Fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probe detection and treatment. ZIF-8 is used as a carrier, curcumin and tetra (4-carboxyphenyl) porphin are packaged in ZIF-8 at the same time to obtain Z-C-T, the outer layer of Z-C-T is coated with a layer of mouse breast cancer cell membrane through an ultrasonic method to obtain Z-C-T (at) CM, and the synthesis method is simple. The prepared Z-C-T (at) CM fluorescent probe has dual fluorescence emission and can be used for near-infrared proportional fluorescence detection of ONOO <->. The carried CCM has anti-cancer activity, and the carried TCPP has a photodynamic effect. After the tumor microenvironment is degraded, the CCM and the TCPP are released, and the CCM and the TCPP can be used for combined treatment (chemotherapy and photodynamic therapy) of the mouse breast cancer, so that diagnosis and treatment integration is realized. The surface of the Z-C-T (at) CM fluorescent probe is coated with a layer of mouse breast cancer cell membrane, so that targeted treatment of mouse breast tumors is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probe detection and treatment, and particularly relates to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] In tumor progression associated with elevated oxidative stress, peroxynitrite (ONOO - ) levels will be greatly increased, so in the diagnosis and treatment of cancer, ONOO - In vivo monitoring of ONOO is very necessary. - Fluorescent probes for detection include organic small molecules, quantum dots, transition metal complexes, and upconversion nanoparticles. Generally speaking, organic small molecule probes are complex to synthesize, have poor water solubility, and are subject to photobleaching. Quantum dots are usually highly biotoxic, and upconversion nanoparticles often need to be combined with small molecules and functionalized targeting ligands (commonly triphenylphosphine). The design of the synthesis process and the realization of the expected function are relatively complex. In addition, few fluorescent probes can have both ONOO - The detection is also used in the treatment of diseases such as tumors.

[0003] Currently, metal-organic frameworks (MOFs) fluorescent probes have been widely used for intracellular and extracellular sensing and imaging. - Probe, MOF-based ONOO - Probe, ONOO only - The detection method includes the following: ONOO - The sensor is coupled to the MOF surface via Resonance energy transfer enables ONOO in solution and living cells - Detection of aryl borate and ONOO - The rapid response can simultaneously detect intracellular and extracellular peroxynitrite; or synthesize a multifunctional nanotherapy platform of ZIF-67 loaded with zinc oxide and loaded with organic near-infrared probes, which can simultaneously achieve ONOO at the bacterial infection site - However, none of the above three methods can simultaneously achieve the detection and effective sterilization of ONOO - Detection and treatment of tumor diseases. Summary of the invention

[0004] In view of the above problems, the present invention proposes an ONOO that is easy to synthesize, has low toxicity and can realize integrated diagnosis and treatment. - Probe.

[0005] In a first aspect, the present invention provides a method for preparing a fluorescent probe, comprising the following steps:

[0006] Will Zn(NO 3 ) 2 6H 2 O is dissolved to obtain solution A;

[0007] Mix and dissolve 2-methylimidazole, tetrakis(4-carboxyphenyl)porphine and curcumin to obtain solution B;

[0008] The solution A and the solution B are mixed and stirred for reaction under light-proof conditions;

[0009] After the reaction is completed, the product is separated, and the solid obtained after separation is washed until the supernatant becomes colorless and has no fluorescence in an ultraviolet dark box, and then the solid product ZCT is collected;

[0010] The solid product ZCT was made into a ZCT suspension and then mixed with a PBS suspension containing mouse breast cancer cell membrane to obtain a mixed solution C. The obtained mixed solution C was ultrasonically treated in an ice bath, and after ultrasonication, the solid product was collected by centrifugation to obtain a ZCT@CM fluorescent probe.

[0011] Furthermore, when preparing solution A, Zn(NO 3 ) 2 6H 2 The mass volume ratio of O to the solvent is 0.08588-0.17175 g:1 mL, for example, it can be 0.08588 g / mL, 0.10305 g / mL, and 0.17175 g / mL.

[0012] Further, the concentration ranges of 2-methylimidazole, tetrakis(4-carboxyphenyl)porphine and curcumin in the solution B are 0.1601-0.3023 g / mL, 0.0002-0.0024 g / mL and 0.00217-0.00434 g / mL, respectively;

[0013] Exemplarily, the concentration range of 2-methylimidazole can be 0.1601 g / mL, 0.3023 g / mL; the concentration of tetrakis(4-carboxyphenyl)porphine can be 0.0002 g / mL 0.0012 g / mL, 0.0024 g / mL; the concentration of curcumin can be 0.00217 g / mL, 0.00434 g / mL.

[0014] Furthermore, the solvent in the solution A and the solution B is methanol.

[0015] Furthermore, the stirring time during the stirring reaction is 12-18 h, for example, 12 h, 13 h, 15 h, 16 h, 18 h; the stirring temperature is 15-30 ° C, for example, 15 ° C, 20 ° C, 26 ° C, 30 ° C.

[0016] Further, the mixed volume ratio of the ZCT suspension and the PBS suspension containing mouse breast cancer cell membrane in the mixed solution C is 1:4;

[0017] The concentration of the ZCT suspension is 100 μg / mL; the cell membrane in the PBS suspension containing mouse breast cancer cell membrane is 1×10 7 -4×10 7 For example, the number of mouse breast cancer cells can be 1×10 7 pcs, 2×10 7 pcs, 3×10 7 4×10 7 indivual.

[0018] In a second aspect, the present invention provides a fluorescent probe prepared by the preparation method.

[0019] Furthermore, the carrier of the fluorescent probe is ZIF-8, the interior of ZIF-8 encapsulates curcumin and tetrakis(4-carboxyphenyl)porphine, and the outer layer is coated with mouse breast cancer cell membrane.

[0020] In a third aspect, the present invention proposes that the fluorescent probe prepared by the above preparation method is used to detect ONOO in a solution. - Application in the process.

[0021] In a fourth aspect, the present invention provides a fluorescent probe prepared by the above preparation method in ONOO - Applications during in vivo imaging.

[0022] In a fifth aspect, the present invention proposes the use of the fluorescent probe prepared by the above preparation method in the preparation of a drug for treating breast cancer.

[0023] Beneficial effects of the present invention:

[0024] The present invention uses ZIF-8 (zeolite imidazolate framework material) as a carrier, encapsulates curcumin (CCM) and tetrakis (4-carboxyphenyl) porphine (TCPP) in ZIF-8 at the same time to obtain ZCT, and then coats a layer of mouse breast cancer cell membrane on the outer layer by ultrasonic method to obtain the nanomaterial ZCT@CM, and the synthesis method is simple.

[0025] The ZCT@CM fluorescent probe prepared by the present invention has dual fluorescence emission, with maximum emission peaks at 545nm and 610nm, respectively. The maximum wavelength emission has reached the near-infrared region of 650-1000nm, and can be used for ONOO - Near-infrared ratio fluorescence detection. The CCM carried by ZCT@CM has anticancer activity, and the TCPP carried by ZCT@CM has a photodynamic effect. After ZCT@CM is degraded in the tumor microenvironment, it releases CCM and TCPP, which can be used for the combined treatment of mouse breast cancer (chemotherapy and photodynamic therapy) to achieve integrated diagnosis and treatment. A layer of mouse breast cancer cell membrane coated on the surface of the ZCT@CM fluorescent probe helps to achieve targeted treatment of mouse breast tumors. Finally, the targeted combined treatment of ZCT@CM will also induce immunogenic death of cancer cells.

[0026] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1A shows a scanning electron microscope image of the ZCT prepared in an embodiment of the present invention;

[0029] Figure 1B The transmission electron microscope image of ZCT@CM in an embodiment of the present invention is shown;

[0030] Figure 2A The fluorescence emission spectra (λ ex =400nm, slit 5 / 10nm).

[0031] Figure 2B The ultraviolet absorption spectrum (450nm-750nm) of TCPP in the embodiment of the present invention is shown when the content of TCPP in ZCT is different (1.25, 2.5, 5, 10, 20, 40mg / L);

[0032] Figure 2C The standard curve between the concentration and absorbance of TCPP at 550 nm in the embodiment of the present invention is shown;

[0033] Figure 2D The UV absorption spectra of ZCT samples with different proportions in the embodiments of the present invention are shown;

[0034] Figure 3A The results show that the ZCT@CM prepared in Example 1 of the present invention has a significant effect on different analytes (ClO - , CO 3 2- , Cys, DTT, GSH, H 2 O 2 , HCO 3 - , HPO 4 2- , S 2- , SCN - , SO 4 2- ,BuOO - , ·OH,ONOO - , 1 O 2 )’s response result;

[0035] Figure 3B The results show that the ZCT@CM prepared in the present invention has different concentrations of ONOO at 0-120 μmol / L. - The results showed fluorescence enhancement response;

[0036] Figure 3C It shows that the ZCT@CM prepared in the embodiment of the present invention is 545nm / I 610nm with ONOO - Linear relationship graph of concentration;

[0037] Figure 4A It shows that in the embodiment of the present invention, at 0.2W / cm 2 UV-visible absorption spectrum of the mixture of 1,3-diphenylisobenzofuran (DPBF) solution and ZCT@CM suspension changes over time under 660nm light;

[0038] Figure 4B It shows ln(A 0 / A) Linear relationship diagram with laser irradiation time;

[0039] Figure 4C The results of detecting ROS in cells using 2',7'-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe in an embodiment of the present invention are shown;

[0040] Figure 5In the examples of the present invention, (A) 100 μL of 100 μg / mL ZCT@CM suspension was injected intratumorally into tumor-bearing mice (0, 4, 8, 12 days), and near-infrared fluorescence in vivo imaging was performed under a 660 nm laser 30 minutes later; (B) H&E staining of ex vivo tumors of tumor-bearing mice (sacrificed on days 0, 4, 8, 12), with a scale of 50 μm.

[0041] Fig. 6A The photographs of mice in the Control group and different experimental groups at the end of treatment (day 16) in the embodiment of the present invention are shown;

[0042] Figure 6B The representative in vitro tumor images of mice in the Control group and different experimental groups at the end of treatment in the embodiment of the present invention are shown;

[0043] Figure 6C The relative tumor volume curves of 4T1 tumor-bearing mice in each group of mice in the embodiment of the present invention within 16 days are shown;

[0044] Fig.6D The tumor weight at the end of different treatment methods in the embodiments of the present invention is shown;

[0045] Fig. 6E The figure shows the tumor inhibition rate of tumor-bearing mice at the end of different treatment methods in the embodiments of the present invention;

[0046] Fig. 7A The in vitro fluorescence images of the tumor, heart, liver, spleen, lung, and kidney at different time points after subcutaneous injection of ZCT@CM in each group of mice in the embodiment of the present invention are shown;

[0047] Figure 7B shows the change of relative fluorescence intensity of different organs over time in the embodiment of the present invention;

[0048] Figure 7C The distribution of Zn in different organs of mice in each group according to the embodiment of the present invention is shown;

[0049] Fig. 8A The H&E staining and immunofluorescence staining images of CRT, HMGB1, CD4 and CD8 of tumor tissues of different treatment groups in the embodiment of the present invention are shown;

[0050] Figure 8B The relative fluorescence intensity of the H&E staining and immunofluorescence staining images of CRT in the embodiment of the present invention is shown;

[0051] Figure 8C The relative fluorescence intensity of H&E staining and immunofluorescence staining images of HMGB1 in the embodiment of the present invention is shown;

[0052] Fig.8D The relative fluorescence intensity of the H&E staining and immunofluorescence staining images of CD4 in the embodiment of the present invention is shown;

[0053] Fig. 8E The relative fluorescence intensity of the H&E staining and immunofluorescence staining images of CD8 in the examples of the present invention are shown. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] The specific process of preparing ZCT@CM is as follows:

[0057] Weigh 0.03435g Zn(NO 3 ) 2 6H 2 O was dissolved in 200 μL methanol to obtain solution A;

[0058] Weigh 0.66501 g of 2-methylimidazole, 0.00045 g of TCPP (tetrakis(4-carboxyphenyl)porphine), and 0.00955 g of CCM (curcumin) in 2.2 mL of methanol, mix by ultrasonic mixing, and dissolve to obtain solution B;

[0059] Mix solution A and solution B, protect from light, and stir at room temperature for 12 hours at 20°C;

[0060] After the reaction, centrifuge (10000rpm), wash the solid with methanol and ultrapure water 2-3 times respectively until the supernatant becomes colorless and has no fluorescence in the UV dark box (λ ex =365nm), the obtained solid was ZCT, which was dried under vacuum at 85°C overnight.

[0061] ZCT was dispersed in water to prepare a suspension (100 μg / mL, 170 μL) and then mixed with mouse breast cancer cell membranes (1×10 7 The purified ZCT@CM was collected by centrifugation at 4°C (10,000 rpm, 30 min) and washed three times with ultrapure water.

[0062] The purified ZCT@CM was redispersed in PBS suspension to obtain ZCT@CM suspension for use in subsequent experiments.

[0063] The morphology images of ZCT and ZCT@CM obtained in this embodiment are shown in Figure 2. Figure 1A and 1B As shown, Figure 1A The morphology of ZCT under scanning electron microscope. Figure 1B The morphology of ZCT@CM under transmission electron microscopy. It can be seen that the size of ZCT is about 100nm, and the size of ZCT@CM is slightly larger than 100nm.

[0064] Example 2

[0065] The synthesis optimization of ZCT is as follows:

[0066] Take Zn(NO 3 ) 2 6H 2 O is an inorganic precursor, and 2-methylimidazole is used as an organic ligand. Different amounts of two organic molecules TCPP and CCM are dissolved in methanol and stirred at room temperature to synthesize ZCTx (Table 1), where x is the mass of TCPP (mg).

[0067] Table 1 Synthesis feed of ZCT with different organic molecule CCM and TCPP contents

[0068]

[0069]

[0070] In order to optimize the fluorescence properties of ZCT and make it show stronger fluorescence dual emission properties, the amount of TCPP in ZCT was investigated. TCPP was prepared according to the method in Example 1, keeping the other synthetic raw materials the same, and changing the amount of TCPP. The results are as follows Figure 2A As shown in the figure, when the content of TCPP increases (0.4-0.6 mg), the fluorescence double emission of ZCT increases simultaneously, and when the amount of TCPP increases further, the fluorescence double emission of ZCT decreases simultaneously. When the amount of TCPP increases to 3.6 mg and above, the fluorescence emission of the TCPP part in ZCT is slightly enhanced, while the fluorescence emission peak of CCM is not significant and almost unchanged.

[0071] Therefore, the actual doping ratio of TCPP in the composite nanomaterials was further investigated. Figure 2B-2D As shown, Figure 2BThe ultraviolet absorption spectra (450nm-750nm) of TCPP in ZCT with concentrations of 1.25, 2.5, 5, 10, 20, and 40 mg / L are shown in the embodiments of the present invention. Figure 2C is the standard curve between the concentration and absorbance of TCPP at 550nm. Figure 2D The UV absorption spectra of ZCT samples with different proportions are shown in Table 2. The TCPP absorption at 550nm is used to calculate the doping amount of TCPP in samples with different proportions through the standard curve method. The calculation results are shown in Table 2:

[0072] Table 2 Doping percentage of TCPP in samples with different proportions

[0073] sample Doping amount of TCPP (%) ZCT-0.4 2.84±0.3 ZCT-0.6 2.87±0.1 ZCT-0.9 2.29±0.2 ZCT-1.8 2.22±0.4 ZCT-3.6 1.72±0.2

[0074] It can be seen that the doping ratio of TCPP in ZCT-0.6 is the highest, and further increasing the TCPP feed ratio will lead to a decrease in the doping ratio. Based on the above experiments, ZCT-0.6 was finally selected as the sample for subsequent experiments.

[0075] Example 3

[0076] ONOO in solution - Sensor detection:

[0077] In aqueous media, ZCT@CM suspensions were used to analyze different analytes (ClO - , CO 3 2- , Cys, DTT, GSH, H 2 O 2 , HCO 3 - , HPO 4 2- , S 2- , SCN - , SO 4 2- ,BuOO - , ·OH,ONOO - , 1 O 2 ) for testing. The specific steps are as follows:

[0078] A ZCT@CM suspension (PBS, 10 mmol / L, pH = 7.4) with a concentration of 100 μg / mL was prepared. - , CO 3 2- , Cys, DTT, GSH, H 2 O 2 , HCO 3- , HPO 4 2- , S 2- , SCN - , SO 4 2- ,BuOO - , ·OH,ONOO - , 1 O 2 ) was added with 2 mL of the suspension and reacted at room temperature for 5 min before fluorescence detection. 400 nm was used as the excitation wavelength and the fluorescence emission in the range of 420 nm to 750 nm was recorded. The fluorescence emission intensity at 550 nm and 627 nm was used as the basis for quantitative analysis.

[0079] The results are as follows Figure 3A As shown in the figure, it can be seen that ZCT@CM has a significant effect on ONOO - Has a specific fluorescence enhancement response.

[0080] Further increase in ONOO - The fluorescence intensity of ZCT@CM was observed at different concentrations. Figure 3B As shown, it can be seen that ONOO - The concentration of ONOO - The fluorescence dual emission of ZCT@CM increases continuously; with the increase of ONOO - The concentration of ONOO - The fluorescence emission intensity of TCPP continues to increase, while that of CCM continues to decrease. 545nm / I 610nm with ONOO - The concentration of 1-30 μmol / L and 30-120 μmol / L showed a linear relationship, respectively. Figure 3C As shown, the linear equations are:

[0081] I 545nm / I 610nm =0.08[ONOO - ]+0.47 and I 545nm / I 610nm =0.03[ONOO - ]+0.64, detection limit

[0082] is 0.15μmol / L. It can be seen that in ONOO - In a certain concentration range, the fluorescence emission of ZCT@CM increases with the concentration of ONOO -The concentration changes linearly.

[0083] Example 4

[0084] Test of the photodynamic effect of ZCT@CM:

[0085] In order to detect the ROS generation performance of ZCT@CM and the control groups (CCM, TCPP, ZC@CM and ZT@CM), 1,3-diphenylisobenzofuran (DPBF) was used as a common reactive oxygen species scavenger, and the whole process was monitored by UV-visible spectroscopy. ZCT@CM (50 μg / mL) and DPBF (0.5 mmol / L) were dispersed in DMF, and then a 660 nm laser was used at 0.2 W / cm 2 The power intensity is irradiated. The absorbance of DPBF at 415nm is recorded every 40s, which will cause the ultraviolet absorption of DPBF at 415nm to continue to decrease ( Figure 4A ).

[0086] The same experiment was performed with free DPBF and the mixed solution (or suspension) in the dark as the blank control group. The same method (660nm, 0.2W / cm 2 ) was used to detect the ROS generation ability of small molecules CCM and TCPP. A 50μg / mL small molecule solution was mixed with DPBF (0.5mmol / L), and the absorption of DPBF at 415nm was detected every 40s. Laser irradiation of small molecules (TCPP and CCM) and materials will cause the ultraviolet absorption of DPBF at 415nm to continue to decrease. However, for the control group (DPBF+660nm, CCM, TCPP, ZC@CM, ZT@CM and ZCT@CM in the dark), the changes in the spectrum are almost negligible. The absorbance ratio at 415nm (maximum absorbance value A 0 The curve of the ratio of the real-time absorbance value A to the laser irradiation time (t) was fitted with an exponential function: A 0 / A=e kt , and obtain pseudo-first-order 1 O 2 Generate Behavior( Figure 4B ), calculate the k value (i.e., the slope in the figure) to compare the generation efficiency of small molecules and composite nanomaterials, 1 O 2 The generation efficiency is ranked as follows: TCPP>ZC@CM>ZCT@CM>CCM>ZT@CM.

[0087] 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect ROS (reactive oxygen species) in cells. Figure 4C Shown are control group, ZC group, ZT group, ZCT group and ZCT@CM group. Scale bar: 50 μm.

[0088] Example 5

[0089] ZCT@CM for ONOO - In vivo imaging:

[0090] ZCT@CM was used for solid tumor imaging. First, 4T1 cells were inoculated on the right lower back of BABL / c nude mice, and 100 μL of 100 μg / mL ZCT@CM suspension (obtained by diluting the ZCT@CM suspension in Example 1) was injected into tumor-bearing mice at different tumor implantation times (0, 4, 8, and 12 days), and near-infrared fluorescence imaging was performed using a small animal imager.

[0091] The results are as follows Figure 5 As shown, A shows that 100 μL of 100 μg / mL ZCT@CM suspension was injected into the tumor of tumor-bearing mice (0, 4, 8, 12 days). After 30 minutes, near-infrared fluorescence in vivo imaging was performed under a 660nm laser. B shows the stained cells of the in vitro tumors in the mice after the tumor-bearing mice were killed on days 0, 4, 8, and 12. The scale in the figure is: 50 μm. The experimental results show that the fluorescence signal in the tumor-bearing mice increases with the number of days of tumor growth, which confirms that ONOO - The content of ONOO in the body does increase with the growth of tumors. - May serve as a potential tumor biomarker.

[0092] Example 6

[0093] Application of ZCT@CM in tumor treatment of 4T1 tumor-bearing mice:

[0094] The tumor-bearing mice were randomly divided into 5 groups: Control group (no treatment), Control+NIR-I group (660nm, 1.5W / cm 2 , 10min), ZCT group (intratumoral injection of ZCT at a dose of 10mg / kg), ZC-T+NIR-I group (intratumoral injection of ZCT at a dose of 10mg / kg, 3h later with 1.5W / cm 2 The ZCT@CM group (10 mg / kg ZCT@CM was injected intratumorally), and the ZCT@CM+NIR-I group (10 mg / kg ZCT@CM was injected intratumorally, and then 1.5 W / cm 2At different time points of treatment, the body weight and tumor volume of the mice were recorded.

[0095] The results are as follows Figures 6A-6D As shown, Fig. 6A These are photos of mice in each group at the end of treatment (day 16); Figure 6B These are representative in vitro images of tumors in each group of mice at the end of treatment; Figure 6C This is the relative tumor volume curve of 4T1 tumor-bearing mice in group mice within 16 days; Fig.6D is the tumor weight at the end of different treatments; Fig. 6E This is the tumor inhibition rate of tumor-bearing mice at the end of different treatments.

[0096] The experimental results showed that within 16 days, there was no significant change in body weight between the control group and the experimental group, indicating that ZCT@CM has good biological safety.

[0097] During the treatment process, the tumor growth of the Control group and different experimental groups was different. According to the photos of tumor-bearing mice after treatment with different treatment methods, it can be seen that the relative tumor volume and weight of the ZCT@CM+NIR-Ⅰ group were much smaller than those of other groups (Control group, Control+NIR-Ⅰ group, ZCT group, ZC-T+NIR-Ⅰ group, and ZCT@CM group). The mice in the ZCT@CM group were loaded with anticancer active CCM, and the surface was coated with 4T1 cell membrane. It can be seen that it has a significant inhibitory effect on tumor growth without near-infrared light. It is worth noting that the tumor volume and weight of the ZCT@CM+NIR-I group decreased the most, which is similar to the results obtained from the representative photos of mouse ex vivo tumors. In order to further evaluate the metabolism of ZCT@CM during the treatment process, the ex vivo organs of mice in the ZCT@CM+NIR-Ⅰ group at different time points were collected and fluorescent imaging was performed. The results are shown in Figure 2. Figures 7A-7C As shown, Fig. 7A These are in vitro fluorescence images of tumors, heart, liver, spleen, lung, and kidney at different time points after subcutaneous injection of ZCT@CM in each group of mice. Figure 7B is the change of relative fluorescence intensity of different organs over time. Figure 7C The distribution of Zn in different organs of mice in each group is shown in the figure. From the experimental results, it can be seen that about 1 hour after injection, the fluorescence signal began to appear in the tumor site, and the fluorescence intensity was the highest at about 3 hours. After 48 hours, the fluorescence signal in the tumor site was greatly weakened. In addition, only the liver and kidney had very weak fluorescence signals. This shows that after ZCT@CM treatment, it is well metabolized in mice.

[0098] From the above examples, it can be seen that the ZCT@CM proposed in the present invention has dual fluorescence emission, with maximum emission peaks at 545nm and 610nm, respectively, and the maximum wavelength emission has reached the near-infrared region of 650-1000nm, which can be used for ONOO - Near-infrared ratio fluorescence detection. However, existing similar materials have poor performance on ONOO - The detection is often based on a single fluorescence emission with a short wavelength of blue or green light, and is easily affected by biological background fluorescence.

[0099] In addition, the CCM carried by the ZCT@CM proposed in the present invention has anticancer activity, and the TCPP carried by the ZCT@CM has a photodynamic effect. After the ZCT@CM is degraded in the tumor microenvironment, it releases CCM and TCPP, which can achieve dual treatment of chemotherapy and photodynamic therapy.

[0100] The dual treatment of ZCT@CM can induce the immunogenic death of cancer cells. This is because ZCT@CM can stimulate tumor immune response under near-infrared light, and the components on the surface of tumor cells will change, causing the immunogenic death of cancer cells (such as Figures 8A-8E The most important thing is that calreticulin (CRT) on the surface of the endoplasmic reticulum of tumor cells migrates to the surface of the cell membrane ( Fig. 8A ), after ZCT@CM was illuminated with NIR-Ⅰ, the tumor cell surface showed the strongest fluorescence signal, which means the most CRT expression ( Figure 8B In addition, the high mobility group protein B1 (HMGB1) in the cell nucleus is released into the cytoplasm and outside the cell ( Fig. 8A ). When ZCT@CM was irradiated with NIR-Ⅰ, the signal of HMGB1 in the nucleus was reduced to the greatest extent, indicating that the release amount of HMGB1 was the greatest ( Figure 8C ). It can be seen that the immunogenic death caused by ZCT@CM under NIR-Ⅰ light is the highest. Mature T cells express CD4 and CD8 cells. After ZCT@CM is irradiated by laser, the expression of CD4 and CD8 cells is the highest, such as Fig. 8A and 8D The red fluorescence signal representing CD4 cells is the strongest in the control group and the experimental group. Fig. 8A and 8E The red fluorescence signal of CD8 cells represented in the middle was the strongest in the control group and the experimental group. The expression of CD4 and CD8 cells promoted the intratumoral infiltration of T cells.

[0101] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a fluorescent probe, characterized in that: The following steps are involved: Dissolve Zn(NO3)2·6H2O to obtain solution A; Mix and dissolve 2-methylimidazole, tetrakis(4-carboxyphenyl)porphine and curcumin to obtain solution B; The solution A and the solution B are mixed and stirred for reaction under light-proof conditions; After the reaction is completed, the product is separated, and the solid obtained after separation is washed until the supernatant becomes colorless and has no fluorescence in an ultraviolet dark box, and then the solid product ZCT is collected; The solid product ZCT was made into a ZCT suspension and then mixed with a PBS suspension containing mouse breast cancer cell membrane to obtain a mixed solution C. The mixed solution C was ultrasonically treated in an ice bath, and after the ultrasonic treatment, the solid product was collected by centrifugation to obtain a ZCT@CM fluorescent probe.

2. The method for preparing a fluorescent probe according to claim 1, characterized in that: When preparing solution A, the mass volume ratio of Zn(NO3)2·6H2O and solvent is 0.08588-0.17175g:1mL.

3. The method for preparing a fluorescent probe according to claim 1, characterized in that: The concentration ranges of 2-methylimidazole, tetrakis(4-carboxyphenyl)porphine and curcumin in the solution B are 0.1601-0.3023 g / mL, 0.0002-0.0024 g / mL and 0.00217-0.00434 g / mL, respectively.

4. The method for preparing a fluorescent probe according to any one of claims 1 to 3, characterized in that: The solvent in the solution A and the solution B is methanol.

5. The method for preparing a fluorescent probe according to claim 1, characterized in that: The stirring time is 12-18h, and the stirring temperature is 15-30℃.

6. The method for preparing a fluorescent probe according to claim 1, characterized in that: The volume ratio of the ZCT suspension and the PBS suspension containing mouse breast cancer cell membrane in the mixed solution C is 1:4; Wherein, the concentration of the ZCT suspension is 100 μg / mL; The mouse breast cancer cell membrane in the PBS suspension containing the mouse breast cancer cell membrane is 1×10 7 -4×10 7 extracted from mouse breast cancer cells.

7. A fluorescent probe, characterized in that The method is prepared by any one of claims 1 to 6.

8. The fluorescent probe according to claim 7, characterized in that The carrier of the fluorescent probe is ZIF-8, the interior of ZIF-8 encapsulates curcumin and tetrakis(4-carboxyphenyl)porphine, and the outer layer is coated with mouse breast cancer cell membrane.

9. Use of the fluorescent probe prepared by the method according to any one of claims 1 to 6 in detecting peroxynitrite in a solution.

10. A fluorescent probe prepared according to the method of any one of claims 1 to 6 in ONOO - Applications during in vivo imaging.

11. Use of the fluorescent probe prepared by the method according to any one of claims 1 to 6 in the preparation of a drug for treating breast cancer.

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