Synthesis of GLP-1-PEI-PFA

[0073] (1) Weigh 30.923 mg of the above-obtained Mal-PEI-PFA polymer into a 1.5 mL EP tube, add 1 mL of ultrapure water and sonicate to dissolve it.

[0074] (2) Weigh 10 mg of glucagon-like peptide-1 (GLP-1) and dissolve it in 1 mL of ultrapure water. The amino acid sequence of GLP-1 is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC (as shown in SEQ ID NO.1).

[0075] (3) Slowly add solution (2) to solution (1) while stirring. The total volume is 2 mL. Let it react overnight for 16 h.

[0076] (4) After the reaction is complete, transfer the reaction solution to a 50 mL centrifuge tube and freeze it at -80 ℃ for 3 h.

[0077] (5) After freezing, place the sample in a vacuum freeze dryer and freeze dry for 30-33 h. After freeze drying, the sample is a white fluffy solid, which is the GLP-1-PEI-PFA polymer. Weigh the obtained solid and store it at -20 ℃ or for subsequent experiments.

[0078] 1.2 Full-wavelength scanning characterization of GLP-1, Mal-PEI-PFA and GLP-1-PEI-PFA polymers

[0079] Take 500 μL of each of 2.5 mg / mL GLP-1, Mal-PEI-PFA, and GLP-1-PEI-PFA and place them in a cuvette. Use ddH2O as a blank control. Scan the wavelength range of 250-450 nm using a full-wavelength UV-Vis spectrophotometer and record the light absorption curves of each sample.

[0080] 1.3 Cytotoxicity detection of PEI, PEI-PFA, and GLP-1-PEI-PFA

[0081] The MTT assay was used to investigate the toxic effects of different concentrations of PEI, PEI-PFA, and GLP-1-PEI-PFA polymers on MIN6 cells. After digestion and counting of MIN6 cells in logarithmic growth phase, the cells were divided into groups of 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL in 96-well plates and cultured at 37°C in a 5% CO2 incubator until cell adhesion occurred. Polymer stock solutions (10 mg / mL) of PEI, PEI-PFA, and GLP-1-PEI-PFA were prepared using DEPC water and serially diluted with fresh medium containing 10% FBS to final concentrations of 0, 125, 250, 500, 1000, and 2000 nM. The original medium in each well was discarded, and different concentrations of polymer solution (100 μL per well) were added, with three replicates per group. Incubation continued for 48 h. The drug solution was discarded, and 100 μL of fresh serum-free medium and 20 μL of MTT solution (5 mg / mL) were added to each well. After incubation in the dark for 3 h, the liquid in the wells was discarded, and 100 μL of DMSO was added to each well. The plates were then shaken for 8 min in a microplate reader, and the absorbance at 490 nm was measured to calculate cell viability.

[0082] 1.4 Detection of erythrocyte hemolysis by PEI, PEI-PFA, and GLP-1-PEI-PFA

[0083] Fresh anticoagulated whole blood from mice was collected and centrifuged at 2000 rpm for 10 min. The upper plasma and leukocyte layers were discarded, and the basal red blood cell pellet was retained. The red blood cells were resuspended by gently pipetting with 3-5 times the volume of pre-cooled physiological saline, centrifuged again, and the supernatant was discarded. The washing was repeated 3-4 times until the supernatant was clear. Two portions of the washed red blood cell pellet were taken and mixed with 9 portions of physiological saline to prepare a 2% red blood cell suspension. PEI, PEI-PFA, and GLP-1-PEI-PFA polymers were diluted with physiological saline to concentration gradients of 2.5, 5, and 10 μM, respectively. 900 μL of each polymer solution was added to 100 μL of the 2% red blood cell suspension and gently mixed. Physiological saline was used as a negative control, and deionized water as a positive control. The mixture was incubated at 37℃ for 1 h, gently shaking to mix every 15 min. After incubation, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant was transferred to a 96-well plate. The absorbance (OD value) was measured at 545 nm. Each group has 3 replicates, and the experiment is repeated 3 times. The hemolysis rate is calculated using the following formula:

[0084]

[0085] 2 Results and Analysis

[0086] 2.1 Synthesis of PEI-PFA and GLP-1-PEI-PFA polymers

[0087] like Figure 1 As shown, under the action of EDC and NHS crosslinking agents, PEI and HFA were acylated to obtain PEI-PFA, which is the first step of the reaction; the second step is to use Sulfo-SMCC as a crosslinking agent to react with the amino group of PEI on the PEI-PFA polymer to obtain maleimide-functionalized Mal-PEI-PFA polymer; then the thiol group at the C-terminus of GLP-1 peptide is reacted with the maleimide group of Mal-PEI-PLA polymer to obtain GLP-1-PEI-PFA polymer.

[0088] 2.2 Full-wavelength scanning characterization of GLP-1-PEI-PFA polymer

[0089] like Figure 2As shown, the free GLP-1 peptide exhibits a characteristic protein absorption peak at 280 nm with an absorbance of 0.536, indicating that the GLP-1 peptide has characteristic UV absorption at this wavelength. The Mal-PEI-PFA polymer generally shows low absorbance values ​​and no obvious characteristic absorption peak in the 250-450 nm range. However, the GLP-1-PEI-PFA polymer shows a distinct characteristic absorption peak at 280 nm with an absorbance of 0.133. Its peak shape is basically the same as that of the free GLP-1 peptide, but the absorbance value is lower, which may be due to the relative concentration dilution after the GLP-1 peptide is coupled to the Mal-PEI-PFA carrier. These results indicate that the GLP-1 receptor-targeting peptide was successfully ligated to the Mal-PEI-PFA carrier, and the GLP-1-PEI-PFA polymer was successfully prepared.

[0090] 2.3 Cytotoxicity detection of PEI, PEI-PFA, and GLP-1-PEI-PFA polymers

[0091] As polymer concentration increased, the survival rates of both cell types decreased with increasing concentration of the three polymers, exhibiting concentration-dependent cytotoxicity. Figure 3 At the same concentration, the unmodified PEI polymer exhibited the greatest cytotoxicity, reducing the survival rate of MIN6 cells to approximately 10.87% at a concentration of 2000 nM. The PEI-PFA polymer showed reduced toxicity compared to PEI, while the GLP-1-PEI-PFA polymer exhibited the lowest toxicity, maintaining a MIN6 cell survival rate of approximately 76.10% at a concentration of 2000 nM. In the low concentration range (125-500 nM), GLP-1-PEI-PFA had the least impact on MIN6 cell survival, with cell viability remaining above 90%. These results indicate that GLP-1 receptor-targeting peptide modification significantly reduced the cytotoxicity of PEI-PFA polymers and improved their biocompatibility.

[0092] 2.4 Detection of erythrocyte hemolysis by PEI, PEI-PFA and GLP-1-PEI-PFA

[0093] As the polymer concentration increased, the hemolysis rate in all groups showed an increasing trend. Figure 4 (a) and Figure 4(b) Unmodified PEI polymers exhibited high hemolysis rates at all concentrations, exceeding 5% at 2.5 μM and reaching as high as 24.97% at 10 μM, indicating poor blood compatibility. The hemolysis rate of PEI-PFA polymers was lower than that of PEI, but significant hemolysis still occurred at higher concentrations (5, 10 μM). GLP-1-PEI-PFA polymers showed the lowest hemolysis rates at all concentrations, consistently below 5%, significantly lower than the PEI and PEI-PFA groups at the same concentrations. These results indicate that GLP-1 peptide modification significantly improved the blood compatibility of PEI-PFA polymers and effectively reduced the risk of hemolysis.

[0094] Example 2: Complexation ability of GLP-1-PEI-PFA polymer on siRNA and characterization of GLP-1-PEI-PFA@siRNA nanocomposite

[0095] The siDYRK1A sequence used in this embodiment (shown in SEQ ID NO.2) is: S: GGAUGGAUCGGUAUGAAAUTT; AS: AUUUCAUACCGAUCCAUCCTT (shown in SEQ ID NO.3, where U is replaced with T in the sequence list), manufactured by GenePharma.

[0096] 1 Experimental Methods

[0097] 1.1 Detection of RNA complexation ability of PEI-PFA and GLP-1-PEI-PFA polymers

[0098] PEI-PFA, Mal-PEI-PFA, and GLP-1-PEI-PFA were mixed with siDYRK1A (5 μM) at different N / P ratios (0.5:1, 1:1, 2:1, 3:1, 5:1, 7.5:1, 10:1, 12.5:1, and 20:1) in EP tubes and incubated at room temperature for 30 min to allow for complete complexation. Then, DEPC water was added to bring the total volume to 10 μL, and 6× DNA Loading buffer was added and mixed thoroughly. A 1% agarose gel (containing nucleic acid dye) was prepared, and each sample was subjected to gel electrophoresis. The electrophoresis results were recorded using a gel imaging system to evaluate the complexation ability of each vector with siDYRK1A at different N / P ratios.

[0099] 1.2 Zeta potential and particle size determination of PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA nanocomposites

[0100] Based on the above experimental results, PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA polymers were prepared at N / P ratios of 5:1 and 7.5:1, respectively, and particle size and potential were measured using a Malvern laser particle size analyzer.

[0101] 1.3 RNA protection of PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA nanocomplex

[0102] PEI-PFA and GLP-1-PEI-PFA were mixed with siRNA at N / P ratios of 5:1 and 7.5:1, respectively. After complexation at room temperature for 30 min, DEPC water was added to make up to 10 μL of the mixture. 1 μL of RNase A (1 mg / mL) was added to the corresponding tube, followed by shaking and centrifugation, and incubation at 37 ℃ for 2 h. After incubation, 2 μL of 6× DNA Loading buffer or 6× DNA Loading buffer containing 1% SDS was added to the corresponding tube according to the grouping requirements. 1% agarose gels (containing nucleic acid dyes) were prepared, and samples from each group were subjected to gel electrophoresis. The results were recorded using a gel imaging system to evaluate the protective ability of different nanocomplexes against siRNA in the presence of RNase A.

[0103] 1.4 Transmission electron microscopy of GLP-1-PEI-PFA@siDYRK1A

[0104] GLP-1-PEI-PFA and siDYRK1A were complexed at a N / P ratio of 7.5:1 for 30 min. The mixture was then slowly added dropwise to a copper grid and allowed to stand at room temperature for 2-5 min. Excess liquid was then absorbed with filter paper. Phosphotungstic acid solution was added for staining for 20 s, and excess stain was absorbed with filter paper. The sample was then allowed to air dry at room temperature. The sample was observed under a transmission electron microscope, and multiple fields of view were randomly selected for photographing. Representative images were recorded.

[0105] 2 Results and Analysis

[0106] 2.1 Gel retardation assay for the complexation conditions of polymers PEI-PFA, Mal-PEI-PFA, and GLP-1-PEI-PFA with siRNA

[0107] like Figure 5 As shown in (a), when the N / P ratio of PEI-PFA to siRNA is 0.5:1 to 3:1, different degrees of free siRNA bands can be seen, indicating that the complexation is incomplete; when the N / P ratio reaches 5:1 or above, the siRNA bands completely disappear and are all retained in the sample well, indicating that PEI-PFA can completely complex siRNA when the N / P ratio is ≥5:1. Figure 5 (b) and Figure 5(c) shows that Mal-PEI-PFA and GLP-1-PEI-PFA can completely complex with siRNA when the N / P ratio is ≥7.5:1. These results indicate that the three polymers PEI-PFA, Mal-PEI-PFA, and GLP-1-PEI-PFA can all effectively complex with siRNA, with minimum N / P ratios of 5:1, 7.5:1, and 7.5:1, respectively, required for complete complexation.

[0108] 2.2 Potential and particle size of PEI-PFA, GLP-1-PEI-PFA, PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA nanocomposites

[0109] Figure 6 (a) Particle size distribution results showed that the hydrated particle size of the PEI-PFA blank vector was 78.44±2.90 nm, and after complexation with siRNA, the particle size of PEI-PFA@siRNA increased to 95.81±1.51 nm. The hydrated particle size of the GLP-1-PEI-PFA blank vector was 82.66±0.55 nm, and after complexation with siRNA, the particle size of GLP-1-PEI-PFA@siRNA was 89.86±2.87 nm. The particle size distribution of all nanoparticles showed a unimodal distribution, indicating good dispersibility.

[0110] Figure 6 (b) Zeta potential measurements showed that the PEI-PFA blank vector carried a strong positive charge with a potential of 73.96 ± 7.28 mV. After complexing with the negatively charged siRNA, the potential of PEI-PFA@siRNA significantly decreased to 24.91 ± 0.69 mV. The potential of the GLP-1-PEI-PFA blank vector was 58.15 ± 0.44 mV, significantly lower than that of unmodified PEI-PFA, indicating that the modification of the GLP-1 peptide effectively shielded part of the positive charge of PEI. After complexing with siRNA, the potential of GLP-1-PEI-PFA@siRNA further decreased to 47.33 ± 0.71 mV.

[0111] The results showed that the prepared PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA nanocomposites both had suitable nanoscale particle sizes (80-100 nm) and positively charged surfaces, which are beneficial for cellular uptake and subsequent in vivo application.

[0112] 2.3 RNA protection of PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA nanocomplex

[0113] like Figure 7As shown, the band of free siRNA completely disappeared after incubation with RNase A for 2 h, indicating that free siRNA is highly susceptible to ribozyme degradation. However, after treatment with RNase A under the same conditions, clear siRNA bands were still observed after the addition of SDS to dissociate the complexes of PEI-PFA@siRNA and GLP-1-PEI-PFA@siRNA. These results demonstrate that the PEI-PFA and GLP-1-PEI-PFA vectors can effectively protect siRNA from ribozyme degradation and significantly enhance its enzymatic stability, laying the foundation for subsequent in vivo applications.

[0114] 2.4 Transmission electron microscopy characterization of GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0115] like Figure 8 As shown, the prepared nanocomposite exhibits spherical or near-spherical particles under an electron microscope. The particles are uniform in size, regular in morphology, and well-dispersed, with no obvious aggregation. The diameter of the nanoparticles in multiple random fields of view ranged from approximately 50 to 80 nm, which is consistent with the hydrated particle size determined by dynamic light scattering. These results demonstrate the successful preparation of the GLP-1-PEI-PFA@siDYRK1A nanocomposite, exhibiting good uniformity and suitable nanoscale particle size.

[0116] Example 3: Study on the delivery efficiency of siRNA by polymer GLP-1-PEI-PFA

[0117] 1 Experimental Methods

[0118] 1.1 Detection of cellular uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0119] 1.1.1 Flow cytometry analysis of cell uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0120] After digesting and counting MIN6 cells in the logarithmic growth phase, the cells were divided into groups of 5 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well in 12-well plates and cultured at 37 °C in a 5% CO2 incubator. Free siDYRK1A, PEI-PFA@siDYRK1A, and GLP-1-PEI-PFA@siDYRK1A (using Cy5-labeled siDYRK1A) were prepared in the dark and replenished with DMEM basal medium. The original medium was discarded, and serum-free DMEM basal medium containing GLP-1-PEI-PFA@siDYRK1A nanocomplex with Cy5-labeled siRNA was added to each well. Free siDYRK1A and PEI-PFA@siDYRK1A nanocomplex served as controls, and a blank control group was also included. Six hours after drug administration, FBS was added to bring the final concentration to 10%, and the final concentration of siRNA to 50 nM. After 12 h and 24 h of incubation, the drug-containing medium was discarded, and the cells were washed twice with pre-cooled PBS. Cells were collected by digesting them with an appropriate amount of trypsin in each well, resuspending them in PBS, filtering them through a 200-mesh sieve, and then detecting the fluorescence intensity of the Cy5 channel using flow cytometry (excitation wavelength 633 nm, emission wavelength 660 nm). 10,000 cells were collected from each sample, and the average fluorescence intensity was analyzed using FlowJo software to assess the cell internalization efficiency of the nanocomposite.

[0121] 1.1.2 Observation of cell uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite using laser confocal microscopy

[0122] After digesting and counting MIN6 cells in the logarithmic growth phase, the cells were divided into groups of 2.5 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well on 12-well spreaders and cultured in a 37 ℃, 5% CO2 incubator. Free siDYRK1A, PEI-PFA@siDYRK1A, and GLP-1-PEI-PFA@siDYRK1A (using Cy5-labeled siDYRK1A) were prepared in the dark. The group without siRNA served as the blank control, and commercial liposomes served as the positive control (LNP@siDYRK1A). After treatment with the drug for 12 h as described in "1.1.1 Flow Cytometry Detection of Cell Uptake of GLP-1-PEI-PFA@siDYRK1A Nanocomplex", the culture medium was discarded, and the cells were washed three times with 1 mL of pre-cooled PBS each time. 1 mL of wheat germ lectin (WGA, 5 μg / mL) was added to each well, and the cells were incubated at room temperature in the dark for 20 min to label the cell membrane; the staining solution was discarded, and the cells were washed three times with PBS. Add 1 mL of 4% paraformaldehyde to each well and fix for 10 min at room temperature; discard the fixative and wash three times with PBS. Add 500 μL of 0.1% Triton X-100 to each well and perforate the membrane for 10 min at room temperature; discard the perforation solution and wash three times with PBS. Add 400 μL of DAPI staining solution (10 μg / mL) to each well and incubate at room temperature in the dark for 5 min to label cell nuclei; discard the staining solution and wash three times with PBS. Mount with anti-fluorescence quencher and fix with nail polish. Observe using a laser confocal microscope. DAPI excitation wavelength is 405 nm (emission wavelength 460 nm), WGA excitation wavelength is 488 nm (emission wavelength 520 nm), and Cy5 excitation wavelength is 633 nm (emission wavelength 660 nm). Multiple fields of view are randomly selected to acquire images to evaluate the distribution and internalization of the nanocomplex in cells. 200 cells are selected from each group, and ImageJ is used to quantitatively analyze the nucleus diameter of each group.

[0123] 1.2 Detection of DYRK1A knockout in cells using GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0124] 1.2.1 RT-qPCR analysis of DYRK1A gene expression level in cells treated with GLP-1-PEI-PFA@siDYRK1A

[0125] After digesting and counting MIN6 cells in the logarithmic growth phase, the cells were divided into groups of 5 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 mcg / mL in 12 wells and cultured at 37 °C in a 5% CO2 incubator. Free siDYRK1A, PEI-PFA@siDYRK1A, and GLP-1-PEI-PFA@siDYRK1A were prepared. The group without siRNA served as the blank control (Blank), and commercial liposomes served as the positive control (LNP@siDYRK1A). After treatment with siRNA for 24 h as described in "1.1.1 Flow Cytometry Detection of Cell Uptake of GLP-1-PEI-PFA@siDYRK1A Nanocomposite," total RNA was extracted from the cells using a high-purity RNA extraction kit according to the manufacturer's instructions. TransScript ®II All-in-One First-Strand cDNA Synthesis Supermix reverse transcription to cDNA. (Using...) PerfectStart PCR analysis was performed using ®Green qPCR SuperMix on a QuantStudio™ 5 Real-Time PCR instrument according to the manufacturer's instructions, specifically: 94 ℃ pre-denaturation for 30 s, 94 ℃ denaturation for 5 s, 58 ℃ annealing for 15 s, 72 ℃ extension for 10 s, for 45 cycles. Each sample was analyzed in triplicate. Primers were purchased from Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 2 below.

[0126] Table 2. Primer sequences for RT-qPCR detection of the DYRK1A gene.

[0127]

[0128] 1.2.2 Western Blot analysis of DYRK1A protein expression level in cells treated with GLP-1-PEI-PFA@siDYRK1A

[0129] After digesting and counting MIN6 cells in the logarithmic growth phase, the cells were divided into groups of 2.5 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 μL in 6-well plates and cultured overnight at 37 °C in a 5% CO2 incubator to allow them to adhere. Free siDYRK1A, PEI-PFA@siDYRK1A, and GLP-1-PEI-PFA@siDYRK1A were prepared. The group without siRNA served as the blank control (Blank), and commercial liposomes served as the positive control (LNP@siDYRK1A). After treatment with the drug as described in "1.1.1 Flow Cytometry Detection of Cell Uptake of GLP-1-PEI-PFA@siDYRK1A Nanocomplex", the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. A suitable amount of trypsin was added to each well to digest and collect the cells. The pellet was resuspended in cell lysis buffer and lysed on ice for 30 min, vortexing every 10 min during lysis. The cells were centrifuged at 4 °C and 12,000 rpm for 15 min, and the supernatant was collected as total protein. After determining protein concentration using the BCA method, an equal volume of protein sample was added to 5×SDS loading buffer and heated in a 100 °C metal bath for 10 min to denature the protein. Samples from each group were separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane using wet transfer. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. After blocking, the membrane was washed three times with TBST for 10 min each time, and DYRK1A primary antibody (dilution 1:700) and β-actin internal control primary antibody (dilution 1:10000) were added, followed by incubation overnight at 4 °C. The next day, the membrane was washed three times with TBST for 10 min each time, and HRP-labeled secondary antibody (dilution 1:5000) was added, followed by incubation at room temperature for 2 h. After washing three more times with TBST, ECL chemiluminescence was used for color development, and the bands were recorded using a gel imaging system. ImageJ software was used for quantitative analysis of the band gray values, and the relative expression level of DYRK1A protein was calculated using β-actin as an internal control.

[0130] 2 Results and Analysis

[0131] 2.1 Cellular uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0132] 2.1.1 Flow cytometry analysis of cell uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0133] Quantitative analysis results of the mean fluorescence intensity (MFI) of Cy5 are as follows: Figure 9 As shown, the fluorescence intensity of cells treated with GLP-1-PEI-PFA@siDYRK1A was significantly higher than that of the PEI-PFA@siDYRK1A group at both 12 h and 24 h (** p <0.01, *** pThe fluorescence intensity of both groups increased in a time-dependent manner (<0.001). GLP-1 modification significantly enhanced the internalization efficiency of the nanocomposite in β cells, laying the foundation for its efficient targeted delivery of siRNA.

[0134] 2.1.2 Observation of cell uptake of GLP-1-PEI-PFA@siDYRK1A nanocomposite using laser confocal microscopy

[0135] like Figure 10 As shown in Figure a, no Cy5 fluorescence signal was detected in the blank control group (Blank) and the free siDYRK1A group, indicating no siRNA internalization. No obvious Cy5 fluorescence signal was also observed in the PEI-PFA@siDYRK1A group and the positive control LNP@siDYRK1A group, suggesting low internalization efficiency of the unmodified vector. However, clear Cy5 red fluorescence signal was observed in the GLP-1-PEI-PFA@siDYRK1A treated group, mainly distributed in the cytoplasm, and well co-localized with cell membrane WGA staining and nuclear DAPI staining, significantly higher than in other groups. Image J quantitatively analyzed the nuclear diameter of each group. Figure 10 The results showed that the nuclear diameter of cells in the Blank control group was approximately 6.12 μm; the nuclear diameter of cells in the free siDYRK1A group and the PEI-PFA@siDYRK1A group was not significantly different from that in the Blank group; the nuclear diameter of cells in the LNP@siDYRK1A group was slightly increased to approximately 7.01 μm; while the nuclear diameter of cells in the GLP-1-PEI-PFA@siDYRK1A treatment group was significantly increased to 9.01 μm, which was significantly higher than that of the other groups (***p<0.001).

[0136] 2.2.1 RT-qPCR analysis of DYRK1A gene expression level in cells treated with GLP-1-PEI-PFA@siDYRK1A

[0137] The expression levels of the DYRK1A gene in cells after different treatments are as follows: Figure 11 As shown. In MIN6 cells, the relative expression level of DYRK1A mRNA in the blank control group (Blank) was set to 1. The expression of DYRK1A mRNA in the free siDYRK1A group decreased slightly, the expression in the PEI-PFA@siDYRK1A treatment group decreased to approximately 0.43, while the expression in the GLP-1-PEI-PFA@siDYRK1A treatment group significantly decreased to approximately 0.20, with a knockdown efficiency of approximately 80%, showing a highly significant difference compared to the blank group and other treatment groups (***). p<0.001). In the LNP@siDYRK1A positive control group, DYRK1A mRNA expression decreased to approximately 0.25, with a knockdown efficiency of about 75%, which was not significantly different from the GLP-1-PEI-PFA@siDYRK1A group. The GLP-1-PEI-PFA@siDYRK1A nanocomposite was able to efficiently silence DYRK1A gene expression in MIN6 cells, with a knockdown efficiency significantly superior to the unmodified vector group and comparable to the positive control LNP group.

[0138] 2.2.2 Western Blot analysis of DYRK1A protein expression level in cells treated with GLP-1-PEI-PFA@siDYRK1A

[0139] The expression levels of DYRK1A protein in cells after different treatments are as follows: Figure 12 (a) and Figure 12 As shown in (b). Compared with the blank control group, there was no significant change in DYRK1A protein expression in the free siDYRK1A group; DYRK1A protein expression in the PEI-PFA@siDYRK1A group decreased slightly, with a relative expression level of 0.88±0.05; while DYRK1A protein expression in the GLP-1-PEI-PFA@siDYRK1A treatment group was significantly reduced, with a relative expression level of 0.56±0.06, and a knockdown efficiency of approximately 44%, which was significantly different from the blank group (**). p <0.01). In the LNP@siDYRK1A positive control group, DYRK1A protein expression decreased to 0.43±0.08, with a knockdown efficiency of approximately 57%, which was not statistically different from the GLP-1-PEI-PFA@siDYRK1A treatment group. The results indicate that the GLP-1-PEI-PFA@siDYRK1A nanocomposite can efficiently silence DYRK1A expression at the protein level, comparable to the positive control LNP@siDYRK1A group.

[0140] Example 4: Evaluation of the biological activity of GLP-1-PEI-PFA@siDYRK1A nanocomposite

[0141] 1 Experimental Methods

[0142] 1.1 Ki67 staining to detect the effect of GLP-1-PEI-PFA@siDYRK1A nanocomplex on β-cell proliferation

[0143] After digesting and counting MIN6 cells in the logarithmic growth phase, the cells were divided into groups of 5 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well on 12-well spreaders and cultured at 37°C in a 5% CO2 incubator. Free siDYRK1A, PEI-PFA@siDYRK1A, GLP-1-PEI-PFA@siDYRK1A, and GLP-1-PEI-PFA@siNC (siNC sequence: S: UUCUCCGAACGUGUCACGUTT; AS: ACGUGACACGUUCGGAGAATT, manufactured by GenePharma) (SEQ ID NO. 8-9) DMEM basal medium was prepared and supplemented. The original medium was discarded, and serum-free DMEM basal medium containing the siRNA-containing GLP-1-PEI-PFA@siDYRK1A nanocomplex was added to each well. Free siDYRK1A, PEI-PFA@siDYRK1A, and GLP-1-PEI-PFA@siNC nanocomplexes were used as controls, and a blank control group was also set up. Six hours after administration, DMEM basal medium containing FBS (final concentration of 10%) or DMEM high-sugar high-lipid medium (simulating glycolipid toxicity) was added, with a final concentration of 100 nM for siRNA.

[0144] Forty-eight hours after drug administration, the culture medium was discarded, and staining was performed according to the Beyotime Ki67 cell proliferation assay kit (immunofluorescence method, red, rabbit monoclonal antibody) instructions. The slides were mounted with an anti-fluorescence quencher and fixed with nail polish. Observation was performed using a laser confocal microscope. Ki67 cells exhibited red fluorescence (excitation wavelength 550 nm, emission wavelength 570 nm), and the cell nuclei showed blue fluorescence (excitation wavelength 364 nm, emission wavelength 454 nm).

[0145] 1.2 EdU staining to detect the effect of GLP-1-PEI-PFA@siDYRK1A nanocomplex on MIN6 cell proliferation.

[0146] The administration method was the same as in "1.1 Ki67 staining to detect the effect of GLP-1-PEI-PFA@siDYRK1A nanocomposite on β-cell proliferation". Forty-eight hours after administration, the culture medium was discarded, and staining was performed according to the BBI EdU cell proliferation assay kit instructions. Slides were mounted with an anti-fluorescence quencher and fixed with nail polish. Observation was performed using a laser confocal microscope. EdU-positive cells showed red fluorescence (excitation wavelength 541 nm, emission wavelength 567 nm), and cell nuclei showed blue fluorescence (excitation wavelength 350 nm, emission wavelength 461 nm). At least 200 cells were randomly selected from each group, and the EdU-positive cell rate was calculated (number of EdU-positive cells / total number of cells × 100%).

[0147] 1.3 In vivo pharmacodynamic study of GLP-1-PEI-PFA@siDYRK1A nanocomposite in a mouse model of type 2 diabetes

[0148] 1.3.1 Model Construction and Drug Administration

[0149] Male C57BL / 6J mice, aged 6-8 weeks and weighing 18-22 g, were selected and housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50-60%, with a 12-hour light-dark cycle and free access to food and water. After one week of acclimatization, the mice were randomly divided into a control group and a model group. Mice in the model group were fed a high-fat diet (containing 60% fat) for 4 weeks to induce insulin resistance. After 4 weeks, the mice in the model group were fasted for 12 hours but allowed free access to water, and then intraperitoneally injected with streptozotocin (STZ, prepared with 0.1 M citrate buffer, pH 4.5 before use) at a dose of 40 mg / kg for 4 consecutive days. Seven days after the last injection, fasting blood glucose was measured by tail vein sampling. Mice with a random blood glucose level ≥16.7 mmol / L that remained stable for one week were considered to have successfully developed type 2 diabetes and were included in subsequent experiments.

[0150] Mice were randomly divided into 5 groups (n=3 per group) based on blood glucose and body weight: a model control group (T2DM), a free siDYRK1A group, a PEI-PFA@siDYRK1A group, a GLP-1-PEI-PFA@siDYRK1A group, and a normal control group (Control). Mice in all groups received siDYRK1A at a dose of 1 mg / kg via tail vein injection every 3 days for a total of 5 administrations (days 0, 3, 6, 9, and 12). The Control and T2DM groups received an equal volume of saline. Mice in all groups continued to be fed the appropriate diet during the administration period.

[0151] 1.3.2 Weight and Random Glucose Monitoring

[0152] Mice in each group were weighed and their weight recorded before administration (day 0) and on days 2, 5, 8, 11, 14, 17, 20, 23, 26, and 29 after administration. Blood was also collected from the tail vein at the above time points, and random blood glucose levels were measured using a glucometer. Weight change curves and random blood glucose change curves were plotted.

[0153] 1.3.3 Intraperitoneal glucose tolerance test (IPGTT)

[0154] On day 21 after drug administration, mice in each group were fasted for 12 hours but allowed free access to water. Fasting blood glucose (0 min) was measured by blood collection from the tail vein. Glucose solution (2 g / kg) was injected intraperitoneally, and blood glucose values ​​were measured by blood collection from the tail vein at 15, 30, 60, 90, and 120 min after injection. Blood glucose-time curves were plotted, and the area under the curve (AUC) was calculated.

[0155] 1.3.4 Insulin Tolerance Test (ITT)

[0156] Six days after the IPGTT test, mice in each group were fasted for 6 hours but allowed free access to water. Blood was collected from the tail vein to measure basal blood glucose (0 min). Insulin (0.75 U / kg) was injected intraperitoneally, and blood glucose levels were measured from the tail vein at 15, 30, 60, 90, and 120 min after injection. Blood glucose-time curves were plotted, and the area under the curve (AUC) was calculated.

[0157] 1.3.5 Serum ELISA was used to detect glycated hemoglobin (GHb) and insulin (INS) in mice of each group.

[0158] Thirty days after drug administration, mice were sampled. Blood was collected and incubated overnight at 4°C, then centrifuged at 3000 rpm for 15 min at 4°C to separate serum, which was then stored at -80°C for later use. Glycated hemoglobin (GHb) and insulin (INS) in each group of mice were detected using ELISA, following the instructions of the Lunchangshuo ELISA kit.

[0159] 2 Results and Analysis

[0160] 2.1 Ki67 staining to detect the effect of GLP-1-PEI-PFA@siDYRK1A nanocomposite on β-cell proliferation

[0161] like Figure 13As shown in the left and right figures, the Ki67 red fluorescence signal in the blank control group (Blank) was weak; there was no significant difference in Ki67 between the free siDYRK1A group and the PEI-PFA@siDYRK1A group and the Blank group, with comparable red fluorescence signal intensity; Ki67 in the GLP-1-PEI-PFA@siNC negative control group also showed no significant enhancement; while the red fluorescence signal of Ki67 in the GLP-1-PEI-PFA@siDYRK1A treatment group was significantly enhanced. Following glycolipid toxicity (SP+Glu) stimulation, the red fluorescence signal of Ki67 cells was significantly reduced compared to normal culture conditions. The red fluorescence signal in the SP+Glu+siDYRK1A and SP+Glu+PEI-PFA@siDYRK1A groups was slightly increased compared to the SP+Glu group, but not significantly. The SP+Glu+PEI-PFA@siNC and SP+Glu+GLP-1-PEI-PFA@siNC groups showed no significant difference compared to the SP+Glu group. However, the red fluorescence signal in the SP+Glu+GLP-1-PEI-PFA@siDYRK1A treatment group was significantly enhanced, partially recovering to near-normal culture levels. These results indicate that the GLP-1-PEI-PFA@siDYRK1A nanocomplex can significantly promote MIN6 cell proliferation under both normal culture conditions and glycolipid toxicity stress.

[0162] 2.2 EdU staining to detect the effect of GLP-1-PEI-PFA@siDYRK1A nanocomplex on MIN6 cell proliferation

[0163] Figure 14 Image a shows representative EdU staining images of cells in each group under normal culture conditions. A small number of EdU-positive cells were observed in the blank control group (Blank); the number of EdU-positive cells in the free siDYRK1A group and the PEI-PFA@siDYRK1A group was not significantly different from that in the Blank group; the number of EdU-positive cells in the GLP-1-PEI-PFA@siNC negative control group also did not increase significantly; while the number of EdU-positive cells in the GLP-1-PEI-PFA@siDYRK1A treatment group increased significantly, and the red fluorescence signal was significantly enhanced.

[0164] Figure 14b shows representative EdU staining images of cells in each group under glycolipid toxicity stimulation. The number of EdU-positive cells in the glycolipid toxicity model group (SP+Glu) was significantly reduced compared with that under normal culture conditions; the number of EdU-positive cells in the SP+Glu+siDYRK1A group and the SP+Glu+PEI-PFA@siDYRK1A group was slightly increased compared with the SP+Glu group, but not significantly; the number of EdU-positive cells in the SP+Glu+PEI-PFA@siNC group and the SP+Glu+GLP-1-PEI-PFA@siNC group was not significantly different from that in the SP+Glu group; while the number of EdU-positive cells in the SP+Glu+GLP-1-PEI-PFA@siDYRK1A treatment group was significantly increased, the red fluorescence signal was significantly enhanced, and some cells recovered to near normal culture levels.

[0165] Figure 14 c and d show the quantitative analysis results of EdU-positive cell rates in each group under normal culture conditions and under glycolipid toxicity stimulation conditions, respectively. Under normal culture conditions, the EdU-positive cell rate in the Blank group was approximately 0.84%; the rates in the free siDYRK1A group and the PEI-PFA@siDYRK1A group were 6.27% and 25.68%, respectively, which were higher than those in the Blank group; the rate in the GLP-1-PEI-PFA@siNC group was 2.17%; while the EdU-positive cell rate in the GLP-1-PEI-PFA@siDYRK1A group significantly increased to 34.92%, which was significantly different from that in the Blank group (***). p <0.001). Under glycolipid toxicity stimulation, the EdU-positive cell rate in the SP+Glu group decreased to 0.53%; the rates in the SP+Glu+siDYRK1A group and the SP+Glu+PEI-PFA@siDYRK1A group were 9.46% and 17.40%, respectively; the rate in the SP+Glu+GLP-1-PEI-PFA@siNC group was approximately 0.54%; while the EdU-positive cell rate in the SP+Glu+GLP-1-PEI-PFA@siDYRK1A group significantly increased to 28.21%, showing a highly significant difference compared to the SP+Glu group (***). p <0.001).

[0166] The above results indicate that the GLP-1-PEI-PFA@siDYRK1A nanocomposite can significantly promote the proliferation of MIN6 cells under both normal culture conditions and glycolipid toxicity stress.

[0167] 2.3 In vivo pharmacodynamic study of GLP-1-PEI-PFA@siDYRK1A nanocomposite in a mouse model of type 2 diabetes

[0168] Figure 15Figure a shows the weight change curves of mice in each group during the treatment period. The weight of mice in the normal control group (Control) increased steadily; the weight of mice in the type 2 diabetes model group (T2DM) decreased slightly; there was no significant difference in weight between the treatment groups and the T2DM group, indicating that the treatment drugs had no significant adverse effects on the weight of mice.

[0169] Figure 15 b shows the random blood glucose changes in each group of mice during treatment. In the T2DM group, random blood glucose remained consistently high (approximately 20-30 mmol / L); the blood glucose in the free siDYRK1A treatment group showed no significant decrease compared to the T2DM group; the blood glucose in the PEI-PFA@siDYRK1A treatment group decreased slightly, but the difference was not significant; while the blood glucose in the GLP-1-PEI-PFA@siDYRK1A treatment group decreased significantly from day 8, reaching approximately 14 mmol / L by the end of treatment (day 29), showing a significant difference compared to the T2DM group (***). p <0.001).

[0170] Figure 15 c and d show the quantitative analysis of blood glucose curves and area under the curve (AUC) of mice in each group after the intraperitoneal glucose tolerance test (IPGTT) following treatment. The IPGTT blood glucose curves of mice in the T2DM group were significantly elevated, with an AUC value of approximately 2965.33. The IPGTT curves of the free siDYRK1A group and the PEI-PFA@siDYRK1A group showed slight improvement, with AUC values ​​decreasing to approximately 2933 and 2773.67, respectively. However, the IPGTT blood glucose curve of the GLP-1-PEI-PFA@siDYRK1A treatment group showed significant improvement, with an AUC value decreasing to approximately 2325, which was significantly different from the T2DM group (***). p <0.001).

[0171] Figure 15 e and f represent the quantitative analysis of the insulin tolerance test (ITT) blood glucose curves and area under the curve (AUC) of mice in each group after treatment. The ITT blood glucose curves of mice in the T2DM group decreased slowly, with an AUC value of approximately 909.8. The ITT curves of the free siDYRK1A group and the PEI-PFA@siDYRK1A group showed slight improvement, with AUC values ​​decreasing to approximately 871.6 and 819.53, respectively. However, the ITT blood glucose curve of the GLP-1-PEI-PFA@siDYRK1A treatment group showed significant improvement, with an AUC value decreasing to approximately 668.27, which was significantly different from the T2DM group (*). p <0.05).

[0172] Figure 15g and h represent the ELISA results of serum glycated hemoglobin and insulin levels in mice after treatment, respectively. In the T2DM group, GHb levels were significantly increased (approximately 2.93 μg / mL), and INS levels were significantly decreased (approximately 158.00 IU / L). In the free siDYRK1A group and the PEI-PFA@siDYRK1A group, GHb and INS levels showed slight improvement, but the differences were not significant. However, in the GLP-1-PEI-PFA@siDYRK1A treatment group, GHb levels significantly decreased to approximately 1.66 μg / mL, and INS levels significantly increased to approximately 227.91 IU / L, both showing significant differences compared to the T2DM group (**). p <0.01, *** p <0.001).

[0173] The above results demonstrate that the GLP-1-PEI-PFA@siDYRK1A nanocomposite exhibits significant hypoglycemic effects and improves glucose tolerance and insulin sensitivity in a type 2 diabetic mouse model. Its therapeutic effect is significantly superior to the unmodified PEI-PFA group and the free siDYRK1A group. This study provides strong in vivo pharmacodynamic evidence for the application of GLP-1-PEI-PFA@siDYRK1A nanocomposite as a gene therapy drug for type 2 diabetes. The above description is merely a preferred embodiment of the present invention.

Claims

1. A nucleic acid delivery system targeting pancreatic β cells, characterized in that, Nanocomplexes containing cationic polymers modified with targeted ligands and siRNA; The targeting ligand is glucagon-like peptide-1; the amino acid sequence of glucagon-like peptide-1 is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC; The cationic polymer is a PEI-perfluorobutyric acid polymer; The siRNA is a siRNA that silences DYRK1A expression; In the PEI-perfluorobutyric acid polymer, PEI and perfluorobutyric acid are coupled through an acylation reaction; The glucagon-like peptide-1 is coupled to PEI-perfluorobutyric acid polymer via a cross-linking agent; The crosslinking agent is Sulfo-SMCC.

2. The nucleic acid delivery system targeting pancreatic β cells according to claim 1, characterized in that, The sequence of the siRNA expressing the silenced DYRK1A is shown in SEQ ID NO.

2.

3. The nucleic acid delivery system targeting pancreatic β cells according to claim 1, characterized in that, The nanocomposite has a particle size of 50–100 nm and a zeta potential of +20–+60 mV; the targeted ligand-modified cationic polymer and siRNA form a nanocomposite through electrostatic self-assembly, wherein the N / P ratio is 5–10:

1.

4. A pharmaceutical composition, characterized in that, The invention comprises the nucleic acid delivery system targeting pancreatic β cells as described in any one of claims 1 to 3, and pharmaceutically acceptable excipients.

5. The use of the nucleic acid delivery system targeting pancreatic β cells according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating type 2 diabetes.

6. The application according to claim 5, characterized in that, The treatment for type 2 diabetes includes at least one of the following: lowering blood glucose, improving glucose tolerance, improving insulin sensitivity, and promoting the proliferation of pancreatic β cells.

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