SiRNA nano-drug carrier and nano anti-tumor drug

Abstract

The invention belongs to the technical field of tumor targeted drug carriers, and discloses a siRNA nano-drug carrier, the active ingredient of the siRNA nano-drug carrier is a nano-particle formed by loading siRNA on a triblock copolymer, the middle block of the triblock copolymer is poly (beta-amino ester), one terminal block is polyethylene glycol, and the other terminal block is polyethylene glycol. One terminal block is poly (3-acrylamido phenylboronic acid) pinacol ester, and the other terminal block is poly (3-acrylamido phenylboronic acid) pinacol ester, and the molecular weight of the poly (3-acrylamido phenylboronic acid) pinacol ester is 3-7 kDa. According to the siRNA nano-drug carrier, siRNA can be prevented from being degraded by nuclease in blood, the in-vivo circulation time of siRNA and Gboxin drugs is greatly prolonged, the permeability of tumor tissues is improved, and the siRNA nano-drug carrier is effectively endocytosed by tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor-targeted drug carriers and relates to a siRNA nano drug carrier and a nano anti-tumor drug. Background Art

[0002] In recent years, as gene therapy research deepens on the mechanisms of disease development and progression, nucleic acid drugs, as an emerging conservative and personalized therapeutic approach, have become a research hotspot. Unlike small molecule or antibody drugs, which rely on recognizing the complex three-dimensional structure of proteins, siRNA exerts its therapeutic effects through precise base pairing with mRNA. Many diseases that are inaccessible to small molecule or monoclonal antibody drugs can be specifically targeted with siRNA. siRNA therapy offers advantages such as a short development cycle, low-dose efficacy, minimal side effects, and low drug resistance, showing broad application prospects in disease treatment. However, siRNA is unstable in the body and has difficulty crossing epithelial cells. Oral administration has not yet been widely used in cancer treatment, and subcutaneous administration is limited by the lipophilicity of siRNA and the size of the carrier. Intravenous administration has become the preferred route for siRNA therapy, but a suitable delivery system is required to protect the exposed siRNA from degradation by enzymes in the serum. The negative surface charge of siRNA makes it difficult for it to cross the similarly negatively charged cell membrane. In healthy tissues, vascular endothelial cells are tightly connected, significantly reducing drug leakage through the vessel wall. However, within tumors, discontinuous endothelial tissue and rapid angiogenesis accelerate drug leakage, which may lead to the risk of increased interstitial fluid pressure. Researchers have made many optimizations to the structure of siRNA and its delivery system, such as polyethylene glycol and other polymer modifications, to reduce siRNA clearance and prolong circulation time.

[0003] PBAE (poly(β-amino ester)) is a low-toxicity, high-yield, non-viral delivery vector. Its ability to bind to nucleic acid drugs is a key factor affecting the success of drug delivery and preventing drug degradation after intravenous injection. The terminal secondary amine group plays an important role in gene delivery, loading siRNA through electrostatic interaction to form a dense nanodrug. PBAE with the introduction of disulfide bonds (reducible bonds that can be cleaved or converted into thiol groups when exposed to a reducing environment) breaks under the high level of glutathione in the tumor, triggering the release of siRNA drugs. In an in situ model of glioblastoma (GBM), it was found that disulfide-modified PBAE / siRNA accelerated the release of siRNA when entering the reducing environment of the cytosol.

[0004] For the treatment of central nervous system (CNS) diseases, the primary challenge in drug delivery lies in how to enable therapeutic drugs to cross the BBB and precisely target specific tissues or cells within the brain. In recent years, polymeric non-viral vectors, by complexing or conjugating with siRNA drugs, have been shown to effectively overcome extracellular and intracellular barriers and enter the tumor microenvironment. Therefore, the development of polymeric non-viral vectors with excellent biocompatibility, stability, and targeting capabilities is a current research hotspot. Summary of the Invention

[0005] The present invention addresses the technical problems existing in current non-viral vectors for siRNA targeted therapy and provides a siRNA nanodrug carrier. The carrier uses a PAPBAE-PBAE-PEG triblock copolymer as a basic unit and forms nanoparticles through self-assembly. Angiopep-2 polypeptide modification gives the carrier the ability to cross the blood-brain barrier (BBB) ​​and target gliomas, thereby achieving safe and efficient targeted synergistic therapy for human gliomas.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an siRNA nanodrug carrier, the active ingredient of which is nanoparticles formed by a triblock copolymer loaded with siRNA, wherein the middle block of the triblock copolymer is poly(β-amino ester) (PBAE), one end block is polyethylene glycol (PEG), and the other end block is poly (3-acrylamidophenylboronic acid pinacol ester) (PAPBAE), and the molecular weight of the poly (3-acrylamidophenylboronic acid pinacol ester) is 3 to 7 kDa.

[0008] In the above technical solution, the triblock copolymer is poly 3-acrylamidophenylboronic acid pinacol ester 3k -Poly(β-amino ester) 5k -Polyethylene glycol 5k, poly (3-acrylamidophenylboronic acid pinacol ester) 5k -Poly(β-amino ester) 5k -Polyethylene glycol 5k or poly (3-acrylamidophenylboronic acid pinacol ester) 7k -Poly(β-amino ester) 5k -Polyethylene glycol 5k.

[0009] In the above technical solution, the synthesis steps of the poly (3-acrylamidophenylboronic acid pinacol ester) (PAPBAE) are as follows: 3-acrylamidophenylboronic acid pinacol ester monomer, 4-cyano-4-[[(dodecylthio)thiocarbonyl]thio]pentanoic acid, and azobisisobutyronitrile are dissolved in 1,4-dioxane, and the mixture is polymerized at 70° C. for 48 hours to obtain poly (3-acrylamidophenylboronic acid pinacol ester).

[0010] In the above technical solution, the molar ratio of 3-acrylamidephenylboronic acid pinacol ester, 4-cyano-4-[[(dodecylthio)thiocarbonyl]thio]pentanoic acid, and azobisisobutyronitrile is 14-32:1:0.2. Depending on the amount of APBAE monomer used, PAPBAE polymers with different molecular weights can be obtained.

[0011] In the above technical solution, the synthesis steps of the poly(β-amino ester) (PBAE) are as follows: 1,4-butanediol diacrylate and 4-amino-1-butanol are weighed according to a molar ratio of 1.05:1, dissolved in dimethyl sulfoxide, heated in an oil bath at 90°C, reacted for 24 hours, cystamine dihydrochloride was added, heated in an oil bath at 60°C, continued to react for 24 hours, and dialyzed and freeze-dried to obtain a PBAE polymer.

[0012] In the above technical solution, the synthesis steps of the triblock copolymer are as follows: PBAE, PAPBAE, MAL-PEG 5000 -COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), triethylamine (TEA) were weighed in a molar ratio of 1:2:2:8:8:16; first, the carboxyl group was activated, and PAPBAE, MAL-PEG 5000 -COOH, EDC, and NHS were dissolved in dichloromethane (DCM) and reacted at room temperature for 24 hours. PBAE-SS-NH2 and TEA were then added for amidation, and the reaction continued at room temperature for another 24 hours. The triblock copolymer was obtained by dialysis, centrifugation, and freeze-drying. Different molecular weights of triblock copolymers can be obtained depending on the amount of PAPBAE used.

[0013] In the above technical solution, the siRNA is selected from siRNA of STAT3 gene, and the sequence of siRNA of STAT3 gene is shown as SEQ ID NO.1-2.

[0014] In the above technical solution, the molar ratio of the triblock copolymer to siRNA is 50-200:1, preferably 50:1.

[0015] In the above technical solution, the nanoparticles are further chemically modified or ligand-modified, the ligand is preferably Angiopep-2, and the chemical modification group is preferably an azide group.

[0016] In the above technical scheme, the synthesis steps of the nano drug carrier modified with the ligand Angiopep-2 and loaded with siRNA and tumor suppressor Gboxin are as follows: weigh the triblock copolymer and dissolve it in THF solution to prepare a 30 mg / mL solution; dissolve siRNA in diethyl pyrocarbonate (DEPC) water to prepare a 1 mg / mL solution for standby use; dissolve the tumor suppressor Gboxin in DMSO solution to prepare a 10 mg / mL solution for standby use; dissolve the Angiopep-2 polypeptide in DEPC water to prepare a 10 mg / mL solution for standby use; add the triblock copolymer, siRNA and Gboxin solution in proportion to a sodium acetate solution with a pH of 5.0, mix well, and let it stand for 30 minutes. After adding a rotor, use magnetic stirring for 2-3 hours to evaporate the THF solution or use dialysis to remove the DMSO organic solvent, and self-assemble to form nanoparticles through hydrophilic and hydrophobic forces; finally, add the Angiopep-2 polypeptide solution to prepare a nano drug with targeting function.

[0017] In a second aspect, the present invention provides a nano anti-tumor drug, the active ingredient of which is the nanoparticles formed by the above-mentioned siRNA nano drug carrier loaded with the anti-tumor drug.

[0018] In the above technical solution, the anti-tumor drug includes a tumor inhibitor, preferably Gboxin which inhibits the growth of glioblastoma.

[0019] In the above technical solution, the above anti-tumor drug further includes one or more pharmaceutically acceptable excipients.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention synthesizes a novel siRNA nanodrug carrier, which uses a PAPBAE-PBAE-PEG triblock copolymer as a basic unit and self-assembles into spherical nanoparticles through hydrophilic and hydrophobic forces. Angiopep-2 polypeptide modification gives it the ability to cross the blood-brain barrier (BBB) ​​and target gliomas. This nanodrug carrier can prevent siRNA from being degraded by nucleases in the blood, greatly prolong the in vivo circulation time of siRNA and Gboxin drugs, improve tumor tissue permeability, and effectively be internalized by tumor cells. The intracellular environment can promote the cleavage of the nanocarrier to release Gboxin and siRNA. Gboxin inhibits mitochondrial function by inhibiting the oxidative phosphorylation pathway, synergizes with siRNA to inhibit tumor cell metabolism, and induces tumor cell apoptosis. The nanodrug carrier of the present invention is expected to solve many key problems faced by nanocarrier targeted delivery, such as short in vivo circulation time, lack of tumor specificity, low small molecule / nucleic acid drug load, poor tumor tissue penetration, low tumor cell uptake, and slow release in the tumor microenvironment, ultimately achieving safe and efficient targeted synergistic treatment of human brain gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The synthetic route of the triblock copolymer of the present invention is shown in FIG.

[0023] Figure 2 This is the H NMR spectrum of the 3 kDa PAPBAE polymer in Example 1.

[0024] Figure 3 This is the H NMR spectrum of the 5 kDa PBAE polymer in Example 1.

[0025] Figure 4 Schematic diagram of the design of the nanomedicine modified with the ligand Angiopep-2 and loaded with siRNA and Gboxin of the present invention.

[0026] Figure 5 are the particle size, potential and morphology of siRNA nanomedicine, Figure 5 (a) Gel electrophoresis results of siRNA nanomedicines prepared from triblock copolymers with molecular weights of 13 kDa, 15 kDa, and 17 kDa; Figure 5 (b) The particle size of siRNA nanomedicine was determined by dynamic light scattering method; Figure 5 (c) Zeta potential of siRNA nanomedicine was measured by dynamic light scattering method; Figure 5 (d) The morphology of siRNA nanodrug (13kDa) taken by TEM.

[0027] Figure 6 To detect the changes in particle size of siRNA nanomedicine after being placed under different conditions by DLS, including: Figure 6 (a) DLS method was used to detect the particle size changes of different siRNA nanomedicines at 25°C for 7 days; Figure 6 (b) The particle size change of siRNA nanomedicine at 37°C for 7 days.

[0028] Figure 7 is the reduction response ability result of siRNA nanomedicine, where: Figure 7 (a) The particle size of siRNA nanomedicines detected by DLS after being placed under different conditions and for different time periods; Figure 7 (b) The cumulative amount of Cy5-labeled siRNA released from Cy5-labeled siRNA nanoparticles in vitro under different conditions and time was measured using a microplate reader. The results are expressed as mean ± standard deviation (n=3).

[0029] Figure 8 To characterize the in vitro cellular uptake of siRNA nanoparticles, including: Figure 8(a) is the flow cytometry results of U251-Luc tumor cells incubated with Free siSTAT3, siRNA NPs (13kDa) and Ang-siRNA NPs (13kDa) nanodrugs for 8 hours; Figure (b) is the flow cytometry results of U251-Luc tumor cells incubated with Angiopep-2 targeted nanodrugs of different molecular weights for 8 hours.

[0030] Figure 9 Fluorescence confocal microscopy was used to detect the in vitro cellular uptake ability of Cy5-labeled siRNA nanomedicines with different molecular weights. The scale bar is 100 μm.

[0031] Figure 10 The results of the in vitro simulated BBB penetration experiment are as follows: Figure 10 (a) Schematic diagram of the BBB in vitro model established using bEnd.3 cells; Figure 10 (b) The efficiency of Cy5-labeled siRNA nanomedicine incubated U251-Luc tumor cells crossing the BBB within 24 h.

[0032] Figure 11 To construct an in vitro 3D tumor spheroid model to test the tumor penetration depth of siRNA nanomedicine, Figure 11 (a) Schematic diagram of the in vitro 3D tumor sphere model established using U251-Luc cells; Figure 11 (b) Confocal images of the penetration depth of Cy5-labeled siRNA nanomedicines with different molecular weights incubated with U251-Luc tumor cells for 8 h at the 3D tumor sphere level. The scale bar is 200 μm.

[0033] Figure 12 The in vitro anti-tumor effect of siRNA nanomedicine, including: Figure 12 (a) MTT assay to detect the cell viability of U251-Luc cells after incubation with nanomedicine for 48 h; Figure 12 (b) Flow cytometry was used to detect the cell apoptosis of U251-Luc cells after incubation with nanomedicine for 48 h.

[0034] Figure 13 The in vitro gene silencing effect of siRNA nanomedicine, including: Figure 13 (a) Detection of STAT3 mRNA level by real-time fluorescence quantitative PCR; Figure 13 (b) The effect of STAT3 protein expression level was verified by Western blotting experiment; Figure 13 (c) Statistics of three Western blotting results.

[0035] Figure 14In vivo pharmacokinetics of Ang-siScr and Ang-siSTAT3 nanomedicines.

[0036] Figure 15 Results of in vivo imaging and biodistribution experiments of siRNA nanomedicines, including: Figure 15 (a) Fluorescence imaging results of Ang-siScr, siSTAT3, and Ang-siSTAT3 nanodrug in situ in U251-Luc tumor-bearing mice at different time points; Figure 15 (b) Stereoscopic imaging of the main organs of orthotopic tumor-bearing mice after tail vein injection of Ang-siScr, siSTAT3, and Ang-siSTAT3 NPs; Figure 15 (c) Statistical graph showing the quantitative results of Ang-siScr, siSTAT3, and Cy5-stained siRNA of Ang-siSTAT3 in different organs of mice, expressed as relative injected dose per gram of organ tissue (%ID / g).

[0037] Figure 16 The results of siRNA nanomedicine's tumor inhibition on U251-Luc orthotopic tumor-bearing nude mice are shown below: Figure 16 (a) To establish a U251-Luc orthotopic tumor-bearing mouse model, nanomedicines (siRNA: 2 mg / kg, Gboxin: 5 mg / kg) were injected into the tail vein on days 8, 11, 14, 17, and 19, and in vivo fluorescence imaging of the mice was performed. Figure 16 (b) Quantitative analysis of fluorescence imaging results; Figure 16 (c) The body weight changes of mice during the administration period; Figure 16 (d) is the survival period of mice during the administration period. The results are expressed as mean ± standard deviation (n=6).

[0038] Figure 17 These are the H&E staining results of the main organs of orthotopic tumor-bearing nude mice after siRNA nanodrug treatment (scale bar is 100 μm).

[0039] Figure 18 These are the results of blood routine and blood biochemistry analysis after siRNA nanodrug treatment.

[0040] Figure 19 is the mRNA level of pro-inflammatory factors, including: Figure 19 (a) The mRNA expression levels of IL-1β, IL-6, and TNF-α in the liver of mice on the 2nd and 14th days after tail vein injection of the drug; Figure 19 (b) The mRNA expression levels of IL-1β, IL-6, and TNF-α in the kidneys of mice on days 2 and 14 after tail vein injection of the drug. The results are expressed as mean ± standard deviation (n=3). DETAILED DESCRIPTION

[0041] The following examples are used to illustrate the present invention but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified.

[0042] Example 1 Preparation of siRNA Nanomedicine

[0043] like Figure 1 Schematic diagram of the synthesis route of triblock copolymers shown.

[0044] 1. Preparation of PAPBAE polymer

[0045]

[0046] A 50 mL reaction tube was prepared and purged with N2 for 30 minutes through a double-chamber tube to remove air. APBAE (3-acrylamidophenylboronic acid pinacol ester, TCI) (150 mg, 0.549 mmol), RAFT reagent (4-cyano-4-[[(dodecylthio)thiocarbonyl]thio]pentanoic acid, J&K) (15.829 mg, 0.0392 mmol), and initiator AIBN (azobisisobutyronitrile, Aladdin) (1.287 mg, 0.00784 mmol) were added to 5 mL of 1,4-dioxane (Tianjin Fuyu). The reaction solution was evacuated 2-3 times through a double-chamber tube and then purged with N2. The reaction system was then incubated in dimethyl silicone oil (oil bath) at 70°C for 48 hours. The reaction system was then rapidly cooled on ice and exposed to air. The solution was dissolved with 10 mL of THF and precipitated three times in icy n-hexane. Filter and dry in a vacuum oven to obtain poly (3-acrylamidophenylboronic acid pinacol ester) polymer (PAPBAE) with a molecular weight of 3 kDa. Figure 2 shown.

[0047] The synthesis of 5kDa and 7kDa PAPBAE polymers follows the same process as above and will not be described in detail. The GPC results of 3kDa, 5kDa, and 7kDa PAPBAE polymers are shown in Table 1.

[0048] Table 1 GPC results of PAPBAE polymer

[0049]

[0050]

[0051] 2. Synthesis of PBAE polymer

[0052]

[0053] Prepare a 50mL reaction tube and purge it with N2 through a double-row tube for 30 minutes to remove air. Take 1,4-butanediol diacrylate (B&K) (1000 mg, 951 μL, 5.045 mmol) and 4-amino-1-butanol (B&K) (428 mg, 443 μL, 4.805 mmol), dissolve in 2.86mL of dimethyl sulfoxide reagent (Tianjin Fuyu), react in dimethyl silicone oil (oil bath) at 90℃ for 24h, add cystamine dihydrochloride (B&K) (1.2 g, 0.005 mmol), and then stir at 60℃. The reaction was continued for 24 hours after heating in an oil bath. The reaction system was then placed on ice for rapid cooling and exposed to air. The reaction was dialyzed using a 500 kDa dialysis bag for 24 hours, with purified water replaced every 2 hours to remove the reaction solvent and unreacted cystamine dihydrochloride. After the dialysis was completed, the dialyzed product was placed in a 50 mL EP tube, covered with tin foil, and placed in a -80 ° C refrigerator for pre-freezing and then vacuum freeze-dried. After 48 hours, a light yellow oily PBAE polymer with a molecular weight of 5 kDa was obtained. Its H NMR spectrum is shown below. Figure 3 shown.

[0054] 3. Synthesis of PAPBAE-PBAE-PEG triblock copolymer

[0055] Prepare a 50 mL reaction tube and purge it with N2 for 30 min through a double-row tube to remove air. Weigh PAPBAE (100 mg, 0.02 mmol, 3 kDa), MAL-PEG 5k -COOH (Shanghai Pengshuo) (100 mg, 0.02 mmol, 5 kDa), EDC (B&K) (12.42 mg, 0.08 mmol), NHS (Aladdin) (9.21 mg, 0.08 mmol) were dissolved in 10 ml of dichloromethane (Tianjin Fuyu), and reacted at room temperature for 24 h. PBAE (40 mg, 0.01 mmol) and TEA (B&K) (16.21 mg, 0.16 mmol) were added, and the reaction was continued at room temperature for 24 h. After dialysis, centrifugation, and freeze-drying, PAPBAE with a molecular weight of 13 kDa was obtained. 3k -PBAE 5k -PEG 5k triblock copolymer.

[0056] 15kDa and 17kDa PAPBAE 5k -PBAE 5k -PEG 5k 、PAPBAE 7k -PBAE 5k -PEG 5k The synthesis method of the triblock copolymer refers to the above process and will not be described in detail.

[0057] 4. Preparation of siRNA Nanomedicine

[0058] like Figure 4 Schematic diagram of the design of siRNA nanomedicine shown.

[0059] (1) Triblock copolymer-loaded siRNA nanomedicine

[0060] Weigh 13kDa PAPBAE 3k -PBAE 5k -PEG 5k (30 mg, 2.3077 mmol) was dissolved in 1000 μL THF solution and mixed. STAT3-siRNA (500 μg, 75.7576 μmol, denoted as siSTAT3) was dissolved in 500 μL DEPC water and mixed. 369 μL of pH 5.0 sodium acetate solution was added to the EP tube, 5.28 μL of siSTAT3 solution was added to the EP tube, and 34.7 μL of PAPBAE was added. 3k -PBAE 5k -PEG 5k The solution was added to an EP tube. After mixing, the solution was placed on ice and allowed to stand for 30 minutes. After standing, it was stirred for 2 hours to allow the organic solvent to evaporate. The triblock copolymer self-assembled through hydrophilic and hydrophobic interactions to form siRNA-loaded nanoparticles, designated siSTAT3 (13 kDa).

[0061] The sequence of STAT3-siRNA (denoted as siSTAT3) is as follows:

[0062] Sense(5'-3'): 5'-GGAUCUAGAACAGAAAAUGTT-3' (SEQ ID NO. 1);

[0063] Antisense (5'-3'): 5'-CAUUUUCUGUUCUAGAUCCTG-3' (SEQ ID NO. 2).

[0064] The Cy5-siRNA sequence is as follows:

[0065] Sense(5'-3'):5'-UUCUCCGAACGUGUCACGUdtdt-3';

[0066] Antisense(5'-3'): 5'-ACGUGACACGUUCGGAGAAdtdt-3'.

[0067] The sequence of Scramble-siRNA (denoted as siScr) is as follows:

[0068] Sense(5'-3'):5'-UUCUCCGAACGUGUCACGUdtdt-3';

[0069] Antisense(5'-3'): 5'-ACGUGACACGUUCGGAGAAdtdt-3'.

[0070] The siSTAT3 (15 kDa) and siSTAT3 (17 kDa) nanoparticles prepared using 15 kDa and 17 kDa PAPBAE-PBAE-PEG triblock copolymers followed the above process, and the molar ratios of the triblock copolymer and siSTAT3 were adjusted to 50:1, 75:1, 150:1, and 200:1, respectively, which will not be repeated here.

[0071] siSTAT3 without triblock copolymer loading was used as a control, designated as Free siSTAT3. Gboxin was added to Free siSTAT3, designated as Free siSTAT3+Gboxin.

[0072] (2) Angiopep-2 peptide-modified triblock copolymer loaded with siRNA nanopharmaceuticals

[0073] Dissolve Angiopep-2 peptide in DEPC water to prepare a 10 mg / mL solution for later use. 3k -PBAE 5k -PEG 5k 5.8 μL of Angiopep-2 peptide solution was added to an EP tube containing a siSTAT3 nanodrug prepared from a triblock copolymer to create the Ang-siSTAT3 (13 kDa) nanodrug. Similarly, Ang-siSTAT3 (15 kDa) and Ang-siSTAT3 (17 kDa) nanodrugs were prepared. Similarly, Ang-siScr (13 kDa), Ang-siScr (15 kDa), and Ang-siScr (17 kDa) nanodrugs were prepared as controls.

[0074] (3) Angiopep-2 peptide-modified triblock copolymer loaded with siRNA+Gboxin nanomedicine

[0075] The tumor suppressor Gboxin (1 mg, 25.4479 mmol) was dissolved in 100 μL DMSO solution to prepare a 10 mg / mL solution for later use. 2.73 μL of Gboxin solution was then added to the EP tube in step (3) to prepare the Ang-siSTAT3@Gboxin (13 kDa) nanodrug. Similarly, Ang-siSTAT3@Gboxin (15 kDa) and Ang-siSTAT3@Gboxin (17 kDa) were prepared. Similarly, Ang-siScr@Gboxin (13 kDa), Ang-siScr@Gboxin (15 kDa), and Ang-siScr@Gboxin (17 kDa) nanodrugs were prepared as controls.

[0076] Example 2 Characterization of the physicochemical properties of siRNA nanomedicine

[0077] (1) Weigh agarose powder and add it to a conical flask, add TBE (Tris-borate-EDTA, TBE) buffer to prepare 2% agarose gel. Place the conical flask in a microwave and heat until boiling. Take out the conical flask and shake to mix. Repeat three times. When the solution is about 60°C, add nucleic acid dye and mix thoroughly. Pour the gel into the prepared gel plate, insert a comb and wait for the gel to completely solidify before pulling it out vertically to avoid tearing the pores. Add 10μL of siSTAT3 nanodrug solution prepared in Example 1 to the gel wells, place the gel plate in the electrophoresis tank, and add TBE buffer to submerge the gel block. After confirming that the electrode direction is correct, set the voltage to 35V for electrophoresis. End after 30 minutes and use a fully automatic gel imaging analysis system to observe RNA migration.

[0078] like Figure 5 As shown in (a): Compared with free siRNA, the nanodrug has no free siRNA band in the nucleic acid gel pores, indicating that when the molar ratio of PAPBAE-PBAE-PEG polymer to siRNA is greater than or equal to 50:1, siRNA can be completely loaded into the polymer. A molar ratio of PAPBAE-PBAE-PEG polymer to siRNA of 100:1 was selected for subsequent experiments.

[0079] (2) 100 μL of Ang-siSTAT3 NPs (13 kDa), Ang-siSTAT3 NPs (15 kDa), and Ang-siSTAT3 NPs (17 kDa) solutions prepared in Example 1 were placed in a quartz dish and placed in a nanoparticle size and zeta potential analyzer. The corresponding solution dispersion was selected and the detection angle was set to 173°. Each siRNA nanodrug sample was measured three times continuously. The particle size and dispersion coefficient of the nanodrug were measured by dynamic light scattering. 600 μL of solution from each group was added to a dedicated U-shaped potential dish, and the electrophoretic mobility was measured by dynamic light scattering to calculate the zeta unit.

[0080] like Figure 5 (b) shows the particle sizes of Ang-siSTAT3 NPs with three molecular weights: 80.3 nm, 94.3 nm, and 103 nm, and PDIs of 0.14, 0.15, and 0.20, respectively. The nanoparticles exhibited relatively uniform particle size and good dispersion. As the molecular weight of the triblock copolymer increased, the resulting nanoparticle size also increased.

[0081] like Figure 5 (c) shows that the Zeta potentials of the three molecular weight nanodrugs formed by triblock copolymer and Ang-siSTAT3 are 4.77mV, 6.84mV, and 9.98mV, respectively.

[0082] (3) Take 10 μL of the Ang-siSTAT3 NPs (13 kDa) solution prepared in Example 1 and mix thoroughly with 5 μL of 1% uranyl acetate solution. Clamp the copper mesh with tweezers to ensure that the front side is facing up. Take 10 μL of the mixed nanodrug and add it to the copper mesh. Dry at room temperature. Wash the copper mesh with ultrapure water, let it stand for 5 minutes, and then gently absorb the excess liquid along its edge with filter paper. Repeat this three times. After the copper mesh is fully dried, transfer it to a clean filter paper surface and characterize the morphology of the siRNA nanodrug using transmission electron microscopy.

[0083] like Figure 5 (d) is shown as follows: The image clearly shows that the morphology of Ang-siSTAT3 NPs (13kDa) is spherical particles, which is basically consistent with the particle size measured by DLS, indicating the successful synthesis of siRNA nanodrugs.

[0084] (4) The Ang-siSTAT3 NPs (13 kDa), Ang-siSTAT3 NPs (15 kDa), and Ang-siSTAT3 NPs (17 kDa) solutions prepared in Example 1 were placed in PBS solution at room temperature (25°C) and physiological temperature (37°C) for 7 days, respectively. The results were as follows: Figure 6As shown, the particle size of Ang-siSTAT3 nanodrugs of three molecular weights remained essentially unchanged over 7 days at both room temperature (25°C) and physiological temperature (37°C), with a PDI consistently below 0.25, demonstrating that siRNA nanodrugs maintain structural and functional stability. This stability promotes nanodrug accumulation in GBM lesions through enhanced permeation and retention, thereby improving therapeutic efficacy.

[0085] (5) In order to verify whether the prepared Ang-siSTAT3 nanomedicine has reduction response ability, Ang-siSTAT3 NPs

[0086] (13kDa) as an example, dithiothreitol (DTT) and H2O2 were added to construct an in vitro tumor microenvironment, and the cells were placed in PBS solution for 12 hours and 24 hours respectively; Ang-siSTAT3 NPs (13kDa) were placed in PBS solution for 24 hours as a control. Figure 7 As shown in (a): The particle size of Ang-siSTAT3 NPs in the control group remained almost unchanged after 24 hours, indicating that the structure of Ang-siSTAT3 nanomedicine is stable under physiological conditions; while the particle size of Ang-siSTAT3 nanomedicine in the experimental group increased from 65nm to 267nm. Figure 7 As shown in (b): The cumulative release rate of Ang-siSTAT3 NPs in the experimental group reached 92.0% after 36 hours under the action of DTT and H2O2, while the release rate of Ang-siSTAT3 NPs in the control group (denoted as Cy5-siRNA in the figure) was only 10.1% at the same time.

[0087] Example 3 Flow cytometry and laser confocal microscopy to detect cell endocytosis

[0088] In the flow cytometry test, U251-Luc cells were plated at 5×10 5 The cells were seeded in 6-well plates at a density of 100 μL each, placed in a cell culture incubator and incubated for 24 hours, then the culture medium was removed and washed twice with PBS solution. 2000 μL of Free siSTAT3, siSTAT3 NPs (13 kDa) and Ang-siSTAT3 NPs (13 kDa) prepared in Example 1 were added respectively, with the final concentration of siRNA in each group being 100 nM. The cells were placed in a cell culture incubator and incubated for 8 hours, then the culture medium was discarded, washed with PBS solution, digested with EDTA-free trypsin and centrifuged, and the cells were blown into a single-cell suspension with PBS solution, added to an EP tube and marked. The EP tube was inserted into an ice box and stored away from light. The difference in cellular uptake efficiency between the targeted peptide-modified nanomedicine and the non-targeted peptide-modified nanomedicine was detected by flow cytometry within 1 hour. As Figure 8As shown in (a), the Cy5 fluorescence of the free Free siSTAT3 group was the weakest, and the fluorescence intensity of Ang-siSTAT3 NPs (13kDa) was increased by about 1.7 times compared with the fluorescence intensity of siSTAT3 NPs (13kDa), and significantly increased by about 3.6 times compared with the free Free siSTAT3 group, indicating that targeted modified nanomedicines can enhance the endocytic ability of cells.

[0089] The same method was used to detect the differences in cellular uptake efficiency of the nanomedicines modified with targeting peptides of different molecular weights, Ang-siSTAT3 (13kDa), Ang-siSTAT3 (15kDa), and Ang-siSTAT3 (17kDa), prepared in Example 1. Figure 8 As shown in (b), compared with free siSTAT3, Ang-siSTAT3 NPs (13kDa), Ang-siSTAT3NPs (15kDa), and Ang-siSTAT3 NPs (17kDa) can be effectively taken up by cells, among which Ang-siSTAT3 NPs (13kDa) is most efficiently internalized by cells and exhibits the strongest fluorescence signal.

[0090] In laser confocal microscopy, sterile glass slides were disinfected with alcohol and sterilized with UV light and then placed in a 12-well plate. U251-Luc cells were seeded into the plate with the glass slides at a seeding density of 1 × 10 cells per well. 5 cells. Place in a cell culture incubator and incubate for 24 hours to remove the culture medium, and wash twice with PBS solution. 2000 μL of FreesiSTAT3, Ang-siSTAT3 NPs (13kDa), Ang-siSTAT3 NPs (15kDa) and Ang-siSTAT3 NPs (17kDa) prepared in Example 1 were added respectively, and the final concentration of siRNA in each group was 100nM. Place in a cell culture incubator and continue to incubate for 8 hours, then discard the culture medium, wash with PBS solution, add 1mL of 4% paraformaldehyde solution to each well to fix the cells, and discard after 10-15 minutes. Wash 3 times with PBS solution, add 500 μL of cytoskeletal dye Actin-Tracker Green-488 (microfilament green fluorescent probe) to each well, and stain for 30 minutes in the dark at room temperature. Wash 3 times with PBS solution, add 500 μL of 10 μg / mL cell nucleus staining solution Hoechst33342 to each well, discard after 5 minutes, and wash 2-3 times with PBS solution, 3-5 minutes each time. Add about 10 μL of anti-fluorescence quencher on the coverslip, take out the round slide with tweezers, slowly place it on the slide at an angle, and absorb the excess sealing agent with absorbent paper. Use confocal microscope to observe the image, and ensure that the Cy5 fluorescence detection channel parameters of each group of samples are consistent during imaging. Figure 9 As shown, the fluorescence signal of the free siSTAT3 group was weak, while the Ang-siSTAT3 NPs (13kDa) group showed dense fluorescent aggregates in the cytoplasm and perinuclear regions. As the polymer molecular weight increased from 13kDa to 17kDa, the intracellular fluorescence intensity gradually weakened, which may be related to the particle size and surface charge of the nanodrug. Therefore, compared with free siSTAT3, the nanodrug formed by loading siRNA with triblock copolymers can significantly improve the siRNA delivery efficiency, and the Ang-siSTAT3 NPs (13kDa) group had the highest delivery efficiency.

[0091] Example 4 Detection of the ability to cross the blood-brain barrier

[0092] In order to evaluate the ability of polymer nanomedicines to cross the blood-brain barrier, a blood-brain barrier model was constructed in vitro using mouse brain microvascular endothelial bEnd.3 cells to conduct BBB penetration experiments. Figure 10 (a) The bEnd.3 cells were digested and centrifuged, and complete culture medium was added to make a single cell suspension. 200 μL of cell suspension was added to each chamber, and each chamber was 5×10 4 cells, and 800 μL of complete culture medium was added to the lower chamber. When the bEnd.3 cells formed a dense monolayer of cells, they could be used as an in vitro blood-brain barrier model for experiments. The upper culture medium of the chamber was removed, and the cells were washed twice with PBS solution. Free siSTAT3, Ang-siSTAT3 NPs (13kDa), Ang-siSTAT3 NPs (15kDa) and Ang-siSTAT3 NPs (17kDa) prepared in Example 1 were prepared respectively, and 200 μL of the mixed nanomedicine was added to the top of each chamber. The final concentration of siRNA in each group was 100 nM. The cells were placed in a cell culture incubator and continued to incubate. At the time points of 1, 2, 4, 8, 12 and 24 h, 300 μL of culture medium was taken out from the bottom of the chamber and placed in a 1.5 mL EP tube, marked, and the same volume of culture medium was added. After the experiment, the fluorescence intensity value of the sample was measured using a microplate reader (Ex = 630 nm, Em = 670 nm), and a Cy5-siRNA standard curve was drawn.

[0093] Figure 10(b) shows the results of an in vitro simulated BBB penetration experiment. As can be seen, the cumulative transport of Cy5-siRNA over 24 hours in the Ang-siSTAT3 NPs (13kDa), Ang-siSTAT3 NPs (15kDa), and Ang-siSTAT3 NPs (17kDa) groups was 9.4%, 7.7%, and 6.2%, respectively. The cumulative transport in the free siSTAT3 group was 4.2% over the same period. This suggests that the Angiopep-2 ligand specifically binds to the LRP1 receptor on the surface of BBB endothelial cells, activating receptor-mediated transcytosis. Due to their smaller particle size, Ang-siSTAT3 NPs (13kDa) are more likely to achieve efficient penetration through tight junctions of the blood-brain barrier or through carrier-mediated transport pathways.

[0094] In the three-dimensional brain tumor spheroid penetration assay, U251-Luc cells were plated at 5×10 cells per well. 5 The density of the cells was inoculated into a transparent U-bottom, ultra-low adsorption 96-well plate. Figure 11 As shown in (a). Place in a cell culture incubator and incubate for 48 hours. When the diameter of the tumor spheres formed by the cells reaches about 500 μm, remove the culture medium, wash twice with PBS solution, prepare FreesiSTAT3, Ang-siSTAT3 NPs (13kDa), Ang-siSTAT3 NPs (15kDa), and Ang-siSTAT3 NPs (17kDa) prepared in Example 1, add 100 μL of mixed nanomedicine to each well, and the final concentration of siRNA in each group is 100 nM. After continuing to incubate in a cell culture incubator for 8 hours, discard the culture medium and wash three times with PBS solution. Add 1 mL of 4% paraformaldehyde solution to each well to fix the cells, discard it after 20 minutes, and wash twice with PBS solution. Add 10 μm of DAPI solution to each well for staining for 15 minutes, then wash twice with PBS solution, aspirate the tumor spheres and place them in a confocal dish. Use a laser confocal microscope to take tomographic images of 3D brain tumor spheres at intervals of 10 μm. As shown Figure 11 As shown, the fluorescence intensity of Cy5 gradually decreases with increasing scanning depth. At the same depth, the larger the molecular weight of the nanodrug, the lower the Cy5 fluorescence intensity. Ang-siSTAT3 NPs (13 kDa) exhibited the strongest fluorescence signal and penetrated more deeply into the tumor core, suggesting that smaller particle size may facilitate nanodrug migration to the core by enhancing diffusion efficiency. Ang-siSTAT3 NPs (13 kDa) exhibit higher cellular uptake, more efficient blood-brain barrier penetration, and enhanced tumor penetration. Therefore, nanodrugs formulated with 13 kDa triblock copolymers were selected for subsequent in vitro and in vivo studies.

[0095] Example 5 Detection of cytotoxicity, gene silencing, and apoptosis-inducing abilities of siRNA nanomedicines

[0096] (1) In vitro anti-tumor effects of siRNA nanomedicines

[0097] In the MTT assay, U251-Luc cells were cultured at a rate of 1.5 × 10 4 The cells were seeded at a density of 100 μg / mL in a 96-well plate. After incubation in a cell culture incubator for 24 hours, the culture medium was removed. PBS, Free siSTAT3+Gboxin, Ang-siScr@Gboxin NPs (13kDa), therapeutic Ang-siSTAT3 NPs (13kDa), Ang-siSTAT3@GboxinNPs (100nM, 13kDa) and Ang-siSTAT3@Gboxin NPs (200nM, 13kDa) prepared in Example 1 were prepared separately, and 100 μL of the mixed nanomedicine was added to each well. The final concentration of each group of siRNA was 100nM or 200nM. The cells were placed in a cell culture incubator and incubated for 48 hours. After removal, 20 μL of MTT solution (5 mg / mL, prepared in PBS solution) was added to each well in the clean bench in the dark and incubated for 4 hours. The solution in the wells was aspirated to terminate the incubation. 100 μL of DMSO solution was added to each well and the wells were placed on a shaker with low speed for 10 minutes to allow the formazan to fully dissolve. The absorbance of formazan was measured at 492 nm using a multifunctional microplate reader and analyzed using Graphpad Pism software. Figure 12 As shown in (a), after 48 hours of treatment with Ang-siSTAT3@Gboxin (100 nM) and Ang-siSTAT3@Gboxin (200 nM), cell death rates were 37.3% and 49.6%, respectively, significantly higher than the 23.3% in the free siSTAT3 group, 17.1% in the Ang-siSTAT3@Gboxin NPs group, and 1.4% in the Ang-siSTAT3 NPs group, and in a concentration-dependent manner. This result suggests that the nano-co-delivery vehicle can enhance the killing efficiency of tumor cells by enhancing the intracellular accumulation of siRNA and Gboxin.

[0098] U251-Luc cells were cultured at a rate of 5 × 10 5Cells were seeded in a 96-well plate at a density of 10 cells / well. After incubation in a cell culture incubator for 24 hours, the culture medium was removed and the cells were washed twice with PBS solution. 2000 μL of PBS and Free siSTAT3+Gboxin, Ang-siScr@Gboxin NPs (13 kDa), therapeutic Ang-siSTAT3 NPs (13 kDa), Ang-siSTAT3@Gboxin NPs (100 nM, 13 kDa) and Ang-siSTAT3@Gboxin NPs (200 nM, 13 kDa) prepared in Example 1 were added, and the final concentration of siRNA was 100 nM or 200 nM. The cells were incubated in a cell culture incubator for another 48 hours, the culture medium was discarded, and the cells were washed with PBS solution. After digestion with EDTA-free trypsin, the cells were centrifuged and washed twice with PBS solution. The cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature. Add 500μL of 1×Binding Buffer to the EP tube and pipette to mix the cells. Then add 5μL of AnnexinV-FITC solution and 5μL of Propidium Iodide solution. Use normal cells that have not been treated with drugs to digest and collect them and divide them into three parts, namely a blank tube and two single-stained tubes. Use 500μL of 1×Binding Buffer to resuspend the cells in the blank tube to evaluate the level of autofluorescence. Add 5μL of AnnexinV-FITC or 5μL of Propidium Iodide to the single-stained tube for staining to determine the boundaries of each cell population. Keep away from light at room temperature for 10 minutes and detect and analyze on the machine within 1 hour. Figure 12 As shown in (b), after the cells were treated with Ang-siSTAT3@Gboxin (100nM) and Ang-siSTAT3@Gboxin (200nM) for 48h, the cell apoptosis rates were 25.3% and 36.6%, respectively, which were significantly higher than those of the free siSTAT3+Gboxin group (15.3%), the Ang-siScr@Gboxin NPs group (13.9%) and the Ang-siSTAT3 NPs group (9.3%), and were dose-dependent.

[0099] From the analysis of the above-mentioned MTT cell viability test and flow cytometry cell apoptosis test results, the peptide-modified nanocarrier improves the cell uptake efficiency through receptor-mediated endocytosis, ensures the delivery of siRNA into the cell to achieve specific silencing of related genes and induce cell apoptosis. At the same time, Gboxin specifically binds to the mitochondrial oxidative phosphorylation complex in the cell, inhibiting the activity of ATP synthase, and can be used as an effective anti-tumor small molecule agent, providing an experimental basis for the nano-combination treatment strategy of brain glioma.

[0100] (2) Gene silencing specificity of siRNA nanomedicines

[0101] To evaluate the gene silencing specificity of siRNA nanomedicine, U251-Luc tumor cells were treated with siSTAT3 nanomedicine, and the expression level of STAT3 mRNA in U251-Luc cells was detected by real-time fluorescence quantitative PCR. 5 The cells were seeded at a density of 100 μL / well in a 6-well plate and incubated overnight at 37°C in a 5% CO2 incubator. The old culture medium was removed and 200 μL of Free siSTAT3+Gboxin, Ang-siScr NPs (13 kDa), Ang-siSTAT3 NPs (100 nM, 13 kDa), and Ang-siSTAT3 NPs (200 nM, 13 kDa) prepared in Example 1 were added to each well to a final siRNA concentration of 100 nM or 200 nM. The cells were then cultured in an incubator for 48 h, all culture medium was removed, the cells were rinsed three times with PBS buffer, trypsin was added for digestion, and the connection between the cells became loose. An appropriate amount of culture medium was added to terminate the digestion, the cells were transferred to a centrifuge tube for centrifugation, the upper layer solution was removed, the cells were resuspended with PBS and centrifuged. The total mRNA was purified using a cell / bacteria total mRNA kit (Takara Tiangen Biotechnology Co., Ltd.), the concentration of the purified mRNA was determined, and the mRNA was reverse transcribed into cDNA using a reverse transcription kit. The amount of STAT3 and GAPDH was then quantified by real-time polymerase chain reaction. The amount of STAT3 gene was normalized to the amount of GAPDH gene. The expression of STAT3 at the protein level was detected by Western blotting.

[0102] like Figure 13 As shown in (a), after the action of Ang-siSTAT3 NPs (100nM) and Ang-siSTAT3 NPs (200nM), STAT3 mRNA decreased by approximately 46.0% and 61.0%, respectively, showing a dose-dependent inhibitory effect, while the STAT3 mRNA of Free siSTAT3+Gboxin and the nonspecific negative control Ang-siScr NPs had no significant difference from that of the control group, and there was almost no STAT3 mRNA gene silencing effect.

[0103] like Figure 13As shown in (c), treatment of U251-Luc cells with Ang-siSTAT3 NPs (100 nM) and Ang-siSTAT3 NPs (200 nM) reduced STAT3 protein expression by approximately 34.0% and 56.0%, respectively, demonstrating a dose-dependent inhibitory effect. However, the STAT3 protein levels of free siSTAT3+Gboxin and Ang-siScr were not significantly different from those of the control group, consistent with the mRNA silencing effect. This indicates that the triblock copolymer nanomedicine can efficiently deliver siRNA into cells and significantly inhibit STAT3 protein expression. However, free siSTAT3+Gboxin is unable to achieve functional gene silencing due to insufficient cellular uptake and lysosomal degradation. Ang-siScr lacks sequence specificity, indicating that STAT3 silencing is strictly sequence-specific. The efficiency and specificity of siRNA nanomedicine gene silencing were verified at both the transcriptional and protein levels.

[0104] Example 6 Detection of siRNA Distribution in Organisms

[0105] BALB / c mice aged 6-8 weeks and of similar weight were randomly divided into two groups, each containing three mice. The mice were housed under a 12-h light-dark cycle, at 20-24°C, and at a relative humidity of 45-65%. All housing conditions and experimental procedures were strictly in accordance with the requirements of the Animal Care Committee of the Medical School of our institution. The two groups of BALB / c mice were injected via the tail vein with 200 μL of Cy5-labeled Ang-siScr (13 kDa) (Free Cy5-siRNA in the figure) and Ang-siSTAT3 NPs (13 kDa) prepared in Example 1, at a dose of 1 mg / kg per mouse. After injection, fix the mice at 1, 5, 10, 30, 60, 120, 240 and 360 minutes, collect about 50 μL of blood from the canthus of the mouse using a capillary tube, store the blood sample in an EP tube and protect from light, immediately add 600 μL of 1% Triton X-100 lysis buffer, dissolve and mix evenly by ultrasonication, incubate at 4°C overnight, and centrifuge at 15000 rpm for 30 minutes. After centrifugation, take the supernatant of the sample into a 96-well plate, use a multifunctional microplate reader to measure the fluorescence intensity value of Cy5 in the supernatant, thereby determining the content of siRNA in the blood at each time point and calculating the half-life of the drug in the body. Figure 14 As shown, the free Cy5-siRNA had the shortest half-life in mice, at 3.9 minutes, and was eliminated very quickly. The half-life of Ang-siSTAT3 NPs was 42.9 minutes. These results indicate that the nano-delivery vector constructed with the synthesized triblock copolymer significantly prolonged the circulation time of siRNA in the blood.

[0106] For the biodistribution study of siRNA nanomedicines, mice bearing orthotopic U251-Luc tumors of uniform size and in good growth condition were selected. 200 μL of Ang-siScr (13 kDa) (Free Cy5-siRNA in the figure), siSTAT3 NPs (13 kDa), and Ang-siSTAT3 NPs (13 kDa) were injected via the tail vein, with three mice per group receiving a dose of 1 mg / kg of Cy5-siRNA per mouse. Eight hours after injection, the mice were anesthetized and sacrificed. The heart, liver, spleen, lungs, kidneys, and brain were removed, washed with PBS, and weighed. These tissues were placed on black paper in the order of brain, heart, liver, spleen, lungs, and kidneys. Cy5-siRNA fluorescence and bioluminescence imaging of the corresponding organs was performed using a small animal in vivo imaging device. Following imaging, the tissues were placed in a homogenizer tube containing 600 μL of 1% Triton X-100 lysis buffer and incubated overnight. The tissues were then ground using a tissue grinder to completely fragment them. Centrifuge at room temperature and 15,000 rpm for 30 minutes. Take 100 μL of the upper homogenate and use a multifunctional microplate reader (Ex = 630 nm, Em = 670 nm) to measure the Cy5-siRNA fluorescence intensity in the sample. Substitute the standard curve to calculate the Cy5-siRNA content in each tissue. Next, anesthetize the mice and dissect them 8 hours after the tail vein injection of the drug to evaluate the distribution of Cy5-siRNA in different tissues. Figure 15 As shown in (a), the Cy5 fluorescence intensity of the Ang-siSTAT3 NPs group in the mouse brain tumor region was consistently higher than that of the STAT3-siRNA NPs group and the free Cy5-siRNA group, demonstrating significant brain accumulation and retention. The fluorescence intensity of the Ang-siSTAT3 NPs group reached its maximum 8 hours after injection, and some accumulation in the brain was still present at 24 hours, with signal intensity consistently higher than that of the Ang-siSTAT3 NPs group and the Free Cy5-siRNA group. The Free Cy5-siRNA group, however, accumulated primarily in the kidneys. These results demonstrate that Angiopep-2-modified nanomedicines significantly enhance the nanocarrier's ability to cross the blood-brain barrier, resulting in higher accumulation in brain tumors.

[0107] from Figure 15As shown in (b) and (c), mice injected with Ang-siSTAT3 NPs via the tail vein showed significantly higher Cy5-siRNA accumulation in the liver, spleen, lung, and brain than the other two groups. Ang-siSTAT3 NPs accumulated 2.1 times more in brain tumors than siSTAT3 NPs. Free Cy5-siRNA is readily degraded by nucleases in vivo and rapidly metabolized and cleared through the kidneys, exhibiting only negligible fluorescence intensity in the brain, with an accumulation of only 0.6%, while accumulation in the kidneys reached a high of 23.2%. When imaging the three-dimensional organs, the Cy5 fluorescence signal overlapped with the autofluorescence of the tumor. These results suggest that free Cy5-siRNA has difficulty crossing the BBB to reach brain tumors, and that STAT3-siRNA NPs have limited ability to reach brain tumors. Nanodrugs formed with triblock copolymers can prolong their in vivo circulation, while nanodrugs modified with Angiopep-2 exhibit strong BBB penetration and accumulate in brain tumors.

[0108] Example 7 Detection of the anti-tumor effect of siRNA nanomedicine in vivo

[0109] U251-Luc cells were orthotopically implanted in BALB / c nude mice, and tumor size was measured using an in vivo imaging device. U251-Luc orthotopically tumor-bearing mice with uniform tumor size and good growth were randomly divided into five groups: PBS, Free siSTAT3+Gboxin, Ang-siScr@Gboxin NPs (13kDa), Ang-siSTAT3 NPs (13kDa), and Ang-siSTAT3@Gboxin NPs (13kDa) prepared in Example 1. Each mouse was injected with 200 μL of nanomedicine (siRNA: 2 mg / kg and Gboxin: 5 mg / kg) via the tail vein. The drugs were administered every two days, and the mouse weight was recorded. Subsequently, each mouse was intraperitoneally injected with 200 μL of D-luciferin saline solution, and the autofluorescence intensity of the tumor was observed using a small animal imaging device. The treatment was terminated after 5 doses of administration. One animal was randomly selected from each group and sacrificed after anesthesia. The brain, heart, liver, spleen, lung, and kidney tissues were removed, washed with PBS solution, and weighed and recorded. The tissues were stored in paraformaldehyde solution at room temperature for subsequent H&E staining, CC3, STAT3, and Ki67 detection experiments.

[0110] After five treatments, the bioluminescence intensity of the head tumor cells was monitored to determine the relative size of the tumors. As shown in Figure 16(a), the autofluorescence intensity of the tumor sites in the PBS group and the Free siSTAT3+Gboxin group showed no significant difference, indicating little inhibitory effect on tumor growth. However, the Ang-siSTAT3@Gboxin NPs and Ang-siSTAT3 NPs groups showed some tumor growth inhibition compared to the PBS group. This may be because the nanocarriers can cross the BBB and target tumor tissue, releasing siSTAT3 and Gboxin, where each exerts its effects. After treatment with the Ang-siSTAT3@Gboxin nanodrug, the bioluminescence of the tumors in the mice was significantly weaker, indicating that the nanodrug can cross the BBB and target tumor tissue, where the released siSTAT3 and Gboxin drugs accumulate in the tumor and collectively inhibit tumor cell growth. Further investigation is needed to determine the specific mechanism of action.

[0111] The luminescence of tumor tissue in mice was quantified and the results were as follows: Figure 16 As shown in (b), differences began to appear after the third imaging. The bioluminescence intensity of the tumors in the mice imaged at each time was quantified and normalized. The results showed that the relative fluorescence intensity of the PBS control group and the Free siSTAT3+Gboxin group continued to increase, approximately 13 times that of the Ang-siSTAT3@GboxinNPs group, 1.5 times that of the Ang-siScr@GboxinNPs group, and 2 times that of the Ang-siSTAT3NPs group. This further verified that siSTAT3 and Gboxin each have limited inhibitory effects on tumors. When used in combination, they significantly inhibited tumor cell growth, while the Ang-siScr@GboxinNPs and Ang-siSTAT3NPs groups had limited inhibitory effects on tumor growth. The Free siSTAT3+Gboxin group without nanocarrier delivery did not inhibit tumor growth in mice after administration.

[0112] The results of body weight changes of mice during treatment were as follows Figure 16As shown in (c), the weight of mice in the Ang-siSTAT3@Gboxin NPs group showed no downward trend, while the weight of mice in the Ang-siSTAT3@Gboxin NPs and Ang-siSTAT3 NPs groups also showed a downward trend. The mice in the PBS and Free siSTAT3+Gboxin groups showed the most significant weight loss and were in poorer condition. This phenomenon may be due to the rapid growth of tumor cells in the brain, leading to brain dysfunction and affecting the normal physiological functions of the mice. This was also confirmed by autopsy of the mice, with larger brain tumor volumes in the PBS and Free siSTAT3+Gboxin groups.

[0113] Survival curves such as Figure 16 As shown in (d), mice treated with Ang-siSTAT3@Gboxin NPs had a higher survival rate with a median survival time of 46 days, which was significantly longer than 21 days in the PBS group, 22 days in the Free siSTAT3+Gboxin group, 35 days in the Ang-siScr@Gboxin NPs group, and 32 days in the Ang-siSTAT3 NPs group.

[0114] After 5 treatment cycles, one mouse was randomly selected from each group and anesthetized and killed, and the heart, liver, spleen, lung, and kidney tissues were stained with H&E. Figure 17 As shown, compared with the PBS control group, each nanodrug group effectively inhibited the growth of brain glioma while having no obvious toxic side effects on the main organs of mice, and no inflammatory infiltration or necrotic lesions were observed, indicating that the nanodrug has good in vivo biosafety.

[0115] Example 8 Biosafety Evaluation

[0116] In order to evaluate the in vivo safety of the nanomedicine, PBS solution and Ang-siSTAT3@Gboxin NPs (13 kDa) were injected into BALB / c mice via the tail vein, and blood samples were collected for routine blood tests and biochemical analysis at predetermined time points after administration. Figure 18As shown, the blood routine indicators in the PBS control group and the Ang-siSTAT3@Gboxin NPs experimental group were essentially the same, indicating that the nanomaterial did not induce systemic inflammation or hematopoietic dysfunction in mice. Liver function indicators ALT, AST, and ALP showed no significant differences between the experimental and control groups, indicating no hepatocellular damage. Renal function indicators UREA, CREA, and UA also remained within the normal physiological range, with no significant differences between the two groups, indicating that the nanomaterial did not increase the metabolic burden on the kidneys. Body weight changes in mice in both groups also remained consistent during the dosing period. Blood biochemistry and blood routine indicators further confirmed the good biosafety of Ang-siSTAT3 NPs in vivo.

[0117] To systematically evaluate the effects of nanomedicine on liver and kidney function and potential inflammatory responses, PBS solution and Ang-siSTAT3@Gboxin NPs were injected into BALB / c mice via the tail vein. The mice were then sacrificed on day 1 and day 14, respectively, and liver and kidney tissues were collected. Fluorescence quantitative PCR was used to detect the mRNA levels of TNF-α, IL-6, and IL-1β. Figure 19 As shown, the expression of tissue inflammatory factors in the liver and kidneys of mice treated with PBS solution and Ang-siSTAT3@Gboxin NPs via tail vein injection was at the same level, with no statistically significant difference. This experiment confirmed at the molecular level that the nanomedicine had minimal effect on liver and kidney function, further demonstrating its good in vivo biocompatibility.

[0118] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A siRNA nano drug carrier, characterized in that The active ingredient is nanoparticles formed by a triblock copolymer loaded with siRNA. The middle block of the triblock copolymer is poly(β-amino ester), one end block is polyethylene glycol, and the other end block is poly(3-acrylamidophenylboronic acid pinacol ester). The molecular weight of the poly(3-acrylamidophenylboronic acid pinacol ester) is 3 to 7 kDa.

2. The siRNA nano drug carrier according to claim 1, characterized in that The triblock copolymer is poly (3-acrylamidophenylboronic acid pinacol ester) 3k -poly(β-amino ester) 5k -Polyethylene glycol 5k , poly (3-acrylamidophenylboronic acid pinacol ester) 5k -poly(β-amino ester) 5k -Polyethylene glycol 5k or poly (3-acrylamidophenylboronic acid pinacol ester) 7k -poly(β-amino ester) 5k -Polyethylene glycol 5k .

3. The siRNA nano drug carrier according to claim 1 or 2, characterized in that The synthesis steps of the poly (3-acrylamidophenylboronic acid pinacol ester) are as follows: 3-acrylamidophenylboronic acid pinacol ester monomer, 4-cyano-4-[[(dodecylthio)thiocarbonyl]thio]pentanoic acid, and azobisisobutyronitrile are dissolved in 1,4-dioxane, and the mixture is polymerized at 70° C. for 48 hours to obtain the poly (3-acrylamidophenylboronic acid pinacol ester).

4. The siRNA nano drug carrier according to claim 3, characterized in that The molar ratio of the 3-acrylamide phenylboronic acid pinacol ester, 4-cyano-4-[[(dodecylthio)thiocarbonyl]thio]pentanoic acid, and azobisisobutyronitrile is 14-32:1:0.

2.

5. The siRNA nano drug carrier according to claim 1, characterized in that The siRNA is selected from siRNA of STAT3 gene, and the sequence of siRNA of STAT3 gene is shown as SEQ ID NO.1-2.

6. The siRNA nano drug carrier according to claim 1 or 5, characterized in that The molar ratio of the triblock copolymer to the siRNA is 50-200:

1.

7. The siRNA nano drug carrier according to claim 1, characterized in that The nanoparticles were also modified with Angiopep-2.

8. A nano anti-tumor drug, characterized in that: The active ingredient is nanoparticles formed by the siRNA nano drug carrier loaded with anti-tumor drugs according to any one of claims 1 to 7.

9. The nano anti-tumor drug according to claim 8, characterized in that: The anti-tumor drugs include tumor suppressors.

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