Nucleic acid-metal oxide nanoparticle with Yolk-Shell structure as well as preparation method and application of nucleic acid-metal oxide nanoparticle

Abstract

The invention discloses a nucleic acid-metal oxide nanoparticle with a Yolk-Shell structure as well as a preparation method and application of the nucleic acid-metal oxide nanoparticle, and belongs to the crossing field of a nanotechnology and nano biomedicine. The method comprises the following steps: firstly, adding DNA into water, then adding a FeCl2. 4H2O solution to obtain a mixed solution, carrying out vortex treatment for 10 seconds, then carrying out metal bath reaction, cooling to room temperature after the reaction is finished, centrifuging, collecting a centrifugal precipitate, cleaning, and resuspending in deionized water to obtain a first solution; and adding hexamethylenetetramine into the first solution, rotating on a rotary shaking table, then reacting in a metal bath, cooling to room temperature after the reaction is finished, centrifuging, collecting centrifugal precipitate, and cleaning with deionized water to obtain the nucleic acid-metal oxide nanoparticles with the Yolk-Shell structure. According to the present invention, through the synergistic effect of the metal ion-DNA coordination driven self-assembly and the oxidation reduction process, the controllable construction of the Yolk-Shell structure nucleic acid-metal oxide nanoparticles is achieved; the prepared nucleic acid-metal oxide nanoparticles with the Yolk-Shell structure can be applied to preparation of nucleic acid delivery drugs.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nanotechnology and nanobiomedicine, and more specifically to a Yolk-Shell structured nucleic acid-metal oxide nanoparticle, its preparation method, and its application. Background Technology

[0002] Nucleic acids, especially DNA, are ideal functional elements due to their excellent molecular recognition capabilities, ease of synthesis and modification, and good biocompatibility. Significant progress has been made in the controllable synthesis of inorganic nanoparticles (NPs) and their assemblies with well-defined structures using DNA as a programmable element. One strategy involves assembling DNA-mediated pre-modified complementary DNA-linked NPs to form NP superstructures linked by double-stranded DNA. However, this strategy typically requires precise control of the DNA-coupled valence state of the NPs, a time-consuming process with low yields. Furthermore, due to the difficulty in achieving efficient surface DNA modification of other types of NPs (such as iron oxide NPs), this method is mainly limited to the assembly of gold nanoparticles (AuNPs). Another strategy abandons the Watson-Crick hybridization assembly principle, using single-stranded DNA as a capping agent to directly synthesize inorganic NPs, including quantum dots, AuNPs, silver NPs, and bimetallic NPs. However, such research focuses primarily on the morphology regulation of inorganic NPs and is rarely used to construct NP superstructures.

[0003] In recent years, yolk-shell nanostructures (YSNs) have attracted significant attention due to their unique core-cavity-shell three-layer design, which has not only made them a frontier in materials science research but also demonstrated enormous application potential in fields such as catalysis, energy storage, and biomedicine. Particularly in the biomedical field, their internal cavity can serve as an ideal drug delivery repository, while the outer porous shell allows for the diffusion of biomolecules and protects the internal functional core from environmental damage. More importantly, they allow for the integration of different functional components (such as metal nanoparticles, metal oxides, and organic frameworks) into a single nanosystem, achieving multifunctional synergistic effects.

[0004] However, existing technologies still face a series of severe challenges in constructing multifunctional YSNs for the biomedical field: First, traditional methods (such as selective etching and template methods) often involve harsh conditions (high-temperature calcination, use of strong acids and bases), which can easily damage the activity of biomolecules and make it difficult to introduce condition-sensitive biological functional components such as nucleic acids. At the same time, the synthesis is complex, cumbersome, and inefficient. Second, although YSNs can also be prepared through Kirkendall effect or Ostwald ripening, this is limited to YSNs with the core and shell made of the same material. In addition, efficient, stable, and controllable binding with biomacromolecules with specific biological functions (such as nucleic acids and antibodies) is a technical challenge. Simple surface physical adsorption often suffers from problems such as weak binding, disordered orientation, and easy functional inactivation.

[0005] Therefore, how to prepare a Yolk-Shell structured nucleic acid-metal oxide nanoparticle using a simple solution method is an urgent problem to be solved in this field. Summary of the Invention

[0006] To address the aforementioned issues, this invention proposes a method for preparing Yolk-Shell structured nucleic acid-metal oxide nanoparticles mediated by single-stranded DNA, based on a metal ion-DNA coordination-driven self-assembly strategy combined with a redox process.

[0007] To achieve the above objectives, this invention first provides a method for preparing Yolk-Shell structured nucleic acid-metal oxide nanoparticles, comprising the following steps: S1, DNA is added to water, and then FeCl2·4H2O aqueous solution is added to obtain a mixed solution. After vortexing for 10s, the mixture is placed in a metal bath for reaction. After the reaction is completed, the mixture is cooled to room temperature, centrifuged, and the centrifuged precipitate is collected. The precipitate is washed with deionized water and then resuspended in deionized water to obtain the first solution.

[0008] S2, add hexamethylenetetramine to the first solution, rotate on a rotary shaker, then react in a metal bath. After the reaction is complete, cool to room temperature, centrifuge, collect the centrifuged precipitate, wash with deionized water to obtain the Yolk-Shell structured nucleic acid-metal oxide nanoparticles.

[0009] Preferably, in step S1, the DNA added to the water is CpG DNA, and the amount used is 15 μL, added to 555 μL of water; the amount of FeCl2·4H2O aqueous solution used is 30 μL; the DNA and Fe in the mixed solution... 2+ The molar ratio is 1:40.

[0010] Preferably, the temperature of the metal bath reaction in step S1 is 95°C and the time is 3 hours.

[0011] Preferably, the centrifugation speed in step S1 is 4000 rpm and the time is 5 min; the amount of deionized water used for resuspension is 600 μL.

[0012] Preferably, the concentration of hexamethylenetetramine in step S2 is 20 mmol / L and the amount used is 15 μL; the rotation speed on the rotary shaker is 30 rpm and the time is 10 min.

[0013] Preferably, in step S2, the temperature of the metal bath is 95°C and the time is 3 hours; the centrifugation speed is 4000 rpm and the time is 5 minutes.

[0014] Secondly, the present invention also provides a Yolk-Shell structured nucleic acid-metal oxide nanoparticle prepared by the method described above. The Yolk-Shell structured nucleic acid-metal oxide nanoparticle is a regular sphere with a shell thickness of 30 nm, a void thickness of 100 nm, and a core diameter of 100 nm. The Yolk-Shell structured nucleic acid-metal oxide nanoparticle is a nanoparticle with a rough surface, uniform size, and monodisperse structure.

[0015] Finally, this invention also provides the application of the Yolk-Shell structured nucleic acid-metal oxide nanoparticles prepared by the above method in the field of catalysis.

[0016] And the application of Yolk-Shell structured nucleic acid-metal oxide nanoparticles prepared by the above method in the preparation of nucleic acid delivery drugs.

[0017] As can be seen from the above technical solution, this invention discloses a Yolk-Shell structured nucleic acid-metal oxide nanoparticle, its preparation method, and its application. Compared with the prior art, its advantages are as follows: 1) This application realizes the controllable construction of Yolk-Shell structured nucleic acid-metal oxide nanoparticles through the synergistic effect of metal ion-DNA coordination-driven self-assembly and redox processes.

[0018] 2) This application innovatively achieves the simple synthesis of CpG-Fe3O4 nanoparticles (CFe-YSNPs) with an egg yolk-shell structure using CpG DNA, enabling efficient carrier-free co-delivery of the immune adjuvant CpG DNA molecules and Fe3O4 nanoparticles with significantly different physicochemical properties. Under the action of an external magnetic field, CFe-YSNPs can actively remain at the tumor site, achieving sustained tumor accumulation and thus significantly enhancing the anti-tumor effect.

[0019] 3) In addition, the MRI visibility of CFe-YSNPs supports non-invasive tracking of nanoparticle accumulation behavior and can dynamically assess the impact of magnetic targeting on the distribution and clearance of nanoparticles within tumors.

[0020] 4) This invention not only establishes a new paradigm for single-stranded DNA-guided superstructure assembly, but also expands the application fields of DNA nanotechnology and metal-DNA coordination chemistry. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 Transmission electron microscope image of Yolk-Shell structured nucleic acid-metal oxide nanoparticles prepared by the method of this invention.

[0023] Figure 2 Analysis of the catalytic Fenton reaction activity of Yolk-Shell structured nucleic acid-metal oxide nanoparticles prepared by the method of this invention.

[0024] Figure 3 The method of this invention prepares Yolk-Shell structured nucleic acid-metal oxide nanoparticles that enhance laser confocal imaging and flow cytometry analysis of DNA endocytosis. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Preparation of Yolk-Shell structured nucleic acid-metal oxide nanoparticles Example 1 (1) Add 15 μL of CpG DNA to 555 μL of water, and then add 30 μL of FeCl2·4H2O aqueous solution, wherein the CpG DNA and Fe... 2+The molar ratio was 1:40. After vortexing for 10 s at room temperature, the mixture was placed in a 95℃ metal bath for 3 h. After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 4000 rpm for 5 minutes. The precipitate was collected and washed three times with deionized water. Then it was resuspended in 600 μL of deionized water to obtain the first solution.

[0027] (2) Add 15 μL of hexamethylenetetramine (HMTA) with a concentration of 20 mmol / L to the first solution, and then place it on a rotating shaker and rotate it at 30 rpm for 10 minutes; then place the system in a metal bath at 95℃ and continue to react for 3 h, centrifuge at 4000 rpm for 5 minutes, collect the centrifuged precipitate, and wash it three times with deionized water to obtain Yolk-Shell structured nucleic acid-metal oxide nanoparticles, denoted as CFe-YSNPs.

[0028] The Yolk-Shell structured nucleic acid-metal oxide nanoparticles prepared in this application were observed by transmission electron microscopy. The results are shown in [Figure number missing]. Figure 1 The images clearly show that CFe-YSNPs are regularly spherical with a concentrated size distribution, and their unique yolk-shell structure can be observed from the contrast between light and dark areas, with a shell thickness of approximately 30 nm, a void thickness of approximately 100 nm, and a core diameter of approximately 100 nm; Scanning electron microscope images ( Figure 1 c) The CFe-YSNPs prepared on the surface were monodisperse NPs with rough surfaces and uniform sizes; high-angle annular dark-field scanning-transmission electron microscopy images and X-ray energy dispersive spectroscopy surface scan results ( Figure 1 d) indicates that the prepared NPs are composed of four elements: Fe, P, O, and N.

[0029] Catalytic Fenton reaction (1) The CFe-YSNPs prepared in Example 1 were dispersed in deionized water at a concentration of 1 mg / mL and ultrasonically treated for 1-3 minutes to obtain an aqueous solution of CFe-YSNPs. (2) Add 40 μL of 100 mmol / L 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) to a 1.5 mL centrifuge tube. The test is divided into four groups: the first group is H2O2 (20 μL, 5 mmol / L), the second group is CFe-YSNPs (25 μg), the third group is a mixture of CFe-YSNPs (25 μg) and H2O2 (20 μL, 5 mmol / L) (pH 7.4), and the fourth group is a mixture of CFe-YSNPs (25 μg) and H2O2 (20 μL, 5 mmol / L) (pH 5.5). After the above systems are mixed evenly, vortex the centrifuge tubes and wait for 3-5 minutes. Use a quartz capillary tube to analyze the above mixed solution for electron spin resonance spectroscopy to detect the generation of hydroxyl radicals and verify the catalytic activity of Fenton reaction.

[0030] See results Figure 2 As shown in the figure, this invention investigated the free radical generation behavior of CFe-YSNPs in a simulated tumor microacidic environment (pH 5.5) using electron paramagnetic resonance (EPR) spectroscopy. The results showed that the generation of hydroxyl radicals (•OH) strictly depends on the coexistence of CFe-YSNPs and H2O2 under weakly acidic conditions: no significant EPR signal was detected in a physiological environment of pH 7.4, or when either component was absent. This phenomenon reveals that CFe-YSNPs possess a pH-dependent dual catalytic mechanism: its Fe3O4 shell first exerts peroxidase-like activity, catalyzing the generation of •OH from H2O2; as the material gradually dissociates in an acidic environment, the released ferrous ions further catalyze the continued generation of •OH through the Fenton reaction, thereby significantly enhancing the oxidative stress effect.

[0031] Example 2 (1) Add 15 μL of Cy5 fluorescently labeled CpG DNA to 555 μL of water, and then add 30 μL of FeCl2·4H2O aqueous solution. The Cy5 fluorescently labeled DNA and FeCl2·4H2O are mixed. 2+ The molar ratio was 1:40. After vortexing for 10 s at room temperature, the mixture was placed in a 95℃ metal bath for 3 h. After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 4000 rpm for 5 minutes. The precipitate was collected and washed three times with deionized water. Then it was resuspended in 600 μL of deionized water to obtain the first solution.

[0032] (2) Add 15 μL of hexamethylenetetramine (HMTA) with a concentration of 20 mmol / L to the first solution, and then place it on a rotating shaker and rotate it at 30 rpm for 10 minutes; then place the system in a metal bath at 95℃ and continue to react for 3 h, centrifuge at 4000 rpm for 5 minutes, collect the centrifuged precipitate, and wash it three times with deionized water to obtain Yolk-Shell structured nucleic acid-metal oxide nanoparticles, denoted as Cy5-CFe-YSNPs.

[0033] Laser confocal imaging and flow cytometry quantitative analysis experiment (1) Preparation of complete culture medium: Add 10% fetal bovine serum, 1% penicillin and antibiotics and 1% glutamine to DMEM medium, mix well and set aside; add 10% fetal bovine serum and 1% penicillin and antibiotics to 1640 medium, mix well and set aside. RAW264.7 macrophages were cultured in the above-prepared DMEM complete culture medium at 37 ℃ in a humidified incubator containing 5% CO2; 4T1 cells were cultured in the above-prepared 1640 complete culture medium at 37 ℃ in a humidified incubator containing 5% CO2.

[0034] (2) RAW264.7 macrophages or 4T1 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 / 25 mm in 35 mm confocal dishes. Once the cells had grown and covered approximately 70-80% of the bottom area, the culture medium was removed, and the cells were washed 2-3 times with pre-warmed PBS. Opti-MEM serum-depleted medium containing Cy5-CFe-YSNPs (DNA concentration 200 nM) was then added. Subsequently, the cells were incubated for 1 h, 2 h, 4 h, and 8 h, respectively. The culture medium containing nanoparticles was removed at each incubator, and the cells were washed 2-3 times with pre-warmed PBS. The nuclei were then stained with Hoechst nuclear blue fluorescent dye, followed by confocal imaging to detect intracellular Cy5 fluorescence signals and verify the intracellular delivery performance of CpG DNA molecules.

[0035] (3) In flow cytometry experiments, RAW264.7 macrophages or 4T1 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of / wells in six-well plates, and subsequent incubation and procedures were the same as those described in (2) above for imaging experiments. Finally, the cells were washed 2-3 times with PBS, digested with 250 μL / well trypsin, and dispersed in 200 μL of PBS containing 1% FBS for flow cytometry quantitative analysis.

[0036] Test results are shown Figure 3Laser confocal imaging of the DNA-metal oxide yolk-shell nanostructure (3a) shows that cells incubated with Cy5-CFe-YSNPs exhibit significantly enhanced fluorescence signals compared to cells incubated with Cy5-CpG alone; flow cytometry quantitative analysis ( Figure 3 (b, c) shows that the fluorescence signal of the Cy5-CFe-YSNPs group was increased by about 15.3 times compared with the Cy5-CpG group, proving that CFE-YSNPs can deliver DNA into cells.

Claims

1. A method for preparing Yolk-Shell structured nucleic acid-metal oxide nanoparticle, characterized in that, The method comprises the following steps: S1, adding DNA into water, then adding an aqueous solution of FeCl2·4H2O to obtain a mixed solution, vortexing for 10 s, then reacting in a metal bath, cooling to room temperature after the reaction, centrifuging, collecting the centrifugal precipitate, washing with deionized water, then resuspending in deionized water to obtain a first solution; S2, adding hexamethylenetetramine into the first solution, rotating on a rotary shaker, then reacting in a metal bath, cooling to room temperature after the reaction, centrifuging, collecting the centrifugal precipitate, washing with deionized water to obtain the Yolk-Shell structure nucleic acid-metal oxide nanoparticle.

2. The production method according to claim 1, characterized by, The DNA added into water in step S1 is CpG DNA, and the amount is 15 μL added into 555 μL of water; The amount of the aqueous solution of FeCl2·4H2O is 30 μL; DNA and Fe in the mixed solution 2+ The molar ratio is 1:

40.

3. The preparation method according to claim 1, characterized in that, The temperature of the metal bath reaction in step S1 is 95℃, and the time is 3 h.

4. The method of claim 1, wherein, The speed of the centrifugation in step S1 is 4000 rpm, and the time is 5 min; The amount of deionized water for resuspension is 600 μL.

5. The preparation method according to claim 1, characterized in that, The concentration of the hexamethylenetetramine in step S2 is 20 mmol / L, and the amount is 15 μL; The speed of the rotation on the rotary shaker in step S2 is 30 r / min, and the time is 10 min.

6. The method of claim 1, wherein, The temperature of the metal bath in step S2 is 95℃, and the time is 3 h; The speed of the centrifugation is 4000 rpm, and the time is 5 min.

7. Yolk-Shell structured nucleic acid-metal oxide nanoparticles prepared according to the method of claim 1, wherein, The Yolk-Shell structure nucleic acid-metal oxide nanoparticle is a regular spherical nanoparticle, the shell thickness is 30 nm, the gap thickness is 100 nm, and the core diameter is 100 nm; the Yolk-Shell structure nucleic acid-metal oxide nanoparticle is a surface roughness, size uniformity, and monodisperse nanoparticle.

8. The Yolk-Shell structure nucleic acid-metal oxide nanoparticle prepared by the method of claim 1 is applied to catalyze Fenton reaction.

9. The Yolk-Shell structure nucleic acid-metal oxide nanoparticle prepared by the method of claim 1 is applied to prepare a nucleic acid delivery drug.

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