Preparation of hypoxia response albumin supramolecular nano drug delivery system and application of hypoxia response albumin supramolecular nano drug delivery system in hypoxia tumor treatment

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to preparation of a hypoxia response albumin supramolecular nano drug delivery system and application of the hypoxia response albumin supramolecular nano drug delivery system in hypoxia tumor treatment. The nano drug delivery system comprises an albumin supramolecular nano carrier taking an azo calix [4] arene derivative as a drug carrying main body, a mitochondrial targeting photosensitizer and an autophagy inhibitor. The system is prepared through a light click chemical reaction between double bonds of azo calixarene and albumin subjected to reduction treatment, and has the hypoxia response characteristic. Meanwhile, the nano drug delivery system can initiate an active oxygen storm effect and inhibit the growth of hypoxic tumors in a hypoxic environment and under external stimulation of red light, and has a fluorescence imaging function. In addition, the novel drug delivery system is mild in preparation process, can load multiple drug combinations at the same time, has a diagnosis and treatment integrated function, and has a wide application prospect in the field of hypoxia tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation of a hypoxia-responsive albumin supramolecular nano-drug delivery system and its application in hypoxic tumor treatment. Background Art

[0002] Hypoxia (pO2≤2.5mmHg) is a common phenomenon in 50-60% of solid tumors. Due to the disordered vascular structure and high interstitial pressure inside hypoxic tumors, the drug enrichment efficiency in tumor tissue is low, the systemic toxicity and side effects are large, and the therapeutic effect is poor. In addition, the hypoxic microenvironment promotes the metabolic reprogramming, angiogenesis, and resistance to traditional therapies of tumors. Photodynamic therapy, as a new anti-tumor treatment method, kills tumor cells by activating photosensitizers to generate reactive oxygen species through light. However, traditional photosensitizers (such as Chlorin e6) are extremely dependent on oxygen to generate reactive oxygen species, which greatly reduces their efficacy in hypoxic tumors. At the same time, metabolic adaptations in hypoxic tumors (such as enhanced mitochondrial autophagy) allow tumor cells to weaken oxidative stress by clearing damaged mitochondria. Therefore, more effective hypoxic tumor treatment options are urgently needed to overcome the limitations of traditional treatments and optimize the treatment effect.

[0003] In recent years, nano-drug delivery systems have shown great potential in tumor treatment due to their excellent targeting, drug loading capacity and controlled release properties. In particular, in hypoxic tumors, nano-delivery systems can effectively enhance the enrichment of drugs in tumor tissues and significantly improve the therapeutic effect. At present, a variety of therapeutic platforms have been developed for the hypoxic microenvironment of tumors. Hypoxia-responsive tumor drug delivery strategies mainly include: (1) Covalent prodrugs: Anti-tumor drugs are covalently linked to hypoxia-responsive groups (such as nitro, quinone, azo, etc.) to form prodrugs. In a hypoxic environment, the responsive groups are reduced by reductase to release the drugs; although the strategy of linking hypoxia-responsive units to photosensitizers to form prodrugs has achieved certain therapeutic effects, this covalent linking strategy faces many challenges, such as time-consuming and complex synthesis and purification processes and potential activity changes and toxicity caused by covalent linking. (2) Nano-drug delivery systems: Anti-tumor drugs are loaded using hypoxia-responsive nano-drug delivery systems. For example, azocalix[4]arene (AC4A) has shown potential as a drug delivery carrier due to its high drug binding affinity and ability to release drugs in response to hypoxia. However, this type of hypoxia-responsive material is easily cleared quickly in the body and has poor biocompatibility, which weakens its overall therapeutic effect. In view of the above shortcomings, it is urgent to optimize its structure and function to improve its feasibility in clinical application. Summary of the invention

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a hypoxia-responsive albumin supramolecular nano-drug delivery system loaded with hydroxychloroquine (HCQ) and mitochondrial-targeted photosensitizer-thiomethylated Nile blue derivative (SMNB) for the treatment of hypoxic tumors. The nano-drug delivery system can responsively release drugs in hypoxic tumor tissues, significantly increase the enrichment of drugs in tumor tissues, and reduce systemic toxic side effects. Among them, the photosensitizer (SMNB) can target mitochondria and produce a large amount of reactive oxygen species without relying on oxygen, thereby enhancing the therapeutic effect, overcoming the problem that traditional photosensitizers rely on oxygen to limit the efficacy. In addition, the reactive oxygen species generated by the nano-drug delivery system under the action of laser can induce mitochondrial damage and autophagy, while hydroxychloroquine (HCQ) blocks mitochondrial autophagy. Through this dual action, reactive oxygen storms can be induced, and ultimately tumor cell death can be induced.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system, the method comprising the following steps:

[0007] S1. Preparation of azocalix[4]arene derivative (AC4A):

[0008] S11, dissolving mono-Boc-polyethylene glycol (n)-diamine in dichloromethane, then adding methacrylic anhydride and triethylamine in sequence, stirring at room temperature, concentrating the reaction solution in vacuo, then dissolving it in ethyl acetate, and washing the ethyl acetate solution with hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively, drying, concentrating in vacuo, and then performing column chromatography separation to obtain a colorless oil, dissolving the colorless oil in dichloromethane, then adding trifluoroacetic acid thereto, stirring at room temperature, and concentrating the reaction solution in vacuo to obtain a yellow oily product, i.e., polyethylene glycol (n)-monomethyl acrylamide;

[0009] S12, dissolving 1-monocarboxy trisulfonic acid azocalix[4]arene in dimethylformamide, then sequentially adding a basic catalyst and a condensing agent for activation reaction, then adding polyethylene glycol (n)-monomethyl acrylamide to the above reaction system, and continuously stirring the reaction. After the reaction is completed, removing the solvent to obtain a crude product, and the crude product is recrystallized with water and methanol to finally obtain a product represented by formula (I);

[0010]

[0011] S2. Preparation of reduced albumin: dissolving albumin in a solvent, adding a reducing agent and stirring the mixture at room temperature to reduce the disulfide bonds to thiol groups;

[0012] S3. Preparation of albumin material modified by azocalixarene: Compound 1: (AC4A), albumin material treated with a reducing agent and a photoinitiator are dissolved in water or a phosphate buffer solution with a pH of 7.4, reacted under ultraviolet light, and then purified by dialysis and dried to obtain the target product;

[0013] S4. Synthesis of Thionile Blue Derivative (SMNB):

[0014] S41. Aluminum sulfate, sodium thiosulfate and zinc chloride are added to N,N-diethyl-p-phenylenediamine in sequence. Potassium dichromate is slowly added under ice bath conditions, and the reaction is stopped when the reaction mixture becomes a viscous substance. The reaction product is filtered and washed to obtain a crude product. The crude product is refluxed in methanol and then filtered to obtain 2-amino-5-diethylaminobenzene sodium thiosulfate shown in formula (II);

[0015]

[0016] S42, dissolve the above-mentioned 2-amino-5-diethylaminobenzene sodium thiosulfate (Compound II) and naphthylamine in DMSO, and then add potassium dichromate to react. After stirring and mixing at room temperature, transfer the mixture to methanol, add hydrochloric acid, and then stir and react at room temperature. After the reaction is completed, remove the solvent, and slowly pour the remaining solution into a saturated sodium chloride solution to precipitate a dark blue solid. Collect the solid and dry it, and then purify it by silica gel column chromatography to obtain a dark blue solid product shown in formula (III), which is a thio-nile blue derivative (SMNB);

[0017]

[0018] S5. Dissolve the thio-Nile blue derivative (SMNB) and hydroxychloroquine (HCQ) of S4 in a solvent, then add the albumin material modified with the azocalix aromatic derivative of S3, stir and react at room temperature, and then dialyze to obtain the hypoxia-responsive albumin supramolecular nanodrug delivery system.

[0019] Preferably, in S11, the molar ratio of the mono-Boc-polyethylene glycol (3)-diamine, methacrylic anhydride and triethylamine is 1:1-1.2:1.2-1.5; and the first stirring time at room temperature is 20-30 hours.

[0020] Preferably, in S11, the volume ratio of dichloromethane to trifluoroacetic acid is 9:1-12:1; and the second stirring time at room temperature is 3-8 hours.

[0021] Preferably, in S12, the alkaline catalyst is any one of diisopropylethylamine (DIPEA) and dimethylaminopyridine (DMAP); the condensing agent is any one of (7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (TBTU).

[0022] Preferably, the activation reaction time is not less than 30 minutes.

[0023] Preferably, in step S12, the molar ratio of the 1-monocarboxytrisulfonic acid azocalix[4]arene to polyethylene glycol (n)-monomethyl acrylamide is 1:1.5-1:2.0, and n in polyethylene glycol (n)-monomethyl acrylamide is an integer of 3-10.

[0024] Preferably, in step S1, the duration of the continuous stirring reaction is 12-24 hours.

[0025] Preferably, in S2, the albumin is any one of human serum albumin, bovine serum albumin and whey albumin; and the reducing agent is any one of reduced glutathione, dithiothreitol and tris(2-carbonylethyl)phosphine hydrochloride.

[0026] Preferably, in S2, the molar ratio of albumin to reducing agent is 1:40-1:120.

[0027] Preferably, in S2, the solvent used is any one of ultrapure water and phosphate buffer solution.

[0028] Preferably, in S2, the stirring reaction is carried out for 6-12 hours.

[0029] Preferably, in S3, the molar ratio of albumin to AC4A is 1:20-1:60, and the reaction time is 12-24 hours.

[0030] Preferably, in S3, the photoinitiator is (2,2-bis(hydroxymethyl)propionic acid), and the reaction under ultraviolet light is a reaction under ultraviolet light with a wavelength of 365 nm.

[0031] Preferably, in S41, the molar ratio of the N,N-diethyl-p-phenylenediamine to aluminum sulfate or zinc chloride is 1:1.05-1:1.2; the molar ratio of the N,N-diethyl-p-phenylenediamine to sodium thiosulfate is 1:2.2-1:2.5; the molar ratio of the N,N-diethyl-p-phenylenediamine to potassium dichromate is 1:0.5-1:1.2.

[0032] Preferably, in S41, the reaction time is 1-3 hours; and the reflux time is 2-4 hours.

[0033] Preferably, in S42, the molar ratio of 2-amino-5-diethylaminobenzene sodium thiosulfate to naphthylamine is 1:1.2-1:1.5.

[0034] Preferably, in S42, the stirring reaction time is 30-50 minutes.

[0035] Preferably, in S42, the dosage of hydrochloric acid added is 20-30 mL of 2 mol / L hydrochloric acid.

[0036] Preferably, in S42, the solvent used for purification by silica gel column chromatography is CH2Cl2 / CH3OH=10:1, (v / v).

[0037] The second aspect of the present invention provides a hypoxia-responsive albumin supramolecular nano drug delivery system prepared by the preparation method described in the first aspect.

[0038] The third aspect of the present invention provides the use of the hypoxia-responsive albumin supramolecular nano-drug delivery system described in the second aspect in the preparation of an integrated anti-hypoxia tumor diagnosis and treatment drug.

[0039] Preferably, the anti-hypoxic tumor diagnosis and treatment integrated drug is a laser-assisted anti-hypoxic tumor diagnosis and treatment integrated drug.

[0040] After entering the body, the nano drug delivery system prepared by the method of the present invention can bind to the albumin receptors (such as gp60) highly expressed on the surface of tumor cells through the active targeting mechanism of albumin, thereby achieving precise delivery and long-term circulation to the tumor site. At the same time, the nano drug delivery system can release hydroxychloroquine (HCQ) and thio-Nile blue derivatives (SMNB) in response to the hypoxic microenvironment of the tumor to achieve fluorescence imaging. In addition, the mitochondrial targeted photosensitizer (SMNB) can generate a large amount of reactive oxygen species independently of oxygen under 660nm laser irradiation, damaging mitochondria. Hydroxychloroquine (HCQ) inhibits autophagy by alkalizing lysosomes, inducing a reactive oxygen storm in the cell, and ultimately leading to tumor cell death.

[0041] Preferably, the hypoxic tumor includes (but is not limited to) breast cancer, melanoma, pancreatic cancer, cervical cancer, prostate cancer, colon cancer, and ovarian cancer.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention discloses a hypoxia-responsive albumin supramolecular nano drug delivery system, which includes an albumin supramolecular carrier with an azocalix[4]arene derivative as the drug carrier, a mitochondrial targeted photosensitizer (such as a thiomethylated Nile blue derivative) and an autophagy inhibitor (such as hydroxychloroquine). The system is prepared by a photo-click chemical reaction between the double bonds of the azocalixarene and the free thiol groups of the reduced albumin, and has hypoxia-responsive characteristics. At the same time, the nano drug delivery system can induce an active oxygen storm effect under hypoxic environment and external stimulation of red light, inhibit the growth of hypoxic tumors, and has a fluorescence imaging function, and is expected to be used in the preparation of anti-hypoxic tumor diagnosis and treatment integrated drugs. In addition, the preparation process of the new drug delivery system is mild, and multiple drug combinations can be loaded at the same time, and it has the function of integrated diagnosis and treatment. The nano drug delivery system of the present invention expands the application scope of albumin nanotechnology, making it widely used in the field of hypoxic tumor treatment. In general, the present invention has the following advantages:

[0044] (1) Highly efficient targeted and oxygen-independent treatment: The present invention utilizes the active targeting mechanism and EPR effect of albumin to achieve precise delivery to the tumor site, improve the enrichment of drugs in tumor tissues, and reduce systemic toxic side effects. In addition, the mitochondrial targeted photosensitizer (SMNB) in the nanosystem can generate reactive oxygen species independently of oxygen in a hypoxic environment, overcoming the limitations of traditional photodynamic therapy in hypoxic tumors and enhancing the therapeutic effect.

[0045] (2) Dual action mechanism enhances therapeutic effect: The present invention combines mitochondrial targeted photosensitizer (SMNB) and autophagy inhibitor (HCQ) to achieve synergistic enhancement. The photosensitizer can produce reactive oxygen species under laser irradiation to induce mitochondrial damage, while HCQ can block the autophagy function of tumor cells and prevent cells from weakening oxidative stress by removing damaged mitochondria. This dual action effectively induces reactive oxygen storm, thereby significantly enhancing the anti-tumor effect.

[0046] (3) Hypoxia response and integrated diagnosis and treatment: Through the combination of hypoxia-responsive azocalixarene and albumin, the nano drug delivery system can trigger drug release in the hypoxic microenvironment of the tumor, and at the same time has fluorescence imaging function, forming an integrated diagnosis and treatment platform. This feature makes the treatment more accurate and efficient, significantly improving its application value in the treatment of hypoxic tumors.

[0047] (4) Simple and efficient preparation process: The preparation process of the present invention is mild, does not require complicated operations and high temperature and high pressure conditions, can load a variety of drug combinations in proportion, and is suitable for large-scale production and application. Albumin carriers have good biocompatibility and long-term circulation time, further improving the safety and efficacy of treatment, while saving energy consumption and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1The synthetic flow chart of azocalix[4]arene derivative (AC4A);

[0049] Figure 2 This is the H NMR spectrum of azocalix[4]arene derivative (AC4A) (DMSO-d6, 400 MHz, 25°C);

[0050] Figure 3 is a synthetic flow chart of thio-Nile blue derivatives (SMNB) (n=1);

[0051] Figure 4 This is the H NMR spectrum of thio-Nile blue derivative (SMNB) (Chloroform-d, 400 MHz, 25°C);

[0052] Figure 5 The reaction principle and UV absorption spectrum of azocalix[4]arene derivative (AC4A) and azocalixarene-modified albumin material (HSA-AC4A); (a) schematic diagram of the molecular structure of the HSA-AC4A chain; (b) UV absorption spectrum of AC4A in PBS; (c) standard curve of UV absorption and concentration of AC4A at 362nm; (d) UV absorption spectrum of HSA-AC4A (125μg / mL);

[0053] Figure 6 TEM images and particle size distribution diagrams of various hypoxia-responsive albumin supramolecular nanodrug delivery systems; (a) HC4; (b) HC4H; (c) SHC4; (d) SHC4H;

[0054] Figure 7 Results of fluorescence quenching (a) and fluorescence recovery (b) of hypoxia-responsive albumin supramolecular nano-drug delivery system;

[0055] Figure 8 Schematic diagram of the principle of drug loading and release of hypoxia-responsive albumin supramolecular nanocarriers;

[0056] Fig. 9 The UV absorption spectra of HCQ and SMNB (a) and the standard curve (b), and the result of releasing HCQ and SMNB in ​​response to hypoxia (c);

[0057] Fig.10 UV absorption spectra of the hypoxia-responsive albumin supramolecular nano-drug delivery system using DHR123 probe to detect the ability to produce reactive oxygen species; (a) SNH2C4H produces reactive oxygen species under normoxic and laser irradiation conditions; (b) SNH2C4H produces reactive oxygen species under hypoxic laser irradiation conditions;

[0058] Fig.11The results of the hypoxia-responsive albumin supramolecular nano-drug delivery system SHC4 and SHC4H co-localization experiments in mitochondria and lysosomes (SMNB and PBS were used as controls);

[0059] Fig.12 The results of the hypoxia-responsive albumin supramolecular nano-drug delivery system SHC4H alkalinizing the lysosomes of hypoxic tumor cells (B16 and MCF-7) are shown;

[0060] Fig.13 For DCFH-DA (total active oxygen indicator), DHR123 (O2 ·- The experimental results of detecting the generation of reactive oxygen species by hypoxia-responsive albumin supramolecular nano-drug delivery system SHC4 and SHC4H using ·OH fluorescence indicator (indicator) and HPF (·OH fluorescence indicator) in B16 and MCF-7 cells (SMNB, HCQ, SPIN2 and PBS were used as controls);

[0061] Fig.14 The results of the cytotoxicity experiments of hypoxia-responsive albumin supramolecular nano-drug delivery systems HC4H, SHC4 and SHC4H on hypoxic B16 and MCF-7 cells with or without laser irradiation (with SMNB, HCQ and PBS as controls); (a) B16; (b) MCF-7;

[0062] Fig.15 The curves of the changes of tumor volume over time in tumor-bearing mice after treatment with hypoxia-responsive albumin supramolecular nanodrug delivery systems HC4H, SHC4 and SHC4H;

[0063] Fig.16 Results of routine blood tests (a) and blood biochemistry (b) analysis of tumor-bearing mice after treatment with the hypoxia-responsive albumin supramolecular nanodrug delivery system SHC4H; HM represents healthy mice, and TM represents tumor-bearing mice. DETAILED DESCRIPTION

[0064] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0065] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0066] Example: Preparation of hypoxia-responsive albumin supramolecular nano-drug delivery system (SHC4H)

[0067] 1. Synthesis of azocalix[4]arene derivatives (Compound Ⅰ, AC4A)

[0068] Its synthetic route is Figure 1 As shown, the specific synthesis method is:

[0069] Weigh mono-Boc-polyethylene glycol (3)-diamine (1 g, 2.97 mmol, CAS: 1235514-18-7, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and dissolve it in 30 mL of dichloromethane. Then add methacrylic anhydride (0.55 mL, 3.46 mmol) and triethylamine (0.55 mL, 3.95 mmol) in sequence and stir at room temperature for 24 hours. Concentrate in vacuo until there is no solvent. Then dissolve the reaction product in ethyl acetate (40 mL). Wash the ethyl acetate solution three times with hydrochloric acid (2 mol / L), saturated sodium bicarbonate aqueous solution and saturated sodium chloride aqueous solution respectively. Then add anhydrous magnesium sulfate and dry overnight. After vacuum concentration, separate by column chromatography (CH2Cl2 / CH3OH=10:1, v / v) to obtain 590 mg of colorless oil with a yield of 48%. The colorless oil was dissolved in dichloromethane (10 mL), trifluoroacetic acid (1 mL) was added thereto, and the mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated in vacuo to obtain a yellow oily product (ie, polyethylene glycol (3)-monomethylacrylamide).

[0070] 1-monocarboxytrisulfonic acid azocalix[4]arene (200 mg, 0.18 mmol) (for the specific synthesis method, refer to the literature "Lu, Lilin, et al." Highly selective chromogenic ionophores for the recognition of chromium(III)based on a water-soluble azocalixarene derivative." Analytica Chimica Acta 535.1-2 (2005): 183-187.") was dissolved in DMF (20 mL), and then DIPEA (44 mg, 0.34 mmol) and HATU (78 mg, 0.21 mmol) were added in sequence, and stirred at room temperature for activation for 30 minutes. Then polyethylene glycol (3)-monomethyl acrylamide (90 mg, 0.35 mmol) was added to the above reaction system, and the resulting mixture was stirred at room temperature (300 rpm) for 12 hours. After the reaction, the reaction product was concentrated in vacuo until there was no solvent, and the crude product was recrystallized with water and methanol (water:methanol=1:4, v:v) to obtain a brick red powder (Compound I). The H NMR spectrum of the product is as follows Figure 2 As shown, the H NMR spectrum data are as follows:

[0071] 1H NMR (400MHz, DMSO-d6) δ7.95 (d, J=8.2Hz, 2H), 7.89–7.76 (m, 10H), 7.76–7.66 (m, 12H), 5.63 (s, 1H) ,5.29(s,1H),4.14(s,4H),3.82(s,4H),3.65–3.32(m,14H),3.24(d,J=5.6Hz,2H),1.82(s,3H)ppm. 13 C NMR(100MHz,DMSO-d6)δ167.68,165.86,158.58,153.66,152.13,149.06,144.87,139.87,135.18 ,130.39,128.48,126.65,123.97,121.79,121.68,119.26,70.14,69.64,69.02,31.78,18.70ppm.

[0072] The product structure is:

[0073]

[0074] 2. Synthesis of Thionile Blue Derivatives (SMNB)

[0075] Its synthetic route is Figure 3 As shown, the specific synthesis method comprises the following steps:

[0076] (1) Synthesis of 2-amino-5-diethylaminophenyl sodium thiosulfate: N,N,-diethyl-p-phenylenediamine (0.50 g, 3.05 mmol) was added to 20 mL of aqueous solution containing aluminum sulfate (2.08 g, 3.23 mmol), and sodium thiosulfate (1.11 g, 7.00 mmol) and zinc chloride (0.44 g, 3.19 mmol) were added to the reaction mixture in a stirring state. Potassium dichromate (0.25 g, 0.84 mmol) was slowly added over 20 minutes, and a viscous substance was obtained after stirring in an ice bath for 2 hours. After filtering, the crude product was washed with acetone to obtain a crude product, which was refluxed in methanol for 3 hours and filtered to obtain a dark gray solid (i.e., compound II: 2-amino-5-diethylaminophenyl sodium thiosulfate).

[0077] (2) The above-obtained 2-amino-5-diethylaminophenyl sodium thiosulfate (1.02 g, 3.70 mmol) and naphthylamine (0.80 g, 5.07 mmol) were dissolved in DMSO, potassium dichromate (1.20 g, 4.05 mmol) was added, and the mixture was stirred at room temperature for 20 minutes. Subsequently, the mixture was transferred to 200 mL of methanol, and 20 mL of hydrochloric acid (2 mol / L) was added, and the reaction was stirred at room temperature for 40 minutes. After the reaction was completed, the solvent was removed under reduced pressure, and the remaining solution was slowly poured into 100 mL of saturated sodium chloride to precipitate a dark blue solid. After being collected and dried, the obtained crude product was purified by silica gel column chromatography (CH2Cl2 / CH3OH=10:1, v / v) to obtain a dark blue solid (Compound III, SMNB) with a yield of 21.46%. The hydrogen nuclear magnetic resonance spectrum of the product is as follows: Figure 4 As shown, the H NMR spectrum data are as follows:

[0078] 1 H NMR(400MHz,Chloroform-d)δ11.70(s,1H),9.23(d,J=8.0Hz,1H),8.89(dd,J=7.9,1.6Hz,1H),7.90(d,J=9.3Hz,1H),7.83–7.69(m,2H ),7.09(dd,J=9.4,2.6Hz,1H),6.84(s,1H),6.78(d,J=2.7Hz,1H),3.61(q,J=7.1Hz,4H),3.40(d,J=4.7Hz,3H),1.35(t,J=7.1Hz,6H).

[0079] The product structure is:

[0080]

[0081] 3. Treat albumin molecules with reducing agents to expose the thiol groups in the molecules: Dissolve human serum albumin (10 mg) and glutathione (3.35 mg) in a phosphate buffered saline solution at pH = 7.4, and stir at room temperature for 12 hours. After the reaction solution is dialyzed for 24 hours using a dialysis bag (MWCO = 2000), it is freeze-dried to obtain the albumin material with exposed thiol groups (Human serum albumin, HSA).

[0082] 4. Preparation of albumin material modified by azocalixarene (HSA-AC4A): Compound I (20 mg), albumin treated with a reducing agent (24 mg) and 2-bis(hydroxymethyl)propionic acid (20 mg) were dissolved in ultrapure water (20 mL) and reacted under 365 nm ultraviolet light for 24 hours. The reaction solution was dialyzed for 24 hours (MWCO = 2000) and freeze-dried to obtain about 25 mg of red solid powder. The structure of the product HSA-AC4A after the photo-click chemistry reaction between the thiol group of albumin and the double bond of the azocalix[4]arene derivative (AC4A) is as follows: Figure 5 As shown in a.

[0083] AC4A was prepared into solutions of different concentrations (2, 4, 8, 10 and 12 μM), and the UV absorption spectra of the different solutions were measured. The results are shown in Figure 5 As shown in b, AC4A has characteristic absorption at 362nm, and the degree-dependent UV standard curve of AC4A is established (such as Figure 5 c). The UV absorption spectrum of HSA-AC4A was detected, and the results were as follows Figure 5 As shown in d, HSA-AC4A (125 μg / mL) showed characteristic ultraviolet absorption of AC4A, proving that the modification was successful. According to the ultraviolet absorbance-concentration standard curve, the modification rate of AC4A in HSA-AC4A was determined to be 17.1%.

[0084] 5. Preparation of hypoxia-responsive albumin supramolecular nano-drug delivery system:

[0085] 100 μL of hydroxychloroquine (HCQ, 100 μM) phosphate buffered saline solution, 100 μL of SMNB (100 μM) phosphate buffered saline solution, 10 μL of SMNB (10 μM) and 100 μL of HCQ (100 μM) phosphate buffered saline solution were respectively added to 2 mL of phosphate buffered saline solution (0.3 mg / mL) of azocalixarene-modified albumin material obtained in the previous step, and reacted at room temperature for 2 hours. Then, a series of hypoxia-responsive albumin supramolecular nanodrug delivery systems (HC4: hypoxia-responsive albumin supramolecular nanodrug delivery system without drug loading; HC4H: hypoxia-responsive albumin supramolecular nanocarrier loaded with hydroxychloroquine; SHC4: hypoxia-responsive albumin supramolecular nanocarrier loaded with SMNB; SHC4H: hypoxia-responsive albumin supramolecular nanocarrier loaded with SMNB and HCQ) were purified by dialysis (MWCO=2000) for 12 hours. The morphology and particle size distribution of the hypoxia-responsive albumin supramolecular nanocarrier are shown in Figure 2. Figure 6 shown.

[0086] In the present invention, we used sodium dithiosulfate to simulate the azoreductase overexpressed in hypoxic tumors. SHC4H (SMNB: 10 μM) was reduced by sodium dithiosulfate (100 μM) to obtain SNH2C4H; SHC4 (SMNB: 10 μM) was reduced by sodium dithiosulfate (100 μM) to obtain SNH2C4.

[0087] Experimental example: Performance test of hypoxia-responsive albumin supramolecular nano-drug delivery system

[0088] (1) Perform fluorescence ON / OFF test on albumin material modified with azocalixarene. The specific method is as follows:

[0089] The azocalixarene-modified albumin material (HSA-AC4A) was prepared into aqueous solutions with concentrations of 1, 2, 3, 4, and 5 μM / L, and dropped into SMNB solution (10 μM), and the fluorescence emission spectra of the mixtures were recorded.

[0090] Test results such as Figure 7 As shown in the figure, as the concentration of azocalixarene-modified albumin material increases, the fluorescence of SMNB is gradually quenched, which is because the photoinduction of SMNB by AC4A has an electron transfer mechanism. When AC4A is reduced to NH2C4A by azoreductase, the fluorescence of SMNB is "turned on", and this property can be used for hypoxia response fluorescence imaging.

[0091] (2) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in the example was tested for hypoxia-responsive drug release performance, and the specific method was as follows:

[0092] The azo bonds in the hypoxia-responsive albumin supramolecular nano-drug delivery system will be broken by various reductases in the hypoxic environment, thereby releasing the drug (e.g. Figure 8 ). HCQ and SMNB were prepared into aqueous solutions with concentrations of 0.5, 1, 2, 4, 6, 8, and 10 μM / L, respectively, and the UV absorption spectra of different solutions were recorded. Rat liver microsomes (40 μg / mL) and NADPH (40 μM) were added to HC4H (HSA-AC4A: 10 μM; HCQ: 10 μM) or SHC4 (HSA-AC4A: 10 μM; SMNB: 10 μM), respectively, and incubated at 37°C under hypoxic conditions (1% O2). 0.5 mL of the solution was taken at different time points and centrifuged for 5 minutes using an ultrafiltration centrifuge tube (MWCO = 3000), the concentrated solution was collected, and its UV absorbance at 342 nm (HCQ) or 625 nm (SMNB) was measured, and the released HCQ / SMNB (such as) was quantified based on the concentration-UV standard curve of HCQ or SMNB. Fig. 9 This process was repeated three times at each time point.

[0093] The results are as follows Fig. 9 As shown in Figure c, under hypoxia simulation conditions, the cumulative release of HCQ and SMNB exceeded 75% within 15 minutes, while almost no drug release was observed under normoxic conditions. These results indicate that the nano drug delivery system has the performance of hypoxia-responsive drug release.

[0094] (3) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example was tested for its in vitro solution-level reactive oxygen generation performance. The specific method is as follows:

[0095] Detection of O2 in solution using fluorescent probe DHR123 ·- Generation. A mixed solution of SHC4H (SMNB: 10 μM) and sodium dithiosulfate (100 μM) or a mixed solution of SHC4 (SMNB: 10 μM) and sodium dithiosulfate (100 μM) was mixed with an aqueous solution of DHR123 (10 μM). To simulate hypoxic conditions, the aqueous solution containing supramolecular nanoparticles was purged with argon for 5 minutes and maintained in a hypoxic state during irradiation. The fluorescence intensity values ​​(525 nm) of different drug solutions irradiated with 660 nm laser for different times were recorded using a fluorescence spectrophotometer.

[0096] Test results such as Fig.10 As shown, SHNH2C4H (a mixed solution of HSA-AC4A reduced by sodium dithiosulfate and SMNB, HCQ) produces O2 under anoxic conditions. ·- There was no significant difference compared with normoxic conditions, indicating that SHC4H produces O2 independently of oxygen concentration. ·- .

[0097] (4) The hypoxia-responsive release of SMNB and the mitochondrial targeting performance of the hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in the example were tested. The specific method is as follows:

[0098] MCF-7 and B16 cells were cultured at 10 5The density of cells / well was inoculated into the confocal dish and incubated overnight until adhered. The culture medium was replaced with a medium containing drugs in gradient concentrations. The drugs used were SMNB and the hypoxia-responsive albumin supramolecular nanodrug delivery system (SHC4 or SHC4H, containing 50 nM SMNB) prepared in the embodiment. After culturing for 12 hours under hypoxic conditions (1% O2), it was replaced with a culture medium containing Mito-Tracker Green (100 nM) or Lyso-Tracker Green (50 nM) and pre-incubated at 37°C for 30 minutes. After labeling the cell nucleus with DAPI, it was observed by confocal laser scanning microscopy. The excitation / emission wavelengths of Mito-Tracker Green were 490 / 516 nm, Lyso-TrackerGreen was 504 nm / 511 nm, DAPI was 340 / 488 nm, and SMNB was 633 / 650-700 nm.

[0099] Test results such as Fig.11 As shown, the hypoxia-responsive albumin supramolecular drug delivery system can release SMNB in ​​response to hypoxia, and SMNB exhibits a targeting effect on mitochondria.

[0100] (5) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example was tested for its lysosomal alkalinization performance. The specific method is as follows:

[0101] MCF-7 and B16 cells were cultured at 10 5 The density of cells / well was inoculated into the confocal dish and incubated overnight until adhered. The culture medium was replaced with a drug-containing culture medium, and the drug used was the hypoxia-responsive albumin supramolecular nano-drug delivery system (SHC4H, containing 10 μM HCQ) prepared in the example, and cultured under hypoxic conditions (1% O2) for 6, 12 and 24 hours. Subsequently, the culture medium containing Lyso-Tracker Green was replaced and incubated for 30 minutes, and the cell samples were observed by confocal laser scanning microscopy.

[0102] Test results such as Fig.12 As shown, SHC4H exhibits excellent lysosomal alkalinization performance.

[0103] (6) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example was tested for its performance in generating reactive oxygen species through electron transfer independently of oxygen, and the results were recorded as follows:

[0104] MCF-7 and B16 cells were cultured at 10 5The cells were seeded into the confocal dish at a density of 100 cells / well and incubated overnight until adhered. The culture medium was replaced with a medium containing a gradient concentration of drugs. The drugs used were a photosensitizer that requires oxygen to produce reactive oxygen - silicon (IV) phthalocyanine di(trihexylsilyl oxide) (SPIN2, CAS: 92396-89-9, purchased from Shanghai Nafu Biotechnology Co., Ltd.) and the hypoxia-responsive albumin supramolecular nanodrug delivery system prepared in the example (SHC4, SHC4H, containing 50 nM SMNB). After culturing for 24 hours under normoxic (21% O2) or hypoxic (1% O2) conditions, the cells were incubated with DCFH-DA (10 μM), dihydrorhodamine 123 (DHR123, 10 μM) or hydroxyphenylfluorescein (HPF, 10 μM) at 37°C for 30 minutes. The cell samples were then irradiated with 660 nm laser (100 mW / cm 2 ; 5 minutes), and the cell nucleus was marked with DAPI. Finally, the cells were observed and images were collected by confocal laser scanning microscopy, with the excitation / emission wavelengths of DAPI at 358 / 461 nm and the excitation / emission wavelengths of DCFH-DA, DHR123 and HPF at 481 / 535 nm.

[0105] Test results such as Fig.13 As shown, after SHC4H was treated with 660nm laser, it showed excellent ROS generation ability even in hypoxic environment.

[0106] (7) Cytotoxicity test of the hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example was performed using B16 and MCF-7 cells. The specific method is as follows:

[0107] B16 cells were cultured at 5 × 10 3 The cells were inoculated into a 96-well plate at a density of 100 cells / well. After overnight incubation to allow them to adhere to the wall, the culture medium was replaced with a culture medium containing gradient concentrations of drugs. The drugs used were a series of hypoxia-responsive albumin supramolecular nano-drug delivery systems (HC4H, SHC4, SHC4H; containing 0.25 μM SMNB, and / or 10 μM HCQ) and two guest drugs (SMNB: 0.25 μM; HCQ: 10 μM) prepared in the examples. After 24 hours of culture, the culture medium was replaced with a normal 1640 culture medium containing 10% fetal bovine serum, and the cells that needed to be illuminated were placed under a 660 nm laser (100 mW / cm 2 ; 5 minutes). After incubation for another 2 hours, the culture medium was removed and rinsed with phosphate buffer solution. 100 μL of cell culture medium containing 10% CCK-8 reagent was added and incubated for 1 hour, and the absorbance value of the sample at a wavelength of 450 nm was tested.

[0108] The test steps based on MCF-7 cells are the same as those based on B16 cells except for the different cell types and drug concentrations. The drugs used in the MCF-7 cell line are a series of hypoxia-responsive albumin supramolecular nano-drug delivery systems (HC4H, SHC4, SHC4H; containing 0.10 μM SMNB and / or 10 μM HCQ) prepared in the examples and two guest drugs (SMNB: 0.10 μM; HCQ: 10 μM).

[0109] Test results such as Fig.14 As shown, SHC4H has little effect on cell activity under normoxic conditions and 660nm laser irradiation, because SHC4H does not release drugs under normoxic conditions. However, under hypoxic conditions and 660nm laser irradiation, SHC4H showed a significant cell proliferation inhibitory effect. This shows that SHC4H in the present invention can only exert its toxic effect under hypoxic conditions and specific red light irradiation. Compared with the control group treated with SHC4+hv (hv represents 660nm laser irradiation), SHC4H+hv showed a more significant cell proliferation inhibitory effect. The calculation results show that the synergistic parameter Q value of SHC4H combined with laser irradiation is greater than 1.15 (Q MCF-7 =1.29, Q B16 =1.23), which indicates that the combined use of SHC4H and laser irradiation has a significant synergistic inhibitory effect on MCF-7 and B16 cells.

[0110] The calculation formula of combination index (Q) value is:

[0111]

[0112] Among them, V(a+b) represents the inhibition rate of combined treatment of A and B, while Va and Vb represent the inhibition rates of A and B alone, respectively (Q < 0.85 represents antagonism, 0.85 ≤ Q < 1.15 represents additive effect, and Q ≥ 1.15 represents synergistic effect).

[0113] (8) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example 1 was tested for its tumor growth inhibition effect and biosafety on B16 / MCF-7 tumor-bearing mice. The specific method is as follows:

[0114] B16 cells (5×10 5 cells dispersed in 100 μL PBS), MCF-7 cells (1×10 6 The cells were dispersed in 100 μL PBS and implanted subcutaneously into the thigh of female BALB / c nude mice to establish a subcutaneous xenograft melanoma / breast cancer tumor model.

[0115] When the tumor volume of BALB / c nude mice grows to 150-180mm 3 Afterwards, the mice were randomly divided into six groups (n=6 mice in each group): PBS group, SMNB+HCQ+hv group, HC4H group, SHC4+hv group, SHC4H group and SHC4H+hv group. The hypoxia-responsive albumin supramolecular nanodrug delivery system and the corresponding pure drug prepared in Example 1 were implemented by tail vein injection for 4 consecutive times. Among them, the dosage of B16 tumor model was SMNB: 0.3mg / Kg, HCQ: 30mg / Kg. The dosage of MCF-7 tumor model was SMNB: 0.3mg / Kg, HCQ: 30mg / Kg. 24 hours after the first two administrations, the hypoxia-responsive albumin supramolecular nanodrug delivery system and the corresponding pure drug were implemented by tail vein injection for 4 consecutive times. Among them, the dosage of B16 tumor model was SMNB: 0.3mg / Kg, HCQ: 30mg / Kg. 2 , 5 minutes) to irradiate the tumor site. Tumor size and mouse weight were recorded every day to evaluate the tumor treatment effect. At the end of treatment, blood samples were collected from healthy mice, tumor-bearing mice treated with PBS, and tumor-bearing mice treated with SHC4H+hv (n=3 mice per group) for routine blood tests and blood biochemistry analysis.

[0116] like Fig.15 As shown, in the two tumor models, the tumor growth rates of the HC4H group and the SHC4H group were similar to those of the PBS group, indicating that the inhibitory effect of HCQ alone on tumors was limited. Compared with the HCQ+SMNB+hv group, the SHC4+hv group and the SHC4H+hv group significantly inhibited tumor growth, further demonstrating the superior targeting efficacy of the hypoxia-responsive albumin supramolecular nanodrug delivery carrier of the present invention on tumors. In particular, the SHC4H+hv group exhibited a more excellent anti-tumor effect than the SHC4+hv group, fully demonstrating the excellent application potential of the SHC4H nanodrug delivery system combined with laser irradiation in the present invention in the treatment of hypoxic tumors. In addition, as Fig.16 As shown, there was no significant difference in the blood routine and biochemical analysis results between healthy mice and tumor mice cured by SHC4H+hv, indicating that the preparation of the present invention has excellent biosafety.

[0117] In summary, the hypoxia-responsive albumin supramolecular nano-drug delivery system co-loaded with hydroxychloroquine (HCQ) and mitochondrial-targeted photosensitizer (SMNB) prepared by the present invention can, on the one hand, actively target tumor tissues, responsively release drugs in hypoxic tumor tissues, significantly increase the enrichment of drugs in tumor tissues, and reduce systemic toxic side effects; on the other hand, the photosensitizer (SMNB) can target mitochondria under laser irradiation and produce a large amount of reactive oxygen species independently of oxygen. Hydroxychloroquine (HCQ) can disrupt hypoxic tumor metabolism and block mitochondrial autophagy, and induce reactive oxygen storms through this dual action, ultimately inducing tumor cell death. Therefore, the nano-drug delivery system of the present invention significantly improves the enrichment and anti-tumor efficacy of drugs in tumors through hypoxia response and dual action mechanisms, and expands the application potential of albumin nanotechnology in the treatment of hypoxic tumors.

[0118] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system, characterized in that: The following steps are involved: S1. Preparation of azocalix[4]arene derivatives: S11, dissolving mono-Boc-polyethylene glycol (n)-diamine in dichloromethane, then adding methacrylic anhydride and triethylamine in sequence, stirring at room temperature, concentrating the reaction solution in vacuo, then dissolving it in ethyl acetate, and washing the ethyl acetate solution with hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution, respectively, drying, concentrating in vacuo, and then performing column chromatography separation to obtain a colorless oil, dissolving the colorless oil in dichloromethane, then adding trifluoroacetic acid thereto, stirring at room temperature, and concentrating the reaction solution in vacuo to obtain a yellow oily product, i.e., polyethylene glycol (n)-monomethyl acrylamide; S12, dissolving 1-monocarboxytrisulfonic acid azocalix[4]arene in dimethylformamide, then sequentially adding a basic catalyst and a condensing agent for activation reaction, then adding polyethylene glycol (n)-monomethyl acrylamide to the above reaction system, and continuously stirring the reaction, after the reaction is completed, removing the solvent to obtain a crude product, and the crude product is recrystallized with water and methanol to finally obtain a product represented by formula (I); S2. Preparation of reduced albumin: dissolving albumin in a solvent, adding a reducing agent and stirring the mixture at room temperature to reduce the disulfide bonds to thiol groups; S3. Preparation of albumin material modified by azocalixarene: dissolving an azocalix[4]arene derivative, albumin material treated with a reducing agent and a photoinitiator in water or a phosphate buffer solution with a pH of 7.4, reacting under ultraviolet light, and then dialysis purification and drying to obtain the target product; S4. Synthesis of Thionile Blue Derivatives: S41, aluminum sulfate, sodium thiosulfate and zinc chloride are added to N,N-diethyl-p-phenylenediamine in sequence, potassium dichromate is slowly added under ice bath conditions, and then the reaction mixture is stirred to become a viscous substance, which is filtered and washed to obtain a crude product, and the crude product is refluxed in methanol and then filtered to obtain 2-amino-5-diethylaminobenzene sodium thiosulfate represented by formula (II); S42, dissolving the above-mentioned 2-amino-5-diethylaminobenzene sodium thiosulfate and naphthylamine in DMSO, adding potassium dichromate, stirring and mixing at room temperature, transferring the mixture into methanol, adding hydrochloric acid, and then stirring and reacting at room temperature. After the reaction is completed, removing the solvent, and slowly pouring the remaining solution into a saturated sodium chloride solution to precipitate a dark blue solid, collecting the solid and drying it, and then purifying it by silica gel column chromatography to obtain a dark blue solid product shown in formula (III), which is a thio-nile blue derivative; S5. Dissolve the thio-nile blue derivative and hydroxychloroquine of S4 in a solvent, add the azocalixarene-modified albumin material of S3, stir and react at room temperature, and then dialyze to obtain the hypoxia-responsive albumin supramolecular nanodrug delivery system.

2. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In S12, the alkaline catalyst is any one of diisopropylethylamine and dimethylaminopyridine; the condensing agent is any one of (7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

3. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In step S12, the molar ratio of the 1-monocarboxytrisulfonic acid azocalix[4]arene to polyethylene glycol (n)-monomethyl acrylamide is 1:1.5-1:2.0, and n in polyethylene glycol (n)-monomethyl acrylamide is an integer of 3-10.

4. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In S2, the albumin is any one of human serum albumin, bovine serum albumin and whey albumin; the reducing agent is any one of reduced glutathione, dithiothreitol and tris(2-carbonylethyl)phosphine hydrochloride.

5. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In S3, the molar ratio of albumin to azocalix[4]arene derivative is 1:20-1:60, and the reaction time is 12-24 hours.

6. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In S3, the photoinitiator is 2,2-bis(hydroxymethyl)propionic acid, and the reaction under ultraviolet light is carried out under ultraviolet light with a wavelength of 365 nm.

7. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In S41, the molar ratio of N,N-diethyl-p-phenylenediamine to aluminum sulfate or zinc chloride is 1:1.05-1:1.2; the molar ratio of N,N-diethyl-p-phenylenediamine to sodium thiosulfate is 1:2.2-1:2.5; the molar ratio of N,N-diethyl-p-phenylenediamine to potassium dichromate is 1:0.5-1:1.

2.

8. The method for preparing a hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 1, characterized in that: In S42, the molar ratio of 2-amino-5-diethylaminobenzene sodium thiosulfate to naphthylamine is 1:1.2-1:1.

5.

9. A hypoxia-responsive albumin supramolecular nano-drug delivery system prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the hypoxia-responsive albumin supramolecular nano-drug delivery system according to claim 9 in the preparation of an integrated anti-hypoxia tumor diagnosis and treatment drug.

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