Near-infrared light activated hypoxia responsive supramolecular nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared light activated hypoxia responsive supramolecular nano-drug as well as a preparation method and application thereof. The supramolecular nano-drug is prepared from tetraamino porphyrin-azobenzene 4, tirapazamine and anionic water-soluble biphenyl expanded column [6] arene in a mass ratio of 1: 1: 0.188. The near-infrared light activated hypoxia responsive supramolecular nano-drug can be used for treating colon cancer and has good stability, the preparation method is simple and efficient, and a good experimental platform is provided for obtaining a hypoxia responsive prodrug with colon cancer targeting and photodynamic therapy-chemotherapy integrated treatment. Compared with the prior art, the near-infrared light activated hypoxia responsive supramolecular nano-drug consumes oxygen through photodynamic therapy to improve the hypoxia degree in tumor cells, the photodynamic therapy and hypoxia activated chemotherapy are combined, and the anti-tumor treatment effect is greatly improved through synergistic therapy of the photodynamic therapy and hypoxia activated chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a hypoxia-responsive supramolecular nanomedicine activated by near-infrared light, and a preparation method and application thereof. Background Art

[0002] As a common feature of most solid tumors, hypoxia in the tumor site is manifested by an average oxygen concentration of less than 10 mmHg, while the oxygen concentration in normal tissue is about 70 mmHg. This hypoxia phenomenon is induced by the rapid proliferation of tumor cells, strong energy requirements, active oxidative metabolism, and limited oxygen supply caused by abnormal vascular structure. Tumor hypoxia plays a central role in tumor progression and resistance to treatment, making it a primary target for cancer treatment. Therefore, hypoxia-responsive nanocarriers based on azobenzene derivatives have attracted increasing attention in hypoxia imaging and treatment because they can be cleaved by reductase under hypoxic conditions. However, given the uneven distribution of hypoxia in tumor tissues, hypoxia-responsive nanocarriers are difficult to completely kill tumor cells, especially those close to blood vessels, which may trigger potential tumor recurrence.

[0003] The combination of photodynamic therapy (PDT) and hypoxia-responsive nanocarriers presents a promising approach to effectively address the above challenges and enhance cancer treatment. PDT uses photosensitizers (PSs) to consume oxygen and produce harmful reactive oxygen species (ROS) when illuminated. Oxygen consumption can lead to aggravated tumor hypoxia, which in turn impairs the therapeutic efficacy of PDT. Therefore, the cascade technology of PDT-induced hypoxia aggravation and subsequent drug release can significantly enhance the synergistic clearance of cancer cells, thereby amplifying the effect of combined treatment.

[0004] Literature search found that Kim's research group at Pohang University of Science and Technology in South Korea prepared hypoxia-responsive amphiphilic block copolymers by linking polyethylene glycol and polylysine with azobenzene in the paper (Immunostimulation of tumor microenvironment by targeting tumor-associated macrophages with hypoxia-responsive nanocomplex for enhanced anti-tumor therapy, Journal of Controlled Release 2022, 343, 78-88) for loading poly I-cells for cancer treatment. However, this system can only rely on the hypoxic environment of the cancer cells themselves for activation, and the uneven distribution of hypoxia in tumor tissues will greatly limit the efficacy of the system, making it difficult to transform and apply it in clinical practice. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a near-infrared light-activated hypoxia-responsive supramolecular nanodrug and a preparation method and application thereof, so as to solve the problems in the prior art that it is difficult to effectively load oxygen scavengers and hypoxia-responsive prodrugs at the same time, and the hypoxia-responsive prodrugs have low efficacy.

[0006] The present invention is achieved through the following technical solutions:

[0007] A near-infrared light-activated hypoxia-responsive supramolecular nanomedicine, wherein the supramolecular nanomedicine is prepared from tetraaminoporphyrin-azobenzene 4, tirapazamine and anionic water-soluble biphenyl extended column [6] aromatic hydrocarbons in a mass ratio of 1:1:0.188; wherein the structural formula of the tetraaminoporphyrin-azobenzene 4 is shown in the following formula I:

[0008]

[0009] Preferably, the particle size of the supramolecular nanomedicine is less than 200 nm.

[0010] The method for preparing the hypoxia-responsive supramolecular nanomedicine activated by near-infrared light comprises the following steps:

[0011] Step 1) preparing tetraaminoporphyrin-azobenzene 4;

[0012] Step 2) Tetraaminoporphyrin-azobenzene 4 and tirapazamine are dissolved in N,N-dimethylformamide solvent in proportion, and then a deionized water solution of anionic water-soluble biphenyl extended column [6] aromatic hydrocarbon is added dropwise, stirred, and dialyzed to obtain the product.

[0013] Preferably, the process of preparing tetraaminoporphyrin-azobenzene 4 in step 1) is as shown in reaction formula II:

[0014]

[0015] The specific steps are as follows:

[0016] Take 34 mg of tetraaminoporphyrin and 113 mg of azobenzene-4-benzoic acid and dissolve them in 5 mL of N,N-dimethylformamide, add 153 mg of 4-dimethylaminopyridine and 233 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and react at room temperature in the dark for 24 hours; after the reaction is completed, add deionized water to quench the reaction, filter, and wash repeatedly with deionized water for 3 times. After the solid crude product obtained after filtration is extracted with dichloromethane for 3 days, the obtained solid is placed in a vacuum drying oven and dried overnight to obtain the product.

[0017] Preferably, in step 2), the concentration of tetraaminoporphyrin-azobenzene 4 is 5.0 mg / mL; the concentration of tirapazamine is 5.0 mg / mL; and the concentration of anionic water-soluble biphenyl extended column [6] aromatic hydrocarbon is 0.94 mg / mL.

[0018] Preferably, in step 2), the volume ratio of the N,N-dimethylformamide to the deionized water is 1:10.

[0019] Preferably, the dialysis conditions in step 2) are: using a dialysis bag with a molecular weight of 500, 500 mL of deionized water × 1 time / 8 hours, and dialysis for 24 hours.

[0020] Application of the above-mentioned near-infrared light-activated hypoxia-responsive supramolecular nanomedicine, or the near-infrared light-activated hypoxia-responsive supramolecular nanomedicine prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0021] Preferably, the tumor is colon cancer.

[0022] An anti-tumor pharmaceutical composition comprises the above-mentioned near-infrared light-activated hypoxia-responsive supramolecular nanomedicine, or the near-infrared light-activated hypoxia-responsive supramolecular nanomedicine prepared by the above-mentioned preparation method.

[0023] The beneficial effects of the present invention are as follows:

[0024] (1) In the components of the hypoxia-responsive supramolecular nanomedicine activated by near-infrared light of the present invention, tetraaminoporphyrin is activated under near-infrared light conditions (light intensity 0.5 W / cm 2 , wavelength 650nm, illumination time 5min), can consume oxygen, enhance the hypoxia level in tumor cells, and generate reactive oxygen species, kill tumor cells, and achieve photodynamic therapy; azobenzene breaks in the enhanced hypoxic environment in the tumor, and in the acidic environment in the tumor, the host-guest complex weakens, the nanodrug disassembles, releases and activates tirapazamine (TPZ), which is toxic to tumor cells and achieves chemotherapy. The supramolecular nanodrug is used in the integrated photodynamic therapy-chemotherapy treatment system for colon cancer cells. The operation is simple, and only two intravenous injections and two illuminations are required to achieve effective treatment of colon cancer tumors, which has important clinical application prospects.

[0025] (2) The near-infrared light-activated hypoxia-responsive supramolecular nanomedicine of the present invention can be used for the treatment of colon cancer, has good stability, and the preparation method is simple and efficient, providing a good experimental platform for obtaining hypoxia-responsive prodrugs with colon cancer targeting and integrated photodynamic therapy-chemotherapy. Compared with the prior art, the near-infrared light-activated hypoxia-responsive supramolecular nanomedicine of the present invention consumes oxygen through photodynamic therapy to increase the degree of hypoxia in tumor cells, combines photodynamic therapy with hypoxia-activated chemotherapy, and greatly improves the effect of anti-tumor treatment through the synergistic treatment of the two combined therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the NMR spectrum of tetraaminoporphyrin-azobenzene 4 in Example 1;

[0027] Figure 2 Schematic diagram of the preparation of hypoxia-responsive supramolecular nanomedicine activated by near-infrared light in Example 1;

[0028] Figure 3 The dynamic light scattering spectrum of the hypoxia-responsive supramolecular nanomedicine activated by near-infrared light in Example 1;

[0029] Figure 4 The effects of the hypoxia-responsive supramolecular nanomedicine activated by near-infrared light in Example 2 on colon cancer cells in a hypoxic environment under the conditions of no light irradiation and near-infrared light irradiation, respectively;

[0030] Figure 5 The effects of the hypoxia-responsive supramolecular nanomedicine activated by near-infrared light in Example 2 on colon cancer cells in a normal oxygen environment under the conditions of no light irradiation and near-infrared light irradiation respectively;

[0031] Figure 6 The effect of each group of drugs in Example 3 on the tumor size of nude mice bearing colon cancer tumors over time;

[0032] Figure 7 The figure shows the effects of each group of drugs in Example 3 on the body weight of nude mice bearing colon cancer tumors over time. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] The experimental animals in the following examples are nude mice, which were provided by the Experimental Animal Center of Nantong University.

[0037] Example 1 Preparation of near-infrared light-activated hypoxia-responsive supramolecular nanomedicine (SN-TAPP / TPZ)

[0038] 1. Preparation of Tetraaminoporphyrin-azobenzene 4 (TAPP-Azo4)

[0039] The structural formula of TAPP-Azo4 is shown in Formula I below:

[0040]

[0041] Its preparation process is shown in reaction formula II:

[0042]

[0043] The specific steps are as follows:

[0044] Azobenzene-4-benzoic acid (Azo-COOH) was obtained by referring to the existing literature (Chem. Commun., 2017, 53, 9462-9465). 34 mg of tetraaminoporphyrin (TAPP) and 113 mg of Azo-COOH were dissolved in 5 mL of N, N-dimethylformamide (DMF), and 153 mg of 4-dimethylaminopyridine (DMAP) and 233 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added to react at room temperature in the dark for 24 hours. After the reaction was completed, deionized water was added to quench the reaction, filtered, and washed repeatedly with deionized water 3 times. The solid crude product obtained after filtration was placed in a Soxhlet extractor and extracted with dichloromethane for 3 days, and the obtained solid was placed in a vacuum drying oven and dried overnight to obtain TAPP-Azo4 0.23 mg, with a yield of 53%.

[0045] The TAPP-Azo4 prepared in this example is as follows Figure 1 As shown, the detailed peak positions are attributed to: 1 H-NMR (400MHz, DMSO-d6, TMS), δ (ppm) = 10.94 (s, 4H, CONHPh), 8.96 (d, 8H, PhNNCCH), 8.34 (t, 16H, COPhNNCCH, COCCH), 8.2 7(d,8H,PhNNCCHCH),8.13(d,8H,CONHCCH),8.01(d,8H,CONHCCHCHCC),7.67(d,12H,NNCCHCHCH,NCCH),-2.84(s,2H,NH).

[0046] 2. Preparation of SN-TAPP / TPZ

[0047] (1) Anionic water-soluble biphenyl-extended columnar [6] aromatics were prepared with reference to existing literature (CN114939175B).

[0048] (2) Figure 2 As shown, 5 mg TAPP-Azo4 and 5.0 mg tirapazamine (TPZ) were dissolved in 1.0 mL DMF, then stirred in the dark for 6 h, and then 10 mL of deionized water solution of 9.4 mg anionic water-soluble biphenyl extended column [6] aromatic hydrocarbon (AWBpP6) was added dropwise, and stirred overnight. Finally, the mixed solution was transferred to a dialysis bag (MWCO 500Da) and dialyzed with ultrapure water for 24 h, and the dialysis bag was renewed every 4 h. The hypoxia-responsive supramolecular nanomedicine (SN-TAPP / TPZ) activated by near-infrared light was obtained.

[0049] The dynamic light scattering spectrum of SN-TAPP / TPZ prepared in this example is as follows: Figure 3 As shown, the number average particle size is 125±1.9 nm and the PDI is 0.11±0.02.

[0050] Example 2 Effect of SN-TAPP / TPZ on Colon Cancer Cells

[0051] 1. Experimental steps

[0052] The SN-TAPP / TPZ prepared in Example 1 was prepared into a cell culture medium with tirapazamine (TPZ) concentrations of 2, 4, 8, 12, 16, and 20 μg / mL, and cultured with colon cancer cells (HCT116) in a hypoxic (Hypoxia) or normal oxygen (Normoxia) environment for 6 hours. Among them, for the light-exposed group, it was irradiated with a near-infrared laser for 5 minutes (650nm, 0.5W / cm 2 ) and continue culturing for 48h.

[0053] 2. Experimental results

[0054] The cell activity test was performed using the MTT method. Figure 4 , 5 As shown, SN-TAPP / TPZ under the conditions of no light (NIR(-) or Light(-)) or near-infrared light irradiation (NIR(+) or Light(+)) under hypoxic environment ( Figure 4 ) were more toxic than those in normal oxygen environment ( Figure 5 ) is highly toxic; under near-infrared light irradiation, the toxicity of SN-TAPP / TPZ in an oxygen-deficient environment is the greatest.

[0055] The experimental results of this example show that SN-TAPP / TPZ exhibits better anti-tumor effects on colon cancer cells under light conditions.

[0056] Example 3 Effect of SN-TAPP / TPZ on HCT116 Colon Cancer Tumor Growth

[0057] 1. Experimental steps

[0058] The mice inoculated with HCT116 tumors were divided into 5 groups as follows:

[0059] PBS group: injection of normal saline (5 mg / kg) via tail vein;

[0060] TPZ group: injection of tirapazamine (5 mg / kg) via tail vein;

[0061] TAPP+NIR group: 8 hours after the tail vein injection of tetraaminoporphyrin (2.9 mg / kg), the mice were irradiated with near-infrared laser;

[0062] SN-TAPP / TPZ group: SN-TAPP / TPZ (24.5 mg / kg) injected into the tail vein;

[0063] SN-TAPP / TPZ+NIR group: 8 hours after the tail vein injection of SN-TAPP / TPZ (24.5 mg / kg), the mice were irradiated with near-infrared laser.

[0064] The mice in the above groups were injected with drugs once on day 0 and day 6, and 8 hours after injection, the TAPP+NIR group and SN-TAPP / TPZ+NIR group were illuminated for 5 min (650 nm, 0.5 W / cm 2 ), and the mice were weighed and the tumor volumes were measured every 3 days.

[0065] 2. Experimental results

[0066] The experimental results are as follows Figure 6 , 7 As shown, for HCT116 tumors, free tirapazamine TPZ group and TAPP+NIR group had almost no inhibitory effect on the tumor, SN-TAPP / TPZ group had a more obvious inhibitory effect on the tumor, and the growth of tumors in all mice in the SN-TAPP / TPZ+NIR group was significantly inhibited ( Figure 6 , P<0.01), and had little effect on the body weight of mice ( Figure 7 ).

[0067] The experimental results of this example show that the SN-TAPP / TPZ of the present invention can be used for the treatment of colon cancer (HCT116), has good stability, and can achieve effective inhibition and treatment of HCT116 subcutaneous tumors after two injections through the tail vein and two illuminations in mice. Among the components of SN-TAPP / TPZ, tirapazamine is used for chemotherapy, tetraaminoporphyrin is used for photodynamic therapy, and hypoxia-activated chemotherapy and photodynamic therapy work synergistically to achieve effective treatment of colon cancer tumors, which has important clinical application prospects.

[0068] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A near-infrared light-activated hypoxia-responsive supramolecular nanomedicine, characterized in that: The supramolecular nanomedicine is prepared from tetraaminoporphyrin-azobenzene 4, tirapazamine and anionic water-soluble biphenyl extended column [6] aromatic hydrocarbons in a mass ratio of 1:1:0.188; wherein the structural formula of the tetraaminoporphyrin-azobenzene 4 is shown in the following formula I:

2. The hypoxia-responsive supramolecular nanomedicine activated by near-infrared light according to claim 1, characterized in that: The particle size of the supramolecular nanomedicine is less than 200 nm.

3. The method for preparing a near-infrared light-activated hypoxia-responsive supramolecular nanomedicine according to claim 1 or 2, characterized in that: The following steps are involved: Step 1) preparing tetraaminoporphyrin-azobenzene 4; Step 2) Tetraaminoporphyrin-azobenzene 4 and tirapazamine are dissolved in N,N-dimethylformamide solvent in proportion, and then a deionized water solution of anionic water-soluble biphenyl extended column [6] aromatic hydrocarbon is added dropwise, stirred, and dialyzed to obtain the product.

4. The method for preparing a near-infrared light-activated hypoxia-responsive supramolecular nanomedicine according to claim 3, characterized in that: The process of preparing tetraaminoporphyrin-azobenzene 4 in step 1) is shown in reaction formula II: The specific steps are as follows: Take 34 mg of tetraaminoporphyrin and 113 mg of azobenzene-4-benzoic acid and dissolve them in 5 mL of N,N-dimethylformamide, add 153 mg of 4-dimethylaminopyridine and 233 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and react at room temperature in the dark for 24 hours; after the reaction is completed, add deionized water to quench the reaction, filter, and wash repeatedly with deionized water for 3 times. After the solid crude product obtained after filtration is extracted with dichloromethane for 3 days, the obtained solid is placed in a vacuum drying oven and dried overnight to obtain the product.

5. The method for preparing a near-infrared light-activated hypoxia-responsive supramolecular nanomedicine according to claim 3, characterized in that: Step 2) The concentration of tetraaminoporphyrin-azobenzene 4 is 5.0 mg / mL; the concentration of tirapazamine is 5.0 mg / mL; and the concentration of anionic water-soluble biphenyl extended column [6] aromatic hydrocarbon is 0.94 mg / mL.

6. The method for preparing a near-infrared light-activated hypoxia-responsive supramolecular nanomedicine according to claim 3, characterized in that: Step 2) The volume ratio of the N,N-dimethylformamide to the deionized water is 1:

10.

7. The method for preparing a hypoxia-responsive supramolecular nanomedicine activated by near-infrared light according to claim 3, characterized in that: Step 2) The dialysis conditions are: using a dialysis bag with a molecular weight of 500, 500 mL of deionized water × 1 time / 8 hours, and dialysis for 24 hours.

8. Use of the near-infrared light-activated hypoxia-responsive supramolecular nanodrug as claimed in claim 1 or 2, or the near-infrared light-activated hypoxia-responsive supramolecular nanodrug prepared by the preparation method according to any one of claims 3 to 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor is colon cancer.

10. An anti-tumor pharmaceutical composition, characterized in that: It comprises the near-infrared light-activated hypoxia-responsive supramolecular nanomedicine as described in claim 1 or 2, or the near-infrared light-activated hypoxia-responsive supramolecular nanomedicine prepared by the preparation method according to any one of claims 3-7.

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