Molecular probe for detecting living copper ions as well as preparation method and application of molecular probe

Abstract

The invention discloses a molecular probe for in-vivo copper ion detection and a preparation method and application thereof, the chemical structural formula of the molecular probe is as follows: # imgabs0 #, the molecular probe provided by the invention can specifically respond to copper ions in a tumor subacid environment so as to cause change of an absorption spectrum, and a photoacoustic signal is started so as to detect the copper ions in the tumor subacid environment. Normal tissues and tumor tissues can be accurately distinguished, and accurate detection of the copper ions in the living tumor is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical synthesis, and in particular to a molecular probe for detecting copper ions in living bodies, and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer. Due to its high risk of recurrence and lack of effective therapeutic targets, it has always been known as the "most toxic" breast cancer. Its characteristics are that the results of immunohistochemical examination of cancer tissue show that estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene HER-2 are all negative. This type of breast cancer accounts for 10.0%-20.8% of all breast cancer pathological types, has special biological behavior and clinical pathological characteristics, and has a worse prognosis than other types. Accurate identification of triple-negative breast cancer is of great value for the subsequent formulation of personalized treatment plans and precise implementation of treatment. At present, the diagnosis of triple-negative breast cancer usually adopts a combination of multiple diagnostic methods, including pathological examination and genetic testing, ductoscopy, breast ultrasound, magnetic resonance imaging (MRI), etc. Pathological examination and genetic testing usually require puncture or surgical operation to obtain tumor tissue, which will inevitably cause physical trauma to patients, and the subsequent detection of tumor immunohistochemical staining, gene sequencing and other processing procedures are complicated. Although imaging examinations have the advantages of being non-invasive, intuitive and clear, highly sensitive, and assisting in diagnosis in the diagnosis of cancer, the currently used imaging methods such as MRI, breast ultrasound, and breast mammography still have certain limitations. For example, although MRI has a high sensitivity, its specificity is relatively low, and breast ultrasound has a relatively low resolution. It cannot accurately detect or clearly display tiny breast lesions, especially early, small TNBC tumors, which can easily lead to missed diagnosis. Breast mammography has certain radiation damage, and lumps near the periphery of the breast may not be detected due to the limitation of the projection position. In addition, due to the variability of breast imaging characteristics, false positives sometimes occur.

[0003] Therefore, there is an urgent need for an imaging detection method that is non-invasive, sensitive, and specific, and can quickly present the results of the examination. Photoacoustic imaging technology is a new type of non-invasive and non-ionizing optical imaging technology that has emerged in recent years. Due to the combination of the characteristics of pure optical tissue imaging and pure ultrasonic tissue imaging, photoacoustic imaging can achieve both micron-level resolution and centimeter-level penetration depth, and has the advantages of high chemical selectivity and real-time imaging. It is expected to fill the shortcomings of clinical imaging technology and provide new opportunities for the diagnosis of triple-negative breast cancer. In terms of probe design, triple-negative breast cancer lacks overexpression of estrogen receptors (ER, PR, HER-2), and there is currently no clinically approved targeted agent. Therefore, it is difficult to achieve accurate detection of triple-negative breast cancer through conventional molecular targeted design ideas. How to improve the imaging sensitivity of photoacoustic probes and discover new triple-negative breast cancer markers is the key to solving the above problems. Summary of the invention

[0004] Changes in cell metabolism are closely related to the occurrence and development of cancer. Compared with normal tissue cells, tumors take up a large amount of glucose and break down glucose into lactic acid through glycolysis, forming a weakly acidic tumor microenvironment. In addition, the latest research has found that triple-negative breast cancer cells have a higher copper ion concentration than normal cells. In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a molecular probe and its preparation method and application, aiming to solve the problems of low sensitivity and specificity in the existing triple-negative breast cancer detection.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect of the present invention, a molecular probe for detecting copper ions in vivo is provided, wherein the chemical structural formula of the molecular probe is as follows:

[0007] A second aspect of the present invention provides a method for preparing a molecular probe, the method comprising the following steps:

[0008] Mixing N-(2-bromomethyl)phthalimide and di(2-pyrrolyl)amine to undergo a first substitution reaction to obtain a first product, and subjecting the first product to a hydrolysis reaction to obtain a first intermediate;

[0009] Mixing 2,3,3-trimethyl-3H-indole with 3-iodopropyl acetate to undergo a second substitution reaction to obtain a second product, and mixing the second product with 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde to undergo a condensation reaction to obtain a second intermediate;

[0010] The first intermediate and the second intermediate are mixed to undergo a third substitution reaction to obtain a third intermediate;

[0011] causing the third intermediate to undergo ester hydrolysis reaction and ring closure reaction to obtain the molecular probe;

[0012] The structural formulas of the first intermediate, the second intermediate, the third intermediate and the molecular probe are as follows:

[0013]

[0014] According to a third aspect of the present invention, a nanoprobe is provided. The nanoprobe comprises nanoparticles and an amphiphilic polymer coated on the outside of the nanoparticles. The nanoparticles comprise a reference dye and the above-mentioned molecular probe.

[0015] Preferably, in the nanoprobe, the mass ratio of the molecular probe, the reference dye and the amphiphilic polymer is 1:(0.5-1.0):(5-15).

[0016] Preferably, the amphiphilic polymer is selected from one or more of phospholipid-methoxypolyethylene glycol, α-hydrogen-ω-hydroxy poly(ethylene oxide) a-poly(propylene oxide) b-poly(ethylene oxide) c] block copolymer, and polyethylene glycol-polylactic acid.

[0017] Preferably, the reference dye is selected from one or more of 5,5'-dichloro-11-diphenylamino-3,3'-diethyl-10,12-vinylthiotricarbocyanine perchlorate, IR-780 iodide, and indocyanine green.

[0018] Preferably, the nanoprobe is spherical in shape, and has a diameter of 22-32 nm.

[0019] A fourth aspect of the present invention provides a method for preparing the above-mentioned nanoprobe, the preparation method comprising the following steps:

[0020] The molecular probe, reference dye and amphiphilic polymer are dissolved in an organic solvent and stirred to obtain the nanoprobe.

[0021] The fifth aspect of the present invention provides the use of the above-mentioned molecular probe and / or the isomers of the molecular probe in the preparation of tumor diagnostic products;

[0022] Or, use of the above-mentioned nanoprobe in the preparation of tumor diagnostic products.

[0023] Preferably, the tumor is triple-negative breast cancer.

[0024] Beneficial effects: The present invention provides a molecular probe for detecting copper ions in vivo and its preparation method and application. The molecular probe has dual response characteristics of copper ions and acid. Its activation principle is as follows: Figure 1As shown, when the molecular probe is nano-sized and injected into the tumor, the acid response group of the molecular probe is converted from a closed-loop form to an open-loop form in the slightly acidic environment of the tumor (pH <7), and the absorption intensity at about 610nm gradually increases with the decrease of pH. In addition, the molecular probe has a copper ion response characteristic, which causes its absorption peak to further red-shift from 610nm to about 700nm, and the absorption intensity increases with the increase of copper ion concentration. Since the photoacoustic signal is proportional to the absorption intensity, the photoacoustic signal is also enhanced. The pH of the normal tissue microenvironment is 7.4, and the acid response group cannot be open-looped, so the near-infrared light absorption that cannot be detected in normal tissue. With the reference dye of non-responsiveness as a reference, the nanoprobe comprising the above-mentioned molecule provided by the present invention can quantify the change of the photoacoustic signal, which helps to accurately distinguish normal tissue from tumor tissue, high copper ion concentration tumor tissue from other tumor tissues. The nanoprobe is also wrapped with an amphiphilic polymer, which can greatly improve its stability, and can achieve accurate detection through photoacoustic imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the activation principle of the molecular probe provided by the present invention.

[0026] Figure 2 This is a synthetic route map of the molecular probe prepared in Example 1 of the present invention.

[0027] Figure 3 This is the synthesis route of the nanoprobe prepared in Example 2 of the present invention.

[0028] Figure 4 The pH response absorption spectrum (a) and copper ion response absorption spectrum (b) of the molecular probe prepared in Example 1 of the present invention.

[0029] Figure 5 (a) and (b) the hydrodynamic particle size diagram of the nanoprobe prepared in Example 2 of the present invention.

[0030] Figure 6 The pH response absorption spectrum of the nanoprobe prepared in Example 2 of the present invention (a) and the absorption spectrum under different copper ion concentration conditions (b).

[0031] Figure 7 The photoacoustic imaging image (a) and the quantitative result (b) of the nanoprobe prepared in Example 2 of the present invention.

[0032] Figure 8 The figure is a graph showing the toxicity test results of the nanoprobe prepared in Example 2 of the present invention on MDA-MB-231 cells at different concentrations.

[0033] Fig. 9This is the photoacoustic imaging image and quantitative result of the nanoprobe prepared in Example 2 of the present invention in an animal body. DETAILED DESCRIPTION

[0034] The present invention provides a molecular probe for detecting copper ions in vivo, a molecular probe and a preparation method and application thereof. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] The present invention provides a molecular probe, the chemical structure of which is as follows:

[0036]

[0037] The present invention provides a method for preparing a molecular probe, which comprises the following steps:

[0038] Mixing N-(2-bromomethyl)phthalimide and di(2-pyrrolyl)amine to undergo a first substitution reaction to obtain a first product, and subjecting the first product to a hydrolysis reaction to obtain a first intermediate;

[0039] Mixing 2,3,3-trimethyl-3H-indole with 3-iodopropyl acetate to undergo a second substitution reaction to obtain a second product, and mixing the second product with 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde to undergo a condensation reaction to obtain a second intermediate;

[0040] The first intermediate and the second intermediate are mixed to undergo a third substitution reaction to obtain a third intermediate;

[0041] causing the third intermediate to undergo ester hydrolysis reaction and ring closure reaction to obtain the molecular probe;

[0042] The structural formulas of the first intermediate, the second intermediate, the third intermediate and the molecular probe are as follows:

[0043]

[0044] In some embodiments, the temperature of the second substitution reaction is 50-150°C, and the time is 24-72h; the temperature of the second substitution reaction can be 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, and the time can be 24h, 30h, 35h, 40h, 45h, 50h, 55h, 60h, 65h, 70h, 72h;

[0045] The temperature of the condensation reaction is 50-100°C, and the time is 2-12h; the temperature of the condensation reaction can be 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, and the time can be 2h, 4h, 8h, 10h, 12h;

[0046] The solvent used in the second substitution reaction is one or more of acetonitrile, N,N-dimethylformamide, dichloromethane, chloroform, toluene, and o-dichlorobenzene;

[0047] The solvent used in the condensation reaction is one or more of acetic anhydride, anhydrous methanol, anhydrous ethanol, and anhydrous N,N-dimethylformamide.

[0048] In some embodiments, the temperature of the ring-closing reaction is 0-50°C, and the time is 2-12h; the temperature of the ring-closing reaction can be 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and the time can be 2h, 4h, 8h, 10h, 12h;

[0049] The solvent used in the ring-closing reaction is one or more of dichloromethane, methanol, ethanol, chloroform and N,N-dimethylformamide.

[0050] In some embodiments, the preparation method of the above molecular probe comprises the following steps:

[0051] Under the protection of an inert gas, N-(2-bromomethyl)phthalimide and potassium carbonate are dissolved in di(2-pyrrolyl)amine, and di(2-pyrrolyl)amine is then added dropwise to cause a first substitution reaction to obtain a first product, and hydrazine hydrate is added to the first product to cause a hydrolysis reaction of the first product to obtain a first intermediate;

[0052] Mixing 2,3,3-trimethyl-3H-indole with 3-iodopropyl acetate to undergo a second substitution reaction to obtain a second product, and then adding sodium acetate to the second product and 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde to undergo a condensation reaction to obtain a second intermediate;

[0053] mixing the first intermediate and the second intermediate to obtain a third intermediate;

[0054] Potassium carbonate and dichloromethane are sequentially added to the third intermediate to cause the third intermediate to undergo ester hydrolysis reaction and ring closure reaction to obtain the molecular probe.

[0055] The embodiment of the present invention provides a nanoprobe, wherein the nanoprobe includes nanoparticles and an amphiphilic polymer coated outside the nanoparticles, wherein the nanoparticles include a reference dye and the above-mentioned molecular probe. The imaging signal intensity of the reference dye is only related to the accumulation amount of the nanoparticles and has nothing to do with pH or copper ion concentration. Therefore, by ratiometric imaging of the molecular probe and the reference dye, the changes in pH and copper ion concentration can be better quantified.

[0056] In some embodiments, the nanoprobe can be a fluorescent imaging probe or a photoacoustic imaging probe.

[0057] In some embodiments, in the nanoprobe, the mass ratio of the molecular probe, the reference dye, and the amphiphilic polymer is 1:(0.5-1.0):(5-15).

[0058] This ratio range can ensure that the nanoprobes have better nanosize and uniformity.

[0059] In some preferred embodiments, the mass ratio of the molecular probe, the reference dye and the amphiphilic polymer is 1:0.5:10.

[0060] In some embodiments, the amphiphilic polymer is selected from one or more of phospholipid-methoxypolyethylene glycol, poloxamer 407, and polyethylene glycol-polylactic acid.

[0061] The phospholipid-methoxy polyethylene glycol in this embodiment can be distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 2000 means that the molecular weight of polyethylene glycol is 2000. The main function of the amphiphilic polymer is to coat the molecular probe with the reference dye to make it a water-soluble nanoprobe. The addition of the amphiphilic polymer improves the water solubility and stability of the nanoprobe.

[0062] In some embodiments, the reference dye is selected from one or more of 5,5'-dichloro-11-diphenylamino-3,3'-diethyl-10,12-vinylthiotricarbocyanine perchlorate, IR-780 iodide, and indocyanine green, but is not limited thereto.

[0063] In some embodiments, the nanoprobe is spherical in shape, and the diameter of the nanoprobe is 22-32 nm. The diameter of the nanoprobe can be 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm.

[0064] Nanoprobes within this diameter range have higher permeability and retention (EPR) effect in solid tumors.

[0065] The present invention provides a method for preparing the nanoprobe, which comprises the following steps:

[0066] The molecular probe, reference dye and amphiphilic polymer are dissolved in an organic solvent and stirred to obtain the nanoprobe.

[0067] In some embodiments, the method for preparing the nanoprobe includes the following steps: dissolving the molecular probe, reference dye and amphiphilic polymer in an organic solvent to obtain a mixed solution, then dropping the mixed solution into deionized water, stirring at 1000 rpm for 30 minutes at room temperature, then using nitrogen bubbling to remove the organic solvent, and then washing to obtain the nanoprobe.

[0068] In some embodiments, the organic solvent includes one or both of tetrahydrofuran and dichloromethane.

[0069] The embodiments of the present invention provide the use of the above-mentioned molecular probe and / or the isomers of the molecular probe in the preparation of tumor diagnosis products;

[0070] Or, use of the above-mentioned nanoprobe in the preparation of tumor diagnostic products.

[0071] It should be noted that the isomers of the molecular probe may be pharmaceutically acceptable enantiomers, diastereomers, and tautomers.

[0072] In some embodiments, the dosage form of the tumor diagnostic product is a capsule, a tablet, an oral preparation, an injection, a suppository, a spray or an ointment.

[0073] In some embodiments, the tumor is triple-negative breast cancer.

[0074] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and are only for illustrating the present invention but not limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0075] Example 1

[0076] Preparation of molecular probes, such as Figure 2 As shown, the following steps are included:

[0077] Synthesis of the first intermediate: Under nitrogen protection, 14g N-(2-bromoethyl)phthalimide and 16.8g potassium carbonate were dissolved in a two-necked reaction bottle containing 30mL dimethylformamide (DMF), and then 5mL di(2-pyrrolyl)amine was slowly added and heated to 95°C for reflux overnight. After the reaction was completed, it was cooled to room temperature, and the reaction mixture was poured into ice water and allowed to stand for 2h to obtain a crude product. After filtering and washing with ice water, the crude product was vacuum dried to obtain 12.2g of a yellow-brown solid product. It was then dissolved in 104.2mL of anhydrous methanol, 300mL of hydrazine hydrate solution was added, and heated to reflux for 4h. A white solid precipitate was obtained. After cooling to room temperature, concentrated hydrochloric acid was added, and the filtrate was obtained by filtering after 1h. The pH value of the filtrate was adjusted to 10 with 1M sodium hydroxide solution. The solution was extracted 5 times with ether, the organic phase was dried with magnesium sulfate, and the solvent was spin-dried to obtain a yellow oily first intermediate with a yield of 26%. 1 H NMR (400 MHz, CDCl 3 )δ8.55(d,J=4Hz,2H),7.65(t,J=8Hz,2H),7.48(d,J=4Hz,2H),7.16(t,J=8Hz,2H),3.86(s,4H),2.83(m,2H),2.72(m,2H),2.19(s,2H).

[0078] Synthesis of the second intermediate: 4.71 mmol of 3-iodoacetic acid propyl ester was added dropwise to an acetonitrile solution containing 3.14 mmol of 2,3,3-trimethyl-3H-indole, and the reaction system was refluxed for 1 day. After the reaction was completed, acetonitrile was removed, and then a mixed solvent of 22.5 mL of methanol (MeOH) and 2.5 mL of dichloromethane (DCM) was added. 10 mL of cold ether was added to the above solution, and the precipitated crystals formed were collected, washed with 3 mL of cold ether for 3 times, and vacuum dried to obtain 1.055 g of light brown solid with a yield of 87%. 1 H NMR (400 MHz, CDCl 3 )δ7.73(m,1H),7.59(m,3H),4.89(t,J=8Hz,2H),4.23(t,J=4Hz,2H),3.17(s,3H),2.38(m,2H),2.00(s,3H),1.67(s,6H). Then, under nitrogen protection, the above 1.3mmol of light brown solid and 0.65mmol of 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde were added to 5mL of acetic anhydride, and then 1.3mmol of sodium acetate was added, heated to 70°C, and reacted for 2h. After the reaction was completed, the obtained mixture was poured into cold ether, and the precipitate was collected to obtain 491mg of the second intermediate as a green solid with a yield of 96%.

[0079] 1 H NMR(500MHz,DMSO-d6)δ8.29(d,J=10Hz,2H),7.66(d,J=10Hz,2H),7.45(m,4H),7.30(m,2H),6.36(d,J=15Hz,2H), 4.30(t,J=7Hz,4H),4.09(t,J=6Hz,4H),2.72(t,J=5Hz,4H),2.08(m,4H),1.94(s,6H),1.86(m,2H),1.69(s,12H).

[0080] Synthesis of the third intermediate: Under nitrogen protection, first dissolve 0.60mmol of sodium hydride and 0.30mmol of the first intermediate in 2mL of DMF and stir for 20min under 0℃ ice bath conditions. Then add 1mL of DMF solution containing 0.10mmol of the second intermediate dropwise to obtain a mixed solution. The above mixed solution was stirred overnight at 50℃. When the color of the mixed solution changed from green to blue, the mixed solution was poured into ice water. Extract with DCM 5 times, collect and spin-dry the organic phase. Purify with silica gel column chromatography (developing agent ratio: dichloromethane / methanol = 97:3) to obtain a blue solid third intermediate with a yield of 46%.

[0081] 1 H NMR(500MHz,MeOD)δ8.47(d,J=5Hz,2H),7.80(td,J 1 =5Hz,J 2 =1.5Hz,2H),7.71(d,J=15Hz,2H),7.51(d,J=5Hz,2H),7.29(m,6H),7.02(m ,4H),5.80(d,J=15Hz,2H),5.34(t,J=5Hz,1H),4.14(t,J=5Hz,4H),4.02(t ,J=5Hz,8H),3.80(t,J=5Hz,2H),2.98(t,J=5Hz,2H),2.62(t,J=5Hz,4H),2.11(m,4H),2.01(s,6H),1.83(m,2H),1.53(s,12H); HRMS (ESI, positive ion mode)calcd for C 54 H 65 N 6 O 4 + 861.5062, found 861.50178; (negative ion mode) calcd for I - 126.9050, found 126.90382.

[0082] Synthesis of molecular probe: Under room temperature, 0.1 mmol of the third intermediate was dissolved in 2 mL of methanol to obtain a mixed solution. Then 0.2 mmol of potassium carbonate was added to the mixed solution, stirred for 1 hour, methanol was removed, and 10 mL of dichloromethane was added. The above solution was washed twice with 10 mL of saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Purification was performed by silica gel column chromatography (developing solvent ratio: dichloromethane / methanol = 95:5) to obtain a blue solid molecular probe with a yield of 20%.

[0083] 1 H NMR(500MHz,MeOD)δ8.48(d,J=5Hz,2H),7.81(td,J 1 =5Hz,J 2 =1.5Hz,2H),7.71(d,J=15Hz,2H),7.52(d,J=5Hz,2H),7.30(m,6H),7.03(m,4H),5.88(d,J=15Hz,2H),5.34(t,J=5Hz,1H),5.40(m,8H),3.79(t,J=5Hz,2H),3.66(t,J=5Hz,4H),2.97(t,J=10Hz,2H),2.60(t,J=10Hz,4H),1.95(m,4H),1.81(m,2H),1.52(s,12H); HRMS (ESI, positive ion mode) calcd for C 50 H 60 N 6 O 2 776.4778, found[M]861.50178.

[0084] Example 2

[0085] Preparation of nanoprobes, such as Figure 3 As shown, the method comprises the following steps: 1 mg of the molecular probe prepared in Example 1, 0.5 mg of IR140, and 10 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) are completely dissolved in tetrahydrofuran, and mixed thoroughly to obtain a mixed solution; then the mixed solution is added dropwise to 15 mL of deionized water, and stirred at 1000 rpm for 30 min to form a homogeneous phase. The above solution is then bubbled with nitrogen at a low speed to remove the residual tetrahydrofuran to obtain a transparent aqueous solution. The aqueous solution is filtered through a syringe-driven needle filter (0.22 μm), and centrifuged at 4° C. and 3500 rpm for 5 min using a 30 KDa ultrafiltration tube, and washed repeatedly 3 times to obtain the nanoprobe.

[0086] Example 3 Spectral Testing of Molecular Probes

[0087] The molecular probe prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a molecular probe mother solution with a concentration of 1 mM. 20 μL of the molecular probe mother solution was added to 980 μL of PBS buffer solutions with different pH values. The absorption spectra of the molecular probe under different pH conditions were obtained by UV-visible spectrophotometry ( Figure 4 ).

[0088] The UV absorption spectrum of the molecular probe changes with pH Figure 4 As shown in (a), it can be seen from the figure that the ultraviolet absorption intensity of the molecular probe at around 610 nm increases as the pH decreases.

[0089] Take 20 μL of molecular probe stock solution and add it to 980 μL of PBS buffer with different copper ion concentrations. Test it with UV-visible spectrophotometer to obtain the absorption spectra of molecular probe under different copper ion concentrations. Figure 4 As shown in (b), the absorption peak of the molecular probe at around 610 nm gradually red-shifts to around 700 nm as the copper ion concentration increases.

[0090] Example 4 Characterization of particle size and morphology of nanoprobes

[0091] The hydrated particle size and dispersion of the nanoprobes prepared in Example 2 were tested using a dynamic light scattering particle size analyzer (DLS). Figure 5 As shown in (a), the hydrated particle size of the nanoprobe is about 27±5 nm. The particle size and morphology of the nanoprobe were characterized using a transmission electron microscope (TEM). Figure 5 As shown in (b).

[0092] Example 5 Spectral Test of Nanoprobe

[0093] A nanoprobe solution with a concentration of 6 μM was prepared using PBS buffer (the nanoprobe was the nanoprobe prepared in Example 2), and a small amount of concentrated hydrochloric acid was added dropwise thereto to adjust the pH of the solution from pH 10 to pH 5.5. The absorption spectra of the nanoprobe under different pH conditions were obtained by UV-visible spectrophotometer testing ( Figure 6 ). The UV absorption spectrum of the nanoprobe as pH changes is shown in Figure 6 As shown in (a), it can be seen from the figure that the absorption intensity of the nanoprobe around 610nm increases with the decrease of pH, while the absorption peak around 830nm remains basically unchanged.

[0094] A nanoprobe solution with a concentration of 6 μM was prepared using PBS buffer (the nanoprobe was the nanoprobe prepared in Example 2), and a 0-15 μM copper ion solution was added dropwise thereto. The absorption spectra of the nanoprobe under different copper ion concentrations were obtained by UV-visible spectrophotometry ( Figure 6 ).from Figure 6 As shown in (b), the absorption peak of the nanoprobe at around 610 nm gradually red-shifts to around 700 nm as the copper ion concentration increases, while the absorption peak at around 830 nm remains basically unchanged.

[0095] Example 6 In vitro photoacoustic performance test of nanoprobes

[0096] The nanoprobe solution with a concentration of 500 μM was prepared using PBS buffer of pH 7.4 and pH 6, respectively (the nanoprobe was the nanoprobe prepared in Example 2), and three solution conditions were set, namely pH 7.4, pH 7.4+Cu 2+ and pH 6+Cu 2+ , the photoacoustic signal intensity of the nanoprobe under the above three different conditions was measured by photoacoustic imaging test ( Figure 7 ),like Figure 7 As shown in (a) and (b), the nanoprobe has a maximum photoacoustic signal at 720 nm under copper ion and acidic conditions, which is 2.48 times higher than that of the pH 7.4 group and 2.5 times higher than that of the pH 7.4+Cu 2+ The group increased by 1.56 times, indicating that the nanoprobe has good dual response performance to acidity and copper ions in photoacoustics.

[0097] Example 7 Evaluation of detection effect at the cell level

[0098] To measure the cytotoxicity of the nanoprobe prepared in Example 2 to MDA-MB-231 cells at different concentrations, MDA-MB-231 cells were cultured in the presence of 0, 1.25, 2.5, 5, 10, 15 and 20 μM nanoprobes (prepared in Example 2) for 12 hours, and the cytotoxicity was measured. The results are as follows: Figure 8 As shown, the cell survival rates were all above 80%, indicating that the materials had low cytotoxicity.

[0099] Example 8 In vivo photoacoustic performance test of nanoprobes

[0100] A group of tumor-bearing nude mice (n=3) were selected as the experimental group. Photoacoustic imaging was performed on the left thigh muscle and right tumor of the nude mice, and then 50 μL of a 6.5 mM nanoprobe solution (the nanoprobe was the nanoprobe prepared in Example 2) was injected into the left thigh muscle and right tumor of the nude mice, respectively. Photoacoustic imaging was performed 1 hour later. The results are as follows: Fig. 9As shown, compared with muscle, the ratio of 720nm to 820nm photoacoustic of the nanoprobe at the tumor site increased by 1.8 times, indicating that its photoacoustic is responsive to the tumor microenvironment, proving the feasibility of the nanoprobe to visualize high copper ion concentration and slightly acidic tumor microenvironment at the living level.

[0101] In summary, the nanoprobe for detecting triple-negative breast cancer provided by the present invention can achieve efficient and accurate tumor detection guided by photoacoustic imaging. By introducing pH and copper ion response groups, it has better specificity. At the same time, this tumor-specific activation property gives the nanoprobe a higher imaging signal-to-noise ratio, which is conducive to accurate detection.

[0102] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A molecular probe for detecting copper ions in vivo, characterized in that: The chemical structural formula of the molecular probe is as follows:

2. A method for preparing a molecular probe for detecting copper ions in vivo, characterized in that: The preparation method comprises the following steps: Mixing N-(2-bromomethyl)phthalimide and di(2-pyrrolyl)amine to undergo a first substitution reaction to obtain a first product, and subjecting the first product to a hydrolysis reaction to obtain a first intermediate; Mixing 2,3,3-trimethyl-3H-indole with 3-iodopropyl acetate to undergo a second substitution reaction to obtain a second product, and mixing the second product with 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde to undergo a condensation reaction to obtain a second intermediate; The first intermediate and the second intermediate are mixed to undergo a third substitution reaction to obtain a third intermediate; causing the third intermediate to undergo ester hydrolysis reaction and ring closure reaction to obtain the molecular probe; The structural formulas of the first intermediate, the second intermediate, the third intermediate and the molecular probe are as follows:

3. A nanoprobe, characterized in that: The nanoprobe comprises nanoparticles and an amphiphilic polymer coated outside the nanoparticles, and the nanoparticles comprise a reference dye and the molecular probe according to claim 1.

4. The nanoprobe according to claim 3, characterized in that: In the nanoprobe, the mass ratio of the molecular probe, the reference dye and the amphiphilic polymer is 1:(0.5-1.0):(5-15).

5. The nanoprobe according to claim 3, characterized in that: The amphiphilic polymer is selected from one or more of phospholipid-methoxy polyethylene glycol, α-hydrogen-ω-hydroxy poly(oxyethylene) a-poly(oxypropylene) b-poly(oxyethylene) c] block copolymer, and polyethylene glycol-polylactic acid.

6. The nanoprobe according to claim 3, characterized in that: The reference dye is selected from one or more of 5,5'-dichloro-11-diphenylamino-3,3'-diethyl-10,12-vinylthiotricarbocyanine perchlorate, IR-780 iodide, and indocyanine green.

7. The nanoprobe according to claim 3, characterized in that: The nanoprobe has a spherical shape and a diameter of 22-32 nm.

8. A method for preparing the nanoprobe according to claim 3, characterized in that: The preparation method comprises the following steps: The molecular probe, reference dye and amphiphilic polymer are dissolved in an organic solvent and stirred to obtain the nanoprobe.

9. Use of the molecular probe according to claim 1 and / or the isomers of the molecular probe in the preparation of tumor diagnostic products; Or, use of the nanoprobe according to any one of claims 3 to 7 in the preparation of tumor diagnostic products.

10. The use according to claim 9, characterized in that: The tumor is triple-negative breast cancer.

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