DNA origami-based siRNA targeted delivery system as well as preparation method and application thereof

Abstract

The invention discloses an siRNA targeted delivery system based on DNA origami as well as a preparation method and application of the siRNA targeted delivery system. The siRNA targeted delivery system based on DNA origami comprises a DNA nano origami tube carrier, siRNA loaded in a tube cavity of the DNA nano origami tube carrier and targeted molecule-ssDNA conjugates modified at two ends of the DNA nano origami tube carrier, wherein the DNA nano origami tube carrier is mainly formed by self-assembling a bracket chain, a staple chain, a siRNA capture chain, an aptamer chain and a fastening chain. According to the siRNA targeted delivery system based on DNA origami provided by the invention, on one hand, long-acting protection of siRNA in serum and slow degradation and release in cells are realized by utilizing the tubular closed structure characteristic of the DNA nano origami tube carrier, the drug action time is prolonged, and the drug administration frequency is reduced; on the other hand, by modifying targeting molecules on the DNA nano origami tube carrier, the effects of inducing penetration of the blood brain barrier and targeted focus delivery can be achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a DNA origami-based siRNA targeted delivery system as well as a preparation method and application thereof. BACKGROUND

[0002] As a gene silencing tool, small interfering RNA (siRNA) has great potential in the field of precision medicine. siRNA gene therapy exerts effects by specifically silencing pathogenic genes, and has shown significant therapeutic potential in various diseases, including genetic diseases, tumors, infectious diseases, and neurodegenerative diseases, etc., due to its precise targeting of pathogenic genes. siRNA silences mRNA through base complementary pairing, and performs gene silencing without changing DNA, so as to prevent irreversible genetic changes and has excellent safety. However, siRNA has the following defects: (1) naked siRNA is easily degraded by serum nucleases, with a half-life of only minutes, so that its retention time in the body is extremely short, and it needs to be protected by an external carrier; (2) naked siRNA is difficult to penetrate the cell membrane and biological barriers (such as blood-brain barrier (BBB), blood-tumor barrier, etc.); (3) naked siRNA is difficult to target the lesion site, and has a tissue off-target effect.

[0003] Alzheimer's disease (AD) is a degenerative nervous system disease caused by the neurotoxicity of abnormal deposition of beta-amyloid and tau protein. GSK-3β is a key molecule in the pathogenesis of AD, which can regulate microglial phagocytosis and autophagy, and can also regulate the generation of AD key pathological protein molecules beta-amyloid and phosphorylated tau protein. However, there are limitations in using GSK-3β siRNA to treat AD, mainly in the following aspects: the retention time of siRNA in blood is extremely short, and there is a blood-brain barrier and its accompanying physical and biochemical barriers, which limit the entry of siRNA and macromolecular drugs into brain tissue; siRNA cannot target AD lesions, and there is a risk of off-target gene silencing. SUMMARY

[0004] The main purpose of the present application is to provide a DNA origami-based siRNA targeted delivery system to solve at least one of the above technical problems.

[0005] According to one aspect of the present application, a DNA origami-based siRNA targeted delivery system is provided, which comprises a DNA nano origami tube carrier, siRNA loaded in the lumen thereof, and a targeting molecule-ssDNA conjugate modified at both ends thereof; wherein the DNA nano origami tube carrier is mainly formed by self-assembly of a scaffold chain, a staple chain (Staples chain), an siRNA capture chain, an aptamer chain, and a locker chain.

[0006] The application constructs a DNA nano origami tube carrier with single-stranded DNA (ssDNA) as the core. The carrier has natural biocompatibility, low immunogenicity, high biological safety, can improve the cell uptake efficiency of siRNA, optimize the pharmacokinetic characteristics, and reduce the metabolic waste of liver and kidney functions. On the basis of the scaffold chain and the Staples chain, the siRNA capture chain and the aptamer chain are further designed to load siRNA and target molecules, and the scaffold chain, the staple chain, the siRNA capture chain and the aptamer chain can be self-assembled into a planar rectangular DNA nano origami sheet through annealing, and the planar rectangular DNA nano origami sheet has an extension chain that can be partially complementary to the sense strand of siRNA and an extension chain that can be partially or completely complementary to the ssDNA part of the target molecule-ssDNA conjugate. Finally, the planar rectangular DNA nano origami sheet is curled into a tubular shape by using the Locker chain, and the siRNA is loaded in the lumen of the DNA nano origami tube. The DNA nano origami tube carrier provided by the application can realize efficient loading and stable wrapping of siRNA, and directional modification of target molecules.

[0007] The siRNA targeted delivery system based on DNA origami provided by the application can realize long-term protection of siRNA in serum and slow release of intracellular degradation, prolong the drug action time, and reduce the drug administration frequency by utilizing the tubular closed structure characteristics of the DNA nano origami tube carrier. On the other hand, by modifying the target molecules on the DNA nano origami tube carrier, the effects of inducing penetration of the blood-brain barrier and targeted lesion delivery can be achieved.

[0008] The M13 plasmid is a circular DNA single strand, which is the genomic DNA of M13 bacteriophage. In the process of DNA origami construction, it is usually used as the scaffold chain of DNA origami. The staple chain is a short single-stranded DNA artificially designed and synthesized, usually containing 20-60 bases, and is designed to be hybridized and complementary to multiple discontinuous regions on the scaffold chain. The scaffold chain and dozens to hundreds of staple chains can be self-assembled into a DNA nano origami sheet with a planar rectangular structure through base complementary pairing. In the application, the scaffold chain is the M13mp18 bacteriophage genomic DNA with a size of 7249 bp (the specific sequence is shown in GenBank: X02513.1), and the staple chain is mainly designed according to the literature "A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo (DOI: 10.1038 / nbt.4071)".

[0009] In some embodiments, the nucleotide sequence of the siRNA capture strand comprises, in order from the 5' end to the 3' end, a nucleotide sequence partially complementary to the siRNA sense strand and a nucleotide sequence of a staple strand. In this way, the siRNA can be efficiently loaded by base complementary pairing. Meanwhile, the number of siRNA capture strands and the connection sites can be controlled as needed to achieve precise control of the loading amount and loading position of the siRNA.

[0010] In some embodiments, the nucleotide sequence of the aptamer strand comprises, in order from the 5' end to the 3' end, a nucleotide sequence of a staple strand and a nucleotide sequence fully or partially complementary to the ssDNA portion of the targeting molecule-ssDNA conjugate. In this way, the targeting molecule can be loaded on the DNA nanofold tube carrier by base complementary pairing, and the loading amount and modification position of the targeting molecule can be precisely controlled by controlling the number of aptamer strands and the connection sites. Preferably, the targeting molecule is modified at both ends of the DNA nanofold tube carrier, thereby facilitating the induction of penetration of the blood-brain barrier and the effect of targeted lesion delivery.

[0011] In some embodiments, the siRNA can be a GSK-3β siRNA, the nucleotide sequence of the sense strand of which comprises a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand of which comprises a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 2.

[0012] In some embodiments, deoxyribonucleotides dTdT can be overhanging at the 3' end of the nucleotide sequences of the sense and antisense strands of the GSK-3β siRNA.

[0013] In some embodiments, the GSK-3β siRNA capture strand can have 32, and the nucleotide sequences thereof are shown in SEQ ID NO: 7~38, respectively.

[0014] In some embodiments, the targeting molecule can be selected from at least one of an angiotensin-derived peptide (ANG peptide), transferrin, lactoferrin, and a QSH peptide; wherein the amino acid sequence of the QSH peptide is shown in SEQ ID NO: 3, and the amino acid sequence of the ANG peptide is shown in SEQ ID NO: 5.

[0015] In some embodiments, the targeting molecule is an ANG peptide and a QSH peptide. The synergistic effect between the ANG peptide and the QSH peptide can form a "penetration-targeting" bifunctional system, in which the ANG peptide mediates efficient penetration of the BBB, and the QSH peptide specifically binds to the Aβ aggregation lesion, breaking through the double barrier of siRNA brain delivery, thereby the synergistic effect of the two can significantly improve the therapeutic effect of GSK-3β siRNA in treating AD.

[0016] In some embodiments, the targeting molecule is coupled to the 5' end of the ssDNA, whereby the targeting molecule can be loaded on the DNA origami carrier by the partial or complete complementarity between the ssDNA and the aptamer strand.

[0017] In some embodiments, the nucleotide sequence of the ssDNA in the ANG peptide-ssDNA conjugate is shown in SEQ ID NO: 6.

[0018] In some embodiments, the nucleotide sequence of the ssDNA in the QSH peptide-ssDNA conjugate is shown in SEQ ID NO: 4.

[0019] In some embodiments, the aptamer strand has 10 strands, and the nucleotide sequences of the 10 strands are shown in SEQ ID NO: 39-48, respectively.

[0020] The number of fastening strands is 2n, wherein n is a natural number and is greater than or equal to 1. A pair of fastening strands is mainly designed for two staple strands I and II located on the opposite long edges of the planar rectangular DNA origami sheet, wherein the nucleotide sequence of one fastening strand is designed to sequentially include the nucleotide sequence of the 5' end of the staple strand I and the nucleotide sequence of the 3' end of the staple strand II from 5' end to 3' end; and the nucleotide sequence of the other fastening strand is designed to sequentially include the nucleotide sequence of the 5' end of the staple strand II and the nucleotide sequence of the 3' end of the staple strand I from 5' end to 3' end. Thus, a pair of fastening strands can form a "lock" to realize the curling of the planar rectangular DNA origami sheet to form a DNA origami tube. The specific number of fastening strands can be designed as needed.

[0021] In some embodiments, the fastening strands have 6 pairs of 12 strands, and the nucleotide sequences of the 12 strands are shown in SEQ ID NO: 49-60, respectively.

[0022] The application provides a DNA origami-based GSK-3β siRNA targeted delivery system, which uses a DNA origami tube as a carrier, efficiently loads GSK-3β siRNA and encapsulates it in the lumen; the ends of the tubular structure are modified with ANG peptide and QSH peptide, respectively, to mediate blood-brain barrier penetration and Aβ lesion targeting, respectively, and the three of GSK-3β siRNA, ANG peptide and QSH peptide can synergistically achieve precise brain delivery, lesion accumulation and long-acting sustained-release treatment of GSK-3β siRNA. The DNA origami-based GSK-3β siRNA targeted delivery system provided by the application can be applied to the treatment of AD or to the preparation of a drug for treating AD.

[0023] According to another aspect of the present invention, a method for preparing the above-described DNA origami-based siRNA targeted delivery system is provided, comprising the following steps: The scaffold strand, staple strand, siRNA capture strand, and aptamer strand were added to a Mg-containing container. 2+ In TAE buffer, the mixture was annealed from 95°C to 15°C to obtain planar rectangular DNA nano-originated sheets; Planar rectangular DNA nanoparticles and siRNA were mixed and annealed from 45°C to 25°C. Then, a binding strand was added, and the mixture was annealed from 37°C to 15°C to obtain DNA nanoparticle tubes. Finally, the targeting molecule-ssDNA conjugate was added, and the mixture was annealed from 37°C to 15°C to obtain the final product.

[0024] In some implementations, the molar ratio of the scaffold chain, any staple chain, any siRNA capture chain, any aptamer chain, and any fastening chain is 1:1:1:1:(1~20).

[0025] In some implementations, the molar ratio of the scaffold chain, any staple chain, any siRNA capture chain, any aptamer chain, and any fastening chain is 1:1:1:1:20.

[0026] In some implementations, the molar ratio of siRNA to any siRNA capture strand is (5~10):1.

[0027] In some implementations, the molar ratio of siRNA to any one of the siRNA capture strands is 10:1.

[0028] In some implementations, the molar ratio of the target molecule-ssDNA conjugate to any aptamer chain is 1:1.

[0029] In some embodiments, Mg 2+ The TAE buffer is mainly prepared from tris(hydroxymethyl)aminomethane (Tris) or its salt, ethylenediaminetetraacetic acid (EDTA), acetic acid and magnesium acetate.

[0030] In some implementations, Mg 2+ The TAE buffer has a pH of 8.0 and consists of 40 mM Tris, 20 mM acetate, 2 mM EDTA and 12.5 mM magnesium acetate. Attached Figure Description

[0031] Figure 1The results show the synthesis and characterization of the DNA nanoparticle-based siRNA targeted delivery system. (A) is a schematic diagram of the synthetic route for the DNA nanoparticle-loaded siRNA. (B) shows the agarose gel electrophoresis results of the M13 plasmid, unloaded DNA nanoparticles, and unloaded DNA nanoparticles. (C) shows the morphological characterization of the unloaded DNA nanoparticles (top) and unloaded DNA nanoparticles (bottom) using atomic force microscopy (AFM) and transmission electron microscopy (TEM). (D) shows the particle size results of the unloaded DNA nanoparticles and unloaded DNA nanoparticles detected by DLS. (E) shows the polyacrylamide gel electrophoresis results of the unloaded DNA nanoparticles, unloaded DNA nanoparticles, and DNA nanoparticles loaded with GSK-3β siRNA. (F) is a schematic diagram of the experimental procedure for verifying the protective effect of DNA nanoparticles on siRNA. (G) shows the results of GSK-3β siRNA loaded after co-incubation with serum for 24 h. Fluorescence emission spectra of DNA nano-origami sheets containing siRNA and DNA nano-origami tubes loaded with GSK-3β siRNA; Figure 2 The results validated the protective and sustained-release effects of DNA nano-origami tubes on siRNA in cells. (A) shows the D / A ratio of DNA nano-origami sheets and DNA nano-origami tubes loaded with GSK-3β siRNA after co-incubation with HT22 and BV2 cells for different time points, with a scale bar of 5 μm; (B) shows the statistical distribution of the D / A ratio of DNA nano-origami sheets loaded with GSK-3β siRNA at corresponding time points in HT22 cells; (C) shows the statistical distribution of the D / A ratio of DNA nano-origami tubes loaded with GSK-3β siRNA at corresponding time points in HT22 cells; (D) shows the statistical distribution of the D / A ratio of DNA nano-origami sheets loaded with GSK-3β siRNA at corresponding time points in BV2 cells; and (E) shows the statistical distribution of the D / A ratio of DNA nano-origami tubes loaded with GSK-3β siRNA at corresponding time points in BV2 cells. Figure 3 Figure 1 shows the in vitro evaluation results of the ability of the DNA nano-origami tube-based siRNA targeted delivery system to penetrate the BBB and be taken up by cells. (A) shows the experimental groups; (B) is a schematic diagram of the Transwell model experiment; (C) shows the flow cytometry results of the fluorescence intensity of Cy5 in HT22 cells; (D) shows the flow cytometry results of the fluorescence intensity of Cy5 in BV2 cells; (E) is a representative confocal image of the uptake of different Cy5-labeled drugs by HT22 and BV2 cells in the Transwell-BBB model, with a scale bar of 10 μm; (F) is a statistical analysis of the fluorescence intensity in (E). Figure 4 Figure 1 shows the in vivo evaluation results of the ability of the DNA nano-origami tube-based siRNA targeted delivery system to penetrate the BBB and target lesions. (A) is a schematic diagram of grouping; (B) is a representative in vivo image of C57 mice injected with Cy5-labeled DON or DON-A at different time points after injection; (C) is the change curve of Cy5 fluorescence intensity in the brain of C57 mice injected with Cy5-labeled DON or DON-A at different time points after injection; (D) is a representative ex vivo brain image and fluorescence intensity quantification result of C57 mice injected with Cy5-labeled DON or DON-A 6 h after injection; (E) is a representative ex vivo image and fluorescence intensity quantification result of the heart, liver, spleen, lung, and kidney tissues of C57 mice injected with Cy5-labeled DON or DON-A 6 h after injection; (F) is a representative confocal image of the distribution of Aβ in the hippocampus of 5×FAD mice. (Scale bar: 50) μm; (G) is a representative confocal image of the distribution of Aβ in the cortical brain tissue of 5×FAD mice, where the scale bar is 50 μm; Figure 5 Figure 1 shows the results of an in vitro gene silencing effect evaluation experiment of a DNA origami-based siRNA targeted delivery system under the Transwell-BBB model. (A) is a schematic diagram of the experimental grouping and cell model design; (B) is a schematic diagram of the mechanism; (C) shows the inhibition of Aβ plaque aggregation after co-incubation with different drugs; (D) shows the flow cytometry and quantitative analysis results of BV2's phagocytic uptake of Aβ42-FITC after different drug treatments; (E) is a representative TEM image of BV2 under the Transwell-BBB model, scale bar 1 nm; (F) shows the Western blot results of the expression of GSK-3β, p62, and LC3B (LC3-I, LC3-II) proteins in BV2 cells after different drug treatments; (GI) shows the quantitative analysis results of (F); (J) is a representative confocal image of the distribution of LC3B in BV2 cells after different drug treatments, scale bar 10 nm. μm; (K) represents the quantitative analysis result of LC3B fluorescence intensity in (J); Figure 6 Figure showing the results of GSK-3β mRNA expression level detection; Figure 7 The results of flow cytometry analysis of the fluorescence intensity of Aβ-FITC in BV2 cells after treatment with different drugs. Figure 8Representative confocal images and quantitative analysis results of the distribution of LC3B, Caspase-3 and Aβ in brain tissue after different drug treatments are shown. Among them, (A) is a representative confocal image of the distribution of LC3B, Caspase-3 and Aβ in the brain tissue of mice in each group, scale bar, 20 μm; (BD) are the quantitative analysis results of the fluorescence intensity of LC3B, Caspase-3 and Aβ in (A), respectively. Figure 9 Figures showing the behavioral results of mice in different groups after different drug treatments: (A) Grouping for experimental drug administration; (B) Schematic diagram of experimental design; (C) Representative figure of the water maze experiment; (D) Result of the number of times mice crossed the target platform in the water maze experiment; (E) Result of the time mice spent in the platform quadrant in the water maze experiment; (F) Result of the percentage of times mice entered the new arm in the Y-maze novel arm experiment; (G) Result of the percentage of time mice spent entering the new arm in the Y-maze novel arm experiment; (H) Result of the preference index in the new object recognition experiment; (I) Schematic diagram of the Y-maze novel arm experiment; (J) Representative figure of the Y-maze novel arm experiment; (K) Schematic diagram of the new object recognition experiment; (L) Representative figure of the new object recognition experiment. Figure 10 HE staining images of major organs of normal mice treated with PBS or siGSK-3β@DON-AQ on days 1 and 7, scale bar, 50 μm; Figure 11 Results of routine blood tests on normal mice treated with PBS or siGSK-3β@DON-AQ on days 1 and 7. Figure 12 Results of blood biochemical parameters in normal mice treated with PBS or siGSK-3β@DON-AQ on days 1 and 7. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0033] In this invention, the M13 plasmid used was purchased from BIORuler, USA, catalog number B3003-50 pmol; the staple strand, siRNA capture strand, aptamer strand, fastening strand, GSK-3βsiRNA, ANG peptide-ssDNA conjugate and QSH peptide-ssDNA conjugate were all synthesized by Sangon Biotech (Shanghai, China).

[0034] In the experimental examples of this invention, the relevant experimental data were statistically analyzed using GraphPad Prism 9 (GraphPad Software, USA). Data were repeated ≥3 times, and results are expressed as mean ± standard deviation (SD). T-tests were used for comparisons between two groups, and one-way or two-way ANOVA was used for comparisons among multiple groups. The symbol "ns" indicates no significance. * express P <0.05, ** express P <0.01, *** express P <0.001, **** express P <0.0001.

[0035] Example 1: Preparation of a DNA origami-based GSK-3β siRNA targeted delivery system (siG@DON-AQ) The materials used to prepare siG@DON-AQ in this embodiment include: M13 plasmid (scaffold chain), 148 staple chains with different sequences, 32 siRNA capture chains, 10 aptamer chains, 12 locker chains, GSK-3β siRNA, ANG peptide-ssDNA conjugate, and QSH peptide-ssDNA conjugate. Among them: The 148 stapled strands with different sequences are the "Staple strands pool" in the literature "A DNA nanorobot functions as a cancertherapeutic in response to a molecular trigger in vivo (DOI:10.1038 / nbt.4071)". The remaining 148 sequences are those excluding sequences 28, 30, 32, 34, 48-49, 51, 53, 55, 57, 59, 72-73, 76, 78, 80, 82, 96-97, 99, 104, 107, 110, 114, 118, 120-121, 135, 137, 139, 141, 144-145, 158, 160, 162, 164, 166, 168-169, 183, 185, 187, and 189 from sequences 13-204 disclosed in (5'-3').

[0036] The nucleotide sequence of the sense strand of GSK-3β siRNA is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2, with deoxyribonucleotide dTdT hanging at the 3' end of both the sense and antisense strands. The specific sequence of GSK-3β siRNA is as follows: GSK-3β siRNA sense strand: 5'-TCCAATCCACTTATCATCTCATTTGUAUUGCAGGACAAGAGAUdTdT-3' GSK-3β siRNA antisense strand: 5'-AUCUCUUGUCCUGCAAUACdTdT-3' In the QSH peptide-ssDNA conjugate, the amino acid sequence of the QSH peptide is shown in SEQ ID NO:3, the nucleotide sequence of the ssDNA is shown in SEQ ID NO:4, and the QSH peptide and ssDNA are coupled by a phosphodiester bond.

[0037] In the ANG peptide-ssDNA conjugate, the amino acid sequence of the ANG peptide is shown in SEQ ID NO:5, the nucleotide sequence of the ssDNA is shown in SEQ ID NO:6, and the ANG peptide and ssDNA are coupled by a phosphodiester bond.

[0038] The nucleotide sequences of the 32 siRNA capture strands are shown in SEQ ID NO:7~38.

[0039] Of the 10 aptamer chains, 5 were used to load the ANG peptide and 5 were used to load the QSH peptide, and their nucleotide sequences are shown in SEQ ID NO:39~48, respectively.

[0040] The nucleotide sequences of the 12 tight chains are shown in SEQ ID NO:49~60.

[0041] The preparation method of siG@DON-AQ includes the following steps: (1) Preparation of planar rectangular DNA nano-origami sheets: In TAE-Mg 2+ Scaffold strands, staple strands, siRNA capture strands, and aptamer strands were added to a buffer solution (40 mM Tris, 20 mM acetate, 2 mM EDTA, and 12.5 mM magnesium acetate, pH 8.0). The final concentration of each of the scaffold strand, any one staple strand, any one siRNA capture strand, and any one aptamer strand was 100 nM. After mixing, the mixture was heated to 95°C and held for 3 min. Then, it was annealed from 95°C to 15°C at a rate of 0.1°C for 10 seconds at a time. The solution was collected and purified by a 100 kDa centrifugal filter to obtain planar rectangular DNA nanosheets.

[0042] (2) Preparation of DNA nano-origami tubes loaded with GSK-3β siRNA (siG@DON): GSK-3β siRNA and the planar rectangular DNA nanoparticles prepared in step (1) were mixed at a molar ratio of 320:1. The mixture was heated to 45°C and held for 5 min. Then, it was annealed from 45°C to 25°C at a rate of 300 seconds per 1°C. Each tight strand was then added at a molar ratio of 20:1 to the planar rectangular DNA nanoparticles. The mixture was heated to 37°C and held for 10 min. Then, it was annealed from 37°C to 15°C at a rate of 600 seconds per 1°C. siG@DON was synthesized by self-assembly.

[0043] A schematic diagram of the synthetic route for DNA nano-origami tubes loaded with siRNA is shown below. Figure 1 As shown in A in the diagram.

[0044] (3) Preparation of DNA nano-origami tubes loaded with ANG peptides and QSH peptides (siG@DON-AQ): Five molar amounts of ANG peptide-ssDNA conjugate and QSH peptide-ssDNA conjugate were added to siG@DON, respectively. After mixing, the mixture was heated to 37°C and held for 5 min. Then, it was annealed from 37°C to 15°C at a rate of 600 seconds per 1°C. The solution was collected and purified by a 100 kDa centrifugal filter to obtain siG@DON-AQ.

[0045] Example 1: Characterization of DNA Nanoparticle Origami Carrier (1) M13 plasmid, unloaded DNA nanoparticles, and unloaded DNA nanoparticle tubes were subjected to agarose gel electrophoresis. The results are as follows: Figure 1 As shown in B in the diagram.

[0046] Because DNA nano-origami tubes are rolled into a tubular structure, their migration rate is faster than that of DNA nano-origami sheets. Therefore, according to the results of agarose gel electrophoresis experiments, DNA nano-origami tubes have been successfully synthesized.

[0047] (2) Atomic force microscopy (AFM) and transmission electron microscopy (TEM) morphology characterization images of unloaded DNA nanofolds and unloaded DNA nanofolds are shown below. Figure 1 As shown in C.

[0048] As shown in the figure, the DNA nano-originated sheets have a regular planar rectangular structure, with a length of about 90-100 nm and a width of about 70-80 nm; the DNA nano-originated tubes have a regular tubular structure, with a length of about 90-100 nm and a width of about 40-50 nm.

[0049] (3) The particle size of unloaded DNA nanoparticles and unloaded DNA nanoparticle tubes was measured using dynamic light scattering DLS (Nano ZS, Malvern, UK). The results are as follows: Figure 1 As shown in D in the diagram.

[0050] The results showed that the particle size of the unloaded DNA nano-origami flakes was approximately 94.3 nm, and the particle size of the unloaded DNA nano-origami tubes was approximately 67.4 nm.

[0051] (4) GSK-3β siRNA was labeled with Cy7 and used to prepare DNA nano-origami tubes loaded with GSK-3β siRNA. Unloaded DNA nano-origami sheets, unloaded DNA nano-origami tubes, and Cy7-labeled DNA nano-origami tubes loaded with GSK-3β siRNA were subjected to polyacrylamide gel electrophoresis (PAGE), and the DNA was stained with GelRed (purchased from Thermo Fisher Scientific, catalog number S33102). The results are as follows. Figure 1 As shown in E in the figure.

[0052] The results showed that the migration rate of DNA nanotubes loaded with GSK-3β siRNA was slower than that of unloaded DNA nanotubes, and Cy7 (red) could overlap with GelRed (green), indicating that GSK-3β siRNA had been successfully loaded onto DNA nanotubes.

[0053] (5) Cy5 and Cy3 were labeled onto the double strand of GSK-3β siRNA, respectively, and used to prepare DNA nano-origami sheets loaded with GSK-3β siRNA and DNA nano-origami tubes loaded with GSK-3β siRNA. For example... Figure 1 As shown in F, DNA nanoparticles loaded with GSK-3β siRNA and DNA nanoparticle tubes loaded with GSK-3β siRNA were added to serum to a final concentration of 20 nM and incubated at 37°C for 24 h. After incubation, the fluorescence emission spectra of the DNA nanoparticles and tubes loaded with GSK-3β siRNA were measured and recorded. The results are as follows: Figure 1 As shown in G.

[0054] The fluorescence resonance energy transfer (FRET) effect between Cy5 and Cy3 can be used to detect the degree of separation of siRNA double strands. A significant FRET effect can be observed when Cy3 and Cy5 are close together. However, when the two fluorophores Cy3 and Cy5 are spatially separated, it leads to increased fluorescence emission from the donor Cy3, decreased fluorescence emission from the acceptor Cy5, and reduced FRET efficiency. The excitation wavelength of Cy3 is 561 nm, with an emission range of 580–620 nm; the excitation wavelength of Cy5 is 561 nm, with an emission range of 650–700 nm.

[0055] Figure 1 The results from G showed that DNA nano-origami tubes had a significantly better protective effect on siRNA than DNA nano-origami sheets, and could effectively improve the retention capacity of siRNA in serum, thus solving the problem of the short half-life of naked siRNA in serum.

[0056] Experiment 2: Verification of the protective and sustained-release effects of DNA nano-origami tube carriers on siRNA in cells. Cy5 and Cy3 were labeled onto the double strand of GSK-3β siRNA, respectively, and used to prepare DNA nanosheets and nanotubes loaded with GSK-3β siRNA. The separation of Cy5 and Cy3 fluorescent molecules on the siRNA double strand was observed using the FRET effect, thus demonstrating the protective effect and sustained-release function of DNA nanotubes and DNA nanosheets on siRNA in cells.

[0057] DNA nanoparticles loaded with GSK-3β siRNA and DNA nanoparticle tubes loaded with GSK-3β siRNA (20 nM) were added to culture medium and co-incubated with HT22 and BV2 cells. Fluorescence imaging was then performed at the cellular level, collecting fluorescence images of the Cy3 and Cy5 channels. The fluorescence intensity ratio of Cy3 to Cy5 (donor-acceptor ratio, D / A ratio) was calculated.

[0058] The results are as follows Figure 2 As shown.

[0059] Depend on Figure 2 The results showed that, compared with DNA nano-origami sheets, the siRNA double strands carried by DNA nano-origami tubes had a lower degree of dissociation under the same incubation time, indicating that DNA nano-origami tubes have a better protective effect on siRNA and have a sustained-release effect.

[0060] Alzheimer's disease (AD) is a chronic condition that often requires long-term, repeated drug administration. The DNA nano-origami tube-based siRNA delivery system provided in this invention allows siRNA to degrade slowly within cells, achieving a sustained-release effect. This not only reduces the frequency of drug administration but also efficiently delivers it into cells to exert its effects, demonstrating significant clinical translational value.

[0061] Experimental Example 3: In vitro evaluation of the ability of a DNA nano-origami tube-based siRNA targeted delivery system to penetrate the BBB and be taken up by cells. (1) Cell culture Neuronal cells (HT22 cells) and microglia (BV2 cells) were placed in complete culture medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin) and incubated in a cell culture incubator at 37°C and 5% CO2 until the cells reached the stable growth phase of the experiment.

[0062] (2) Construction of the Transwell-BBB model This invention constructs a three-layer co-culture model of the blood-brain barrier.

[0063] First, brain microvascular endothelial cells (BMEC), astrocytes, and pericytes were resuscitated and expanded to ensure viability ≥90%. BMECs were cultured in ECM medium containing 5% FBS, ECGF (20 ng / mL), and heparin (10 U / mL); astrocytes and pericytes were cultured in DMEM / F12 medium containing 10% FBS.

[0064] Transwell membranes were coated with type IV collagen or Matrigel: type IV collagen (50 μg / mL) or Matrigel (1:50 dilution) was dissolved in serum-free medium; coating solution (0.33 cm) was added to the inside of the Transwell membrane. 2 Add 50 μL of coating solution to the membrane and incubate overnight at 4°C or 1 h at 37°C; remove excess coating solution, wash twice with PBS, and air dry for later use.

[0065] Seeding order: The bottom layer consists of astrocytes, 2 × 10⁶ cells. 4 Cells / well, cultured at 37℃ for 24-48 h until cells are completely adhered and confluent; the middle layer is pericytes, and pericyte suspension is added to the underside of the Transwell insert membrane at 2.5 × 10⁻⁶. 3 Cells / inserts were incubated in an inverted culture dish at 37°C for 2 h. After pericytes adhered, the insert was placed upright into a receiving plate inoculated with astrocytes. The top layer of BMECs was seeded on the upper side of the Transwell insert membrane at 2 × 10⁶ cells / cm². 4 cells / inserts.

[0066] Co-culture for 5 - 7 days at 37°C and 5% CO₂ using serum-free DMEM medium (Gibco; 11965 - 092), and change the medium every 2 days. Verify the model by regularly measuring the transendothelial electrical resistance (TEER), and correct the TEER value to be stable ≥ 200 Ω·cm 2 That is considered qualified. Specifically, on the 1st, 3rd, 5th, and 7th days of culture, use an EVOM2 resistance meter to detect the TEER value. Insert the electrodes into the upper and lower chambers of the Transwell respectively. After the readings are stable, record the values, and measure each sample 3 times and take the average; calculate the corrected TEER value: TEER (Ω cm²) = (measured value - blank membrane value) × membrane area.

[0067] (3) Cell uptake experiment Replace GSK-3β siRNA with a Cy5-labeled complementary strand and complementary connect it to the RNA capture strand to prepare Cy5-labeled DNA nano-origami tubes (DON), Cy5-labeled DNA nano-origami tubes loaded with QSH peptide (DON-Q), Cy5-labeled DNA nano-origami tubes loaded with ANG peptide (DON-A), and Cy5-labeled DNA nano-origami tubes loaded with ANG peptide and QSH peptide (DON-A-Q).

[0068] As Figure 3 shown in B of 6 Inoculate 10 6 bEnd.3 cells into the Transwell-BBB model insert (upper chamber); inoculate 10

[0069] HT22 cells or BV2 cells into the lower chamber of the Transwell-BBB model and incubate for 24 h; then add PBS, DON, DON-Q, DON-A, DON-A-Q (all 20 nM) to the upper chamber respectively and co-incubate for 6 h; then aspirate the medium and perform the following operations on the cells in the lower chamber: wash three times with PBS, 5 min each time; then fix with 4% paraformaldehyde for 30 min, aspirate the paraformaldehyde, wash three times with PBS, 5 min each time; then incubate with phalloidin for 15 min in the dark, aspirate the phalloidin, wash three times with PBS, 5 min each time; then incubate with DAPI for 5 - 10 min in the dark, aspirate the DAPI, wash three times with PBS, 5 min each time; after aspiration, observe the distribution of Cy5 fluorescence signal in the cells under a confocal microscope (Nikon, Japan) in the dark, and perform quantitative analysis using ImageJ software. Collect the cells in the lower chamber for flow cytometry (BD Biosciences, USA) to detect the fluorescence intensity of Cy5, and use FlowJo v10.8.1 (USA) for data analysis.

[0070] The results are as follows Figure 3 As shown.

[0071] Depend on Figure 3 The results showed that after loading ANG peptide, the DNA nano-origami tubes had a significantly enhanced ability to penetrate the BBB and be taken up by cells. This proves that the DNA origami-based siRNA targeted delivery system provided by the present invention can efficiently penetrate the BBB and be taken up by microglia and neurons at the lesion site.

[0072] Example 4: In vivo evaluation of the ability of a DNA nano-origami tube-based siRNA targeted delivery system to penetrate the BBB and target lesions. The experimental drugs were DON, DON-A and DON-AQ as described in Experimental Example 3.

[0073] C57 mice were used. Cy5-labeled DON and DON-A were injected intravenously at a concentration of 20 nM into the mice via the tail vein. Three mice were injected into each group, with an injection volume of 200 μL. The distribution of Cy5 fluorescence was tracked using an IVIS Spectrum imaging system (PerkinElmer, USA). Six hours after injection, mice were sacrificed, and brain, heart, liver, spleen, lung, and kidney tissues were harvested and visualized using similar imaging parameters. Fluorescence intensity was quantitatively assessed using Living Image 4.3.1 (PerkinElmer, USA).

[0074] Five-fold FAD mice were used. Cy5-labeled DON-A and DON-AQ were injected into the mice via the tail vein at a concentration of 20 nM. Three mice were injected into each group, and the injection volume was 200 μL. Six hours after injection, the mice were sacrificed, and brain tissue was collected, fixed in paraformaldehyde, sectioned, and subjected to Aβ immunofluorescence staining. The colocalization of DON and Aβ in the brain was observed using confocal microscopy.

[0075] The results are as follows Figure 4 As shown.

[0076] Figure 4 The results showed that, in the animal central nervous system, the DNA nano-origami tube drug delivery system loaded with ANG peptides significantly enhanced the ability to penetrate the BBB and reach the brain. Figure 4 In the same time frame, the DNA nano-origami tube drug delivery system loaded with ANG peptides can effectively penetrate the BBB and accumulate in the brain. Figure 4 CD), and showed a significantly reduced liver metabolism ( Figure 4 (E in the text).

[0077] Depend on Figure 4As shown in F and G, in the G3 group of 5×FAD mice given DON-AQ peptide loaded with QSH peptide, the distribution of Cy5 in the hippocampus where Aβ aggregates was significantly greater than that in the G2 group of mice given DON-A without QSH peptide, indicating that QSH peptide has a targeting effect on Aβ lesion areas.

[0078] Experimental Example 5: Evaluation of the in vitro gene silencing effect of a DNA origami-based siRNA targeted delivery system. The experimental drugs were: unloaded DON; siG@DON loaded only with GSK-3β siRNA; siG@DON-Q loaded only with GSK-3β siRNA and QSH peptide; siG@DON-A loaded only with GSK-3β siRNA and ANG peptide; and siG@DON-AQ loaded with GSK-3β siRNA, ANG peptide, and QSH peptide.

[0079] 1. Detection of Aβ plaque aggregation Different drugs (20 nM) and 50 μM Aβ42 solution were added dropwise to a six-well plate and incubated together. After standing for 24 h, the results were observed under a bright-field microscope. Figure 5 As shown in C, the aggregation of Aβ is inhibited after binding to the QSH peptide.

[0080] 2. Evaluation of in vitro gene silencing effect The Transwell-BBB model was constructed in the same way as in Experiment 3.

[0081] (1) Detection of BV2 uptake and phagocytosis of Aβ42-FITC 10 6 10 bEnd.3 cells were seeded in the upper chamber of the Transwell-BBB model; 6 BV2 cells were seeded in the lower chamber of a Transwell-BBB model and incubated to a suitable cell density. 50 μM of Aβ42-FITC was added to the lower chamber, and 20 μM of different drugs were added to the upper chamber. The cells were incubated at 37°C for 24 h. Cells from the lower chamber were collected for flow cytometry analysis to observe the phagocytosis and uptake of Aβ by BV2 cells.

[0082] The results are as follows Figure 5 As shown in D in the diagram.

[0083] (2) Detection of autophagy in BV2 cells under the Transwell model 10 6 10 bEnd.3 cells were seeded in the upper chamber of the Transwell-BBB model; 6BV2 cells were seeded in the lower chamber of a Transwell-BBB model and incubated to a suitable cell density. 20 μM of different drugs were added to the upper chamber and the cells were placed in a 37°C incubator for 6 h. Cells from the lower chamber were collected for TEM observation to observe the autophagosome status of BV2 cells.

[0084] The results are as follows Figure 5 As shown in E in the figure.

[0085] (3) Place 10 6 10 bEnd.3 cells were seeded in the upper chamber of the Transwell-BBB model; 6 BV2 cells were seeded in the lower chamber of a Transwell-BBB model and incubated with Aβ42-FITC (50 μM) for 24 h. Then, PBS, DON, siG@DON, siG@DON-Q, siG@DON-A, and siG@DON-AQ (all 20 nM) were added to the upper chamber and incubated for 6 h. Cells from the lower chamber were collected for the following experiments: 1) Western blot Cell samples were placed on ice and mixed with RIPA lysis buffer (containing 50 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, and 1 mM PMSF). The mixture was then purified by 12000× [method / method - not specified]. g Centrifuge for 15 minutes and collect the supernatant for soluble protein analysis. Protein concentration was determined using the BCA method. Equal amounts of protein from each sample were loaded onto 8–15% SDS-PAGE gels for electrophoresis, followed by transfer to 0.45 μm polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk solution to prevent non-specific binding and incubated sequentially with the corresponding primary antibody and horseradish peroxidase-labeled secondary antibody. Immunoblotting results were visualized using an ECL substrate and chemiluminescence imaging system, and the band signal intensity was quantitatively analyzed using ImageJ software from the National Institutes of Health (NIH).

[0086] The detection results of GSK-3β, p62 and LC3B (LC3-I, LC3-II) proteins are as follows: Figure 5 As shown in F in the figure. The ratios of GSK-3β, LC3-II / LC3-I, and the results of Western blot quantitative analysis of p62 protein are shown in the figure below. Figure 5 As shown in G, H, and I.

[0087] 2) qRT-PCR Total RNA was extracted from BV2 cells using the SteadyPure Rapid RNA Extraction Kit (Accurate Biology, China).

[0088] cDNA synthesis was performed according to the instructions for the Prime Script™ RT kit (Takara Bio, Japan).

[0089] Real-time quantitative PCR (qRT-PCR) was performed using a SYBR Green PCR Master Mix (Takara Bio, Japan) on an ABI StepOne Plus real-time quantitative PCR instrument (Thermo Fisher Scientific, USA). Results were standardized using β-actin (ACTB) gene as an internal reference. Quantitative analysis was performed using the ΔΔCt method, and results from at least three independent replicates were statistically analyzed.

[0090] The primer sequences used for qRT-PCR detection are as follows: GSK-3β forward: 5'-AAGCCGATTTAAGAACCGAGAGC -3'; GSK-3β reverse: 5'-AGAAATACCGCAGTCGGACTAT-3'; ACTB forward:5'-TCGTGCGTGACATTAAGGAGAAGC-3'; ACTB reverse: 5'-TCGTGCGTGACATTAAGGAGAAGC-3'.

[0091] The qRT-PCR reaction program is set as follows: Stage 1 (Pre-denaturation): Hold at 95℃ for 5 minutes; Stage 2 (Cyclic Reaction): Denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds, for a total of 40 cycles; Phase 3 (melting curve analysis): Incubate at 95°C for 15 seconds, incubate at 60°C for 60 seconds, and finally incubate at 95°C for 15 seconds.

[0092] The results of GSK-3β mRNA expression level detection are as follows: Figure 6 As shown.

[0093] 3) LC3B immunofluorescence staining experiment Cells were fixed with paraformaldehyde, sections were stained with LC3B immunofluorescence, the distribution of LC3B in cells was observed using confocal microscopy, and quantitative analysis was performed using ImageJ software.

[0094] The results are as follows Figure 5 J and K are shown in the figure.

[0095] 4) Flow cytometry was used to detect the fluorescence intensity of FITC, and data analysis was performed using FlowJo v10.8.1 (USA). Results are as follows: Figure 7 As shown.

[0096] Depend on Figures 5-7 The results show that the GSK-3β siRNA targeted delivery system based on DNA nano-origami tubes provided by this invention can penetrate the BBB and be taken up by BV2 cells to exert a gene silencing effect under the Transwell-BBB model. It can significantly inhibit the expression of the target gene GSK-3β and initiate the downstream autophagy pathway to promote cell autophagy.

[0097] Experimental Example 6: Evaluation of the in vivo gene silencing effect of a DNA origami-based siRNA targeted delivery system The experimental drug is referenced in Case 5.

[0098] Five-fold FAD mice were used. PBS (G0), DON (G1), siG@DON (G2), siG@DON-A (G3), and siG@DON-AQ (G4) were injected into the mice via the tail vein at a concentration of 20 nM. Three mice were injected into each group, with an injection volume of 200 μL. Six hours after injection, the mice were sacrificed, and brain tissue was collected, fixed in paraformaldehyde, and sectioned for immunofluorescence experiments. The distribution of LC3B, Caspase-3, and Aβ in the brain tissue was observed using confocal microscopy, and quantitative analysis was performed using ImageJ software. WT mice (i.e., C57 mice) were injected with PBS as a control.

[0099] The results are as follows Figure 8 As shown. By Figure 8 The results showed that, compared with the PBS group, DON-AQ significantly promoted autophagy, and the two peptides, ANG peptide and QSH peptide, could synergistically exert therapeutic effects, promote the phagocytosis and degradation of Aβ by microglia, and significantly improve neuronal apoptosis.

[0100] The results of Experiments 5 and 6 show that the GSK-3β siRNA targeted delivery system based on DNA nano-origami tubes provided by this invention can promote autophagy, enhance the clearance of Aβ by microglia, and significantly inhibit neuronal apoptosis by targeting and silencing the GSK-3β gene, thereby improving the pathological progression of AD.

[0101] Experiment Example 7: Behavioral Experiment The experimental drug was the same as in Case 6.

[0102] (1) Morris water maze experiment WT (C57) mice and 5×FAD mice were used. Different groups of mice were injected with the drug, and a control group (PBS) was administered. The drug dose was 20 nM, and the injection volume was 200 μL, administered via tail vein. Eight mice were in each group. Injections were given every 5 days for a total of 6 times, followed by the Morris water maze test. This test involved a circular pool divided into four invisible quadrants, with a platform in one quadrant hidden 1 cm below the water surface. Various colored cues—triangles, circles, squares, and pentagons made of architectural paper—were placed around the pool to provide spatial reference points. An overhead video tracking system recorded the mice's swimming trajectory. The test period was 6 days, including a hidden platform test (days 1-5) and a detection test (day 6). During the hidden platform test, mice were randomly placed in each quadrant of the pool and given 60 seconds to locate the hidden platform. Tests were conducted daily in three different quadrants, with 4-hour intervals between trials, and escape latency was recorded. On the final day, a 24-hour probe test was conducted with the platform removed, allowing the mice to swim freely for 60 seconds. A video tracking system captured their trajectories, and the data was analyzed to assess spatial learning and memory by examining escape latency and swimming speed.

[0103] The results are as follows Figure 9 As shown in C to E.

[0104] (2) Y-maze novel arm exploration experiment WT (C57 mice) and 5×FAD mice were used. Different groups of drugs and a control group (PBS) were injected into the mice. The drug dose was 20 nM and the injection volume was 200 μL. The drugs were injected via the tail vein. There were 8 mice in each group. The injections were given every 5 days for a total of 6 times before training began.

[0105] The procedure for the Y-maze novel arm exploration experiment is as follows: First, complete the experimental preparation. Select a Y-maze with three arms and an angle of 120°. Mark the starting arm, the other arms, and the novel arm with partitions. Adjust the video analysis equipment. Maintain a quiet experimental environment with soft lighting and a room temperature of 22-25℃. One day before the experiment, place the mice in an empty maze for 20-30 minutes to acclimatize. Before the experiment and after each animal's test, wipe the maze with 75% alcohol and allow it to evaporate to remove odor and hair interference. Then, proceed to the training phase. Close the novel arm and place the mouse from the starting arm facing the center. Start the video recording and allow the mouse to freely explore the open arms for 10-15 minutes. Afterward, return the mouse to its cage and clean the maze again. Animals were randomly assigned to different arms in a balanced manner. After training, intervals were set: short-term memory tests were conducted at 1-4 hour intervals, and long-term memory tests at 24-hour intervals, during which the housing environment was kept stable. Then, the testing phase was conducted. The partition separating the new arms was removed, opening all arms. Mice were placed into the maze facing the center from the initial arm, and video was recorded, capturing their exploration behavior for 5 minutes. Entry into an arm was defined as all four limbs or more than 50% of the body entering the arm. After the test, the mice were removed, and the maze was cleaned. Finally, data were recorded and analyzed, including the percentage of time spent exploring the new arm, the percentage of times the mouse entered, and the latency to first entry. A new arm preference index was calculated, and independent samples t-tests or analysis of variance were used to compare differences between groups. P <0.05 indicates a statistically significant difference. Throughout the experiment, interference from sound, light, etc., should be avoided, and the mice should not be touched. If the mice remain still for a long time, the maze wall can be gently tapped for moderate stimulation.

[0106] The results are as follows Figure 9 The F, G, I, and J are shown in the figure.

[0107] (3) New object recognition experiment WT (C57 mice) and 5×FAD mice were used. Different groups of drugs and a control group (PBS) were injected into the mice. The drug dose was 20 nM and the injection volume was 200 μL. The drugs were injected via the tail vein. There were 8 mice in each group. The injection was given once every 5 days for a total of 6 times. After that, the new object recognition experiment was carried out.

[0108] The procedure for the new object recognition experiment is as follows: First, complete the experimental preparation. Select a square open field test chamber of appropriate size. Prepare two sets of non-toxic objects of similar material and size but different shapes. Debug the video analysis equipment. Maintain a quiet environment with uniform lighting and a room temperature of 22-25℃. One to three days before the experiment, place the mice in an empty open field for 5-10 minutes to acclimatize and reduce environmental stress. Before the experiment and after each animal's test, wipe the chamber and objects with 75% alcohol and allow it to evaporate to eliminate odor and hair interference. Then, enter the training phase by placing two identical objects in symmetrical positions in the open field. For familiarization with the object, place the mouse facing the center of the enclosure wall and record its exploration for 5-10 minutes. Afterward, return the mouse to its cage and clean the enclosure. The object positions and types are randomly balanced to avoid spatial bias. After training, set intervals: short-term memory tests every 1-4 hours and long-term memory tests every 24 hours, maintaining a stable environment during this period. Next, conduct the testing phase. Replace one of the familiar objects with a completely new object, keeping the position unchanged. Place the mouse facing the center of the enclosure wall and record its exploration behavior for 5 minutes. Valid exploration is defined as the mouse's nose being within 2 cm of the object and actively sniffing or touching it. Afterward, remove the mouse and thoroughly clean the enclosure. Finally, record and analyze the data, calculating the exploration time for the new and familiar objects, the total exploration time, and the recognition index using independent samples. t Tests or analysis of variance can be used to compare differences between groups. P <0.05 indicates a statistically significant difference. Throughout the experiment, noise, strong light, and other interferences were avoided. Mice were not touched actively. If the animals showed obvious signs of immobility, the box could be gently tapped for mild stimulation to ensure experimental consistency and data reliability.

[0109] The results are as follows Figure 9 H, K, and L are shown in the figure.

[0110] Depend on Figure 9 The results showed that ANG peptide and QSH peptide had a synergistic effect, jointly targeting the lesion site to exert therapeutic effects and significantly improving the cognitive function of mice.

[0111] Experimental Example 8: Biosafety Evaluation C57 mice were randomly divided into two groups (n=6) using a random number table, receiving PBS via tail vein injection and siGSK-3β@DON-AQ injection via tail vein injection, respectively. Compared with the PBS injection group, the brain, heart, kidney, liver, lung, and spleen were stained with hematoxylin and eosin (HE) on days 1 and 7 after siGSK-3β@DON-AQ (20 nM, 200 μL) injection. The biosafety of siGSK-3β@DON-AQ was further evaluated by complete blood count and blood biochemistry.

[0112] The results are as follows Figures 10-12 As shown.

[0113] Compared with the Ctrl group, no adverse effects of siGSK-3β@DON-AQ on organs were found, and blood routine and blood biochemistry related indicators were all within the normal range, indicating that the DNA origami-based GSK-3β siRNA targeted delivery system provided by this invention has high biosafety.

[0114] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A DNA origami-based siRNA targeted delivery system, characterized in that, The DNA nanoorigami tube carrier includes a DNA nanotube carrier, siRNA loaded within its lumen, and a target molecule-ssDNA conjugate modified at both ends. The DNA nanoorigami tube carrier is primarily formed by the self-assembly of a scaffold chain, staple chain, siRNA capture chain, aptamer chain, and fastening chain. The scaffold chain is an M13 plasmid; The nucleotide sequence of the siRNA capture strand includes, from the 5' end to the 3' end, a nucleotide sequence complementary to the positive strand of the siRNA and a staple strand of nucleotide sequence. The nucleotide sequence of the aptamer chain includes, from the 5' end to the 3' end, a staple chain nucleotide sequence and a nucleotide sequence that is partially or completely complementary to the ssDNA in the target molecule-ssDNA conjugate. The scaffold chain, staple chain, siRNA capture chain, and aptamer chain self-assemble to form a planar rectangular DNA nano-origami sheet; The fastening chain is used to roll the planar rectangular DNA nano-origami sheets into DNA nano-origami tubes.

2. The DNA origami-based siRNA targeted delivery system according to claim 1, characterized in that, The siRNA is GSK-3β siRNA, the nucleotide sequence of its sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:1, and the nucleotide sequence of its antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:

2.

3. The DNA origami-based siRNA targeted delivery system according to claim 2, characterized in that, The siRNA capture chain has 32 strands, and their nucleotide sequences are shown in SEQ ID NO:7~38.

4. The DNA origami-based siRNA targeted delivery system according to any one of claims 1 to 3, characterized in that, In the target molecule-ssDNA conjugate, the target molecule is selected from at least one of ANG peptide, transferrin, lactoferrin, and QSH peptide; wherein the amino acid sequence of the QSH peptide is shown in SEQ ID NO:

3.

5. The DNA origami-based siRNA targeted delivery system according to claim 4, characterized in that, In the ANG peptide-ssDNA conjugate, the amino acid sequence of the ANG peptide is shown in SEQ ID NO:5, and the nucleotide sequence of the ssDNA is shown in SEQ ID NO:6; in the QSH peptide-ssDNA conjugate, the nucleotide sequence of the ssDNA is shown in SEQ ID NO:

4.

6. The DNA origami-based siRNA targeted delivery system according to claim 5, characterized in that, The aptamer chain has 10 strands, and their nucleotide sequences are shown in SEQ ID NO:39~48.

7. The DNA origami-based siRNA targeted delivery system according to claim 1, characterized in that, The fastening chain has 12 strands, and their nucleotide sequences are shown in SEQ ID NO:49~60.

8. The method for preparing the DNA origami-based siRNA targeted delivery system according to any one of claims 1 to 7, characterized in that, Includes the following steps: The scaffold strand, staple strand, siRNA capture strand, and aptamer strand were added to a Mg-containing container. 2+ In TAE buffer, the mixture was annealed from 95°C to 15°C to obtain planar rectangular DNA nano-originated sheets; Planar rectangular DNA nanoparticles and siRNA were mixed and annealed from 45°C to 25°C. Then, a binding strand was added, and the mixture was annealed from 37°C to 15°C to obtain DNA nanoparticle tubes. Finally, the targeting molecule-ssDNA conjugate was added, and the mixture was annealed from 37°C to 15°C to obtain the final product.

9. The preparation method according to claim 8, characterized in that, The molar ratio of the scaffold chain, any staple chain, any siRNA capture chain, any aptamer chain, and any fastening chain is 1:1:1:1:(1~20); the molar ratio of the siRNA to any siRNA capture chain is (5~10):1; and the molar ratio of the target molecule-ssDNA conjugate to any aptamer chain is 1:

1.

10. The use of the DNA origami-based siRNA targeted delivery system according to any one of claims 2 to 7 in the preparation of a medicament for treating Alzheimer's disease.

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