Sound-sensitive supramolecular spherical nucleic acid as well as preparation method and application thereof

Abstract

The invention relates to sound-sensitive supramolecular spherical nucleic acid as well as a preparation method and application thereof. The sound-sensitive supramolecular spherical nucleic acid is mainly applied to immunotherapy of oral leukoplakia. The supramolecular spherical nucleic acid is formed by a dendrimer core modified by a sound-sensitive agent and beta-CD and a nucleic acid shell modified by ferrocene through a subject-object recognition effect. Compared with a traditional treatment method, the spherical nucleic acid prepared by the invention not only has more excellent sonodynamic effect and immune activation ability, but also can realize transdermal drug delivery of oral cavity diseased tissues. The transdermal administration route can not only improve the drug enrichment effect of the focus part and realize precise treatment, but also reduce the drug metabolism burden and potential toxic and side effects. The invention provides a novel oral leukoplakia treatment means which has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of polymer materials and biomedical technology, and in particular to a sonosensitive supramolecular spherical nucleic acid and a preparation method and application thereof. Background Art

[0002] Oral cancer (most commonly of the tongue) is the sixth most common cancer worldwide. Oral squamous cell carcinoma (OSC) accounts for over 90% of all oral malignancies and is a common malignancy with a poor prognosis, with 250,000 cases recorded annually. Most OSCs develop from oral precancerous lesions, collectively referred to as potentially malignant conditions. Oral leukoplakia (OLK), a common precancerous lesion in oral mucus, is a precursor to oral cancer and carries a high risk of developing cancer. Therefore, effectively reversing the immunosuppression of OLK and preventing its progression to cancer has been a major challenge in medical research.

[0003] While traditional treatments such as surgical resection, radiotherapy, and chemotherapy have demonstrated some efficacy, they still face limitations, such as postoperative recurrence and poor therapeutic efficacy. Therefore, finding new and effective treatments is an urgent challenge. Conventional assembly methods based on electrostatic adsorption hinder the controlled release of nucleic acid drugs.

[0004] Therefore, there is an urgent need to explore a new method of nucleic acid delivery that can simultaneously achieve efficient loading and controllable release of nucleic acid drugs. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to provide a supramolecular spherical nucleic acid nanomaterial that can achieve efficient loading and controlled release of nucleic acid drugs.

[0006] In a first aspect of the present invention, a method for preparing a sonosensitizing supramolecular spherical nucleic acid is provided, the method comprising: using a 5th generation polyamidoamine (G5 PAMAM) dendrimer modified with β-CD and loaded with a sonosensitizer as a core, and using a ferrocene-modified CpG DNA and an anti-PD-L1 DNA aptamer sequence as an outer shell, to form the sonosensitizing supramolecular spherical nucleic acid through host-guest supramolecular recognition;

[0007] The surface of the fifth generation polyamide-amine (G5 PAMAM) dendrimer contains -NH2.

[0008] In another preferred embodiment, the sonosensitizer is selected from the following group: indocyanine green (ICG), dihydrochlorin (Ce6), protoporphyrin IX (PpIX), titanium dioxide (TiO2), black phosphorus (BP), zinc oxide (ZnO) or bismuth sulfide (Bi2S3).

[0009] In another preferred embodiment, the ferrocene-modified CpG DNA sequence is shown in SEQ ID NO: 1.

[0010] In another preferred embodiment, the sequence of the ferrocene-modified anti-PD-L1 DNA aptamer is shown in SEQ ID NO: 2.

[0011] In another preferred embodiment, the method comprises the following steps:

[0012] (S1) Preparation of a sonosensitizing supramolecular spherical nucleic acid core: coupling a fifth-generation polyamidoamine (G5PAMAM) dendrimer modified with a sonosensitizer with β-cyclodextrin (β-CD), followed by acetylation to obtain the sonosensitizing supramolecular spherical nucleic acid core.

[0013] Wherein, the surface of the fifth generation polyamidoamine (G5 PAMAM) dendrimer contains -NH2;

[0014] (S2) preparing a sonogenic supramolecular spherical nucleic acid shell: synthesizing a ferrocene (Fc)-modified CpG DNA sequence (SEQ ID NO: 1) and an anti-PD-L1 DNA aptamer sequence (SEQ ID NO: 2) as the sonogenic supramolecular spherical nucleic acid shell using a DNA synthesizer;

[0015] (S3) Assembling the sonosensitive supramolecular spherical nucleic acid core and the sonosensitive supramolecular spherical nucleic acid shell to obtain the sonosensitive supramolecular spherical nucleic acid.

[0016] In another preferred embodiment, the step (S1) further comprises the following steps:

[0017] (S1-1) The fifth generation polyamidoamine (G5 PAMAM) dendrimer was terminally modified with a sonosensitizer to obtain G5.NH2-ICG by dialysis and freeze drying.

[0018] Wherein, the surface of the fifth generation polyamidoamine (G5 PAMAM) dendrimer contains -NH2;

[0019] (S1-2) β-cyclodextrin (β-CD) is coupled with G5.NH2-ICG through host-guest recognition to form G5.NH2-ICG-CD;

[0020] (S1-3) Acetylation of G5.NH2-ICG-CD to obtain the G5.NHAc-ICG-CD supramolecular spherical nucleic acid core.

[0021] In another preferred embodiment, the step (S3) further comprises the following steps:

[0022] The ferrocene (Fc)-modified CpG DNA and the anti-PD-L1 DNA in step (S2) are mixed at a molar ratio of 1:1, and assembled with the supramolecular spherical nucleic acid core obtained in step (S1) through host-guest supramolecular recognition to obtain the sonogenic supramolecular spherical nucleic acid.

[0023] In another preferred embodiment, the method comprises the following steps:

[0024] A. Preparation of sonogenic supramolecular spherical nucleic acid cores:

[0025] (A1) The fifth generation polyamidoamine (G5 PAMAM) dendrimer was modified with the sonosensitizer indocyanine green (ICG) at the end, and the sonosensitizer-modified G5 PAMAM dendrimer (G5.NH2-ICG) was obtained by dialysis and freeze-drying.

[0026] Wherein, the surface of the fifth generation polyamidoamine (G5 PAMAM) dendrimer contains -NH2;

[0027] (A2) coupling β-cyclodextrin (β-CD) with the G5.NH2-ICG via host-guest recognition to form a β-CD-modified and sonosensitizer-loaded G5 PAMAM dendrimer (G5.NH2-ICG-CD);

[0028] (A3) acetylation of the G5.NH2-ICG-CD to obtain the supramolecular spherical nucleic acid core (G5.NHAc-ICG-CD);

[0029] B. Preparation of ferrocene-modified nucleic acid immune adjuvant CpG and nucleic acid aptamer PD-L1:

[0030] A ferrocene (Fc)-modified CpG DNA sequence (SEQ ID NO: 1) and an anti-PD-L1 DNA aptamer sequence (SEQ ID NO: 2) were synthesized using a DNA synthesizer;

[0031] C. Assembly of sonogenic supramolecular spherical nucleic acids:

[0032] The ferrocene (Fc)-modified CpG DNA and the ferrocene (Fc)-modified anti-PD-L1 DNA aptamer in step B were mixed at a molar ratio of 1:1 and co-assembled with the G5.NHAc-ICG-CD obtained in step A through host-guest supramolecular recognition. Unbound DNA was removed by ultrafiltration to obtain the sonogenic supramolecular spherical nucleic acid (sSNA-CpG / AptPD-L1).

[0033] In another preferred embodiment, in step (A1), the molar ratio of the indocyanine green (ICG) to the G5 PAMAM dendrimer is 5-20:1.

[0034] In another preferred embodiment, in step (A2), the molar ratio of β-CD to G5.NH2-ICG is 5-20:1.

[0035] In another preferred embodiment, in step (A2), in the acetylation treatment, the molar ratio of triethylamine:acetic anhydride:the β-CD-modified and sonosensitizer-loaded G5 PAMAM dendrimer (G5.NH2-ICG-CD) is 50-100:50-100:1.

[0036] In another preferred embodiment, in step (C), the molar ratio of the ferrocene (Fc)-modified CpG DNA to the supramolecular spherical nucleic acid core (G5.NHAc-ICG-CD) is 10-20:1.

[0037] In another preferred embodiment, in step (C), the molar ratio of the ferrocene (Fc)-modified anti-PD-L1 DNA aptamer to the supramolecular spherical nucleic acid core (G5.NHAc-ICG-CD) is 10-20:1.

[0038] In a second aspect of the present invention, a sonic-sensitive supramolecular spherical nucleic acid is provided. The sonic-sensitive supramolecular spherical nucleic acid is prepared by the method described in the first aspect of the present invention.

[0039] In another preferred embodiment, the sonogenic supramolecular spherical nucleic acid comprises the following structure:

[0040] (1) Core, including β-CD-modified and ICG-loaded G5 PAMAM dendrimer;

[0041] (2) Shell, including ferrocene-modified CpG DNA and anti-PD-L1 DNA aptamer;

[0042] The core and the shell are assembled to form a stable spherical structure through host-guest supramolecular recognition.

[0043] In another preferred embodiment, the particle size of the sonogenic supramolecular spherical nucleic acid is 10-100 nm, preferably 20-80 nm, and more preferably 30-35 nm.

[0044] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising: (1) the sonosensitive supramolecular spherical nucleic acid prepared by the method of the first aspect of the present invention or the sonosensitive supramolecular spherical nucleic acid as described in the second aspect of the present invention; and

[0045] (2) Medical dressings.

[0046] In another preferred embodiment, the volume ratio of the sonogenic supramolecular spherical nucleic acid to the dressing is 1-5:2.

[0047] In another preferred embodiment, the medical dressing is selected from the following group: wound dressing, gel dressing, or a combination thereof.

[0048] In another preferred embodiment, the gel dressing includes: thermosensitive hydroxybutyl chitosan wound dressing, chitosan / β-sodium glycerophosphate gel, hydroxyethyl cellulose gel, polyvinyl alcohol gel, polyethylene glycol gel, polyacrylic acid gel, polyurethane gel, gelatin, sodium alginate gel, chitosan gel or hyaluronic acid gel.

[0049] In another preferred embodiment, the pharmaceutical composition is in the form of oral or parenteral administration.

[0050] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0051] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0052] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of transdermal patch, injection, inhalant, tincture, powder, granule, capsule, oral solution, tablet, pill, suspension, emulsion, lozenge, or pill.

[0053] In another preferred embodiment, the pharmaceutical composition is in the form of a transdermal patch.

[0054] In another preferred embodiment, the pharmaceutical composition is used to treat oral leukoplakia.

[0055] In another preferred embodiment, the pharmaceutical composition is used to treat oral leukoplakia via transdermal administration.

[0056] In a fourth aspect of the present invention, there is provided a use of a sonogenic supramolecular spherical nucleic acid in the preparation of a drug for treating tumors and / or oral leukoplakia.

[0057] In another preferred embodiment, the tumor is a tumor that requires ultrasound treatment.

[0058] In another preferred embodiment, the tumor includes hematological tumors and solid tumors.

[0059] In another preferred embodiment, the solid tumor is selected from the group consisting of oral cancer, malignant mesothelioma, gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, lung cancer, bladder cancer, ovarian cancer, triple-negative breast cancer, cervical cancer, endometrial cancer, uterine serous carcinoma, colon cancer, prostate cancer, laryngeal cancer, tonsil cancer, or a combination thereof.

[0060] In another preferred embodiment, the drug is a drug for ultrasonic treatment of oral leukoplakia.

[0061] In another preferred embodiment, the frequency of the ultrasonic treatment for oral leukoplakia is 1-2 MHz, and the action time is 1-10 minutes.

[0062] In another preferred embodiment, the treatment of oral leukoplakia includes inhibiting the abnormal proliferation of oral leukoplakia and preventing its further deterioration to oral squamous cell carcinoma.

[0063] In another preferred embodiment, the sonically sensitive supramolecular spherical nucleic acid is used for a purpose selected from the group consisting of:

[0064] (1) Inducing immunogenic cell death of oral leukoplakia cells by releasing reactive oxygen species (ROS) in response to ultrasound; and / or

[0065] (2) Release CpG immune adjuvant and PD-L1 aptamer, reverse the immunosuppressive microenvironment, and activate dendritic cells and T cell immune responses.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0067] Compared with the prior art, the present invention has the following technical effects:

[0068] (1) The supramolecular spherical nucleic acid nanomaterial provided by the present invention is a multifunctional nanoassembly precisely constructed based on the host-guest recognition of macrocyclic molecules. Therefore, the number, composition, and ratio of functional nucleic acid molecules on its surface can be easily and precisely controlled. The present invention uses a dendrimer macromolecule modified with β-CD and loaded with a sonosensitizer as the core and a programmable nucleic acid sequence modified with ferrocene as the shell. Under host-guest recognition, a highly efficient and sequence-adjustable sonosensitivity supramolecular spherical nucleic acid is formed.

[0069] (2) By efficiently co-assembling CpG immune adjuvants and PD-L1 aptamers on the surface of sonosensitizer-modified dendrimers, the stability and bioavailability of nucleic acid drugs are improved and their controlled release is achieved. The supramolecular spherical nucleic acid designed in the present invention has both the immune adjuvant CpG oligonucleotide sequence and the immune checkpoint blocker PD-L1 aptamer oligonucleotide sequence. It not only has the biological activity of a vaccine adjuvant but also has the immune checkpoint blocking ability. The reactive oxygen generated by the sonodynamic effect of the supramolecular spherical nucleic acid triggers the dissociation of the supramolecular host and guest in the supramolecular spherical nucleic acid. On the one hand, the CpG sequence is released, the "vaccine" is generated in situ, the function of antigen presenting cells is enhanced, and the immune system is activated; on the other hand, the PD-L1 blocker is released, which binds to PD-L1 on the surface of white spot cells, antagonizes the immunosuppressive effect mediated by the PD-1 / PD-L1 signaling pathway, and maintains the continuous activation state of T lymphocytes. However, the immunomodulator R848 in the comparative document only indirectly enhances the tumor immune response by repolarizing M2 macrophages to M1 macrophages, and the immune activation effect is limited.

[0070] (3) Based on the reactive oxygen species produced by the sonodynamic effect, oral leukoplakia cells are induced to undergo immunogenic death, while at the same time stimulating the release of CpG immune adjuvants and PD-L1 aptamers, activating the immune system and reversing the immunosuppression of oral leukoplakia, thereby inhibiting the growth and carcinogenesis of oral leukoplakia.

[0071] (4) The supramolecular spherical nucleic acid designed in this invention has excellent transdermal ability and can be applied to the vitiligo lesions without the need for systemic administration. It can not only enhance the local drug enrichment effect and effectively treat local lesions, but also reduce the drug metabolic burden and potential toxic side effects.

[0072] (5) The supramolecular spherical nucleic acid provided by the present invention is suitable for use in a moist and dynamic oral environment. This patent uses the treatment of oral leukoplakia as an example to curb the occurrence and development of precancerous lesions at the source. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Shown is the preparation process (A) and H NMR spectrum (B) of the G5 dendrimer supramolecular spherical nucleic acid core in one embodiment of the present invention, as well as a schematic diagram of the synthesis of the G5 dendrimer supramolecular spherical nucleic acid (C).

[0074] Figure 2 Atomic force microscopy (TAM) results (A) and height distribution diagram (B) of the supramolecular spherical nucleic acid prepared in one embodiment of the present invention are shown.

[0075] Figure 3 The ESR graph shows the release of singlet oxygen and hydroxyl radicals from the supramolecular spherical nucleic acid prepared in one embodiment of the present invention after ultrasound. The red line represents singlet oxygen ( 1O2) detection results, black represents the detection results of hydroxyl radicals (·OH); US refers to the simple ultrasound group, and sSNA+US refers to the sSNA-CpG / AptPD-L1 supramolecular spherical nucleic acid response group.

[0076] Figure 4 The figure shows a comparison of the penetration effects of supramolecular spherical nucleic acid sSNA-CpG / AptPD-L1 and DNA-CpG / AptPD-L1 on different levels of 3D cell spheroids prepared in one embodiment of the present invention.

[0077] Figure 5 The graph shows the production of reactive oxygen species (ROS) in oral leukoplakia cells by the supramolecular spherical nucleic acid prepared in one embodiment of the present invention and different experimental groups after ultrasound response (green fluorescence is a ROS probe).

[0078] Figure 6 Flow cytometric graph showing the activation of dendritic cells by supramolecular spherical nucleic acid prepared in one embodiment of the present invention and different experimental groups after transwell experiment to verify oral leukoplakia cells after treatment with materials and ultrasound.

[0079] Figure 7 This figure shows an experimental protocol for the treatment of oral leukoplakia in mice induced by 4NQO and lipopolysaccharide (LPS) using supramolecular spherical nucleic acids prepared in one embodiment of the present invention. C57BL / 6 mice were given a combined drink containing 4NQO (100 μg / mL) and LPS (10 μg / mL) from week 0 to week 14, and pure water from week 14 to week 18. Different drugs were also administered epidermally.

[0080] Figure 8 Shown is a graph showing the therapeutic effects of supramolecular spherical nucleic acids prepared in one embodiment of the present invention and different treatment groups on oral leukoplakia in mice.

[0081] Figure 9 Shown is the supramolecular spherical nucleic acid prepared in one embodiment of the present invention and the T cell phenotype analysis of the spleen after the treatment of oral leukoplakia in mice in different treatment groups.

[0082] Figure 10 Shown are the H&E staining results of the tongue of mice after the treatment of oral leukoplakia using supramolecular spherical nucleic acids prepared in one embodiment of the present invention and different treatment groups. DETAILED DESCRIPTION

[0083] To make the purpose, technical solutions, beneficial effects, and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0084] the term

[0085] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0086] As used herein, the term “including” or “comprising” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0087] As used herein, the terms "supramolecular spherical nucleic acid", "supramolecular spherical nucleic acid nanomaterial", and "sonosensitive supramolecular spherical nucleic acid" are used interchangeably and all refer to the sonosensitive supramolecular spherical nucleic acid prepared by the method described in the first aspect of the present invention.

[0088] Fifth generation polyamidoamine (G5 PAMAM) dendrimers

[0089] The fifth-generation polyamidoamine (G5 PAMAM) dendrimer is a highly branched synthetic polymer belonging to the polyamidoamine (PAMAM) dendrimer family. Known for its precise three-dimensional structure, rich surface functional groups, and excellent biocompatibility, it is widely used in drug delivery, gene therapy, bioimaging, and nanomaterials science. G5 PAMAM typically uses ethylenediamine (EDA) as its core, and its dendritic structure is constructed through a stepwise polymerization reaction. Each generation of PAMAM increases the number of branches. G5 PAMAM has five generations of branches, with the number of branches increasing exponentially with each generation, ultimately forming a highly symmetrical spherical structure. It has a molecular weight of approximately 25,000-30,000 Da and a diameter of approximately 12-15 nm. G5 PAMAMs typically have amino (-NH2) or carboxyl (-COOH) groups at their termini, but can also be modified to incorporate other groups (such as hydroxyl-OH or phenylboronic acid-PBA). Due to the complex structure of PAMAM dendrimers, their precise molecular formula is usually not given directly, but rather their repeating units and terminal groups are described. The basic structure of G5 PAMAM can be represented as follows: (1) Core: ethylenediamine (NH2-CH2-CH2-NH2); (2) Branch unit: acrylamide (-CH2-CH2-CO-NH-) repeating structure; (3) Terminal: 32 amino groups (G5-NH2) or hydroxyl groups (G5-OH).

[0090] G5 PAMAM is usually synthesized by stepwise polymerization or alternating polymerization methods: (1) step-growth method: starting from the ethylenediamine core, acrylamide monomers are added generation by generation to form a dendritic structure; (2) free radical polymerization (such as ATRP or RAFT): used to more precisely control the molecular weight and distribution; (3) post-modification: different functional groups (such as PEG, fluorescent labels, targeting ligands, etc.) can be introduced through chemical reactions after synthesis.

[0091] N,N'-Carbonyldiimidazole (CDI)

[0092] CDI (N,N'-carbonyldiimidazole) is a commonly used carboxyl or hydroxyl activating reagent that reacts with the primary hydroxyl group (C6-OH) of β-CD to form an imidazole carbonate intermediate (β-CD-O-CO-Im). This intermediate readily undergoes substitution reactions with amino (-NH2) or hydroxyl (-OH) groups to form stable amide or ester bonds. The structural formula of CDI (CAS No. 530-62-1) is shown below:

[0093]

[0094] CpG immune adjuvant

[0095] CpG oligonucleotides (CpG ODNs) are synthetic DNA fragments containing an unmethylated cytosine-guanine dinucleotide (CpG motif), where "CpG" represents a cytosine and a guanine linked together. These molecules play a vital role in the immune system because they can mimic the DNA sequences of bacteria and viruses, triggering the body's immune response. They can activate the immune system through Toll-like receptor 9 (TLR9), enhancing the antibody response and cellular immune response to vaccines.

[0096] The ferrocene nucleic acid immune adjuvant CpG provided by the present invention combines the immunostimulatory activity of CpG with the redox properties of ferrocene, and can be used to enhance vaccine immune responses or develop new immunotherapy strategies.

[0097] Ferrocene modification

[0098] Ferrocene is an organometallic compound with a sandwich structure and aromatic properties. It is chemically stable and easily modified. Modification methods include: (1) cyclopentadienyl substitution reaction, where the cyclopentadienyl ring of ferrocene can undergo electrophilic substitution reactions (such as acylation, sulfonation, alkylation, etc.) to produce mono- or di-substituted derivatives; (2) PEGylation modification, where ferrocene can be conjugated with polyethylene glycol (PEG), such as Ad-PEG-Ferrocene, to improve water solubility and biocompatibility.

[0099] sonosensitizers

[0100] Sonosensitizers are compounds that generate reactive oxygen species (ROS) in response to ultrasound to kill tumor cells. Indocyanine green (ICG) is a common organic dye used not only in photodynamic therapy (PDT), but also as a sonosensitizer, and is being studied for sonodynamic therapy (SDT).

[0101] ICG (Indocyanine Green)

[0102] ICG is a near-infrared (NIR) fluorescent dye with an absorption peak at 780 nm. It exhibits excellent photothermal conversion and ROS generation capabilities. ICG can generate reactive oxygen species (ROS) under ultrasound. To improve the stability and targeting of ICG, researchers often encapsulate it in nanocarriers, such as polydopamine (PDA)-coated LAP nanoparticles (ICG / LAP-PDA-PEG-RGD NPs), to enhance the combined effects of photothermal and photodynamic therapy. ICG can also be used for photoacoustic imaging (PA) and combined with photothermal therapy (PTT) and photodynamic therapy (PDT) to achieve integrated diagnosis and treatment.

[0103] The sonosensitive supramolecular spherical nucleic acid of the present invention

[0104] Host-guest recognition based on macrocyclic molecules can be used to construct multifunctional nanoassemblies. β-cyclodextrin (β-CD) is one of the most widely used host molecules in the preparation of supramolecular nanocarriers. Previous studies have demonstrated that stimuli-responsive host-guest complexes exhibit reversible dissociation and association upon application of external stimuli, providing further possibilities for constructing supramolecular systems with controlled drug release. The host-guest interaction based on β-CD not only enables the efficient assembly of multiple building blocks onto a nanoplatform but also enables responsive release of each module. Furthermore, dendrimers, a class of highly branched, nanoscale, monodisperse polymers with a stable three-dimensional spherical structure, are excellent materials for the core of spherical nucleic acid backbones. Therefore, using β-CD-modified dendrimers as the core and a ferrocene-modified programmable nucleic acid shell, sequence-tunable stimuli-responsive supramolecular spherical nucleic acids can be efficiently constructed under host-guest recognition to activate the immune system and reverse the immunosuppressive microenvironment of oral leukoplakia. Ultrasound, with its deep tissue penetration, was used as the stimulation mechanism, and the sonodynamic effect of ICG was exploited to induce immunogenic cell death in oral leukoplakia cells. Subsequently, antigen-presenting cells recognize vitiligo antigens, process them, and present them to T cells, activating T cell adaptive immune responses. Furthermore, the reactive oxygen species generated by the sonodynamic effect trigger the dissociation of supramolecular hosts and guests in supramolecular spherical nucleic acids. On the one hand, this releases CpG sequences, generating a "vaccine" in situ and enhancing the function of antigen-presenting cells. On the other hand, it releases PD-L1 nucleic acid aptamers, which bind to PD-L1 on the surface of vitiligo cells, antagonizing the immunosuppressive effects mediated by the PD-1 / PD-L1 signaling pathway and maintaining the continuous activation of T lymphocytes. However, achieving safe and effective oral vitiligo immunotherapy from both "activating the immune system" and "reversing immunosuppression" approaches has yet to be explored.

[0105] The supramolecular spherical nucleic acid designed in this invention has excellent transdermal ability and can be applied to the oral leukoplakia lesions without the need for systemic administration. This not only enhances the local accumulation of drugs, effectively treating localized lesions, but also reduces the metabolic burden of drugs and potential toxic side effects.

[0106] The present invention provides a sonogenic supramolecular spherical nucleic acid nanomaterial and a preparation method thereof, which can effectively reverse the immunosuppression of oral leukoplakia through sonogenic effect and immune activation, activate the immune system, and inhibit its deterioration to oral squamous cell carcinoma.

[0107] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial sources unless otherwise specified.

[0108] Example 1: Preparation of supramolecular spherical nucleic acids

[0109] This example designs a special supramolecular spherical nucleic acid with a core composed of a dendrimer modified with β-CD and loaded with a sonosensitizer, and a shell composed of a programmable nucleic acid sequence modified with ferrocene. This allows for efficient sequence-tunable sonosensitivity through host-guest recognition. The specific steps are as follows:

[0110] 1. Preparation of Sonogenic Supramolecular Spherical Nucleic Acid Cores

[0111] See also Figure 1 A, The sonosensitizer ICG was modified at the end of the G5 PAMAM dendrimer. 10 mg of G5. PAMAM dendrimer and 5.1 mg of ICG-NHS were dissolved in DMSO, respectively, and then stirred at room temperature for 24 hours. After the reaction, the solution was dialyzed in ultrapure water using a dialysis bag (molecular weight cutoff of 1000) for three days, with the water changed three times per day. The product in the dialysis bag was collected and freeze-dried to obtain powdered G5.NH2-ICG.

[0112] To further modify β-CD, 8.7 mg of β-CD and 12.47 mg of CDI were weighed and dissolved in DMSO. The CDI solution was then added dropwise to the β-CD solution under stirring for activation for 6 hours. The activated β-CD solution was then added dropwise to a solution of 10 mg of G5.NH2-ICG, also dissolved in DMSO. After stirring for 48 hours, the product was collected into a dialysis bag (molecular weight cutoff 5000) and dialyzed against distilled water for three days (three times per day). The product was then collected from the dialysis bag and freeze-dried to obtain powdered G5.NH2-ICG-CD.

[0113] Dissolve 10 mg of G5.NH2-ICG-CD in 3 mL of ultrapure water, add triethylamine (25 μL) dropwise, and stir at room temperature for 30 minutes. Then, add acetic anhydride (17 μL) dropwise to the solution and continue stirring at room temperature for 24 hours. After the reaction is complete, collect the product and dialyze it in ultrapure water for three days using a dialysis bag with a molecular weight cutoff of 5000. Then, collect the product in the dialysis bag and freeze-dry it to obtain a powdered G5.NHAc-ICG-CD supramolecular spherical nucleic acid core. Its H NMR spectrum is as follows: Figure 1 As shown in Figure B, the characteristic peaks at chemical shifts of 2.2-3.4 ppm represent the proton peaks of the methylene groups on the G5 backbone, the characteristic peaks at chemical shifts of 3.5-4.0 ppm and 5.0 ppm represent the proton peaks of the CD molecular structure, and the characteristic peak at chemical shifts of 7.5-8.0 ppm represents the proton peak of the naphthalene ring in the ICG molecular structure. These peaks confirm that β-CD and ICG are successfully coupled to the G5 surface. After adding triethylamine (25 μL) dropwise to G5.NH2-ICG-CD and stirring at room temperature for 30 minutes, acetic anhydride (17 μL) was added dropwise to the solution and stirred at room temperature for 24 hours. After acetylation, a characteristic peak of the methyl group of the acetyl group appeared at a chemical shift of 1.8 ppm. Area integration of the characteristic peaks of G5, β-CD, ICG, and acetyl groups revealed that each G5 surface was modified with 6.9 ICG molecules, 15.9 β-CD molecules, and 45 acetyl groups.

[0114] 2. Preparation of ferrocene-modified nucleic acid immune adjuvant CpG and nucleic acid aptamer PD-L1

[0115] Using a DNA synthesizer, nucleic acid immune adjuvant CpG DNA and nucleic acid aptamer anti-PD-L1 DNA were prepared according to the nucleic acid sequences shown below. The specific nucleic acid sequences (5'-3') are as follows:

[0116] Ferrocene-modified CpG DNA sequence (abbreviated as Fc-CpG):

[0117] Fc-TTTTTTTTCGTACGATTTTTCGTACGATTTTTCGTACGATTTTTCGTACGA (SEQ ID NO: 1)

[0118] Ferrocene-modified anti-PD-L1 DNA sequence (abbreviated as Fc-Apt PD-L1 ):

[0119] Fc-TTTTTTACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT (SEQ ID NO: 2)

[0120] 3. Preparation of Sonogenic Supramolecular Spherical Nucleic Acids

[0121] See also Figure 1 C, ferrocene-modified CpG sequence and PD-L1 aptamer sequence (i.e., Fc-CpG and Fc-Apt PD-L1 ) were mixed at a molar ratio of 1:1, and then the two were mixed with G5.NHAc-ICG-CD and stirred for 12 hours, and then the unbound DNA was separated and removed by ultrafiltration to obtain sSNA-CpG / Apt PD-L1 The AFM image of the sonosensitive supramolecular spherical nucleic acid is shown below. Figure 2 As shown in A, supramolecular spherical nucleic acids are all spherical structures with an average height of about 33 nm ( Figure 2 B).

[0122] 4. Preparation of Thermosensitive Hydroxybutyl Chitosan Wound Dressing Containing Supramolecular Spherical Nucleic Acids

[0123] The supramolecular spherical nucleic acid (0.1 mg) prepared in step 3 above was mixed with a commercially available thermosensitive hydroxybutyl chitosan wound dressing at a volume ratio of 3:2 to prepare a dressing that is highly effective in treating oral leukoplakia.

[0124] Example 2: Acoustic Dynamic Performance Experiment

[0125] Sonodynamic therapy (SDT) is an anti-tumor or antibacterial treatment strategy that combines ultrasound and sonosensitizers. Its core mechanism is to induce cell damage by generating reactive oxygen species (ROS, such as hydroxyl radicals ·OH and singlet oxygen¹O2) through the sonodynamic effect.

[0126] Based on the supramolecular spherical nucleic acid prepared in Example 1 above, this example uses electron spin resonance (ESR) technology to detect the sonodynamic properties of the supramolecular spherical nucleic acid. The specific process is as follows:

[0127] ESR technology combined with TEMP (for the detection of singlet oxygen free radicals 1 O2) and DMPO (for detecting hydroxyl radicals ·OH) were used to analyze the types of reactive oxygen species (ROS) generated. The specific steps are as follows: the ultrasound therapy device (Nu-Tek) probe was coated with the corresponding coupling agent and then placed in a solution containing 1 mg / mL sSNA-CpG / Apt PD-L1The supramolecular spherical nucleic acid solution was ultrasonicated at a frequency of 1 MHz. After ultrasonication for 5 minutes, 30 μL of sSNA-CpG / Apt with a concentration of 1 mg / mL was first taken. PD-L1 Supramolecular spherical nucleic acids were mixed with 50 μL of TEMP (100 mM), and then 30 μL of sSNA-CpG / Apt at a concentration of 1 mg / mL was taken out from the solution bottle. PD-L1 Supramolecular spherical nucleic acids were mixed with 30 µL of DMPO (100 mM deionized water / methanol as solvent). After mixing evenly, a certain amount of the mixture was aspirated using a capillary tube. After being covered with a quartz tube, the mixture was placed in the EPR sample chamber for testing of singlet oxygen radicals and hydroxyl radicals. Finally, the characteristic peak signals were detected using an ESR spectrometer.

[0128] Figure 3 The results shown show that sSNA-CpG / Apt PD-L1 Supramolecular spherical nucleic acids can effectively generate singlet oxygen radicals and hydroxyl radicals through ultrasound response.

[0129] Example 3: Verification of the in vitro penetration effect of supramolecular spherical nucleic acids on oral leukoplakia cells

[0130] This example constructs 3D cell spheroids to simulate and test the transdermal effect of the supramolecular spherical nucleic acid of the present invention on oral leukoplakia. The specific experimental process is as follows:

[0131] Leuk-1 cells were seeded into 12 wells of a 96-well plate with ultra-low attachment, with 500 cells per well. The cells were cultured in a cell culture incubator (5% CO2, 37°C) for 2-3 days until the cells grew into solid 3D cell spheres with a diameter of approximately 200 μm. PD-L1 and sSNA-CpG / Apt PD-L1 After the solution was mixed with GelRed for 1 h, the DNA-CpG / Apt PD-L1 Add sSNA-CpG / Apt stained with GelRed to wells 1 to 6 PD-L1 Add to wells 7 to 12 and incubate with 3D cell spheroids for 4 h respectively. Carefully remove the cell spheroids into centrifuge tubes and allow the cell spheroids to settle to the bottom of the centrifuge tubes. Then remove and discard the culture medium containing the nanomaterials. Gently wash three times with sterile PBS. Then, place the cell spheroids of each group into laser confocal dishes respectively, and use laser confocal microscopy to perform layer scanning to observe the penetration effect of each group of nanomaterials on the 3D cell spheroids.

[0132] Figure 4The results showed that simple DNA sequences were difficult to penetrate into cell spheres, while supramolecular spherical nucleic acids constructed by supramolecular chemistry were able to achieve deep penetration into 3D cell spheres, so supramolecular spherical nucleic acids could achieve good transdermal effects.

[0133] Example 4: Sonodynamic Effects of Supramolecular Spherical Nucleic Acids on Oral Leukoplakia Cells in Vitro

[0134] This example uses cell experiments to test the sonodynamic therapeutic effect of the supramolecular spherical nucleic acid of the present invention. The specific experimental process is as follows:

[0135] Leuk-1 cells were seeded into 12-well plates at a rate of 1×10 5 The cells were placed in a cell culture incubator (5% CO2, 37 ℃) for 12 hours to allow the cells to adhere to the wall and grow. PD-L1 , sSNA-NC,sSNA-CpG, sSNA-Apt PD-L1 The culture medium was ultrasonicated at a frequency of 1 MHz for 5 minutes using an ultrasonic therapy device, and then the cells were incubated with the above-mentioned grouped nanomaterials under 5% CO2 and 37°C for 6 hours. After culture, the culture medium was poured out, the cells were washed three times with sterile PBS, and the reactive oxygen probe DCFH-DA was added for staining, and incubated at 37°C for 20-30 minutes. Then the staining solution was poured out, the residual staining solution was washed away with PBS, 500 μL of 2.5% glutaraldehyde solution was added to fix the cells for 15-20 minutes, and then the fixative was poured out, PBS was added to wash 3 times, and DAPI was added to stain the nuclei of B16 cells for 15-20 minutes. After the staining was completed, the cells were washed 3 times with PBS, and then 200 μL of PBS was added to soak the bottom of each well to maintain the good morphology of the cells. The expression of reactive oxygen in the cells was observed under a fluorescence microscope using the green fluorescence of the probe. The results are shown in the figure. Figure 5 As shown, the supramolecular spherical nucleic acids containing sonosensitizers all produced obvious green fluorescence after ultrasonic treatment, while the experimental group without ultrasound had no obvious green fluorescence, indicating that the supramolecular spherical nucleic acids can produce more reactive oxygen species through ultrasonic stimulation.

[0136] Example 5: Verification of the immune effect of supramolecular spherical nucleic acids on oral leukoplakia cells in vitro

[0137] This example uses cell experiments to test the immunogenicity of the supramolecular spherical nucleic acid of the present invention and the immune activation effect of the material itself through Example 3. The specific experimental process is as follows:

[0138] This example designed a transwell experiment to determine the immunogenic death of leukoplakia cells by determining whether dendritic cells (DCs) express maturation factors. First, Leuk-1 cells were plated at 2×10 cells per well. 5 The cells were seeded with 1 mL of culture medium in the upper chamber of a 6-well plate with a 0.4 μm polycarbonate porous membrane and placed in a 5% CO2, 37°C incubator for 12 h to allow the cells to adhere to the wall and grow. The cell culture medium in the upper chamber was then replaced with a medium containing a certain concentration of sSNA-CpG / Apt PD-L1 ,sSNA-NC,sSNA-CpG, sSNA-Apt PD-L1 The cells were cultured in a 1 MHz ultrasound machine for 5 minutes, and then incubated with the above-mentioned nanomaterials for 12 hours at 5% CO2 and 37°C. PBS-treated cells served as the control group. 5 The cells were seeded at a density of 1 mL of culture medium in the wells of the lower chamber and incubated for 24 h. Subsequently, DCs were trypsinized and collected by centrifugation. They were stained with CD86 and CD80 flow cytometry antibodies for 15 min in the dark. After staining, the cells were washed with PBS by centrifugation and resuspended in 0.2 mL of PBS for flow cytometric analysis.

[0139] The results are as follows Figure 6 As shown, supramolecular spherical nucleic acids containing CpG sequences can activate dendritic cells to a certain extent and produce immune effects without ultrasonic treatment, and the activation effect is higher after ultrasound, which proves that the reactive oxygen species generated by the sonodynamic effect further activates the immune effects of dendritic cells through the damage-associated molecular patterns released by immunogenic death.

[0140] Example 6: In vivo experimental testing of the therapeutic effect of sonogenic supramolecular spherical nucleic acids on oral leukoplakia

[0141] This example uses animal experiments to test the therapeutic effect of the sonogenic supramolecular spherical nucleic acid of the present invention on oral leukoplakia. The specific experimental process is as follows:

[0142] Male C57BL / 6 mice aged 4 to 6 weeks were randomly divided into seven groups, with 5 to 6 mice in each group. They were given a combined drinking water containing 4NQO (100 μg / mL, (4-nitroquinoline-1-oxide) is a chemical inducer commonly used to induce oral precancerous lesions such as leukoplakia in animal models) and LPS (10 μg / mL) from week 0 to week 14, and pure water ( Figure 7 ), and different drugs are administered through epidermal delivery, and the specific groups are as follows:

[0143] (1) Pure water group (Normal group)

[0144] (2) PBS group (4NQO+LPS+PBS)

[0145] (3) PBS+US group (4NQO+LPS+ultrasound treatment)

[0146] (4) sSNA-CpG / Apt PD-L1 Group (4NQO+LPS+ sSNA-CpG / Apt PD-L1 )

[0147] (5) sSNA-NC+US group (4NQO+LPS+ sSNA-NC+ultrasound treatment)

[0148] (6) sSNA-CpG+US group (4NQO+LPS+ sSNA-CpG+ultrasound treatment)

[0149] (7) sSNA-Apt PD-L1 + US group (4NQO+LPS+ sSNA-Apt PD-L1 +Ultrasound therapy)

[0150] (8) sSNA-CpG / Apt PD-L1 +US group (4NQO+LPS+ sSNA-CpG / Apt PD-L1 +Ultrasound therapy)

[0151] All grouped materials were mixed with thermosensitive hydroxybutyl chitosan wound dressing at a volume ratio of 3:2 and then applied to the surface of the mouse tongue.

[0152] During this period, the weight changes and vitiligo treatment status of the mice were recorded weekly. All mice were sacrificed at week 18, and their tongues were photographed. The spleens of the mice were dissected, cut into small pieces, ground on a 40 μm cell sieve, and suspended in DMEM medium. After centrifugation (1000 rpm, 5 minutes), the pieces were resuspended in DMEM medium. Subsequently, the single-cell suspension was passed through a nylon wool column to finally obtain splenic T cells. Next, the extracted T cells were labeled with anti-CD4-PE / anti-CD8-FITC flow cytometry antibodies, respectively. Finally, the phenotypic populations of T cells with different staining treatments were analyzed by flow cytometry.

[0153] Histopathological analysis of mouse tongues was performed according to standard procedures for hematoxylin and eosin (H&E) staining. Mouse tongues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections before H&E staining. The sections were scanned using a 3DHISTECH slide scanning system, and images of mouse tongues were processed using CaseViewer image processing software.

[0154] The results are as follows Figure 8 As shown in the figure, after modeling, the mouse tongue had obvious white patches. The untreated PBS group had undergone a certain degree of canceration and transformed into oral squamous cell carcinoma. After treatment with the sSNA-NC+US group, the oral leukoplakia was improved to a certain extent due to the killing effect of reactive oxygen species generated by sonodynamics on the oral leukoplakia. The sSNA-CpG / Apt PD-L1 The leukoplakia in the group after treatment was also slightly relieved, indicating that the combined treatment of nucleic acid immune adjuvant CpG and PD-L1 aptamer can activate the body's immune response to oral leukoplakia. PD-L1 The +US group showed a good therapeutic effect on leukoplakia, proving that the sonographic supramolecular spherical nucleic acid of the present invention can responsively dissociate the nucleic acid immune adjuvant CpG and PD-L1 aptamer after ultrasonic stimulation, thereby activating immunity and reversing the immunosuppressive microenvironment of the leukoplakia lesion site. At the same time, the reactive oxygen species generated by sonodynamics has an immunogenic death effect on leukoplakia cells, thereby enhancing the immunotherapy against oral leukoplakia.

[0155] Given that supramolecular spherical nucleic acids have a good immune activation effect on the treatment of oral leukoplakia, the T cells in the spleen were analyzed by flow cytometry, and the results were as follows: Figure 9 As shown, sSNA-CpG / Apt PD-L1 group, sSNA-NC+US group, sSNA-Apt PD-L1 CD4+US group and sSNA-CpG+US group + 、CD8 + The expression levels of T cells increased to a certain extent, indicating that the nucleic acid immune adjuvant CpG and PD-L1 aptamer have the ability to activate T cells, and the immunogenic death caused by the reactive oxygen species generated by ultrasound can activate immunity, while sSNA-CpG / Apt PD-L1 The combined immunotherapy of the +US group can activate T cell immunity to the greatest extent and improve the therapeutic effect on oral leukoplakia.

[0156] The results of H&E staining ( Figure 10 ) showed that the modeling group, i.e. oral leukoplakia, had obvious hyperplasia of the tongue dorsum epithelium, obvious granular layer, thickening of the spinous layer, and proliferation of basal cells. PD-L1 group, sSNA-NC+US group, sSNA-Apt PD-L1 The treatment of the +US group and the sSNA-CpG+US group alleviated abnormal proliferation to a certain extent, but the sSNA-CpG / Apt PD-L1 The +US group combined with immunotherapy had the most obvious therapeutic effect and suppressed the abnormal proliferation of the spinous layer to the greatest extent.

[0157] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A method for preparing a sonosensitive supramolecular spherical nucleic acid, characterized in that: The method comprises: using a fifth-generation polyamidoamine (G5 PAMAM) dendrimer modified with β-CD and loaded with a sonosensitizer as a core, and using a ferrocene-modified CpG DNA and an anti-PD-L1 DNA aptamer sequence as an outer shell, to form the sonosensitizing supramolecular spherical nucleic acid through host-guest supramolecular recognition; The surface of the fifth generation polyamide-amine (G5 PAMAM) dendrimer contains -NH2.

2. The preparation method according to claim 1, characterized in that The sonosensitizer is selected from the following group: indocyanine green (ICG), dihydrochlorin (Ce6), protoporphyrin IX (PpIX), titanium dioxide (TiO2), black phosphorus (BP), zinc oxide (ZnO) or bismuth sulfide (Bi2S3).

3. The preparation method according to claim 1, characterized in that The ferrocene-modified CpG DNA sequence is shown in SEQ ID NO: 1; the ferrocene-modified anti-PD-L1 DNA aptamer sequence is shown in SEQ ID NO:

2.

4. The preparation method according to claim 1, characterized in that The method further comprises the following steps: A. Preparation of sonogenic supramolecular spherical nucleic acid cores: (A1) The fifth generation polyamidoamine (G5 PAMAM) dendrimer was modified with the sonosensitizer indocyanine green (ICG) at the end, and the sonosensitizer-modified G5 PAMAM dendrimer (G5.NH2-ICG) was obtained by dialysis and freeze-drying. Wherein, the surface of the fifth generation polyamidoamine (G5 PAMAM) dendrimer contains -NH2; (A2) coupling β-cyclodextrin (β-CD) with the G5.NH2-ICG via host-guest recognition to form a β-CD-modified and sonosensitizer-loaded G5 PAMAM dendrimer (G5.NH2-ICG-CD); (A3) acetylation of the G5.NH2-ICG-CD to obtain the supramolecular spherical nucleic acid core (G5.NHAc-ICG-CD); B. Preparation of ferrocene-modified nucleic acid immune adjuvant CpG and nucleic acid aptamer PD-L1: A ferrocene (Fc)-modified CpG DNA sequence (SEQ ID NO: 1) and an anti-PD-L1 DNA aptamer sequence (SEQ ID NO: 2) were synthesized using a DNA synthesizer; C. Assembly of sonogenic supramolecular spherical nucleic acids: The ferrocene (Fc)-modified CpG DNA and the ferrocene (Fc)-modified anti-PD-L1 DNA aptamer in step B were mixed at a molar ratio of 1:1, and co-assembled with the G5.NHAc-ICG-CD obtained in step A through host-guest supramolecular recognition. Unbound DNA was removed by ultrafiltration to obtain the sonogenic supramolecular spherical nucleic acid (sSNA-CpG / AptPD-L1).

5. The preparation method according to claim 4, characterized in that In step (A1), the molar ratio of the indocyanine green (ICG) to the G5 PAMAM dendrimer is 5-20:1; In step (A2), the molar ratio of β-CD to G5.NH2-ICG is 5-20:1, and in the acetylation treatment, the molar ratio of triethylamine:acetic anhydride:the β-CD-modified and sonosensitizer-loaded G5 PAMAM dendrimer (G5.NH2-ICG-CD) is 50-100:50-100:

1. In step (C), the molar ratio of the ferrocene (Fc)-modified CpG DNA to the supramolecular spherical nucleic acid core (G5.NHAc-ICG-CD) is 10-20:1, and the molar ratio of the ferrocene (Fc)-modified anti-PD-L1 DNA aptamer to the supramolecular spherical nucleic acid core (G5.NHAc-ICG-CD) is 10-20:

1.

6. A sonosensitive supramolecular spherical nucleic acid, characterized in that: The sonogenic supramolecular spherical nucleic acid is prepared by the method according to claim 1.

7. The sonosensitive supramolecular spherical nucleic acid according to claim 6, characterized in that The particle size of the sonogenic supramolecular spherical nucleic acid is 10-100 nm, preferably 20-80 nm, and more preferably 30-35 nm.

8. A pharmaceutical composition, characterized in that include: (1) The sonosensitive supramolecular spherical nucleic acid prepared by the method of claim 1 or the sonosensitive supramolecular spherical nucleic acid according to claim 6; and (2) Medical dressing; the medical dressing is selected from the following group: wound dressing, gel dressing, or a combination thereof.

9. Use of a sonogenic supramolecular spherical nucleic acid in the preparation of a drug for treating tumors and / or oral leukoplakia.

10. The use according to claim 9, characterized in that The tumor is selected from the group consisting of oral cancer, malignant mesothelioma, gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, lung cancer, bladder cancer, ovarian cancer, triple-negative breast cancer, cervical cancer, endometrial cancer, uterine serous carcinoma, colon cancer, prostate cancer, laryngeal cancer, tonsil cancer, or a combination thereof; The medicine is a medicine for ultrasonic treatment of oral leukoplakia; The sonosensitive supramolecular spherical nucleic acid is used for a purpose selected from the group consisting of: (1) Inducing immunogenic cell death of oral leukoplakia cells by releasing reactive oxygen species (ROS) in response to ultrasound; and / or (2) Release CpG immune adjuvant and PD-L1 aptamer, reverse the immunosuppressive microenvironment, and activate dendritic cells and T cell immune responses.

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