Cathepsin B activated fluorescent probe, nanoparticles and application

Abstract

The invention discloses a cathepsin B activated fluorescent probe which comprises a dicyanoisosulfone fluorescent report unit and a cathepsin B specific restriction enzyme cutting site GFLG peptide chain. When CTSB specifically cuts a GFLG peptide chain, a DCO molecule with an amino group with relatively strong electron donating ability is generated, so that the fluorescence emission behavior of the probe is rapidly activated through a one-step enzyme catalysis reaction. The invention also provides a preparation method of the cathepsin B activated fluorescent probe. The invention also provides a nano particle for packaging the cathepsin B activated fluorescent probe, and the nano particle is simple in preparation process and mild in reaction condition. The cathepsin B activated fluorescent probe is prepared into nanoparticles, and the nanoparticles can realize accumulation of the material in tumor tissues and accurate imaging in apoptotic tumor cells by enhancing the permeation retention effect, have better biocompatibility and can be used for detecting the apoptotic tumor cells.

Description

Technical Field

[0001] The invention relates to the fields of fluorescent probes and biotechnology, and in particular to a cathepsin B activated fluorescent probe, nanoparticles and applications. Background Art

[0002] Apoptosis is one of the ways to disintegrate and ablate tumor tissues in many tumor treatment methods, including radiotherapy, chemotherapy, phototherapy, hormone therapy, and gene therapy, in order to achieve the purpose of cure. The exploration of apoptosis pathways is related to the timely and accurate evaluation of the effect of tumor treatment. Mitochondria play a core role in apoptosis. They activate Caspase 3 by releasing molecules such as cytochrome c and SMAC, thereby promoting apoptosis. Studies have shown that the pro-apoptotic function of mitochondria is regulated by BCL-2 family proteins. BCL-2 family proteins include pro-apoptotic factors (such as Bid) and anti-apoptotic factors (such as BCL-x), which work together to regulate cell apoptosis and survival. In tumor cells, anti-apoptotic factors are overexpressed, which inhibits the pro-apoptotic function of mitochondria and makes cells more tolerant to apoptosis. During effective treatment, lysosomes selectively release some protein factors (such as proteases, esterases, etc.) into the cytoplasm. Among them, the released cathepsin (Cathepsin, CTS) can reduce the inhibition of anti-apoptotic factors on mitochondrial activation of intracellular apoptotic enzymes and promotion of apoptosis, and plays an important role in the process of apoptosis. Therefore, cathepsins have the potential to detect apoptotic cells. However, due to their overexpression in tumor cell lysosomes, CTS has long been used as a tumor targeting unit and simply plays the role of a biomarker.

[0003] Taking cathepsin B (CTSB) as an example, reference 1 (Ju Hee Ryu, Kwangmeyung Kim. Non-invasive optical imaging of cathepsin B with activatable fluorogenicnanoprobes in various metastatic models. Biomaterials., 35, 2302-2311, 2014, doi: 10.1016 / j.biomaterials.2013.11.080) discloses a new nanoprobe called CB-CNP, which can specifically detect CTSB. The nanoprobe consists of a self-quenching fluorescent peptide probe and tumor-targeted glycol chitosan nanoparticles (CNPs). In the absence of CTSB, the fluorescence of CB-CNP is strongly suppressed, but in the presence of CTSB, CB-CNP emits a strong fluorescent signal due to its specific cleavage of the peptide probe. The nanoprobe is used as a non-invasive molecular imaging tool for detecting and depicting metastatic tumors.

[0004] Reference 2 (Ji Young Yhee, Kwangmeyung Kim.Optical Imaging of Cancer-Related Proteases Using Near-Infrared Fluorescence Matrix Metalloproteinase-Sensitive and Cathepsin B-Sensitive Probes.Theranostics, 2, 179-189, 2012, doi: 10.7150 / thno.3716) discloses an imaging nanoprobe based on polymer nanoparticles for detecting CTSB and MMP, two cancer-related enzymes. The protease-sensitive peptide sequence contained in the probe ensures its specificity. The CTSB probe can restore fluorescence after entering the cytoplasm, which helps to evaluate the cytoplasmic targeted delivery effect of nanoparticles in vivo. The MMP probe shows higher specificity in in vivo and tissue studies. This study provides accurate information for peptide-based optical probes, which helps their application in cancer diagnosis.

[0005] In these studies, researchers developed fluorescent probes that specifically target the enzyme and applied them to tumor detection. These probes can accurately identify and track tumor cells, providing an effective tool for early diagnosis and treatment of cancer. However, at present, there is a lack of CTSB fluorescent probes for detecting the results of tumor cell apoptosis. This also suggests that the development of new probes to monitor the role of CTSB in the process of cell apoptosis may provide new perspectives and methods for cancer treatment. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a cathepsin B-activated fluorescent probe, which achieves high-precision imaging of tumor cell apoptosis by responding to the specific activation of cathepsin B.

[0007] A cathepsin B activated fluorescent probe comprises a dicyanoisosulfone fluorescent reporter unit and a cathepsin B specific enzyme cleavage site GFLG peptide chain, and its structure is as follows:

[0008]

[0009] In the present invention, a cathepsin B activated fluorescent probe (A-DCO) is composed of a dicyanoisosulfone (DCO) fluorescent reporter unit and a cathepsin B (CTSB) specific cleavage site Gly-Phe-Leu-Gly (GFLG) peptide chain. The formation of an amide bond weakens the electron-donating ability of the DCO amino group, so that the fluorescence emission behavior of A-DCO remains "off", and A-DCO does not emit light in the initial state. Figure 1 As shown, when CTSB specifically cuts the GFLG polypeptide chain, a DCO molecule with an amino group with strong electron-donating ability is generated, so that the fluorescence emission behavior of the probe is rapidly activated through a one-step enzyme-catalyzed reaction.

[0010] The present invention also provides a method for preparing the above-mentioned cathepsin B activated fluorescent probe. The preparation method is simple, and A-DCO is obtained by subjecting the synthesized DCO to an amidation reaction with the GFLG peptide chain.

[0011] A method for preparing a cathepsin B activated fluorescent probe comprises the following steps:

[0012] (1) adding isophorone and malononitrile under nitrogen protection, then adding sodium acetate as a catalyst and ethanol as a solvent to obtain a solution, stirring and heating to reflux reaction, and after the reaction is completed, extracting and purifying to obtain compound 1;

[0013] (2) dissolving the compound 1 obtained in step (1) and p-acetamidobenzaldehyde in toluene, adding piperidine as a catalyst, and after the reaction is completed, concentrating to remove toluene, and washing with acetonitrile to obtain compound 2;

[0014] (3) dissolving the compound 2 obtained in step (2) in ethanol, then adding concentrated hydrochloric acid, heating and stirring the mixture, and after the reaction is completed, treating the mixture to obtain the compound DCO;

[0015] (4) The compound DCO obtained in step (3) is reacted with the GFLG peptide chain to synthesize A-DCO through an amidation reaction.

[0016] The specific synthetic route is as follows:

[0017]

[0018] The present invention also provides a nanoparticle encapsulating the above-mentioned cathepsin B activated fluorescent probe, and the nanoparticle can realize the accumulation of materials in tumor tissues and the precise imaging in apoptotic tumor cells by utilizing the enhanced permeation retention effect.

[0019] A nanoparticle encapsulating a cathepsin B activated fluorescent probe comprises an amphiphilic polymer and the cathepsin B activated fluorescent probe, wherein the cathepsin B activated fluorescent probe is wrapped on the amphiphilic polymer.

[0020] By taking advantage of the hydrophilic and hydrophobic properties of amphiphilic polymers, A-DCO can be self-assembled in aqueous solution to form nanoparticles after being wrapped on the amphiphilic polymers. The nanoparticles can achieve the accumulation of materials in tumor tissues and accurate imaging in apoptotic tumor cells by utilizing the enhanced permeability retention effect, and have good biocompatibility and can be used for the detection of apoptotic tumor cells.

[0021] Preferably, the amphiphilic polymer is a block copolymer containing polyoxyethylene and polyoxypropylene.

[0022] The polyoxypropylene (PPO) block exhibits hydrophobic properties in aqueous solution, while the polyoxyethylene (PEO) block is water-affinity, making the entire molecule amphiphilic. This amphiphilic polymer can self-assemble into nanoparticles in water.

[0023] More preferably, the block copolymer containing polyoxyethylene and polyoxypropylene is F127.

[0024] F127 is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene. The polyoxypropylene block in the middle is hydrophobic, and the polyoxyethylene blocks at both ends are hydrophilic. It is an amphiphilic polymer.

[0025] Preferably, the mass ratio of the amphiphilic polymer to the cathepsin B-activated fluorescent probe is 15-20:1.

[0026] Preferably, the amphiphilic polymer can effectively encapsulate the A-DCO molecules, thereby significantly enhancing the solubility of A-DCO in water.

[0027] The present invention also provides a method for preparing the nanoparticles. The A-DCO molecules are prepared into nanoparticles by a nano-coprecipitation method. The method only requires one step and is simple to prepare.

[0028] A method for preparing nanoparticles encapsulating a cathepsin B activated fluorescent probe comprises the following steps: dissolving A-DCO in an organic solvent, adding the solution dropwise to an amphiphilic polymer solution, and simultaneously performing ultrasound to obtain a uniform dispersion, filtering the dispersion to remove impurities, and freeze-drying the dispersion to obtain nanoparticles encapsulating the cathepsin B activated fluorescent probe.

[0029] Preferably, the organic solvent is tetrahydrofuran, acetonitrile or chloroform.

[0030] More preferably, the organic solvent is tetrahydrofuran, and the solution of the amphiphilic polymer is an aqueous solution of the amphiphilic polymer F127.

[0031] Preferably, the amphiphilic polymer F127 is rapidly mixed with an organic solvent and water to supersaturate the organic solvent, thereby promoting the formation and growth of hydrophobic nanoparticles. The prepared nanoparticles have a narrow particle size range and a relatively uniform morphology.

[0032] The present invention also provides the use of the nanoparticles encapsulating the cathepsin B activated fluorescent probe in detecting apoptotic tumor cells.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The cathepsin B-activated fluorescent probe provided by the present invention can achieve high-precision imaging of tumor cell apoptosis by responding to the specific activation of cathepsin B.

[0035] (2) The present invention obtains a nanoparticle encapsulating a cathepsin B-activated fluorescent probe, the preparation process of which is simple and the reaction conditions are mild.

[0036] (3) The cathepsin B-activated fluorescent probe is made into nanoparticles, which can utilize the enhanced permeability retention effect to achieve the accumulation of materials in tumor tissues and precise imaging in apoptotic tumor cells. The nanoparticles have good biocompatibility and can be used for the detection of apoptotic tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the principle of cathepsin B-activated fluorescent probe (A-DCO).

[0038] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of compound 1 obtained in Example 1.

[0039] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of compound 2 obtained in Example 1.

[0040] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the compound DCO obtained in Example 1.

[0041] Figure 5 This is the nuclear magnetic resonance hydrogen spectrum of the cathepsin B activated fluorescent probe obtained in Example 1.

[0042] Figure 6 This is the carbon nuclear magnetic resonance spectrum of the cathepsin B activated fluorescent probe obtained in Example 1.

[0043] Figure 7 This is the mass spectrometry of the cathepsin B activated fluorescent probe obtained in Example 1.

[0044] Figure 8 The optical properties of the nanoparticles (A-DCONPs) encapsulating the cathepsin B activated fluorescent probe prepared in Example 2 are characterized as follows:

[0045] (a) is the ultraviolet absorption spectra of the cathepsin B activated fluorescent probe prepared in Example 1 and the A-DCONPs prepared in Example 2;

[0046] (b) Fluorescence emission spectra of A-DCO group, A-DCO NPs+CTSB group and DCO group;

[0047] (c) and (d) are the absorption spectra and standard curves of A-DCO NPs with different concentrations, respectively.

[0048] Fig. 9 is the characterization result of A-DCO NPs, where

[0049] (a) is the particle size stability test diagram of A-DCO NPs;

[0050] (b) Zeta potential diagram of A-DCO and F127;

[0051] (c) Transmission electron microscopy image of A-DCO NPs.

[0052] Fig.10 This is the fluorescence detection diagram of ADO on L929 cells and 4T1 cells, where:

[0053] (a) Fluorescence images of L929 cells and 4T1 cells cultured with 1 μM A-DCO for 0.5 h, 2 h, and 3 h;

[0054] (b) is the fluorescence intensity statistics of (a).

[0055] Fig.11 Figure 2 is the fluorescence detection diagram of ADO NPs on L929 cells and 4T1 cells, where:

[0056] (a) Fluorescence images of 1 μM A-DCO NPs cultured with L929 cells and 4T1 cells for 0.5 h, 2 h, and 3 h.

[0057] (b) is the fluorescence intensity statistics of (a).

[0058] Fig.12 In vitro release curves of A-DCO NPs at pH 6.5 and 4.7.

[0059] Fig.13 Fluorescence images of 4T1 cells and MCF-7 cells incubated with serum-free culture medium mixed with A-DCO NPs and DOX (scale bar: 200 μm).

[0060] Fig.14 Laser confocal microscopy images of A-DCO NPs localization in lysosomes of 4T1 cells (scale bar, 10 μm).

[0061] Fig.15 Laser confocal microscopy images of A-DCO NPs localized in the lysosomes of 4T1 cells and MCF-7 cells (the scale bar in figure a is 50 μm, and the scale bar in figure b is 10 μm).

[0062] Fig.16 Fluorescence images of MCF-7ADR cells incubated with serum-free culture medium containing A-DCO NPs mixed with DOX and LCL161 (scale bar: 200 μm).

[0063] Fig.17 This is the detection diagram of cell apoptosis induced by A-DCO NPs under co-incubation of DOX, IR1048 and LCL161, where:

[0064] (a)-(c) are flow cytometric histograms of A-DCO NPs under the action of DOX, IR1048 and LCL161, respectively;

[0065] (d) is the apoptosis diagram of IR1048 at different concentrations;

[0066] (e) is the linear regression curve of the fluorescence intensity of A-DCO NPs changing with the apoptosis rate. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.

[0068] The raw materials used in the present invention are all commercially available.

[0069] Among them, the amphiphilic polymer F127 used in the embodiment was purchased from Anaiji Chemical, and its structural formula is Among them, x=100, y=60.

[0070] Example 1: Preparation of Cathepsin B Activated Fluorescent Probe

[0071] (1) Preparation of Compound 1

[0072] Isophorone (8 mL, 53.4 mmol) and malononitrile (3.5 mL, 55.6 mmol) were added to a double-necked flask protected by nitrogen, and then 30 mL of ethanol and 3 g of sodium acetate were added, and refluxed and stirred at 50 ° C for 12 hours. After the reaction was completed, solid impurities were removed by filtration, and then ethanol was removed by vacuum distillation, and the organic phase was extracted with dichloromethane and water. The crude product was further purified by gel chromatography (PE / CH2Cl2, 10:1, v / v) to obtain a white solid compound 1 (6.79 g, 73%). Using deuterated chloroform as solvent, the nuclear magnetic resonance hydrogen spectrum of compound 1 was tested as follows Figure 2 As shown, 1 H NMR (500MHz, CDCl3, 298K) δ (ppm): 6.62 (s, 1H), 2.51 (s, 2H), 2.17 (s, 2H), 2.02 (s, 3H), 1.01 (s, 6H).

[0073] (2) Preparation of Compound 2

[0074] Compound 1 (6.13 g, 32.91 mmol) and 4-acetamidobenzaldehyde (5.37 g, 32.91 mmol) were dissolved in toluene. Then 0.1 mL of piperidine was added as a catalyst. The reaction mixture was stirred at 120°C for 3 hours. After the reaction was completed, it was washed three times with acetonitrile and filtered. 5.69 g of pure orange-red product was obtained with a yield of 85.82%. Using deuterated DMSO as solvent, the H NMR spectrum of compound 2 was obtained as shown in the following figure: Figure 3 As shown, 1H NMR (500MHz, DMSO, 298K) δ (ppm): 10.12 (s, 1H), 7.63 (d, J = 4.7Hz, 4H), 7.27 (d, J = 18.7Hz, 2H), 6.84 (s, 1H), 2.61 (s, 2H), 2.54 (s, 2H), 1.02 (s, 6H).

[0075] (3) Preparation of compound DCO

[0076] Compound 2 (5 g, 15.09 mmol) was dissolved in 40 mL of ethanol, and then 30 mL of concentrated hydrochloric acid (18 mol / L) was added. The reaction mixture was stirred at 105 °C for 10 hours. Part of the reaction mixture was transferred to a separation funnel, and then water and sodium bicarbonate were added until there were no bubbles. Ethyl acetate was added for extraction, anhydrous magnesium sulfate was added to remove moisture, and the upper layer of the extract was dried on a rotary evaporator. The extraction mixture was purified by gel chromatography (petroleum ether / ethyl acetate = 1:3, v / v) to obtain a red-black product (3.81 g, 62.5%). Using deuterated chloroform as solvent, the nuclear magnetic resonance hydrogen spectrum of dicyanoisosulfone DCO was tested as shown in the following figure Figure 4 As shown; 1 H NMR (500MHz, CDCl3) δ (ppm): 7.60 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.14 (d, J = 8.0 Hz, 2H), 2.50 (s, 1H).

[0077] (4) Preparation of A-DCO

[0078] DCO (2.89 g, 10 mmol), GFLG peptide chain (5.65 g, 15 mmol), 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (11.4 g, 30 mmol) and N,N-diisopropylethylamine (3.88 g, 30 mmol) were dissolved in 50 mL of anhydrous N,N-dimethylformamide, stirred at room temperature for 8 hours, extracted with ethyl acetate, then washed with water 3 times, and then anhydrous magnesium sulfate was added to remove moisture, and the upper layer of the extract was dried by rotary evaporator. The extraction mixture was purified by gel chromatography (petroleum ether / ethyl acetate = 1:3, v / v) to obtain a red product A-DCO (2.13 g, 32%). The A-DCO nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, and mass spectrum were tested as shown below: Figure 5-7 shown. 1H NMR (500MHz, DMSO-d6, 298K) δ (ppm): 10.42 (s, 1H), 10.35 (s, 1H), 8.87 (d, J = 7. 9Hz,1H),7.69(d,J=9.6Hz,4H),7.63(s,1H),7.24(s,5H),6.86(s,1H),6.75(s, 1H),6.39(d,J=13.3Hz,1H),4.50(s,1H),4.11(s,1H),3.14(s,3H),2.97(s,2H) ,1.76(s,2H),1.62(s,4H),1.47(s,2H),1.24(s,3H),1.03(s,6H),0.91(s,3H). 13 C NMR (126MHz, DMSO-d6, 298K) δ (ppm): 170.75, 170.30, 168.48, 157.40, 156.59, 140.46, 139.35, 137.79, 135.18, 131.71, 129.99, 129.16, 128.8 8,127.52,122.77,119.86,119.33,114.42,113.61,77.42,76.26,53.6 5,40.80,38.63,37.39,33.79,32.10,27.89,25.63.ESI-HRMS[A-DCO+H] + :calcd.for[C 38 H 44 N6O5] + 664.34, found 664.80.

[0079] Example 2: Preparation of Nanoparticles Encapsulating Cathepsin B Activated Fluorescent Probe

[0080] A-DCO (2 mg) and amphiphilic polymer F127 (30 mg) prepared in Example 1 were dissolved in 1.5 mL tetrahydrofuran and 20 mL deionized water, respectively, and dispersed evenly by ultrasound. The tetrahydrofuran solution of A-DCO was then added dropwise to the F127 aqueous solution, and ultrasound with a power of 26 W was used to promote the folding and dispersion of the droplets to obtain a uniformly dispersed nanoparticle dispersion; finally, a water filter was used to filter out some impurities, and the obtained solution was placed in a freeze dryer to dry, thereby obtaining nanoparticles encapsulating the cathepsin B activated fluorescent probe (A-DCO NPs, referred to as NPs).

[0081] Sample analysis

[0082] 1. Optical performance test of A-DCO and A-DCO NPs

[0083] A-DCO and A-DCO NPs were dissolved in DMSO and deionized water, respectively, and characterized by UV spectrophotometer absorption spectra, as shown in Figure 8 As shown in (a), both A-DCO and A-DCO NPs themselves can show strong absorption in the visible light range of 400-450nm.

[0084] A-DCO and DCO were dissolved in water respectively, CTSB was pre-incubated with DTT and EDTA, and then mixed with the aqueous solution of A-DCO, and the emission spectrum was characterized by fluorescence spectrometer, such as Figure 8 As shown in (b), when CTSB is not added to A-DCO, A-DCO has no obvious fluorescence. When CTSB is present, it can specifically cut the GFLG polypeptide chain on A-DCO to generate DCO molecules with amino groups with strong electron-donating ability. Therefore, the maximum fluorescence emission wavelengths of the A-DCO+CTSB group and the DCO group are around 660nm, showing near-infrared fluorescence.

[0085] A-DCO NPs were prepared into solutions with different concentrations and their UV absorption spectra were measured. Figure 8 As shown in (c) and (d), the maximum absorption wavelength of A-DCO NPs of different concentrations is 400-450nm, and as the concentration increases, the light intensity shows a linear relationship with the concentration.

[0086] 2. Characterization of A-DCO NPs prepared in Example 2

[0087] The stability of A-DCO NPs in water was analyzed by dynamic light scattering. Fig. 9 As shown in (a), after the nanoparticles were incubated in water for 84 hours, the particle size did not change significantly, indicating good stability.

[0088] The zeta potential of A-DCO NPs and amphiphilic polymer F127 was measured as Fig. 9 As shown in (b), A-DCO NPs have a higher zeta potential than the amphiphilic polymer F127, indicating that A-DCO forms A-DCO NPs after being encapsulated by F127, and its stability is improved.

[0089] The morphology of A-DCO NPs was analyzed by TEM. Fig. 9 As shown in (c), the A-DCO NPs have uniform particle size, good dispersion and high sphericity.

[0090] 3. Selectivity of A-DCO prepared in Example 1 and A-DCO NPs prepared in Example 2 for normal cells and tumor cells

[0091] A-DCO (1 μM) prepared in DMEM was co-cultured with L929 cells and 4T1 cells for 0.5 h, 2 h, and 3 h, respectively. Images were then taken under the green channel using a FL microscope, and fluorescence intensity statistics were performed using Image J. For comparison, A-DCO NPs prepared in DMEM were also co-cultured with L929 cells and 4T1 cells for 0.5 h, 2 h, and 3 h, respectively, and then analyzed using a fluorescence microscope.

[0092] Fig.10 and Fig.11 The following are the fluorescence detection images of A-DCO and A-DCO NPs on L929 cells and 4T1 cells, respectively. A-DCO shows obvious green fluorescence in mouse breast cancer cells 4T1, but almost no fluorescence in normal mouse fibroblasts L929. A-DCO NPs have no obvious fluorescence for both types of cells, indicating that A-DCO is selective for normal cells and tumor cells.

[0093] 4. In vitro release test of A-DCO NPs prepared in Example 2 under different pH conditions The drug release behavior of A-DCO NPs was studied using dialysis. A-DCO NPs solution (1 mL, 1 mM) was added to a disposable dialysis bag (molecular weight cutoff: 10000 Da). The dialysis bag containing the A-DCO NPs solution was then immersed in an aqueous solution (5 mL) with a pH value of 6.5 and 7.45, respectively, and placed on an oscillator (300 rpm) for oscillation. Each sample was repeated three times independently. 2 ml of the release solution was taken out at a specific time point and poured back after the measurement. The absorbance at a wavelength of 433 nm was measured using a UV spectrophotometer. The release amount was calculated according to the A-DCO standard curve, and the drug release kinetics curve was drawn to calculate the drug release amount of A-DCO.

[0094] Fig.12 Figure 3 is the in vitro release graph of A-DCO NPs under pH 6.5 and 7.45 conditions. Under the condition of pH 6.5 simulating the extracellular microenvironment of tumor cells, the drug release amount of A-DCO in A-DCO NPs gradually increased with time. Under the condition of pH 7.45 simulating the normal body fluid environment, the drug release amount of A-DCO in A-DCO NPs did not change significantly.

[0095] V. Use of A-DCO NPs prepared in Example 2 for detection of apoptotic cells

[0096] 1. Detection of A-DCO NPs prepared in Example 2 in apoptotic cells

[0097] (1) 4T1 and MCF-7 cells

[0098] a. Use DOX to simulate cell apoptosis

[0099] 4T1 cells and MCF7 cells were cultured at approximately 2×10 4 The cells were incubated at a density of 1.54 cells / well in an 8-well plate. After 24 h of incubation, the culture medium was replaced with serum-free medium (DMEM) containing 5 μM A-DCO NPs alone or 5 μM A-DCO NPs + 5 μM chemotherapeutic drug DOX, respectively. Fluorescence microscopy was used to image the cells after 0.5 h and 2 h of incubation.

[0100] Fig.13 The fluorescence images of serum-free culture medium mixed with A-DCO NPs and DOX after incubation in 4T1 cells and MCF-7 cells (scale bar is 200μm). A-DCO NPs have almost no green fluorescence when the chemotherapy drug DOX is not added, but after adding DOX, obvious green fluorescence is produced, indicating that the cells will undergo apoptosis under the action of the chemotherapy drug DOX, prompting CTSB to escape from the lysosome into the cytoplasm, and under the action of the acidic environment of tumor cells, A-DCO NPs dissociate and release free probe molecules, which are then specifically cut by CTSB entering the cytoplasm to produce the fluorescent group DCO, which is the green fluorescence in the figure.

[0101] b. Lysosomal localization test

[0102] ① 4T1 cells were cultured at a rate of about 2×10 4 The cells were incubated at a density of 1.54 cells / well in an 8-well plate. After incubation for 24 h, the culture medium was replaced with a serum-free culture medium (DMEM) containing 5 μM A-DCO NPs, 5 μM A-DCO NPs + 5 μM LCL161 in a 1:1 ratio. After incubation for 3 h, the lysosomes were co-incubated and stained with the lysosomal fluorescent probe LysoSensorTM Yellow / Blue DND-160. After 15 min, the cells were washed twice with PBS, and the cell membrane was co-incubated and stained with the cell membrane probe DID. After 30 min, the cells were washed twice with PBS and imaged using a laser confocal microscope (CLSM).

[0103] Fig.14This is a laser confocal microscopy image of A-DCO NPs localized in the lysosomes of 4T1 cells (scale bar is 10 μm). After verifying the apoptotic effect of the chemotherapy drug DOX on 4T1 and MCF-7 cells, we used LCL161, a small molecule inducer of apoptosis, to verify that apoptosis induced by different drugs can activate the probe fluorescence. As shown in the figure, in the culture medium supplemented with LCL161, apoptosis causes CTSB to escape from the lysosome into the cytoplasm, and under the action of the acidic environment of tumor cells, A-DCO in A-DCO NPs is released into the cytoplasm in a free form, and is recognized and cut by CTSB in the cytoplasm to produce the fluorescent group DCO. All these signals are in the cell, indicating that the fluorescence emission behavior of the nanoparticles is mediated by the physiological activities in the cell, avoiding nonspecific interference.

[0104] ②4T1 and MCF-7 cells were cultured at a rate of approximately 2×10 4 The cells were incubated at a density of 1.54 cells / well in an 8-well plate. After 24 h of incubation, the culture medium was replaced with a serum-free medium (DMEM) containing 10 μM A-DCO NPs or 10 μM A-DCO NPs + 5 μM DOX in a 1:1 ratio. After 3 h of incubation, the lysosomes were co-incubated and stained with the lysosomal fluorescent probe LysoSensorTM Yellow / Blue DND-160. After 15 min, the cells were washed twice with PBS and imaged using a laser confocal microscope (CLSM).

[0105] Fig.15 Laser confocal microscopy images of A-DCO NPs localized in the lysosomes of 4T1 cells and MCF-7 cells (the scale bar in figure a is 50 μm, and the scale bar in figure b is 10 μm). Fig.15 As shown in (a), in 4T1 cells and MCF-7 cells, after the addition of DOX, cell apoptosis promotes CTSB to escape from lysosomes into the cytoplasm. Under the action of the acidic environment of tumor cells, A-DCO in A-DCONPs is released into the cytoplasm in a free form and is recognized and cut by CTSB in the cytoplasm to produce green fluorescence of DCO, as shown in Figure 2. Fig.15 As shown in (b), the green fluorescence belonging to DCO does not completely coincide with the lysosome position, proving that it is the lysosomal escape of CTSB caused by apoptosis that activates the fluorescence of the nanoparticles.

[0106] (2) It was confirmed in MCF-7ADR cells that only apoptosis can activate nanoparticles to produce fluorescence

[0107] MCF-7ADR cells are a type of human breast cancer cell line that is resistant to doxorubicin. The chemotherapy drug DOX alone will not cause apoptosis of the cells. Only under the action of other types of pro-apoptotic drugs will MCF-7ADR cells apoptosis. 4 The cells were incubated at a density of 500 cells / well in an 8-well plate. After 24 h of incubation, the culture medium was replaced with serum-free culture medium (DMEM) containing 5 μM A-DCO NPs alone, 5 μM A-DCO NPs + 5 μM DOX, and 5 μM A-DCO NPs + 5 μM DOX + 5 μM LCL161, respectively. Fluorescence microscopy was used to image the cells after 0.5 h and 2 h of incubation.

[0108] Fig.16 The fluorescence image of MCF-7ADR cells after incubation with serum-free culture medium mixed with A-DCO NPs, DOX and LCL161 (scale bar is 200 μm). As shown in the figure, no fluorescence was observed in the group co-incubated with A-DCO NPs and DOX, indicating that the cells under this condition did not undergo apoptosis. MCF-7ADR cells underwent apoptosis only after the addition of LCL161, which caused CTSB to escape from lysosomes and enter the cytoplasm, activating the free A-DCO dissociated from A-DCO NPs and producing the green fluorescence in the figure.

[0109] 6. Detection of apoptosis-induced fluorescence by flow cytometry

[0110] In vitro apoptosis assays were performed to evaluate the pro-apoptotic effects of A-DCO NPs incubated with DOX, IR1048, and LCL161, respectively. 4T1 cells were seeded into 12-well plates, then stained with Annexin V-FITC / PI for 10 minutes, washed with PBS, and harvested for flow cytometry analysis, which served as the control group. Thereafter, DOX, IR1048, and LCL161 were added to other 12-well plates seeded with 4T1 cells, and the IR1048 group was subjected to near-infrared irradiation (1064 nm, 1.0 w / cm 2 2 minutes), 4 hours later, the cells were washed twice with PBS and A-DCO NPs were added, and the fluorescence intensity histogram of the nanoparticles was observed at 660nm. On this basis, different concentrations of IR1048 (from 0.54μM to 2.7μM) were selected, and after near-infrared light irradiation, they were incubated for another 4 hours, and then Annexin V-FITC / PI was added to stain the cells for 10 minutes, and the cells were washed with PBS and harvested for flow cytometric analysis to determine the percentage of cell apoptosis caused by different concentrations of IR1048. Then, A-DCONPs were added at the corresponding concentrations, and the fluorescence histogram of the nanoparticles was collected at 660nm, and a linear relationship diagram of fluorescence intensity to apoptosis percentage was drawn.

[0111] Fig.17 (a)-(c) in the figure are the detection images of cell apoptosis induced by A-DCO NPs after incubation with DOX, IR1048 and LCL161, respectively. It can be seen that the fluorescence intensity of A-DCO NPs mixed with different drugs is higher than that of the single drug group and A-DCO NPs. Fig.17 As shown in (d) and (e), different concentrations of IR1048 can lead to different degrees of cell apoptosis, and the apoptosis rate is linearly correlated with the fluorescence intensity of A-DCO NPs. This further indicates that A-DCO NPs have the ability to detect tumor cell apoptosis to a certain extent.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cathepsin B-activated fluorescent probe, characterized in that: It includes a dicyanoisosulfone fluorescent reporter unit and a cathepsin B specific enzyme cleavage site GFLG peptide chain, and its structure is as follows:

2. The method for preparing the cathepsin B-activated fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) adding isophorone and malononitrile under nitrogen protection, then adding sodium acetate as a catalyst and ethanol as a solvent to obtain a solution, stirring and heating to reflux reaction, and after the reaction is completed, extracting and purifying to obtain compound 1; (2) dissolving the compound 1 obtained in step (1) and p-acetamidobenzaldehyde in toluene, adding piperidine as a catalyst, and after the reaction is completed, concentrating to remove toluene, and washing with acetonitrile to obtain compound 2; (3) dissolving the compound 2 obtained in step (2) in ethanol, then adding concentrated hydrochloric acid, heating and stirring the mixture, and after the reaction is completed, treating the mixture to obtain the compound DCO; (4) The compound DCO obtained in step (3) was subjected to an amidation reaction with the GFLG peptide chain to synthesize a cathepsin B-activated fluorescent probe.

3. A nanoparticle encapsulating a cathepsin B-activated fluorescent probe, characterized in that: The nanoparticles include an amphiphilic polymer and the cathepsin B activated fluorescent probe according to claim 1, wherein the cathepsin B activated fluorescent probe is wrapped on the amphiphilic polymer.

4. The nanoparticles encapsulating cathepsin B-activated fluorescent probe according to claim 3, characterized in that: The amphiphilic polymer is a block copolymer containing polyoxyethylene and polyoxypropylene.

5. The nanoparticles encapsulating cathepsin B-activated fluorescent probe according to claim 3, characterized in that: The mass ratio of the amphiphilic polymer to the cathepsin B activated fluorescent probe is 15-20:

1.

6. The method for preparing nanoparticles encapsulating cathepsin B-activated fluorescent probe according to any one of claims 3 to 5, characterized in that: The following steps are involved: The cathepsin B activated fluorescent probe is dissolved in an organic solvent and then added dropwise to a solution of an amphiphilic polymer, assisted by ultrasound, to obtain a uniform dispersion. The dispersion is filtered to remove impurities and freeze-dried to obtain nanoparticles encapsulating the cathepsin B activated fluorescent probe.

7. The method for preparing nanoparticles encapsulating cathepsin B-activated fluorescent probe according to claim 6, characterized in that: The organic solvent is tetrahydrofuran, acetonitrile or chloroform.

8. The method for preparing nanoparticles encapsulating cathepsin B-activated fluorescent probe according to claim 6, characterized in that: The organic solvent is tetrahydrofuran, and the solution of the amphiphilic polymer is an aqueous solution of the amphiphilic polymer F127.

9. Use of the nanoparticles encapsulating cathepsin B-activated fluorescent probe according to any one of claims 3 to 5 in detecting apoptotic tumor cells.

Images