A nano-drug for catalyzing hydrogen sulfide in situ in a tumor and preparation and application thereof

Abstract

The application discloses a kind of tumor in situ catalytic production of hydrogen sulfide nano-drug and its preparation and application, belong to medical technology field.The application is with nano covalent organic framework TfpPA as main body structure, then bond hydrogen sulfide donor small molecule S-benzoyl sulfenamide is prepared to obtain nano-drug, realizes tumor in situ catalytic production H2S.Nano-drug of the application can be delivered H2S to tumor site, induces tumor ICD, while down-regulating IDO1 expression and inhibiting IDO1 activity, to trigger specific anti-tumor immune response to kill tumor, and reverse the immunosuppressive environment of tumor Sensitization immunotherapy effect, finally reach the effect of inhibiting tumor metastasis and recurrence.

Description

Technical Field

[0001] This invention relates to a nanomedicine for in-situ catalytic production of hydrogen sulfide from tumors, its preparation and application, and belongs to the field of pharmaceutical technology. Background Technology

[0002] In recent years, tumor immunotherapy has become a hot topic in basic and clinical research. However, due to insensitivity to immune checkpoint blockers, poor immunogenicity at the tumor site, and the presence of a strong immunosuppressive environment, cancer treatment using the body's own immune system often fails to achieve a sustained response rate. On the one hand, tumor-infiltrating anti-tumor lymphocytes may be limited by various immunosuppressive mechanisms, such as the inhibitory effect of regulatory T cells (Tregs) and the induction of inhibitory checkpoint receptor expression. On the other hand, cancer cells with high mutation rates can produce neoantigens that dendritic cells (DCs) cannot recognize or recognize poorly, thereby evading the immune response. In addition, the acidic and hypoxic microenvironment within the tumor reduces lymphocyte infiltration and enhances immune escape, also affecting the efficacy of tumor immunotherapy. Therefore, to achieve effective cancer immunotherapy, it is necessary to promote antigen production to improve tumor immunogenicity while overcoming tumor immunosuppression to enhance the immune response rate.

[0003] Inducing immunogenic cell death (ICD) in cancer cells is a promising approach to achieving the aforementioned goals because it can simultaneously elicit adjuvant and antigenicity from dying cells, effectively promoting both innate and adaptive immunity, thereby sensitizing the efficacy of tumor immunotherapy. During ICD in tumor cells, dying tumor cells release tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs). However, while enhancing tumor immunogenicity, ICD also acts as a double-edged sword, activating the immunosuppressive environment of the tumor and thus weakening the anti-tumor immune effect induced by ICD. The effect of ICD on the immunosuppressive microenvironment is due to the fact that interferon-γ (IFN-γ, a potent anti-tumor cytokine that inhibits cancer cell growth) secreted by effector T cells can induce transcriptional activation of the indoleamine 2,3-dioxygenase (IDO1) promoter, leading to upregulation of IDO1 expression in tumor cells and dendritic cells (DCs). As a negative regulatory response, the immunometabolic pathway of IDO1 is opposite to the immunogenic effect of ICD. High levels of IDO1 in tumor cells and dendritic cells (DCs) can consume catalytic tryptophan (TRP) to convert it into excess kynurenine (KYN), causing DCs to tend toward an immunosuppressive phenotype and promoting Treg differentiation, resulting in an enhanced immunosuppressive environment in the tumor.

[0004] H2S is a third gaseous signaling molecule that plays a crucial role in tumor development. Related studies have shown that high concentrations of exogenous H2S can inhibit the function of cytochrome c oxidase (COX), thereby suppressing oxidative phosphorylation (OXPHOS) and generating excessive reactive oxygen species (ROS). This disrupts the redox homeostasis of tumor cells, leading to cellular "chemical asphyxiation." Simultaneously, excessive ROS may induce oxidative damage and intracellular oxidative damage (ICD) in tumor cells, activating tumor danger signaling pathways to release DAMPs, providing potential antigen stimulation to the immune system and enhancing specific anti-tumor immune responses. Furthermore, literature has demonstrated that H2S downregulates IDO1 expression by blocking the NF-κB and STAT3 pathways, and inhibits IDO1 activity through H2S / NO crosstalk, reducing tryptophan metabolism and kynurenine production, inducing T effector cells, and inhibiting MDSCs, thus effectively suppressing tumor development. Therefore, delivering H2S to the tumor site is expected to successfully induce tumor ICD, while downregulating IDO1 expression and inhibiting IDO1 activity, which is a potential breakthrough for enhancing tumor immunotherapy.

[0005] Currently, methods for delivering H2S mainly include direct inhalation, inorganic sulfide salts, and small molecule H2S donors. Direct inhalation is the most direct method for administering exogenous H2S, but due to the malodorous nature of the gas and its lack of targeting, direct inhalation of H2S is difficult to achieve in clinical settings. Inorganic sulfide salts are currently the most widely used delivery strategy in biological and preclinical research; however, the release of H2S from sulfide salts is uncontrolled and difficult to release at the target site, failing to achieve effective drug concentrations. This necessitates high-dose administration, causing H2S concentrations in blood and tissues to spike to supraphysiological levels, posing a health risk. Small molecule H2S donors have recently received widespread attention, but they still suffer from poor stability, low solubility, weak targeting, and short half-life.

[0006] In recent years, covalent organic frameworks (COFs) have attracted widespread research attention as a new class of crystalline porous polymers, especially nanoscale COFs (nCOFs), which have rapidly emerged in cancer therapy. Currently, some literature uses nCOFs as drug carriers to load and deliver drugs for targeted therapy; however, due to the physical interaction between the carrier and the drug, their stability is relatively poor.

[0007] Therefore, how to use covalent organic frameworks as carriers to deliver H2S in order to achieve better tumor treatment results is an urgent problem to be solved. Summary of the Invention

[0008] [Technical Issues]

[0009] Currently, methods for delivering H2S suffer from poor stability, low solubility, weak targeting, and short half-life.

[0010] [Technical Solution]

[0011] To address the aforementioned issues, this invention utilizes the nano-covalent organic framework TfpPA as the main structure, subsequently bonding it with the hydrogen sulfide donor small molecule S-benzoylthiohydroxylamine to prepare a nanomedicine, achieving in-situ catalytic production of H2S from tumors. This nanomedicine can deliver H2S to the tumor site, inducing tumor ICD, simultaneously downregulating IDO1 expression and inhibiting IDO1 activity, thereby triggering a specific anti-tumor immune response to kill tumor cells. Furthermore, it reverses the immunosuppressive environment of the tumor, enhancing the efficacy of tumor immunotherapy, ultimately inhibiting tumor metastasis and recurrence.

[0012] The first objective of this invention is to provide a method for preparing nanomedicines for in-situ catalytic hydrogen sulfide production from tumors, comprising the following steps:

[0013] The covalent organic framework TfpPA and S-benzoylthiohydroxylamine STA were dispersed in dichloromethane for reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain a nanomedicine for in-situ catalytic hydrogen sulfide production in tumors.

[0014] In one embodiment of the present invention, the covalent organic framework TfpPA is made from 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde and 1,4-phenylenediamine.

[0015] In one embodiment of the present invention, the method for preparing the covalent organic framework TfpPA is as follows:

[0016] 2,4,6-Trihydroxybenzene-1,3,5-tricarboxaldehyde (Tfp) and 1,4-phenylenediamine (PA) were added to a mixed solvent of butanol and 1,2-dichlorobenzene to obtain a mixture. The mixture was sonicated for 20 minutes, and then an aqueous acetic acid solution was added. The mixture was degassed by a freeze-pump-thaw cycle and heated under vacuum. After the reaction was completed, the mixture was cooled, washed, and dried to obtain the covalent organic framework TfpPA.

[0017] The molar ratio of 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde to 1,4-phenylenediamine is 2:3.

[0018] In a mixed solvent of butanol and 1,2-dichlorobenzene, the volume ratio of butanol to 1,2-dichlorobenzene is 1:1.

[0019] The ratio of 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde, mixed solvent, and aqueous acetic acid solution was 0.3 mmol: 3 mL: 0.5 mL.

[0020] The concentration of the acetic acid aqueous solution is 3 mol / L;

[0021] The degassing cycle of freezing-pumping-thawing is repeated 3 times;

[0022] Vacuum-sealed heating involves sealing under vacuum and heating at 120°C for 3 days.

[0023] Cooling means cooling to room temperature;

[0024] The washing process involved washing with anhydrous tetrahydrofuran, anhydrous acetone, and anhydrous dichloromethane, respectively.

[0025] Drying was carried out under vacuum at 120°C for 12 hours;

[0026] The covalent organic framework TfpPA is a dark red powder.

[0027] In one embodiment of the present invention, the mass ratio of the covalent organic framework TfpPA to S-benzoylthiohydroxylamine STA is 100:20-30.

[0028] In one embodiment of the present invention, the ratio of the covalent organic framework TfpPA to dichloromethane is 100 mg: 5 mL.

[0029] In one embodiment of the present invention, the reaction is carried out at 120°C for 12 hours.

[0030] In one embodiment of the present invention, the reaction involves the amino group of S-benzoylthiohydroxylamine STA being linked to the carbonyl group (or aldehyde group) of a covalent organic framework.

[0031] In one embodiment of the present invention, the centrifugation is performed at 8000 rpm, the washing is done with ethanol, and the drying is performed at 60°C.

[0032] The second objective of this invention is to prepare a nanomedicine for in-situ catalytic hydrogen sulfide production in tumors using the method described herein.

[0033] In one embodiment of the present invention, the nanomedicine has a size of approximately 120 nm.

[0034] In one embodiment of the present invention, the nanomedicine is based on a nano-covalent organic framework, and then bonded with a small molecule of hydrogen sulfide donor to achieve in-situ catalytic production of H2S in tumors.

[0035] The third objective of this invention is the application of the nanomedicine described herein in the preparation of drugs for treating tumors.

[0036] [Beneficial Effects]

[0037] (1) The nanomedicine described in this invention is based on a nano-covalent organic framework, which can achieve efficient H2S production in situ of tumors, induce tumor ICD, downregulate IDO1 expression and inhibit IDO1 activity, and achieve a strong inhibitory effect on primary tumors and distant metastases.

[0038] (2) In this invention, after the small molecule H2S donor S-benzoylthiohydroxylamine is bonded to the main structure covalent organic framework TfpPA, H2S can be released under the action of high content of glutathione at the tumor site, thereby achieving the effect of targeted release.

[0039] (3) The method for preparing nanomedicine described in this invention is simple, has good dispersibility and stability, and is beneficial for the biological application of H2S carrier delivery in vivo.

[0040] (4) The nanomedicine for in-situ catalytic hydrogen sulfide production in tumors described in this invention provides new ideas and guidance for enhancing the research and application of tumor immunotherapy, and provides a research basis for clinical application. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the synthesis of nanomedicines for in-situ catalytic hydrogen sulfide production from tumors.

[0042] Figure 2 Infrared test results for TfpPA and TfpPA-STA.

[0043] Figure 3 The results are for particle size testing of TfpPA-STA.

[0044] Figure 4 This is an electron microscope image of TfpPA-STA.

[0045] Figure 5 Characterization of the ability of TfpPA-STA to produce H2S in vitro.

[0046] Figure 6 Characterization of TfpPA-STA-induced reactive oxygen species production in tumor cells.

[0047] Figure 7 Analysis of TfpPA-STA-induced immunogenic cell death in tumor cells, where A represents CALR and B represents HMGB1.

[0048] Figure 8 Analysis of TfpPA-STA's inhibition of tumor IDO1 expression.

[0049] Figure 9 Characterization of the in vitro H2S production capacity of nanomedicines prepared with different amounts of S-benzoylthiohydroxylamine (STA).

[0050] Figure 10 This describes the synthesis process of the drug in Comparative Example 2. Detailed Implementation

[0051] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0052] Test method:

[0053] 1. Ability to produce H2S in vitro:

[0054] Add 2 μmol of free cysteine ​​to 2 mL of 10 mg / mL TfpPA-STA aqueous solution and stir at room temperature; collect the reaction aliquot (1 mL) into 2 mL EP tubes every 5 minutes, wherein each EP tube contains 100 μL of zinc acetate (1% w / v) and 600 μL of trichloroacetic acid (10% w / v) to obtain a mixture;

[0055] Centrifuge the mixture at 5000g for 10 minutes and filter the precipitate; transfer 1.2mL of the clear solution to another 2mL EP tube; add 20mM 150μL of N,N-dimethyl-p-phenylenediamine sulfate solution (solvent: 7.2mol / L HCl solution), then add 30mM 150μL of FeCl3 solution (solvent: 1.2mol / L HCl solution), and measure the absorbance of the aliquots at 670nm. Then calculate the H2S concentration of each sample according to the standard curve.

[0056] 2. Induction of reactive oxygen species production in tumor cells:

[0057] 10 4 Four 4T1 cells (mouse-derived breast cancer cells) were seeded in 24-well plates. The 4T1 cells were incubated with S-arylthioxime covalent organic framework TfpPA-STA at 37°C for 4 hours (drug concentration 1.0 mg / mL). Then, fresh medium containing 10 μmol / L 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was added and the cells were incubated for another 30 min. The cells were washed, fixed, and stained with DAPI. The changes in reactive oxygen species levels in the cells were observed using laser confocal microscopy.

[0058] 3. Induction of immunogenic cell death in tumor cells:

[0059] 4T1 cells (mouse-derived breast cancer cells) were incubated with S-arylthioxime covalent organic framework TfpPA-STA at 37°C for 4 hours (drug concentration 1.0 mg / mL), and then stained with antibodies against DAMP markers CALR and HMGB1.

[0060] 4. Inhibits tumor IDO1 expression:

[0061] A mouse subcutaneous tumor model was constructed, with tumors approximately 100 mm in volume. 3 PBS, TfpPA, and TfpPA-STA (drug dose 100 μg, drug concentration 1 mg / mL) were injected into the tumor. After 48 h, the tumor was removed and frozen sections were prepared. The tumor sections were stained with IDO1 antibody (recombinant anti-indoleamine 2,3-dioxygenase antibody, ab277522, Abcom) and donkey anti-rabbit IgG H&L (Alexa Fluor@488) secondary antibody.

[0062] Raw materials used in the examples:

[0063] The preparation method of the covalent organic framework TfpPA is as follows:

[0064] 0.3 mmol (63 mg) of 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde (Tfp) and 0.45 mmol (48.6 mg) of 1,4-phenylenediamine (PA) were added to a Pyrex tube containing 1.5 mL of butanol and 1.5 mL of 1,2-dichlorobenzene to obtain a mixture;

[0065] The mixture was sonicated for 20 minutes, then 0.5 mL of 3 mol / L acetic acid aqueous solution was added, and the mixture was degassed three times by a freeze-pump-thaw cycle. The mixture was then sealed under vacuum and heated at 120°C for three days.

[0066] After the reaction was completed, the mixture was cooled to room temperature and washed with anhydrous tetrahydrofuran, anhydrous acetone and anhydrous dichloromethane respectively.

[0067] Finally, the powder was dried at 120°C under vacuum for 12 hours to obtain a deep red powder, namely the covalent organic framework TfpPA.

[0068] Example 1

[0069] A method for preparing nanomedicines for in-situ catalytic hydrogen sulfide production in tumors, comprising the following steps:

[0070] 100 mg of covalent organic framework TfpPA and 25 mg of S-benzoylthiohydroxylamine STA were dispersed in 5 mL of dichloromethane and reacted at 120 °C for 12 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm, the supernatant was removed, and 1 mL of ethanol was added for washing three times. The mixture was then dried overnight in an oven at 60 °C to obtain the nanomedicine S-aromatic thiooxime covalent organic framework TfpPA-STA, which is used for in-situ catalytic hydrogen sulfide production in tumors.

[0071] The performance of the obtained nanomedicine S-aromatic thiooxime covalent organic framework TfpPA-STA was tested, and the test results are as follows;

[0072] (1) Infrared characterization:

[0073] TfpPA and TfpPA-STA were analyzed using an infrared analyzer, and the infrared results are as follows: Figure 2 .

[0074] from Figure 2 It can be seen that both the TfpPA and TfpPA-STA spectra exhibit a common characteristic C=O vibrational band (1583 cm⁻¹). -1 ), C=C vibration zone (1451cm) -1 ) and CN vibration belt (1256cm) -1 This confirms the formation of its skeletal structure. In the TfpPA-STA spectrum, at 1656 cm⁻¹... -1 The observation of a unique C=N vibration band provides direct evidence for the reaction of small molecule STA with TfpPA to form an imine bond.

[0075] (2) Size characterization:

[0076] Particle size results as follows Figure 3 The electron microscopy results are as follows Figure 4 .

[0077] from Figure 3 and Figure 4 It can be seen that the particle size of nanomedicine is approximately 120 nm.

[0078] (3) Characterization of in vitro H2S production capacity:

[0079] Characterization of in vitro H2S production capacity, such as Figure 5 .

[0080] from Figure 5 It can be seen that the ability to produce H2S in vitro reaches its maximum value (30 μM) at 15-20 min, and then maintains a high concentration (above 15 μM) for 2 hours.

[0081] (4) Characterization of tumor cell reactive oxygen species production:

[0082] The results are as follows Figure 6 ,from Figure 6 It can be seen that after treatment with TfpPA-STA, the intracellular reactive oxygen species (ROS) content of cells increased significantly, reaching 36 times that of PBS treatment. This confirms that tumor cells experience an increase in intracellular ROS content under the influence of H2S.

[0083] (5) Analysis of the induction of immunogenic cell death in tumor cells:

[0084] Statistical analysis of confocal images yielded the following results: Figure 7 As shown, the exposure of CALR on the cell surface increased and the signal was significantly enhanced after TfpPA-STA treatment, which was 3.8 times that of PBS treatment. Figure 7 (A); simultaneously, the retention of HMGB1 in the nucleus decreased, and the signal weakened, with a reduction of 59% compared to PBS treatment. Figure 7 (B) There was a significant release. The above results demonstrate that H2S generated by the S-arylthioxime covalent organic framework TfpPA-STA can significantly enhance the ROS level of tumor cells and induce CALR exposure and HMGB1 release, leading to immunogenic cell death in tumor cells.

[0085] (6) Analysis of inhibition of tumor IDO1 expression:

[0086] The fluorescence signal of IDO1 in frozen sections of tumor was analyzed and statistically analyzed, and the results are as follows: Figure 8 As shown, the fluorescence signal intensity in tumors treated with TfpPA-STA was significantly reduced, decreasing by 81% compared to PBS treatment; confirming that TfpPA-STA can effectively inhibit IDO1 expression in tumors. These results demonstrate that H2S generated by the S-arylthioxime covalent organic framework TfpPA-STA can significantly inhibit IDO1 expression in tumors, reducing the impact of the tumor immunosuppressive microenvironment on tumor immunotherapy.

[0087] Example 2

[0088] The dosage of S-benzoylthiohydroxylamine (STA) in Example 1 was adjusted to 10 mg, 15 mg, 20 mg, and 30 mg, while other aspects remained the same as in Example 1. The resulting nanomedicines were designated as NP1, NP2, NP3, and NP4, respectively.

[0089] The obtained nanomedicine was tested, and the test results are as follows: Figure 9 :

[0090] from Figure 9It can be seen that the H2S release amounts at 20 min were approximately 13.1, 21.0, 27.7, and 32.4 μM, respectively. The H2S release amount increased with the increase of STA amount, but the H2S release amount did not differ much when the STA amount was 20-30 mg.

[0091] Comparative Example 1

[0092] The covalent organic framework TfpPA in Example 1 was changed to the metal-organic framework ZIF-8, while everything else remained the same as in Example 1.

[0093] The results showed that S-benzoylthiohydroxylamine (STA) had too low a drug loading capacity and insufficient ability to produce H2S in vitro, which could not meet the requirements of related experiments at the cellular and animal levels.

[0094] Comparative Example 2

[0095] The S-benzoylthiohydroxylamine (STA) in Example 1 was replaced with BHA, while all other aspects remained the same as in Example 1. The synthesis process is as follows: Figure 10 .

[0096] The results showed that the obtained nanomedicine TfpPA-BHA could not catalyze the production of H2S.

[0097] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing nanomedicines for in-situ catalytic hydrogen sulfide production in tumors, characterized in that, Includes the following steps: The covalent organic framework TfpPA and S-benzoylthiohydroxylamine STA were dispersed in dichloromethane and reacted. After the reaction, the mixture was centrifuged, washed, and dried to obtain a nanomedicine for in-situ catalytic hydrogen sulfide production in tumors. The structure of the covalent organic framework TfpPA is as follows:

2. The method according to claim 1, characterized in that, The mass ratio of the covalent organic framework TfpPA to S-benzoylthiohydroxylamine STA is 100:20-30.

3. The method according to claim 1, characterized in that, The ratio of the covalent organic framework TfpPA to dichloromethane is 100 mg: 5 mL.

4. The method according to claim 1, characterized in that, The reaction was carried out at 120°C for 12 hours.

5. The method according to claim 1, characterized in that, The covalent organic framework TfpPA is made from 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde and 1,4-phenylenediamine.

6. The method according to claim 1, characterized in that, The preparation method of the covalent organic framework TfpPA is as follows: 2,4,6-Trihydroxybenzene-1,3,5-tricarboxaldehyde (Tfp) and 1,4-phenylenediamine (PA) were added to a mixed solvent of butanol and 1,2-dichlorobenzene to obtain a mixture. The mixture was sonicated for 20 minutes, and then an aqueous acetic acid solution was added. The mixture was degassed by a freeze-pump-thaw cycle and heated under vacuum. After the reaction was completed, the mixture was cooled, washed, and dried to obtain the covalent organic framework TfpPA.

7. The method according to claim 6, characterized in that, The molar ratio of 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde to 1,4-phenylenediamine is 2:3; the volume ratio of butanol to 1,2-dichlorobenzene in the mixed solvent of butanol and 1,2-dichlorobenzene is 1:

1.

8. The method according to claim 6, characterized in that, The ratio of 2,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde, mixed solvent, and aqueous acetic acid solution is 0.3 mmol: 3 mL: 0.5 mL.

9. The nanomedicine for in-situ catalytic hydrogen sulfide production in tumors prepared by the method according to any one of claims 1-8.

10. The use of the nanomedicine according to claim 9 in the preparation of a tumor treatment drug.

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