PH-NTR cascade response type nanoprobe, preparation method and application

Abstract

The invention relates to the technical field of fluorescent probes, and discloses a pH-NTR cascade response type nanoprobe, a preparation method and application, the nanoprobe is formed by encapsulating an NTR response type fluorescent probe CyBrN by a liposome self-assembled by a pH response type molecule DCPA, and the nanoprobe can be subjected to structural dissociation in a tumor acidic microenvironment and realize controllable release of probe molecules, so that the pH-NTR cascade response type nanoprobe can be used for detecting tumor tumors. The released probe can be specifically recognized by overexpressed nitroreductase in tumor cells and is subjected to reduction activation, so that a near-infrared fluorescence signal is recovered, and accurate positioning of the tumor cells is realized; meanwhile, the released probe efficiently generates active oxygen under laser irradiation, precise photodynamic killing of tumor cells is achieved, and the probe can be used as a PDT photosensitizer.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to a pH-NTR cascade-responsive nanoprobe, its preparation method, and its application. Background Technology

[0002] Cancer has always been a major disease threatening global human health. Researchers proposed the hypothesis of hypoxic regions within tumors as early as 1955, and subsequent experiments have shown that tumor cells have lower oxygen concentrations than normal cells. Because tumor hypoxia is closely related to the malignant phenotype of tumor cells, resistance to cancer treatment, and mortality rates in cancer patients, the development of effective hypoxia detection methods for cancer treatment and diagnosis is urgently needed. Hypoxia is an important characteristic of the tumor microenvironment, and its detection can predict and treat treatment responses. Currently, the main methods for detecting tumor hypoxia include: histological analysis, oxygen electrode analysis, DNA breakage analysis, nuclear magnetic resonance analysis, nuclear medicine detection methods, and hypoxia probe imaging. Hypoxia probe imaging analysis has significant advantages over the previous four methods due to its high sensitivity and simplicity. Within hypoxic cells, hypoxia probes can be reduced to generate or release fluorescent substances, thus achieving the purpose of detecting hypoxia.

[0003] Nitroreductases (NTRs) are a class of typical hypoxia-related enzymes that reduce aromatic nitro compounds to their corresponding amino derivatives. NTRs are overexpressed under hypoxic conditions and can serve as effective biomarkers of hypoxia. Numerous studies have shown that NTRs are overexpressed in various solid tumor tissues, and their activity levels are closely related to the degree of tumor hypoxia. Based on this characteristic, NTRs have become an important biomarker for hypoxic tumors and an ideal target for achieving tumor-specific diagnosis and controlled treatment.

[0004] Intracellular pH is a crucial cellular parameter, closely related to vital processes such as receptor-mediated signal transduction, enzyme activity, cell proliferation and apoptosis, and ion transport. Under normal physiological conditions, the pH level within human cells is... + The concentration was 40 nmol / L (pH = 7.4), with only slight variations within a very small range (7.35-7.45). Studies have shown that abnormal pH fluctuations are often associated with cellular dysfunction and some diseases, such as kidney poisoning, Alzheimer's disease, emphysema, inflammation, and cancer. The pH range of tumor cells is approximately 5.8–7.7, about 0.5 units lower than that of normal cells. Studies have shown that the acidic environment in tumors is related to the ion transporter Na+. + / H + Exchanger (NHE) and H +The pH of tumor cells is related to processes such as the lactate cotransporter system. Therefore, the acidity of tumor cells differs from that of normal tissues, which can serve as a starting point for targeted drug design and tumor diagnosis. Currently, methods for detecting pH mainly include microelectrode methods, nuclear magnetic resonance (NMR), absorption spectroscopy, and fluorescence spectroscopy. Among these, fluorescence technology has advantages such as high sensitivity, non-invasiveness, and the ability to perform direct in-situ detection, making it a promising technology for pH detection in the intracellular microenvironment.

[0005] In summary, researchers primarily locate tumor cells in the body by exploiting the hypoxia and weakly acidic pH of the cellular microenvironment. However, most reported fluorescent probes can only detect hypoxic environments or pH levels individually, which limits the accuracy of tumor cell localization.

[0006] Photodynamic therapy (PDT) is a minimally invasive treatment method that utilizes the synergistic effects of photosensitizers, specific wavelengths of light, and oxygen to generate reactive oxygen species (ROS) through photochemical reactions, thereby killing diseased tissues (such as tumor cells and precancerous lesions). PDT has shown great potential in cancer treatment due to its advantages of being non-invasive, precisely targeted, and highly reproducible. However, traditional photosensitizers generally suffer from poor photostability, insufficient targeting, and decreased efficacy in the hypoxic tumor microenvironment, limiting the widespread application of PDT in the clinical treatment of solid tumors. Summary of the Invention

[0007] To address the problems mentioned in the background art, this invention employs a strategy of encapsulating near-infrared photosensitizing molecules with pH-responsive liposomes to provide a pH-NTR cascade-responsive nanoprobe. This nanoprobe can undergo structural dissociation in the acidic microenvironment of tumors, enabling the controlled release of probe molecules. The released probe can be specifically recognized and reactivated by overexpressed nitroreductases (NTRs) in tumor cells, thereby restoring the near-infrared fluorescence signal and achieving precise localization of tumor cells. Simultaneously, the released probe efficiently generates reactive oxygen species (ROS) under laser irradiation, achieving precise photodynamic killing of tumor cells, and can be used as a PDT photosensitizer.

[0008] To achieve the above-mentioned technical effects, the present invention provides the following technical solution.

[0009] In a first aspect, the present invention provides a pH-NTR cascade-responsive nanoprobe, which is composed of a liposome encapsulating an NTR-responsive fluorescent probe of the structure shown in formula (II) through self-assembly of a pH-responsive molecule of formula (I).

[0010] (I);

[0011] (II).

[0012] The NTR-responsive fluorescent probe provided by this invention uses a hemicyanine-based near-infrared photosensitive molecule as its core structure. A p-nitrobenzyl protecting group is introduced at the end of the probe molecule to achieve specific recognition and controllable activation of nitroreductase (NTR). Then, a pH-responsive molecule self-assembles into liposomes to encapsulate the NTR-responsive fluorescent probe. The liposomes can sense changes in the pH value of the surrounding environment and undergo structural changes, thereby specifically releasing the encapsulated probe in the acidic tumor microenvironment.

[0013] Furthermore, the diameter of the pH-NTR cascade-responsive nanoprobe is ≤130 nm. Nanoprobes in this size range can achieve passive targeted accumulation in tumor tissue through the EPR effect (Enhanced Permeability and Retention Effect), which is beneficial for increasing the accumulation and retention time of the probe at the tumor site. Simultaneously, they can more effectively penetrate tumor tissue and reduce in vivo clearance, thus improving the efficacy of photodynamic therapy.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned pH-NTR cascade-responsive nanoprobe, which includes the following steps: Step 1: Prepare the NTR-responsive fluorescent probe CyBrN with the structure shown in formula (II) according to the synthetic route shown in reaction formula (III); specifically, IR780 iodide and 5-bromo-1,3-benzenediol undergo a nucleophilic substitution reaction in the presence of potassium carbonate to obtain compound CyBr, and compound CyBr and 4-nitrobenzyl bromide undergo a nucleophilic substitution reaction in the presence of potassium carbonate to obtain the NTR-responsive fluorescent probe CyBrN with the structure shown in formula (II);

[0015] (III); IR780 iodide is a cationic cyanine dye. By undergoing a nucleophilic substitution reaction with 5-bromo-1,3-benzenediol, the 5-bromo-1,3-benzenediol structural unit is linked to the IR780 backbone to obtain compound CyBr, which provides a phenolic hydroxyl platform for further reactions of IR780. Then, compound CyBr undergoes a nucleophilic substitution reaction with 4-nitrobenzyl bromide to introduce a p-nitrobenzyl protecting group at the end of the molecule, so as to achieve specific recognition and controllable activation of nitroreductase (NTR).

[0016] Step 2: Prepare the pH-responsive molecule DCPA with the structure shown in formula (I) according to the synthetic route shown in reaction formula (IV); specifically, Boc-glutaric acid and hexadecanoic acid undergo esterification reaction under the action of EDCI and DMAP to obtain compound DC-1. Compound DC-1 undergoes acid deprotection reaction with trifluoroacetic acid and then undergoes amidation reaction with isonicotinic acid under the action of EDCI, HOBt and DIPEA to obtain compound DC-2. Compound DC-2 undergoes substitution reaction with bromoacetic acid to obtain the pH-responsive liposome DCPA with the structure shown in formula (I);

[0017] (Ⅳ); In the above synthetic route of DCPA, EDCI is a condensing agent, whose full name is (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride); DMAP is an acylation reaction catalyst, whose full name is 4-dimethylaminopyridine; HOBt is an activated ester catalyst, whose full name is 1-hydroxybenzotriazole; DIPEA is an organic base used to neutralize the acid produced in the reaction, whose full name is N,N-diisopropylethylamine.

[0018] The above-mentioned synthetic route of DCPA starts from Boc-glutaric acid and gradually introduces hydrophobic chains, active linkers and aromatic ring skeletons in the following four steps, finally obtaining a bifunctional amphiphilic molecule with an ingenious structure. Due to the definite amphiphilicity of this molecule, it can form a vesicle nanostructure to achieve the encapsulation of core material.

[0019] (1) Esterification: Connected to hexadecyl alcohol, introducing a hydrophobic tail chain; (2) Deprotection: Removing the Boc group to expose the amino group; (3) Amide formation: ligation with isonicotinic acid to introduce a pyridine ring head group; (4) Alkylation: Quaternization of the pyridine ring introduces a carboxylate group to form a zwitterion head group.

[0020] Because the head group of the synthesized DCPA is a quaternized isonicotinamide (pyridinium ion) with an additional carboxylate group, it is an amphoteric ion. In neutral or alkaline environments (such as blood pH = 7.4), its head group maintains its amphoteric form, exhibiting specific polarity and hydration. When the environmental pH is significantly decreased (such as entering the tumor microenvironment pH = 6.5), the carboxylate group (-COO) on the head group... - The molecules are protonated and transformed into neutral carboxyl groups (-COOH). This change drastically alters the polarity, hydration capacity, and electrostatic interactions of the molecular head, thereby triggering a change in membrane structure and enabling pH-triggered probe release.

[0021] Step 3: pH-responsive molecule DCPA self-assembles liposomes to encapsulate NTR-responsive fluorescent probe CyBrN to obtain the pH-NTR cascade-responsive nanoprobe.

[0022] Further, the operation method of step 1 is as follows: 5-bromo-1,3-benzenediol and potassium carbonate are dissolved in acetonitrile (ACN, a polar aprotic solvent) and stirred at 20-25°C under a nitrogen atmosphere for 30 min. Then, a dichloromethane solution containing IR780 iodide is added and heated at 50°C for 4 h. After solvent removal (removal by vacuum evaporation) and purification by silica gel column chromatography (using dichloromethane and methanol as eluents), compound CyBr is obtained. Compound CyBr, 4-nitrobenzyl bromide and potassium carbonate are dissolved in acetonitrile and heated at 50°C for 4 h. After solvent removal (removal by vacuum evaporation) and purification by silica gel column chromatography (using dichloromethane and methanol as eluents), NTR-responsive fluorescent probe CyBrN with the structure shown in formula (II) is obtained.

[0023] Further, the operation method of step 2 is as follows: Boc-glutaric acid and hexadecyl alcohol are dissolved in dichloromethane (DCM), EDCI and DMAP are added, and the mixture is stirred at 20-25℃ for 5 h. After solvent removal (evaporation under reduced pressure) and purification by silica gel column chromatography (using petroleum ether and ethyl acetate as eluents), compound DC-1 is obtained; compound DC-1 is dissolved in dichloromethane, trifluoroacetic acid (TFA) is added, and after the reaction is complete, the mixture is evaporated to dryness and then dissolved in dichloromethane (DCM). Isonicotinic acid, EDCI, HOBt and DIPEA are added, and the mixture is reacted at 20-25℃ for 5 h. After solvent removal (evaporation under reduced pressure) and purification by silica gel column chromatography (using petroleum ether and ethyl acetate as eluents), compound DC-2 is obtained; compound DC-2 and bromoacetic acid are dissolved in acetonitrile (ACN) and refluxed at 80℃ for 8 h. h, after solvent removal (removal by vacuum evaporation) and silica gel column chromatography purification (using dichloromethane and methanol as eluents), yielded pH-responsive liposomes DCPA with the structure shown in formula (Ⅰ).

[0024] Furthermore, the encapsulation method in step 3 employs a co-solvent method, and ultrasonic treatment is used to ensure that the diameter of the pH-NTR cascade-responsive nanoprobe is ≤130 nm. Even further, the operation method in step 3 is as follows: the NTR-responsive fluorescent probe prepared in step 1 and the pH-responsive liposome DCPA prepared in step 2 are added to THF solvent and mixed evenly to obtain a mixed solution. The mixed solution is ultrasonically treated for 10 min and then injected into deionized water. The THF solvent is removed by vacuum evaporation and stirred filtration. Further, the ultrasonic frequency for the ultrasonic treatment is 20 kHz, and the ultrasonic power is 65 W; the vacuum evaporation is performed using a rotary evaporator, with the water bath temperature set to 50℃ and the rotation speed to 80 rpm; the stirred filtration is performed using a 30 K ultrafiltration tube, centrifuged at 3500 rpm for 20 min.

[0025] Thirdly, the present invention provides the application of the above-mentioned pH-NTR cascade-responsive nanoprobe in the preparation of tumor detection reagents.

[0026] Fourthly, the present invention provides the application of the above-mentioned pH-NTR cascade-responsive nanoprobe in the preparation of PDT photosensitizers.

[0027] The pH-NTR cascade-responsive nanoprobe provided by this invention has good biocompatibility and stability. It is designed by encapsulating NTR-responsive fluorescent probes with pH-sensitive liposomes, which can achieve precise release of the probes in the acidic microenvironment of tumors. The released NTR-responsive fluorescent probes have near-infrared fluorescence emission optical properties and exhibit excellent selectivity and sensitivity to NTR. The probes can be specifically reduced under the action of NTR, resulting in enhanced fluorescence signals and activation of photosensitivity, thereby realizing specific imaging and photodynamic therapy of tumor tissues.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The nanoprobe provided by the present invention greatly improves the accuracy of tumor cell imaging through the combined effect of pH and NTR; (2) The pH-NTR cascade response mechanism constructed by the nanoprobe provided by the present invention can specifically release near-infrared photosensitive molecules in the acidic microenvironment of tumors, thereby reducing the phototoxic reaction of normal tissues; the released probe efficiently generates reactive oxygen species (ROS) under laser irradiation, achieving precise photodynamic killing of tumor cells, and can be used as a PDT photosensitizer.

[0029] (3) The nanoprobes provided by the present invention have an average particle size of no more than 130 nm, and can achieve passive enrichment at the tumor site through the EPR effect, thereby further improving the efficiency and imaging sensitivity of photodynamic therapy. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0031] Figure 1 The 1H NMR spectrum of CyBrN, an NTR-responsive fluorescent probe prepared in Example 1; Figure 2 The 1H NMR spectrum of the pH-responsive molecule DCPA prepared in Example 2; Figure 3 The fluorescence spectra of the probe CyBrN prepared in Example 1 after reaction with different concentrations of nitroreductase are shown. Figure 4 The graph shows the linear response of the probe CyBrN prepared in Example 1 to different concentrations of NTR in terms of fluorescence intensity. Figure 5 The selectivity diagram of the probe CyBrN prepared in Example 1; Figure 6 A schematic diagram of the particle size distribution of the pH-NTR cascade-responsive nanoprobe CyBrN NPs prepared in Example 4; Figure 7 The curve showing the change in particle size of the CyBrN NPs nanoprobes prepared in Example 4 over storage time; Figure 8 The diagram shows the in vitro release behavior of the CyBrN NPs nanoprobes prepared in Example 4; Figure 9 The bar chart shows the MTT test results of the probe CyBrN prepared in Example 1 and the probe CyBrN NPs prepared in Example 4 under normal oxygen and hypoxia conditions, after laser treatment and without laser treatment. Figure 10 Live and dead cell staining images of probe CyBrN prepared in Example 1 and probe CyBrN NPs prepared in Example 4. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the reagents and equipment used in the following examples can be purchased through legitimate commercial channels.

[0034] The IR780 iodide used in the following examples has the CAS number 207399-07-3 and is manufactured by Bid Pharmaceuticals. Its structural formula is shown in formula (V).

[0035]

[0036] (V).

[0037] Example 1: Preparation of NTR-responsive fluorescent probe CyBrN The preparation method is as follows: (1) Synthesis of compound CyBr: 5-bromo-1,3-benzenediol (189 mg, 1 mmol) and K2CO3 (138 mg, 1 mmol) were placed in a flask containing ACN (5 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 30 min. IR780 (333 mg, 0.5 mmol) from CH2Cl2 (3 mL) was introduced into the mixture using a syringe, and the reaction mixture was heated at 50 °C for 4 h. The solvent was removed by vacuum evaporation, and the compound CyBr (184 mg, 75%) was obtained by silica gel column chromatography (eluent: CH2Cl2 / MeOH = 10:1, v / v).

[0038] (2) Synthesis of compound CyBrN: CyBr (310 mg, 0.5 mmol) and K2CO3 (103 mg, 0.75 mmol) were placed in a flask containing ACN (5 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 15 min. 4-Nitrobenzyl bromide (108 mg, 0.5 mmol) from ACN (2 mL) was introduced into the mixture using a syringe, and the reaction mixture was heated at 50 °C for 4 h. CyBrN (150 mg, 40%) was obtained by silica gel column chromatography (eluent: CH2Cl2 / MeOH = 8:1, v / v).

[0039] The room temperature mentioned above is the laboratory temperature, 20-25℃.

[0040] The above synthesis reaction process is shown in reaction formula (Ⅲ).

[0041]

[0042] (III).

[0043] The compound CyBrN prepared above was analyzed by NMR, and the results are shown in [reference needed]. Figure 1 As shown, the specific data is as follows: 1 H NMR (600 MHz, CD3OD) 8.72 (d, J = 15.1 Hz, 1H), 8.30 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 7.3 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 32.4 Hz, 2H), 7.41 (s, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.09(d, J = 2.4 Hz, 1H), 6.64 (d, J = 15.1 Hz, 1H), 5.40 (s, 2H), 4.41 (t, J = 7.4Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.72 (t, J = 6.2 Hz, 2H), 1.99 – 1.93 (m,4H), 1.83 (s, 6H), 1.09 (t, J = 7.4 Hz, 3H).

[0044] from Figure 1 As can be seen from the above data, the NMR analysis results of the NTR-responsive fluorescent probe CyBrN prepared in Example 1 are consistent with the theoretical results of the structure shown in Formula (II).

[0045] Example 2: Preparation of pH-responsive molecular DCPA The preparation method is as follows: (1) Synthesis of compound DC-1: Boc-glutaric acid (1.00 g, 4.04 mmol) and hexadecyl alcohol (1.96 g, 8.08 mmol) were dissolved in DCM (30 mL), and EDCI (1.54 g, 8.08 mmol) and DMAP (0.25 g, 2.02 mmol) were added. After stirring at room temperature for 5 h, the solvent was removed by vacuum evaporation, and compound DC-1 (2.11 g, 75%) was purified by silica gel column chromatography (PE / EA = 8:1, v / v).

[0046] (2) Synthesis of compound DC-2: Compound DC-1 (2 g, 2.87 mmol) was dissolved in DCM (20 mL), and 5 mL of trifluoroacetic acid was added. After the reaction was completed, the solution was evaporated to dryness and dissolved in DCM (30 mL). Isonicotinic acid (0.32 g, 2.58 mmol), EDCI (0.66 g, 3.44 mmol), HOBt (0.64 g, 3.44 mmol) and DIPEA (0.89 g, 6.88 mmol) were added. The reaction was carried out at room temperature for 5 h. The solvent was removed by vacuum evaporation and the compound DC-2 (0.72 g, 60%) was purified by silica gel column chromatography (PE / EA = 1:1, v / v).

[0047] (3) Synthesis of compound DCPA: Compound DC-2 (0.50 g, 0.71 mmol) and bromoacetic acid (0.26 g, 1.92 mmol) were placed in a flask containing ACN (10 mL) and refluxed at 80 °C for 8 h. The solvent was removed by vacuum evaporation and purified by silica gel column chromatography (CH2Cl2 / MeOH = 15:1, v / v) to obtain compound DCPA (0.32 g, 60%).

[0048] The room temperature mentioned above is the laboratory temperature, 20-25℃.

[0049] The above synthesis reaction process is shown in reaction formula (Ⅳ).

[0050]

[0051] (Ⅳ).

[0052] The compound DCPA prepared above was analyzed by NMR, and the results are shown in [reference needed]. Figure 2 As shown, the specific data is as follows: 1H NMR (600 MHz, CDCl3) δ 9.42 (d, J = 7.3 Hz, 1H), 9.21 (d, J = 6.2Hz, 2H), 8.66 (d, J = 6.2 Hz, 2H), 4.66 (d, J = 7.6 Hz, 1H), 4.64 (s, 2H), 4.12 (t, J = 6.8 Hz, 2H), 4.04 (t, J = 6.8 Hz, 2H), 2.55 (t, J = 7.0 Hz, 2H), 2.43 – 2.33 (m, 2H), 1.62 (dt, J = 31.2, 7.2 Hz, 4H), 1.25 (d, J = 3.5 Hz, 52H), 0.87 (t, J = 6.9 Hz, 6H).

[0053] from Figure 2 As can be seen from the above data, the NMR analysis results of the pH-responsive molecule DCPA prepared in Example 2 are consistent with the theoretical results of the structure shown in Formula (I).

[0054] Example 3: Fluorescence spectroscopy and selectivity experiment of CyBrN in response to nitroreductase The nitroreductase-responsive fluorescent probe CyBrN prepared in Example 1 was subjected to fluorescence spectroscopy analysis to obtain the fluorescence spectra of CyBrN reacting with different concentrations of nitroreductase.

[0055] Figure 3 The fluorescence spectra of the interaction between CyBrN and NTR were shown. The fluorescent probe concentration was 5 μM, and the NTR concentrations were 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μg / mL, respectively. The PBS buffer was pH 7.4 and contained 250 μM NADH. The fluorescence intensity was measured using a fluorometer (Ex / Em = 660 / 710 nm). The results showed that the probe was almost non-fluorescent without NTR; after the addition of NTR, a significant near-infrared emission peak appeared at 710 nm. The mechanism may be that in the presence of the coenzyme NADH, NTR reduces the nitro group of the probe molecule to hydroxylamine or amino groups, followed by 1,6-rearrangement and elimination reactions, releasing a fluorophore and thus producing near-infrared fluorescence. The fluorescence intensity of the probe increased with increasing NTR concentration, and the fluorescence intensity increased by about 5 times at 0.6 μg / mL, which can be used for NTR detection.

[0056] Figure 4 The fluorescence intensity showed a good linear relationship with NTR concentration, demonstrating a linear relationship between CyBrN and NTR concentration (R0). 2=0.9963), and the detection limit is 30 ng / mL, indicating that the probe can detect NTR with high sensitivity and reliability.

[0057] The CyBrN solution prepared in Example 1 was diluted to 5 µM with PBS buffer (pH = 7.4, containing 250 μM NADH) for later use. The diluted CyBrN solution was divided into 22 groups (3 mL per group), with group 1 serving as a blank and the other 20 groups containing the interfering agent (Na2+). + K + Ca 2+ Mg 2+ Zn 2+ OH•, NO2 - Cl - SO4 2- The concentrations of GSH, Cys, Arg, Glu, Hcy, Lys, CE, NQO1, NADH, ALP, and APN were 50-500 μM. NTR (0.6 μg / mL) was added to group 22. The fluorescence intensity was measured using a fluorometer (Ex / Em = 660 / 710 nm). The results are shown in [link to results]. Figure 5 As shown.

[0058] Figure 5 The fluorescence changes in the solution after the addition of other interfering substances were not significant, but the fluorescence was significantly enhanced after the addition of NTR. This indicates that the nitroreductase-responsive probe CyBrN provided by this invention exhibits good selectivity for other possible interfering substances in the biological system and only specifically responds to NTR.

[0059] Example 4: Preparation of pH-NTR cascade-responsive nanoprobes CyBrN NPs (1) The preparation method is as follows: A THF mixture (1 mL) containing CyBrN (prepared in Example 1, 1 mg / mL) and DCPA (prepared in Example 2, 10 mg / mL) was continuously and vigorously sonicated at 65 W and then rapidly injected into 10 mL of distilled deionized water. After sonicating the mixture for 5 min, THF was slowly removed using a rotary evaporator (50°C water bath, 80 rpm) to obtain a coarse nanoparticle suspension. This suspension was first filtered through a 0.45 μm microporous membrane to remove larger aggregates, and then centrifuged at 3500 rpm for 20 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa for concentration and buffer replacement, thereby effectively removing residual organic solvents and unencapsulated drugs. Finally, the purified CyBrN NPs solution was concentrated to 1 mg / mL and stored at 4°C protected from light.

[0060] (2) Particle size detection: The particle size of the CyBrN NPs solution was determined using dynamic light scattering (DLS) technology. The results are shown in the figure. Figure 6 As shown. From Figure 6 It can be concluded that the average diameter of the particles in the CyBrN NPs solution prepared in this embodiment is 123.1 nm, the PDI is 0.189, and the particle size distribution is uniform.

[0061] (3) Stability study: See Figure 7 The CyBrN NPs solution prepared in this embodiment maintained a relatively stable particle size over a period of 15 days.

[0062] (4) Calculation of drug loading content (DLC) and encapsulation efficiency (EE): According to the formula Calculate drug loading, formula Calculate the encapsulation efficiency. Where A represents the weight of CyBrN in the probe nanoparticles, B represents the weight of the CyBrN NPs solution, and C represents the weight of the probe CyBrN added to prepare the probe nanoparticles.

[0063] The CyBrN NPs solution was used to establish a standard absorption curve and obtain the A value. The DLC and EE of the CyBrN NPs nanoprobes prepared in this embodiment reached 6.9% and 82%, respectively.

[0064] (5) In vitro release behavior of nanoprobes: One mL of the prepared CyBrN NPs nanoparticles was placed in a dialysis bag (MV = 3500) and immersed in 30 mL of phosphate-buffered saline (PBS, pH = 5.5, 6.5, or 7.4) at a constant temperature of 37°C to simulate the acidic microenvironment of tumors and the normal physiological environment. The system was continuously shaken on a constant speed shaker (100 rpm) to maintain homogeneous mixing of the solution. Samples were taken at predetermined time intervals (0, 1, 2, 4, 8, 12, 16, 24, 32, and 48 h), and the release medium was replenished with an equal volume of fresh PBS. After filtration through a 0.22 μm filter membrane, the probe release was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: a C18 reversed-phase column (4.6 × 250 mm, 5 μm); mobile phase: methanol and water (75:25 v / v); flow rate: 1.0 mL / min. - ¹; The detection wavelength was 600 nm. The cumulative release was calculated based on the standard curve, and release curves were plotted under different pH conditions. See [reference needed]. Figure 8 .

[0065] The results showed that the release rate of the probe was significantly higher at pH 5.5 than at neutral conditions (pH 7.4), indicating that the system has significant acidic pH response characteristics, which is beneficial for the specific release of CyBrN in the tumor microenvironment.

[0066] Example 5: Evaluation of the photodynamic therapy effect of pH-NTR cascade-responsive nanoprobe CyBrN NPs The CyBrN NPs solution prepared in Example 4 was used, and the CyBrN solution prepared in Example 1 (not encapsulated by DCPA) was used as a control. The photodynamic therapy (PDT) effect in in vitro cultured 4T1 cells was evaluated using the MTT assay and Calcein-AM / PI staining method. The experiment was divided into four groups: Control group, CyBrN group, CyBrN NPs group, CyBrN + Light group, and CyBrNNPs + Light group.

[0067] like Figure 9 As shown, under no-light conditions, all groups of cells maintained high viability, indicating that CyBrN and CyBrNNPs have good biocompatibility in both normoxic and hypoxic environments, and their cytotoxicity is negligible. Under normoxic conditions, after laser treatment (660 nm, 30 mW / cm²), the cells were further isolated. 2 After 10 minutes of irradiation, the cell survival rate in the CyBrN group decreased slightly, while the cell survival rate in the CyBrN NPs group decreased significantly, indicating that CyBrN NPs have a stronger photodynamic killing ability. This is because the nanoprobes have higher cellular uptake efficiency and better photostability, thus generating more reactive oxygen species under light irradiation. Under hypoxic conditions, the phototoxicity of the CyBrN NPs group was still significantly higher than that of the CyBrN group, indicating that the nanostructure can effectively promote the accumulation of probes in hypoxic tumor cells and enhance the photodynamic therapy effect through reduction and activation by nitroreductase.

[0068] The cell status was further verified by live / dead cell (Calcein-AM / PI) staining experiments. Figure 10 The results showed that cells in the unilluminated group mainly emitted green fluorescence (indicating survival), while the CyBrN NPs group, after illumination, exhibited significantly enhanced red fluorescence (indicating cell death), with a correspondingly higher proportion of cell death compared to the CyBrN group. This result is consistent with the MTT assay, further demonstrating that CyBrN NPs possess superior photodynamic therapy performance under illumination, highlighting the significant advantages of nanomaterial delivery systems in cell uptake and PDT efficiency.

[0069] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A pH-NTR cascade-responsive nanoprobe, characterized in that: The nanoprobe is composed of a pH-responsive molecule self-assembled into a liposome containing an NTR-responsive fluorescent probe of structure (II). （Ⅰ）； （Ⅱ）。 2. The pH-NTR cascade-responsive nanoprobe according to claim 1, characterized in that: The diameter of the nanoprobe is ≤130 nm.

3. The method for preparing the pH-NTR cascade-responsive nanoprobe according to claim 1 or 2, characterized in that: Includes the following steps: Step 1: Prepare the NTR-responsive fluorescent probe CyBrN with the structure shown in formula (II) according to the synthetic route shown in reaction formula (III): （Ⅲ）； Step 2: Prepare the pH-responsive molecule DCPA with the structure shown in formula (I) according to the synthetic route shown in reaction formula (IV): （Ⅳ）； Step 3: pH-responsive molecule DCPA self-assembles liposomes to encapsulate NTR-responsive fluorescent probe CyBrN to obtain the pH-NTR cascade-responsive nanoprobe.

4. The preparation method according to claim 3, characterized in that: The operation method of step 1 is as follows: 5-bromo-1,3-benzenediol and potassium carbonate are dissolved in acetonitrile and stirred at 20-25℃ under a nitrogen atmosphere for 30 min. Then, a dichloromethane solution containing IR780 iodide is added and heated at 50℃ for 4 h. After solvent removal and silica gel column chromatography purification, compound CyBr is obtained. Compound CyBr, 4-nitrobenzyl bromide and potassium carbonate are dissolved in acetonitrile and heated at 50℃ for 4 h. After solvent removal and silica gel column chromatography purification, NTR-responsive fluorescent probe CyBrN with the structure shown in formula (II) is obtained.

5. The preparation method according to claim 3, characterized in that: The operation method of step 2 is as follows: Boc-glutaric acid and hexadecyl alcohol are dissolved in dichloromethane, EDCI and DMAP are added, and the mixture is stirred at 20-25℃ for 5 h. After solvent removal and silica gel column chromatography purification, compound DC-1 is obtained. Compound DC-1 is dissolved in dichloromethane, trifluoroacetic acid is added, and after the reaction is complete, the mixture is evaporated to dryness and then dissolved in dichloromethane. Isonicotinic acid, EDCI, HOBt and DIPEA are added, and the mixture is reacted at 20-25℃ for 5 h. After solvent removal and silica gel column chromatography purification, compound DC-2 is obtained. Compound DC-2 and bromoacetic acid are dissolved in acetonitrile, and the mixture is refluxed at 80℃ for 8 h. After solvent removal and silica gel column chromatography purification, the pH-responsive molecule DCPA with the structure shown in formula (I) is obtained.

6. The preparation method according to claim 3, characterized in that: The encapsulation method in step 3 employs a co-solvent method, and ultrasonic treatment is used to make the diameter of the pH-NTR cascade-responsive nanoprobe ≤130 nm.

7. The preparation method according to claim 6, characterized in that: The operation method of step 3 is as follows: the NTR-responsive fluorescent probe prepared in step 1 and the pH-responsive molecule DCPA prepared in step 2 are added to THF solvent and mixed evenly to obtain a mixed solution. The mixed solution is ultrasonically treated for 10 min and then injected into deionized water. The THF solvent is removed by vacuum evaporation and stirring filtration.

8. The preparation method according to claim 7, characterized in that: The ultrasonic treatment uses an ultrasonic frequency of 20 kHz and an ultrasonic power of 65 W; the reduced pressure evaporation is performed using a rotary evaporator with a water bath temperature of 50°C and a rotation speed of 80 rpm; the stirred filtration is performed using a 30 K ultrafiltration tube at 3500 rpm for 20 min.

9. The application of the pH-NTR cascade-responsive nanoprobe according to claim 1 or 2, or the pH-NTR cascade-responsive nanoprobe prepared by any one of claims 3 to 8, in the preparation of tumor detection reagents.

10. The application of the pH-NTR cascade-responsive nanoprobe according to claim 1 or 2, or the pH-NTR cascade-responsive nanoprobe prepared by any one of claims 3 to 8, in the preparation of PDT photosensitizers.

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