A nano-diagnostic and therapeutic preparation for accelerating drug release due to hypoxia and its preparation method

Abstract

The present invention discloses a nano-diagnostic and therapeutic preparation for accelerating drug release under hypoxia and a preparation method thereof, and belongs to the field of biomedical technology. In view of the problems existing in photodynamic therapy under the current hypoxic tumor microenvironment, the present invention discloses a nano-diagnostic and therapeutic preparation for accelerating drug release under hypoxia, including: aggregation-induced emission molecule (AIE) probe TPA-TT, hypoxia-responsive prodrug, DSPE-PEG-2000 and immune cell membrane; aggregation-induced emission molecule (AIE) probe TPA-TT and hypoxia-responsive prodrug are co-encapsulated in DSPE-PEG-2000, and finally immune cell membrane is wrapped on its surface. Preparation of aggregation-induced emission molecular probe; Preparation of nano-diagnostic and therapeutic preparation loaded with aggregation-induced emission molecular probe and hypoxia-responsive prodrug; Covering M1 macrophage immune cell membrane on the surface of nano-diagnostic and therapeutic preparation. The present invention provides a scheme for killing tumor cells and promoting cancer cell apoptosis guided by precise imaging, which has both tumor near-infrared fluorescence imaging and photodynamic and chemodynamic synergistic therapeutic performance while specifically targeting cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a nano-diagnostic and therapeutic 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 a high mortality rate and limited treatment options. Among various treatment modalities, chemotherapy is an effective strategy for treating both localized and metastatic cancers in clinical settings. Despite considerable progress in cancer chemotherapy over the past few decades, treatment outcomes remain 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, often fail to discriminate between normal and tumor cells, leading to damage to rapidly proliferating tissues and cells. Unlike the normal physiological environment, the tumor microenvironment (TME) of most solid tumors exhibits unique characteristics, including a slightly acidic pH, elevated glutathione levels, and extreme hypoxia. Scientific evidence has established hypoxia as a major factor in tumor metastasis, angiogenesis, and responsiveness to many therapeutic interventions. Furthermore, the unique hypoxic properties of tumors offer 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 regions but are converted into cytotoxic compounds in hypoxic tumor compartments, thereby selectively exerting their therapeutic effects in the tumor. However, since the hypoxic microenvironment is often shown to be heterogeneous, hindering efficient and rapid prodrug conversion, relying solely on hypoxic oxygen therapy drugs usually fails to achieve optimal anti-tumor efficacy.

[0003] Recently, photodynamic therapy (PDT) has been demonstrated, in which a photosensitizer reacts with oxygen to produce singlet oxygen ( 1 The process of generating oxygen (O2) or reactive oxygen species (ROS) 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 response to the problems existing in current photodynamic therapy under hypoxic tumor microenvironment, the present invention provides a nano-diagnostic and therapeutic preparation for accelerating drug release under 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 the presence of hypoxia, comprising: an aggregation-induced emission (AIE) probe TPA-TT, a hypoxia-responsive prodrug, DSPE-PEG-2000, and an immune cell membrane;

[0008] The aggregation-induced emission (AIE) probe is a molecular probe prepared based on an N-oxide structure. The N-oxide structure molecular probe can generate a large amount of reactive oxygen species 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 a camptothecin prodrug under hypoxia stimulation; the hypoxia-activated camptothecin prodrug is accelerated to convert into the chemotherapy drug camptothecin under 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 comprises the following steps:

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

[0020] Step 2: Dissolve the aggregation-induced emission (AIE) probe TPA-TT, the hypoxia-responsive prodrug, and DSPE-PEG-2000 in tetrahydrofuran as a solvent, add the solution to deionized water, stir overnight, and then centrifuge to obtain a nanotheranostic preparation encapsulating TPA-TT and the hypoxia-responsive prodrug;

[0021] Step 3: Cover the surface of the nano-diagnostic and therapeutic preparation with 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 Stiehler reaction with benzobisthiadiazole, and then an oxidation reaction is carried out with m-chloroperbenzoic acid to obtain a molecular probe prepared based on an N-oxide structure.

[0023] Furthermore, 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.

[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 production of reactive oxygen species 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 locally in solid tumors, leading to a more severe hypoxic environment and accelerating the release of hypoxia-responsive prodrugs. The synergistic treatment of photodynamic and chemodynamic 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 the macrophage membrane, it can not only reduce the phagocytosis of monocytes, but also prolong the circulation time and enhance the targeted aggregation of tumors. Since the α4β1 integrin on macrophages can bind to the vascular cell adhesion molecules on cancer cells, the tumor targeting ability of nanoparticles is improved.

[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 set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a roadmap 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'-(naphthadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine); Figure B is the emission spectrum of N,N'-(naphthadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine); Figure C is the fluorescence spectrum of N,N'-(naphthadiazole-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'-(naphthadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalene-1-amine) molecules under white light irradiation. Figure 2. Fluorescence peak intensity versus water fraction in THF / water mixtures, where I0 and I represent the fluorescence intensities in pure THF and THF / water mixtures with various water fractions, respectively. Figure E shows the relationship between I / I0 and white light irradiation time. I0 and I represent the emission intensity of 2',7'-dichlorofluorescein (DCF) at 525 nm before and after irradiation. Figure F shows the relationship between A / A0 and white light irradiation time. I0 and I represent the absorbance of 2,3,5,6-tetrachloro-1,4-benzoquinone diethyl ester (ABDA) at 378 nm before and after irradiation.

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

[0034] Figure 7 This is the hydrogen nuclear magnetic resonance 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] Figure 9 This is the verification provided in Experimental Example 3 of the present invention of the conversion of a prodrug of camptothecin to 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] Figure 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] Figure 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] Figure 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] Figure 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] Figure 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 into 4T1 (mouse breast cancer cells) tumor cells, Figure B is the uptake of CTNPs and M1-CTNPs into RAW264.7 (mouse mononuclear macrophage leukemia cells) macrophages, and Figure C is the uptake of CTNPs and M1-CTNPs into MCF-10A (mouse normal mammary epithelial cells) cells.

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

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

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

[0045] Figure 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] Figure 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] Figure 20 These are the near-infrared fluorescence imaging results 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] Figure 21 This is a schematic diagram of the 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] Figure 22 This is the situation after different treatment 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 treatment interventions (n=5), and C is a tumor weight diagram of mice after different treatments (n=5).

[0050] Figure 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] Figure 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] Figure 25 These are 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] Figure 26 This is the blood test results of mice provided in Experimental Example 10 of the present invention, wherein Figure A shows the blood routine index and Figure B shows the blood biochemical index. DETAILED DESCRIPTION

[0054] To gain a deeper understanding of the present invention, we will provide a comprehensive and detailed description thereof. However, the present invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a comprehensive understanding of the present disclosure.

[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 those that specifically bind to tumor tissue, or are used to label 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 excitation of photosensitizers under the irradiation of light of a specific wavelength. In the excited state, it will react with oxygen to produce a series of reactive oxygen species, such as free radicals and singlet oxygen. These reactive oxygen species have strong oxidizing properties and can cause strong damage to cell membranes and organelles, thereby inducing apoptosis of tumor cells or damaging 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 the photosensitizer, light penetration depth and maximum laser exposure. Further research and improvement of PDT remains a hot research field and is expected to bring more breakthroughs and progress to clinical treatment.

[0058] The tumor microenvironment (TME) refers to the microenvironment surrounding tumor cells, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and the extracellular matrix. In solid tumors, the rapid growth and expansion of tumor tissue, coupled with an incomplete vascular system within the tumor tissue, lead to insufficient oxygen supply within the tumor tissue, resulting in an overall hypoxic TME. Due to this oxygen deficiency, tumor cells are forced to metabolize energy through anaerobic glycolysis, which results in the accumulation of lactic acid. Simultaneously, ion exchange proteins on the tumor cell membrane continuously transport H+ from the cell to the outside of the cell to prevent autoacidosis. These cellular reactions also contribute to varying degrees to a lower pH in the TME, creating an overall acidic environment. During tumor development and the hypoxic, acidic microenvironment, a large number of cells in the tumor and surrounding tissues undergo apoptosis, releasing cell fragments and chemokines, leading to the infiltration of inflammatory cells and the secretion of inflammatory factors. Furthermore, the development of the tumor itself triggers an immune response, causing inflammatory cells to accumulate in the area and trigger a severe inflammatory response.

[0059] Reactive oxygen species (ROS): A class of highly reactive oxidizing compounds, typically including superoxide anions, hydroxyl radicals, singlet oxygen, and hydrogen peroxide. Small amounts of ROS are produced during cellular metabolism and are normal metabolic products in living organisms. These ROS possess unpaired electrons or contain reactive oxygen-oxygen bonds, exhibiting high reactivity. Excessive ROS can lead to increased intracellular oxidative stress, further triggering cell damage and tissue inflammation, and are closely associated with the development and progression of various diseases. In cancer treatment, the cytotoxicity of ROS can be effectively used to induce apoptosis in tumor cells.

[0060] The Stille reaction, also known as the Stille coupling reaction or Stille coupling reaction, is a palladium-catalyzed cross-coupling reaction between an organotin compound and a halogenated hydrocarbon that does not contain β-hydrogen. The reaction is typically carried out in a dehydrated, deoxygenated solvent and an inert environment. The addition of equistoichiometric amounts of Cu(I) or Mn(II) salts can enhance the specificity and rate of the reaction. Oxygen oxidizes the palladium catalyst and causes the organotin compound to self-couple. Tetrakis(triphenylphosphine)palladium is the most commonly used palladium catalyst; other catalysts include PdCl2(PPh3)2 and PdCl2(MeCN)2. The halogenated hydrocarbon used is typically a vinyl or aryl triflate or a chlorine-, bromine-, or iodine-substituted hydrocarbon.

[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)naphthalen-1-amine. The specific preparation process is as follows: 1.1 g of N-(4-bromophenyl)-N-phenylnaphthalen-1-amine (10 mmol), 1,1'-bis(phenylphosphine)ferroene-palladium(II) dichloride dichloromethane complex (0.245 g, 0.3 mmol), and bis(picalo)diborane (3.81 g, 15 mmol) were added to a 100 mL two-necked round-bottom flask. Under an argon atmosphere, 40 mL of tetrahydrofuran (THF) was added, and the mixture was heated to reflux and stirred for 24 hours. After the reaction, water was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over MgSO4, and 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 N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-1-amine as a white solid product in 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(phenylnaphthalen-1-amine), abbreviated as TPA-TT, was performed as follows: 2.29 g of N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-1-amine (5 mmol), 4,9-dibromonaphtho[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 two-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 the mixture was extracted three times with dichloromethane. The organic phases were combined and dried over MgSO4. After removing the solvent, the residue was purified by silica gel column chromatography (eluent: dichloromethane / n-hexane = 1 / 5) to obtain a dark purple solid product, N,N'-(naphtho[2,3-c][1,2,5]thiadiazole-4,9-diyl(4,1-phenyl))bis(phenylnaphthalen-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] Characterization of the spectral properties of TPA-TT, such as Figure 5 As shown;

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

[0071] The production of ROS and the activity of TPA-TT were evaluated using DCFH-DA and ABDA probes under white light irradiation. 1 O2 ability, showing 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] I. Preparation of camptothecin prodrug. The specific preparation process is shown in Figure 6: 100 mg (0.12 mmol) of CPT and 17.1 mg (0.14 mmol) of 4-dimethylaminopyridine were dissolved in 15 mL of dry dichloromethane (CH2Cl2) in a 50 mL two-necked round-bottom flask and cooled to 0°C. Then, a solution of 30.2 mg (0.14 mmol) of 4-nitrobenzyl chloroformate dissolved in dry dichloromethane was added dropwise. The mixed solution was then stirred at room temperature overnight. After the reaction was completed, the mixture was extracted three times with dichloromethane. The organic layer was separated, dried over anhydrous MgSO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 2) to obtain CPT-NB as a white solid in a 70% yield.

[0075] 2. 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. To test the hypoxic reactivity of camptothecin prodrug, 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). Figure 9 As shown in Figure A, the CPT-NB peak at 28.6 minutes gradually decreases with increasing Na2S2O4 concentration, while a new peak appears and strengthens at 15.2 minutes, indicating that some prodrug has been converted to CPT. At 100 mM Na2S2O4, the peak at 28.6 minutes completely disappears, indicating that the prodrug has been completely converted to free CPT. Figure 9 B is the mass spectrum of the middle portion after prodrug reduction.

[0077] Mass spectrometry 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 after drug release. 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 Figure 13 As shown, the specific preparation process is as follows: a total of 2 mg of TPA-TT, 1 mg of CPT-NB, and 10 mg of DSPE-PEG-2000 were completely dissolved in 1 mL of tetrahydrofuran (THF) at room temperature (10-30°C). Subsequently, the drug solution was introduced into 10 mL of deionized water under rapid stirring. The mixture was stirred overnight at room temperature in a fume hood to eliminate the THF in the solution. The resulting CTNPs were then harvested by centrifugation using a centrifugal filter. To generate M1-CTNPs, freshly prepared CTNPs (2 mL) were combined with 4 mg of M1 macrophage membranes under ultrasonic conditions (100 W, 50 Hz, 10 minutes). The M1-CTNPs were extruded through a polycarbonate membrane, stored at 4°C, and used immediately.

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

[0083] Experimental Example 5

[0084] In vitro cell targeting studies, such as Figure 14 As shown. Nanoparticles generally have poor tumor targeting ability, partly due to poor circulation in the body, which makes them easily cleared by phagocytes as foreign bodies. On the other hand, they lack effective tumor targeting, thus reducing tumor aggregation. To this end, the present invention explores the immune escape and tumor targeting ability of nanoparticles. Compared with CTNPs, M1 macrophage membrane wrapping 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 wrapping successfully reduces the phagocytosis of M1-CTNPs by macrophages. At the same time, after incubation of normal mouse mammary epithelial cells (MCF-10A) 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, e.g. Figure 15 、 Figure 16 、 Figure 17As shown in the figure, under white light irradiation, the M1-CTNPs + light irradiation group exhibited excellent ROS production, significantly enhancing tumorigenicity. Treated 4T1 cells displayed a bright green fluorescence signal, and we also observed that the hypoxia-responsive prodrug also increased TPA-TT's ROS generation. The M1-CTNPs + L group produced the most ROS. This analysis suggests that the M1 macrophage membrane increases nanoparticle internalization by cancer cells, accelerating the release of the hypoxia-responsive prodrug and increasing TPA-TT's ROS generation. This confirms the excellent PDT properties of TPA-TT. Furthermore, increasing M1-CTNP concentrations demonstrated dose-dependent tumorigenicity. Live / dead staining results also showed that 4T1 cells treated with PBS alone displayed a strong calcein staining signal (green fluorescence), indicating viable cells. Cells treated with TNPs and light irradiation exhibited significant PI-stained dead cells (red fluorescence), demonstrating the tumorigenicity of individual PDT. Cytotoxicity was modest when treated with CNPs alone. A significant increase in cell death was observed in cells treated with either CTNPs+L or M1-CTNPs+L, with the latter displaying the highest tumor-suppressive potency, attributed to enhanced tumor cell uptake by the M1 cell membrane coating. In the M1-CTNPs+L group, nearly all 4T1 cells showed signs of cell death, highlighting the synergistic therapeutic impact of the targeted PDT combined with chemotherapy. In the absence of light exposure, TNPs alone exhibited minimal cytotoxicity. These results suggest that the dark toxicity of TPA-TT is negligible. However, upon white light irradiation, the viability of 4T1 cells significantly decreased to approximately 40%, demonstrating the potential of PDT for tumor cell eradication. Following the introduction of a hypoxia-responsive prodrug, the cytotoxicity of CTNPs surpassed that of TNPs under white light irradiation under hypoxic conditions. This enhanced tumor-killing efficacy is attributed to the hypoxia-triggered chemotherapeutic agent. Notably, M1-CTNPs, characterized by both tumor-targeting cell membranes and a combined white light-chemotherapeutic killing effect, exhibited the highest tumor cell inhibition under white light irradiation, demonstrating effective tumor cell killing.

[0087] Experimental Example 7

[0088] In vitro biosafety studies, such as Figure 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 Figure 19, as shown in Figure 20. The fluorescence signal was strongest at the tumor site 24 hours after 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 M1 macrophage membrane coating was better. In addition, even 48 hours 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 M1 macrophage cell membrane can help NPs escape from normal tissues. This is consistent with previous cell experiments, in which M1-CTNPs were less taken up by 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 anti-tumor effect studies, such as Figures 21-24 As shown. PBS, CNPs, TNPs, CTNPs, and M1-CTNPs were intravenously injected three times on day 0, day 2, and day 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). Tumor volume and weight were monitored daily for mice receiving various treatments. All mice were immediately sacrificed after treatment was completed on day 14. Tumors from each group were weighed and photographed, and tumor growth curves, tumor size, and tumor weight were carefully monitored to assess treatment efficacy. Compared to the PBS group, both the CNPs and CTNPs groups showed a modest reduction in tumor size, indicating an antitumor effect induced by the CPT prodrug-induced chemotherapy. Under white light irradiation, the M1 cell membrane-encapsulated CTNPs demonstrated stronger tumor inhibition than other interventions. The tumor inhibition achieved in the M1-CTNPs group was attributed to enhanced accumulation of the nanodrug at the tumor site, resulting in more cancer cells being killed, resulting in superior tumor therapeutic efficacy. Notably, all treatments exhibited good biosafety, as demonstrated by minimal changes in mouse body weight across treatment groups. Next, the tumor volume of mice treated in the different groups over the 14 days of monitoring was plotted. It can be clearly observed that the tumor volume of mice in the final group did not increase significantly, indicating that the M1-CTNPs had a strong tumor inhibitory effect.

[0093] Experimental Example 10

[0094] Biosafety studies, such as Figure 25 and Figure 26As shown. On day 0, day 2, and day 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 analyses 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 preparation.

[0095] Finally, it should be noted that any matters not described in detail in this specification constitute prior art known to those skilled in the art. While the above description of the present invention provides illustrative embodiments to facilitate understanding by those skilled in the art, it should be understood that the present invention is not limited to the scope of the specific embodiments. As long as various modifications are readily apparent to those skilled in the art within the spirit and scope of the present invention as defined and established by the appended claims, all inventions utilizing the present invention are protected.

[0096] Any matters not described in detail in this specification are prior art known to those skilled in the art. Although the above description of the present invention is based on specific embodiments to facilitate understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the specific embodiments. As long as various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, such modifications will be obvious to those skilled in the art, and all inventions and creations utilizing the concepts of the present invention are protected.

Claims

1. A nano-diagnostic and therapeutic preparation for accelerating drug release in the presence of hypoxia, characterized by: 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-methoxypolyethylene glycol-2000; 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 thereof; The immune cell membrane is an M1 macrophage immune cell membrane; The hypoxia-responsive prodrug is produced by the reaction of camptothecin and 4-nitrobenzyl chloroformate.

2. The nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 1, characterized in that: The camptothecin is obtained by converting a camptothecin prodrug under hypoxia stimulation; the camptothecin prodrug is as follows: 。 3. The method for preparing a nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to any one of claims 1 to 2, characterized in that: The following steps are involved: Step 1, preparing aggregation-induced emission molecular probe TPA-TT; Step 2: Dissolving the aggregation-induced emission molecular probe TPA-TT, the hypoxia-responsive prodrug, and DSPE-PEG-2000 in tetrahydrofuran as a solvent, adding the solution to deionized water, stirring overnight, and then centrifuging to obtain a nano-theranostic preparation encapsulating TPA-TT and the hypoxia-responsive prodrug; Step 3: Cover the surface of the nano-diagnostic and therapeutic preparation with immune cell membrane.

4. The method for preparing a nano-diagnostic and therapeutic preparation for accelerating drug release in hypoxia according to claim 3, 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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