Preparation of a hypoxia-responsive albumin supramolecular nanodrug delivery system and its application in hypoxic tumor treatment

Abstract

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 the treatment of hypoxic tumors. The nano-drug delivery system of the present invention includes an albumin supramolecular nano-carrier with an azocalix[4]arene derivative as the drug-carrying body, a mitochondrial-targeted photosensitizer, and an autophagy inhibitor. The system is prepared by a photo-click chemical reaction between the double bond of the azocalixarene and the albumin after reduction treatment, and has hypoxia-responsive characteristics. At the same time, the nano-drug delivery system can trigger an active oxygen storm effect under the external stimulation of red light in a hypoxic environment, inhibit the growth of hypoxic tumors, and has a fluorescence imaging function. In addition, the preparation process of the new drug delivery system is mild, and it can simultaneously load a combination of multiple drugs, and has the function of integrated diagnosis and treatment, and has broad application prospects in the field of hypoxic 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 the treatment of hypoxic tumors. 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 through light to generate reactive oxygen species. 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 treatment effects.

[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. Especially in hypoxic tumors, nano-delivery systems can effectively enhance the accumulation 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 reductases 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 vehicle due to its high drug binding affinity and ability to release drugs in response to hypoxia. However, these hypoxia-responsive materials are easily cleared from the body and have poor biocompatibility, which weakens their overall therapeutic efficacy. In view of these shortcomings, it is urgent to optimize their structure and function to improve their 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 nanodrug delivery system loaded with hydroxychloroquine (HCQ) and a mitochondrial-targeted photosensitizer-thiomethylated Nile blue derivative (SMNB) for the treatment of hypoxic tumors. This nanodrug 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 their efficacy. In addition, the reactive oxygen species generated by this nanodrug delivery system under the action of laser can induce mitochondrial damage and autophagy, while hydroxychloroquine (HCQ) blocks mitochondrial autophagy. Through this dual action, a reactive oxygen storm can be induced, ultimately inducing tumor cell death.

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

[0006] A 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, and then adding methacrylic anhydride and triethylamine in sequence, stirring at room temperature, and 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 separating by column chromatography to obtain a colorless oil, dissolving the colorless oil in dichloromethane, and then adding trifluoroacetic acid thereto, stirring at room temperature, and then concentrating the reaction solution in vacuo to obtain a yellow oily product, i.e., polyethylene glycol (n)-monomethacrylamide;

[0009] 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 recrystallizing the crude product with water and methanol to finally obtain the 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 disulfide bonds to sulfhydryl groups;

[0012] S3. Preparation of azocalixarene-modified albumin material: Compound I: (AC4A), the albumin material treated with a reducing agent, and a photoinitiator are dissolved in water or a phosphate buffer solution at pH 7.4, reacted under ultraviolet light, and then dialyzed, purified, 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 in an ice bath, and the reaction is stopped when the reaction mixture becomes viscous. 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 represented by 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 complete, remove the solvent, and slowly pour the remaining solution into a saturated sodium chloride solution to precipitate a dark blue solid. Collect the solid, dry it, and then purify it by silica gel column chromatography to obtain a dark blue solid product represented by formula (III), which is a thionile 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 azocalixarene 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 irradiation is carried out under ultraviolet light irradiation with a wavelength of 365 nm.

[0031] Preferably, in S41, the molar ratio of the N,N-diethyl-p-phenylenediamine and aluminum sulfate or zinc chloride is 1:1.05-1:1.2; the molar ratio of the N,N-diethyl-p-phenylenediamine and sodium thiosulfate is 1:2.2-1:2.5; and the molar ratio of the N,N-diethyl-p-phenylenediamine and 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 albumin receptors (such as gp60) highly expressed on the surface of tumor cells through the active targeting mechanism of albumin, achieving precise delivery to the tumor site and long-term circulation. 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 alkalinizing 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 photoclick chemical reaction between the double bond of the azocalixarene and the free thiol group of the reduced albumin, and has hypoxia-responsive characteristics. At the same time, the nano drug delivery system can trigger an active oxygen storm effect under hypoxic environment and red light external stimulation, 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 it can simultaneously load a variety of drug combinations and 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 have a wide range of application prospects 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: This invention utilizes the active targeting mechanism of albumin and the EPR effect to achieve precise delivery to the tumor site, improve drug accumulation in tumor tissue, 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 efficacy: The present invention combines a mitochondrial-targeted photosensitizer (SMNB) with an autophagy inhibitor (HCQ) to achieve a synergistic enhancement effect. The photosensitizer can produce reactive oxygen species under laser irradiation, inducing mitochondrial damage, while HCQ can block the autophagy function of tumor cells, preventing cells from weakening oxidative stress by clearing damaged mitochondria. This dual action effectively induces a reactive oxygen storm, thereby significantly enhancing the anti-tumor effect.

[0046] (3) Hypoxia response and integrated diagnosis and treatment: By combining hypoxia-responsive azocalixarene with albumin, the nano-drug delivery system can trigger drug release in the hypoxic microenvironment of the tumor and simultaneously possess fluorescence imaging capabilities, forming an integrated diagnosis and treatment platform. This feature makes treatment more precise and efficient, significantly enhancing its application value in hypoxic tumor treatment.

[0047] (4) Simple and efficient preparation process: The preparation process of the present invention is gentle, does not require complex operations and high temperature and high pressure conditions, can load a variety of drug combinations proportionally, and is suitable for large-scale production applications. The albumin carrier has good biocompatibility and long 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 thionile blue derivatives (SMNB) (n=1);

[0051] Figure 4 This is the H NMR spectrum of thioNile 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 362 nm; (d) UV absorption spectrum of HSA-AC4A (125 μg / mL);

[0053] Figure 6 TEM images and particle size distributions of various hypoxia-responsive albumin supramolecular nano-drug 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] Figure 9 UV absorption spectra of HCQ and SMNB (a) and standard curves (b), as well as the results of HCQ and SMNB release in response to hypoxia (c);

[0057] Figure 10 UV absorption spectra of the hypoxia-responsive albumin supramolecular nano-drug delivery system using the DHR123 probe to detect its 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] Figure 11The results of the co-localization experiment of SHC4 and SHC4H in mitochondria and lysosomes of the hypoxia-responsive albumin supramolecular nano-drug delivery system (SMNB and PBS were used as controls);

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

[0060] Figure 13 For the use of DCFH-DA (total active oxygen indicator), DHR123 (O2 ·- Figure 3. Experimental results of detecting reactive oxygen species generated by hypoxia-responsive albumin supramolecular nano-drug delivery system SHC4 and SHC4H using hydroxyl radical (HydroxyPropanol) and HPF (·OH fluorescence indicator) in B16 and MCF-7 cells (SMNB, HCQ, SPIN2 and PBS were used as controls);

[0061] Figure 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 and without laser irradiation (with SMNB, HCQ, and PBS as controls); (a) B16; (b) MCF-7;

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

[0063] Figure 16 Figure 2 shows the results of blood routine (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 following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may 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 derivative (Compound Ⅰ, AC4A)

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

[0069] Mono-Boc-polyethylene glycol (3)-diamine (1 g, 2.97 mmol, CAS: 1235514-18-7, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was weighed and dissolved in 30 mL of dichloromethane. Methacrylic anhydride (0.55 mL, 3.46 mmol) and triethylamine (0.55 mL, 3.95 mmol) were then added sequentially and stirred at room temperature for 24 hours. The mixture was concentrated in vacuo until free of solvent. The reaction product was then dissolved in ethyl acetate (40 mL). The ethyl acetate solution was washed three times with hydrochloric acid (2 mol / L), saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. Anhydrous magnesium sulfate was then added to dry overnight. After vacuum concentration, the mixture was separated by column chromatography (CH2Cl2 / CH3OH = 10:1, v / v) to obtain 590 mg of a 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 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 structural formula 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 a 20 mL aqueous solution containing aluminum sulfate (2.08 g, 3.23 mmol). Sodium thiosulfate (1.11 g, 7.00 mmol) and zinc chloride (0.44 g, 3.19 mmol) were then added to the reaction mixture in a stirring state. Potassium dichromate (0.25 g, 0.84 mmol) was slowly added over 20 minutes. After stirring in an ice bath for 2 hours, a viscous substance was obtained. After filtration, the crude product was washed with acetone to obtain a crude product. The crude product 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 collection and drying, the 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 H NMR 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 structural formula is:

[0080]

[0081] 3. Treat albumin with a reducing agent to expose intramolecular sulfhydryl groups: Dissolve human serum albumin (10 mg) and glutathione (3.35 mg) in phosphate-buffered saline (pH 7.4) and react at room temperature with stirring for 12 hours. The reaction solution is dialyzed for 24 hours using a dialysis bag (MWCO = 2000) and then lyophilized to obtain sulfhydryl-exposed albumin (HSA).

[0082] 4. Preparation of albumin material modified with azocalixarene (HSA-AC4A): Compound I (20 mg), albumin treated with 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 photoclick 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 2. Figure 5 As shown in b, AC4A has a characteristic absorption at 362nm, and an AC4A degree-dependent UV calibration curve is established (e.g. Figure 5 c). The UV absorption spectrum of HSA-AC4A was detected, and the results were as follows. Figure 5 As shown in Figure d, HSA-AC4A (125 μg / mL) showed characteristic UV absorption of AC4A, proving that the modification was successful. Based on the UV 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 added dropwise to 2 mL of the phosphate buffered saline solution (0.3 mg / mL) of the azocalixarene-modified albumin material obtained in the previous step. After stirring at room temperature for 2 hours, the mixture was purified by dialysis (MWCO = 2000) for 12 hours to obtain 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). The morphology and particle size distribution of the hypoxia-responsive albumin supramolecular nanocarrier are shown in Figure 4. Figure 6 shown.

[0086] In this study, we used sodium dithiosulfate to mimic the overexpressed azoreductase in hypoxic tumors. SHC4H (SMNB: 10 μM) was reduced with sodium dithiosulfate (100 μM) to yield SNH2C4H, while SHC4 (SMNB: 10 μM) was reduced with sodium dithiosulfate (100 μM) to yield SNH2C4.

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

[0088] (1) Fluorescence ON / OFF test of azocalixarene-modified albumin material was performed as follows:

[0089] 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), respectively, and the fluorescence emission spectra of the mixtures were recorded.

[0090] The test results are as follows Figure 7 As shown, as the concentration of azocalixarene-modified albumin increases, the fluorescence of SMNB gradually quenches. This is due to the electron transfer mechanism of AC4A photoinduced SMNB. When AC4A is reduced to NH2C4A by azoreductase, the fluorescence of SMNB "turns on." This property can be used for hypoxia-responsive fluorescence imaging.

[0091] (2) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example 1 was tested for its hypoxia-responsive drug release performance. The specific method is 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 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 HCQ or SMNB concentration-UV standard curve. Figure 9 This process was repeated three times at each time point.

[0093] The results are as follows Figure 9 As shown in c, under hypoxia-simulated 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 ability to release drugs in response to hypoxia.

[0094] (3) The hypoxia-responsive albumin supramolecular nano-drug delivery system prepared in Example 1 was tested for its in vitro solution-level reactive oxygen species generation performance. The specific method is as follows:

[0095] Detection of O2 in solution using fluorescent probe DHR123 ·- Generation of supramolecular nanoparticles. 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 (525 nm) of the different drug solutions was recorded using a fluorescence spectrophotometer after irradiation with a 660 nm laser for different times.

[0096] The test results are as follows Figure 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 Example 1 were tested. The specific method is as follows:

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

[0099] The test results are as follows Figure 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 1 was subjected to alkalinization lysosomal performance test. The specific method is as follows:

[0101] MCF-7 and B16 cells were cultured at 10 5 Cells were seeded onto confocal microplates at a density of 100 μg / well and incubated overnight until adherent. The culture medium was replaced with a drug-containing medium containing the hypoxia-responsive albumin supramolecular nanodrug delivery system (SHC4H, containing 10 μM HCQ) prepared in the Examples. The cells were cultured under hypoxic conditions (1% O₂) for 6, 12, and 24 hours. The culture medium was then replaced with a medium containing Lyso-Tracker Green and incubated for 30 minutes. The cell samples were observed using a confocal laser scanning microscope.

[0102] The test results are as follows Figure 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 independent of oxygen, as recorded below:

[0104] MCF-7 and B16 cells were cultured at 10 5Cells were seeded into confocal dishes at a density of 1000 cells / well and incubated overnight until adhered. The culture medium was replaced with a medium containing gradient concentrations of drugs. The drugs used were a photosensitizer that requires oxygen to produce reactive oxygen species - 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 normoxia (21% O2) or hypoxia (1% O2), 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 nuclei were labeled with DAPI. Finally, the cells were observed and images were collected using a confocal laser scanning microscope with an excitation / emission wavelength of 358 / 461 nm for DAPI and 481 / 535 nm for DCFH-DA, DHR123, and HPF.

[0105] The test results are as follows Figure 13 As shown, after SHC4H was treated with 660 nm laser, it showed excellent ROS generation ability even in hypoxic environment.

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

[0107] B16 cells were cultured at a rate of 5 × 10 3 Cells were seeded into 96-well plates at a density of 100 μg / well and incubated overnight to allow them to adhere. The culture medium was then replaced with a medium containing a gradient of drug concentrations. 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) prepared in the Examples and two guest drugs (SMNB: 0.25 μM; HCQ: 10 μM). After 24 hours of culture, the culture medium was replaced with standard 1640 medium containing 10% fetal bovine serum. The cells requiring illumination were placed under a 660 nm laser (100 mW / cm 2 After an additional 2 h of incubation, the culture medium was removed and the cells were rinsed with phosphate buffered saline. After incubation for 1 h, 100 μL of cell culture medium containing 10% CCK-8 reagent was added and the absorbance of the sample was measured at 450 nm.

[0108] The MCF-7 cell-based assay followed the same testing procedures as the B16 cell-based assay, except for the differences in cell type and drug concentration. The drugs used in the MCF-7 cell line included the hypoxia-responsive albumin supramolecular nanoparticle delivery systems (HC4H, SHC4, SHC4H; containing 0.10 μM SMNB and / or 10 μM HCQ) prepared in the Examples, along with two guest drugs (SMNB: 0.10 μM; HCQ: 10 μM).

[0109] The test results are as follows Figure 14 As shown in the figure, 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 light), 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 the 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 indicates antagonism, 0.85 ≤ Q < 1.15 indicates additive effect, and Q ≥ 1.15 indicates 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 methods are 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 150-180 mm 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 nano-drug delivery system and the corresponding pure drug prepared in Example 1 were implemented by tail vein injection and administered 4 times continuously. 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 nano-drug delivery system and the corresponding pure drug were administered 4 times continuously. 2 The tumor site was irradiated with a 5-minute irradiation (1 minute, 2 minutes). Tumor size and mouse weight were recorded daily to assess the therapeutic efficacy. 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 analysis and biochemical analysis.

[0116] like Figure 15 As shown in the figure, 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 showed a more excellent anti-tumor effect than the SHC4+hv group, which fully demonstrated the excellent application potential of the SHC4H nanodrug delivery system combined with laser irradiation in the treatment of hypoxic tumors. In addition, as Figure 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 prepared by the present invention, which is co-loaded with hydroxychloroquine (HCQ) and mitochondrial-targeted photosensitizer (SMNB), can, on the one hand, actively target tumor tissues and responsively release drugs in hypoxic tumor tissues, significantly increasing the accumulation of drugs in tumor tissues and reducing 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, inducing a reactive oxygen storm through this dual action, and 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. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection 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, and 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. After drying and concentrating in vacuo, separation by column chromatography is performed to obtain a colorless oil. The colorless oil is dissolved in dichloromethane, and trifluoroacetic acid is added thereto. After stirring at room temperature, the reaction solution is concentrated in vacuo to obtain a yellow oily product, i.e., polyethylene glycol n-monomethyl acrylamide; wherein n in the polyethylene glycol n-monomethyl acrylamide is an integer of 3-10; 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-monomethylacrylamide to the above reaction system, and continuously stirring the reaction. After the reaction is completed, removing the solvent to obtain a crude product, and recrystallizing the crude product with water and methanol to finally obtain the 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 disulfide bonds to sulfhydryl groups; S3. Preparation of albumin material modified with azocalixarene: dissolve the azocalix[4]arene derivative, the albumin material treated with a reducing agent, and a photoinitiator in water or a phosphate buffer solution with a pH of 7.4, react 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 sequentially to N,N-diethyl-p-phenylenediamine, and potassium dichromate is slowly added under ice bath conditions. The reaction mixture is then stirred to become a viscous substance, which 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 represented by formula (II); S42, dissolving the above-mentioned 2-amino-5-diethylaminophenyl 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 complete, removing the solvent, and slowly pouring the remaining solution into a saturated sodium chloride solution to precipitate a dark blue solid. The solid is collected and dried, and then purified by silica gel column chromatography to obtain a dark blue solid product represented by formula (III), which is a thionile blue derivative; S5. Dissolve the thio-Nile blue derivative and hydroxychloroquine of S4 in a solvent, then add the azocalixarene-modified albumin material of S3, stir and react at room temperature, and then dialyze to obtain a 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-monomethylacrylamide is 1:1.5-1:2.

0.

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 treated with a reducing agent to the 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; and 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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