Ultrasound-activated blood coagulation targeting tumor selective prodrug as well as preparation method and application thereof

Abstract

The invention provides an ultrasonic activated blood coagulation targeting tumor selective prodrug as well as a preparation method and application thereof. The blood coagulation targeting tumor selective prodrug has a structure as shown in a formula I. The blood coagulation targeting tumor selective prodrug can trigger a blood coagulation cascade reaction by utilizing tumor vascular injury generated by ultrasonic induction so as to realize targeted anchoring of the drug and inhibit off-target diffusion of an active drug; meanwhile, the prodrug is activated by ultrasound, so that the limitation of tumor heterogeneity on the activation of the prodrug can be overcome. According to the technical scheme, selective enrichment of active drugs in tumors can be effectively improved, and toxicity caused by drug off-target is reduced while tumor growth is remarkably inhibited.

Description

Technical Field

[0001] This invention belongs to the field of materials technology, specifically relating to an ultrasound-activated coagulation-targeting tumor-selective prodrug, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy is currently one of the key research directions in cancer treatment. It enhances the tumor immune cycle and weakens the immunosuppressive effects in the tumor microenvironment, thereby improving the tumor immune response to treat cancer. It boasts advantages such as good efficacy and long-lasting effects. Based on different nodes in the tumor immune cycle, immunotherapy includes immune checkpoint blockade therapy, adoptive cell therapy, tumor vaccines, and cytokine therapy, among others. However, due to the heterogeneity of tumors, most immunotherapy regimens also have significant limitations.

[0003] Studies have shown that tumor antigen presentation mechanisms constitute an essential technical element in various immunotherapy regimens during the transition from non-specific to specific immunity. Antigen-presenting cells (APCs) achieve immune regulation by uptake, processing, and presenting antigens, among which mature dendritic cells (DCs) possess the strongest antigen-presenting function. Therefore, immunotherapy based on dendritic cell activation holds significant clinical application potential.

[0004] Toll-like receptors (TLRs) are a class of receptors that mediate inflammatory responses and innate immunity. They are widely expressed across the immune cell spectrum, inducing the secretion of cytokines and chemokines, activating innate immune responses, and mediating the activation of acquired immune responses. TLRs 7 and 8 specifically recognize nucleic acid components in viruses, bacteria, and infected cells, inducing the production of pro-inflammatory factors and type I interferon, and promoting the maturation of antigen-presenting cells such as dendritic cells. Imidazoquinoline (IMDQ), as a highly potent TLR 7 / 8 immune agonist, is widely used as an immune adjuvant and in immunotherapy. However, due to insufficient targeting, IMDQ drugs pose a risk of excessive activation of the systemic immune system after administration, limiting their clinical application. Therefore, it is necessary to develop IMDQ drugs with tumor-site selective accumulation properties to improve therapeutic efficacy and reduce toxic side effects.

[0005] At present, the existing technical means related to IMDQ drugs are mainly based on two strategies: (1) constructing antibody-drug conjugates (ADCs) or peptide-drug conjugates (PDCs) by conjugating targeting molecules. These drugs can specifically bind to highly expressed receptors on the surface of tumor cells or in the tumor microenvironment, and enhance the enrichment of drugs at the tumor site through receptor-mediated targeted delivery mechanisms; (2) constructing selectively activated prodrugs by utilizing the biological differences between tumor tissues and normal tissues in terms of hypoxia level, pH value, and specific enzyme expression levels. These drugs can be specifically activated or released in tumor tissues, increasing the accumulation of active drugs at the tumor site. However, both of the above two strategies have inherent defects: for the targeted drug delivery system, its targeting depends on the passive retention mechanism achieved by the binding of the targeting head to the receptor molecule, and it cannot actively regulate the directional diffusion of drugs towards tumor tissues, thus it is difficult to inhibit the acute immunotoxicity caused by IMDQ drugs; for tumor-selectively activated prodrugs, limited by the heterogeneity of the tumor microenvironment, there are risks of insufficient activation rate and immunotoxicity caused by the extravasation of active drugs. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an ultrasound-activated coagulation-targeted tumor-selective prodrug, its preparation method and application. The prodrug is based on ultrasound induction, can effectively improve the selective enrichment of active drugs in tumors, and reduce the toxicity caused by drug off-target while significantly inhibiting tumor growth.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides an ultrasound-activated coagulation-targeted tumor-selective prodrug, which has the structure of formula I:

[0009]

[0010] Wherein, R1 is selected from substituted or unsubstituted C1-C10 straight-chain alkyl groups, substituted or unsubstituted C3-C10 branched-chain alkyl groups or substituted or unsubstituted C6-C12 aryl groups;

[0011] R2 is selected from substituted or unsubstituted C1-C6 straight-chain alkylene groups, substituted or unsubstituted C3-C6 branched-chain alkylene groups or substituted or unsubstituted C6-C10 arylene groups;

[0012] R3 is selected from substituted or unsubstituted C3-C5 straight-chain alkyl groups or substituted or unsubstituted C3-C5 ether groups;

[0013] a, b, c, d are the contents of repeating units, 0 < a < 1, 0 < b < 1, 0 < c < 1, 0 < d < 1; n is the degree of polymerization, 50 ≤ n ≤ 500.

[0014] Preferably, R1 is selected from substituted or unsubstituted C2-C8 straight-chain alkyl, substituted or unsubstituted C3-C8 branched alkyl, or substituted or unsubstituted C6-C10 aryl.

[0015] Preferably, R2 is selected from substituted or unsubstituted C1-C4 straight-chain alkylene, substituted or unsubstituted C3-C5 branched alkylene, or substituted or unsubstituted C6-C9 aryl.

[0016] Preferably, R3 is selected from substituted or unsubstituted C3-C4 straight-chain alkyl groups or substituted or unsubstituted C3-C4 ether groups.

[0017] Preferably, R1 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, n-heptyl, isoheptyl, n-octyl, phenyl, benzyl, naphthyl, or biphenyl.

[0018] Preferably, R2 is selected from methylene, ethylene, propylene, butylene, phenylene, or benzylene.

[0019] Preferably, R3 is selected from n-propyl, n-butyl, n-pentyl, CH3CH2CH2O-, CH3CH2OCH2-, CH3OCH2CH2- or CH3CH2CH2OCH2-, either individually or in combination.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned coagulation-targeting tumor-selective prodrug, comprising the following steps:

[0021] S1: Polyglutamic acid, imidazoquinoline azide prodrug of formula A and maleimide of formula B are condensed to obtain an intermediate product.

[0022] S2: The intermediate product is subjected to precipitation treatment. The product obtained from precipitation is redissolved and reacted with the coagulation-targeting peptide shown in Formula C. The product is then freeze-dried to obtain the final product.

[0023]

[0024] Preferably, the condensation reaction is carried out in the presence of a solvent and a condensation reagent.

[0025] Preferably, the solvent is selected from non-alcohol / ammonia organic solvents, specifically from any one or more of dimethylformamide, benzene, toluene, carbon tetrachloride, or tetrahydrofuran.

[0026] Preferably, the condensing agent is selected from any one or more of 2,4,6-trichlorobenzoyl chloride, N,`N`-diisopropylcarbodiimide, dicyclohexylcarbodiimide, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0027] Preferably, the condensation reaction is carried out at a temperature of 25°C to 70°C for 2 to 4 days.

[0028] Preferably, the reaction temperature in step S2 is 20℃~40℃, and the time is 2~24h.

[0029] Preferably, the mass ratio of the imidazoquinoline azide prodrug of Formula A, the maleimide of Formula B, and polyglutamic acid is (0.1-0.3):(0.05-0.2):1.

[0030] Preferably, the molar ratio of the coagulation-targeting peptide to polyglutamic acid is (0.5-5):1.

[0031] Thirdly, the present invention provides an ultrasound-activated coagulation-targeting tumor-selective prodrug system, which includes ultrasound conditions and the aforementioned coagulation-targeting tumor-selective prodrug.

[0032] Preferably, the energy of the ultrasound is 1.5–2.5 W / cm². 2 The duty cycle is 20-100%, and the ultrasound time is 2.5-5 minutes.

[0033] Fourthly, the present invention provides the application of the coagulation-targeting tumor-selective prodrug involved in the above-mentioned technical solution in the preparation of a drug for treating cancer.

[0034] Preferably, the cancer includes any one or more of breast cancer, colorectal cancer, liver cancer, melanoma, osteosarcoma, kidney cancer, prostate cancer, ovarian cancer, pancreatic cancer, bladder cancer, thyroid cancer, or nasopharyngeal cancer.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] This invention provides an ultrasound-activated coagulation-targeting tumor-selective prodrug, having the structure of Formula I, with polyglutamic acid as the main chain, which can improve the drug's water solubility and biocompatibility. It carries an imidazoquinoline azide prodrug of Formula A (hereinafter referred to as: IMDQ prodrug), maleimide, and a coagulation-targeting peptide. The IMDQ prodrug can be spatiotemporally and controllably activated under ultrasound, effectively reducing the immunotoxicity of IMDQ drugs. Maleimide can conjugate with antigens in vivo, promoting antigen presentation and enhancing immunotherapy. The coagulation-targeting peptide can specifically bind to activated fibrin, artificially creating targets within the tumor using ultrasound, thus improving drug targeting.

[0037] The coagulation-targeting tumor-selective prodrug provided by this invention can achieve targeted drug anchoring and inhibit off-target diffusion of the active drug by inducing a coagulation cascade reaction through ultrasound-induced tumor vascular damage. Simultaneously, ultrasound activation of the prodrug overcomes the limitations imposed by tumor heterogeneity on prodrug activation. This technical solution can effectively improve the selective enrichment of the active drug in tumors, significantly inhibiting tumor growth while reducing off-target toxicity. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the synthesis and characterization results of the ultrasound-responsive imidazoquinoline azide prodrug described in Example 1.

[0039] Wherein, A is a schematic diagram of the synthesis of the imidazoquinoline azide prodrug, and B is the 1H NMR spectrum before and after synthesis. 1 Comparison of HNMR spectra; C is the carbon NMR spectrum before and after synthesis. 13 C NMR comparison chart;

[0040] Figure 2 The image shows the characterization results of the ultrasound-activated coagulation-targeting tumor-selective prodrug described in Example 2.

[0041] Where A represents an unlinked polypeptide nanoprodrug. 1 H NMR spectra; B represents ultrasound-activated coagulation-targeting tumor-selective prodrugs, unlinked peptide nanoprodrugs, and peptides. 1 H NMR comparison chart;

[0042] Figure 3 This is a graph showing the in vivo toxicity assessment results of the ultrasound-activated coagulation-targeting tumor-selective prodrug described in Example 3;

[0043] In the figure, A to C are graphs showing the changes in the concentration of various cytokines in the blood of healthy / tumor-bearing mice after drug administration; D is graph showing the changes in the body weight of healthy / tumor-bearing mice after drug administration.

[0044] Figure 4 This is a graph showing the in vitro ultrasound activation evaluation results of the ultrasound-activated coagulation-targeting tumor-selective prodrug described in Example 4.

[0045] In this study, A represents the in vitro ultrasound reduction characterization of the coagulation-targeting tumor-selective prodrug AMINP as detected by HPLC; and B represents the flow cytometry analysis of AMINP in vitro ultrasound-activated DC cells.

[0046] Figure 5 The tumor hemorrhage and drug distribution in mice treated with MINP NPs, MINP NPs+US, AMINP NPs, and AMINP NPs+US were plotted at 4 hours.

[0047] Figure 6The image shows the coagulation targeting characterization results of the ultrasound-activated coagulation-targeting tumor-selective prodrug described in Example 5; IMDQ-N3 is a small molecule imidazoquinoline azide prodrug, A`MINP is a non-coagulation-targeting imidazoquinoline azide nanoprodrug with an inactivation target, and AMINP is a coagulation-targeting tumor-selective prodrug.

[0048] Where A corresponds to the size of the blood clot formed after adding different drugs to fresh blood; B corresponds to the drug content in the blood clot; and C corresponds to the content of the active drug IMDQ in different tissues of mice after drug administration.

[0049] Figure 7 This is a graph showing the tumor suppression evaluation results of the ultrasound-activated coagulation-targeting tumor-selective prodrug in the mouse CT26 tumor model described in Example 6.

[0050] In the diagram, A is the experimental protocol roadmap for the tumor suppression experiment; B is the tumor growth curve of mice; C is the weight change of mice; and D is the survival time of mice.

[0051] Figure 8 This is a graph showing the evaluation results of the improvement of the tumor microenvironment in mice by the ultrasound-activated coagulation-targeting tumor-selective prodrug described in Example 7;

[0052] Where A represents intratumoral CD4 + and CD8 + A) is a graph showing the content of T cells; B) is a graph showing the content of activated DC cells within the tumor; C) is a graph showing the content of M1 macrophages within the tumor. Detailed Implementation

[0053] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0054] 4-Amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-ethanol (IMDQ) is a small molecule TLR7 / 8 agonist imidazoquinoline. It has been reported that IMDQ can activate TLR7 / 8 located on the endosomal membrane of immune cells, induce the secretion of cytokines and the maturation of related immune cells, and has the ability to activate and regulate the immune system. However, clinical experiments have shown that IMDQ has extremely strong systemic immunotoxicity, which leads to a narrow therapeutic window for IMDQ and limits its clinical application. The present invention adopts the strategy of preparing prodrugs, modifying the amine group at the active site where IMDQ binds to TLR7 / 8 into a biologically inactive azide group (IMDQ-N3), restricting the action range of active IMDQ. At the same time, the present invention loads a coagulation-targeting peptide and IMDQ-N3 onto a polyglutamic acid nanoplatform, and uses the targeting property of the coagulation-targeting peptide and the long retention effect of nanoparticles at the tumor site to enhance the enrichment of the drug at the tumor site. The present invention uses the high energy and high penetrability of focused ultrasound to destroy tumor blood vessels and create targets; at the same time, it uses ultrasound at a constant power and fixed points to quantitatively activate the drug, avoiding the problem of non-activation of the prodrug probability caused by tumor heterogeneity.

[0055] Specifically, the present invention provides an ultrasound-activated coagulation-targeting tumor-selective prodrug having the structure of formula I:

[0056]

[0057] Wherein, R1 is selected from a substituted or unsubstituted C1-C10 straight-chain alkyl group, a substituted or unsubstituted C3-C10 branched-chain alkyl group or a substituted or unsubstituted C6-C12 aryl group;

[0058] R2 is selected from a substituted or unsubstituted C1-C6 straight-chain alkylene group, a substituted or unsubstituted C3-C6 branched-chain alkylene group or a substituted or unsubstituted C6-C10 arylene group;

[0059] R3 is selected from a substituted or unsubstituted C3-C5 straight-chain alkyl group or a substituted or unsubstituted C3-C5 ether group.

[0060] Here, a, b, c, d represent the content of repeating units, so 0 < a < 1, 0 < b < 1, 0 < c < 1, 0 < d < 1, and a + b + c + d = 1.

[0061] n is the degree of polymerization, 50 ≤ n ≤ 500, such as 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500, etc.

[0062] In some embodiments of the present invention, the coagulation-targeting tumor-selective prodrug has the structure of Formula I, wherein R1 is preferably selected from substituted or unsubstituted C2-C8 straight-chain alkyl, substituted or unsubstituted C3-C8 branched-chain alkyl, or substituted or unsubstituted C6-C10 aryl; R2 is preferably selected from substituted or unsubstituted C1-C4 straight-chain alkylene, substituted or unsubstituted C3-C5 branched-chain alkylene, or substituted or unsubstituted C6-C9 aryl; and R3 is preferably selected from substituted or unsubstituted C3-C4 straight-chain alkyl or substituted or unsubstituted C3-C4 ether.

[0063] In this invention, the substituted group in the above-mentioned "substituted or unsubstituted" can be selected from C0-C4 alcohol hydroxyl groups, ether bonds or C0-C4 aliphatic amino groups.

[0064] In some specific embodiments of the present invention, R1 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, n-heptyl, isoheptyl, n-octyl, phenyl, benzyl, naphthyl, or biphenyl; R2 is selected from methylene, ethylene, propylene, butylene, phenylene, or benzylene; R3 is selected from n-propyl, n-butyl, n-pentyl, CH3CH2CH2O-, CH3CH2OCH2-, CH3OCH2CH2-, or CH3CH2CH2OCH2-, either individually or in combination.

[0065] As can be seen from the structure of Formula I above, this invention uses polyglutamic acid as the main chain structure, while simultaneously incorporating IMDQ-N3, maleimide, and a coagulation-targeting peptide. Polyglutamic acid as the main chain improves the water solubility and biocompatibility of the drug. IMDQ prodrugs can be spatiotemporally activated under ultrasound, effectively reducing the immunotoxicity of IMDQ drugs. Maleimide can conjugate with antigens in the body, promoting antigen presentation and enhancing immunotherapy. The coagulation-targeting peptide can specifically bind to activated fibrin, artificially creating targets within the tumor using ultrasound, thereby improving drug targeting.

[0066] The present invention also provides a method for preparing the above-mentioned coagulation-targeting tumor-selective prodrug, comprising the following steps:

[0067] S1: Polyglutamic acid, imidazoquinoline azide prodrug of formula A and maleimide of formula B are condensed to obtain an intermediate product.

[0068] S2: The intermediate product is subjected to precipitation treatment. The product obtained from precipitation is redissolved and reacted with the coagulation-targeting peptide shown in Formula C. The product is then freeze-dried to obtain the final product.

[0069]

[0070]

[0071] According to the present invention, polyglutamic acid, imidazoquinoline azide prodrug of formula A and maleimide of formula B are first subjected to a condensation reaction to obtain an intermediate product.

[0072] In some embodiments of the present invention, the condensation reaction is carried out in the presence of a solvent and a condensation reagent. The solvent is selected from any one or more of dimethylformamide, benzene, toluene, carbon tetrachloride, or tetrahydrofuran, preferably dimethylformamide; the condensation reagent is selected from any one or more of 2,4,6-trichlorobenzoyl chloride, N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, preferably N,N'-diisopropylcarbodiimide.

[0073] In some specific embodiments of the present invention, polyglutamic acid is preferably dissolved in a solvent, and then the imidazoquinoline azide prodrug of Formula A and maleimide of Formula B are added. The reaction is carried out at 25°C to 70°C for 2 to 4 days, preferably at 35°C to 60°C for 2 to 3 days. The mass of the maleimide of Formula B as a reaction raw material accounts for 5% to 20% of the mass of polyglutamic acid, such as 5%, 8%, 10%, 12%, 15%, 18%, or 20%, etc.; the mass of the imidazoquinoline azide prodrug of Formula A as a reaction raw material accounts for 10% to 30% of the mass of polyglutamic acid, such as 10%, 15%, 20%, 25%, or 30%, etc.

[0074] After obtaining the intermediate product, according to the present invention, the intermediate product is subjected to precipitation treatment, the product obtained by precipitation is redissolved and reacted with the coagulation targeting peptide shown in Formula C, and then freeze-dried to obtain the final product.

[0075] In some embodiments of the present invention, the intermediate product is precipitated in excess diethyl ether, the resulting crude product is dissolved in phosphate buffer solution, a coagulation-targeting peptide is added, and the reaction is carried out at 20°C–40°C for 2–24 h, preferably at 20–30°C for 2–18 h. After the reaction, the product is preferably subjected to salting, dialysis, and lyophilization to obtain a coagulation-targeting tumor-selective prodrug. The molar ratio of the coagulation-targeting peptide to polyglutamic acid is (0.5–5):1, preferably (0.5–3):1, and more preferably (0.5–2):1.

[0076] It is evident that the preparation method of the above-mentioned coagulation-targeting tumor-selective prodrug provided by the present invention is simple, requires no excessive complicated steps, and is easy to realize industrial or commercial production.

[0077] The present invention also provides an ultrasound-activated coagulation-targeting tumor-selective prodrug system, which includes the coagulation-targeting tumor-selective prodrug described above under ultrasound conditions.

[0078] In the above system, the energy of the ultrasound is 1.5–2.5 W / cm². 2 For example, it could be 1.5W / cm 2 16W / cm 2 1.7W / cm 2 1.8W / cm 2 1.9W / cm 2 2.0W / cm 2 2.1W / cm 2 2.2W / cm 2 2.3W / cm 2 2.4W / cm 2 Or 2.5W / cm 2 The duty cycle is 20-100%, preferably 30-80%, and more preferably 50%; the ultrasound time is 2.5-5 min, which can be 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min or 5 min.

[0079] In this invention, the aforementioned ultrasound-activated coagulation-targeting tumor-selective prodrug system can destroy tumor blood vessels through focused ultrasound, triggering a coagulation cascade reaction and effectively initiating targeted drug accumulation. Simultaneously, the ultrasound-activated coagulation-targeting tumor-selective prodrug can increase the content of effective drugs in the tumor, effectively enhancing drug efficacy while inhibiting the side effects caused by excessive activation of the systemic immune system due to drug off-target effects.

[0080] The present invention also provides the application of the above-mentioned coagulation-targeting tumor-selective prodrug in the preparation of a drug for treating cancer.

[0081] The cancers mentioned include any one or more of the following: breast cancer, colorectal cancer, liver cancer, melanoma, osteosarcoma, kidney cancer, prostate cancer, ovarian cancer, pancreatic cancer, bladder cancer, thyroid cancer, or nasopharyngeal cancer.

[0082] To further illustrate the present invention, the following embodiments provide a detailed description. The experimental materials used in the following embodiments of the present invention are all commercially available products.

[0083] Example 1: Preparation of IMDQ-N3

[0084] A schematic diagram of the preparation of IMDQ-N3 is shown below. Figure 1As shown in Figure A. IMDQ-N3 was prepared by reacting fluorosulfonyl azide with IMDQ. Specifically, 286.33 mg IMDQ (1.0 mmol) was dissolved in 10 mL of dimethylformamide and stirred at 0 °C. Then, 10 mL of a methyl tert-butyl ether solution of fluorosulfonyl azide (containing 124.97 mg fluorosulfonyl azide (1.0 mmol)) and 400.476 mg potassium bicarbonate (1.0 mmol) was added, and the reaction was carried out at room temperature for 10 h. After the reaction was complete, 10 mL of ethyl acetate and 40 mL of water were added for precipitation to obtain IMDQ-N3. Using deuterated DMSO as a solvent, the solution was obtained by... 1 H NMR and 13 The structure of IMDQ-N3 was confirmed and characterized by 1C NMR (see [link]). Figure 1 (B, C in the middle).

[0085] Example 2: Preparation of ultrasound-activated coagulation-targeting tumor-selective prodrug, namely, A15-Mal-IMDQ-N3-PLG (AMINP).

[0086] A15 is a polypeptide containing a specific sequence of glutamine. A15 can achieve targeted coagulation through the catalytic action of transglutaminase (conversion of coagulation factor XIII) and fibrin chemical cross-linking.

[0087] Specifically, the preparation steps of AMINP are as follows:

[0088] 300.00 mg of poly(L-glutamic acid) (PLG) was dissolved in 20 mL of dimethylformamide, and 12.2 mg of 4-dimethylaminopyridine (0.1 mmol), 31.21 mg of IMDQ-N3 (0.1 mmol), 28.22 mg of N-(2-hydroxyethyl)maleimide (0.2 mmol), and 126.20 mg of N,N'-diisopropylcarbodiimide (1.0 mmol) were added. The mixture was heated to 60 °C and stirred for 72 h. After precipitation in excess diethyl ether, a targetless nanoprodrug crude product, A15-Mal-IMDQ-PLG, abbreviated as AMINP, was obtained. The crude product was dissolved in 10 mL of phosphate buffer solution, and 50.00 mg of A15 was added. The mixture was reacted at 30 °C for 2 h, followed by salting, dialyzing, and lyophilization to obtain AMINP. Using deuterium-rich water to process Mal-IMDQ-N3-PLG, abbreviated as MINP ( Figure 2 A) and AMINP Figure 2 B) in 1 H NMR characterization confirmed its successful synthesis.

[0089] Example 3: In vivo toxicity assessment of AMINP in mice

[0090] Thirty female BALB / c mice were divided into 10 groups (n=3 per group): a blank control group (injected with PBS), three small molecule control groups (treated with different concentrations of IMDQ, i.e., 10.0 mg / kg, 30.0 mg / kg, and 50.0 mg / kg), three nanoparticle control groups (treated with different concentrations of unmodified A15-Mal-IMDQ-PLG, i.e., 10.0 mg / kg, 30.0 mg / kg, and 50.0 mg / kg as IMDQ), and three experimental groups (treated with different concentrations of AMINP, i.e., 10.0 mg / kg, 30.0 mg / kg, and 50.0 mg / kg as IMDQ). The drugs were administered via tail vein.

[0091] Intravenous injection of AMINP at doses ranging from 10 mg / kg to 50 mg / kg into mice did not cause a significant increase in cytokine concentrations in the mouse blood. Figure 3 (A-C) and no decrease in mouse weight was observed ( Figure 3 (D in the text). Conversely, both the small molecule active drug IMDQ and the unmodified A15-Mal-IMDQ-PLG caused a significant increase in cytokine concentrations in mouse blood and a significant decrease in body weight. These results demonstrate the strong potential of AMINP in avoiding the systemic immunotoxicity of IMDQ-like drugs.

[0092] Example 4: In vitro ultrasound reconstruction assessment of AMINP

[0093] The 10 μM AMINP aqueous solution (calculated as the molar amount of the active drug IMDQ) was divided into four groups, each treated with 2 W / cm² solution. 2 Ultrasounding was performed at 50% duty cycle for 0 min, 1 min, 2 min, and 5 min. The nanomedicine was hydrolyzed using sodium hydroxide, and the ultrasonic reduction effect of AMINP after ultrasounding was detected by high-performance liquid chromatography. The results are as follows: Figure 4 As shown in Figure A. After sonication, IMDQ-N3 on AMINP showed significant reduction, with the reduction product being IMDQ. Simultaneously, the in vitro bioactivity of AMINP after sonication reduction was assessed. Dendritic cells derived from bone marrow of 4-6 week old black mice were added to PBS, or 10 μg / mL of IMDQ or AMINP (calculated as the active drug IMDQ), treated under the specified conditions, and cultured for 24 h. The proportion of mature cells was determined by flow cytometry. The results are shown below. Figure 4 As shown in Figure B, AMINP cannot activate dendritic cells (DCs) without ultrasound. Under ultrasound conditions, AMINP can promote DC maturation. These results indicate that ultrasound can activate AMINP and enable it to function as a TLR7 / 8 agonist.

[0094] Example 5: Evaluation of the ability of ultrasound to destroy tumor blood vessels and the targeting of drugs after ultrasound-induced blood vessel destruction.

[0095] In a Balb / c tumor-bearing mouse model with transplanted CT26 tumors, focused ultrasound (2W / cm²) was used to examine the mouse tumors. 2 (50% duty cycle, 3 min). Fluorescent sections show ( Figure 5 Ultrasound can induce blood clots in tumors. Furthermore, a comparison of two drugs, MINP and AMINP, with and without a target was conducted. Figure 5 ) and in vitro coagulation test ( Figure 6 A, B), drug tissue distribution experiment ( Figure 6 C) indicates that the A15 coagulation-targeting peptide enhances coagulation and, through cross-linking, immobilizes itself in the blood clot, thereby enhancing drug accumulation at the clot site. Drug tissue distribution experiments demonstrate that, compared to small molecules and ineffective target nanoparticles, AMINP exhibits stronger active drug accumulation in tumors and demonstrates good targeting properties.

[0096] Example 6: Evaluation of the tumor-suppressing effect of AMINP on the CT26 tumor model in balb / c mice

[0097] Dosing regimen such as Figure 7 As shown in A, specifically on days 0, 2, and 4, the tumor volume of 100 mmHg was analyzed according to the group. 3 Tumor-bearing mice were injected with the appropriate dose and type of drug via the tail vein, and ultrasound was used as an option. The mice were then sacrificed on day 12, the tumors were isolated, and the tumor-suppressing effects and changes in body weight of the mice over 12 days were compared among the groups.

[0098] Compared to the ultrasound and non-ultrasound groups of PBS, the ultrasound and non-ultrasound groups of the ineffective target nanoprodrug, as well as the non-ultrasound group of AMINP, showed that the combination of AMINP and ultrasound significantly inhibited tumor growth and completely cured some mice (40%). Figure 7 (B in the text). Meanwhile, mice treated with AMINP did not experience weight loss, demonstrating the low toxicity and safety of AMINP. Mouse survival time testing showed (…). Figure 7 The combined use of AMINP and ultrasound significantly prolonged the survival of mice, with 40% of mice surviving for more than 60 days. These results demonstrate the high efficacy and safety of AMINP.

[0099] Example 7: Flow cytometry assessment of changes in the tumor immune microenvironment in mice after treatment

[0100] Mouse tumors isolated during the tumor suppression experiment were ground and isolated into single cells. Antibody staining was performed on the surface antigens of immune cells, and then flow cytometry was used to calculate and compare the proportion of positively stained cells.

[0101] The results showed that AMINP combined with ultrasound significantly increased the number of T cells in mouse tumors ( Figure 8 A in the middle), mature DC cells ( Figure 7 B) and M1 macrophages ( Figure 7 The content of C in the AMINP indicates that the tumor immune microenvironment in mice has been significantly improved, and AMINP combined with ultrasound therapy effectively activated the anti-tumor immune response in mice.

[0102] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An ultrasound-activated coagulation-targeting tumor-selective prodrug, characterized in that, It has the structure of Formula I: Wherein, R1 is selected from a substituted or unsubstituted C1-C10 straight-chain alkyl group, a substituted or unsubstituted C3-C10 branched-chain alkyl group or a substituted or unsubstituted C6-C12 aryl group; R2 is selected from a substituted or unsubstituted C1-C6 straight-chain alkylene group, a substituted or unsubstituted C3-C6 branched-chain alkylene group or a substituted or unsubstituted C6-C10 arylene group; R3 is selected from a substituted or unsubstituted C3-C5 straight-chain alkyl group or a substituted or unsubstituted C3-C5 ether group; a, b, c, d are the contents of repeating units, 0 < a < 1, 0 < b < 1, 0 < c < 1, 0 < d < 1; n is the degree of polymerization, 50 ≤ n ≤ 500.

2. The coagulation-targeting tumor-selective prodrug according to claim 1, characterized in that, The said R1 is selected from a substituted or unsubstituted C2-C8 straight-chain alkyl group, a substituted or unsubstituted C3-C8 branched-chain alkyl group or a substituted or unsubstituted C6-C10 aryl group; R2 is selected from a substituted or unsubstituted C1-C4 straight-chain alkylene group, a substituted or unsubstituted C3-C5 branched-chain alkylene group or a substituted or unsubstituted C6-C9 arylene group; R3 is selected from a substituted or unsubstituted C3-C4 straight-chain alkyl group or a substituted or unsubstituted C3-C4 ether group.

3. The coagulation-targeting tumor-selective prodrug according to claim 1, characterized in that, The said R1 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, n-heptyl, isoheptyl, n-octyl, phenyl, benzyl, naphthyl or biphenyl; The said R2 is selected from methylene, ethylene, propylene, butylene, phenylene or benzylidene; The said R3 is selected from n-propyl, n-butyl, n-pentyl, CH3CH2CH2O-, CH3CH2OCH2-, CH3OCH2CH2- or CH3CH2CH2OCH2- independently or in combination.

4. A method for preparing a coagulation-targeting tumor-selective prodrug as described in claim 1, characterized in that, It includes the following steps: S1: Condensation reaction is carried out on polyglutamic acid, the imidazoquinoline azide prodrug shown in Formula A and the maleimide shown in Formula B to obtain an intermediate product; 5. The preparation method according to claim 4, characterized in that, S2: The intermediate product is subjected to sedimentation treatment. After the product obtained by sedimentation is redissolved, it is reacted with the blood coagulation targeting peptide shown in Formula C and freeze-dried to obtain the product; The said condensation reaction is carried out in the presence of a solvent and a condensation reagent; The said solvent is selected from any one or more of dimethylformamide, benzene, toluene, carbon tetrachloride or tetrahydrofuran; The said condensation reagent is selected from any one or more of 2,4,6-trichlorobenzoyl chloride, N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; The temperature of the said condensation reaction is 25°C to 70°C, and the time is 2 to 4 days; 6. The preparation method according to claim 4 or 5, characterized in that, The temperature of the reaction in step S2 is 20°C to 40°C, and the time is 2 to 24 h. The mass ratio of the imidazoquinoline azide prodrug shown in Formula A, the maleimide shown in Formula B to polyglutamic acid is (0.1 - 0.3):(0.05 - 0.2):1; The molar ratio of the blood coagulation targeting peptide to polyglutamic acid is (0.5 - 5):

1.

7. An ultrasound-activated coagulation-targeting tumor-selective prodrug system, characterized in that, The method includes ultrasound conditions and the coagulation-targeting tumor-selective prodrug prepared according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 6.

8. The system according to claim 7, characterized in that, The energy of the ultrasound is 1.5–2.5 W / cm². 2 The duty cycle is 20-100%, and the ultrasound time is 2.5-5 minutes.

9. The use of the coagulation-targeting tumor-selective prodrug according to any one of claims 1 to 3 or the coagulation-targeting tumor-selective prodrug prepared by the preparation method according to any one of claims 4 to 6 in the preparation of a medicament for treating cancer.

10. The application according to claim 9, characterized in that, The cancers mentioned include any one or more of the following: breast cancer, colorectal cancer, liver cancer, melanoma, osteosarcoma, kidney cancer, prostate cancer, ovarian cancer, pancreatic cancer, bladder cancer, thyroid cancer, or nasopharyngeal cancer.

Images