Preparation of nucleic acid-directed supramolecular semiquinone radical nanoparticles and application thereof in photothermal tumor treatment

Abstract

This invention belongs to the field of nanomaterials technology, specifically relating to the preparation of nucleic acid-guided supramolecular semiquinone radical nanoparticles and their application in tumor photothermal therapy. The nucleic acid-guided supramolecular semiquinone radical nanoparticles provided by this invention are formed by non-covalent bonding of long single-stranded DNA containing repeating G-quadruplex units with polydopamine, and can be used to prepare tumor photothermal therapy reagents. This invention improves both the photothermal conversion capacity and ROS scavenging capacity of the nanomaterials by modifying the structure of polydopamine, achieving a synergistic effect in PTT (photothermal phototherapy).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterials, and particularly relates to preparation of nucleic acid-directed supramolecular semiquinone radical nanoparticles and application thereof in photothermal therapy of tumors. BACKGROUND

[0002] Photothermal therapy (PTT) has attracted extensive attention in recent years due to its ability to convert near-infrared (NIR) laser energy into heat energy through photothermal agents, and effectively induce tumor cell death. However, traditional PTT faces huge contradictory challenges.

[0003] Polydopamine (PDA) is an emerging photothermal material, and PDA is an oligomer formed by dopamine (DA) under oxidation conditions. However, the photothermal absorption intensity of PDA itself is weak, which hinders its further biological application. The patent with the publication number CN107510842A and the invention name of "Preparation method of composite photothermal agent with photothermal effect" discloses that the composite microspheres prepared by PDA and CuS have better photothermal effect than PDA, and can be used for tumor treatment.

[0004] However, only improving the photothermal effect of nanomaterials cannot meet the clinical application, because the photothermal conversion efficiency and the complex immune environment in actual clinical treatment are also important factors to be considered. Low photothermal conversion efficiency will result in less release of damage-associated molecular patterns (DAMPs), thereby limiting the intensity of the immune response activated by PTT, and causing poor immune activation effect. At the same time, rapid heating will cause heat stress in tumor tissue and release a large amount of reactive oxygen species (ROS), and the accumulated ROS will destroy the balance of the redox microenvironment required for the survival and proliferation of immune cells, affecting the function and survival of immune cells (such as T cells) infiltrating the tumor. Disturbed redox environment can trigger an over-activated inflammatory response and further form a vicious feedback cycle of ROS production, thereby significantly weakening the anti-tumor immune response. Although the catechol / semiquinone radical in PDA has high electron / hydrogen atom transfer activity and high oxidation potential, it has ROS scavenging ability, but the inherent uncontrollability of the DA oxidation polymerization process may lead to oxidation of catechol groups and semiquinone radicals, thereby causing loss of electrons to weaken the ROS scavenging ability of PDA.

[0005] In summary, it is necessary to propose a new strategy that can improve the photothermal conversion ability and ROS scavenging effect of PDA composite nanomaterials to meet the needs of the field. SUMMARY

[0006] The purpose of the present application is to provide a nucleic acid-directed supramolecular semiquinone radical nanoparticle preparation and its application in photothermal therapy of tumors, which partially solves or alleviates the above-mentioned deficiencies in the prior art, and specifically includes the following technical solutions.

[0007] Use of a nucleic acid-directed supramolecular semiquinone radical nanoparticle in the preparation of a tumor photothermal therapy reagent, the nucleic acid-directed supramolecular semiquinone radical nanoparticle being formed by non-covalent bonding of long single-stranded DNA containing repeating G-quadruplex units and polydopamine. Specifically, the non-covalent bonding includes π-π stacking (interface interaction between PDA rich in aromatic structure and DNA, namely π-π stacking).

[0008] Further, the tumor photothermal therapy reagent includes experimental reagents, therapeutic reagents, and drugs.

[0009] Therefore, another aspect of the present application also provides use of a nucleic acid-directed supramolecular semiquinone radical nanoparticle in the preparation of a tumor photothermal therapy drug.

[0010] Further, the nucleic acid-directed supramolecular semiquinone radical nanoparticle has a uniform spherical morphology, and a particle size range of 150-200 nm.

[0011] Further, the nucleic acid-directed supramolecular semiquinone radical nanoparticle has dispersibility.

[0012] Further, the dosage form of the tumor photothermal therapy reagent includes injections.

[0013] Further, the dosage form of the tumor photothermal therapy drug includes injections.

[0014] Further, the long single-stranded DNA containing repeating G-quadruplex units is obtained by rolling circle amplification using a short DNA nucleic acid sequence linear template rich in guanine as a starting material; the sequence of the short DNA nucleic acid sequence linear template is shown in SEQ ID NO. 1, and the primer sequence is shown in SEQ ID NO. 2.

[0015] Further, the nucleic acid-directed supramolecular semiquinone radical nanoparticle has synergistically enhanced photo-thermal conversion ability and active oxygen scavenging ability under photo-thermal conditions. The synergistic enhancement is that the photo-thermal conversion ability and active oxygen scavenging ability of the nucleic acid-directed supramolecular semiquinone radical nanoparticle are simultaneously enhanced under photo-thermal conditions.

[0016] Another aspect of the present application also provides a preparation method of a nucleic acid-directed supramolecular semiquinone radical nanoparticle, including the following steps: S01: synthesizing long single-stranded DNA containing repeating G-quadruplex units by rolling circle amplification using a short DNA nucleic acid sequence linear template rich in guanine as a starting material; the sequence of the short DNA nucleic acid sequence linear template is shown in SEQ ID NO. 1, and the primer sequence is shown in SEQ ID NO. 2; S02: The long single-stranded DNA solution containing repeating G-quadruplex units is mixed and reacted with an aqueous dopamine solution, wherein the mass ratio of the long single-stranded DNA solution containing repeating G-quadruplex units to the aqueous dopamine solution is in the range of 0.02-0.05:1; to obtain the nucleic acid-directed supramolecular semiquinone radical nanoparticles.

[0017] Furthermore, the mass ratio of the long single-stranded DNA solution containing repeating G-quadruplex units to the dopamine aqueous solution ranges from 0.03 to 0.04:1.

[0018] Furthermore, the long single-stranded DNA solution containing repeating G-quadruplex units is mixed with the dopamine aqueous solution and then gently spun to induce a reaction.

[0019] Furthermore, the rotation speed is set to 30-50 rpm.

[0020] Specifically, the steps include: S011: Starting with a guanine-rich short-chain DNA nucleic acid sequence linear template, phosphorylate the guanine-rich short-chain DNA nucleic acid sequence linear template to obtain the first reactant solution; S012: Mix the first reactant solution with the primer to obtain the second reactant solution; S013: Mix the second reactant solution with T4 DNA ligase, T4 ligase buffer, human recombinant albumin and ultrapure water to obtain the third reactant solution containing a circular template; S014: The formed circular template is mixed with Phi29 buffer, human recombinant albumin, dNTP mixture, Phi29 DNA polymerase, and ultrapure water to perform a rolling circle amplification reaction to obtain the long single-stranded DNA containing repeating G-quadruplex units.

[0021] Beneficial technical effects: This invention provides a novel application of nucleic acid-guided supramolecular semiquinone radical nanoparticles in photothermal therapy for tumors. Using DNA as the supramolecular host, this invention stabilizes semiquinone radicals generated during dopamine oxidation via non-covalent bonding, forming a "miniature battery" that facilitates electron transfer while significantly narrowing the effective optical band gap and enhancing photothermal performance. Furthermore, by utilizing the complementary base pairing principle of DNA as an interfacial guide, the final polydopamine oligomer exhibits increased orderliness, improving the accessibility of the catechol / diquinone functional groups to the external environment and enhancing the antioxidant capacity of the polydopamine.

[0022] This invention uses only one strategy to improve the photothermal conversion capacity and ROS scavenging capacity of nanomaterials by changing the structure of polydopamine, thereby achieving a synergistic effect in PTT therapy. This strategy avoids: (1) the problem of complex nanoparticle synthesis unit modules and difficult synthesis caused by adding other materials with ROS scavenging function during nanoparticle preparation in order to achieve the purpose of ROS scavenging; (2) the problem of introducing other materials to combine with PDA to achieve better photothermal efficacy (e.g., CuS is introduced in CN107510842A).

[0023] Experimental results confirm that the maximum temperature that the nucleic acid-guided supramolecular semiquinone radical nanoparticles provided by this invention can reach after laser irradiation for 6 min in H2O2 environment is not only unaffected, but is actually increased; at the same time, its radical scavenging activity is also enhanced under near-infrared light irradiation and high temperature (50℃) environment; proving that the material has synergistic enhancement of photothermal conversion ability and reactive oxygen scavenging ability under photothermal conditions. Attached Figure Description

[0024] 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 or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0025] Figure 1 This is a gel electrophoresis image of the product obtained by phosphorylation, circularization and rolling circle amplification reaction of short-chain guanine-rich DNA nucleic acid in one embodiment of the present invention. Figure 2 This is the CD spectrum of the long-chain G-tetrachain final product obtained from RCA prepared in one embodiment of the present invention; Figure 3 This is a transmission electron microscope (TEM) image (scale bar 200 nm) of DNA-guided polydopamine nanospheres in one embodiment of the present invention. Figure 4 This is a HAADF-STEM image and elemental distribution diagram of DNA-guided polydopamine nanospheres in one embodiment of the present invention. Figure 5 The images shown are TEM images of supramolecular composite nanospheres prepared with different ratios of DNA to dopamine in one embodiment of the present invention (a: DA: G4s = 1: 0.015625; b: DA: G4s = 1: 0.0625). Figure 6 This is a schematic diagram illustrating the application principle of DNA-guided polydopamine nanoparticles in ROS scavenging in one embodiment of the present invention; Figure 7 The scavenging ability of DNA-guided polydopamine nanoparticles against DPPH free radicals (A), hydroxyl free radicals (B), and total antioxidant capacity (C) is shown in one embodiment of the present invention. Figure 8 This is a high-resolution XPS spectrum of DNA-guided polydopamine nanoparticles after co-incubation with H2O2 for 4 h in one embodiment of the present invention (A is the CO single bond content in DNA-guided polydopamine nanoparticles; B is the CO single bond content in pure polydopamine; C is the CO single bond content after DNA-guided polydopamine nanoparticles react with hydrogen peroxide solution for 4 h). Figure 9 In one embodiment of the present invention, the bandgap energy is (αhv). 2 Tauc plot of hv (A is the band gap of DNA-guided polydopamine nanoparticles; B is the band gap of pure polydopamine); Figure 10 This is an EPR spectrum of one embodiment of the present invention (the green curve is the EPR spectrum of DNA-guided polydopamine nanoparticles; the red curve is the EPR spectrum of pure polydopamine). Figure 11 This invention verifies the interaction between photothermal performance and ROS scavenging ability in one embodiment (A shows the heating curves of DNA-directed polydopamine nanoparticles of the same concentration and DNA-directed polydopamine nanoparticles co-incubated with H2O2 within 6 min; B shows the scavenging ability of DNA-directed polydopamine nanoparticles of the same concentration and pure polydopamine nanoparticles on DPPH free radicals before and after light irradiation; C shows the scavenging ability of DNA-directed polydopamine nanoparticles of the same concentration and pure polydopamine nanoparticles on DPPH free radicals before and after high-temperature treatment; where PPDANs represent DNA-directed polydopamine nanoparticles; PPDANs represent pure polydopamine nanoparticles). Figure 12 This is a schematic diagram illustrating tumor implantation modeling and drug injection in mice in one embodiment of the present invention; Figure 13 This is an example of the effect of drug injection on a mouse model in one embodiment of the present invention (A is the change in mouse body weight during treatment; B is the change in tumor volume of mice during treatment; C is the change in tumor volume of different groups of mice during treatment; D is an image of tumors in different groups of mice; E is a statistical chart of tumor weight in mice after treatment; F is a section of tumor tissue from different groups of mice). Figure 14This is an immunofluorescence section of mouse tumor tissue in one embodiment of the present invention (A is an iNOS level immunofluorescence section; B is a Ly6G level immunofluorescence section; C is a CD4+ section). + T-cell level immunofluorescence section; D represents CD8. + (Immunofluorescent sections at the T cell level); Figure 15 The photothermal heating curves are shown in one embodiment of the present invention (A is the photothermal heating curve of DNA-guided polydopamine nanoparticles; B is the photothermal heating curve of pure polydopamine nanoparticles). Figure 16 This is an example of the scavenging ability of nanoparticles of the same concentration against DPPH free radicals in one embodiment of the present invention (where PPDANs represent DNA-directed polydopamine nanoparticles; PDANs represent pure polydopamine nanoparticles). Figure 17 This is a schematic diagram of the DNA-guided polydopamine nanosynthesis process in one embodiment of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0028] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0029] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0030] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0031] Definition of the noun: The "supramolecular" described in this invention refers to a complex, organized aggregate composed of two or more molecules bound together by non-covalent interactions, maintaining a certain integrity to possess a defined microscopic structure and macroscopic properties. Specifically, in this invention, it refers to a structure formed by the non-covalent bonding of DNA and PDA molecules.

[0032] Example 1 An example of DNA-guided synthesis of polydopamine nanoparticles (also known as supramolecular composite nanospheres) is presented.

[0033] G-quadruplexes are special nucleic acid secondary structures formed by the folding of DNA or RNA containing continuous G sequences under specific conditions. The four G bases are linked by Hoogsteen hydrogen bonds to form a G-quadruplex planar structure, and then adjacent G-quadruplexes are stacked π-π to form four stable G-quadruplexes. Subsequently, nucleic acid sequences containing G-quadruplexes can be amplified by rolling circle amplification (RCA) to obtain long single-stranded DNA sequences containing repeating G-quadruplexes.

[0034] In the presence of Tris buffer, dopamine can be oxidized under alkaline conditions to form polydopamine.

[0035] Introducing G-quadruplexes during the polymerization of dopamine to form oligomers allows for interaction with the G-quadruplexes primarily through π-π stacking, thereby forming DNA-directed polydopamine nanoparticles via non-covalent bonding. These DNA-directed polydopamine nanoparticles exhibit better total semiquinone radical stability and less disorder in the polydopamine oligomers. (See schematic diagram for synthesis.) Figure 17 The red dots represent free radicals; the number of free radicals increases in the synthesized supramolecular composite nanospheres.

[0036] Example 2 The main reagents involved in this embodiment are: T4 ligase buffer (Nanjing Novizan Biotechnology Co., Ltd.), T4 PNK enzyme (Nanjing Novizan Biotechnology Co., Ltd.), T4 DNA ligase (Nanjing Novizan Biotechnology Co., Ltd.), recombinant albumin (NEB), Phi29 buffer (Nanjing Novizan Biotechnology Co., Ltd.), Phi29 DNA polymerase (Nanjing Novizan Biotechnology Co., Ltd.), dNTP mixture (NEB), Tris buffer (Maclean's).

[0037] A specific method for preparing DNA-guided polydopamine nanoparticles is as follows.

[0038] S01: Synthesize long single-stranded DNA structures containing repeating G-quadruplex units via rolling circle amplification (RCA).

[0039] S011: Phosphorylate the guanine-rich short-chain DNA nucleic acid sequence linear template (hereinafter referred to as the linear template): Mix the linear template, T4 ligase buffer, T4 PNK enzyme, and ultrapure water in a volume ratio of 4:4:1:31. After mixing, incubate at 30°C for 30 minutes, then heat to 60°C and incubate for 20 minutes to obtain the first reaction solution.

[0040] S012: Mix the first reactant solution with the primers (as shown in Table 1) at a volume ratio of 4:1, incubate at 95°C for 5 minutes, and then gradually cool to 25°C at a rate of 1°C / min to obtain the second reactant solution.

[0041] S013: Mix the second reactant solution with T4 DNA ligase, T4 ligase buffer, human recombinant albumin, and ultrapure water in a volume ratio of 25:6:10:10:49, incubate at 16°C for 4 hours, and incubate at 65°C for 20 minutes to obtain the third reactant solution, wherein the third reactant solution contains a circular template.

[0042] S014: RCA: The formed circular template is mixed with Phi29 buffer, human recombinant albumin, dNTP mixture (2mM), Phi29 DNA polymerase, and ultrapure water in a volume ratio of 10:4:4:8:1:9, and reacted at 30°C for 20 minutes, followed by incubation at 65°C for 10 minutes to terminate the reaction, to obtain a long single-stranded DNA structure containing repeating G-quadruplex units.

[0043] The products obtained are analyzed using nucleic acid gel electrophoresis, such as Figure 1 As shown, with each step, the molecular weight of the product increases and the electrophoretic distance decreases. The final long single-stranded DNA structure (referred to as G-quadruplex or G4 complex) containing repeating G-quadruplex units, obtained through rolling circle amplification, exhibits pore retention during gel electrophoresis. The sequences involved are shown in Table 1.

[0044] Table 1 The synthesized G4 chain aqueous solution was mixed with anhydrous ethanol and sodium chloride solution in a ratio of 1:2:0.2 and incubated overnight. The mixture was centrifuged at 13200 rpm, the supernatant was discarded, and 1 mL of 75% ethanol was added. The mixture was then centrifuged again at 13200 rpm to prepare a solution with a concentration of 1 mg / mL. The CD spectral results are as follows... Figure 2 As shown, this indicates the formation of a long-chain G-quadruplex with a stable parallel structure.

[0045] S02: The above-synthesized G4 chain is mixed with an aqueous dopamine solution and reacted to form DNA-directed polydopamine nanoparticles.

[0046] The long-chain G-tetrachain was mixed with an aqueous solution of dopamine and reacted gently at a rotation speed of 30 rpm for 5 minutes. Tris buffer was then added afterward. The mass ratio of the G4 chain aqueous solution to dopamine was 0.03125:1, and the concentration of the Tris buffer was 2.5 mg / mL. -1 The mixture was stirred overnight and centrifuged to obtain supramolecular composite nanospheres at a speed of 12,000 rpm.

[0047] According to the preparation method provided in this embodiment, the mass ratio of G4 chain aqueous solution to dopamine in SO2 is 0.03125:1, and the final morphological characteristics of the resulting supramolecular composite nanospheres are as follows. Figure 3 As shown, the morphology is spherical with a particle size of approximately 150-170 nm, and the dispersion is good. The supramolecular composite nanospheres were characterized by HAADF-STEM, and the results are as follows. Figure 4 As shown, it contains C, N, O, and P elements.

[0048] This embodiment also attempted to react G4 chain aqueous solution with dopamine aqueous solution in other mass ratios, but the synthesized nanospheres did not meet the requirements in terms of particle size or dispersibility.

[0049] like Figure 5 As shown, when the mass ratio is DA: G4s=1:0.015625 (a), the formed nanospheres have a larger particle size, and the larger nanospheres may pose a risk of blockage after injection; when the mass ratio is DA: G4s=1:0.0625 (b), the formed nanospheres have a smaller particle size, but there is obvious agglomeration.

[0050] Example 3 Performance and functional verification of the DNA-directed polydopamine nanoparticles synthesized in Example 2.

[0051] 1. Validation of the photothermal conversion capability of DNA-guided polydopamine nanoparticles (1) Photothermal curve experiment Method: Concentrations of 10, 20, 40, 80, and 120 μg mL were used. -1 DNA-directed polydopamine nanoparticle aqueous suspension and pure polydopamine nanoparticle aqueous suspension were tested at a power density of 1.5 W / cm². -2 The solution was irradiated with an 808nm near-infrared laser for 6 minutes. The solution temperature was recorded every 30 seconds using a digital thermometer equipped with a thermocouple probe.

[0052] Result: See Figure 15 The results showed that DNA-directed polydopamine nanoparticle suspensions exhibited different effects at different concentrations (10-120 μg / mL). -1 The temperature rises by approximately 20°C to 36°C within 6 minutes. Specifically, when PPDA is 80 μg / mL... -1 At that time, the temperature reached 56.1℃ after 6 minutes of irradiation, an increase of 32.8℃; 120μg mL -1 At that time, the temperature reached 58.8℃ after 6 minutes of irradiation, an increase of 35.6℃. As a control, when the PDA concentration was 80 μg / mL... -1 After 6 minutes of irradiation, the temperature reached 44.3℃, an increase of 20℃; 120μg mL -1 After 6 minutes of irradiation, the temperature reached 52.3℃, an increase of 22.4℃. This shows that the PPDA heats up more than the PDA after the same amount of laser irradiation, indicating that its photothermal conversion efficiency is higher.

[0053] 2. Verification of ROS scavenging effect of DNA-guided polydopamine nanoparticles (1) DPPH scavenging experiment Methods: A 0.2 mM DPPH working solution was freshly prepared using anhydrous ethanol (95%). Then, 200 μL of DNA-directed polydopamine nanoparticles of different concentrations were mixed with 0.8 mL of the DPPH working solution. Scavenging activity was evaluated by monitoring the absorbance at 517 nm.

[0054] Result: See Figure 7 A. The results showed that the scavenging ability of DNA-guided polydopamine nanoparticles on DPPH free radicals was concentration-dependent; the higher the concentration of DNA-guided polydopamine nanoparticles, the better the scavenging ability on DPPH free radicals.

[0055] (2) Comparative experiment on DPPH scavenging ability Method: Prepare 0.2 mM DPPH working solution freshly using anhydrous ethanol solution (95%), then add 200 μL and 80 μg mL of the solution.-1 DNA-directed polydopamine nanoparticles and 200 μL, 80 μg mL -1 Pure polydopamine nanoparticles were mixed with 0.8 mL of DPPH working solution. The scavenging activity was evaluated by monitoring the absorbance at 517 nm.

[0056] Result: See Figure 16 The results showed that PPDA had a DPPH scavenging activity of 46.57%, while PDA had a DPPH scavenging activity of 11.98%. DNA-guided polydopamine nanoparticles showed stronger DPPH free radical scavenging ability than pure polydopamine nanoparticles.

[0057] (3) •OH removal experiment Methods: •OH was generated via the Fenton reaction. Solutions containing salicylic acid (SA, 3 μM), H₂O₂ (1 mM), ferrous sulfate (FeSO₄, 0.72 μM), and samples of different concentrations were dissolved in PBS buffer (25 mM, pH 7.4). The mixtures were incubated in the dark for 20 min, and the characteristic absorbance of 2,3-dihydroxybenzoic acid at 510 nm was monitored.

[0058] Result: See Figure 7 B. The results showed that the scavenging ability of DNA-directed polydopamine nanoparticles for •OH was concentration-dependent.

[0059] (4) Total antioxidant capacity experiment Methods: Total antioxidant capacity was determined using a total antioxidant capacity kit.

[0060] Result: See Figure 7 C. The results showed that the total antioxidant capacity of DNA-directed polydopamine nanoparticles increased with increasing concentration, especially at a PPDANs concentration of 120 μg / mL. -1 At that time, the total antioxidant capacity reached approximately 0.195 mmol / L. -1 .

[0061] 3. Verification of the photothermal properties and ROS scavenging interaction of DNA-guided polydopamine nanoparticles (1) Experiment on eliminating the effect of H2O2 on the photothermal properties of DNA-directed polydopamine nanoparticles Methods: DNA-directed polydopamine nanoparticles were incubated with H2O2, followed by exposure to an 808 nm near-infrared laser (1.5 W cm⁻¹). -2 Irradiate for 6 minutes. Record the temperature change every minute during the irradiation period.

[0062] Result: See Figure 11A. The results showed that after the introduction of H2O2, the highest temperature that DNA-guided polydopamine nanoparticles could reach after 6 minutes of laser irradiation was not only unaffected, but was actually increased by about 10°C.

[0063] (2) Experiment on the effect of DNA-directed polydopamine nanoparticles on DPPH free radical scavenging after high temperature treatment or laser irradiation Methods: The scavenging ability of DNA-directed polydopamine nanoparticles against DPPH radicals after laser irradiation for 6 minutes or heat treatment (50℃ for 6 min) was evaluated using a DPPH radical probe. The scavenging activity was assessed by monitoring the decrease in absorbance at 517 nm.

[0064] Result: See Figure 11 Results B and C show that the DPPH radical scavenging activity of DNA-guided polydopamine nanoparticles increased from 41.4% to 50.3%, indicating that near-infrared light irradiation synergistically enhances their free radical scavenging ability. Simultaneously, the effect of temperature on reactive oxygen species (ROS) scavenging function was evaluated by heating on a constant 50°C hot plate. The results confirmed that the free radical scavenging activity of DNA-guided polydopamine nanoparticles was also enhanced at 50°C.

[0065] Example 4 Animal experiments of the DNA-directed polydopamine nanoparticles synthesized in Example 2.

[0066] Methods: Subcutaneous tumors were transplanted into mice to model tumors, and the tumor size was increased to 100 mm. 3 Subsequently, the drug was administered via tail vein injection. Experimental and control groups were established, with the control group receiving an equal volume of PBS; the drug concentration in the control group was 5 mg / kg. The specific groupings were: 1) Control, 2) Control + NIR, 3) PDANs, 4) PDANs + NIR, 5) GONs, 6) GONs + NIR, 7) PPDANs, 8) PPDANs + NIR; where GONs represents graphene oxide nanosheets. Each group of drugs was injected into mice, and 24 h after injection, infrared laser simulation (1.5 W / cm²) was used. 2 6 min) of light exposure. See the diagram for animal drug administration. Figure 12 .

[0067] Result: See Figure 13 and Figure 14 . Figure 13The results showed that the body weight of mice treated with DNA-guided polydopamine nanoparticles remained relatively stable during the experiment. After 12 consecutive days of treatment, the tumor size and weight of the PDANS+NIR, GONS+NIR, and PPDANs+NIR groups were significantly reduced. H&E staining of tumor tissues showed varying degrees of necrosis in all groups, with more severe necrosis in the PDANS+NIR, GONS+NIR, and PPDANs+NIR groups. Furthermore, TUNEL staining revealed a significant increase in the number of apoptotic cells in the PDANS+NIR, GONS+NIR, and PPDANs+NIR groups. Ki67 staining showed that, except for the PPDANs+NIR group, the tumor cells in the other groups still exhibited high proliferative activity.

[0068] Figure 14 In this study, the level of inflammation was verified by immunofluorescence staining of iNOS. Results showed that the iNOS level was significantly reduced in the PPDANs+NIR group. Furthermore, neutrophil levels were differentiated by immunofluorescence staining of Ly6G, with results indicating neutrophil infiltration in both the PPDANs+NIR and GONs+NIR groups, suggesting a significant local inflammatory response. Regarding ROS and inflammatory mediators, CD4 levels were significantly lower after PTT during treatment. + T cells and CD8 + Immunofluorescence staining of T cells was used to detect T cell recruitment. Results showed that the PPDANs+NIR group had higher levels of CD4 compared to the GONs+NIR and PPDANs+NIR groups. + T cells and CD8 + T cell expression.

[0069] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0070] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. The application of nucleic acid-guided supramolecular semiquinone radical nanoparticles in the preparation of tumor photothermal therapy reagents, characterized in that, The nucleic acid-guided supramolecular semiquinone radical nanoparticles are formed by non-covalent bonding of long single-stranded DNA containing repeating G-quadruplex units with polydopamine.

2. The application as described in claim 1, characterized in that, The nucleic acid-guided supramolecular semiquinone radical nanoparticles have a uniform spherical morphology and a particle size range of 150-200 nm.

3. The application as described in claim 1, characterized in that, The nucleic acid-guided supramolecular semiquinone radical nanoparticles are dispersible.

4. The application as described in claim 1, characterized in that, The dosage form of the tumor photothermal therapy reagent includes an injectable form.

5. The application as described in claim 1, characterized in that, The long single-stranded DNA containing repeating G-quadruplex units is obtained by rolling circle amplification starting with a linear template of a short-stranded DNA nucleic acid sequence rich in guanine; the sequence of the linear template of the short-stranded DNA nucleic acid sequence is shown in SEQ ID NO.1, and the primer sequence is shown in SEQ ID NO.

2.

6. The application as described in claim 1, characterized in that, The nucleic acid-guided supramolecular semiquinone radical nanoparticles exhibit synergistic enhancement of photothermal conversion and reactive oxygen species scavenging capabilities under photothermal conditions.

7. A method for preparing nucleic acid-guided supramolecular semiquinone radical nanoparticles, characterized in that, Includes the following steps: S01: Starting with a short-chain DNA nucleic acid sequence linear template rich in guanine, a long single-stranded DNA containing repeating G-quadruplex units is synthesized by rolling circle amplification; the sequence of the short-chain DNA nucleic acid sequence linear template is shown in SEQ ID NO.1, and the primer sequence is shown in SEQ ID NO.2; S02: The long single-stranded DNA solution containing repeating G-quadruplex units is mixed and reacted with an aqueous dopamine solution, wherein the mass ratio of the long single-stranded DNA solution containing repeating G-quadruplex units to the aqueous dopamine solution is in the range of 0.02-0.05:1; to obtain the nucleic acid-directed supramolecular semiquinone radical nanoparticles.

8. The preparation method according to claim 7, characterized in that, The mass ratio of the long single-stranded DNA solution containing repeating G-quadruplex units to the dopamine aqueous solution ranges from 0.03 to 0.04:

1.

9. The preparation method according to claim 7, characterized in that, The long single-stranded DNA solution containing repeating G-quadruplex units is mixed with the dopamine aqueous solution and then gently spun to induce a reaction.

10. The preparation method according to claim 9, characterized in that, The rotation speed is set to 30-50 rpm.

Images