Preparation of novel ferroptosis micelle capable of consuming NADPH (Nicotinamide Adenine Dinucleotide Phosphate)

Abstract

The invention discloses preparation and application of a novel ferroptosis micelle capable of consuming NADPH (Nicotinamide Adenine Dinucleotide Phosphate). The micelle is formed by self-assembly of an amphiphilic copolymer mPEG-Azo-P (Asp-NI)-Ce6, a hydrophilic segment is mPEG and is connected with a hydrophobic segment P (Asp-NI) through an azobenzene connecting bridge with hypoxia response, and the hydrophobic segment contains a nitroimidazole group with dual response to hypoxia and singlet oxygen and is bonded with a photosensitizer Ce6. The micelle can be further used for physically encapsulating a hypoxia activated prodrug terapazamine. The micelle disclosed by the invention can respond to a hypoxia condition in a tumor microenvironment to realize PEG shedding and enhance cellular uptake; meanwhile, singlet oxygen generated by photodynamic therapy is responded, so that micelle disintegration and quick release of the medicine are realized. By combining the Ce6-mediated photodynamic therapy and the PDT-activated TPZ chemotherapy, the synergistic anti-tumor effect is achieved. The invention also provides a preparation method of the micelle, which is simple and convenient to operate and good in reproducibility. The nano platform provides a new strategy for efficient co-delivery of the photosensitizer and the chemotherapeutic drug, and particularly shows a good application prospect in breast cancer treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomaterials, specifically to the preparation of a novel NADPH-consuming ferroptosis micelle for the synergistic delivery of photosensitizers and hypoxia-activating prodrugs, enabling combined photodynamic therapy and chemotherapy. Background Technology

[0002] Cancer is one of the major diseases threatening human health. Traditional surgery, chemotherapy, and radiotherapy have limited efficacy and significant side effects. Photodynamic therapy (PDT), as a non-invasive treatment, has attracted widespread attention due to its low toxicity, high selectivity, and controllability. PDT utilizes photosensitizers activated under specific wavelengths of light to convert oxygen molecules in tissues into reactive oxygen species such as singlet oxygen, which are cytotoxic, thereby killing tumor cells.

[0003] However, the efficacy of phototherapy (PDT) is heavily dependent on the oxygen content of tumor tissue. Hypoxic areas are often present within solid tumors, limiting the effectiveness of PDT. Furthermore, the PDT process itself consumes oxygen, further exacerbating tumor hypoxia and creating a therapeutic paradox. On the other hand, reactive oxygen species have short lifespans and limited diffusion distances, requiring photosensitizers to be efficiently released and concentrated at the tumor site.

[0004] To address these challenges, researchers have attempted to develop various intelligent nanomedicine delivery systems. Utilizing the hypoxic tumor microenvironment to achieve efficient cellular internalization of nanocarriers, on-demand drug release, and synergistic therapy with hypoxia-activated prodrugs (such as telapazaryamine, TPZ) has emerged as a promising strategy. However, designing a multifunctional nanoplatform capable of simultaneously responding to hypoxia and singlet oxygen, and synergistically regulating drug release, cellular uptake, and therapeutic activation remains a challenge. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of the prior art and provide a polymer micelle that can simultaneously respond to hypoxia and singlet oxygen. This micelle can synergistically deliver the photosensitizer Ce6 and the hypoxia-activated prodrug TPZ, thereby achieving enhanced cellular uptake, accelerated drug release, and synergistic anti-tumor effects of PDT and chemotherapy through a "three birds with one stone" strategy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A novel NADPH-consuming iron death micelle, characterized in that the polymeric micelles are formed by the self-assembly of an amphiphilic copolymer mPEG-Azo-P(Asp-NI)-Ce6, wherein: mPEG is the hydrophilic segment; Azo is an azophenyl group, serving as a hypoxia-responsive linking bridge; P(Asp-NI) is a hydrophobic segment in which the side chain of the aspartic acid polymer is connected to a nitroimidazole group, which is responsive to both low oxygen and singlet oxygen. Ce6 is a photosensitizer, chlorine (E6), which is chemically bonded to the polymer chain.

[0007] Furthermore, the micelles physically encapsulate the hypoxia-activated prodrug telapazidine, forming drug-loaded micelles DHM-Ce6@TPZ.

[0008] The micelles have a particle size of 150-200 nm.

[0009] This invention also provides a method for preparing a novel NADPH-consuming iron death micelle, comprising the following steps: 1. Synthesis of mPEG-Azo-P(Asp-NI)-Ce6 polymer: a. By amidation, an azophenyl group is attached to the end of mPEG to obtain mPEG-Azo-NH2.

[0010] b. Using mPEG-Azo-NH2 as an initiator, the ring-opening polymerization of aspartic N-carboxylic anhydride was induced to obtain mPEG-Azo-PAsp.

[0011] c. The nitroimidazolium group is attached to the side chain of polyaspartic acid via ammonolysis to obtain mPEG-Azo-P(Asp-NI).

[0012] d. The photosensitizer Ce6 was linked to the polymer chain via a carbodiimide-mediated amidation reaction to obtain the target polymer mPEG-Azo-P(Asp-NI)-Ce6.

[0013] 2. Preparation of drug-loaded micelles: Drug-loaded micelles were prepared using a thin-film hydration method. The polymer mPEG-Azo-P(Asp-NI)-Ce6 and the drug TPZ were dissolved together in an organic solvent, and a thin film was formed by rotary evaporation. The film was then hydrated with an aqueous solution, stirred, dialyzed, filtered, and lyophilized to obtain the drug-loaded micelles DHM-Ce6@TPZ.

[0014] The present invention also provides the application of the novel NADPH-consuming ferroptosis micelles in the preparation of medicaments for the treatment of breast cancer.

[0015] The beneficial effects of this invention are as follows: 1. Dual Response and Synergistic Therapy: Micelles simultaneously respond to the tumor's intrinsic hypoxic microenvironment and the singlet oxygen generated by PDT. Hypoxia leads to the cleavage of azobenzene and the shedding of PEG, enhancing cellular uptake; singlet oxygen oxidizes nitroimidazole, causing micelle disintegration and rapid drug release. Simultaneously, the oxygen consumption of PDT further activates the chemotherapeutic effect of TPZ, forming a cascade synergistic effect between PDT and chemotherapy.

[0016] 2. "Killing three birds with one stone" functional integration: One system simultaneously solves three key problems: ① Enhances cellular uptake through hypoxia response; ② Achieves rapid on-demand drug release through dual response; ③ Activates chemotherapy by utilizing the side effects (oxygen consumption) of PDT, turning disadvantages into advantages.

[0017] 3. Highly efficient targeting and enrichment: Nanoscale micelles can be enriched at the tumor site through the EPR effect, improving therapeutic efficacy and reducing systemic toxicity.

[0018] 4. Good biocompatibility: Both in vitro and in vivo experiments have demonstrated that this micelle system has low toxicity to normal cells and good biocompatibility. Attached Figure Description

[0019] Figure 1 This diagram illustrates the use of hypoxia- and singlet oxygen-responsive polymeric micelles in a mouse model carrying tumors to integrate photodynamic therapy and chemotherapy.

[0020] Figure 2 Physicochemical properties of hypoxic and singlet oxygen-responsive micelles (n = 3). (A) Hydrodynamic dimensions of DHM-Ce6 and DHM-Ce6@TPZ. Transmission electron microscopy images of DHM-Ce6 (B) and DHM-Ce6@TPZ (C) (scale bar: 500 nm). (D) UV-Vis absorption spectra of Ce6, TPZ, DHM, DHM-Ce6, and DHM-Ce6@TPZ. (E) Normalized absorbance of different formulations at 411 nm DPBF under illumination. (F) After laser treatment (660 nm, 200 mW / cm²). 2 TEM images of DHM-Ce6@TPZ micelles (10 min). Scale bar: 500 nm. (G) UV-Vis spectrum of DHM-Ce6@TPZ treated with sodium sulfite (10 mM). Simulated release of TPZ from DHM-Ce6@TPZ micelles under hypoxic conditions. (H) (10 mM sodium sulfite) and under laser irradiation (I) (660 nm, 200 mW / cm). 2 Release status (10 minutes).

[0021] Figure 3Confocal images of 4T1 cells after incubation with DHM-Ce6@TPZ micelles for 2, 4, and 6 hours under normoxic or hypoxic conditions (scale bar: 20 μm, n=3).

[0022] Figure 4 Survival rates of 4T1 cells under normoxic or hypoxic conditions (n=4) under different formulations. (A) DHM-Ce6@TPZ treatment group without laser irradiation under normoxic or hypoxic conditions. (B) DHM-Ce6 treatment group with laser irradiation under normoxic or hypoxic conditions. (C) DHM-Ce6@TPZ treatment group without laser irradiation under normoxic or hypoxic conditions. (D) Imaging of live and dead cells after treatment with a fixed TPZ dose of 1 μg / mL. Cells were stained with calcein AM (green, live cells) and propidium iodide (red, dead cells) (scale bar: 20 μm).

[0023] Figure 5 Fluorescence imaging of total reactive oxygen species (ROS) in 4T1 cells after treatment with different formulations under different conditions. ROS was detected using the DCFH-DA probe (scale bar: 50 μm), and the TPZ dose was fixed at 1 μg / mL (n=3).

[0024] Figure 6 for Figure 5 Dynamic biodistribution of micelle formulations in 4T1 tumor-bearing mice (n=3). (A) In vivo dynamic fluorescence of Cy5 in 4T1 tumor-bearing mice after intravenous injection of free Cy5 and DHM@Cy5 micelles, lasting up to 24 hours. (B) Dynamic fluorescence quantitative analysis of Cy5 in tumors. (C) Semi-quantitative fluorescence summary of Cy5 levels in healthy organs and tumors at the end of the biodistribution study. ***p<0.001. (D) Ex vivo fluorescence analysis of Cy5 in tumors and other healthy organs 24 hours after administration.

[0025] Figure 7 To assess the in vivo antitumor efficacy of a novel NADPH-depleting ferroptotic micelle (n = 5). (A) Tumor growth inhibition effects of seven formulations. (B) Quantitative analysis of tumor weight. (C) Images of tumors at the end of treatment. (D) Changes in mouse body weight during treatment. (E) TUNEL staining (scale bar: 50 μm) and H&E staining (scale bar: 100 μm) at the end of treatment. **p<0.01; ***p<0.001.

[0026] Figure 8 Histological analysis of major healthy organs (heart, liver, spleen, lungs, and kidneys) at the end of the efficacy study (scale bar: 100 micrometers). Detailed Implementation

[0027] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Example 1 Synthesis of polymer mPEG-Azo-P(Asp-NI)-Ce6 1. Reaction of mPEG-NHS with 4,4'-diaminoazobenzene yields mPEG-Azo-NH2.

[0029] 2. Using mPEG-Azo-NH2 as an initiator, the ring-opening polymerization of BLA-NCA was initiated under anhydrous conditions to obtain mPEG-Azo-PBLA.

[0030] 3. mPEG-Azo-PAsp was obtained by hydrazine protection under alkaline conditions.

[0031] 4. Reaction of mPEG-Azo-PAsp with excess 2-nitroimidazole in the presence of condensing agent EDC / NHS, and attachment of nitroimidazole groups to the carboxyl groups of polyaspartic acid side chains by ammonolysis to obtain mPEG-Azo-P(Asp-NI).

[0032] 5. Ce6, EDC, and NHS were dissolved in formamide to activate their carboxyl groups. Then, a formamide solution of mPEG-Azo-P(Asp-NI) was added, and the reaction was carried out under nitrogen protection for 24 hours. The reaction solution was dialyzed against deionized water for 2 days, and then lyophilized to obtain the final product mPEG-Azo-P(Asp-NI)-Ce6. 1 The structure was confirmed by H-NMR, and its molecular weight was calculated to be approximately 10560 Da.

[0033] Example 2 Preparation of drug-loaded micelles DHM-Ce6@TPZ 30 mg of the polymer synthesized in Example 1 and 3 mg of TPZ were dissolved together in 25 mL of acetonitrile. The solvent was removed by rotary evaporation at 40°C, forming a uniform film at the bottom of the flask. Then, 30 mL of deionized water was added, and the mixture was magnetically stirred at room temperature for 45 minutes to hydrate. The resulting emulsion was centrifuged at 2000 rpm for 5 minutes to remove undispersed impurities. The supernatant was then placed in a dialysis bag (MWCO 3500 Da) and dialyzed against deionized water for 2 days to remove unencapsulated TPZ and organic solvent. Finally, the dialyzed micelle solution was filtered through a 0.45 μm filter membrane and lyophilized to obtain drug-loaded micelles DHM-Ce6@TPZ. The micelle size was determined to be 189 ± 15 nm, the TPZ drug loading was 7.38% (w / w), and the Ce6 binding content was 4.2% (w / w).

[0034] Example 3 In vitro responsive drug release studies of micelles Release experiments were conducted using a Franz diffusion cell. 2 mL of DHM-Ce6@TPZ aqueous solution (2.5 mg / mL) was added to the donor chamber, and the recipient solution was PBS containing 5% SDS.

[0035] Hypoxia response release group: 10 mM Na2S2O4 was added to the recipient fluid to simulate a hypoxic environment.

[0036] Singlet oxygen-responsive release group: Laser irradiation (660 nm, 200 mW / cm) of micelle solution in the donor chamber. 2 After 10 minutes, the release experiment was conducted again.

[0037] Control group: No Na2S2O4 added or no laser irradiation.

[0038] Samples were taken at predetermined time points, and the cumulative release of TPZ was determined using UV-vis. Results are as follows: Figure 2 As shown, under hypoxic or laser irradiation conditions, the release rate and extent of TPZ were significantly higher than those of the control group, demonstrating the stimulus-responsive drug release characteristics of micelles.

[0039] Example 4 Evaluation of in vivo anti-tumor effects Female BALB / c mice were selected and subcutaneously inoculated with 4T1 breast cancer cells. Tumors were allowed to grow to approximately 100 mm in size. 3 At that time, the mice were randomly divided into 6 groups (n=5): 1. PBS + laser 2. Free TPZ + Laser 3. DHM-Ce6 (without laser) 4. DHM-Ce6 + Laser 5. DHM-Ce6@TPZ (No laser) 6. DHM-Ce6@TPZ + Laser All formulations were administered via tail vein injection, with a Ce6 dose of 5 mg / kg. Eight hours post-injection, tumor sites were irradiated in groups requiring phototherapy (660 nm, 200 mW / cm²). 2 (30 minutes). Tumor volume and body weight were measured every two days. Mice were sacrificed after 21 days, and tumors and major organs were collected for histological analysis.

[0040] Result: As Figure 7As shown, group 6 (DHM-Ce6@TPZ + laser) exhibited the most significant tumor inhibition effect, with tumor volume and weight significantly smaller than other groups, and mouse body weight remained stable, indicating low systemic toxicity. H&E and TUNEL staining revealed the most severe tumor cell necrosis and apoptosis in this group. No obvious pathological damage was observed in the staining of major organs. Figure 8 This demonstrates that the nanosystem has good biosafety. This invention, through Example 1, details the controllable synthetic pathway of the target polymer mPEG-Azo-P(Asp-NI)-Ce6. Through a multi-step reaction, a smart polymer integrating a hydrophilic segment (mPEG), a hypoxia-responsive bridge (Azo), a dual-responsive hydrophobic segment (P(Asp-NI)), and a photosensitizer (Ce6) was successfully constructed. 1H-NMR confirmed the chemical structure and calculated the polymer molecular weight to be approximately 10560 Da, demonstrating the feasibility of the synthetic route and the clarity of the product; Example 2: TPZ-loaded micelles DHM-Ce6@TPZ were successfully prepared using the classic thin-film hydration method. This method is mature, reproducible, and easily scalable. The obtained micelles have an ideal nanoscale size (189 ± 15 nm), which is beneficial for enrichment at the tumor site through the EPR effect. The drug delivery system has high drug loading efficiency: TPZ drug loading is 7.38%, and Ce6 binding is 4.2%, ensuring effective delivery of therapeutic components. Example 3: Through in vitro release experiments, it was conclusively demonstrated that the micelles possess the designed dual stimulation response: firstly, a hypoxia response, in a simulated hypoxic environment (Na2S2O4), the release rate and extent of TPZ are significantly higher than the normoxic control group; secondly, a singlet oxygen response, after laser irradiation (660 nm), the release of TPZ is also significantly accelerated. This experiment directly confirms that micelles can achieve on-demand rapid drug release under tumor microenvironment (hypoxia) and external laser triggering (generating singlet oxygen), solving the problems of short ROS diffusion distance and slow drug release in traditional PDT. The animal experiments in Example 4 are key to proving the value of this invention. The results show that group 6 (DHM-Ce6@TPZ + laser) It exhibited the most significant tumor growth inhibition effect, with efficacy far superior to single treatment groups (such as the DHM-Ce6+ laser group with PDT only) or non-responsive treatment groups. This fully demonstrates the success of the cascade synergistic treatment strategy of "PDT-exacerbated hypoxia-activated chemotherapy". Photodynamic therapy not only directly kills tumors, but its oxygen-consuming side effects are also utilized to activate the chemotherapeutic effect of TPZ, forming a positive cycle with the responsive drug release of micelles, producing a synergistic effect of "1+1>>2". Throughout the treatment period, the body weight of mice in each treatment group remained stable, and no obvious systemic toxicity reactions were observed. H&E staining analysis of major organs (heart, liver, spleen, lung, and kidney) did not reveal obvious pathological damage, demonstrating that the nanodelivery system has good biocompatibility and providing important safety evidence for its clinical translation.

[0041] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A novel iron-death micelle that consumes NADPH, characterized in that, It is formed by the self-assembly of an amphiphilic copolymer with the general formula mPEG-Azo-P(Asp-NI)-Ce6; wherein, mPEG is a hydrophilic polyethylene glycol segment; Azo is an azophenyl group, which serves as a low-oxygen responsive connecting bridge; P(Asp-NI) is a hydrophobic segment, which is composed of a polyaspartic acid backbone and nitroimidazole groups connected to its side chains via amide bonds; Ce6 is a photosensitizer chloroe6 connected to the polymer via chemical bonds.

2. The novel iron-death micelles that can consume NADPH according to claim 1, characterized in that, The micelles physically encapsulate the hypoxia-activated prodrug telapazamine.

3. A novel iron-death micelle that consumes NADPH according to claim 1 or 2, characterized in that, The volume-weighted average hydrodynamic particle size of the micelles is 150 nm to 200 nm.

4. A method for preparing a novel NADPH-consuming iron-death micelle as described in claim 1, characterized in that, Includes the following steps: a) React mPEG-NHS with 4,4'-diaminoazobenzene to prepare mPEG-Azo-NH2; b) Using mPEG-Azo-NH2 as an initiator, the ring-opening polymerization of aspartic acid β-benzyl ester N-carboxylic anhydride was initiated to obtain mPEG-Azo-PBLA; c) The mPEG-Azo-PBLA was subjected to hydrazinolysis to remove the benzyl ester protecting group, yielding mPEG-Azo-PAsp; d) React mPEG-Azo-PAsp with 2-nitroimidazole in the presence of a condensing agent to obtain mPEG-Azo-P(Asp-NI); e) After activating the carboxyl group of photosensitizer Ce6, it reacts with the amino group on mPEG-Azo-P(Asp-NI) to obtain the final polymer mPEG-Azo-P(Asp-NI)-Ce6; f) The polymer obtained in step e) is self-assembled in water using a thin-film hydration method to form polymer micelles.

5. A method for preparing drug-loaded micelles as described in claim 2, characterized in that, The polymer mPEG-Azo-P(Asp-NI)-Ce6 described in claim 1 was dissolved together with telapazamin in an organic solvent, and drug-loaded micelles were prepared by thin-film hydration. Then, free drug and organic solvent were removed by dialysis.

6. The method according to claim 5, characterized in that, The organic solvent is acetonitrile.

7. The use of a novel NADPH-consuming ferroptosis micelle as described in any one of claims 1-3 or the drug-loaded micelle as described in claim 2 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The tumor is breast cancer.

9. The application according to claim 7, characterized in that, The antitumor drug is used in combination with photodynamic therapy and chemotherapy.

10. The application according to claim 9, characterized in that, The combined treatment involves activating the photosensitizer Ce6 to generate singlet oxygen through external light source irradiation for photodynamic therapy. Simultaneously, the oxygen-consuming hypoxic microenvironment exacerbated by photodynamic therapy activates the cytotoxicity of telapazarymine and, in conjunction with the dual responsiveness of micelles, enhances cellular uptake and drug release.

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