A nano material with pH / NIR / hypoxia multi-stage response, a preparation method and application thereof in preparation of an anti-tumor drug

Abstract

The application provides a kind of single near-infrared light irradiation simultaneously generates photothermal and photodynamic effect with pH / NIR / hypoxia multistage response nanomaterial (Por-COFs), it has excellent photothermal conversion efficiency and ROS generation capacity, stability and good biocompatibility;And through the porous of Por-COFs, Π-Π effect and electrostatic interaction load hypoxia activated antitumor drug tirapazamine (TPZ), finally with tumor targeting molecule hyaluronic acid HA on the surface of the prepared nanomedicine modification, preparation has pH / NIR / hypoxia multistage response tumor targeting nanomedicine (Por-COF@TPZ / HA). Nanomedicine with Por-COFs as main material when using 808 nm single laser irradiation produces excellent photothermal conversion and a large number of ROS realizes the combined treatment of photothermal therapy and photodynamic therapy, PDT treatment makes tumor microenvironment more hypoxic at the same time and then activates TPZ, realizes the synergistic treatment of photothermal, light and chemotherapy. Surface modification of nanomedicine HA improves the biocompatibility and tumor specificity of drug, and then improves the antitumor effect of nanomedicine.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, specifically relating to a multi-level responsive nanomaterial with pH / NIR / hypoxia (i.e., worm-like nanoporphyrin covalent organic frameworks Por-COFs with photothermal and photodynamic properties), its preparation method, and its application in the preparation of antitumor drugs. Background Technology

[0002] Cancer, especially malignant tumors, poses a serious threat to human health and life. Currently, the efficacy of the three commonly used clinical treatments—surgery, radiotherapy, and chemotherapy (CT)—is unsatisfactory. Multimodal synergistic anti-tumor strategies, combining two or more of these methods such as CT, chemodynamic therapy (CDT), photodynamic therapy (PDT), photothermal therapy, and immunotherapy, can leverage the advantages of different treatment methods. By utilizing different mechanisms of action to synergistically inhibit tumor growth, improve efficacy, and reduce side effects, this approach shows promising clinical application prospects and has become a research hotspot.

[0003] Nanotechnology plays a crucial role in combination therapies, serving as drug carriers and even enabling some nanomaterials to directly treat cancer. Combined photothermal therapy (PTT) and photodynamic therapy (PDT) have shown good efficacy in combating multidrug-resistant and hypoxia-related tumors. However, such systems often require the simultaneous loading of photothermal agents and photosensitizers, complicating the nanosystem and increasing metabolic toxicity. Furthermore, PTT and PDT require laser irradiation with different wavelengths, complicating subsequent experimental procedures. Covalent organic frameworks (COFs) possess unique advantages such as large surface area, tunable porous structure, high stability, low toxicity, and good biocompatibility. Moreover, the excitation wavelengths for PTT and PDT can be tuned by adjusting the conjugation degree of COFs, making COFs, especially nanoscale COFs, excellent candidate materials for biomedical applications in recent years. Based on this, this application was developed. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects of the prior art and provide a multi-level responsive nanomaterial with pH / NIR / oxygen deficiency (i.e., a Por-COFs worm-like nanoporphyrin covalent organic framework with photothermal and photodynamic properties), which has excellent photothermal conversion efficiency and ROS generation capacity, stability and good biocompatibility.

[0005] This invention also provides a method for preparing the above-mentioned pH / NIR / hypoxia multi-level responsive nanomaterials and their application in the preparation of antitumor drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing nanomaterials with pH / NIR / hypoxia multi-level response includes the following steps: 1) Synthesize 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin (TAPP). The preparation process can be carried out according to the literature J. Mater. Chem. B, 2021, 9, 3180; 2) A terephthalaldehyde acetonitrile solution was mixed with a TAPP dichloromethane solution, followed by the addition of ferric chloride acetonitrile solution and acetic acid. The mixture was dissolved by sonication and stirred for 12-16 h at room temperature in the dark to obtain a dark green suspension. After centrifugation and washing, dark green nanomaterials with pH / NIR / hypoxia multi-level response (Por-COFs) were obtained and stored in ultrapure water at 4 °C in the dark. The synthetic route of the pH / NIR / hypoxia multi-level response nanomaterials of this invention is shown below: .

[0007] Specifically, in step 2), 5.00-6.00 mg of terephthalaldehyde (TPAL) is dissolved in 10-15 mL of acetonitrile, and 5-6 mg of TAPP is dissolved in 10-15 mL of dichloromethane. The two solutions are then mixed, followed by the addition of 0.2-0.5 mL of a solution with a concentration of 48-52 mg / mL. −1 Add ferric chloride acetonitrile solution and 5-6 mL of acetic acid. Centrifuge at 12000-16000 rpm for 10-20 min.

[0008] Furthermore, the synthesis of TAPP in step 1) specifically involves: Tetra(4-nitrophenyl)porphyrin (TNPP) was dissolved in concentrated hydrochloric acid and nitrogen gas was passed through it for 15-25 min. Simultaneously, SnCl2∙2H2O was dissolved in concentrated hydrochloric acid and nitrogen gas was passed through it for 20-25 min. The two samples were then mixed and reacted at 80-90℃ for 30-40 min. After cooling to room temperature, the system was placed in an ice-water bath and the reaction continued for 1-2 h. The pH of the system was adjusted to neutral (this can be achieved by adding ammonia). The mixture was filtered, and 4-8% NaOH solution was added to the filtrate. A solid appeared under stirring. After filtration, washing, and vacuum drying, the crude product was obtained. The crude product was further purified in acetone using a Soxhlet extractor to obtain a high-purity purple solid (TAPP).

[0009] More preferably, 1-2 g of tetra(4-nitrophenyl)porphyrin can be dissolved in 45-55 mL of concentrated hydrochloric acid, and 4-5 g of SnCl2∙2H2O can be dissolved in 70-80 mL of concentrated hydrochloric acid.

[0010] This invention provides a multi-level responsive nanomaterial with pH / NIR / oxygen deprivation properties prepared by the above method.

[0011] This invention provides the application of the above-mentioned pH / NIR / hypoxia multilevel responsive nanomaterials in the preparation of antitumor drugs.

[0012] The present invention also provides a nanomedicine (Por-COF@TPZ / HA) comprising the above-mentioned pH / NIR / hypoxia multi-level responsive nanomaterial, which is prepared by the following steps: Dissolve 10 mg of pH / NIR / hypoxia multilevel responsive nanomaterials (Por-COFs) in 4-7 mL of ultrapure water, dissolve 4-6 mg of hypoxia-responsive drug teirazamin (TPZ) in 3-5 mL of ultrapure water, and dissolve 9-12 mg of hyaluronic acid (HA) in 4-7 mL of ultrapure water. Mix the three solutions by sonication until homogeneous, and react at room temperature in the dark for 20-25 h. After centrifugation and washing with ultrapure water, the final product is obtained.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a worm-like nanoporphyrin covalent organic framework (Por-COFs) that simultaneously generates photothermal and photodynamic effects under single near-infrared light irradiation. It exhibits excellent photothermal conversion efficiency, ROS generation capacity, stability, and good biocompatibility. The invention utilizes the porosity, π-π interaction, and electrostatic interaction of the COFs to load the hypoxia-activated antitumor drug teirazamine (TPZ). Finally, the surface of the prepared nanomedicine is modified with the tumor-targeting molecule hyaluronic acid (HA) to prepare a tumor-targeting nanomedicine (Por-COF@TPZ / HA) with multi-level pH / NIR / hypoxia responses. The prepared nanomedicine (Por-COF@TPZ / HA) generates excellent photothermal conversion and abundant ROS under 808 nm single laser irradiation, enabling combined PTT and PDT treatment. PDT treatment simultaneously further hypoxicizes the tumor microenvironment, thereby activating the hypoxic drug TPZ and achieving synergistic treatment of photothermal, photodynamic, and chemotherapy effects. HA surface modification improves the biocompatibility and tumor specificity of the nanomedicine, thus enhancing its antitumor efficacy. Attached Figure Description

[0014] Figure 1 Infrared spectrum of Por-COF@TPZ / HA; Figure 2The UV spectrum of Por-COF@TPZ / HA; Figure 3 TEM image of Por-COF@TPZ / HA; Figure 4 SEM image of Por-COF@TPZ / HA; Figure 5 Photothermal curves of Por-COF@TPZ / HA under 808 nm laser irradiation; Figure 6 ROS generation curve for Por-COF@TPZ / HA irradiated with 808 nm laser; Figure 7 The distribution of Por-COF@TPZ / HA in mice; (A) In vivo fluorescence imaging of mice after injection of Por-COF@TPZ / HA; (B) Relative fluorescence intensity in tumors at different time points after injection; (C) Fluorescence images of various organs 48 h after intravenous injection; (D) Relative fluorescence intensity of fluorescence images of various organs 48 h after intravenous injection. Figure 8 Figure 1 shows the in vitro cytotoxicity of Por-COF@TPZ / HA; (A) Concentration-dependent cytotoxicity of TPZ, Por-COF / HA, and Por-COF@TPZ / HA after 24 h of incubation with B16-F10 cells; (B) Saline+Laser, Por-COF / HA+Laser, and Por-COF@TPZ / HA+Laser (1.0 W cm⁻¹). -2 Concentration-dependent cytotoxicity after incubation with B16-F10 for 24 h (6 min) and 6 min). Detailed Implementation

[0015] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0016] In the following examples, all raw materials used are commercially available products that can be directly purchased or prepared using conventional methods in the art. Room temperature refers to 25±5 °C. Commercially available hydrochloric acid with a mass concentration of 36%-38% was used.

[0017] Example 1 A method for preparing a nanomedicine with pH / NIR / hypoxia multi-level response (Por-COF@TPZ / HA) includes the following steps: 1) Preparation of 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin: Tetra(4-nitrophenyl)porphyrin (TNPP) (1.3 g) was dissolved in 50 mL of concentrated hydrochloric acid, and nitrogen gas was passed through the solution for 20 min. Simultaneously, SnCl₂∙₂H₂O (19.1 mmol, 4.3 g) was dissolved in 70 mL of concentrated hydrochloric acid, and nitrogen gas was passed through the solution for 20 min. The two samples were then mixed and reacted at 80 ºC for 30 min. Heating was stopped, and the reaction mixture was cooled to room temperature. The mixture was then placed in an ice-water bath and the reaction continued for 1 h. Approximately 62.5 mL of ammonia (25%) was added to the reaction mixture to neutralize the concentrated hydrochloric acid and adjust the pH to neutral. A solid was observed, which was filtered through a sintered glass funnel, and the filtrate was collected. 100-120 mL of 5% NaOH solution was added to the filtrate, and the mixture was stirred vigorously. A solid was observed again, which was then filtered and collected. The filter cake was washed three times with distilled water and dried under vacuum at room temperature for 24 h. The crude product was further purified in acetone using a Soxhlet extractor to obtain a high-purity purple solid, 5,10,15,20-tetra(4-aminophenyl)-21H,23H-porphyrin (TAPP).

[0018] 2) Preparation of Por-COFs: Terephthalaldehyde (TPAL) (0.03727 mmol, 5.00 mg) was dissolved in 10 mL of acetonitrile, and purple solid TAPP (0.007409 mmol, 5 mg) was dissolved in 10 mL of dichloromethane. The two solutions were then mixed. Subsequently, 0.2 mL of a 50 mg / mL solution was added. −1 A ferric chloride acetonitrile solution and 5 mL of acetic acid were added. The mixture was completely dissolved under ultrasonic assistance and stirred overnight (12 h) at room temperature in the dark to obtain a dark green suspension. The mixture was then centrifuged (16000 rpm, 15 min) to obtain a dark green precipitate, which was washed with acetonitrile and ultrapure water to obtain dark green Por-COFs worm-like nanomaterials, which were stored in ultrapure water at 4 °C in the dark.

[0019] 3) Preparation of Por-COF@TPZ / HA: 10 mg of Por-COFs were dissolved in 5 mL of ultrapure water, 5 mg of the hypoxia-responsive drug teirazamin (TPZ) was dissolved in 3 mL of ultrapure water, and 10 mg of hyaluronic acid (HA) was dissolved in 5 mL of ultrapure water. The three solutions were ultrasonically mixed and stirred at room temperature in the dark for 24 h. The mixture was then centrifuged (16000 rpm, 15 min) to obtain a dark red precipitate, which was washed three times with ultrapure water to obtain drug-loaded and targeted modified Por-COF@TPZ / HA.

[0020] Detection and Experiment The following describes the characterization and research of the nanomedicine (Por-COF@TPZ / HA) with pH / NIR / hypoxia multi-level response prepared in Example 1 (the multi-level response is reflected in the low pH release of the loading material, the fact that laser irradiation helps drug release, the fact that laser irradiation of the sample can generate heat and ROS, and the fact that the drug is activated by hypoxia).

[0021] The structure of Por-COF@TPZ / HA was characterized by infrared and ultraviolet spectroscopy, and the results are as follows: Figure 1 and 2 As shown. Figure 1 The characteristic peak is at 1626 cm⁻¹. −1 (ν-C=N) and 1301 cm −1 (ν−C−NH) represents the reaction between an amino group and an aldehyde group to form a carbon-nitrogen double bond, confirming the preparation of Por-COFs. (1662 cm⁻¹) −1 (ν-C=O) and 3289 cm −1 (ν-OH) is the characteristic peak of HA's -COOH group, at 3441 cm⁻¹. −1 (ν-NH), 1415 cm −1 (ν-NH) and 727 cm −1 (ν-NH) is the characteristic peak of -NH in TPZ, and the overlap of characteristic peaks verifies the successful preparation of Por-COF@TPZ / HA. Figure 2 The presence of an absorption peak of TPZ at 463 nm confirms the successful loading of TPZ.

[0022] The morphology of Por-COF@TPZ / HA was characterized by TEM and SEM, and the results are shown in [Figure number missing]. Figure 3 and 4 As can be seen from the figure, the Pro-COF@TPZ / HA nanomedicine exhibits good dispersion and relatively uniform particle size, with a length-to-short axis ratio of approximately 10:1. Rod-shaped or worm-shaped nanosystems possess better membrane permeability, therefore the prepared Pro-COFs are promising biomedical materials.

[0023] Testing of photothermal properties: To investigate the photothermal conversion capability of Por-COF@TPZ / HA, tests were performed on water and Por-COF@TPZ / HA aqueous solutions (200 μg / mL). -1 The temperature change during 10 minutes of 808 nm laser irradiation was shown in the results. Figure 5 .from Figure 5 It can be observed that the temperature of Por-COF@TPZ / HA increased by approximately 35.5 ℃, while the water temperature only increased by 5.7 ℃, demonstrating that Por-COF@TPZ / HA has excellent photothermal conversion capabilities and is a promising photothermal therapy drug.

[0024] Detection of ROS generation capability: The generation of ROS in Por-COF@TPZ / HA solution under 808 nm laser irradiation was detected using a 1,3-diphenylisobenzofuran (DPBF) probe. The effect of laser irradiation time on the generation of ROS was also investigated. 1 The effect of O2, in 1 mL of Por-COF@TPZ / HA methanol solution (100 μg / mL) -1 Add 1 mL of DPBF methanol solution (60 μg / mL) to the solution. -1 Irradiation was performed using an 808 nm NIR laser with a power density of 1.0 W / cm². -2 The absorbance of DPBF at 418 nm was recorded using a UV-Vis spectrometer at irradiation times of 2, 4, 6, 8, and 10 minutes. The results are shown in the table below. Figure 6 The analysis results showed that as the irradiation time increased, the absorption intensity of DPBF in the solution at 418 nm gradually decreased. This is because the ROS generated by the light irradiation of Por-COF@TPZ / HA reacts with DPBF, reducing the concentration of DPBF in the system. The results demonstrate that irradiation of Por-COF@TPZ / HA solution with an 808 nm laser can generate a large amount of ROS for photodynamic therapy of cancer.

[0025] Distribution experiment in mice: B16-F10 cells (1×10⁻⁶) 7 (Number of tumor-bearing mice) were injected under the armpits of BALB / c-nu mice to establish a tumor-bearing mouse model. When the tumor volume reached 80-100 mm... 3 Por-COF@TPZ / HA (200 μL, 2 mg mL) -1 Mice were injected via tail vein and then anesthetized with isoflurane. Images were taken of the mice at 2, 4, 6, 8, 24, 36, and 48 hours post-injection. At the last 48 hours, fluorescence imaging of major organs (heart, liver, spleen, lung, kidney, and brain) and tumors was collected from each group of mice to investigate the enrichment and retention of Por-COF@TPZ / HA in organs and tumors. Results are shown in [see attached image]. Figure 7 .

[0026] Figure 7It can be seen that: 4 hours after injection of Por-COF@TPZ / HA, fluorescence began to appear in the tumor site, but the signal was weak. The tumor signal increased over time. The tumor fluorescence signal reached its maximum at 24 hours, while the abdominal fluorescence signal disappeared at 24 hours. The tumor fluorescence signal remained strong until 48 hours after injection. Among the mouse organs and tumors after 48 hours, only the lungs showed a weak fluorescence signal, while the tumor site exhibited strong fluorescence. This indicates that Por-COF@TPZ / HA can accumulate in tumors for a long time. Using the ROI function of the IVIS Lumina XRMS series to quantify the fluorescence signal, 2 hours after injection of TAPP-4mPEG / PTX, the number of photons in the tumor increased over time, reaching a peak at 24 hours. Afterward, the fluorescence intensity of the tumor began to decrease over time. Fluorescence signals were still detectable in the tumor area 48 hours after injection. Only the lungs of the mice showed a weak photon count. This indicates that Por-COF@TPZ / HA can accumulate in the tumor area for a long time.

[0027] In vitro cytotoxicity assay: B16-F10 cells (6000 cells / well) were seeded into 96-well plates and incubated for 24 h. Then, different concentrations (2, 4, 6, 8, 10 μg / mL) were added to the 96-well plates. −1 Samples containing TPZ (100% TPZ) were co-incubated for 24 h with saline + laser, TPZ, Por-COF / HA, Por-COF@TPZ / HA, Por-COF / HA + laser, and Por-COF@TPZ / HA + laser. The wells were then placed under an 808 nm laser (1.0 W cm⁻¹). -2 Irradiate for 6 min, discard the culture medium, and add 50 μL (1 mg / mL) to each well. -1 MTT solution was used. The culture medium was incubated for 4 h, then discarded, and 100 μL of DMSO was added. The 96-well plates were placed in a 37 ℃ oven for 10 min, and the absorbance at 490 nm was measured using a microplate reader to calculate cell viability. Results are as follows: Figure 8 As shown.

[0028] Figure 8 It can be seen that even at a concentration of 10 μg / mL, Por-COF / HA... -1 (TPZ%), cell viability remained around 90%, indicating the biocompatibility and safety of the Por-COF / HA material. The same concentration of Por-COF@TPZ / HA at 10 μg / mL was also observed. -1At TPZ concentration, Por-COF / HA exhibited significant cytotoxicity in B16-F10 tumor cells, with a cell survival rate of approximately 60%, slightly higher than the approximately 45% survival rate at the same TPZ concentration. This is reasonable considering the controlled release of the drug. Furthermore, under near-infrared (NIR) radiation, due to the photothermal conversion and photodynamic effects of Por-COF / HA, cell viability decreased, and significant cancer cell death was observed. At the same concentrations of Por-COF / HA and Por-COF@TPZ / HA under 808 nm irradiation, cell viability was approximately 39% and 24%, respectively, significantly different from the non-NIR group. This indicates that the viability of B16-F10 tumor cells is related to the concentration of Por-COF@TPZ / HA and whether irradiation was performed at 808 nm. The prepared Por-COF@TPZ / HA possesses a highly effective multimodal combination therapy capability, encompassing photodynamic therapy, photothermal therapy, and chemotherapy (PDT / PTT / chemotherapy).

Claims

1. A method for preparing a tumor-targeting nanomedicine Por-COF@TPZ / HA with multi-level pH / NIR / hypoxia response, characterized in that, Includes the following steps: 1) Synthesize TAPP; 2) Mix terephthalaldehyde acetonitrile solution with TAPP dichloromethane solution, then add ferric chloride acetonitrile solution and acetic acid, sonicate to dissolve, stir for 12-16 h at room temperature in the dark to obtain a dark green suspension, centrifuge and wash to obtain a multi-level responsive nanomaterial with pH / NIR / oxygen deficiency. 3) Dissolve 10 mg of pH / NIR / hypoxia multi-level responsive nanomaterials in 4-7 mL of ultrapure water, dissolve 4-6 mg of terazamine in 3-5 mL of ultrapure water, and dissolve 9-12 mg of hyaluronic acid in 4-7 mL of ultrapure water. Mix the three solutions thoroughly and react at room temperature in the dark for 20-25 h. After centrifugation and washing with ultrapure water, the product is obtained. The prepared Por-COF@TPZ / HA nanomedicine has good dispersibility and relatively uniform particle size, with a length-to-short axis ratio of 10:

1.

2. The preparation method according to claim 1, characterized in that, In step 2), dissolve 5.00-6.00 mg of terephthalaldehyde in 10-15 mL of acetonitrile, and dissolve 5-6 mg of TAPP in 10-15 mL of dichloromethane. Then mix the two solutions, and subsequently add 0.2-0.5 mL of a solution with a concentration of 48-52 mg / mL. −1 A solution of ferric chloride and acetonitrile, and 5-6 mL of acetic acid.

3. The preparation method according to claim 1, characterized in that, The synthesis of TAPP in step 1) is specifically as follows: Tetra(4-nitrophenyl)porphyrin was dissolved in concentrated hydrochloric acid and nitrogen gas was passed through for 15-25 min. Simultaneously, SnCl2∙2H2O was dissolved in concentrated hydrochloric acid and nitrogen gas was passed through for 20-25 min. The two samples were then mixed and reacted at 80-90 °C for 30-40 min. After cooling to room temperature, the system was placed in an ice-water bath and the reaction continued for 1-2 h. The pH of the system was adjusted to neutral, and the mixture was filtered. 4-8% NaOH solution was added to the filtrate, and a solid appeared with stirring. After filtration, washing, and vacuum drying, the product was obtained.

4. The preparation method according to claim 3, characterized in that, Dissolve 1-2 g of tetra(4-nitrophenyl)porphyrin in 45-55 mL of concentrated hydrochloric acid, and dissolve 4-5 g of SnCl2∙2H2O in 70-80 mL of concentrated hydrochloric acid.

5. The tumor-targeting nanomedicine Por-COF@TPZ / HA with pH / NIR / hypoxia multi-level response prepared by any one of the methods described in claims 1 to 4.

6. The use of the tumor-targeting nanomedicine Por-COF@TPZ / HA as described in claim 5 in the preparation of antitumor drugs.

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