A molecular probe for in vivo copper ion detection, its preparation method and application

Abstract

This invention discloses a molecular probe for detecting copper ions in vivo, its preparation method, and its application. The chemical structure of the molecular probe is as follows: The molecular probe provided by this invention can specifically respond to copper ions in the slightly acidic environment of tumors, thereby causing changes in the absorption spectrum and activating photoacoustic signals, which helps to accurately distinguish between normal tissues and tumor tissues, and realize the precise detection of copper ions in living tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical synthesis technology, and in particular to a molecular probe for detecting copper ions in vivo, its preparation method, and its application. Background Technology

[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 is often referred to as the "most aggressive" breast cancer. Its defining characteristic is that immunohistochemical examination of the cancer tissue shows negative results for estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene HER-2. This type of breast cancer accounts for 10.0%-20.8% of all breast cancer pathological types, exhibiting unique biological behavior and clinicopathological features, and has a poorer prognosis than other types. Accurate identification of triple-negative breast cancer is crucial for developing personalized treatment plans and implementing precise treatment. Currently, the diagnosis of triple-negative breast cancer typically employs a combination of diagnostic methods, including pathological examination and genetic testing, ductoscopy, breast ultrasound, and magnetic resonance imaging (MRI). Pathological examination and genetic testing usually require obtaining tumor tissue through biopsy or surgery, inevitably causing physical trauma to the patient, and the subsequent processing procedures, such as tumor immunohistochemical staining and gene sequencing, are complex. While imaging examinations offer advantages in cancer diagnosis, such as being non-invasive, providing direct visualization and clarity, high sensitivity, and aiding in diagnosis, current imaging techniques like MRI, breast ultrasound, and mammography still have certain limitations. For example, while MRI has high sensitivity, its specificity is relatively low. Breast ultrasound has relatively low resolution, making it difficult to accurately detect or clearly visualize small breast lesions, especially early-stage, small TNBC tumors, easily leading to missed diagnoses. Mammography involves radiation exposure; masses near the periphery of the breast may not be detected due to limitations in the projection location. Furthermore, the variability in breast imaging characteristics sometimes results in false positives.

[0003] Therefore, there is an urgent need for an imaging method that is non-invasive, sensitive, specific, and can rapidly present examination results. Photoacoustic imaging is a novel non-invasive and non-ionizing optical imaging technology that has emerged in recent years. Combining the characteristics of pure optical tissue imaging and pure ultrasound tissue imaging, photoacoustic imaging can achieve both micrometer-level resolution and centimeter-level penetration depth, and has advantages such as high chemical selectivity and real-time imaging. It is expected to fill the gaps in clinical imaging technology and provide new opportunities for the diagnosis of triple-negative breast cancer. Regarding probe design, triple-negative breast cancer lacks overexpression of estrogen receptors (ER, PR, HER-2), and there are currently no clinically approved targeted agents. Therefore, it is difficult to achieve accurate detection of triple-negative breast cancer through conventional molecular targeting design approaches. Improving the imaging sensitivity of photoacoustic probes while discovering new biomarkers for triple-negative breast cancer is key to solving these problems. Summary of the Invention

[0004] Alterations in cellular metabolism are closely related to the occurrence and development of cancer. Compared to normal tissue cells, tumors take up large amounts of glucose and break it down into lactic acid through glycolysis, forming a weakly acidic tumor microenvironment. Furthermore, recent studies have found that triple-negative breast cancer cells have a higher copper ion concentration than normal cells. In view of the shortcomings of the existing technology, the purpose of this invention is to provide a molecular probe, its preparation method, and its application, aiming to solve the problems of low sensitivity and specificity in current triple-negative breast cancer detection.

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

[0006] In a first aspect, the present invention provides a molecular probe for detecting copper ions in vivo, the chemical structural formula of which 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] N-(2-bromomethyl)phthalimide was mixed with di(2-pyrrolyl)amine to undergo a first substitution reaction to give a first product. The first product was then hydrolyzed to give a first intermediate.

[0009] 2,3,3-trimethyl-3H-indole was mixed with propyl 3-iodoacetate, and a second substitution reaction was carried out to give a second product. The second product was then mixed with 2-chloro-3-(hydroxymethylene)-cyclohex-1-encarbaldehyde, and a condensation reaction was carried out to give a second intermediate.

[0010] The first intermediate is mixed with the second intermediate, and a third substitution reaction occurs to obtain the third intermediate;

[0011] The third intermediate is subjected to ester hydrolysis and ring-closing reactions to obtain the molecular probe.

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

[0013]

[0014] A third aspect of the present invention provides a nanoprobe comprising nanoparticles and an amphiphilic polymer coated on the nanoparticles, wherein the nanoparticles comprise a reference dye and the aforementioned molecular probe.

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

[0016] Preferably, the amphiphilic polymer is selected from one or more of phospholipid-methoxy polyethylene glycol, α-hydro-ω-hydroxypoly(oxyethylene)a-poly(oxypropylene)b-poly(oxyethylene)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-vinylthiatricarbonine perchlorate, IR-780 iodide, and indocyanine green.

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

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

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

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

[0022] Alternatively, the aforementioned nanoprobes may be used in the preparation of tumor diagnostic products.

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

[0024] Beneficial effects: This invention provides a molecular probe for the detection of copper ions in vivo, its preparation method, and its application. This molecular probe exhibits dual response characteristics to both copper ions and acids. Its activation principle is as follows: Figure 1As shown, when the molecular probe is nanoscaled and injected intratumorally, the acid-responsive group of the molecular probe changes from a closed-ring form to an open-ring form in the microacidic environment of the tumor (pH < 7), and the absorption intensity at around 610 nm gradually increases with decreasing pH. Furthermore, the molecular probe exhibits copper ion responsiveness, causing its absorption peak to redshift further from 610 nm to around 700 nm, and the absorption intensity increases with increasing copper ion concentration. Since the photoacoustic signal is proportional to the absorption intensity, the photoacoustic signal also increases accordingly. The pH of the normal tissue microenvironment is 7.4, and the acid-responsive group cannot open the ring, therefore, near-infrared light absorption is undetectable in normal tissue. Using a non-responsive reference dye as a reference, the nanoprobe containing the above-mentioned molecules provided by this invention can quantify changes in the photoacoustic signal, helping to accurately distinguish between normal tissue and tumor tissue, and between tumor tissue with high copper ion concentration and other tumor tissues. The nanoprobe is also encapsulated with an amphiphilic polymer, which greatly improves its stability, enabling precise detection through photoacoustic imaging. Attached Figure Description

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

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

[0027] Figure 3 This is a synthetic route diagram 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 this invention.

[0029] Figure 5 The hydrodynamic particle size distribution (a) and TEM image (b) of the nanoprobe prepared in Example 2 of this invention are shown.

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

[0031] Figure 7 Photoacoustic imaging (a) and quantitative results (b) of the nanoprobe prepared in Example 2 of the present invention.

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

[0033] Figure 9Photoacoustic imaging and quantitative results of the nanoprobe prepared in Example 2 of this invention in an animal. Detailed Implementation

[0034] This invention provides a molecular probe for the detection of copper ions in vivo, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] This invention provides a molecular probe, the chemical structural formula of which is as follows:

[0036]

[0037] This invention provides a method for preparing a molecular probe, the method comprising the following steps:

[0038] N-(2-bromomethyl)phthalimide was mixed with di(2-pyrrolyl)amine to undergo a first substitution reaction to give a first product. The first product was then hydrolyzed to give a first intermediate.

[0039] 2,3,3-trimethyl-3H-indole was mixed with propyl 3-iodoacetate, and a second substitution reaction was carried out to give a second product. The second product was then mixed with 2-chloro-3-(hydroxymethylene)-cyclohex-1-encarbaldehyde, and a condensation reaction was carried out to give a second intermediate.

[0040] The first intermediate is mixed with the second intermediate, and a third substitution reaction occurs to obtain the third intermediate;

[0041] The third intermediate is subjected to ester hydrolysis and ring-closing reactions to obtain the molecular probe.

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

[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, or 72h.

[0045] The condensation reaction is carried out at a temperature of 50-100℃ for 2-12 hours; the condensation reaction temperature can be 50℃, 60℃, 70℃, 80℃, 90℃, or 100℃, and the time can be 2 hours, 4 hours, 8 hours, 10 hours, or 12 hours.

[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 closed-loop reaction is carried out at a temperature of 0-50°C for a time of 2-12 hours; the closed-loop reaction temperature 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 2 hours, 4 hours, 8 hours, 10 hours, or 12 hours.

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

[0050] In some embodiments, the method for preparing the above-mentioned molecular probe includes the following steps:

[0051] Under inert gas protection, N-(2-bromomethyl)phthalimide and potassium carbonate were dissolved in di(2-pyrrole)amine, and then di(2-pyrrole)amine was added dropwise to undergo a first substitution reaction to obtain a first product. Hydrazine hydrate was added to the first product to cause a hydrolysis reaction to obtain a first intermediate.

[0052] 2,3,3-trimethyl-3H-indole was mixed with propyl 3-iodoacetate to undergo a second substitution reaction to give a second product. The second product was then mixed with 2-chloro-3-(hydroxymethylene)-cyclohex-1-encarbaldehyde and sodium acetate was added to undergo a condensation reaction to give a second intermediate.

[0053] The first intermediate is mixed with the second intermediate to obtain the third intermediate;

[0054] Potassium carbonate and dichloromethane are added sequentially to the third intermediate to induce ester hydrolysis and ring-closing reactions, thereby obtaining the molecular probe.

[0055] This invention provides a nanoprobe comprising nanoparticles and an amphiphilic polymer coating the nanoparticles. The nanoparticles include a reference dye and the aforementioned molecular probe. The imaging signal intensity of the reference dye is only related to the accumulation of nanoparticles and is independent of pH or copper ion concentration. Therefore, ratiometric imaging using the molecular probe and reference dye allows for better quantification of changes in pH and copper ion concentration.

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

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

[0058] This range of ratios ensures that the nanoprobes have good nanoscale size and uniformity.

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

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

[0061] In this embodiment, the phospholipid-methoxy polyethylene glycol can be distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), where 2000 indicates that the molecular weight of the polyethylene glycol is 2000. The main function of the amphiphilic polymer is to coat the molecular probe with the reference dye, making 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-vinylthiatricarbonyl perchlorate, IR-780 iodide, and indocyanine green. However, it is not limited thereto.

[0063] In some embodiments, the nanoprobe has a spherical morphology and a diameter of 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, or 32 nm.

[0064] Nanoprobes within this diameter range exhibit high permeability and retention (EPR) effects in solid tumors.

[0065] This invention provides a method for preparing the above-mentioned nanoprobe, the method comprising the following steps:

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

[0067] In some embodiments, the preparation method of 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 adding the mixed solution dropwise to deionized water, stirring at 1000 rpm for 30 min at room temperature, then removing the organic solvent by bubbling with nitrogen, and then washing to obtain the nanoprobe.

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

[0069] This invention provides the application of the above-described molecular probes and / or isomers of the molecular probes in the preparation of tumor diagnostic products;

[0070] Alternatively, the aforementioned nanoprobes may be used in the preparation of tumor diagnostic products.

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

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

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

[0074] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely some embodiments of the present invention, not all embodiments, and are intended only to illustrate the present invention and not to limit it. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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, 14 g of N-(2-bromoethyl)phthalimide and 16.8 g of potassium carbonate were dissolved in a two-necked reaction flask containing 30 mL of dimethylformamide (DMF), and then 5 mL of di(2-pyrrole)amine was slowly added dropwise. The mixture was heated to 95 °C and refluxed overnight. After the reaction was complete, the mixture was cooled to room temperature, and then poured into ice water and allowed to stand for 2 h to obtain a crude product. The crude product was filtered, washed with ice water, and dried under vacuum to obtain 12.2 g of a yellowish-brown solid product. This solid was then dissolved in 104.2 mL of anhydrous methanol, and 300 mL of hydrazine hydrate solution was added. The mixture was heated to reflux for 4 h to obtain a white solid precipitate. After cooling to room temperature, concentrated hydrochloric acid was added, and the mixture was filtered after 1 h to obtain a filtrate. The pH of the filtrate was adjusted to 10 with 1 M sodium hydroxide solution. The solution was extracted five times with diethyl ether, the organic phase was dried with magnesium sulfate, and the solvent was evaporated to obtain a yellow, oily first intermediate in 26% yield. 1 H NMR (400MHz, CDCl3) 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 propyl 3-iodoacetate was added dropwise to an acetonitrile solution containing 3.14 mmol of 2,3,3-trimethyl-3H-indole. The reaction system was refluxed for 1 day. After the reaction was complete, the 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 diethyl ether was then added to the above solution, and the formed precipitate was collected. The precipitate was washed three times with 3 mL of cold diethyl ether and dried under vacuum to give 1.055 g of a light brown solid, with a yield of 87%. 1 ¹H NMR (400MHz, CDCl₃) δ 7.73 (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, 1.3 mmol of the above light brown solid and 0.65 mmol of 2-chloro-3-(hydroxymethylene)-cyclohexyl-1-encarbaldehyde were added to 5 mL of acetic anhydride, followed by the addition of 1.3 mmol of sodium acetate. The mixture was heated to 70 °C and reacted for 2 h. After the reaction was complete, the resulting mixture was poured into cold diethyl ether, and the precipitate was collected to give 491 mg of a green solid second intermediate, with a yield of 96%.

[0079] 1H 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, 0.60 mmol of sodium hydride and 0.30 mmol of the first intermediate were dissolved in 2 mL of DMF and stirred for 20 min at 0 °C in an ice bath. Then, 1 mL of DMF solution containing 0.10 mmol of the second intermediate was added dropwise to obtain a mixed solution. The above mixed solution was stirred overnight at 50 °C. When the color of the mixed solution changed from green to blue, it was poured into ice water. Extracted five times with DCM, the organic phase was collected and evaporated to dryness. Purified by silica gel column chromatography (developing solvent ratio: dichloromethane / methanol = 97:3) to give a blue solid third intermediate in 46% yield.

[0081] 1 H NMR(500MHz,MeOD)δ8.47(d,J=5Hz,2H),7.80(td,J1=5Hz,J2=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 N6O4 + 861.5062, found 861.50178; (Negative ion mode) calcd for I - 126.9050, found 126.90382.

[0082] Synthesis of the molecular probe: At 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, and after stirring for 1 h, the methanol was removed, followed by the addition of 10 mL of dichloromethane. The 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. The solution was then purified 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,J1=5Hz,J2=1.5Hz,2H),7.71(d,J=1 5Hz,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 N6O2 776.4778, found[M]861.50178.

[0084] Example 2

[0085] Preparation of nanoprobes, such as Figure 3 The procedure includes 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 thoroughly mixed 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 solution is then bubbled slowly with nitrogen to remove residual tetrahydrofuran, yielding a clear 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. The washing process is repeated three times to obtain the nanoprobe.

[0086] Example 3: Spectroscopic testing of molecular probes

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

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

[0089] 20 μL of the molecular probe stock solution was added to 980 μL of PBS buffer with different copper ion concentrations. The absorption spectra of the molecular probe under different copper ion concentrations were obtained using a UV-Vis spectrophotometer. 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 concentration of copper ions increases.

[0090] Example 4: Particle size and morphology characterization of nanoprobes

[0091] The hydrated particle size and dispersity of the nanoprobes prepared in Example 2 were tested using a dynamic light scattering particle size analyzer (DLS), and the results are as follows: Figure 5 As shown in (a), the hydrated particle size of the nanoprobe is approximately 27 ± 5 nm. The particle size and morphology of the nanoprobe were characterized using transmission electron microscopy (TEM), and the results are as follows: Figure 5 As shown in (b).

[0092] Example 5: Spectroscopic Testing of Nanoprobes

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

[0094] A 6 μM nanoprobe solution (the nanoprobe prepared in Example 2) was prepared using PBS buffer. Then, a 0-15 μM copper ion solution was added dropwise to the solution. The absorption spectra of the nanoprobe under different copper ion concentrations were obtained using a UV-Vis spectrophotometer. 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 concentration of copper ions increases, while the absorption peak at around 830 nm remains basically unchanged.

[0095] Example 6: In vitro photoacoustic performance testing of nanoprobes

[0096] Nanoprobe solutions (the nanoprobes prepared in Example 2) with a concentration of 500 μM were prepared using PBS buffer at pH 7.4 and pH 6, respectively. Three solution conditions were set: pH 7.4, pH 7.4 + Cu 2+ and pH 6+Cu 2+ The photoacoustic signal intensity of the nanoprobe was measured under the three different conditions described above using photoacoustic imaging. Figure 7 ),like Figure 7 As shown in (a) and (b), the nanoprobe exhibits the 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 higher than that of the pH 7.4+Cu group. 2+ The group improved by 1.56 times, indicating that the nanoprobe has good dual response performance to acid and copper ions in photoacoustics.

[0097] Example 7: Evaluation of Detection Results at the Cell Level

[0098] To measure the cytotoxicity of the nanoprobes prepared in Example 2 against MDA-MB-231 cells at different concentrations, MDA-MB-231 cells were cultured for 12 hours in media containing nanoprobes (prepared in Example 2) at concentrations of 0, 1.25, 2.5, 5, 10, 15, and 20 μM, respectively. The cytotoxicity was then measured, and the results are as follows: Figure 8 As shown, the cell survival rate was above 80%, indicating that the material has low cytotoxicity.

[0099] Example 8: In vivo photoacoustic performance testing 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 the right tumor of the mice, respectively. Then, 50 μL of a 6.5 mM nanoprobe solution (the nanoprobe prepared in Example 2) was injected into the left thigh muscle and the right tumor, respectively. Photoacoustic imaging was performed 1 hour later, and the results are as follows. Figure 9 As shown, compared to muscle, the photoacoustic ratio of 720nm to 820nm at the tumor site was increased by 1.8 times, indicating that its photoacoustic response is tumor microenvironment-responsive, demonstrating the feasibility of visualizing the high copper ion concentration and slightly acidic tumor microenvironment at the in vivo level.

[0101] In summary, the nanoprobe for detecting triple-negative breast cancer provided by this invention enables efficient and accurate tumor detection guided by photoacoustic imaging. By introducing pH and copper ion responsive groups, it exhibits better specificity. Furthermore, this tumor-specific activation characteristic endows the nanoprobe with a higher imaging signal-to-noise ratio, facilitating accurate detection.

[0102] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

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 includes the following steps: N-(2-bromomethyl)phthalimide was mixed with di(2-pyrrolyl)amine to undergo a first substitution reaction to give a first product. The first product was then hydrolyzed to give a first intermediate. 2,3,3-trimethyl-3H-indole was mixed with propyl 3-iodoacetate, and a second substitution reaction was carried out to give a second product. The second product was then mixed with 2-chloro-3-(hydroxymethylene)-cyclohex-1-encarbaldehyde, and a condensation reaction was carried out to give a second intermediate. The first intermediate is mixed with the second intermediate, and a third substitution reaction occurs to obtain the third intermediate; The third intermediate is subjected to ester hydrolysis and ring-closing reactions to obtain the molecular probe. The structural formulas of the first intermediate, the second intermediate, the third intermediate, and the molecular probe are shown below: 。 3. A nanoprobe, characterized in that, The nanoprobe includes nanoparticles and an amphiphilic polymer coated on the nanoparticles, wherein the nanoparticles include a reference dye and the molecular probe of claim 1.

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

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

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

7. The use of the molecular probe according to claim 1 in the preparation of a diagnostic product for triple-negative breast cancer; Alternatively, the use of the nanoprobe according to any one of claims 3-5 in the preparation of a triple-negative breast cancer diagnostic product.

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