Barrel-shaped DNA origami nano-structure / chitosan composite nano-particle, preparation method and application of barrel-shaped DNA origami nano-structure / chitosan composite nano-particle

Abstract

The invention relates to a barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle, a preparation method and application thereof, the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle comprises a barrel-shaped DNA origami nanostructure and chitosan, the barrel-shaped DNA origami nanostructure is used as a carrier, the chitosan is compounded on the barrel-shaped DNA origami nanostructure through electrostatic interaction, and the chitosan is compounded on the barrel-shaped DNA origami nanostructure through electrostatic interaction. And forming the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles with positive electricity. The composite nanoparticle has good biocompatibility and low immunogenicity, has good stability in vivo, and has high transfer efficiency between cells, so that an application basis is provided for further genetic disease and cancer gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle, its preparation method, and its application. Background Technology

[0002] Gene therapy treats diseases by introducing, correcting, or replacing defective genes, and has shown broad application prospects in areas such as protein replacement therapy, treatment of hereditary diseases, and vaccine development. However, because functional genes are negatively charged biomolecules, they cannot easily cross the negatively charged and hydrophobic phospholipid bilayer cell membrane and are easily degraded by nucleases in the blood or cytoplasm. Therefore, naked functional genes cannot achieve safe and efficient protein expression. Thus, gene therapy requires the use of vectors to compress and protect genes to achieve efficient delivery.

[0003] In recent years, liposomes and cationic polymers have become a hot topic in non-viral gene delivery research due to their simple synthesis, multiple functionalization sites, and flexible and controllable structures. However, liposomes and synthesized polymers still have many limitations, such as non-specific distribution, the possibility of PEGylated lipids inducing strong immunogenicity, and in vivo safety, which seriously limit their large-scale clinical application.

[0004] DNA origami structures exhibit good biocompatibility and low immunogenicity under physiological conditions, making them suitable as efficient delivery carriers and showing great promise in biomedical fields such as biosensing, molecular imaging, and targeted therapy. However, due to the inherent negative charge of DNA, it does not easily cross the negatively charged and hydrophobic phospholipid bilayer cell membrane, and it readily binds to proteins in blood or cytoplasm or is degraded by nucleases. Furthermore, as a delivery carrier, the DNA origami structure itself lacks active targeting and efficient transmembrane capabilities. Therefore, current three-dimensional DNA origami cannot achieve safe and efficient gene delivery, which severely limits its practical clinical application and translation. Summary of the Invention

[0005] The purpose of this invention is to provide a barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle, its preparation method, and its application. The obtained composite nanoparticles have good biocompatibility and low immunogenicity, good stability in vivo, and high intercellular delivery efficiency.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle is characterized by comprising a barrel-shaped DNA origami nanostructure and chitosan, with the barrel-shaped DNA origami nanostructure as a carrier, wherein the chitosan is composited onto the barrel-shaped DNA origami nanostructure through electrostatic interaction to form a positively charged barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle. Preferably, the molecular weight of the chitosan is in the range of 3000-20000, and the degree of polymerization (DP) is in the range of n=18~124.

[0007] Preferably, the mass ratio of chitosan to barrel-shaped DNA origami nanostructure is 100~10:10~1.

[0008] Preferably, the barrel-shaped DNA origami nanostructure is prepared by the following steps: 1) The configuration of the nanobuckets was determined based on 2D / 3D image models, and detailed design was completed with the help of Tiamat and caDNAno, professional DNA nanostructure design tools; 2) Based on the configuration designed in step 1), its variants are further constructed, and internal and external functional sites that can be chemically modified are systematically set to obtain unpurified DNA origami nanostructures; 3) The unpurified DNA origami nanostructures were subjected to thermal annealing self-assembly, followed by purification using polyethylene glycol precipitation to effectively remove impurities such as unbound staple chains, thereby obtaining high-purity barrel-shaped DNA origami nanostructures.

[0009] The application of the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles described in this invention in the preparation of gene delivery vectors.

[0010] A gene composite nanoparticle includes the above-mentioned barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle, and a gene loaded thereon, wherein the gene is one of mRNA, siRNA, or plasmid DNA.

[0011] Preferably, the mass ratio of the barrel-shaped DNA origami nanostructure, chitosan, and gene is 100~10:10~1:5~1.

[0012] The application of the gene composite nanoparticles described in this invention in gene therapy drugs.

[0013] This invention discloses a barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle, which uses a barrel-shaped DNA origami nanostructure as a carrier substrate and combines it with the natural substrate chitosan through electrostatic interaction to form a low-immunogenic composite nanoparticle carrier, providing a carrier candidate for clinical gene therapy.

[0014] The barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles described in this invention can effectively overcome the degradation of barrel-shaped DNA origami nanostructures by nucleases, thus improving the stability of the barrel-shaped DNA origami nanostructures in vivo. The formed composite nanoparticles exhibit excellent affinity for genes, and the nanoparticles are uniformly sized and positively charged. In vitro transfection results show that the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles can efficiently deliver genes in various tissue cell lines, providing a basis for further applications in gene therapy for hereditary diseases and cancer.

[0015] The present invention also discloses the above-mentioned barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles. Experiments have confirmed that it is a novel gene delivery vector of DNA origami / chitosan composite nanoparticle system with extremely low cytotoxicity. The accuracy and wide applicability of the invention have been verified in various tissue cells, including human liver cancer cells (HCCLM3), human liver cancer cells (HepG2), and cervical cancer cells (HeLa).

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to combine chitosan with barrel-shaped DNA origami nanostructures to form composite nanoparticles through electrostatic interaction, which are used as gene delivery carriers. Compared with the mainstream commercial cationic polymer transfection reagents such as jetPEI and Lipo3000 in the current field, the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles have good biocompatibility and low immunogenicity, and therefore have greater potential for clinical application and translation. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the barrel-shaped DNA origami nanostructure in Embodiment 1 of the present invention.

[0018] Figure 2 The results of 1.5% agarose gel electrophoresis characterization of the barrel-shaped DNA origami nanostructure in Example 1 of this invention are shown.

[0019] Figure 3 This shows the microstructure of the barrel-shaped DNA origami nanostructure in Example 1 of the present invention.

[0020] Figure 4 The results show the affinity test results of the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles for DNA in Example 1 of this invention.

[0021] Figure 5 The particle size is the size of the complex nanoparticles formed by compressing DNA with the barrel-shaped DNA origami nanostructure / chitosan (CS) complex in Example 1 of this invention.

[0022] Figure 6The zeta potential of the complex nanoparticles formed by compressing DNA using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in Example 1 of this invention is positive, which is beneficial for subsequent cellular uptake.

[0023] Figure 7 This image shows the microstructure of the complex nanoparticles formed by compressing DNA using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in Example 1 of this invention.

[0024] Figure 8 This is a fluorescence photograph of human hepatocellular carcinoma cells (HCCLM3) after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding the green fluorescent protein (GFP) gene.

[0025] Figure 9 This is the result of quantitative analysis of positive cells encoding the green fluorescent protein (GFP) gene after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in human hepatocellular carcinoma cells (HCCLM3) according to Example 1 of this invention.

[0026] Figure 10 To measure the cell viability of cells transfected with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding green fluorescent protein (GFP) in human hepatocellular carcinoma cells (HCCLM3) of the present invention (Example 1).

[0027] Figure 11 This is a fluorescence photograph of human liver cancer cells (HepG2) after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding the green fluorescent protein (GFP) gene, as described in Example 1 of this invention.

[0028] Figure 12 This is the result of quantitative analysis of positive cells encoding the green fluorescent protein (GFP) gene after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in human hepatocellular carcinoma cells (HepG2) according to Example 1 of this invention.

[0029] Figure 13 To measure the cell viability of cells transfected with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding green fluorescent protein (GFP) in human hepatocellular carcinoma cells (HepG2) according to Example 1 of this invention.

[0030] Figure 14 This is a fluorescence photograph of cells transfected with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding the green fluorescent protein (GFP) gene in human cervical cancer cells (HeLa) according to Example 1 of this invention.

[0031] Figure 15This is the quantitative result of positive cells encoding the green fluorescent protein (GFP) gene after transfection with barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles in human cervical cancer cells (HeLa).

[0032] Figure 16 The survival rate of human cervical cancer cells (HeLa) transfected with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding green fluorescent protein (GFP) in Example 1 of this invention. Detailed Implementation

[0033] To further understand the present invention, the present invention will be described below with reference to embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0034] Example 1 The preparation of a barrel-shaped DNA origami nanostructure includes the following steps: 1) Design a barrel-shaped DNA origami structure based on caDNAno software, with the target structure being 40nm high and 40nm in diameter. Select the single-stranded DNA 7560 of M13mp18 phage as the backbone strand, and design and derive the sequences of 250 short DNA strands to construct the barrel-shaped structure. 2) Based on the configuration designed in step 1), its variants are further constructed, and internal and external functional sites that can be chemically modified are systematically set to obtain unpurified DNA origami nanostructures; To synthesize the barrel-shaped DNA origami structure, the DNA origami self-assembly reaction solution was first prepared by uniformly mixing the M13mp18 DNA 7560 backbone strand with 250 short strands as "staple" strands in a molar ratio of 1:10 in 1×TAE / Mg2+ buffer (10mM Tris-Acetate, 1mM EDTA, 10mM Mg(Cl)2), with final concentrations of 10nM and 100nM, respectively. 3) Place the sample in a PCR instrument for annealing. The specific annealing process is as follows: heat at 75℃ for 5 minutes, then cool down to 65℃, and then cool down from 65℃ to 5℃ at a rate of 1℃ / 15 min. The total annealing time is about 15 hours. After annealing, the synthesized barrel-shaped DNA origami structure was purified using polyethylene glycol precipitation to remove excess staple short chains. The specific steps are as follows: Mix the DNA origami self-assembly reaction solution with PEG precipitation buffer at a volume ratio of 1:1, centrifuge at 16,000×g for 25 minutes at room temperature to form a precipitate, carefully aspirate the supernatant and remove the unassembled staple short chains. The PEG precipitation buffer consists of 15% PEG 8000, 10mM Tris-Acetate, 1mM EDTA, 10mM Mg(Cl)2, and 505mM NaCl. The precipitate was resuspended in 1×TAE / Mg2+ buffer, and the absorbance of the purified DNA origami structure solution at 260 nm was measured using Nanodrop. The concentration was calculated based on the absorbance value and the absorbance coefficient.

[0035] Preparation of a barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle: The barrel-shaped DNA origami nanostructure solution prepared above is mixed with a chitosan solution, vortexed at high speed for 30s, and then allowed to stand for 20min to obtain barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle.

[0036] Preparation of a gene composite nanoparticle: The obtained barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle solution is mixed with a gene solution, vortexed at high speed for 30s, and then allowed to stand for 10 min to form gene composite nanoparticles.

[0037] 1.1 Structural Characterization The barrel-shaped DNA origami nanostructure obtained in Example 1 of this invention is as follows: Figure 1 As shown in the figure, it is cylindrical in shape, with a diameter of 40.2 nm and a height of 38.6 nm.

[0038] 1.2 The barrel-shaped DNA origami structure sample prepared in Example 1 of this invention was analyzed using a 1.5% agarose gel. The specific method is as follows: A 1.5% agarose gel was prepared using 0.5×TBE-Mg2+ electrophoresis buffer (0.5× Tris-Borate-EDTA, 10mM Mg(Cl)2).

[0039] The sample was then mixed with 6× DNA loading buffer at a volume ratio of 5:1 and added to the sample well of the gel. The gel was run for 1.5 h at room temperature using a 0.5x TBE-Mg2+ buffer system and a constant voltage of 70 V. The gel was then removed, stained with 3×4S GelRed staining solution, and imaged using a gel imaging system. Results are shown below. Figure 2 .

[0040] from Figure 2As can be seen, the gel electrophoresis results indicate that the barrel-shaped DNA origami nanostructure in Example 1 was successfully prepared. Lane 1 represents the M13mp18 7560 backbone chain, lane 2 represents unpurified barrel-shaped DNA origami, and lane 3 represents purified barrel-shaped DNA origami. The agarose gel electrophoresis images of the M13mp18 7560 backbone chain, unpurified barrel-shaped DNA origami, and purified barrel-shaped DNA origami prepared in Example 1 are shown. Lane 1 represents the M13mp18 7560 backbone chain, lane 2 represents unpurified DNA origami, and lane 3 represents purified DNA origami. It can be seen that the DNA origami structure can be purified by PEG precipitation to remove excess staple short chains.

[0041] 1.3 The barrel-shaped DNA origami structure prepared in Example 1 was characterized by TEM.

[0042] The specific method is as follows: First, dilute the DNA origami structure to 3 nM with 1×TAE / Mg2+ buffer (10 mM Tris-Acetate, 1 mM EDTA, 10 mM Mg(Cl)2). Take 5 μL of the 3 nM DNA origami sample and drop it onto a hydrophilically treated copper grid. After deposition for approximately 3 minutes, blot off the solution on the copper grid with filter paper. Then, add 5 μL of 1×TAE / Mg2+ buffer to the copper grid and immediately blot off the buffer with filter paper to wash the sample. Next, add 5 μL of 2% uranium acetate solution to the copper grid for negative staining of the DNA structure. After 20 seconds, blot off the staining solution with filter paper. Finally, place the copper grid in the air to dry. The dried sample is then imaged using a TEM instrument.

[0043] See results Figure 3 , Figure 3 The image shows a TEM image of the barrel-shaped DNA origami structure prepared in Example 1. The TEM image shows that the barrel-shaped DNA origami has a stable and uniform cylindrical structure. The TEM image of the DNA origami clearly shows uniform and dispersed barrel-shaped nanostructures with a length and diameter of approximately 40 × 40 nm, which is basically consistent with the theoretical size designed using caDNAno software and meets expectations.

[0044] I. Analysis of Performance Study Results The composite nanoparticle solution was diluted to 100 μL with serum-free medium, and then 100 μL of LPoGreen working solution was added. The excitation fluorescence intensity was detected under the conditions of an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Composite nanoparticles were prepared using a similar method. The particle size and surface zeta potential of the composite nanoparticles were measured using dynamic light scattering (DLS), and the morphology of the composite nanoparticles was characterized using TEM.

[0045] Figure 4 The results show the DNA affinity test results of the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles obtained in Example 1. The results confirm that when the mass ratio of barrel-shaped DNA origami nanostructure:chitosan:mRNA is 30:1:1, 60:1:1, 30:2:1, and 60:2:1, the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles exhibit excellent DNA affinity, with a DNA affinity efficiency exceeding 80%, reaching a maximum of 97%. Furthermore, the affinity of the barrel-shaped DNA origami nanostructure alone to the gene is only 20%, and the barrel-shaped DNA origami nanostructure alone has extremely poor stability and cannot effectively compress the gene. Therefore, the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles have a better protective efficiency for DNA.

[0046] When the mass ratios of barrel-shaped DNA origami nanostructures: chitosan: mRNA are 30:1:1, 60:1:1, 30:2:1 and 60:2:1, the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles exhibit excellent DNA affinity properties, with a DNA affinity efficiency exceeding 82%, and the barrel-shaped DNA origami nanostructures alone cannot compress genes.

[0047] Figure 5 This study describes the particle size analysis of the complex nanoparticles formed by compressing DNA using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in Example 1 of this invention. DLS results confirmed that when the mass ratio of barrel-shaped DNA origami nanostructures:chitosan:mRNA was 30:1:1, 60:1:1, 30:2:1, and 60:2:1, the resulting nanoparticles ranged in size from 144 nm to 208 nm. This is beneficial for subsequent cellular uptake of the complex nanoparticles and gene transfection.

[0048] Figure 6 This study presents the zeta potential test of the complex nanoparticles formed by compressing DNA using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in Example 1 of this invention. DLS results confirmed that when the mass ratio of barrel-shaped DNA origami nanostructures:chitosan:mRNA was 30:1:1, 60:1:1, 30:2:1, and 60:2:1, the surface potential of the formed nanoparticles was positive, ranging from +14 mV to 23 mV. The zeta potential of the complex nanoparticles formed by the barrel-shaped DNA origami nanostructures and DNA was negative. Therefore, the barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles are beneficial for subsequent cellular uptake and can promote subsequent gene transfection.

[0049] Figure 7This document describes the microstructure of the complex nanoparticles formed by compressing DNA using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles in Example 1 of this invention. TEM results confirm that when the mass ratio of barrel-shaped DNA origami nanostructures:chitosan:mRNA is 30:1:1, it can compress DNA to form stable spherical particles with a relatively uniform distribution. This further demonstrates the excellent stability of the formed complex nanoparticles, effectively overcoming the limitation of poor in vivo stability of barrel-shaped DNA origami nanostructures and providing the necessary conditions for subsequent gene transfection.

[0050] II. Analysis of Cell Research Results HCCLM3, HepG2, and HeLa cells were cultured at a concentration of 1.0 × 10⁻⁶. 4 Cells were seeded at a density of 2.0 × 10⁶ cells / well in 96-well plates and cultured overnight at 37°C. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C.

[0051] First, a barrel-shaped DNA origami nanostructure solution was mixed with a chitosan solution and vortexed at high speed for 30 seconds, then allowed to stand for 20 minutes to pre-assemble into complex nanoparticles. These pre-assembled nanoparticles were then mixed with a GFP gene solution, vortexed at high speed for 30 seconds, and allowed to stand for 10 minutes to form more complex nanoparticles. This mixture was then added to serum-containing culture medium, thoroughly mixed, and slowly added to cells. Cells were cultured for 24 hours, and cells transfected with green fluorescent protein DNA were observed under a fluorescence microscope. Furthermore, flow cytometry was used to test the mRNA transfection efficiency. Cell viability after transfection with luciferase-encoding DNA was quantitatively evaluated using 10% Alamar Blue solution.

[0052] Figure 8 These are fluorescence images of cells transfected with the complex nanoparticles encoding the green fluorescent protein (GFP) gene, formed by compressing DNA using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles as described in Example 1. The images also demonstrate the qualitative evaluation of DNA transfection performance in HCCLM3 cells. Figure 8 In HCCLM3 cells, when the mass ratio of barrel DNA origami nanostructure to chitosan to mRNA was 30:1:1, 60:1:1, 30:2:1, and 60:2:1, the barrel DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 exhibited higher DNA transfection efficiency, while the barrel DNA origami nanostructure alone could not transfect the GFP gene. This further confirms that chitosan (CS) can significantly improve the stability of the barrel DNA origami nanostructure and enhance its gene transfection.

[0053] Figure 9This is the quantitative result of positive cells transfected with the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles encoding the green fluorescent protein (GFP) gene, as described in Example 1. From... Figure 9 It can be found that in HCCLM3 cells, the highest transfection efficiency of the GFP gene mediated by the barrel DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 exceeded 34.1%.

[0054] Figure 10 This refers to the cell viability rate after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding green fluorescent protein (GFP) in Example 1. From... Figure 10 In HCCLM3 cells, the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 maintained extremely high cell activity, reaching 95%, regardless of the mass ratio. This is mainly due to the excellent biocompatibility and safety of the barrel-shaped DNA origami nanostructure and chitosan (CS) themselves, further confirming that the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles have the potential for clinical gene therapy.

[0055] Figure 11 These are fluorescence images of cells transfected with the green fluorescent protein (GFP) gene using the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles described in Example 1. The images also demonstrate the qualitative evaluation of DNA transfection performance in HepG2 cells. Figure 11 In HepG2 cells, when the mass ratio of barrel DNA origami nanostructure: chitosan: mRNA was 30:1:1, 60:1:1, 30:2:1, and 60:2:1, the barrel DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 exhibited higher DNA transfection efficiency, while the barrel DNA origami nanostructure alone could not transfect the GFP gene. This further confirms that chitosan (CS) can significantly improve the stability of the barrel DNA origami nanostructure and enhance its gene transfection.

[0056] Figure 12 This is the quantitative result of positive cells transfected with the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles encoding the green fluorescent protein (GFP) gene, as described in Example 1. From... Figure 12 It can be observed that in HepG2 cells, the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 efficiently mediated GFP gene transfection.

[0057] Figure 13 This refers to the cell viability rate after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding green fluorescent protein (GFP) in Example 1. From... Figure 13In HepG2 cells, the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 maintained extremely high cell activity, reaching 99.4%, regardless of the mass ratio. This is mainly due to the excellent biocompatibility and safety of the barrel-shaped DNA origami nanostructure and chitosan (CS) themselves, further confirming the potential of the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles for clinical gene therapy.

[0058] Figure 14 This is a fluorescence photograph of cells transfected with the green fluorescent protein (GFP) gene using barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles, as described in Example 1. Figure 14 In HeLa cells, it was found that when the mass ratio of barrel DNA origami nanostructure: chitosan: mRNA was 30:1:1, 60:1:1, 30:2:1 and 60:2:1, the barrel DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 exhibited higher DNA transfection efficiency, while the barrel DNA origami nanostructure alone could not transfect the GFP gene. This further confirms that chitosan (CS) can significantly improve the stability of the barrel DNA origami nanostructure and enhance its gene transfection.

[0059] Figure 15 This is the quantitative result of positive cells transfected with the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles encoding the green fluorescent protein (GFP) gene, as described in Example 1. From... Figure 15 In HeLa cells, the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles from Example 1 efficiently mediated GFP gene transfection, achieving a transfection efficiency of up to 58%, while the transfection efficiency of the barrel-shaped DNA origami nanostructure alone was less than 5%. These results confirm that chitosan (CS) can significantly improve the stability of the barrel-shaped DNA origami nanostructure and significantly enhance its gene transfection.

[0060] Figure 16 This refers to the cell viability rate after transfection with barrel-shaped DNA origami nanostructures / chitosan (CS) composite nanoparticles encoding green fluorescent protein (GFP) in Example 1. From... Figure 16 In HeLa cells, the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles in Example 1 maintained extremely high cell activity, reaching 99.5%, regardless of the mass ratio. This is mainly due to the excellent biocompatibility and safety of the barrel-shaped DNA origami nanostructure and chitosan (CS) themselves, further confirming that the barrel-shaped DNA origami nanostructure / chitosan (CS) composite nanoparticles have the potential for clinical gene therapy.

[0061] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A barrel-shaped DNA origami nanostructure / chitosan composite nanoparticle, characterized in that, It includes a barrel-shaped DNA origami nanostructure and chitosan. The barrel-shaped DNA origami nanostructure serves as a carrier, and the chitosan is electrostatically bonded to the barrel-shaped DNA origami nanostructure to form positively charged barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles.

2. The barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles as described in claim 1, characterized in that, The chitosan has a molecular weight range of 3000-20000 and a degree of polymerization (DP) range of n=18~124.

3. The barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles as described in claim 1, characterized in that, The mass ratio of chitosan to barrel-shaped DNA origami nanostructure is 100~10:10~1.

4. The barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles as described in claim 1, characterized in that, The barrel-shaped DNA origami nanostructure was prepared through the following steps: 1) The configuration of the nanobuckets was determined based on 2D / 3D image models, and detailed design was completed with the help of Tiamat and caDNAno, professional DNA nanostructure design tools; 2) Based on the configuration designed in step 1), its variants are further constructed, and internal and external functional sites that can be chemically modified are systematically set to obtain unpurified DNA origami nanostructures; 3) The unpurified DNA origami nanostructures were subjected to thermal annealing self-assembly, followed by purification using polyethylene glycol precipitation to effectively remove impurities such as unbound staple chains, thereby obtaining high-purity barrel-shaped DNA origami nanostructures.

5. The application of the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles as described in any one of claims 1 to 3 in the preparation of gene delivery vectors.

6. A gene-composite nanoparticle, characterized in that, The invention includes the barrel-shaped DNA origami nanostructure / chitosan composite nanoparticles as described in any one of claims 1 to 3, and the gene loaded thereon, wherein the gene is one of mRNA, siRNA, or plasmid DNA.

7. The gene composite nanoparticles as described in claim 6, characterized in that, The mass ratio of the barrel-shaped DNA origami nanostructure, chitosan, and gene is 100~10:10~1:5~1.

8. The application of the gene composite nanoparticles as described in claim 6 or 7 in gene therapy drugs.

Images