A hypoxia-responsive supramolecular polypeptide nanoprodrug and its preparation method and application

Abstract

The present invention discloses a hypoxia-responsive supramolecular polypeptide nanoprodrug and its preparation method and application. The hypoxia-responsive supramolecular polypeptide nanoprodrug is prepared from tetraaminoporphyrin-poly(L-lysine)-azobenzene nitrogen mustard and a novel water-soluble column [6] aromatic hydrocarbon AWBpP6. The hypoxia-responsive supramolecular polypeptide nanoprodrug of the present invention increases drug loading efficiency, reduces the toxic side effects of nanomedicines, and improves stability in the form of polymer-prodrug. Under near-infrared light conditions, the porphyrin in the prodrug particles can not only release reactive oxygen species to achieve photodynamic therapy, but also consume oxygen in the process, thereby enhancing chemotherapy mediated by the hypoxic environment. By combining chemotherapy and photodynamic therapy in a dual combination therapy, the effect of anti-tumor treatment is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a hypoxia-responsive supramolecular polypeptide nanoprodrug, a preparation method thereof, and an application thereof. Background Art

[0002] Solid tumors are characterized by structural and transient hypoxia, resulting from uncontrolled cell proliferation, abnormal tumor vasculature, and insufficient oxygen supply. Low oxygen levels within tumor tissues promote tumor invasion, metastasis, and drug resistance, leading to overexpression of various physiological enzymes, such as azoreductase, DT-diaphorase, and nitroreductase, which can be considered effective stimulators of therapeutic drugs. Against this backdrop, hypoxia-responsive drug delivery systems have been developed. However, due to the heterogeneous distribution of hypoxia within tumor tissue, hypoxia-responsive drug delivery systems are unable to damage tumor cells with normal oxygen levels, especially those near blood vessels, which can easily lead to tumor recurrence. Therefore, the development of more effective hypoxia-responsive drug delivery systems is urgently needed.

[0003] The combination of photodynamic therapy (PDT) and hypoxia-responsive drug delivery systems is a promising approach because PDT reduces oxygen in tumor tissues by utilizing photosensitizers to catalyze the conversion of oxygen into reactive oxygen species (ROS), which are toxic to cells, under illumination, thereby achieving hypoxic conditions. Therefore, the reduction in intracellular oxygen content caused by PDT and the subsequent hypoxia-responsive drug release combine to eliminate tumor cells through a synergistic effect, improving the therapeutic effect. Furthermore, compared with traditional anticancer therapies, PDT has the advantages of spatiotemporal selectivity, low invasiveness, and low systemic toxicity. Furthermore, chemotherapeutic drugs physically encapsulated in nanoparticles often have the risk of drug leakage in the bloodstream before reaching the tumor site. However, peptide nanoprodrugs that covalently couple anticancer drugs to polymer backbones have the advantages of low toxicity, easily adjustable drug loading, high stability, and good biocompatibility.

[0004] In their study, “Smart hypoxia-responsive transformable and charge-reversible nanoparticles for the deep penetration and tumor microenvironment modulation of pancreatic cancer” (Biomaterials 2022, 287, 121599), Chen Jiang et al. reported a hypoxia-responsive nanoprodrug that utilizes the hypoxic environment of tumor tissue to responsively release drugs and kill tumor cells. However, this nanoprodrug only utilizes the hypoxic properties of tumor tissue itself, and due to the heterogeneity of tumor tissue hypoxia, it is difficult to achieve efficient hypoxia-responsive drug release and complete ablation of the entire tumor tissue, resulting in poor anti-tumor effects and difficulty in clinical translation and application. Summary of the Invention

[0005] In response to the problems of low drug loading efficiency and premature drug leakage in the prior art, as well as the problem that the uneven distribution of the tumor hypoxic environment makes it difficult for hypoxia-responsive drugs to exert their efficacy, the present invention provides a hypoxia-responsive supramolecular polypeptide nanoprodrug and its preparation method and application. Through the form of polymer-prodrug, the drug loading efficiency is increased, the toxic side effects of nanomedicines are reduced, and the stability is improved. Moreover, under near-infrared light conditions, the porphyrin in the prodrug particles can not only release reactive oxygen species to achieve photodynamic therapy, but also consume oxygen in the process, thereby enhancing chemotherapy mediated by the hypoxic environment. In addition, by combining chemotherapy and photodynamic therapy with dual combination therapy, the effect of anti-tumor treatment is greatly improved.

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

[0007] A method for preparing a hypoxia-responsive supramolecular polypeptide nanoprodrug comprises the following steps:

[0008] Step 1) preparing tetraaminoporphyrin-poly(L-lysine)-azobenzene mustard;

[0009] Step 2) preparing a novel water-soluble column [6]arene AWBpP6;

[0010] Step 3) The tetraaminoporphyrin-poly(L-lysine)-azobenzene mustard prepared in step 1) is dissolved in N,N-dimethylformamide solvent and stirred for 12 hours. Then, the novel water-soluble column [6] aromatic hydrocarbon AWBpP6 prepared in step 2) is dissolved in deionized water. Then, the two are added dropwise into a container, stirred at room temperature for 24 hours, and dialyzed to obtain the hypoxia-responsive supramolecular polypeptide nanoprodrug.

[0011] Preferably, the process of preparing tetraaminoporphyrin-poly(L-lysine)-azobenzene mustard in step 1) is as shown in Reaction Scheme 2:

[0012]

[0013] The specific steps are as follows:

[0014] Step A) Tetraaminoporphyrin and lysine are added to a container, followed by addition of N,N-dimethylformamide, and the reaction is carried out at 25° C. for 48 hours. After the reaction is completed, the product TAPP-PZLys4 is obtained by precipitation with ether;

[0015] Step B) The TAPP-PZLs4 obtained in step A) was dissolved in a glacial acetic acid / trifluoroacetic acid mixed solvent, and a hydrobromic acid / glacial acetic acid mixed solvent was added at 0°C, and the reaction was continued for 1.5 hours. After the reaction was completed, the reaction solution was precipitated in diethyl ether and centrifuged three times, and finally dried to obtain the product TAPP-PLL4;

[0016] Step C) The TAPP-PLL4 obtained in step B), nitrogen mustard, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are placed in a container, and then N,N-dimethylformamide is added to react at room temperature overnight. After the reaction is completed, the mixture is precipitated with ether to obtain the tetraaminoporphyrin-poly(L-lysine)-azobenzene nitrogen mustard.

[0017] Preferably, in step 3), the concentration of tetraaminoporphyrin-poly(L-lysine)-azobenzene mustard is 2 mg / mL, and the concentration of the novel water-soluble column [6] aromatic hydrocarbon AWBpP6 is 0.8 mg / mL.

[0018] Preferably, in step 3), the volume ratio of the N,N-dimethylformamide solvent to the deionized water is 1:10.

[0019] Preferably, the dialysis conditions in step 3) are: using a dialysis bag with a molecular weight of 1000, 500 mL of deionized water × 1 time / 8 hours, and dialysis for 48 hours.

[0020] The hypoxia-responsive supramolecular polypeptide nanoprodrug prepared by the above preparation method has a particle size of 198.1±5.8 nm.

[0021] Application of the above hypoxia-responsive supramolecular peptide nanoprodrug in the preparation of anti-tumor drugs.

[0022] Preferably, the tumor is cervical cancer.

[0023] An anti-tumor pharmaceutical composition comprises the above-mentioned hypoxia-responsive supramolecular polypeptide nanoprodrug and pharmaceutically acceptable excipients.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) The present invention efficiently synthesizes a supramolecular prodrug nanoparticle. The hypoxia-responsive supramolecular polypeptide nanoparticle has good biostability in a normal physiological environment and can be rapidly disassembled in the acidic environment of tumor cells and under near-infrared light conditions.

[0026] (2) The hypoxia-responsive supramolecular polypeptide nanoprodrug loaded porphyrin of the present invention can absorb oxygen in cells to produce singlet oxygen under the irradiation of 650nm near-infrared light, which can enhance the killing effect on tumor cells and realize photodynamic therapy.

[0027] (3) The porphyrin component in the hypoxia-responsive supramolecular polypeptide nanoprodrug of the present invention can absorb oxygen in cells under the irradiation of 650nm laser to enhance the hypoxic environment, and can cause azobenzene to break, releasing nitrogen mustard chemotherapy drugs, thereby achieving chemotherapy.

[0028] (4) The hypoxia-responsive supramolecular polypeptide nanoprodrug of the present invention can be used in the integrated chemotherapy-photodynamic therapy technology. It is simple to operate and only requires a single intravenous injection to achieve effective treatment of tumors, which has important clinical application prospects.

[0029] (5) The present invention provides a simple and effective way to prepare supramolecular prodrug nanoparticles, and provides a good experimental platform for obtaining supramolecular prodrug nanoparticles with hypoxia-responsive integrated chemotherapy-photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the NMR spectrum of Azo-CB in Example 1;

[0031] Figure 2 is the NMR spectrum of TAPP-(PLL-Azo-CB)4 in Example 1;

[0032] Figure 3 Schematic diagram of the preparation principle of hypoxia-responsive supramolecular polypeptide nanoprodrug in Example 1;

[0033] Figure 4 This is the dynamic light scattering spectrum of the hypoxia-responsive supramolecular polypeptide nanoprodrug in Example 1;

[0034] Figure 5 This is a transmission electron microscopy image of the hypoxia-responsive supramolecular polypeptide nanoprodrug in Example 1;

[0035] Figure 6The results of the hypoxia-responsive supramolecular peptide nanoprodrug test on HeLa cells in the absence of light in Example 2 are shown;

[0036] Figure 7 These are the results of the cell activity test of the hypoxia-responsive supramolecular polypeptide nanoprodrug on HeLa cells under light conditions in Example 2. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1 Preparation of Hypoxia-Responsive Supramolecular Polypeptide Nanoprodrug

[0040] 1. The preparation of Azo-CB is shown in Reaction Scheme 1. The specific steps are as follows:

[0041]

[0042] (1) Synthesis of nitrogen mustard with reference to existing literature: 4 g of N-phenyldiethanolamine and 5 mL of phosphorus oxychloride were first placed in a 50 mL round-bottom flask. N-phenyldiethanolamine was then slowly added in an ice bath, and the mixture was heated to 110°C and refluxed for 1 h. The mixture was then cooled to room temperature and concentrated by rotary evaporation. The remaining material was dissolved in ethyl acetate and washed three times with water. The organic layer was spin-dried and purified by column chromatography to obtain the product as a light yellow oil (nitrogen mustard) with a yield of 95%.

[0043] (2) 5.3 g of sodium nitrite was dissolved in 30 mL of water, 1 g of 4-aminobenzoic acid was added, and 5 mL of concentrated hydrochloric acid was added thereto. The mixture was placed in a 100 mL round-bottom flask and stirred at room temperature for 20 min. 2 g of the nitrogen mustard obtained in step (1) was then dissolved in ethanol and slowly added to the round-bottom flask. The mixture was reacted at room temperature for 2 h. After the reaction was completed, the mixture was extracted with dichloromethane and water, the organic layer was collected and dried, and then purified by column chromatography to obtain a red solid product (Azo-CB) with a yield of 85%.

[0044] The prepared Azo-CB is as follows Figure 1 As shown, the detailed peak positions are attributed to: 1 H-NMR (400MHz, CDCl3): δ13.1 (m, J=8Hz, 1H, COOPh), 8.09 (d, J=8Hz, 2H, CH), 7.86 (q, J=4Hz, 4H, CH), 3.85 (q, J=6Hz, 8H, CH2).

[0045] 2. Preparation of tetraaminoporphyrin-poly (L-lysine)-azobenzene mustard (TAPP-(PLL-Azo-CB)4), as shown in Reaction Scheme 2, the specific steps are as follows:

[0046]

[0047] (1) 100 mg of tetraaminoporphyrin (TAPP) and 220 mg of lysine were added to a 25 mL round-bottom flask, and then 3 mL of N,N-dimethylformamide (DMF) was added. The temperature was raised to 25 °C and the reaction was carried out in a glove box for 48 h. After the reaction was completed, the product TAPP-PZLys4 was obtained by precipitation with ether with a yield of 45%.

[0048] (2) 100 mg of TAPP-PZLs4 obtained in step (1) was dissolved in 10 mL of a mixed solvent of glacial acetic acid / trifluoroacetic acid (volume ratio of 1:1). 1.1 mL of a mixed solution of hydrobromic acid / glacial acetic acid (33 wt%) was added at 0°C, and the reaction was continued for 1.5 h. After the reaction was completed, the reaction solution was precipitated in 100 mL of diethyl ether and centrifuged three times. Finally, the solution was dried in a vacuum drying oven to obtain a green solid product, TAPP-PLL4, with a yield of 90%.

[0049] (3) 25 mg of TAPP-PLL4 obtained in step (2), 80 mg of nitrogen mustard, 10 mg of N-hydroxysuccinimide (NHS) and 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were placed in a 25 mL round-bottom flask, and then 5 mL of N,N-dimethylformamide (DMF) was added to react at room temperature overnight. After the reaction was completed, the product TAPP-(PLL-Azo-CB)4 was obtained by precipitation with ether with a yield of 51%.

[0050] The TAPP-(PLL-Azo-CB)4 prepared in this example is as follows Figure 2 As shown, the detailed peak positions are attributed to: 1 H-NMR (400MHz, CDCl3): δ8.26 (m, J=46Hz, 192H, (NPh) 30 ,Ph),7.89(m,J=23Hz,60H,(NPh) 30 ,CH),4.26(m,J=20Hz,240H,(CH2) 120 ),3.06(m,J=11Hz,30H,(NHCH) 30 ),2.79(s,J=44Hz,60H,(NHCH2) 30 ),1.54(d,J=46Hz,120H,(CH2) 60 ).

[0051] 3. Preparation of hypoxia-responsive supramolecular polypeptide nanoprodrugs, such as Figure 3 The specific steps are as follows:

[0052] (1) A new water-soluble column [6] aromatic hydrocarbon AWBpP6 was obtained by referring to existing literature (ACS Macro Lett. 2022, 11, 830-834).

[0053] (2) 8 mg of TAPP-(PLL-Azo-CB)4 was dissolved in 1 mL of N,N-dimethylformamide and stirred for 12 h. 2 mg of the novel water-soluble column [6] aromatic hydrocarbon AWBpP6 was dissolved in 10 mL of deionized water and added dropwise thereto, followed by stirring for another 24 h. Afterwards, the solution was placed in a dialysis bag with a molecular weight of 1000 and dialyzed against 500 mL of deionized water for 48 h, with the deionized water being replaced every 8 h. After the dialysis, supramolecular prodrug nanoparticles SPN-TAPP-PCB4 were obtained, which are hypoxia-responsive supramolecular peptide nanoprodrugs.

[0054] The dynamic light scattering spectrum of the prepared SPN-TAPP-PCB4 is as follows: Figure 4 As shown in the figure, the average particle size is 198.1±5.8nm, and the PDI is 0.156±0.05; the transmission electron microscope image is shown in the figure Figure 5 As shown, it is a uniform spherical micelle structure.

[0055] Example 2 Effects of Hypoxia-Responsive Supramolecular Polypeptide Nanoprodrugs on HeLa Cells

[0056] The supramolecular prodrug nanoparticles (SPN-TAPP-PCB4) prepared in Example 1 were formulated in cell culture medium at nitrogen mustard concentrations of 0.1, 0.2, 0.5, 1, 2, and 4 μg / mL, corresponding to SPN-TAPP-PCB4 concentrations of 0.818, 1.63, 4.08, 8.17, 16.3, and 32.47 μg / mL, respectively. HeLa cells (cervical adenocarcinoma) were then cultured for 48 hours under normal or hypoxic conditions. Cell viability was assessed using the MTT assay.

[0057] The results are as follows Figure 6 and Figure 7 As shown, Figure 6 Is cytotoxic under non-illumination conditions, Figure 7 It is cytotoxic under light conditions (650nm near-infrared light). Figure 6 and Figure 7 In the figure, the horizontal axis refers to the concentration of azobenzene mustard.

[0058] like Figure 6As shown in the figure, under the condition of no light, SPN-TAPP-PCB4 exhibited greater cytotoxicity in hypoxic environment. This was because SPN-TAPP-PCB4 could responsively release the chemotherapy drug nitrogen mustard in hypoxic environment to kill HeLa cells.

[0059] like Figure 7 As shown in the results, SPN-TAPP-PCB4 also exhibited greater cytotoxicity in a hypoxic environment under light conditions. This was because the porphyrin in SPN-TAPP-PCB4 could consume oxygen and release reactive oxygen species under light conditions, killing tumor cells. At the same time, the consumption of oxygen enhanced the hypoxic environment in the cells, thereby improving the chemotherapy efficacy mediated by the hypoxic environment.

[0060] The above results indicate that the hypoxia-responsive supramolecular polypeptide nanoprodrug SPN-TAPP-PCB4 of the present invention can exhibit better anti-tumor effect under near-infrared light conditions, achieving a synergistic chemotherapy-photodynamic therapy effect.

[0061] It should be understood that the solutions provided in the above embodiments are for illustrative purposes only and are not intended to limit the present invention. Those skilled in the art may make various modifications and substitutions to the above embodiments without departing from the purpose and spirit of the present invention, and all of these modifications and substitutions shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a hypoxia-responsive supramolecular polypeptide nanoprodrug, characterized in that: The following steps are involved: Step 1) prepare tetraaminoporphyrin-poly (L-lysine)-azobenzene mustard, the process is shown in Reaction Scheme 2: Reaction formula 2; The specific steps are as follows: Step 1-1) Tetraaminoporphyrin and lysine are added to a container, followed by addition of N,N-dimethylformamide, and the reaction is carried out at 25° C. for 48 hours. After the reaction is completed, the product TAPP-PZLys4 is obtained by precipitation with ether; Step 1-2) The TAPP-PZLs4 obtained in step 1-1) was dissolved in a glacial acetic acid / trifluoroacetic acid mixed solvent, and a hydrobromic acid / glacial acetic acid mixed solvent was added at 0°C, and the reaction was continued for 1.5 hours. After the reaction was completed, the reaction solution was precipitated in diethyl ether and centrifuged three times, and finally dried to obtain the product TAPP-PLL4; Step 1-3) TAPP-PLL4 obtained in step 1-2), nitrogen mustard, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are placed in a container, and then N,N-dimethylformamide is added to react overnight at room temperature. After the reaction, the mixture is precipitated with diethyl ether to obtain; Step 2) preparing a water-soluble column [6]arene AWBpP6; Step 3) The tetraaminoporphyrin-poly(L-lysine)-azobenzene mustard prepared in step 1) is dissolved in N,N-dimethylformamide solvent and stirred for 12 hours. The water-soluble column [6] aromatic hydrocarbon AWBpP6 prepared in step 2) is then dissolved in deionized water. The two are then added dropwise into a container. After stirring at room temperature for 24 hours, the hypoxia-responsive supramolecular polypeptide nanoprodrug is obtained by dialysis.

2. The method for preparing a hypoxia-responsive supramolecular polypeptide nanoprodrug according to claim 1, characterized in that: In step 3), the concentration of tetraaminoporphyrin-poly(L-lysine)-azobenzene mustard is 2 mg / mL, and the concentration of the water-soluble column [6] aromatic hydrocarbon AWBpP6 is 0.8 mg / mL.

3. The method for preparing a hypoxia-responsive supramolecular polypeptide nanoprodrug according to claim 1, characterized in that: In step 3), the volume ratio of the N,N-dimethylformamide solvent to the deionized water is 1:

10.

4. The method for preparing a hypoxia-responsive supramolecular polypeptide nanoprodrug according to claim 1, characterized in that: Step 3) The dialysis conditions are as follows: using a dialysis bag with a molecular weight of 1000, 500 mL of deionized water × 1 time / 8 hours, and dialysis for 48 hours.

5. The hypoxia-responsive supramolecular polypeptide nanoprodrug prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The particle size of the hypoxia-responsive supramolecular polypeptide nanoprodrug is 198.1±5.8 nm.

6. Use of the hypoxia-responsive supramolecular polypeptide nanoprodrug according to claim 5 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that The tumor is cervical cancer.

8. An anti-tumor pharmaceutical composition, characterized in that: The invention comprises the hypoxia-responsive supramolecular polypeptide nanoprodrug as claimed in claim 5, and pharmaceutically acceptable excipients.

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