Nanometer preparation HBMn-FA, and preparation method and application thereof

Abstract

This invention discloses a nano-formulation HBMn-FA, its preparation method, and its application. The method includes: dissolving and dispersing a heme chloride alkaline solution in a PLGA / PLGA-PEG / DCM solution and sonicating it to obtain a mixed emulsion A; dissolving and dispersing an aqueous MnCl2 solution and an aqueous BSO solution in a PLGA / PLGA-PEG / DCM solution and sonicating them to obtain a mixed emulsion B; mixing the mixed emulsion A and emulsion B and sonicating them, then adding an aqueous serum albumin (BSA) solution and sonicating, stirring, centrifuging to collect microparticles, redissolving them in deionized water, adding polyethyleneimine and stirring, then adding DSPE-PEG-FA dropwise and stirring, centrifuging, and washing to obtain the nano-formulation HBMn-FA. This formulation can effectively exert ferroptosis, generate toxic ROS, trigger mitochondrial DNA release, and achieve endogenous signal specific activation of the STING pathway to exert anti-tumor therapeutic effects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing a nano-formulation HBMn-FA. Background Technology

[0002] Cellular DNA has long been considered a potential signaling molecule by the host's innate immune system, but the mechanisms by which the immune system recognizes these danger signals remain unclear. Studies have shown that the DNA sensor ring GMP-AMP synthase (cGAS) acts as a bridge between the host's sensing of cytoplasmic DNA and immune defense. Interferon gene stimulating factor (STING), a key linker molecule located downstream of the DNA sensing pathway, is crucial for the sustained induction of anti-tumor immunity through stimulation of type I interferon and other cytokines. However, non-specific activation of STING can induce widespread inflammatory responses, which is detrimental to its clinical application. STING agonists, such as cGAMP and cyclic dinucleotides (CDNs), have shown promising efficacy in enhancing immune stimulation and anti-tumor immune responses; however, their large molecular weight and strong polarity make them difficult to penetrate cell membranes, leading to insufficient cellular uptake. Furthermore, their phosphodiester bonds are easily hydrolyzed by enzymes, resulting in metabolic instability and off-target toxicity, which limits their further clinical application. Encouragingly, the cGAS-STING pathway can sense not only exogenous danger signals from viruses and bacteria, but also endogenous signals (such as dsDNA) from damaged mitochondria and debris from dead tumor cells. Unlike nuclear DNA, mitochondrial DNA (mtDNA), lacking histone protection, is more susceptible to damage from disruptive molecules (such as reactive oxygen species, ROS), promoting its release. Furthermore, to enhance cGAS's sensitivity to DNA, promoting the subsequent synthesis of the second messenger cGAMP and increasing the affinity of cGAMP for STING are also key to specifically activating the cGAS-STING pathway. Currently, numerous studies report Mn 2+ It can enhance the sensitivity of cGAS to dsDNA and increase the affinity of cGAMP for STING, thereby amplifying the activation of the STING pathway. At the same time, ferroptosis is a unique mode of tumor cell death induced by lipid peroxidation, which provides a large amount of intracellular ROS that causes mitochondrial DNA damage and release, as well as the release of cytoplasmic dsDNA from dead cell debris, thereby bidirectionally activating the cGAS-STING pathway.

[0003] Given the clinical limitations of exogenous STING agonists, precisely targeting the release of endogenous signaling DNA to specifically activate the STING pathway is crucial. Therefore, it is necessary to develop a nanoformulation capable of effectively triggering ferroptosis and releasing endogenous signaling DNA to activate the STING pathway for highly efficient tumor therapy. Summary of the Invention

[0004] The purpose of this invention is to provide a nano-formulation HBMn-FA, its preparation method, and its application. This nano-therapeutic formulation can effectively exert ferroptosis, generate toxic ROS, trigger mitochondrial DNA release, and achieve endogenous signal specific activation of the STING pathway to exert anti-tumor therapeutic effects.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a method for preparing the nano-formulation HBMn-FA is provided, the method comprising:

[0007] Heme chloride was dissolved in an aqueous solution of ammonium carbonate and sonicated to obtain an alkaline solution of heme chloride.

[0008] PLGA and PLGA-PEG were dissolved together in dichloromethane to obtain a PLGA / PLGA-PEG / DCM solution.

[0009] The alkaline solution of heme chloride was dissolved and dispersed in the PLGA / PLGA-PEG / DCM solution and sonicated to obtain mixed emulsion A;

[0010] MnCl2 aqueous solution and BSO aqueous solution were dissolved and dispersed together in the PLGA / PLGA-PEG / DCM solution and sonicated to obtain mixed emulsion B;

[0011] The mixed emulsion A and the mixed emulsion B are mixed and then sonicated to obtain a mixed solution C;

[0012] The mixed solution C was added to an aqueous solution of serum albumin BSA and sonicated. Then it was added dropwise to a deionized aqueous solution and stirred. After the organic solvent had completely evaporated, it was centrifuged and the microparticles were collected.

[0013] The microparticles were redissolved in deionized water, followed by the addition of polyethyleneimine and stirring. Then, DSPE-PEG-FA was added dropwise and stirred. After centrifugation and washing, the nano-formulation HBMn-FA was obtained.

[0014] Furthermore, the concentration range of the alkaline heme chloride solution is 4–8 mg / mL, and the concentration of the ammonium carbonate aqueous solution is 0.312–0.625 M.

[0015] Furthermore, the ratio of the mass of PLGA, the mass of PLGA-PEG, to the volume of dichloromethane is (133-399) mg : (50-100) mg : (25-35) mL.

[0016] Further, the volume ratio of the heme chloride alkaline solution to the PLGA / PLGA-PEG / DCM solution is (125-175):(15-24).

[0017] Further, the volume ratio of the MnCl2, the BSO and the PLGA / PLGA-PEG / DCM is (0.2-0.4):(0.2-0.4):(2.4-3.6).

[0018] Further, the volume ratio of the mixed emulsion A to the mixed emulsion B is (0.5-1.0):(2-4).

[0019] Furthermore, the nanoparticles are redissolved in deionized water to achieve a concentration of 1–5 mg / mL.

[0020] Furthermore, the mass ratio of the polyethyleneimine to the DSPE-PEG-FA is (1-3):(2-10).

[0021] In a second aspect of the invention, a nano-formulation HBMn-FA prepared using the method is provided.

[0022] In a third aspect of the invention, the use of the aforementioned nanoformulation HBMn-FA in the preparation of a medicament for antitumor immunotherapy is provided.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] This invention provides a nano-formulation HBMn-FA and its preparation method, which utilizes a biocompatible PLGA polymer to simultaneously encapsulate a highly efficient iron-containing catalyst Hemin, a GSH biosynthesis inhibitor BSO, and a cGAS-STING activator Mn. 2+ Then, the nano-formulation HBMn-FA was prepared by modification with DSPE-PEG-FA, which can effectively improve the targeting precision of the therapeutic agent and reduce damage to normal tissues. Simultaneously, under the combined action of Hemin and BSO, it induces an increase in intracellular ROS levels in tumor cells, triggering severe mitochondrial stress, ultimately inducing the release of mtDNA into the cytoplasm and activating the STING pathway. Furthermore, the Mn released by the nano-therapeutic agent... 2+This can increase the sensitivity of cGAS to mtDNA, promote the synthesis of the subsequent second messenger cGAMP, and enhance the affinity of cGAMP for STING, further expanding the activation of the cGAS-STING pathway, thereby achieving highly efficient tumor therapy. Triggering STING pathway activation based on ferroptosis, a unique cell death mechanism, can not only effectively trigger the release of mtDNA from tumor cells to activate the STING pathway, but also cause tumor cell damage, releasing tumor cell fragments that can further activate the STING pathway in antigen-presenting cells, thus bidirectionally activating the STING pathway and inducing an immune response. This endogenous signal activation of the STING pathway not only overcomes the difficulty of delivering STING agonists but also achieves a specific immune response, resulting in highly efficient and low-toxicity anti-tumor effects and enhanced tumor immunotherapy efficacy. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The antitumor mechanism of HBMn-FA nanoparticles.

[0027] Figure 2 Characterization of HBMn-FA nanoformulation.

[0028] Figure 3 Cytotoxicity of HBMn-FA nanoparticle formulation.

[0029] Figure 4 This triggers the release of endogenous signals and activation of the STING pathway in HBMn-FA nanoformulations.

[0030] Figure 5 To assess the tumor-targeting ability and biocompatibility of HBMn-FA nanoparticle formulations in mice.

[0031] Figure 6 In vivo antitumor effects and immunological analysis of HBMn-FA nanoparticle formulation.

[0032] Figure 7 Analysis of the in vivo anti-transfer properties of HBMn-FA nanoformulation.

[0033] Figure 8 The in vivo antitumor efficacy of HBMn-FA nanoparticles combined with immune checkpoint therapy. Detailed Implementation

[0034] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0035] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, 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 pertains. In the event of any conflict, this specification shall prevail.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0037] To solve the technical problem of this invention, the overall concept of this invention is as follows:

[0038] According to a typical embodiment of the present invention, a method for preparing the nano-formulation HBMn-FA is provided, the method comprising:

[0039] Step S1: Dissolve heme chloride in an aqueous solution of ammonium carbonate and sonicate to obtain an alkaline solution of heme chloride;

[0040] In step S1

[0041] The concentration range of the alkaline solution of heme chloride is 4–8 mg / mL, and the concentration of the aqueous solution of ammonium carbonate is 0.312–0.625 M.

[0042] Hemin chloride is insoluble in neutral aqueous solution but readily soluble in alkaline aqueous solution. To ensure complete dissolution, the ultrasonic power is 20%–80%, the ultrasonic duration is 1–4 seconds, followed by a 3–6 second pause. If the concentration of the alkaline hemin chloride solution is less than 4 mg / mL, it cannot effectively trigger intracellular ROS and hinders the effective release of mitochondrial DNA; if it is greater than 8 mg / mL, it cannot be completely dissolved and cannot effectively form uniformly sized nanoparticles.

[0043] Step S2: Dissolve PLGA and PLGA-PEG together in dichloromethane to obtain a PLGA / PLGA-PEG / DCM solution;

[0044] In step S2

[0045] The ratio of the mass of PLGA, the mass of PLGA-PEG, and the volume of dichloromethane is (133–399) mg : (50–100) mg : (25–35) mL. This ratio range is beneficial for forming nanoparticles with uniform size and good stability.

[0046] Step S3: Dissolve and disperse the alkaline solution of heme chloride in the PLGA / PLGA-PEG / DCM solution and sonicate to obtain mixed emulsion A;

[0047] In step S3

[0048] The volume ratio of the heme chloride alkaline solution to the PLGA / PLGA-PEG / DCM solution is (125-175):(15-24).

[0049] The ultrasonic treatment time is 1 to 5 minutes; the ultrasonic power is 20% to 80%, the ultrasonic treatment lasts for 1 to 4 seconds, and the pause lasts for 3 to 6 seconds.

[0050] Step S4: Dissolve and disperse the MnCl2 aqueous solution and BSO aqueous solution together in the PLGA / PLGA-PEG / DCM solution and sonicate to obtain mixed emulsion B;

[0051] In step S4

[0052] The volume ratio of the MnCl2 aqueous solution, the BSO aqueous solution, and the PLGA / PLGA-PEG / DCM is (0.2-0.4):(0.2-0.4):(2.4-3.6).

[0053] The ultrasonic treatment time is 1 to 5 minutes; the ultrasonic power is 20% to 80%, the ultrasonic treatment lasts for 1 to 4 seconds, and the pause lasts for 3 to 6 seconds.

[0054] The concentration of the MnCl2 aqueous solution is 0.6M to 1.0M; the concentration of the BSO aqueous solution is 5 to 10 mg / mL.

[0055] If the concentration of the MnCl2 aqueous solution is too low, it will hinder the nanoparticles from properly absorbing Mn. 2+ The effective loading capacity is too high, which cannot effectively play the role of accelerating the activation of the STING pathway in cells. If it is too high, the final synthesized nanoparticles will have high toxicity and side effects, which is not conducive to in vivo application.

[0056] If the concentration of the GSH biosynthesis inhibitor BSO is too low, it will not be conducive to the effective loading of BSO by the nanoparticles, and will not be able to effectively exert the effect of inhibiting GSH synthesis in cells, thereby weakening ferroptosis. If it is too high, it will result in the final synthesized nanoparticles having high toxicity and side effects, which is not conducive to in vivo application.

[0057] In one specific implementation method, 3.4–4.6 g of manganese chloride (MnCl2) is dissolved in deionized water to obtain an aqueous solution of MnCl2 with a concentration of 0.6 M–1.0 M, while 100–200 mg of BSO is dissolved in deionized water to obtain an aqueous solution with a concentration of 5–10 mg / mL. -1 The BSO solution should be stored at 4°C before use.

[0058] Step S5: Mix the mixed emulsion A and the mixed emulsion B and then sonicate to obtain a mixed solution C;

[0059] The volume ratio of the mixed emulsion A to the mixed emulsion B is (0.5-1.0):(2-4).

[0060] The ultrasonic treatment time is 1 to 5 minutes; the ultrasonic power is 20% to 80%, the ultrasonic treatment lasts for 1 to 4 seconds, and the pause lasts for 3 to 6 seconds.

[0061] Step S6: Add the mixed solution C to the serum albumin BSA aqueous solution and sonicate. Then add it dropwise to the deionized aqueous solution and stir. After the organic solvent has completely evaporated, centrifuge and collect the microparticles.

[0062] The mass fraction of the bovine serum albumin (BSA) aqueous solution is 1–4 wt%.

[0063] The volume ratio of the mixed solution C to the serum albumin BSA aqueous solution is (3-5):(5-8);

[0064] The ultrasonic power is 20%–80%, the ultrasonic time is 1–4 seconds, and the pause time is 3–6 seconds; the centrifugation speed is 12000–16000 r / m, and the centrifugation time is 5–10 min.

[0065] Step S7: The microparticles are redissolved in deionized water, followed by the addition of polyethyleneimine and stirring. Then, DSPE-PEG-FA is added dropwise and stirred. After centrifugation and washing, the nano-formulation HBMn-FA is obtained.

[0066] In step S7

[0067] The nanoparticles are redissolved in deionized water to a concentration of 1–5 mg / mL.

[0068] The mass ratio of the polyethyleneimine to the DSPE-PEG-FA is (1-3):(2-10).

[0069] If too little DSPE-PEG-FA is added, it will reduce the tumor targeting of the final nanoparticles and reduce the stability of the nanoparticles. If too much is added, it will cause the nanoparticles to easily aggregate and reduce their dispersibility, thereby affecting the treatment efficiency.

[0070] This invention provides a method for preparing the nano-formulation HBMn-FA, such as... Figure 1 As shown, the antitumor mechanism involves the simultaneous encapsulation of a highly efficient iron-containing catalyst Hemin, a GSH biosynthesis inhibitor BSO, and a cGAS-STING activator Mn using a biocompatible PLGA polymer. 2+ Then, it was modified with DSPE-PEG-FA to effectively improve the targeting precision of the therapeutic agent and reduce damage to normal tissues. Simultaneously, under the combined action of Hemin and BSO, it induced an increase in intracellular ROS levels in tumor cells, triggering severe mitochondrial stress, ultimately inducing the release of mtDNA into the cytoplasm and activating the STING pathway. Furthermore, the Mn released by the nanotherapy formulation... 2+ It can increase the sensitivity of cGAS to mtDNA, promote the synthesis of the subsequent second messenger cGAMP, and enhance the affinity of cGAMP for STING, further expanding the activation of the cGAS-STING pathway, thereby triggering innate anti-tumor immunotherapy. Furthermore, ferroptosis can also cause tumor cell damage, releasing tumor cell debris into the tumor microenvironment to further activate the STING pathway in antigen-presenting cells, thus bidirectionally activating the STING pathway and inducing an immune response. This endogenous signaling activation of the STING pathway not only overcomes the difficulty of delivering STING agonists but also achieves a specific immune response, resulting in highly effective and low-toxicity anti-tumor effects and enhanced tumor immunotherapy efficacy.

[0071] This biosafe nano-formulation's "targeted-STING activation-immunotherapy" strategy overcomes the shortcomings of traditional nano-therapeutic agents, such as poor precision and a single treatment mode, and achieves highly efficient and precise targeted therapy for systemic anti-tumor treatment.

[0072] According to another typical embodiment of the present invention, a nano-formulation HBMn-FA obtained by the method is provided.

[0073] According to another typical embodiment of the present invention, the application of the nano-formulation HBMn-FA in the preparation of a drug for anti-tumor immunotherapy is provided.

[0074] The nano-formulation HBMn-FA and its preparation method of this application will be described in detail below with reference to examples, comparative examples and experimental data.

[0075] Example 1: HBMn-FA nano-formulation and its preparation method

[0076] I. The preparation method of HBMn-FA nano-formulation provided in the embodiments of the present invention includes the following steps:

[0077] 1. Dissolve 1.5g of hemin chloride in 0.625M ammonium carbonate solution (NH4)2CO3 and sonicate until fully dissolved to obtain a concentration of 8mg / mL. -1 Prepare an alkaline solution of Hemin and store at 4°C.

[0078] 2. Dissolve 3.5g of manganese chloride (MnCl2) in deionized water to obtain a 1M MnCl2 aqueous solution. Simultaneously, dissolve 200mg of BSO in deionized water to obtain a 10mg / mL solution. -1 The BSO solution was prepared and stored at 4°C.

[0079] 3. Dissolve 399 mg PLGA and 100 mg PLGA-PEG in 30 mL of dichloromethane, sonicate until fully dissolved, and store at 4 °C.

[0080] 4. Dissolve and disperse 125 μL of Hemin / (NH4)2CO3 from step 1 above in 2.4 mL of PLGA / PLGA-PEG / DCM, and sonicate for 1 min using a cell disruptor to obtain a mixed emulsion A containing Hemin / (NH4)2CO3; sonication power 50%, sonication for 3 s, pause for 6 s;

[0081] 5. Dissolve and disperse 400 μL of MnCl2 and 400 μL of BSO from step 2 above in 3.6 mL of PLGA / PLGA-PEG / DCM, and sonicate for 1 min using a cell disruptor to obtain a solution containing Mn. 2+ / BSO mixed emulsion B; ultrasonic power 50%, ultrasonic for 3 seconds, stop for 6 seconds;

[0082] 6. Mix the mixed emulsion A obtained in 4 and the mixed emulsion B obtained in 5, and then sonicate again for 1 minute to obtain mixed solution C. The sonication power is 50%, and the sonication time is 3 seconds followed by a 6-second pause.

[0083] 7. Quickly add 5 mL of 2 wt% bovine serum albumin (BSA) aqueous solution to the mixed solution C obtained in step 6, sonicate again for 2 min, then slowly add it dropwise to 30 mL of deionized water solution, and stir magnetically overnight; after the organic solvent has completely evaporated, centrifuge using a refrigerated high-speed centrifuge and collect the particles; sonicate at 50% power for 3-4 seconds, pause for 5-6 seconds; centrifuge at 12000 r / m for 10 min.

[0084] 8. The nanoparticles described in 7 are redissolved in deionized water to a concentration of 5 mg / mL. Then, 1 mg of polyethyleneimine (PEI) is added to the solution and stirred for 15 min. Then, 10 mg of DSPE-PEG-FA is added dropwise and stirred again for 6 h. After centrifugation and washing, the nanoparticles are characterized by the fact that PEI makes the surface positively charged, which facilitates the modification of the target molecule DSPE-PEG-FA.

[0085] II. Structural Characterization and Performance Testing of HBMn-FA Nanoformulations

[0086] The morphology and size of the HBMn-FA nanoformulation in Example 1 were characterized using transmission electron microscopy (TEM); Figure 2 As shown in AC, the HBMn-FA nanoparticles exhibit a regular morphology and a size of approximately 200 nm. The elemental mapping distribution indicates that the nanoparticles successfully encapsulated Mn. 2+ BSO and Hemin. The particle size distribution of the prepared HBMn-FA nanoparticles was determined using dynamic scattering (DLS); such as Figure 2 As shown in Figure DF, the drug loading capacity of the prepared HBMn-FA nanoformulation for Hemin and BSO, as well as the drug release behavior, were determined using ultraviolet spectrophotometry and high-performance liquid chromatography. The results further demonstrate that this nanoformulation exhibits excellent drug loading efficiency and pH-responsive drug release behavior.

[0087] Comparative Example 1: HBMn Nanoparticle Formulation and Its Preparation Method

[0088] In this comparative example, the PLGA polymer simultaneously encapsulates the highly efficient iron-containing catalyst Hemin, the GSH biosynthesis inhibitor BSO, and the cGAS-STING activator Mn. 2+ However, it was not targeted by DSPE-PEG-FA; the specific preparation method is as follows:

[0089] 1. Dissolve 1.5g of hemin chloride in 0.625M ammonium carbonate solution (NH4)2CO3 and sonicate until fully dissolved to obtain a concentration of 8mg / mL. -1 Prepare an alkaline solution of Hemin and store at 4°C.

[0090] 2. Dissolve 3.5g of manganese chloride (MnCl2) in deionized water to obtain a 1M MnCl2 aqueous solution. Simultaneously, dissolve 200mg of BSO in deionized water to obtain a 10mg / mL solution. -1 The BSO solution was prepared and stored at 4°C.

[0091] 3. Dissolve 399 mg PLGA and 100 mg PLGA-PEG in 30 mL of dichloromethane, sonicate until fully dissolved, and store at 4 °C.

[0092] 4. Dissolve and disperse 125 μL of Hemin / (NH4)2CO3 from step 1 above in 2.4 mL of PLGA / PLGA-PEG / DCM, and sonicate for 1 min using a cell disruptor to obtain a mixed emulsion A containing Hemin / (NH4)2CO3; sonication power 50%, sonication for 3 s, pause for 6 s;

[0093] 5. Dissolve and disperse 400 μL of MnCl2 and 400 μL of BSO from step 2 above in 3.6 mL of PLGA / PLGA-PEG / DCM, and sonicate for 1 min using a cell disruptor to obtain a solution containing Mn. 2+ / BSO mixed emulsion B; ultrasonic power 50%, ultrasonic for 3 seconds, stop for 6 seconds;

[0094] 6. Mix the mixed emulsion A obtained in 4 and the mixed emulsion B obtained in 5, and then sonicate again for 1 minute to obtain mixed solution C. The sonication power is 50%, and the sonication time is 3 seconds followed by a 6-second pause.

[0095] 7. Quickly add 5 mL of 2 wt% bovine serum albumin (BSA) aqueous solution to the mixed solution C obtained in step 6, sonicate again for 2 min, then slowly add it dropwise to 30 mL of deionized water solution, and stir magnetically overnight; after the organic solvent has completely evaporated, centrifuge using a refrigerated high-speed centrifuge to collect the HBMn particles; sonication power 50%, sonication for 3-4 s, pause for 5-6 s; centrifugation speed 12000 r / m, centrifugation time 10 min.

[0096] Comparative Example 2: MnPs Nanoparticles and Their Preparation Methods

[0097] In this comparative example, the PLGA polymer only encapsulated the cGAS-STING activator Mn. 2+ The other steps are the same as in Example 1, and the specific preparation method is as follows:

[0098] 1. Dissolve 3.5g of manganese chloride (MnCl2) in deionized water to obtain a 1M MnCl2 aqueous solution, and store at 4℃.

[0099] 2. Dissolve 399 mg PLGA and 100 mg PLGA-PEG in 30 mL of dichloromethane, sonicate until fully dissolved, and store at 4 °C;

[0100] 3. Disperse 400 μL of MnCl2 from step 1 above in 3.6 mL of PLGA / PLGA-PEG / DCM, and sonicate for 1 min using a cell disruptor to obtain a solution containing Mn. 2+ The mixed emulsion; ultrasonic power 50%, ultrasonic for 3 seconds, pause for 6 seconds;

[0101] 4. Quickly add 5 mL of 2 wt% bovine serum albumin (BSA) aqueous solution to the mixed solution obtained in step 3, sonicate again for 2 min, then slowly add it dropwise to 30 mL of deionized water solution, and stir magnetically overnight; after the organic solvent has completely evaporated, centrifuge using a refrigerated high-speed centrifuge to collect the MnPs particles; sonication power 50%, sonication for 3-4 s, pause for 5-6 s; centrifugation speed 12000 r / m, centrifugation time 10 min.

[0102] Comparative Example 3: HMn Nanoparticle Formulation and Its Preparation Method

[0103] In this comparative example, the PLGA polymer simultaneously encapsulates the highly efficient iron-containing catalyst Hemin and the cGAS-STING activator Mn. 2+ The specific preparation method is as follows:

[0104] 1. Dissolve 1.5g of hemin chloride in 0.625M ammonium carbonate solution (NH4)2CO3 and sonicate until fully dissolved to obtain a concentration of 8mg / mL. -1 Prepare an alkaline solution of Hemin and store at 4°C.

[0105] 2. Dissolve 3.5g of manganese chloride (MnCl2) in deionized water to obtain a 1M MnCl2 aqueous solution, and store at 4℃.

[0106] 3. Dissolve 399 mg PLGA and 100 mg PLGA-PEG in 30 mL of dichloromethane, sonicate until fully dissolved, and store at 4 °C.

[0107] 4. Dissolve and disperse 125 μL of Hemin / (NH4)2CO3 from step 1 and 400 μL of MnCl2 from step 2 in 3.6 mL of PLGA / PLGA-PEG / DCM, and sonicate for 1 min using a cell disruptor to obtain a mixed emulsion containing Hemin / MnCl2; sonication power 50%, sonication for 3 s, pause for 6 s;

[0108] 5. Quickly add 5 mL of 2 wt% bovine serum albumin (BSA) aqueous solution to the mixed solution C obtained in step 4, sonicate again for 2 min, then slowly add it dropwise to 30 mL of deionized water solution, and stir magnetically overnight; after the organic solvent has completely evaporated, centrifuge using a refrigerated high-speed centrifuge to collect the HMn particles; sonication power 50%, sonication for 3-4 s, pause for 5-6 s; centrifugation speed 12000 r / m, centrifugation time 10 min.

[0109] Application Example 1: In vitro application of HBMn-FA nano-formulation

[0110] The in vitro antitumor properties of the nano-formulation were systematically studied by co-culturing 4T1 tumor cells with the HBMn-FA nano-formulation prepared in Example 1.

[0111] Tumor cytotoxicity. First, 4T1, CT26, and B16 cells were seeded in 96-well plates (3 × 10⁶ cells / well). 4 Cells / well were cultured in 1640 RPMI medium for 24 h. Different concentrations of nano-formulation were cultured with cells for 24 h, then 20 μL MTT was added, and incubation was continued for 4 h. Absorbance at 540 nm was measured using a microplate reader. The toxicity of different materials at different concentrations to 4T1, CT26, and B16 tumor cells was determined. Figure 3 As shown in AC, the HBMn-FA nano-formulation exhibits strong killing effect on 4T1 tumor cells at (Hemin = 9.02 ug / ml, BSO = 4.66 ug / ml), with a cell survival rate of less than 12%, indicating that the HBMn-FA nano-formulation can effectively target tumor cells and trigger tumor cell ferroptosis.

[0112] Tumor cell ferroptosis. To verify that tumor cells die via ferroptosis, 4T1 cells were first seeded in 6-well plates (4 × 10⁶ cells / well). 4 Cells / well were cultured and incubated in 1640 RPMI medium for 24 h. HBMn-FA nanoparticles were co-cultured with 4T1 cells for 24 h, and then intracellular Fe was measured. 2+ Content. For example... Figure 3 As shown in DF, Fe in tumor cells 2+ The levels of GSH were significantly increased in the HBMn-FA group, indicating that the iron-containing catalyst Hemin effectively entered tumor cells. Simultaneously, the levels of GSH within tumor cells were significantly decreased, further demonstrating that the GSH synthesis inhibitor BSO can effectively enter tumor cells to inhibit GSH synthesis, thereby further inhibiting GPX4. This triggers both the classical ferroptosis pathway (BSO-GSH-GPX4) mediated by HBMn-FA and the non-classical ferroptosis pathway (Hemin-HMOX1-Fe). 2+ .like Figure 3 As shown in G, further observation using Bio-TEM revealed that the mitochondrial morphology in the control group was basically normal, while in the 4T1 tumor cells treated with HBMn and HBMn-FA, a large number of mitochondrial fragments and many disordered mitochondrial cristae were observed, indicating that the ferroptosis induced by these nanotherapy agents leads to severe mitochondrial damage.

[0113] Mitochondria are key participants in innate immunity, and intracellular ROS readily affect them, inducing mitochondrial stress. The large amounts of ROS produced intracellularly during ferroptosis can cause a certain degree of mitochondrial stress. Therefore, this study uses the mitochondrial-specific ROS marker MitoSOX to monitor superoxide levels in the mitochondria of 4T1 tumor cells after stimulation with various nanotherapeutic agents. (As...) Figure 4 As shown in Figure A, the HB-FA, HBMn, and HBMn-FA treatment groups exhibited significantly enhanced MitoSOX signaling, indicating the production of mitochondrial ROS. Furthermore, due to the lack of histone protection, mtDNA is more susceptible to mROS attack. As... Figure 4 As shown in BD, the amount of cytoplasmic dsDNA (cDNA) significantly increased, while the amount of mtDNA significantly decreased, demonstrating that ferroptosis-mediated mitochondrial oxidative stress can trigger the release of mtDNA into the cytoplasm. The presence of intracellular DNA is crucial for activating the intracellular STING pathway; cGAS is considered a major cytoplasmic DNA sensor, playing a vital role in DNA-mediated innate immune pathways. cGAS binds to DNA, catalyzing the synthesis of cGAMP from ATP and GTP, which subsequently binds to and activates the STING pathway. As... Figure 4 As shown in Figure E, in 4T1 tumor cells treated with the nanoparticle formulation, the expression of downstream proteins related to the cGAS-STING pathway, such as phos-STING, phos-TBK1, phos-IRF3, phos-p65, and IFN-β, was significantly increased, indicating that ferroptosis-mediated mitochondrial oxidative stress can activate the cGAS-STING pathway in tumor cells. Meanwhile, the expression of STING pathway-related proteins in the HBMn-FA group was significantly better than that in the HB-FA group, further demonstrating that Mn... 2+ The combined action of released mtDNA can amplify the activation of the cGAS-STING pathway. More importantly, the STING pathway occurs not only in tumor cells but also in antigen-presenting cells (such as dendritic cells). To further investigate whether tumor-derived cytoplasmic dsDNA from tumor cells that die from ferroptosis can also activate the cGAS-STING pathway in dendritic cells, we collected the supernatant from co-culturing HBMn-FA and 4T1 tumor cells and further co-incubated them with DC2.4 cells. As... Figure 4 As shown in Figure F, the expression of phosphorylated STING, TBK1, IRF3, and IFN-β was also significantly increased in DC2.4 cells, indicating that tumor-derived cytoplasmic dsDNA from HBMn-FA-induced tumor cell death can activate the cGAS-STING pathway in DCs. In summary, ferroptosis-induced mitochondrial oxidative stress can generate and release large amounts of mtDNA to activate cGAS, thereby catalyzing the synthesis of the second messenger cGAMP. Simultaneously released Mn... 2+This further enhanced the sensitivity of cGAS to mtDNA, increased the activity of related enzymes, and enhanced the binding affinity of cGAMP-STING, thereby amplifying the activation of the STING pathway in tumor cells and DC cells.

[0114] Application Example 2: In vivo tumor targeting ability and biocompatibility of HBMn-FA nano-formulation

[0115] This invention uses Balb / c mice as the model animal to construct a 4T1 tumor-bearing mouse model, and systematically studies the in vivo tumor-targeting ability and biocompatibility of the active material after tail vein injection; including:

[0116] (1) Tumor-targeted enrichment

[0117] When the mouse tumor grew to 120 mm 3 At approximately 10:00 AM, Cy5.5-labeled PLGA nanoparticles (HBMn) were used as a control group. The targeting of Cy5.5-labeled HBMn-FA nanoparticles in 4T1 tumor-bearing mice was investigated using an IVIS imaging system. Mice were injected via tail vein and anesthetized at predetermined time points (0, 2, 4, 8, 12, 24, 36 h) using a Maestro in vivo fluorescence imaging system for observation. Figure 5 A, over time, within 4 hours of HBMn-FA administration, strong fluorescent signals appeared at the tumor sites in mice, indicating strong tumor accumulation in the tumor tissue. As... Figure 5 As shown in Figure B, the quantitative fluorescence intensity of isolated organs further demonstrates that nano-formulations have excellent tumor targeting properties.

[0118] (2) Biosafety Analysis

[0119] 119. A comprehensive evaluation of various indicators in mice after material treatment was conducted, including blood biochemistry analysis, immunotoxicity, and biocompatibility, to assess the biosafety and clinical application potential of the nano-formulation constructed in this invention. For example... Figure 5 As shown in C and D, after injection of bioactive materials, the various indicators of the mice were not significantly different from those of the normal control mice, indicating that the nano-formulation has good biosafety and clinical application potential.

[0120] Application Example 3: In vivo immune response and antitumor properties of HBMn-FA nanoparticles and related research.

[0121] This invention uses Balb / c mice to construct a 4T1 tumor-bearing mouse model and systematically studies the therapeutic effect of nano-formulations on tumors after tail vein injection. When the tumor grows to 80 mm... 3Mice bearing tumors were randomly divided into groups, and the corresponding materials were injected into the mice via the tail vein on days 1, 3, 6, and 9. Changes in mouse body weight (measured using a balance) and tumor size (measured using calipers for the long diameter (L) and short diameter (W) of the tumor) were recorded every two days. Tumor volume was calculated using Still's formula: V = W² × L / 2. On day 9 of treatment, some mice were sacrificed, and tumor lymph nodes and tumor tissue were collected for immunological analysis.

[0122] Simultaneously, comparative examples were set up including Control (PBS), MnPs (preparation method as described in Comparative Example 2 above), HMn (preparation method as described in Comparative Example 3 above), HBMn (preparation method as described in Comparative Example 1 above), and HB-FA (containing Hemin, BSO, and DSPE-PEG-FA but lacking Mn). 2+ The cGAS-STING activator Mn is not encapsulated during the preparation process. 2+ Other steps are the same as in Example 1;

[0123] like Figure 6 As shown in AC, compared with other control materials (control materials include Control (PBS), MnPs, HMn, HBMn, and HB-FA), tumor growth was significantly inhibited and tumor volume was significantly smaller in tumor-bearing mice treated with HBMn-FA nanoparticles. This indicates that HBMn-FA nanoparticles can effectively inhibit the growth of breast cancer tumors and have good anti-tumor efficacy. Simultaneously, the survival time of tumor-bearing mice was significantly prolonged. Figure 6 As shown in Figure D, the percentage of mature dendritic cells (DCs) in tumor lymph nodes was detected by flow cytometry. Compared with the control group (11.6%), the MnPs, HMn, and HB-FA treatment groups only moderately accelerated the maturation of DCs in lymph nodes (14.7%, 16.4%, and 18.8%, respectively), while the HBMn-FA treatment group had the highest DC maturation rate, reaching 35.3%. This indicates that the nanoparticles successfully activated the potent STING pathway, triggering an anti-tumor immune response. Figure 6 As shown in E and F, tumor infiltration CD8 in the HBMn group and the HBMn-FA group. + The percentages of T cells were 3.15 times and 4.28 times that of the control group, respectively, while the MnPs group, HMn group, and HB-FA group showed tumor infiltration CD8. + The percentages of T cells were only 1.32 times, 1.62 times, and 2.10 times that of the control group, respectively. Similarly, compared with other groups, the HBMn-FA treatment group showed significantly higher tumor infiltration CD4 counts. + The percentage of T cells also increased significantly. This further proves that Mn 2+ The presence of [a substance] greatly promotes the activation of the STING pathway, triggering a powerful anti-tumor immune response. For example... Figure 6As shown in G, CD4 in tumor tissue after different treatments + T cells and CD8 + Immunofluorescence staining of T cells further supported the results of flow cytometry analysis. For example... Figure 6 As shown in H and I, the secretion of TNF-α and IFN-γ was significantly increased in tumors treated with HBMn-FA, indicating that the STING pathway activated by the HBMn-FA nanoparticles is conducive to the secretion of large amounts of anti-tumor cytokines TNF-α and IFN-γ, thereby enhancing tumor immunotherapy.

[0124] Application Example 4: In vivo anti-tumor metastasis study of HBMn-FA nano-formulation

[0125] This invention uses Balb / c mice as a model animal to establish a tumor metastasis model by tail vein injection of 4T1 breast cancer cells. The therapeutic effect of nano-formulation on tumor metastasis after tail vein injection is systematically studied. When the tumor grows to 50mm... 3 In this study, tumor-bearing mice were randomly divided into groups, and the corresponding materials were injected into the mice via the tail vein on days 1, 3, 6, and 9. On day 8, 4T1 breast cancer cells were injected via the tail vein to investigate the immune response induced by the nanoparticles and their ability to resist tumor metastasis. Changes in mouse body weight (measured using a balance) and tumor size (measured using calipers for the long diameter (L) and short diameter (W) of the tumor) were recorded every two days. Tumor volume was calculated using the Still's formula: V = W² × L / 2. After treatment, some mice were sacrificed, and their lungs were collected for observation.

[0126] like Figure 7 As shown in AC, HBMn-FA treatment significantly suppressed 4T1 subcutaneous tumors and effectively inhibited lung metastasis. Notably, the HBMn-FA treatment group showed only scattered lung metastatic nodules, while the Control-, MnPs-, and HB-FA treatment groups all showed numerous metastatic nodules. Compared to the nanoparticle treatment groups in Comparative Examples 1-3, the nanoparticle treatment group in Example 1 significantly suppressed tumor growth. Furthermore, H&E staining of lung tissue further revealed numerous metastatic lesions with granulation in the lung tissue of mice in all groups except the HBMn-FA group. These results indicate that HBMn-FA activation of the STING pathway-mediated immune response effectively inhibits subcutaneous tumor growth and metastasis.

[0127] Application Example 5: Study on the enhanced in vivo antitumor properties of HBMn-FA nano-formulation combined with immune checkpoint inhibitors

[0128] This invention uses Balb / c mice as a model animal to construct a bilateral subcutaneous tumor model and systematically studies the therapeutic effect of nano-formulations administered via tail vein injection on proximal and distal tumors. 4T1 cells were subcutaneously injected into the right and left sides of each Balb / c mouse on days 0 and 7, respectively, to establish a bilateral tumor model. On day 7, the 4T1 tumor-bearing mice (with primary tumors approximately 80 mm)... 3 The distal tumor was 0mm. 3 Mice were randomly divided into 5 groups (14 mice / group): 1) control, 2) HB-FA, 3) HB-FA + αPD-1, 4) HBMn-FA, 5) HBMn-FA + αPD-1. Different nanoparticle formulations (HB-FA and HBMn-FA, respectively) were intravenously injected on days 8, 10, and 13, and αPD-1 (20 μg / mouse) was intravenously injected on days 9, 11, and 16. Changes in mouse body weight (measured using a balance) and tumor size (measured using calipers for the long diameter (L) and short diameter (W) of the tumor) were recorded every two days. Tumor volume was calculated using Still's formula: V = W² × L / ². Patient body weight, primary tumor and distant tumor volume, and survival status were monitored every other day for 60 days.

[0129] As Figure 8 As shown in AC, HBMn-FA+αPD-1 treatment significantly inhibited the growth of primary tumors in mice, and the growth of distant tumors was also more effectively inhibited, indicating that combined αPD-1 immunotherapy can effectively enhance the systemic anti-tumor ability of HBMn-FA. In contrast, mice treated with HB-FA and HB-FA+αPD-1 did not achieve such excellent inhibitory effects on primary and distant tumors, further confirming that the STING pathway activated by HBMn-FA is beneficial for promoting anti-tumor immunotherapy. Meanwhile, the average body weight of mice in different treatments did not change significantly during treatment, and 85.7% of HBMn-FA+αPD-1 mice survived for more than 44 days after tumor cell inoculation, while mice in the control group and HB-FA treatment group died within 40 days. This indicates that the STING pathway activated by HBMn-FA combined with immune checkpoint therapy can induce a stronger systemic anti-tumor immune response.

[0130] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0131] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0132] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a nanofomulation HBMn-FA, characterized by, The method comprises: dissolving hemin chloride in an aqueous ammonium carbonate solution and ultrasonicating to obtain a hemin chloride basic solution; dissolving PLGA and PLGA-PEG together in dichloromethane to obtain a PLGA / PLGA-PEG / DCM solution; dispersing the hemin chloride basic solution in the PLGA / PLGA-PEG / DCM solution and ultrasonicating to obtain a mixed emulsion A; dispersing an aqueous MnCl2 solution and an aqueous BSO solution together in the PLGA / PLGA-PEG / DCM solution and ultrasonicating to obtain a mixed emulsion B; mixing the mixed emulsion A and the mixed emulsion B and ultrasonicating to obtain a mixed solution C; adding the mixed solution C into an aqueous bovine serum albumin (BSA) solution and ultrasonicating, then adding dropwise into an aqueous deionized solution and stirring, centrifuging after the organic solvent is completely volatilized, and collecting microparticles; resolubilizing the microparticles in deionized water, then adding polyethyleneimine and stirring, then adding dropwise DSPE-PEG-FA and stirring, and centrifuging and washing to obtain a nano-preparation HBMn-FA.

2. The production method according to claim 1, characterized by, The concentration of the hemin chloride basic solution ranges from 4 to 8 mg / mL, and the concentration of the aqueous ammonium carbonate solution ranges from 0.312 to 0.625 M.

3. The production method according to claim 1, characterized by, The ratio of the mass of the PLGA, the mass of the PLGA-PEG, and the volume of the dichloromethane is (133-399) mg : (50-100) mg : (25-35) mL.

4. The method of claim 1, wherein, The volume ratio of the mixed emulsion A and the mixed emulsion B is (0.5-1.0) : (2-4).

5. The preparation method according to claim 1, characterized in that, The mass ratio of the polyethyleneimine and the DSPE-PEG-FA is (1-3) : (2-10).

6. A nano-preparation HBMn-FA prepared by the preparation method of any one of claims 1-5.

7. The nano-preparation HBMn-FA of claim 6 for use in the preparation of a medicament for immunotherapy against breast cancer.

8. The nano-preparation HBMn-FA of claim 6 and αPD-1 for use in the preparation of a medicament for immunotherapy against breast cancer.

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