Nanometer diagnosis and treatment preparation for accelerating drug release of hypoxia and preparation method of nanometer diagnosis and treatment preparation

Abstract

The invention discloses a nanometer diagnosis and treatment preparation for hypoxia accelerated drug release and a preparation method thereof, and belongs to the technical field of biomedicine. In order to solve the problems existing in photodynamic therapy in an existing hypoxia tumor microenvironment, the nano diagnosis and treatment preparation for accelerating drug release in hypoxia, disclosed by the invention, comprises an aggregation-induced emission (AIE) molecule probe TPA-TT, a hypoxia response prodrug, DSPE-PEG-2000 and an immune cell membrane; an aggregation-induced emission molecule (AIE) probe TPA-TT and a hypoxia response prodrug are jointly wrapped in DSPE-PEG-2000, and finally the surface of the DSPE-PEG-2000 is wrapped with an immune cell membrane. Preparing an aggregation-induced emission molecular probe; preparing a nano diagnosis and treatment preparation loaded with an aggregation-induced emission molecular probe and a hypoxia response prodrug; the surface of the nano diagnosis and treatment preparation is covered with an M1 macrophage immune cell membrane. The invention provides a scheme for killing tumor cells and promoting apoptosis of cancer cells under the guidance of accurate imaging, and has tumor near-infrared fluorescence imaging and photodynamic and chemical dynamic synergistic treatment performance while specifically targeting the cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a nano-diagnosis and treatment preparation for accelerating drug release due to hypoxia and a preparation method thereof. Background Art

[0002] Despite significant advances in medical technology, cancer remains a disease with high mortality and limited treatment options. Among various treatment modalities, chemotherapy is an effective strategy for treating localized and metastatic cancers in clinical settings. Despite considerable progress in tumor chemotherapy over the past few decades, treatment outcomes are still limited by the emergence of drug resistance and severe side effects caused by toxicity to normal cells. Traditional small molecule chemotherapeutics, such as doxorubicin and cisplatin, are often unable to distinguish between normal cells and tumor cells, resulting in damage to rapidly proliferating tissues and cells. Unlike the normal physiological environment, the tumor microenvironment (TME) of most solid tumors presents unique characteristics, including weakly acidic pH and high glutathione values ​​as well as extreme hypoxia. Scientific evidence has identified hypoxia as a major factor in tumor metastasis, angiogenesis, and intervention in many treatments. At the same time, the unique hypoxic properties of tumors provide opportunities for targeted cancer therapy. To achieve precision medicine, hypoxia-activated prodrugs have been developed as a promising strategy. These prodrugs remain stable and nontoxic in normoxic areas, but are converted into cytotoxic compounds in hypoxic tumor areas, thereby selectively exerting drug effects in tumors. However, since the hypoxic microenvironment is usually shown to be heterogeneous, hindering effective and rapid prodrug conversion, drugs that rely solely on hypoxia usually fail to achieve optimal anti-tumor efficacy.

[0003] Recently, photodynamic therapy (PDT), in which a photosensitizer reacts with oxygen to produce singlet oxygen ( 1 O2) or reactive oxygen species (ROS), which can directly destroy tumor cells. Aggregation-induced emission is a new photophysical phenomenon that enables high-performance emission and imaging. However, due to the rapid growth and oxygen consumption of tumor cells, the microenvironment of many malignant solid tumors is severely hypoxic, which reduces the performance of photodynamic therapy for solid tumors.

[0004] Therefore, how to utilize the hypoxic characteristics of the tumor microenvironment and the advantages of photodynamic therapy to provide a diagnostic and therapeutic preparation that can not only achieve accurate bioimaging and real-time tumor diagnosis, but also exert a powerful anti-tumor effect through photodynamic therapy and chemotherapy is a technical problem that urgently needs to be solved. Summary of the invention

[0005] In view of the problems existing in the current photodynamic therapy under hypoxic tumor microenvironment, the present invention provides a nano-diagnostic and therapeutic preparation for accelerating drug release by hypoxia and a preparation method thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia, the nano-diagnostic and therapeutic preparation comprising: aggregation-induced emission molecule (AIE) probe TPA-TT, hypoxia-responsive prodrug, DSPE-PEG-2000 and immune cell membrane;

[0008] The aggregation-induced emission (AIE) probe is a molecular probe prepared based on an N-oxide structure, and the molecular probe of the N-oxide structure can generate a large amount of reactive oxygen under white light stimulation. The structural formula of the aggregation-induced emission (AIE) probe TPA-TT is as follows:

[0009]

[0010] The DSPE-PEG-2000 is 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000.

[0011] Furthermore, the aggregation-induced emission (AIE) probe TPA-TT and the hypoxia-responsive prodrug are co-encapsulated in DSPE-PEG-2000, and finally the immune cell membrane is encapsulated on the surface of the DSPE-PEG-2000.

[0012] Furthermore, the immune cell membrane is an M1 macrophage immune cell membrane.

[0013] Furthermore, the hypoxia-responsive prodrug is produced by the reaction of camptothecin and 4-nitrobenzyl chloroformate.

[0014] Furthermore, the camptothecin is obtained by converting the camptothecin prodrug under hypoxia stimulation; the hypoxia-activated camptothecin prodrug will accelerate the conversion into the chemotherapy drug camptothecin in the environment of probe oxygen consumption and tumor hypoxia, achieving a good tumor treatment effect. The camptothecin prodrug is as follows:

[0015]

[0016] The mechanism of conversion of camptothecin prodrug to camptothecin is as follows:

[0017]

[0018] A method for preparing a nano-diagnostic and therapeutic preparation for accelerating drug release under hypoxia, comprising the following steps:

[0019] Step 1, preparing aggregation-induced emission (AIE) probe TPA-TT;

[0020] Step 2, using tetrahydrofuran as a solvent to dissolve the aggregation-induced emission (AIE) probe TPA-TT, the hypoxia-responsive prodrug and DSPE-PEG-2000, adding the solution into deionized water, stirring overnight and then centrifuging to obtain a nano-diagnostic preparation encapsulating TPA-TT and the hypoxia-responsive prodrug;

[0021] Step 3: Cover the surface of the nano-diagnostic and therapeutic preparation with an immune cell membrane.

[0022] Furthermore, in step 1, the specific method for preparing the aggregation-induced emission (AIE) probe TPA-TT is: a halogenated N,N-diethylaniline derivative undergoes a Stieler reaction with benzobisthiadiazole, and then undergoes an oxidation reaction with m-chloroperbenzoic acid to obtain a molecular probe prepared based on an N-oxide structure.

[0023] Furthermore, in step 3, the specific method of covering the immune cell membrane on the surface of the nano-diagnostic and therapeutic preparation is: covering the immune cell membrane on the surface of the nano-diagnostic and therapeutic preparation by ultrasonic co-incubation.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The fluorescence imaging of the nano-diagnostic and therapeutic preparations of the present invention in vivo helps to sensitively depict the tumor area and provide accurate guidance for tumor treatment. The generation of reactive oxygen induced by white light can promote the apoptosis of tumor cells, and combined with the release of camptothecin triggered by hypoxia, improve the effect of anti-tumor treatment. The photodynamic effect consumes oxygen in the local area of ​​solid tumors, leading to a more serious hypoxic environment and accelerating the release of hypoxia-responsive prodrugs. Photodynamic and chemodynamic synergistic therapy can promote cancer cell apoptosis and inhibit tumor growth. When the surface of the nano-diagnostic and therapeutic preparation is covered with immune cell membranes, especially macrophage membranes, under the mediation of macrophage membranes, it can not only reduce the phagocytosis of monocytes, but also prolong the circulation time and enhance the targeted aggregation of tumors, because the α4β1 integrin on macrophages can bind to the vascular cell adhesion molecules on cancer cells, thereby improving the tumor targeting ability of nanoparticles.

[0026] The nano-diagnostic and therapeutic preparation provided by the present invention proposes a new strategy to achieve hypoxia-activated drug conversion, providing a new solution for achieving precise imaging-guided tumor treatment.

[0027] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a route map for the preparation and synthesis of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) provided in Experimental Example 1 of the present invention.

[0029] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) provided in Experimental Example 1 of the present invention.

[0030] Figure 3 This is the carbon NMR spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) provided in Experimental Example 1 of the present invention.

[0031] Figure 4 This is a high-resolution mass spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) provided in Experimental Example 1 of the present invention.

[0032] Figure 5 It is a spectral property characterization diagram provided by Experimental Example 2 of the present invention; wherein, Figure A is the absorption spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine); Figure B is the emission spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine); Figure C is the fluorescence spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) molecules in THF / water mixtures with various water fractions; Figure D is the fluorescence spectrum of N,N'-(naphthothiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) molecules under white light irradiation. Figure 1 is a graph of fluorescence peak intensity versus water fraction in THF / water mixtures, where I0 and I are the fluorescence intensities in pure THF and THF / water mixtures with various water fractions, respectively; Figure E is the relationship between I / I0 and white light irradiation time, where I0 and I represent the emission intensity of 2',7'-dichlorofluorescein (DCF) at 525nm before and after light irradiation; Figure F is the relationship between A / A0 and white light irradiation time, where I0 and I represent the absorbance of 2,3,5,6-tetrachloro-1,4-benzoquinone diethyl ester (ABDA) at 378nm before and after light irradiation.

[0033] Figure 6 This is the synthesis route of the camptothecin prodrug provided in Experimental Example 3 of the present invention.

[0034] Figure 7 This is the nuclear magnetic resonance hydrogen spectrum provided by Experimental Example 3 of the present invention.

[0035] Figure 8This is the carbon nuclear magnetic resonance spectrum provided by Experimental Example 3 of the present invention.

[0036] Fig. 9 This is the verification provided in Experimental Example 3 of the present invention that the camptothecin prodrug is converted into camptothecin under hypoxic conditions simulated by sodium dithionite. Figure A is a high performance liquid chromatogram, and Figure B is a mass spectrum of the intermediate during the prodrug conversion process.

[0037] Fig.10 This is the hydrogen spectrum provided in Experimental Example 3 of the present invention for verifying the conversion of camptothecin prodrug into camptothecin.

[0038] Fig.11 This is the carbon spectrum provided in Experimental Example 3 of the present invention for verifying the conversion of camptothecin prodrug into camptothecin.

[0039] Fig.12 This is the mass spectrum provided in Experimental Example 3 of the present invention for verifying the conversion of camptothecin prodrug into camptothecin.

[0040] Fig.13 This is the change in particle size and morphology of the nano-diagnostic and therapeutic preparation provided in Experimental Example 4 of the present invention before and after wrapping the macrophage membrane; wherein, Figure A is the TEM image and particle size image of the nano-diagnostic and therapeutic preparation (CTNPs) not wrapped in the macrophage membrane, and Figure B is the TEM image and particle size image of the nano-diagnostic and therapeutic preparation (M1-CTNPs) wrapped in the macrophage membrane.

[0041] Fig.14 This is the in vitro targeting study provided by Experimental Example 5 of the present invention; wherein, Figure A is a diagram of the uptake of CTNPs and M1-CTNPs by 4T1 (mouse breast cancer cells) tumor cells, Figure B is the uptake of CTNPs and M1-CTNPs by RAW264.7 (mouse mononuclear macrophage leukemia cells) macrophages, and Figure C is the uptake of CTNPs and M1-CTNPs by MCF-10A (mouse normal mammary epithelial cells) cells.

[0042] Fig.15 The ROS staining monitoring of 4T1 cells treated with different formulations for 24 hours under hypoxic conditions is provided in Experimental Example 6 of the present invention. Scale bar: 50 μm, where 2',7'-dichlorofluorescein (2',7'-Dichlorofluorescein, referred to as DCF) is used.

[0043] Fig.16 The live-dead staining monitoring of 4T1 cells treated in different ways for 24 hours under hypoxic conditions is provided in Experimental Example 6 of the present invention. Scale bar: 100 μm.

[0044] Fig.17This is a graph showing the CCK-8 assay results of the killing effects of different concentrations of M1-CTNPs on 4T1 tumor cells under hypoxic conditions provided in Experimental Example 6 of the present invention.

[0045] Fig.18 This is a biosafety test diagram of different concentrations of M1-CTNPs in vitro in mice provided in Experimental Example 7 of the present invention.

[0046] Fig.19 These are the near-infrared imaging results of tumor-bearing mice at different times after injection of PBS, CTNPs and M1-CTNPs nanoparticles provided in Experimental Example 8 of the present invention.

[0047] Fig. 20 These are the results of near-infrared fluorescence imaging of the internal organs of tumor-bearing mice after injection of PBS, CTNPs and M1-CTNPs nanoparticles, as provided in Experimental Example 8 of the present invention.

[0048] Fig.21 This is a schematic diagram of a timetable for evaluating the therapeutic effects of various intervention measures in 4T1 tumor-bearing mice provided in Experimental Example 9 of the present invention.

[0049] Fig. 22 This is the situation after different therapeutic interventions provided in Experimental Example 9 of the present invention; wherein A is a tumor growth curve diagram (n=5), B is a tumor growth picture after different therapeutic interventions (n=5), and C is a tumor weight diagram of mice after different treatments (n=5).

[0050] Fig.23 This is a Tunel immunofluorescence staining image of tumor sections after different treatment interventions provided in Experimental Example 9 of the present invention, scale bar: 50 μm.

[0051] Fig.24 These are pathological sections of tumor tissues in each group after different treatment interventions provided in Experimental Example 9 of the present invention (n=5).

[0052] Fig.25 These are the histopathological sections of the heart, liver, spleen, lung and kidney of mice in each group after different treatment interventions provided in Experimental Example 10 of the present invention (n=3).

[0053] Fig.26 This is the blood test results of mice provided in Experimental Example 10 of the present invention, wherein Figure A is the blood routine index and Figure B is the blood biochemical index. DETAILED DESCRIPTION

[0054] In order to gain a deeper understanding of the present invention, we will provide a comprehensive and detailed description of the present invention. However, the present invention has multiple implementations and is not limited to the specific examples listed herein. The presentation of these examples is intended to deepen the comprehensive understanding of the disclosure of the present invention.

[0055] Explanation of terms

[0056] Fluorescence imaging (NIR-I FI): is a fluorescence imaging technology based on the near-infrared (NIR) light region, with a wavelength range of approximately 700 to 900 nanometers. Within this wavelength range, biological tissues have low light scattering and absorption, and good tissue depth penetration, making it suitable for in vivo imaging. NIR-I FI usually uses fluorescent probes or fluorescent markers, such as specific binding to tumor tissue, or for marking specific cells or molecules. When these fluorescent probes or markers are excited, they emit NIR-I fluorescence signals, which can be captured and recorded by imaging equipment. Near-infrared fluorescence imaging has a wide range of applications in the biomedical field, including tumor diagnosis, tumor microenvironment research, and tumor treatment monitoring.

[0057] Photodynamic therapy (PDT): It is a widely used treatment method in the medical field. Its principle is based on the fact that photosensitizers are excited under light of a specific wavelength. In the excited state, they react with oxygen to produce a series of reactive oxygen species, such as free radicals and singlet oxygen. These reactive oxides have strong oxidizing properties and can cause strong damage to cell membranes and organelles, thereby inducing apoptosis of tumor cells or causing damage to other diseased tissues. The appropriate dosage, irradiation time and irradiation intensity can be selected according to the needs and the severity of the disease. Therefore, the advantages of PDT are its non-invasiveness, selectivity and high specificity. It only causes damage to diseased tissues and has almost no side effects on normal tissues. Like PTT, PDT is still limited by photosensitizers, light penetration depth and maximum laser exposure. Further research and improvement of PDT is still a hot research field and is expected to bring more breakthroughs and progress to clinical treatment.

[0058] Tumor microenvironment (TME): refers to the surrounding microenvironment of tumor cells, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules and extracellular matrix. In solid tumors, due to the rapid growth of tumor tissue, high expansion and incomplete vascular system inside the tumor tissue, these will lead to insufficient oxygen supply in the tumor tissue, and the tumor microenvironment presents the characteristics of overall hypoxia. Due to insufficient oxygen supply, tumor cells can only metabolize energy through anaerobic glycolysis, which will cause the accumulation of lactic acid; at the same time, the ion exchange proteins on the tumor cell membrane are also continuously transporting H+ inside the cell to the outside of the cell to avoid autoacidosis. These cellular reactions also cause the pH of the tumor microenvironment to decrease to varying degrees, presenting an acidic environment as a whole. In the development of tumors and hypoxia, acidic microenvironment, a large number of tumor tissues and peripheral tissue cells will undergo apoptosis, release cell fragments and chemokines, leading to inflammatory cell infiltration and secretion of inflammatory factors. At the same time, the development of the tumor itself will also trigger the immune response of the immune system, causing inflammatory cells to gather in the area and trigger a severe inflammatory response.

[0059] Reactive oxygen species (ROS): A class of highly active oxidizing compounds, usually including superoxide anions, hydroxyl radicals, singlet oxygen, hydrogen peroxide, etc. A small amount of ROS is produced during cell metabolism and is a normal metabolite in the body. These ROS have unpaired electrons or contain active oxygen-oxygen bonds and show high reactivity. Excessive ROS can lead to increased oxidative stress in cells, which in turn causes cell damage and tissue inflammation, and is closely related to the occurrence and development of a variety of diseases. In cancer treatment, the cytotoxicity of reactive oxygen species can effectively induce apoptosis of tumor cells.

[0060] Stille reaction, also known as Stille coupling reaction, is a cross-coupling reaction between organotin compounds and halogenated hydrocarbons without β-hydrogen under palladium catalysis. The reaction is generally carried out in a dehydrated and deoxygenated solvent and an inert environment. Equivalent amounts of Cu(I) or Mn(II) salts can improve the specificity and reaction rate of the reaction. Oxygen will oxidize the palladium catalyst and cause the organotin compound to self-couple. Tetrakis(triphenylphosphine)palladium is the most commonly used palladium catalyst, and other catalysts include: PdCl2(PPh3)2, PdCl2(MeCN)2, etc. The halogenated hydrocarbons used are generally vinyl or aryl trifluoromethanesulfonates or chlorine, bromine, or iodine hydrocarbons.

[0061] Example 1

[0062] Preparation of TPA-TT, the synthetic route is as follows Figure 1 shown.

[0063] 1. Preparation of N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalene-1-amine, the specific preparation process is as follows: 1.1 g of N-(4-bromophenyl)-N-phenylnaphthalene-1-amine (10 mmol) and 1,1'-bis(phenylphosphine)ferroene-palladium(II) dichloride dichloromethane complex (0.245 g, 0.3 mmol) and bis(picallo)diborane (3.81 g, 15 mmol) were added to a 100 mL double-necked round-bottom flask. 40 mL of tetrahydrofuran (THF) was added under argon atmosphere, and the mixture was heated to reflux and stirred for 24 hours. After the reaction was completed, water was added and extracted three times with dichloromethane. The organic phases were combined, dried over MgSO4, and then the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / n-hexane = 1 / 2) to give a white solid product N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-1-amine with a yield of 76%.

[0064] 2. Preparation of N,N'-(naphtho[2,3-c][1,2,5]thiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine), referred to as TPA-TT, the specific preparation process is as follows: 2.29 g of N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalene-1-amine (5 mmol) and 4,9-dibromonaphthalene[2,3-c][1,2,5]thiadiazole (1.67 g, 2 mmol), tetrakis(phenylphosphine)palladium (60 mg, 0.05 mmol) and sodium carbonate (2.07 g, 15 mmol) were added to a 100 mL double-necked round-bottom flask. The flask was evacuated and purged with argon three times. Then 40 mL of tetrahydrofuran (THF) and 10 mL of water were added, and the mixture was heated to reflux and stirred in the absence of light for 48 hours. After the reaction, water was added and extracted three times with dichloromethane. The organic phases were combined and dried with MgSO4. After the solvent was removed, the residue was purified by silica gel column chromatography (eluent: dichloromethane / n-hexane = 1 / 5) to obtain a black purple solid product N,N'-(naphtho[2,3-c][1,2,5]thiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) with a yield of 62%.

[0065] like Figure 2 As shown, 1H NMR (500MHz, CDCl3): δ8.26(dd,J=5.6,3.2Hz,1H),8.19–8.13(m,1H),7.84(dt,J=7.9,1.6Hz,1H),7.75(dt,J=7.9,1.1H z,1H),7.60–7.43(m,7H),7.32–7.24(m,4H),7.19(dt,J=7.2,0.8Hz,1H),7.14–7.08(m,2H),7.04(tt,J=7.8,1.5Hz,1H).

[0066] like Figure 3 As shown, 13 C NMR (125MHz, CDCl3) δ155.44,147.10,145.83,142.24,134.75,132.81,131.52,130.63,129.15,128.98,1 28.53,128.28,127.23,126.85,126.21,126.09,125.77,124.72,124.11,124.08,123.77,123.57,123.23.

[0067] like Figure 4 As shown, HRMS (MALDI-TOF, m / z): [M+H] + calcd.forC 54 H 36 N4S, 772.2661; found, 772.2647.

[0068] Example 2

[0069] The spectral properties of TPA-TT are characterized by Figure 5 As shown;

[0070] Photophysical properties of TPA-TT: In THF, the maximum absorption wavelength of TPA-TT is 538nm; the maximum emission wavelength is 730nm.

[0071] The production of ROS and 1 O2's ability to show excellent ROS and 1 O2 production capacity.

[0072] Experimental Example 3

[0073] Characterization of the transformation of camptothecin prodrugs under hypoxic conditions, such as Figure 6-Figure 9 shown.

[0074] 1. Preparation of camptothecin prodrug, the specific preparation process is shown in Figure 6: 100 mg (0.12 mmol) CPT and 17.1 mg (0.14 mmol) 4-dimethylaminopyridine were dissolved in 15 mL dry dichloromethane (CH2Cl2), placed in a 50 mL double-necked round-bottom flask, and cooled to 0°C. Then, 30.2 mg (0.14 mmol) of 4-nitrobenzyl chloroformate solution dissolved in dry dichloromethane was added dropwise. Then, the mixed solution was stirred at room temperature overnight. After the reaction was completed, it was extracted three times with dichloromethane. The organic layer was separated, dried with anhydrous MgSO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 2) to obtain white solid CPT-NB with a yield of 70%.

[0075] 2. The NMR carbon spectrum of camptothecin prodrug, such as Figure 7 and Figure 8 As shown, 1 H NMR(500MHz, CDCl3)δ8.21(d,J=2.1Hz,1H),7.96–7.91(m,1H),7.79–7.73(m,1H),7.67(td,J=8.0,1.1 Hz,1H),7.47–7.40(m,1H),7.19(t,J=1.0Hz,1H),6.86(dd,J=10.6,9.2Hz,1H),6.33(dq,J=10.6,1.1H z,1H),6.17(tq,J=6.9,1.0Hz,1H),5.14–5.05(m,2H),4.92(s,2H),4.76(dq,J=1.8,0.9Hz,2H),4.47( dt,J=9.2,8.3Hz,1H),2.61–2.51(m,1H),2.43–2.33(m,1H),2.23–2.09(m,2H),1.05(t,J=8.3Hz,3H). 13 CNMR(125MHz, CDCl3)δ169.91,158.77,154.43,153.60,149.20,145.42,145.16,132.25,131.17,130.76,129.78,129.61–12 9.33(m),128.50,126.52,123.73,119.09(d,J=3.1Hz),98.26,81.93(d,J=15.5Hz),67.51,67.31,51.03,31.02,25.05,7.60.

[0076] 3. Test of the hypoxic reactivity of camptothecin prodrugs. Sodium dithionite (Na2S2O4) solution with different concentrations was used to simulate hypoxic conditions, and the reaction system was monitored by high performance liquid chromatography (HPLC). Fig. 9 As shown in A, with the increase of Na2S2O4 concentration, the peak of CPT-NB at 28.6 minutes gradually decreased, while a new peak appeared and strengthened at 15.2 minutes, indicating that part of the prodrug has been converted to CPT. At 100mM Na2S2O4 concentration, the peak at 28.6 minutes completely disappeared, indicating that the prodrug has been completely converted to free CPT. Fig. 9 B is the mass spectrum of the intermediate after prodrug reduction.

[0077] Mass spectral data: HRMS (MALDI-TOF, m / z): [M+H] + calcd.forC 28 H 24 N3O6, 498.1665; found, 498.1665.

[0078] Figure 10-12 It shows that after the conversion of camptothecin prodrug, it is proved that camptothecin is released, and the NMR and mass spectra are as follows:

[0079] 1 H NMR (500MHz, CDCl3) δ8.30 (dd, J=5.6, 3.2Hz, 1H), 8.07-8.02 (m, 1H), 7.81 (ddt, J=11.1, 7.9, 1.5Hz, 2H), 7.73-7. 68(m,2H),7.61-7.45(m,4H),7.29-7.21(m,4H),7.12(ddd,J=23.1,7.3,1.4Hz,3H),7.02(tt,J=7.6,1.4Hz,1H). 13 CNMR(125MHz, CDCl3)δ154.23,146.79,145.65,142.58,134.82,132.29,131.33,130.42,129.14,128.74,128.43,1 28.19,127.11(d,J=16.6Hz),126.49(d,J=15.5Hz),124.39,124.22,123.80,123.70,123.52.HRMS(ESI)m / z:calcd for C 20 H 16 N2O4[M+H] + 349.1184, found 349.1187.

[0080] Experimental Example 4

[0081] Preparation of nano-theranostics, such as Fig.13 As shown, the specific preparation process is as follows: a total of 2 mg TPA-TT, 1 mg CPT-NB and 10 mg DSPE-PEG-2000 are completely dissolved in 1 mL tetrahydrofuran (THF) at room temperature 10-30 ° C. Subsequently, the drug solution is introduced into 10 mL deionized water under rapid stirring. The mixed solution is placed in a fume hood and stirred at room temperature overnight to eliminate THF in the solution. The resulting CTNPs are then harvested by centrifugation using a centrifugal filter. In order to produce M1-CTNPs, freshly prepared CTNPs (2 mL) are combined with 4 mg M1 macrophage membranes under ultrasonic conditions (100 W, 50 Hz, 10 minutes), extruded with a polycarbonate membrane to produce M1-CTNPs, stored at 4 ° C and used quickly.

[0082] The results of transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements showed that it was a spherical structure with an average diameter of about 88nm; M1-CTNPs was a spherical structure with an average diameter of about 112nm.

[0083] Experimental Example 5

[0084] In vitro cell targeting studies, such as Fig.14 As shown. Nanoparticles usually have poor tumor targeting ability, partly due to poor in vivo circulation, which makes them easily removed by phagocytes as foreign bodies. On the other hand, they lack effective tumor targeting, thus reducing tumor aggregation. For this reason, the present invention explores the immune escape and tumor targeting ability of nanoparticles. Compared with CTNPs, M1 macrophage membrane encapsulation can greatly improve the tumor targeting ability of M1-CTNPs. The macrophage uptake of M1-CTNPs is much lower than that of CTNPs. This is because the M1 macrophage membrane encapsulation successfully reduces the phagocytosis of M1-CTNPs by macrophages. At the same time, after incubation of normal mammary epithelial cells (MCF-10A) of mice with M1-CTNPs, their fluorescence signal was significantly weakened and weaker than that of CTNPs, indicating that M1-CTNPs have excellent targeting ability for cancer cells and will not target healthy epithelial cells.

[0085] Experimental Example 6

[0086] In vitro cell studies, such as Fig.15 , Fig.16 , Fig.17As shown. Under white light irradiation, the M1-CTNPs+light irradiation group showed a fairly excellent ROS production ability, greatly improving the tumor killing ability. The treated 4T1 cells showed a bright green fluorescence signal and we also observed that the hypoxia-responsive prodrug also increased the ROS generation ability of TPA-TT. We can observe that the M1-CTNPs+L group produced the most ROS. The analysis is because the M1 macrophage membrane increases the internalization of nanoparticles by cancer cells, accelerates the release of hypoxia-responsive prodrugs, and increases the ROS generation ability of TPA-TT. The excellent PDT properties of TPA-TT were confirmed. In addition, with the increase in the concentration of M1-CTNPs, a dose-dependent tumor killing ability was also shown. The results of cell live and dead staining also showed that 4T1 cells treated with PBS alone showed a strong calcein staining signal (green fluorescence), indicating live cells. Cells treated with TNPs and light irradiation showed significant PI-stained dead cells (red fluorescence), indicating the tumor killing effect of individual PDT. The cytotoxicity of cells treated with CNPs alone was general. A significant increase in cell death was observed in cells treated with CTNPs+L or M1-CTNPs+L, with the latter showing the highest tumor-suppressive ability, due to the M1 cell membrane coating facilitating tumor cell uptake. In the M1-CTNPs+L group, almost all 4T1 cells showed signs of cell death, emphasizing the synergistic therapeutic impact of the combination of targeted PDT and chemotherapy. In the absence of light exposure, TNPs alone showed minimal cytotoxicity. These results indicate that the dark toxicity of TPA-TT is negligible. However, after white light irradiation, the survival rate of 4T1 cells was significantly reduced to about 40%, indicating the potential of PDT in tumor cell eradication. After the introduction of hypoxia-responsive prodrugs, it was observed that the cytotoxicity of CTNPs exceeded that of TNPs under white light irradiation under hypoxic conditions. This enhanced tumor-killing efficacy is attributed to the chemotherapeutic agent triggered by hypoxia. Notably, M1-CTNPs, characterized by tumor-targeted cell membranes and the killing effect of white light-chemotherapy combination, showed the highest inhibition of tumor cells under white light irradiation and could effectively kill tumor cells.

[0087] Experimental Example 7

[0088] In vitro biosafety studies, such as Fig.18 When the concentration of M1-CTNPs ranged from 0 to 100 μg mL -1 All blood samples showed relatively low hemolysis rates (<6%), indicating good blood compatibility. These results suggest the biosafety of M1-CTNPs.

[0089] Experimental Example 8

[0090] In vivo tumor fluorescence imaging studies, such as Fig.19, as shown in 20. The fluorescence signal was strongest at the tumor site at 24h of administration. The fluorescence signal of mice treated with M1-CTNPs was higher than that of CTNPs, indicating that the tumor accumulation ability mediated by the membrane coating of M1 macrophages was better. In addition, even 48h after administration, a strong NIR fluorescence signal could still be observed at the tumor site, indicating that NPs have long-term in vivo tumor imaging capabilities. Compared with CTNPs, the tumors of mice treated with M1-CTNPs showed higher NIR fluorescence signals, while the accumulation of NPs in the liver and spleen was reduced. This may be because the cell membrane of M1 macrophages can help NPs escape from normal tissues. This is consistent with previous cell experiments, where M1-CTNPs were less taken up in RAW264.7 cells. The above experimental results demonstrate the effective accumulation of M1-CTNPs at the tumor site.

[0091] Experimental Example 9

[0092] In vivo antitumor effect studies, such as Figures 21 to 24 As shown. 200 μL of PBS, CNPs, TNPs, CTNPs, and M1-CTNPs were intravenously injected three times on days 0, 2, and 4, respectively. 24 hours after each injection, 4T1 tumor-bearing mice in the “TNPs+L,” “CTNPs+L,” and “M1-CTNPs+L” groups were irradiated with white light (1 W·cm -2 , 10 minutes). The tumor volume and weight of mice undergoing various treatments were monitored daily. All mice were killed immediately after the completion of treatment on day 14. Tumors from each group were weighed and photographed, and tumor growth curves, tumor size, and tumor weight were carefully monitored to evaluate the efficacy of treatment. Compared with the PBS group, both the CNPs group and the CTNPs group showed a moderate reduction in tumor size, indicating the anti-tumor effect of chemotherapy induced by CPT prodrug. Under white light irradiation, the CTNPs wrapped by the M1 cell membrane showed a stronger tumor inhibition ability than other interventions. The tumor inhibition achieved in the M1-CTNPs group was attributed to the enhanced accumulation of nanomedicines at the tumor site, which killed more cancer cells, resulting in excellent tumor treatment effects. It is worth noting that all treatments showed good biosafety, as evidenced by the minimal changes in the weight of mice in each treatment group. Next, the tumor volume of mice treated in different groups over the 14 days of monitoring was plotted. We can clearly observe that the tumor volume of mice in the final group did not increase significantly, indicating that M1-CTNPs had a good inhibitory effect on tumors.

[0093] Experimental Example 10

[0094] Biosafety studies, such as Fig.25 and Fig.26As shown. On days 0, 2, and 4, "CNPs", "M1-CTNPs", "TNPs+L", "CTNPs+L", and "M1-CTNPs+L" were intravenously administered to healthy mice (n=3 mice per group). PBS-treated healthy mice served as controls (n=3 mice). On day 10, all mice were killed, and the main organs (heart, liver, spleen, lungs, and kidneys) of the mice were collected for paraffin-embedded sections, followed by hematoxylin and eosin (H&E) staining. In addition, the results of blood routine and blood biochemical analysis also showed that there were no abnormalities in the mice in the various treatment groups, proving the safety of the M1-CTNPs nanodiagnostic and therapeutic preparations.

[0095] Finally, it should be noted that the contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. Although the illustrative specific embodiments of the present invention are described above to facilitate the understanding of the present invention by the technical personnel in the field, it should be clear that the present invention is not limited to the scope of the specific embodiments. For the ordinary technicians in the field, as long as the various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0096] The contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. Although the illustrative specific embodiments of the present invention are described above to facilitate the understanding of the present invention by the technical personnel in the field, it should be clear that the present invention is not limited to the scope of the specific embodiments. For the ordinary technical personnel in the field, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

Claims

1. A nano-diagnostic and therapeutic preparation for accelerating drug release under hypoxia, characterized in that: The nano-diagnostic and therapeutic preparation comprises: aggregation-induced luminescence molecular probe TPA-TT, hypoxia-responsive prodrug, DSPE-PEG-2000 and immune cell membrane; The structural formula of the aggregation-induced emission (AIE) probe TPA-TT is as follows: The DSPE-PEG-2000 is 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000.

2. The nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 1, characterized in that: The aggregation-induced luminescence molecular probe TPA-TT and the hypoxia-responsive prodrug are co-encapsulated in DSPE-PEG-2000, and finally the immune cell membrane is encapsulated on the surface of the DSPE-PEG-2000.

3. The nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 1, characterized in that: The immune cell membrane is an M1 macrophage immune cell membrane.

4. The nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 1, characterized in that: The hypoxia responsive prodrug is produced by the reaction of camptothecin and 4-nitrobenzyl chloroformate.

5. The nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 4, characterized in that: The camptothecin is obtained by converting a camptothecin prodrug under hypoxia stimulation; the camptothecin prodrug is as follows:

6. The method for preparing a nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1, preparing aggregation-induced emission molecular probe TPA-TT; Step 2, using tetrahydrofuran as a solvent to dissolve the aggregation-induced emission molecular probe TPA-TT, the hypoxia-responsive prodrug and DSPE-PEG-2000, adding the solution into deionized water, stirring overnight and then centrifuging to obtain a nano-diagnostic and therapeutic preparation encapsulating TPA-TT and the hypoxia-responsive prodrug; Step 3: Cover the surface of the nano-diagnostic and therapeutic preparation with an immune cell membrane.

7. The method for preparing a nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 6, characterized in that: The specific method for preparing the aggregation-induced emission molecular probe TPA-TT in step 1 is: a halogenated N,N-diethylaniline derivative undergoes a Stieler reaction with benzobithiadiazole, followed by an oxidation reaction with m-chloroperbenzoic acid to obtain a molecular probe prepared based on an N-oxide structure.

8. The method for preparing a nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 6, characterized in that: The specific method of step 3, covering the immune cell membrane on the surface of the nano-diagnostic and therapeutic preparation is: covering the immune cell membrane on the surface of the nano-diagnostic and therapeutic preparation by ultrasonic co-incubation.

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