Gambogic acid-manganese nanoparticles and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to gambogic acid-manganese nanoparticles and application thereof. The gambogic acid-manganese nanoparticles are mainly obtained by combining a complex of gambogic acid and divalent manganese ions with an oxidation-sensitive high-molecular polymer; a monomer for preparing the oxidation-sensitive high-molecular polymer comprises an ROS responsive structural unit with a thioketal group; monomers for preparing the oxidation-sensitive high-molecular polymer further comprise 1, 2, 4, 5-cyclohexane tetracarboxylic dianhydride and a water-soluble group.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medicines, in particular to gambogic acid-manganese nanoparticles and application thereof. BACKGROUND

[0002] As the core hub of cytosolic DNA sensing, the cGAS-STING signaling axis mediates innate immune responses by catalyzing the synthesis of the second messenger cGAMP, and plays a key role in monitoring abnormal cytosolic DNA (including pathogen-derived double-stranded DNA, mitochondria-damaged self DNA and tumor cell apoptosis-derived genomic DNA). This pathway triggers the TBK1-IRF3 phosphorylation cascade by activating the STING adaptor protein, and finally induces the expression of type I interferons and pro-inflammatory factors, which not only constitutes an important defense line against pathogen invasion, but also plays multiple biological functions in tumor immune surveillance, autoimmune disease occurrence and aging-related inflammation regulation. Its precise spatiotemporal activation mechanism has become a research hotspot for immune intervention strategies.

[0003] Gambogic acid (GBA) is a natural product of xanthone isolated from the resin of Garcinia hanburyi. Its unique cage-like polycyclic skeleton endows it with multiple biological activities. Studies have found that gambogic acid mainly induces cell apoptosis and inhibits angiogenesis through regulating cell signaling pathways to exert anti-tumor effects. Its molecular mechanism of action may include: 1) as a stabilizer of p53 protein, it activates the transcription of p53-dependent pro-apoptotic genes (such as Bax and PUMA) by inhibiting MDM2-mediated ubiquitination and degradation, triggering a mitochondrial-mediated apoptosis cascade; 2) it directly activates Caspase-3 zymogen and executes the apoptosis program by cleaving key substrates such as PARP-1; 3) it inhibits the PI3K / Akt signaling axis by competing for the ATP pocket, blocks the downstream mTOR-S6K1 survival pathway, and targets the ERK1 / 2 and p38 subfamilies in the MAPK signaling network, inhibiting the cell cycle progression (G1 / S phase arrest) and EMT phenotype conversion of tumor cells. Gambogic acid has shown significant anti-tumor immunotherapy potential due to its multi-target pharmacological activity. However, its inherent physicochemical defects (such as poor solubility caused by hydrophobic groups, metabolic instability and low bioavailability caused by lactone ring structure, etc.) have seriously hindered its clinical translation process.

[0004] Manganese (II) (Mn²⁺) as a life-essential transition metal ion, plays a catalytic cofactor role in a variety of metalloenzymes in the organism. Numerous studies have found that manganese ions (Mn²⁺) and their complexes show great potential in anti-tumor research, especially in activating the cGAS-STING pathway to enhance immunotherapy. Specific manganese complexes can interact with STING through their unique structure to act as STING agonists, activate the downstream IFN signaling pathway, and promote the apoptosis of tumor cells. SUMMARY

[0005] The present application encompasses the following technical solutions: One aspect of the present application relates to a gambogic acid-manganese nanoparticle, which is mainly obtained by combining a complex of gambogic acid and divalent manganese ions with an oxidation-sensitive high polymer; The monomers for preparing the oxidation-sensitive high polymer include ROS-responsive structural units with a thioalkylidene group; the monomers for preparing the oxidation-sensitive high polymer also contain 1,2,4,5-cyclohexanetetracarboxylic dianhydride and a water-soluble group.

[0006] Another aspect of the present application relates to the use of the gambogic acid-manganese nanoparticle as described above as a cGAS-STING pathway agonist.

[0007] Still another aspect of the present application relates to the use of the gambogic acid-manganese nanoparticle as described above in the preparation of a medicament for treating a cGAS-STING pathway disorder-related disease; the cGAS-STING pathway disorder-related disease is at least one of cancer, autoimmune disease, and viral infection.

[0008] Still another aspect of the present application relates to a preparation method of the gambogic acid-manganese nanoparticle as described above, comprising: a) preparing a solution by mixing gambogic acid and manganese ions at a molar ratio of 1:(3-10) to form a gambogic acid-manganese complex; b) mixing the obtained gambogic acid-manganese complex with the oxidation-sensitive high polymer to obtain an organic phase solution; c) uniformly dispersing the organic phase solution in water to obtain a mixed solution; d) performing dialysis treatment on the mixed solution, filtering, and obtaining the gambogic acid-manganese nanoparticle.

[0009] Still another aspect of the present application relates to a pharmaceutical composition containing the gambogic acid-manganese nanoparticle as described above and a pharmaceutically acceptable carrier.

[0010] The disclosure innovatively constructs a manganese (II) gallate nanodelivery system based on metal-organic coordination chemistry. The core is formed by the coordination self-assembly of Mn2+ and the carboxyl group of gallate. The surface is modified by an oxidation-sensitive amphiphilic polymer to construct a core-shell nanostructure with uniform particle size. The nanocarrier effectively loads hydrophobic drugs through the hydrophobic core, which not only enhances the water solubility and stability of gallate, but also improves its targeting, bioavailability and persistence by the characteristics of the nanocarrier. At the same time, it effectively activates the cGAS-STING pathway and enhances the immune response, which has significant anti-tumor and anti-viral potential. This new complex provides a new direction for the research of cGAS-STING pathway agonists and an innovative treatment plan for related immunotherapy applications. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0012] Figure 1 The synthesis diagram and nuclear magnetic resonance spectrum of the oxidation-sensitive polymer provided by one embodiment of the present disclosure.

[0013] Figure 2 The particle size distribution test results of GBA NP and GBA-Mn NP provided by one embodiment of the present disclosure.

[0014] Figure 3 The particle size stability test results of GBA NP and GBA-Mn NP provided by one embodiment of the present disclosure.

[0015] Figure 4 The coordination structure stability test results of GBA-Mn NP provided by one embodiment of the present disclosure.

[0016] Figure 5 The transmission electron microscopy (TEM) images of GBA NP and GBA-Mn NP provided by one embodiment of the present disclosure.

[0017] Figure 6 The in vitro simulated release results of GBA-Mn NP.

[0018] Figure 7 The results of the inhibition of the proliferation activity of GBA-Mn NP on mouse colon cancer cells MC38, human ovarian cancer cells SK-OV-3 and mouse breast cancer cells 4T1.

[0019] Figure 8 The inhibitory effects of GBA and GBA-Mn NP on mouse embryonic fibroblast 3T3 and mouse breast cancer cells 4T1 are shown in the figure.

[0020] Figure 9 Figure showing the effect of GBA-Mn NP on apoptosis in mouse breast cancer cells 4T1.

[0021] Figure 10 The results of detecting the activation effect of GBA-Mn NP on Caspase-3 expression are shown in the figure.

[0022] Figure 11 Figure showing the regulatory effects of GBA-Mn NP on the expression of Bid, Bcl-2, and Caspase-3.

[0023] Figure 12 The results of detecting the activation effect of GBA-Mn and GBA-Mn NP on Caspase-3 expression are shown in the figure.

[0024] Figure 13 Figure showing the results of detecting the effects of GBA-Mn NP on the expression of p-STING, p-IRF3, p-TBK1 and γ-H2AX proteins.

[0025] Figure 14 Uptake efficiency of GBA-Mn NP in mouse breast cancer cells 4T1. Detailed Implementation

[0026] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0027] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0028] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0029] The choice of the conjunctive range of the terms "and / or", "or / and", "and / or" used herein includes any one of the two or more related listed items, and also includes any and all combinations of the related listed items, including a combination of any two related listed items, a combination of any more related listed items, or a combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes a combination of any two or any three of A, B, C and D, and also includes a four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0030] The terms "containing", "including" and "comprising" used in the present application are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0031] The numerical ranges used in the present application expressed in endpoints include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0032] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It is also intended to cover a specific number, e.g. about 20 includes 20.

[0033] Further, in describing representative embodiments of the present application, the specification can have presented the method and / or process of the present application as a particular sequence of steps. However, to the extent that the method or process depends on the performance of such steps, the method or process should not be limited to the

[0034] In the present application, the concentration values are intended to include fluctuations within a certain range. For example, they can fluctuate within a corresponding accuracy range. For example, 2% can fluctuate within a range of ±0.1%. For values that are relatively large or do not need to be controlled too precisely, the values are also intended to include larger fluctuations. For example, 100 mM can fluctuate within a range of ±1%, ±2%, ±5%, etc. For molecular weights, the values are intended to include fluctuations of ±10%.

[0035] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0036] In the present application, the descriptions such as "a plurality of", "a plurality of kinds", etc. refer to a number greater than or equal to 2 unless otherwise specified.

[0037] In the present application, the technical features described in an open form include both closed technical solutions consisting of listed features and open technical solutions including listed features.

[0038] In the present application, "preferably", "more preferably", "even more preferably", "suitably" only describe embodiments or examples with better effects, and should be understood as not constituting a limitation on the protection scope of the present application. In the present application, "optionally", "optional", "may" refer to the presence or absence, i.e., refer to any one selected from the two parallel schemes of "yes" or "no". If multiple "optionally" appear in a technical solution, each "optionally" is independent of each other unless otherwise specified, and there is no contradictory relationship or mutual restriction.

[0039] In the present application, "GBA" represents a gambogic acid small molecule; GBA NP represents a nanoparticle obtained by combining GBA with an oxidation-sensitive high polymer; "GBA-Mn" represents a gambogic acid-manganese small molecule; and "GBA-Mn NP" represents a nanoparticle obtained by combining GBA-Mn with an oxidation-sensitive high polymer.

[0040] In the present application, "treatment" generally refers to a process or means for partial or complete remission, inhibition, improvement, delay, stabilization or alleviation of a disease or pathological condition suffered by a subject. The term not only covers the cure of a disease, but also includes the alleviation of symptoms, the delay of disease progression, the prevention of related complications, etc. For example, treatment can include administration, surgery, physical intervention, biological therapy or gene therapy, etc.

[0041] As used herein, "therapeutically effective" and "effective dose" refer to a substance or amount that causes a desired biological activity or effect.

[0042] The terms "subject" or "patient" are used interchangeably unless indicated, and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Thus, as used herein, the terms "subject" or "patient" mean any mammalian patient or subject to whom a gallocatechin-manganese nanoparticle of the present disclosure can be administered.

[0043] A first aspect of the present application relates to a gallocatechin-manganese nanoparticle, which is mainly obtained by combining a complex of gallocatechin and divalent manganese ion with an oxidation-sensitive high-molecular polymer; The monomer for preparing the oxidation-sensitive high-molecular polymer comprises a ROS-responsive structural unit with a thiolactone group; the monomer for preparing the oxidation-sensitive high-molecular polymer further comprises 1,2,4,5-cyclohexanetetracarboxylic dianhydride and a water-soluble group.

[0044] Traditional drug delivery systems mostly use nanocarriers such as liposomes and albumin to improve the water solubility and biocompatibility of chemotherapeutic drugs. However, such carriers generally lack the ability to respond to the tumor microenvironment, making it difficult to achieve precise tumor-targeted delivery and treatment, and easily causing systemic toxic side effects. The nanoparticle system designed in the present disclosure can respond to the active oxygen (ROS) enriched in the tumor microenvironment, achieve selective release of drugs, enhance the treatment effect on tumors, and significantly reduce the toxicity to normal tissues.

[0045] In addition, the present application unexpectedly found that GBA-Mn NPs have a strong activation effect on the Caspase-3 system compared to GBA NPs, while Mn 2+ However, Mn NPs have no such effect. Thus, they have a stronger killing effect on tumor cells.

[0046] As the ROS-responsive structural unit with a thiolactone group, one or more of TK-COOH, TK-NH2, Вос-TK-NH2, Fmoc-TK-NH2, Biotin-TK-NH2, FA-TK-NH2, FITC-TK-NH2, NH2-TK-NH2, COOH-ТK-COOH and OH-ТK-OH can be listed. In some embodiments, the ROS-responsive structural unit with a thiolactone group is OH-TK-OH 【2,2-(propane-2,2-diylbis sulfanediyl) bisethanol】.

[0047] In some embodiments, the water-soluble group comprises a methoxy PEG hydroxyl group. Its molecular weight is preferably 4000-6000, for example, 4000, 5000, 6000; and more preferably 5000, for example, mPEG5000-OH.

[0048] In some embodiments, the oxidation-sensitive high-molecular polymer is: ; wherein m = 50-150; n = 5-20.

[0049] m is the degree of polymerization, i.e. the average number of water-soluble groups contained in the polymer macromolecular chain, corresponding to the molecular weight of said water-soluble groups. In some embodiments, m is selected to have a value of 50-150, for example 60, 70, 80, 90, 100, 110, 120, 130, 140. For example, when the water-soluble group is a methoxy PEG hydroxyl group and has a molecular weight of 5000, m is specifically 112.

[0050] In some embodiments, n is selected to have a value of 5-20, for example 8, 12, 16, 20.

[0051] In some embodiments, the particle size thereof is 50 nm-300 nm, preferably 80 nm-120 nm.

[0052] In some embodiments, the mass ratio of said gallic acid-manganese complex to said oxidation-sensitive high polymer is 1:(5-20), for example 1:10, 1:15, 1:20.

[0053] The second aspect of the present application relates to the use of the gallic acid-manganese nanoparticle as described above as a cGAS-STING pathway agonist.

[0054] The third aspect of the present application relates to the use of the gallic acid-manganese nanoparticle as described above in the preparation of a medicament for treating a disease related to the disorder of the cGAS-STING pathway.

[0055] Because of the key role of cGAS-STING pathway in host immune response, the pharmacological regulation of its activity has been widely recognized as a potential broad-spectrum immunotherapy strategy for treating pathogen infection and tumors. Studies have found that STING signal activation helps to promote dendritic cell maturation and antigen cross-presentation, enhance CD8+ T cell priming and function (Diamond et al., 2011; Woo et al., 2014). In addition, STING not only plays a role in immune cells, but also can induce the expression of adhesion molecules (such as VCAM-1, ICAM-1, etc.) in vascular endothelial cells, improve tumor vascular permeability, promote T cell infiltration and vascular normalization, and enhance the ability of immune cells to enter the tumor microenvironment (Campisi et al., 2020; Yang et al., 2019). STING agonists (such as ADU-S100) can also induce the formation of tumor-localized tertiary lymphoid structures (TLS), support T cell activation, B cell affinity maturation and antibody production, thereby maintaining and enhancing anti-tumor immune responses (Chelvanambi et al., 2021; Martinet et al. 2012). Low-dose STING agonists combined with PD-1 antibodies show synergistic anti-tumor effects in melanoma, breast cancer and other models (Sivick et al., 2018). It is worth noting that excessive activation of STING can also induce autoimmune diseases, such as SAVI syndrome, which is related to acquired functional mutations of the STING gene (such as R284S, G166E), and is characterized by persistent IFN signal activation and inflammatory response (Liu et al., 2014; Clarke et al., 2016; Konno et al., 2018; König et al., 2017). In addition, even in tumors with loss of function of STING signaling, cGAMP derived from cancer cells can activate STING+ immune cells in the tumor microenvironment through paracrine, inducing anti-tumor responses (Campisi et al., 2020). These studies demonstrate that the pharmacological activation of STING is a promising immunotherapy method for treating viral infections and cancer. Therefore, according to the knowledge of those skilled in the art, in some embodiments, the disease related to the dysregulation of the cGAS-STING pathway is at least one of cancer, autoimmune disease, and viral infection.

[0056] In preferred embodiments, the cancer is a solid tumor. In some specific embodiments, the cancer is breast cancer.

[0057] The preparation method of the gambogic acid-manganese nanoparticles can be obtained by any means. As a preferred exemplary scheme, the fourth aspect of the present application relates to a preparation method of the gambogic acid-manganese nanoparticles as described above, comprising: a) preparing a solution of gambogic acid and manganese ions in a molar ratio of 1:(3-10), and mixing to form a gambogic acid-manganese complex; b) mixing the gambogic acid-manganese complex obtained with the oxidation-sensitive high-molecular polymer to obtain an organic phase solution; c) uniformly dispersing the organic phase solution in water to obtain a mixed solution; d) dialyzing the mixed solution, filtering, and obtaining gambogic acid-manganese nanoparticles.

[0058] In step a), the manganese ions are in excess relative to the gambogic acid, and more preferably, the molar ratio of the gambogic acid in the gambogic acid solution to the manganese ions in the manganese ion solution is 1:(3-10).

[0059] In step a), the preparation method of mixing the gambogic acid solution and the manganese ion solution is commonly used in the art. For example, the solvent used for the gambogic acid solution is an organic solvent; the organic solvent used can include, but is not limited to, dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform, and other organic solvents that can dissolve gambogic acid. For example, the manganese ion solution includes water, methanol, ethanol, dimethyl sulfoxide, and other polar solvents that can dissolve manganese-containing salts. The dissolution process can be carried out at room temperature, and the dissolution can be accelerated by magnetic stirring, vortexing, ultrasonication, or the like.

[0060] An exemplary method of step a) is: dissolving gambogic acid and manganese ions in an organic solvent in a molar ratio of 1:(3-10), and fully mixing and stirring for 8 hours.

[0061] The mixing or dispersing method used in the present disclosure can be selected from stirring, vortexing, ultrasonication, or a combination thereof. The solid-liquid separation method used in the present disclosure can be selected from evaporation, distillation, centrifugation, filtration, or a combination thereof. The centrifugation speed can be 3000 r / min-4000 r / min; the filter pore size for filtration can be 0.22 μm-0.45 μm.

[0062] In step b), the mass ratio of the gambogic acid-manganese complex to the oxidation-sensitive high-molecular polymer is 1:(5-20).

[0063] In step c), the water can be selected from distilled water, deionized water, and reverse osmosis water.

[0064] In step d), the water used for dialysis can be selected from distilled water, deionized water, and reverse osmosis water.

[0065] In some embodiments, in step c), ultrasonication and stirring are used for uniform dispersion.

[0066] A fifth aspect of the present disclosure relates to a pharmaceutical composition containing the gambogic acid-manganese nanoparticles as described above and a pharmaceutically acceptable carrier.

[0067] The term "pharmaceutically acceptable" as used herein defines a compound, material, composition, or agent that is, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio. The term can be used interchangeably with "pharmacologically acceptable."

[0068] The term "pharmaceutically acceptable carrier" as used herein defines a pharmacologically acceptable material, composition or vehicle, such as a liquid, solid filler, diluent, excipient, solution, or packaging material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and not injurious to the patient. The following materials can be used as pharmaceutically acceptable carriers: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as methyl cellulose and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, safflower oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol (mannitol), mannitol; (12) esters, such as ethyl oleate and ethyl laureate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions and (21) other nontoxic compatible substances used in pharmaceutical formulations.

[0069] The pharmaceutically acceptable carrier can include physiologically acceptable agents to increase stability, increase solubility, or increase the ability of the nanoparticles described in the present disclosure to be absorbed. The selection of the pharmaceutically acceptable carrier, including the physiologically acceptable agents, depends on the mode of administration of the agents. The preparation of the formulation or the combination of agents can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The combination of agents (formulation) can also be a liposome or other polymeric matrix, and the nanoparticles or conjugates are incorporated therein. For example, a liposome or other lipid composition is a non-toxic, biocompatible, metabolizable carrier, and is easy to prepare and use.

[0070] The pharmaceutical compositions can be administered by any of a variety of routes of administration including oral administration (e.g., dissolved in water or non- water solutions or suspensions, tablets, capsules, including sprinkle capsules and gel capsules, pills, powders, granules, pastes) and parenteral administration. As used herein, "parenteral administration" refers to modes of administration other than enteral and topical administration, and typically involves injection, including, but not limited to, intratumoral, intravenous, intramuscular, intercostal, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinous, intrasternal, intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include nanosized particles in sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, sterile powders which can be reconstituted into sterile injectable solutions or dispersions. The pharmaceutical compositions can include sterilizing agents, buffers, bacteriostatic agents, solutes which render the compositions isotonic with the blood of the intended recipient, suspending agents, thickening agents, or

[0071] Embodiments of the present application will be described in detail with reference to the following Examples. It is to be understood that these Examples are intended to be illustrative only and the scope of the present application is not intended to be limited to the particular Examples presented. In the following Examples, unless otherwise indicated, the experimental procedures are performed according to the guidelines set forth in the present application, according to standard procedures in the art, or according to other procedures known in the art or according to the manufacturer's instructions.

[0072] In the following specific examples, the amount of the raw material components are measured with a certain degree of precision, which can vary slightly unless otherwise specified. The temperature and time parameters are allowed to vary within acceptable limits due to instrument testing or operational precision.

[0073] Example 1 Preparation of GBA-Mn NPs (1) Gambogic acid (GBA) is added to a reactor and dissolved in an organic solvent to obtain solution A; ((Z)-4-((1S,3aR,5S,11R,14aS)-8-Hydroxy-2,2,11-trimethyl-13-(3-methylbut-2-en-1-yl)-11-(4-methylpent-3-en-1-yl)-4,7-dioxo-1,2,5,7-tetrahydro-11H-1,5-methanofuro[3,2-g]pyrano[3,2-b]xanthen-3a(4H)-yl)-2-methylbut-2-enoic acid) is added to a reactor and dissolved in an organic solvent to obtain solution A; The organic solvent used includes, but is not limited to, dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform, etc.

[0074] The dissolving process is carried out at room temperature, and the dissolving can be accelerated by magnetic stirring, vortex, ultrasonic, etc.

[0075] (2) Dissolve the manganese salt in the solvent to obtain solution B; The manganese-containing salt used is acetate of divalent manganese and its crystal hydrate.

[0076] The solvent used includes distilled water, methanol, ethanol, dimethyl sulfoxide, etc. polar solvents capable of dissolving the manganese-containing salt.

[0077] The dissolving process is carried out at room temperature, and the dissolving can be accelerated by magnetic stirring, vortex, ultrasonic, etc.

[0078] (3) Slowly drop solution B into solution A, and react for 8 hours; The molar ratio of manganese ions in solution A and solution B to GBA is 3:1-10:1, so that the metal ions are in excess.

[0079] The dropping process is carried out at room temperature, and the process can be accelerated by magnetic stirring, vortex, ultrasonic, etc.

[0080] (4) Oxidation-sensitive polymer synthesis method: weigh monomer A (0.2465 g, 1.1 mmol) and monomer B (0.196 g, 1 mmol) and dissolve them in 1 mL of super-dry DMF, and react at 50°C for 12 h. Then add mPEG5000-OH (0.5 g, 0.1 mmol) to the reaction system, and react at 50°C for 12 h. Add the reacted liquid to a dialysis bag with a molecular weight cut-off of 8000, and dialyze for 48 h. Freeze-dry to obtain the product. The 1H NMR (400 MHZ, deuterated DMSO) spectrum and synthesis path are shown in Figure 1 .

[0081] (5) Add the oxidation-sensitive polymer to the organic solvent of the GBA-Mn complex to obtain an organic phase solution; slowly drop the organic phase solution into 10 times the volume of deionized water solution at room temperature; then transfer the liquid to a dialysis bag with a MWCO of 3500, dialyze in deionized water for 3 days, and change the water every 6 h; then centrifuge (3500 r / min) and filter (0.22 μm) to obtain GBA-Mn NP nanoparticles.

[0082] The oxidation-sensitive polymer used is NPs.

[0083] The mass ratio of the GBA-Mn complex to NPs is 1:(5-20).

[0084] The volume of the organic solvent used is 2%-10% of the deionized water solution, preferably 4%-6%.

[0085] To make the particle size meet the preparation requirements, the time of adding the organic phase solution into the deionized water solution is controlled to be one drop per second, and the rotation speed is controlled to be 300-600 rpm.

[0086] Example 2 Material characterization of GBA-Mn NPs The above-prepared GBA NPs and GBA-Mn NPs were diluted 5 times with deionized water, respectively, and the particle size was measured by dynamic light scattering instrument, as shown in Figure 2 The average particle size of GBA NPs was 107.27 nm, and the average particle size of GBA-Mn NPs was 119.07 nm. The change of particle size was continuously monitored within 5 days, as shown in Figure 3 The particle sizes of GBA NPs and GBA-Mn NPs remained basically unchanged during this period, indicating that both GBA NPs and GBA-Mn NPs had good dispersion stability. After diluting GBA-Mn NPs 4 times with deionized water, the spectral changes within 5 days were measured by UV-Vis absorption spectrum, as shown in Figure 4 The position and intensity of the absorption peak did not change significantly within 5 days, indicating that the coordination structure had good stability. The morphology and structure of GBA NPs and GBA-Mn NPs were observed by field emission transmission electron microscopy, as shown in Figure 5 Both of them showed uniform distribution and clear spherical structure.

[0087] Example 3 Drug release of GBA-Mn NPs in simulated oxidative environment of hydrogen peroxide The release data of GBA-Mn NPs in simulated normal environment and 10 mM hydrogen peroxide simulated oxidative stress environment were tested by dialysis method, wherein the molecular weight of the dialysis bag was 10 kDa, the liquid volume ratio of the inside and outside of the liquid was 1:100, the temperature was set to 37℃, 2% tween 80 was added to the outside liquid to achieve the leakage condition, and sampling was performed at 0, 1, 2, 4, 8, 12, 24, 48 h to calculate the release ratio.

[0088] The results are shown in Figure 6 Within 24 hours, the cumulative release amount under normal conditions was only 14%, while the release amount under oxidative stress conditions increased significantly to 55%. This indicates that GBA-Mn NPs have good oxidative stress response characteristics and can achieve rapid drug release in high ROS environment, which helps to improve its therapeutic selectivity and efficiency in the tumor microenvironment.

[0089] Example 4 Inhibition of cancer cell proliferation by GBA-Mn NPs The CCK-8 method was used to evaluate the inhibitory effect of GBA-Mn NPs on the proliferation of cancer cells. Mouse colon cancer cells MC38, human ovarian cancer cells SK-OV-3, and mouse breast cancer cells 4T1 were seeded in a 96-well plate at a density of 8000 cells per well. SK-OV-3 and 4T1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin 100x (P / S), and MC38 cells were cultured in DMEM high-glucose medium containing 10% FBS and 1% P / S. The cells were incubated at 37°C and 5% CO2 for 12 hours. Diluted GBA, GBA NPs, GBA-Mn, and GBA-Mn NPs were added to the different cell culture systems. The final concentrations of the drugs in the SK-OV-3 culture system were 4, 2, 1, 0.5, 0.25, 0.125, 0.0125, and 0.00125 µM, in the MC38 culture system were 1.5, 0.75, 0.375, 0.1875, 0.09375, and 0.009375 µM, and in the 4T1 culture system were 1.5, 0.75, 0.375, 0.1875, 0.09375, and 0.009375 µM. All cells were incubated at 37°C and 5% CO2 for 24 hours. 10% CCK-8 solution was added to each well, and the incubation was continued under the same conditions for 4 hours. The absorbance value of each well was measured at 450 nm using a microplate reader. Cell activity was represented by the absorbance ratio of the experimental group to the control group.

[0090] The results of CCK-8 are shown in Figure 7 The results show that the half-maximal inhibitory concentration (IC 50 ) of GBA, GBA NPs, GBA-Mn, and GBA-Mn NPs on SK-OV-3 were 0.6223, 0.4632, 0.3762, and 0.2539 µM, respectively; the IC 50 of MC38 were 0.3575, 0.3402, 0.3084, and 0.2774 µM, respectively; and the IC 50 of 4T1 were 0.3197, 0.2644, 0.2798, and 0.2512 µM, respectively. The toxicity of GBA-Mn NPs on cells was slightly higher than that of the other three groups, indicating that the modification of Mn may enhance the anti-tumor efficacy of GBA NPs and improve the toxic effect on cancer cells, and the inhibitory effect is concentration-dependent.

[0091] Example 5 Inhibitory effect of GBA-Mn NPs on mouse embryonic fibroblast 3T3 and mouse breast cancer cell 4T1 The inhibitory effect of GBA-Mn NPs on mouse embryonic fibroblast 3T3 and mouse breast cancer cell 4T1 was evaluated according to the method of Example 4. The results are shown in Table 1. Figure 8 It was shown that the IC50 of free GBA on 3T3 and 4T1 cells were 0.6928 µM and 0.6371 µM, respectively, with no obvious selectivity in toxicity; while the IC50 of GBA-Mn NPs on 3T3 and 4T1 were 4.318 µM and 0.2830 µM, respectively, showing the characteristics of significantly reduced toxicity on normal cells and significantly enhanced toxicity on tumor cells. This result indicates that after being encapsulated by oxidation-sensitive polymer, GBA-Mn NPs can be rapidly degraded and release active drugs in the high ROS environment of tumor cells, while remaining stable in normal cells, thereby effectively reducing side effects and improving tumor targeting, and showing good selective anti-tumor potential. 50 It was shown that the IC50 of free GBA on 3T3 and 4T1 cells were 0.6928 µM and 0.6371 µM, respectively, with no obvious selectivity in toxicity; while the IC50 of GBA-Mn NPs on 3T3 and 4T1 were 4.318 µM and 0.2830 µM, respectively, showing the characteristics of significantly reduced toxicity on normal cells and significantly enhanced toxicity on tumor cells. This result indicates that after being encapsulated by oxidation-sensitive polymer, GBA-Mn NPs can be rapidly degraded and release active drugs in the high ROS environment of tumor cells, while remaining stable in normal cells, thereby effectively reducing side effects and improving tumor targeting, and showing good selective anti-tumor potential.

[0092] Example 6 Apoptosis of mouse breast cancer cell 4T1 induced by GBA-Mn NPs (1) Annexin V-FITC / PI double staining method for evaluating the effect of GBA-Mn NPs on apoptosis of mouse breast cancer 4T1 cells Flow cytometry combined with Annexin V-FITC and PI double staining method was used to evaluate the induction of GBA-Mn NPs on apoptosis of 4T1 mouse breast cancer cells. Mouse breast cancer cells 4T1 were seeded in a 6-well plate at a density of 0.8 million cells per well and cultured at 37°C, 5% CO2 for 12 hours. 2 mL of RPMI 1640 complete medium containing 1 µM manganese acetate, 0.6 µM GBA NPs, 0.6 µM GBA-Mn NPs and no nanoparticles, respectively, were added to each well, and the culture was continued at 37°C, 5% CO2 for 24 hours. The cell culture solution was aspirated into a 1.5 mL EP tube, and the cells were washed with PBS buffer, and an appropriate amount of trypsin digestion solution was used to digest the cells at 37°C for 5 minutes, and the digestion reaction was terminated using cell culture solution. The cell suspension was transferred to a 1.5 mL EP tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS buffer. 195 µL of Annexin V-FITC conjugate, 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) staining solution were added and mixed gently, and incubated at room temperature for 30 minutes in the dark. After incubation, the supernatant was discarded by centrifugation, and the cells were washed twice with PBS buffer. Finally, the cells were resuspended with PBS buffer and subjected to flow cytometry detection. Through Annexin V and PI double staining, the early apoptosis, late apoptosis and necrosis of the cells can be quantitatively analyzed.

[0093] The results are shown in Table 1. Figure 9As shown, the negative control group and 1 µM Mn(OAc)2treatment had little effect on the growth of 4T1 cells; while the cells treated with 0.6 µM GBA NPs and 0.6 µM GBA-Mn NPs showed obvious apoptosis, and the degree of apoptosis in the GBA-Mn NPs group was more significant. This indicates that GBA-Mn NPs can effectively induce the apoptosis of mouse breast cancer cells 4T1, and its effect is strong in concentration-dependent manner.

[0094] (2) Flow cytometry detection of the effect of GBA-Mn NPs on Caspase-3 activity in mouse breast cancer cells 4T1 Flow cytometry was used to detect the effect of GBA-Mn NPs on Caspase-3 activity in mouse breast cancer cells 4T1. Mouse breast cancer cells 4T1 were treated according to the method in 1. The cells treated with GreenNuc™ Caspase-3 Substrate specific fluorescent labeling reagent were stained, and the activation of Caspase-3 was detected by flow cytometry. Flow cytometry analysis of the proportion of Caspase-3 positive cells and the change of fluorescence intensity quantitatively evaluated the degree of activation of Caspase-3 activity by GBA-Mn NPs.

[0095] The experimental results are shown in Figure 10 As shown, the Caspase-3 activity in the negative control group and the 1 µM Mn(OAc)2treatment group did not change significantly, and the activation of Caspase-3 in the cells was low, indicating that these two groups of treatment had weak effect on the apoptosis of mouse breast cancer cells 4T1. In contrast, both the 0.6 µM GBA NP treatment group and the 0.6 µM GBA-Mn NP treatment group significantly increased the activity of Caspase-3. In particular, the GBA-Mn NP group, the proportion of Caspase-3 positive cells increased significantly, and the fluorescence intensity also increased significantly, indicating that GBA-Mn NPs can strongly activate Caspase-3 in mouse breast cancer cells 4T1. This result shows that GBA-Mn NPs effectively induce the apoptosis of breast cancer cells by activating the Caspase-3 pathway, suggesting that it has potential anti-cancer effect, further supporting its potential as a therapeutic strategy for cancer immunotherapy.

[0096] (3) Western Blot detection of the effect of GBA-Mn NPs on the expression of apoptosis-related proteins Bcl-2 / Bid / Caspase-3 in mouse breast cancer cells 4T1 The effect of GBA-Mn NPs on the expression of apoptosis-related proteins Bcl-2 / Bid / Caspase-3 in mouse breast cancer cells 4T1 was analyzed by Western Blot (WB). Mouse breast cancer cells 4T1 were seeded in 6 cm cell culture dishes at a density of 1.5 million cells per well and cultured at 37°C, 5% CO2 for 12 hours. 5 mL of RPMI 1640 complete medium containing final concentrations of 1 µM manganese acetate, 0.6 µM GBA NPs and 0.6 µM GBA-Mn NPs, and no nanoparticles were added to each culture dish, and the culture was continued at 37°C, 5% CO2 for 24 hours. Cell total protein was extracted using RIPA lysis buffer (RIPA lysis buffer: PMSF: phosphatase inhibitor A liquid + B liquid = 100:1:1). The protein concentration was determined by BCA method, and 20 µg of protein sample was used for SDS-PAGE electrophoresis to separate the protein, and the separated protein was transferred to a 0.45 µm PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours to prevent non-specific binding, and the membrane was washed with TBS-T three times for 5 minutes each time. The specific primary antibody against Caspase-3, Bcl-2, Bid was incubated at 4°C overnight. After washing the membrane with TBS-T, the HRP-labeled secondary antibody was incubated at room temperature for 2 hours, and then developed by ECL method and detected by chemiluminescence imaging system.

[0097] The results are shown in Figure 11 The expression levels of Cleaved-caspase-3 and Bid proteins were significantly up-regulated, and the expression level of Bcl-2 protein was significantly down-regulated in the GBA-Mn NP treatment group. This result indicates that GBA-Mn NPs can effectively promote the apoptosis of 4T1 cells by activating the apoptosis signaling pathway, further supporting the potential application value of GBA-Mn NPs in breast cancer treatment.

[0098] Example 7 Effect of GBA-Mn, GBA-Mn NPs on Caspase-3 activity of mouse breast cancer cells 4T1 According to the method in Example 6 (2), the effect of GBA-Mn, GBA-Mn NPs on Caspase-3 activity of mouse breast cancer cells 4T1 was evaluated.

[0099] The results are shown in Figure 12 The proportion of Caspase-3 positive cells in the GBA-Mn NP group was significantly increased, indicating that after being wrapped with an oxidation-sensitive polymer, the delivery efficiency of GBA-Mn in tumor cells and the ability to activate the Caspase-3-mediated apoptosis pathway were significantly enhanced. The above results indicate that GBA-Mn NPs have stronger ability to induce apoptosis of 4T1 cells, which helps to further improve the anti-tumor efficacy.

[0100] Example 8 GBA-Mn NP activates cGAS-STING signaling pathway The expression of cGAS-STING signaling pathway related proteins and the expression of DNA damage marker γ-H2AX in mouse breast cancer cells 4T1 were detected by WB method, aiming to further explore the mechanism of GBA-Mn NP in immune regulation. By analyzing the phosphorylation status of STING, TBK1, IRF3 and other key proteins, as well as the expression changes of DNA damage marker γ-H2AX, the role of GBA-Mn NP in activating immune response and promoting DNA damage repair response can be evaluated.

[0101] The results are shown in Figure 13 GBA-Mn NP significantly up-regulated the expression of STING protein phosphorylation (P-STING), TBK1 protein phosphorylation (P-TBK1), and IRF3 protein phosphorylation (P-IRF3) compared with the other three groups. These results indicate that GBA-Mn NP activates the cGAS-STING signaling pathway, induces downstream immune response and inflammatory response, activates TBK1 and IRF3 phosphorylation, and promotes anti-tumor immune response. At the same time, the up-regulation of γ-H2AX further indicates that GBA-Mn NP enhances the immune surveillance of tumor cells by inducing DNA damage, and promotes the elimination of tumor cells. In summary, the mechanism of GBA-Mn NP in tumor immunotherapy involves the activation of cGAS-STING signaling pathway and the enhancement of DNA damage response, further revealing its potential immune regulation function, suggesting its potential as an anti-tumor immunotherapy.

[0102] Example 9 Endocytosis efficiency of GBA-Mn NP in mouse breast cancer cells 4T1 Flow cytometry was used to evaluate the distribution and endocytosis efficiency of GBA-Mn NP in cells. Mouse breast cancer cells 4T1 were seeded in a 6-well plate at a density of 800,000 cells per well and cultured for 12 hours. Fluorescent dye Cy5-labeled GBA-Mn NP was co-cultured with mouse breast cancer cells 4T1. Cell samples were collected at 1 hour, 4 hours and 7 hours, respectively. The fluorescence signal intensity of Cy5 in 4T1 cells was detected by flow cytometry.

[0103] The experimental results are shown in Figure 14 As the incubation time increased, the intracellular fluorescence intensity gradually increased, indicating that the endocytosis process of GBA-Mn NP was time-dependent, and reached a high fluorescence intensity at 7 hours, further confirming that the nanoparticles could effectively enter the cells and accumulate over time.

[0104] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but should not be understood as a limitation on the patent scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A gambogic acid-manganese nanoparticle, which is obtained by combining a complex of gambogic acid and divalent manganese ion with an oxidation-sensitive high polymer; The monomer for preparing the oxidation-sensitive high polymer comprises a ROS-responsive structural unit with a thiolactone group; and the monomer for preparing the oxidation-sensitive high polymer further comprises 1,2,4,5-cyclohexanetetracarboxylic dianhydride and a water-soluble group.

2. The gambogic acid-manganese nanoparticle according to claim 1, wherein the ROS-responsive structural unit with a thiolactone group is OH-TK-OH.

3. The gambogic acid-manganese nanoparticle according to claim 1, wherein the water-soluble group comprises a methoxy PEG hydroxyl group.

4. The gallic acid-manganese nanoparticle of claim 1, wherein the oxidation-sensitive high polymer is: ; wherein m = 50-150; n = 5-20.

5. The gambogic acid-manganese nanoparticle according to any one of claims 1 to 4, which has a particle size of 50 nm to 300 nm, preferably 80 nm to 120 nm.

6. Use of the gambogic acid-manganese nanoparticle according to any one of claims 1 to 5 as a cGAS-STING pathway agonist.

7. Use of the gambogic acid-manganese nanoparticle according to any one of claims 1 to 5 in the preparation of a medicament for treating a disease related to cGAS-STING pathway disorder; the disease related to cGAS-STING pathway disorder is at least one of cancer, autoimmune disease and viral infection.

8. The use according to claim 7, wherein the cancer is a solid tumor; preferably breast cancer.

9. A preparation method of the gambogic acid-manganese nanoparticle according to any one of claims 1 to 5, comprising: a) preparing a solution of gambogic acid and manganese ions at a molar ratio of 1:(3-10), and forming a gambogic acid-manganese complex after mixing; b) mixing the obtained gambogic acid-manganese complex with the oxidation-sensitive high polymer to obtain an organic phase solution; c) uniformly dispersing the organic phase solution in water to obtain a mixed solution; d) performing dialysis treatment on the mixed solution, filtering, and obtaining the gambogic acid-manganese nanoparticle.

10. A pharmaceutical composition comprising the gambogic acid-manganese nanoparticle according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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