Iron death agonist, hypoxia-responsive nano co-delivery system and application thereof

Abstract

This invention belongs to the field of drug preparation technology, specifically relating to a ferroptosis agonist, a hypoxia-responsive nanocomposite delivery system, and their applications. This invention synthesizes a novel ferroptosis agonist, RSL3-ClAc, by introducing a chloroacetyl fragment into the RSL3 molecule, significantly enhancing its binding affinity to GPX4 and thus improving ferroptosis induction efficiency. Simultaneously, RSL3-ClAc is self-assembled with the hypoxia-responsive photosensitizer TCy5-NO2 to construct a nanocomposite drug delivery system. This nanocomposite drug delivery system utilizes the EPR effect and hypoxia-responsive characteristics of the nanocarrier to effectively penetrate the tumor barrier and precisely locate lesions. In the hypoxic tumor microenvironment, TCy5-NO2 photosensitizer activation is triggered by nitroreductase (NTR), simultaneously releasing RSL3-ClAc to enhance the ferroptosis induction effect.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, specifically relating to a ferroptosis agonist, a hypoxia-responsive nano-co-delivery system, and their applications. Background Technology

[0002] Ferroprelation is a novel iron-dependent, non-apoptotic form of programmed cell death, distinct from apoptosis, necrosis, and autophagy. It is characterized by increased intracellular iron levels, excessive lipid peroxidation, and an imbalance in redox levels, ultimately leading to cell death. Since its initial definition in 2012, ferroptosis has garnered significant attention due to its close association with various diseases, particularly in the field of anti-tumor research, where its unique cell death mechanism makes it a highly promising area of ​​study.

[0003] Glutathione peroxidase 4 (GPX4) plays a central regulatory role in ferroptosis. GPX4 belongs to the glutathione peroxidase family and is a selenocysteine-containing selenoprotein. Its main function is to clear intracellular iron-dependent lipid peroxides, thereby protecting cells from ferroptosis. Under normal circumstances, the Glu / cysteine ​​transporter (XC...) - By exchanging cysteine ​​for glutamate, GPX4 provides the raw materials for the synthesis of glutathione (GSH). GSH, as a donor of reduced glutathione from GPX4, works synergistically with GPX4 to maintain intracellular redox balance. Once a key component of this antioxidant system is disrupted, such as by the XC system... - Impaired function or reduced GPX4 activity leads to the continuous accumulation of intracellular lipid peroxides, which in turn triggers ferroptosis.

[0004] In cancer treatment, inducing ferroptosis in tumor cells is an emerging strategy. In recent years, significant progress has been made in the development of anti-tumor drugs targeting the ferroptosis pathway. RSL3, as an important ferroptosis inducer, covalently binds to the GPX4 protein, inhibiting its enzymatic activity, disrupting intracellular redox balance, and inducing ferroptosis in tumor cells, thereby inhibiting tumor growth. However, existing ferroptosis inducers such as RSL3 have some limitations, such as low selectivity, unsatisfactory efficacy in in vivo tumor treatment, and the potential for drug tolerance with long-term administration, which severely restricts their widespread application in cancer treatment. Therefore, developing novel GPX4 inhibitors with higher selectivity and efficacy has become an urgent scientific problem to be solved, and is of great significance for promoting the application of ferroptosis in anticancer therapy.

[0005] Ferroprelation, as an emerging anticancer strategy, has attracted much attention due to its unique mechanism of action. GPX4 is a key enzyme mediating ferroptosis, and the GSH-GPX4 antioxidant system plays a central role in the ferroptosis pathway. While the existing ferroptosis inducer RSL3 can covalently bind to the GPX4 protein and inhibit its function, it suffers from low selectivity and unsatisfactory in vivo antitumor effects. Therefore, developing novel GPX4 inhibitors with high selectivity and efficacy is an urgent scientific challenge. Summary of the Invention

[0006] In view of this, the present invention proposes a ferroptosis agonist RSL3-ClAc molecule, which combines nanodelivery technology to solve the problems of poor water solubility, low bioavailability and poor clinical efficacy of the existing anticancer drug RSL3.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: One objective of this invention is to provide a ferroptosis agonist, RSL3-ClAc, wherein the chemical structural formula of RSL3-ClAc is: .

[0008] This invention synthesizes a novel ferroptosis agonist, RSL3-ClAc, by introducing a chloroacetyl fragment into the RSL3 molecule, which significantly enhances its binding ability to GPX4 and thus improves the ferroptosis induction efficiency.

[0009] Photodynamic therapy (PDT), as an emerging cancer treatment method, delivers photosensitizers to the tumor site and activates them with near-infrared radiation, generating reactive oxygen species (ROS) to induce tumor cell apoptosis and necrosis. However, the hypoxic tumor microenvironment limits the efficacy of PDT. Studies have shown that combining PDT with ferroptosis induction therapy can overcome the limitations of single therapy and enhance anti-tumor effects. For example, ROS generated during ferroptosis can alleviate the hypoxia problem of PDT, while ROS generated by phototherapy can effectively promote ferroptosis. In addition, the local thermal effect induced by phototherapy can increase the permeability of cancer cells, improve drug uptake, and thus reduce the required drug dosage. RSL3 has low water solubility, poor biocompatibility, and serious side effects, which limits its application. Utilizing nanoprodrugs to deliver insoluble drugs to cancer cells is an attractive strategy. Small molecule self-assembled nanomedicines can effectively trigger the release of active drugs in the tumor microenvironment, with significant advantages such as large drug loading, few side effects, and minimal tissue damage.

[0010] Therefore, a second objective of this invention is to provide an oxygen-deficiency-responsive nanocomposite delivery system, which is self-assembled from the RSL3-ClAc and the photosensitizer TCy5-NO2 in a nano-encapsulation material.

[0011] Furthermore, the mass ratio of RSL3-ClAc to photosensitizer TCy5-NO2 is 10~15:1.

[0012] Furthermore, the nano-encapsulating material is PEG. 1000 .

[0013] Furthermore, the particle size of the hypoxia-responsive nanocomposite delivery system is 180~200 nm.

[0014] The third objective of this invention is to provide a method for preparing the aforementioned hypoxia-responsive nanocomposite delivery system, comprising the following steps: RSL3-ClAc and photosensitizer TCy5-NO2 were mixed in DMSO and injected into an ultrasonically vibrating aqueous phase. The resulting clear nanoparticle solution was dialyzed to remove organic solvents and then further concentrated by ultrafiltration. The resulting nanoparticles were mixed with nano-encapsulated materials, ultrasonicated, dialyzed, and concentrated by ultrafiltration to obtain the hypoxia-responsive nano-co-delivery system.

[0015] Furthermore, the mass ratio of the nanoparticles to the nano-encapsulating material is 1:8~12.

[0016] The fourth objective of this invention is to provide the application of the hypoxia-responsive nano-co-delivery system in the preparation of antitumor drugs.

[0017] Furthermore, the tumor includes colorectal cancer.

[0018] The fifth objective of this invention is to provide the application of the hypoxia-responsive nanocomposite delivery system in tumor imaging products.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention synthesizes a novel ferroptosis inducer, RSL3-ClAc, enhancing its binding affinity to GPX4 and improving ferroptosis induction efficiency. RSL3-ClAc is then self-assembled with the hypoxia-responsive photosensitizer TCy5-NO2 to construct a nanomedicine delivery system. Utilizing the EPR effect and hypoxia-responsive properties of the nanocarrier, this system can overcome the tumor barrier and precisely locate lesions. Figure 1 Finally, through the synergistic effect of enhanced ferroptosis and photodynamic therapy, the growth of colorectal cancer was significantly reduced and the survival rate of mice was improved.

[0020] This invention utilizes molecular design and nano-encapsulation technology to modify RSL3 with chloroacetyl groups, thereby significantly improving the drug targeting of tumor drug delivery systems and enhancing the therapeutic effect of colorectal cancer, providing a new and effective strategy for tumor diagnosis and treatment. Attached Figure Description

[0021] Figure 1A schematic diagram illustrating the mechanism of hypoxia-responsive nanocommunotransmission system in the synergistic treatment of colorectal cancer ferroptosis and photodynamic therapy.

[0022] Figure 2 This is a synthetic route diagram for RSL3-ClAc.

[0023] Figure 3 The image shows the proton NMR spectrum of RSL3-ClAc.

[0024] Figure 4 This is the carbon NMR spectrum of RSL3-ClAc.

[0025] Figure 5 For the molecular performance evaluation of RSL3-ClAc. A: Effects of different concentrations of RSL3-ClAc and RSL3 on the viability of CT26, 4T1, and B16 cells. B: Changes in GPX4 protein expression levels in CT26, 4T1, and B16 cells after treatment with RSL3-ClAc and RSL3. C: Molecular interactions between RSL3-ClAc, RSL3, and GPX4 protein at different concentrations.

[0026] Figure 6 Properties of RC / TCN@PEG nanoparticles. A: TEM image of RC / TCN@PEG nanoparticles. B: Fluorescence imaging of RC / TCN@PEG in the presence of NTR. C: ROS generation of RC / TCN@PEG in the presence of NTR.

[0027] Figure 7 To verify the cytotoxic effects of nanoparticles in vitro. A: IncuCyte live-cell imaging was used to monitor the cytotoxic effects of different nanoparticles at different concentrations on CT-26 and 4T1 cells under hypoxic / noroxic conditions. B: IncuCyte live-cell imaging was used to monitor the cytotoxic effects of different nanoparticles at different concentrations on CT-26 and 4T1 cells under conditions with and without laser excitation.

[0028] Figure 8 Visualization of hypoxia induction at the tumor site after administration of TCy5-NO2 and RC / TCN@PEG. A: Schematic diagram of CT-26 cell in situ tumor bearing in mouse abdomen and drug administration. B: In vivo imaging of mice within 12 h after drug administration. C: Quantitative fluorescence signal intensity at different time points in the tumor site (Right) and normal mammary gland site (Left) after RC / TCN@PEG administration. D: Quantitative fluorescence signal intensity at different time points in the tumor site (Right) and normal mammary gland site (Left) after TCy5-NO2 administration.

[0029] Figure 9This study validates the antitumor properties of nanoparticles in mice. A: Schematic diagram of CT26 cell tumor formation in the groin region of mice and drug administration. B: Tumor size after different drug administration treatments. C: Tumor weight after different drug administration treatments. D: Tumor volume after different drug administration treatments. E: Changes in body weight after different drug administration treatments. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] Compound 1a in the following examples is L-tryptophan methyl ester, CAS No.: 4299-70-1, and compound 1b is 4-(chloroacetoxy)benzaldehyde, CAS No.: 444286-13-9. The photosensitizer TCy5-NO2 is disclosed in the literature: Han, F.; Abbas Abedi, SA; He, S.; Zhang, H.; Long, S.; Zhou, X.; Chanmungkalakul, S.; Ma, H.; Sun, W.; Liu, X.; Du, J.; Fan, J.; Peng, X. Aryl-modified pentamethyl cyanine dyes at the C2′position: a tunable platform for activatable photosensitizers. Adv. Sci. 2024, 11, 2305761 DOI: 10.1002 / advs.202305761

[0032] Example 1: Synthesis and Characterization of RSL3-ClAc 1. Experimental Methods (1) Synthesis of compound 1 Compound 1b (1.09 g, 5 mmol) and compound 1a (990 mg, 5 mmol) were dissolved in anhydrous dichloromethane (50 mL) in a 100 mL round-bottom flask and stirred at room temperature for 5 min. Trifluoroacetic acid (40 μL, 0.5 mmol) was then added dropwise and refluxed for 60 min. The formation of Schiff bases was monitored by TLC. After the reactants were confirmed to be converted to Schiff bases, trifluoroacetic acid (1140 μL, 0.5 mmol) was added, and the mixture was refluxed and stirred overnight. After the reaction was complete, the mixture was cooled to room temperature, quenched with saturated NaHCO3 solution, and neutralized to a slightly alkaline state. The mixture was extracted with dichloromethane, and the combined organic phases were washed twice with saturated brine (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain compound 1.

[0033] (2) Synthesis of compound RSL3-ClAc Compound 1 was dissolved in anhydrous dichloromethane (30 mL) in a 100 mL round-bottom flask, and sodium bicarbonate (1.1 eq) was added. The mixture was stirred at room temperature for 5 min. Chloroacetyl chloride (1.0 eq) was added dropwise under ice bath conditions. The reaction was monitored by TLC. If compound 1 was not completely reacted, chloroacetyl chloride (0.5 eq) was added until the reaction was complete. The reaction was quenched with distilled water. The mixture was extracted with ethyl acetate, and the organic phases were combined and washed twice with saturated brine (50 mL). After drying with anhydrous Na2SO4, the mixture was concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1 (v / v), containing 1% triethylamine) and separated by thin-layer chromatography on silica gel preparative plates to obtain the corresponding final product RSL3-ClAc.

[0034] Figure 2 This is the synthetic route diagram for RSL3-ClAc. Figure 3 The image shows the proton NMR spectrum of RSL3-ClAc. Figure 4 This is the carbon NMR spectrum of RSL3-ClAc.

[0035] 2. Experimental Results This embodiment obtains the RSL3-ClAc molecule through chemical synthesis. Figure 3 and Figure 4 The structure of the compound was identified by nuclear magnetic resonance spectroscopy, confirming the successful formation of the compound.

[0036] Figure 5 Performance evaluation of RSL3-ClAc molecules. Figure 5A represents the cell viability of CT26, 4T1, and B16 cells cultured at different concentrations of RSL3-ClAc and RSL3 after 48 hours. The results show that both RSL3-ClAc and RSL3 significantly reduced cell viability, and RSL3-ClAc exhibited stronger cytotoxicity compared to RSL3. Figure 5 As shown in Figure B, the immunoblotting results of the protein showed that RSL3-ClAc and RSL3 treatment significantly reduced the expression level of GPX4 protein, and the effect of RSL3-ClAc was more significant. This indicates that RSL3-ClAc induces ferroptosis in cells by inhibiting the expression of GPX4 protein. Figure 5 Using surface plasmon resonance (SPR) technology, C observed that the binding ability of RSL3-ClAc to GPX4 increased with increasing concentration, further indicating that RSL3-ClAc can effectively bind to the GPX4 protein.

[0037] Example 2: Preparation and performance evaluation of an oxygen-deficiency-responsive nanocomposite delivery system 1. Experimental Methods (1) Preparation of hypoxia-responsive nanocomposite delivery system The nanoparticles RSL3 / TCy5-NO2@PEG and RSL3-ClAc / TCy5-NO2@PEG were prepared by nanoprecipitation.

[0038] RSL3-ClAc and TCy5-NO2 were dissolved separately in DMSO and mixed at a molar ratio of RSL3-ClAc:TCy5-NO2 = 10:1. The mixture was then injected at a uniform rate into ultrapure water subjected to ultrasonic oscillation (frequency 45 kHz, temperature 40 °C). After sonication for 2 hours, the nanoparticle mixture was placed in a dialysis bag (MW3000) and dialyzed in ultrapure water to remove organic solvents and small organic molecules. Subsequently, the nanoparticle solution was further concentrated by ultrafiltration to obtain RSL3-ClAc / C2-NO2 nanoparticles. These nanoparticles were then mixed with PEG1000 (mass ratio 1:10), ultrasonicated, dialyzed, and concentrated by ultrafiltration (under the same conditions). The resulting nanoparticles were stored in a refrigerator for subsequent experiments, thus obtaining the hypoxia-responsive nanocomposite delivery system RSL3-ClAc / T-NO2@PEG, abbreviated as RC / C-NO2@PEG or RC / TCN@PEG.

[0039] The preparation method of RSL3 / TCy5-NO2@PEG is basically the same as that of RSL3-ClAc / TCy5-NO2@PEG, except that RSL3-ClAc is replaced with RSL3. RSL3 / TCy5-NO2@PEG will be abbreviated as R / C-NO2@PEG or R / TCN@PEG.

[0040] (0) Performance Evaluation Figure 6 Properties of RSL3-ClAc / TCy5-NO2@PEG nanoparticles. Figure 6 A represents the morphological characteristics of the nanoparticles observed using transmission electron microscopy. The prepared nanoparticles exhibit a regular spherical structure with an average particle size of 190±10 nm. Figure 6 B-mode imaging, using an IVIS imaging system, showed that the fluorescence intensity of the nanoparticles was significantly enhanced in the presence of NTR, demonstrating that the photosensitizer TCy5-NO2 was effectively activated. Figure 6 The C-value was evaluated for nanoparticles under 650 nm laser irradiation and non-irradiation conditions in the presence of NTR (nitroreductase), with the addition of TEMPO (radical scavenger) to reflect the different ROS generated by the drugs. 1 The results showed that nanoparticles can efficiently generate ROS under NTR induction.

[0041] 2. Experimental Results Figure 7 To verify the cytotoxic effect of nanoparticles in vitro and in cells. Experimental results are as follows: Figure 8 As shown in A and 8B, simple illumination (650 nm, 100 mW / cm²) 2 Treatment with hypoxia (20 min) or anaerobic conditions (2% O2) had no significant effect on cell growth; however, under the same light parameters, cell viability gradually decreased with increasing nanoparticle concentration, indicating that photodynamic therapy can effectively inhibit the proliferation of CT-26 and B16 cells. Simultaneously, at the same nanoparticle concentration, cell viability under hypoxic conditions was lower than under normal conditions, suggesting that the hypoxic environment enhanced the photodynamic effect of the photosensitizer.

[0042] Figure 8 Visualization of hypoxia induction in tumor sites by TCy5-NO2 and RC / TCN@PEG. (Example) Figure 8 As shown in Figure A, we established a 4T1 orthotopic breast cancer BALB / c mouse model and administered the drug. When the tumor volume reached 50 mm... 3 At the same time, free TCy5-NO2 (5 mg / kg) and RC / TCN@PEG (5 mg / kg) were injected into the adjacent breast tissue and normal breast tissue, respectively. Figure 8 Figure B shows the results of evaluating the biodistribution of nanoparticles in vivo using the IVIS in vivo imaging system. Dynamic fluorescence distribution of nanoparticles at 1, 2, 4, 6, 8, and 12 h was imaged using a 716 nm excitation filter (exposure time: 100 ms). The experiment showed that fluorescence at the tumor site in mice was stronger than that at the normal mammary gland site, indicating that hypoxia in the tumor tissue activated the photosensitizer, such as... Figure 8 As shown in C and D.

[0043] Figure 9 To verify the antitumor properties of nanoparticles in mice. Figure 9 A represents the establishment and drug administration process for a tumor-bearing mouse model. CT-26 tumor-bearing BALB / c mice were established, and the tumor volume was measured to be 50 mm². 3 Mice were randomly divided into 7 groups (n=7). PBS+L, RSL3-ClAc, TCy5-NO2+L, R / TCN@PEG, RC / TCN@PEG, R / TCN@PEG+L, and RC / TCN@PEG+L were administered intravenously on days 0, 3, 6, 9, and 12 (5 mg / kg). Three hours after injection, all mice in the PBS+L, TCy5-NO2+L, R / TCN@PEG+L, and RC / TCN@PEG+L groups received near-infrared irradiation for 10 min (λex = 650 nm; 100 mW / cm²). 2 Body weight and tumor volume were measured at 0, 3, 6, 9, 12, and 15 days. Mice were euthanized on day 15, and cancerous tissue and major organs were collected. Body weight records of cancerous tissue were preserved. The results showed that the combined treatment with two drugs had a better inhibitory effect than single-drug therapy, and the tumor-suppressive ability was significantly improved after the drugs were co-assembled (BE in Figure 9).

[0044] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0045] 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 both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A hypoxia-responsive nanocomposite delivery system, characterized in that, It is self-assembled from the ferroptosis agonist RSL3-ClAc and the photosensitizer TCy5-NO2 in a nano-supported material, wherein the nano-supported material is PEG1000, and the chemical structural formula of RSL3-ClAc is as follows: 。 2. The hypoxia-responsive nanocomposite delivery system according to claim 1, characterized in that, The molar ratio of RSL3-ClAc to photosensitizer TCy5-NO2 is 10~15:

1.

3. The hypoxia-responsive nanocomposite delivery system according to claim 2, characterized in that, The particle size of the hypoxia-responsive nanocomposite delivery system is 180~200 nm.

4. A method for preparing an oxygen-responsive nanocomposite delivery system according to any one of claims 1 to 3, characterized in that, Includes the following steps: RSL3-ClAc and photosensitizer TCy5-NO2 were mixed in DMSO and injected into an ultrasonically vibrating aqueous phase. The resulting clear nanoparticle solution was dialyzed to remove organic solvents and then further concentrated by ultrafiltration. The resulting nanoparticles were mixed with nano-encapsulated materials, ultrasonicated, dialyzed, and concentrated by ultrafiltration to obtain the hypoxia-responsive nano-co-delivery system.

5. The method for preparing a hypoxia-responsive nanocomposite delivery system according to claim 4, characterized in that, The mass ratio of the nanoparticles to the nano-encapsulated material is 1:8~12.

6. The application of the hypoxia-responsive nano-co-delivery system according to any one of claims 1 to 3 in the preparation of antitumor drugs, characterized in that, The tumor is colorectal cancer.

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