Dendritic drug-drug conjugate-based hypoxia-responsive core-crosslinked supramolecular nano prodrug as well as preparation method and application of dendritic drug-drug conjugate-based hypoxia-responsive core-crosslinked supramolecular nano prodrug

Abstract

The invention discloses a hypoxia-responsive core-crosslinked supramolecular nano prodrug based on a dendritic drug-drug conjugate as well as a preparation method and application of the supramolecular nano prodrug. The supramolecular nano prodrug is prepared from polyethylene glycol-poly (glutamic acid-cyclodextrin) and IR806-(azobenzene-nitrogen mustard) 4 in a mass ratio of 2: 1. The hypoxia-responsive core-crosslinked supramolecular nano prodrug based on the dendritic drug-drug conjugate is simple and effective in preparation process, the hypoxia-responsive core-crosslinked supramolecular nano prodrug comprises nitrogen mustard chemotherapy, IR806 photo-thermal therapy and photodynamic therapy, chemotherapy, photo-thermal therapy and photodynamic therapy cooperate for treatment, a new thought is provided for anti-tumor integrated treatment, and the hypoxia-responsive core-crosslinked supramolecular nano prodrug has broad application prospects. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate, and a preparation method and application thereof. Background Art

[0002] Nanoprodrugs are established through various coupling methods such as polymer-drug conjugates and drug-drug conjugates, and have attracted increasing attention due to their high drug loading efficiency and low potential systemic toxicity. Drug-drug conjugates are usually synthesized by coupling two or more drug molecules through stimulus-response bonds. Under the corresponding stimulus, the drug conjugate will decompose and release the activated drug. In particular, due to their unique dendritic structure and multivalent synergy (due to their highly branched but precisely controllable structure), dendritic macromolecules are expected to greatly improve their performance when used in drug-drug conjugates. However, dendritic drug-drug conjugates still have certain obstacles in the construction of nanoprodrugs, such as complex synthesis process, uncontrolled drug release behavior, and limited selection of anticancer drugs. In view of this, the introduction of macrocyclic molecules with cavities may provide a feasible strategy to improve the above problems, that is, encapsulating dendritic drug-drug conjugates through host-guest complexation. In this context, supramolecular dendritic drug-drug conjugates show great potential in the development of ideal nanomedicines.

[0003] In addition, many solid tumors have a hypoxic environment due to rapid cell proliferation and insufficient angiogenesis. Inspired by this key feature, a series of bioreductive prodrugs containing hypoxia-responsive linking groups have been used for hypoxia imaging and treatment. Among them, the most widely used functional group in hypoxia-responsive nanomedicines is azobenzene (Azo), which can be reduced to anilino groups in the presence of nicotinamide adenine dinucleotide phosphate and azoreductase in the hypoxic tumor microenvironment. In addition, photodynamic therapy (PDT) is often used in combination with hypoxia-responsive nanomedicines because PDT can promote the conversion of oxygen into cytotoxic reactive oxygen species (ROS) under light irradiation by using photosensitizers, thereby exacerbating the potential degree of hypoxia.

[0004] Literature search found that Liu Jianyong's research group at Fuzhou University in my country linked the dipyrromethyl boron photothermal agent with the chemotherapy drug camptothecin through the hypoxia-responsive group azobenzene in the paper (A Hypoxia-Activated Prodrug Conjugated with a BODIPY-Based Photothermal Agent for Imaging-Guided Chemo-Photothermal Combination Therapy, ACS Appl. Mater. Interfaces 2022, 14, 40546-40558) to prepare a hypoxia-responsive prodrug for combined cancer treatment. However, the loaded photosensitizer dipyrromethyl boron cannot consume oxygen, and thus cannot enhance the hypoxia level of the tumor, so it cannot efficiently activate the chemotherapy drug camptothecin through hypoxia, making it difficult to transform and apply in clinical practice. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate and a preparation method and application thereof, so as to solve the current problems of difficulty in effectively loading oxygen scavengers and hypoxia-responsive prodrugs at the same time, as well as the preparation and loading of dendritic drug-drug conjugates.

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

[0007] A hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate, wherein the supramolecular nanoprodrug is prepared from polyethylene glycol-poly(glutamic acid-cyclodextrin) and IR806-(azobenzene-nitrogen mustard) 4 in a mass ratio of 2:1; wherein the structural formula of the polyethylene glycol-poly(glutamic acid-cyclodextrin) is shown in the following formula I:

[0008]

[0009] In formula I: n is 20;

[0010] The structural formula of IR806-(azobenzene-nitrogen mustard) 4 is shown in the following formula III:

[0011]

[0012] Preferably, the particle size of the supramolecular nanoprodrug is 205.3±0.96 nm.

[0013] The preparation method of the above-mentioned hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate comprises the following steps:

[0014] Step 1) preparing polyethylene glycol-poly(glutamic acid-cyclodextrin);

[0015] Step 2) Preparation of IR806-(azobenzene-nitrogen mustard) 4;

[0016] Step 3) polyethylene glycol-poly(glutamic acid-cyclodextrin) and IR806-(azobenzene-nitrogen mustard) 4 are dissolved in N,N-dimethylformamide solvent according to proportion, and then a deionized water solution is added, stirred, and dialyzed to obtain the product.

[0017] Preferably, the process of preparing polyethylene glycol-poly(glutamic acid-cyclodextrin) in step 1) is as shown in reaction formula II:

[0018]

[0019] The specific steps are as follows:

[0020] Step 1-1) 187 mg of polyethylene glycol-amino and 300 mg of Bn-Glu-NCA were dissolved in 5 mL of dry N,N-dimethylformamide and stirred continuously at room temperature for 48 h under a nitrogen atmosphere; then ether was added three times to precipitate the reaction mixture, and the white precipitate was collected by centrifugation; the precipitate was dried in a vacuum oven overnight to obtain PEG-PBGlu;

[0021] Step 1-2) 100 mg of PEG-PBGlu was dissolved in 20 mL of 1 M NaOH solution and vigorously stirred at 100° C. under reflux overnight; the resulting reaction mixture was placed in a dialysis bag and dialyzed with deionized water for 48 h. After dialysis, the mixture was freeze-dried to obtain PEG-PGlu;

[0022] Step 1-3) 50 mg PEG-PGlu, 12 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 5 mg 4-dimethylaminopyridine and 106.3 mg β-cyclodextrin solution were dissolved in 5 mL dry N,N-dimethylformamide and stirred at room temperature for 24 h; after the reaction was completed, the reaction solution was added to diethyl ether for precipitation, and centrifuged to obtain a white solid precipitate, which was the polyethylene glycol-poly(glutamic acid-cyclodextrin).

[0023] Preferably, the process of preparing IR806-(azobenzene-nitrogen mustard) 4 in step 2) is as shown in reaction formula IV:

[0024]

[0025] The specific steps are as follows:

[0026] Step 2-1) 456.3 mg of azobenzene-nitrogen mustard-carboxyl and 760 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in 10 mL of dry N,N-dimethylformamide and stirred at 0°C for 5 h to obtain a mixture A; then 348 mg of N,N-diisopropylethylamine was added to the mixture A and heated to room temperature to obtain a mixture B; 105.6 mg of Boc-D2 solution was gradually added to 5 mL of dry N,N-dimethylformamide, and mixture B was added and stirred for 48 h; water was added to terminate the reaction, followed by extraction with dichloromethane; the organic phase was dried over sodium sulfate, and the solvent was removed under reduced pressure; finally, the crude product was purified by silica gel column chromatography to obtain Boc-D2-(Azo-CB)4;

[0027] Step 2-2) 130 mg of Boc-D2-(Azo-CB)4 was dissolved in 1 mL of dichloromethane, and then 1 mL of trifluoroacetic acid was slowly added and stirred for 2 h. The solution was then evaporated to remove the solvent and dried in vacuo to obtain an orange-red solid. Then 80 mg of IR806 and 58 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in 5 mL of dry N,N-dimethylformamide and stirred at 0°C for 3 h to obtain solution C. 0.5 mL of N,N-diisopropylethylamine was added to solution C and heated to room temperature to obtain solution D; 217.5 mg of the orange-red solid was dissolved in 2 mL of dry N,N-dimethylformamide, and then slowly added dropwise to solution D, and stirring was continued for 24 hours to obtain solution E; finally, solution E was placed in a dialysis tube, first dialyzed with dimethyl sulfoxide for 24 hours, and then dialyzed with ultrapure water for 24 hours, and after freeze-drying, the IR806-(azobenzene-nitrogen mustard) 4 was obtained.

[0028] Preferably, in step 3), the concentration of the polyethylene glycol-poly(glutamic acid-cyclodextrin) is 4.0 mg / mL; the concentration of the IR806-(azobenzene-nitrogen mustard) 4 is 2.0 mg / mL.

[0029] Preferably, in step 3), the volume ratio of the N,N-dimethylformamide to the deionized water is 1:5.

[0030] Preferably, the dialysis conditions in step 3) are: using a dialysis bag with a molecular weight of 3500, 500 mL of deionized water × 1 time / 8 hours, and dialysis for 24 hours.

[0031] The above-mentioned hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate, or the use of the above-mentioned hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0032] An anti-tumor pharmaceutical composition comprises the above-mentioned hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate, or the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate prepared by the above-mentioned preparation method, and pharmaceutically acceptable excipients.

[0033] The beneficial effects of the present invention are as follows:

[0034] (1) The component IR806 in the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate of the present invention is irradiated with near-infrared light (light intensity 1 W / cm 2 , wavelength 808nm, illumination time 5min), can quickly heat up, kill tumor cells and achieve photothermal therapy.

[0035] (2) The component IR806 in the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate of the present invention was irradiated with near-infrared light (light intensity 1 W / cm 2 , wavelength 808nm, illumination time 5min), can consume oxygen, enhance the hypoxia level in tumor cells, and generate reactive oxygen species, killing tumor cells and achieving photodynamic therapy.

[0036] (3) In the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugates of the present invention, azobenzene breaks in the enhanced hypoxia environment in the tumor, the nanodrug disassembles, and the chemotherapeutic drug nitrogen mustard is released, which is toxic to tumor cells and achieves chemotherapy.

[0037] (4) The present invention efficiently synthesizes a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate, providing a simple and effective way to prepare the supramolecular nanoprodrug. At the same time, the technical operation of using the supramolecular nanoprodrug for the integrated photothermal therapy-photodynamic therapy-chemotherapy of liver cancer cells is simple, requiring only two intravenous injections and two irradiations to achieve effective treatment of liver cancer tumors, and has important clinical application prospects. The present invention provides a good experimental platform for obtaining hypoxia-responsive prodrugs for integrated photothermal therapy-photodynamic therapy-chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the NMR spectrum of polyethylene glycol-poly(γ-benzyl-glutamic acid) in Example 1;

[0039] Figure 2 is the NMR spectrum of polyethylene glycol-poly(glutamic acid-cyclodextrin) in Example 1;

[0040] Figure 3 is the NMR spectrum of tert-butyloxycarbonyl-linked 2-(azobenzene-nitrogen mustard) 4 in Example 1;

[0041] Figure 4 is the NMR spectrum of IR806-(azobenzene-nitrogen mustard) 4 in Example 1;

[0042] Figure 5 Schematic diagram of the preparation of hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugates in Example 1;

[0043] Figure 6 The dynamic light scattering spectrum of the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on the dendritic drug-drug conjugate in Example 1;

[0044] Figure 7 The effects of the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugates in Example 2 on liver cancer cells in normal oxygen and hypoxic environments under dark conditions;

[0045] Figure 8 The effects of the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugates in Example 2 on liver cancer cells in normal oxygen and hypoxic environments under light conditions;

[0046] Fig. 9 The effect of each group of drugs in Example 3 on the tumor size of nude mice bearing liver cancer tumors over time;

[0047] Fig.10 The figure shows the effects of each group of drugs in Example 3 on the body weight of nude mice bearing liver cancer tumors over time. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0051] The experimental animals in the following examples are nude mice, which were provided by the Experimental Animal Center of Nantong University.

[0052] Example 1 Preparation of hypoxia-responsive core-crosslinked supramolecular nanoprodrug (CSN-IR806 / CB) based on dendritic drug-drug conjugates

[0053] 1. Preparation of polyethylene glycol-poly(glutamic acid-cyclodextrin) (PEG-PGlu-CD)

[0054] The structural formula of PEG-PGlu-CD is shown in Formula I below:

[0055]

[0056] In formula I: n is 20.

[0057] Its preparation process is shown in reaction formula II:

[0058]

[0059] The specific steps are as follows:

[0060] (1) Preparation of polyethylene glycol-poly(γ-benzyl-glutamic acid) (PEG-PBGlu)

[0061] Take 187 mg of polyethylene glycol-amino and 300 mg of γ-benzyl-glutamic acid NCA (Bn-Glu-NCA) and dissolve them in 5 mL of dry N,N-dimethylformamide (DMF), and stir them continuously at room temperature for 48 hours under nitrogen atmosphere. Then add ether (20 mL) three times to precipitate the reaction mixture, and collect the white precipitate by centrifugation. The precipitate is dried in a vacuum oven overnight to obtain PEG-PBGlu196.6 mg, with a yield of 86.4%.

[0062] The PEG-PBGlu prepared in this example is as follows Figure 1 As shown, the detailed peak positions are attributed to: 1 H-NMR (400MHz, DMSO-d6): δ=7.37-7.17(m,205H,phH),5.09-4.9(m,82H,phCH2O),4.32-3.77(m,41H,COCHNH),3 .58-3.43(m,368H,O(CH2)2O),3.24(s,3H,OCH3),2.47-2.3(m,82H,OOCCH2),2.07-1.8(m,82H,(CO)CH(NH)CH2).

[0063] (2) Preparation of PEG-PGlu-CD

[0064] PEG-PBGlu (100 mg) was dissolved in 20 mL 1M NaOH solution and stirred vigorously at 100 ° C reflux overnight. The resulting reaction mixture was placed in a dialysis bag and dialyzed with deionized water for 48 h. After dialysis, lyophilization was performed to obtain 70.4 mg of polyethylene glycol-polyglutamic acid (PEG-PGlu) with a yield of 85%. Subsequently, 50 mg of PEG-PGlu, 12 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 5 mg of 4-dimethylaminopyridine (DMAP) and 106.3 mg of β-cyclodextrin solution were dissolved in 5 mL of dry DMF and stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was added to diethyl ether (20 mL) for precipitation, and centrifugation was performed to obtain 91.96 mg of white solid precipitate PEG-PGlu-CD with a yield of 35.3%.

[0065] The PEG-PGlu-CD prepared in this example is as follows Figure 2 As shown, the detailed peak positions are attributed to: 1 H-NMR (400MHz, DMSO-d6): δ=5.78-5.75(d,35H,OCHO),4.84-4.8(m,60H,CHOH),4.52-4.47(t,40H,COOCH2,CHCH2OH),3.67- 3.52(m,24H,COCHNH,CONHCH2),3.39-3.26(m,512H,OCH2,CH2OH),3.0-2.93(m,60H,OCH(CH)2),2.72-2.7(m,44H,OCOCH2).

[0066] 2. Preparation of IR806-(Azobenzene-nitrogen mustard) 4 (IR806-(Azo-CB) 4)

[0067] The structural formula of IR806-(Azo-CB)4 is shown in the following formula III:

[0068]

[0069] Its preparation process is shown in reaction formula IV:

[0070]

[0071] The specific steps are as follows:

[0072] (1) Preparation of tert-butyloxycarbonyl-linked 2-(azobenzene-nitrogen mustard) 4 (Boc-D2-(Azo-CB)4)

[0073] With reference to existing literature (Science China Materials. 2018; 61: 1475-1483), tert-butyloxycarbonyl-link 2 (Boc-D2) was obtained, and with reference to existing literature (Adv. Healthcare Mater. 2023, 2303308), azobenzene-nitrogen mustard-carboxyl (Azo-CB-COOH) was obtained. 456.3 mg Azo-CB-COOH and 760 mg 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were dissolved in 10 mL of dry DMF and stirred at 0 ° C for 5 h to obtain mixture A. Then 348 mg N,N-diisopropylethylamine (DIPEA) was added to mixture A and heated to room temperature to obtain mixture B. Boc-D2 solution (105.6 mg) was gradually added to 5 mL of dry DMF, and mixture B was added and stirred for 48 h. Water was added to terminate the reaction, followed by extraction with dichloromethane (DCM). The organic phase was dried over sodium sulfate, and then the solvent was removed under reduced pressure. Finally, the crude product was purified by silica gel column chromatography to obtain 128.3 mg of Boc-D2-(Azo-CB)4 with a yield of 46.2%.

[0074] The Boc-D2-(Azo-CB)4 prepared in this example is as follows Figure 3 As shown, the detailed peak positions are attributed to: 1 H-NMR (400MHz, DMSO-d6): δ (ppm) = 7.9-7.88 (d, 8H, ClCH2CH2NPh), 7.81-7.77 (d, 8H, ClCH2CH2NPhNNPh), 7.77-7.71 (d,8H,ClCH2CH2NPhNNPh),6.72-6.62(d,8H,ClCH2CH2NPh),3.8-3.7(m,16H,ClCH2),3.69-3.57(m,16H,ClCH2CH2),

[0075] 3.53-3.35(m,H,(CH3)3COCONHCH2,(CH3)3COCONH(CH2)2NCH2,CONHCH2,

[0076] PhCONHCH2,PhCONH(CH2)2NHCOCH2),2.66-2.16(m,H,

[0077] (CH3)3COCONHCH2CH2,(CH3)3CONH(CH2)2NCH2CH2,

[0078] (CH3)3COCONH(CH2)2NCH2CH2,NHCOCH2,NHCOCH2CH2NCH2),1.3(s,9H,(CH3)3).

[0079] (2) Preparation of IR806-(Azo-CB)4

[0080] IR806 was obtained with reference to existing literature (ACS Macro Lett. 2022; 11: 830-834). First, Boc-D2-(Azo-CB)4 (130 mg) was dissolved in 1 mL of dichloromethane, and trifluoroacetic acid (1 mL) was slowly added and stirred for 2 h. The solution was then evaporated to remove the solvent and dried in vacuo to obtain an orange-red solid. IR806 (80 mg) and HATU (58 mg) were then dissolved in 5 mL of dry DMF and stirred at 0 ° C for 3 h to obtain solution C. DIPEA (0.5 mL) was added to solution C and heated to room temperature to obtain solution D. The orange-red solid (217.5 mg) obtained above was dissolved in 2 mL of dry DMF, and then slowly added dropwise to solution D, and stirring was continued for 24 h to obtain solution E. Finally, solution E was placed in a dialysis tube (MWCO 2000Da), dialyzed against dimethyl sulfoxide for 24 hours, and then dialyzed against ultrapure water for 24 hours. After freeze-drying, 242.67 mg of IR806-(Azo-CB)4 was obtained with a yield of 86.3%.

[0081] IR806-(Azo-CB)4 prepared in this example is as follows Figure 4 As shown, the detailed peak positions are attributed to: 1H-NMR (400MHz, CDCl3): δ=8.8-8.7(m,5H,PhCONH),8.67-8.5(m,3H,(CH3)2CPh,SPh),8.25-8 .14(m,2H,NPh,(CH3)2CPh),8.0-7.96(d,8H,NNPhN),7.88-7.78(m,18H,CONH(CH2)2,NHCOPhN N),7.56-7.51(d,2H,SPh),7.46-7.34(m,3H,(CH3)2Ph,CH3(CH2)2NPh),7.28-7.16(m,1H,CH3 (CH2)2NPh),7.0-6.91(m,5H,(Cl(CH2)2)2NPh,SCCHCH),6.4-6.3(m,2H,SCCHCH),4.89-4.81( m,1H,SCCHCH),4.2-4.0(m,2H,SCCHCHCH),3.9-3.84(m,16H,ClCH2CH2),3.84-3.77(m,16H,C lCH2),3.37-3.3(m,22H,SPhCONHCH2,SPhCONH(CH2)2NCH2,NCH2CH2CONH),3.3-3.2(m,8H,NNP hCONHCH2),2.71-2.59(m,12H,NHCOCH2),2.3-2.15(m,6H,NHCH2CH2N,SPhCONHCH2CH2),1.8-1 .67(m,2H,CH3CH2),1.46-1.32(s,12H,PhCCH3),1.23-1.19(m,2H,CH3CH2),1.12-1.1(m,2H,N + CH2),0.97-0.87(m,6H,N(CH2)2CH3).

[0082] 3. Preparation of CSN-IR806 / CB

[0083] like Figure 5 As shown, PEG-PGlu-CD (4.0 mg) and IR806-(Azo-CB)4 (2.0 mg) were dissolved in DMF (1.0 mL), then stirred for 12 h, and then 5 mL of ultrapure water was slowly added and stirred for another 12 h. Finally, the mixed solution was transferred to a dialysis bag (MWCO 3500 Da) and dialyzed with ultrapure water for 24 h. The dialysis bag was renewed every 4 h to obtain a hypoxia-responsive core-crosslinked supramolecular nanoprodrug (CSN-IR806 / CB) based on a dendritic drug-drug conjugate.

[0084] The dynamic light scattering spectrum of CSN-IR806 / CB prepared in this example is as follows: Figure 6 As shown, the number average particle size is 205.3±0.96 nm and the PDI is 0.145±0.03.

[0085] Example 2 Effect of CSN-IR806 / CB on liver cancer cells

[0086] 1. Experimental steps

[0087] The CSN-IR806 / CB prepared in Example 1 was prepared with a cell culture medium containing nitrogen mustard (CB) at concentrations of 0.2, 0.5, 1, 2, 4, and 8 μg / mL, and cultured with HepG2 cells (liver cancer cells) in a hypoxic (Hypoxia) or normal oxygen (Normoxia) environment for 6 hours. For the illumination group, a near-infrared laser was used to illuminate the cells for 5 minutes (808 nm, 1 W / cm 2 ) and continue culturing for 48h.

[0088] 2. Experimental results

[0089] The cell activity test was performed using the MTT method. Figure 7 , 8 As shown, in the absence of light (Dark) or with near-infrared light irradiation (Light), the cytotoxicity of CSN-IR806 / CB in a hypoxic environment is greater than that in a normal oxygen environment; under near-infrared light irradiation, the toxicity of CSN-IR806 / CB in a hypoxic environment is the greatest.

[0090] The experimental results of this example show that CSN-IR806 / CB exhibits better anti-tumor effects on liver cancer cells under light conditions.

[0091] Example 3 Effect of CSN-IR806 / CB on HepG2 liver cancer tumor growth

[0092] 1. Experimental steps

[0093] The mice inoculated with HepG2 tumors were divided into 5 groups as follows:

[0094] PBS group: injection of normal saline (5 mg / kg) via tail vein;

[0095] CB group: injection of nitrogen mustard (5 mg / kg) via tail vein;

[0096] IR806+L group: IR806 (4.2 mg / kg) was injected into the tail vein for 12 hours, and then irradiated with near-infrared laser;

[0097] CSN-IR806 / CB group: CSN-IR806 / CB (39.4 mg / kg) was injected into the tail vein;

[0098] CSN-IR806 / CB+L group: 12 hours after the tail vein injection of CSN-IR806 / CB (39.4 mg / kg), the mice were irradiated with near-infrared laser.

[0099] The mice in the above groups were injected with drugs once on day 0 and day 5, and 12 hours after injection, the IR806+L group and CSN-IR806 / CB+L group were illuminated for 5 min (808 nm, 1 W / cm 2 ), and the mice were weighed and the tumor volumes were measured every 2 days.

[0100] 2. Experimental results

[0101] The results are as follows Fig. 9 , 10 As shown, for HepG2 tumors, the free nitrogen mustard CB group had almost no inhibitory effect on the tumor, the IR806+L group had a slight inhibitory effect on the tumor, and the CSN-IR806 / CB group had a more obvious inhibitory effect on the tumor. The growth of subcutaneous tumors in all mice in the CSN-IR806 / CB+L group was significantly inhibited ( Fig. 9 , P<0.01), and had little effect on the body weight of mice ( Fig.10 ).

[0102] The experimental results of this embodiment show that the CSN-IR806 / CB of the present invention is injected into mice through the tail vein, and can maintain good stability during the blood circulation process, and then enriched in the tumor tissue through the EPR effect. After entering the tumor cells, CSN-IR806 / CB is irradiated with near-infrared light. Under the action of light, the temperature of IR806 rises and kills the tumor cells, realizing photothermal therapy; at the same time, it can also consume oxygen to produce reactive oxygen, kill cells, and realize photodynamic therapy; consuming oxygen can enhance the degree of hypoxia in the cells, so that the hypoxia-responsive azobenzene link breaks, releasing nitrogen mustard to kill tumor cells, and realizing chemotherapy. In summary, the CSN-IR806 / CB of the present invention can be used for the integrated treatment of photothermal therapy, photodynamic therapy, and chemotherapy of liver cancer cells, and has important clinical application prospects.

[0103] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugates, characterized in that: The supramolecular nanoprodrug is prepared from polyethylene glycol-poly(glutamic acid-cyclodextrin) and IR806-(azobenzene-nitrogen mustard) 4 in a mass ratio of 2:1; wherein the structural formula of the polyethylene glycol-poly(glutamic acid-cyclodextrin) is shown in the following formula I: In formula I: n is 20; The structural formula of IR806-(azobenzene-nitrogen mustard) 4 is shown in the following formula III:

2. The hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate according to claim 1, characterized in that: The particle size of the supramolecular nanoprodrug is 205.3±0.96 nm.

3. A method for preparing a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate according to claim 1 or 2, characterized in that: The following steps are involved: Step 1) preparing polyethylene glycol-poly(glutamic acid-cyclodextrin); Step 2) Preparation of IR806-(azobenzene-nitrogen mustard) 4; Step 3) polyethylene glycol-poly(glutamic acid-cyclodextrin) and IR806-(azobenzene-nitrogen mustard) 4 are dissolved in N,N-dimethylformamide solvent according to proportion, and then a deionized water solution is added, stirred, and dialyzed to obtain the product.

4. The method for preparing a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate according to claim 3, characterized in that: The process of preparing polyethylene glycol-poly(glutamic acid-cyclodextrin) in step 1) is shown in reaction formula II: The specific steps are as follows: Step 1-1) 187 mg of polyethylene glycol-amino and 300 mg of Bn-Glu-NCA were dissolved in 5 mL of dry N,N-dimethylformamide and stirred continuously at room temperature for 48 h under a nitrogen atmosphere; then ether was added three times to precipitate the reaction mixture, and the white precipitate was collected by centrifugation; the precipitate was dried in a vacuum oven overnight to obtain PEG-PBGlu; Step 1-2) 100 mg of PEG-PBGlu was dissolved in 20 mL of 1M NaOH solution and vigorously stirred at reflux at 100°C overnight; the resulting reaction mixture was placed in a dialysis bag, dialyzed with deionized water for 48 h, and lyophilized after dialysis to obtain PEG-PGlu; Step 1-3) 50 mg PEG-PGlu, 12 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 5 mg 4-dimethylaminopyridine and 106.3 mg β-cyclodextrin solution were dissolved in 5 mL dry N,N-dimethylformamide and stirred at room temperature for 24 h; after the reaction was completed, the reaction solution was added to diethyl ether for precipitation, and centrifuged to obtain a white solid precipitate, which was the polyethylene glycol-poly(glutamic acid-cyclodextrin).

5. The method for preparing a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate according to claim 3, characterized in that: Step 2) The process of preparing IR806-(azobenzene-nitrogen mustard) 4 is shown in reaction formula IV: The specific steps are as follows: Step 2-1) 456.3 mg of azobenzene-nitrogen mustard-carboxyl and 760 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in 10 mL of dry N,N-dimethylformamide and stirred at 0°C for 5 h to obtain a mixture A; then 348 mg of N,N-diisopropylethylamine was added to the mixture A and heated to room temperature to obtain a mixture B; 105.6 mg of Boc-D2 solution was gradually added to 5 mL of dry N,N-dimethylformamide, and mixture B was added and stirred for 48 h; water was added to terminate the reaction, followed by extraction with dichloromethane; the organic phase was dried over sodium sulfate, and the solvent was removed under reduced pressure; finally, the crude product was purified by silica gel column chromatography to obtain Boc-D2-(Azo-CB)4; Step 2-2) 130 mg of Boc-D2-(Azo-CB)4 was dissolved in 1 mL of dichloromethane, and then 1 mL of trifluoroacetic acid was slowly added and stirred for 2 h. The solution was then evaporated to remove the solvent and dried in vacuo to obtain an orange-red solid. Then 80 mg of IR806 and 58 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in 5 mL of dry N,N-dimethylformamide and stirred at 0°C for 3 h to obtain solution C. 0.5 mL of N,N-diisopropylethylamine was added to solution C and heated to room temperature to obtain solution D; 217.5 mg of the orange-red solid was dissolved in 2 mL of dry N,N-dimethylformamide, and then slowly added dropwise to solution D, and stirring was continued for 24 hours to obtain solution E; finally, solution E was placed in a dialysis tube, first dialyzed with dimethyl sulfoxide for 24 hours, and then dialyzed with ultrapure water for 24 hours, and after freeze-drying, the IR806-(azobenzene-nitrogen mustard) 4 was obtained.

6. The method for preparing a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate according to claim 3, characterized in that: Step 3) The concentration of the polyethylene glycol-poly(glutamic acid-cyclodextrin) is 4.0 mg / mL; the concentration of the IR806-(azobenzene-nitrogen mustard) 4 is 2.0 mg / mL.

7. The method for preparing a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate according to claim 3, characterized in that: Step 3) The volume ratio of the N,N-dimethylformamide to the deionized water is 1:

5.

8. The method for preparing a hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate according to claim 3, characterized in that: Step 3) The dialysis conditions are: using a dialysis bag with a molecular weight of 3500, 500 mL of deionized water × 1 time / 8 hours, and dialysis for 24 hours.

9. Use of the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate as claimed in claim 1 or 2, or the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on a dendritic drug-drug conjugate prepared by the preparation method according to any one of claims 3 to 8 in the preparation of anti-tumor drugs.

10. An anti-tumor pharmaceutical composition, characterized in that: It comprises the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate as described in claim 1 or 2, or the hypoxia-responsive core-crosslinked supramolecular nanoprodrug based on dendritic drug-drug conjugate prepared by the preparation method according to any one of claims 3 to 8, and pharmaceutically acceptable excipients.

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