Enzyme response near-infrared two-region fluorescence / computed tomography dual-mode imaging probe based on gold nano-clusters and preparation method and application of enzyme response near-infrared two-region fluorescence / computed tomography dual-mode imaging probe

Abstract

The invention discloses an enzyme response near-infrared two-region fluorescence / computed tomography dual-mode imaging probe based on a gold nano-cluster and a preparation method and application of the enzyme response near-infrared two-region fluorescence / computed tomography dual-mode imaging probe. The probe comprises: a gold nanocluster capable of simultaneously providing near-infrared two-region fluorescence and computed tomography imaging signals; the polypeptide molecule is modified on the surface of the material. The polypeptide comprises a cathepsin B response sequence, biotin with a targeting function and a click reaction unit 2-cyanobenzothiazole. The probe disclosed by the invention is subjected to enzyme digestion and trigger click reaction under the action of highly expressed CTSB in liver cancer cells, so that the gold nanoclusters are subjected to cross-linked aggregation at tumor parts, the retention time of the probe is prolonged, NIR-II fluorescence and CT imaging effects are remarkably enhanced, and high-sensitivity detection on liver cancer is realized.

Description

Technical Field

[0001] This invention relates to an enzyme-responsive near-infrared fluorescence / computed tomography dual-modal imaging probe based on gold nanoclusters, its preparation method and application, belonging to the field of nanobiomaterials. Background Technology

[0002] Timely identification and accurate in vivo measurement of small tumors are crucial for clinical staging, surgical margin design, and efficacy monitoring. Currently, multi-phase enhanced computed tomography (CT) is widely used in clinical practice for diagnosis, but its sensitivity to sub-centimeter tumors is limited. The contrast effects of different phases (e.g., arterial phase, portal venous phase, and delayed phase) are often inconsistent; some small tumors may only appear briefly in one phase, while appearing as isodense images in other phases, thus reducing the contrast between the tumor and surrounding liver tissue and affecting detection sensitivity and measurement reliability. Compared to CT, fluorescence imaging offers higher molecular sensitivity. Especially under near-infrared window (NIR-II, 1000–1700 nm) imaging conditions, due to reduced tissue scattering and decreased autofluorescence signal, deeper imaging can be achieved, producing higher tumor-background contrast. However, residual photon scattering still leads to a decrease in spatial resolution, making NIR-II fluorescence imaging less accurate than CT in anatomical localization. Based on this, the NIR-II / CT dual-modal imaging strategy, which combines the advantages of both, is expected to provide reliable anatomical information while maintaining high molecular sensitivity, thereby significantly improving the early detection capability of small hepatocellular carcinoma lesions.

[0003] In recent years, in-situ self-assembly technology has been recognized as an effective signal amplification technique in tumor imaging. Small molecule precursors (such as organic molecules or nanoparticles) can self-assemble under the influence of the tumor microenvironment (such as acidic environment, redox substances, and specific enzymes), forming larger nanostructures at the lesion site. This strategy can effectively delay systemic clearance, prolong intratumoral retention time, and significantly improve imaging contrast without the need for large amounts or high doses of imaging agents. Commonly used self-assembly mechanisms mostly rely on click chemistry reactions, among which the 2-cyanobenzothiazole-cysteine ​​(CBT-Cys) reaction has attracted much attention due to its good biocompatibility and rapid reaction kinetics. This reaction has been designed to respond to a variety of tumor-associated enzymes (such as cathepsin B, matrix metalloproteinases, and aminopeptidase) and applied to the construction of multimodal probes, including fluorescence, Raman, photoacoustic, magnetic resonance, and CT imaging.

[0004] However, there are currently no near-infrared II fluorescence / computed tomography (NIR-II / CT) dual-modal imaging probes targeting liver cancer-related enzyme responses that have been applied clinically. Significant research and application gaps remain in this field. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an enzyme-responsive near-infrared fluorescence / computed tomography dual-modal imaging probe based on gold nanoclusters, its preparation method and application, which improves the sensitivity of detecting small lesions in liver cancer.

[0006] Technical Solution: To solve the above-mentioned technical problems, the present invention provides an enzyme-responsive near-infrared fluorescence / computed tomography dual-modal imaging probe based on gold nanoclusters. The probe comprises: a gold nanocluster with 25 or more gold atoms and a polypeptide molecule grafted onto the surface of the gold nanocluster; the polypeptide molecule has an azide group and a targeting group biotin; the probe can detect in situ lesions with a minimum diameter of 1-4 mm.

[0007] The gold nanoclusters are ligand-protected gold nanoclusters with a size of sub-2 nanometers.

[0008] The gold nanoclusters include diphenylcyclooctyne-modified gold nanoclusters. Only diphenylcyclooctyne-modified gold nanoclusters can click-link with peptides containing azide groups.

[0009] The amino acid sequence of the polypeptide molecule is Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT.

[0010] In this process, Val-Cit in the polypeptide molecule is selectively cleaved as a specific substrate of CTSB; while Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT can achieve the self-assembly of gold nanoclusters through CBT-Cys click reaction.

[0011] The present invention also provides a method for preparing the enzyme-responsive near-infrared II fluorescence / computed tomography dual-modal imaging probe based on gold nanoclusters, comprising the following steps: (1) Preparation of gold nanoclusters modified with diphenylcyclooctyne: Mercaptohexanoic acid solution and cysteine ​​solution were added to ultrapure water and mixed. Chloroauric acid solution was added dropwise under stirring, followed by sodium hydroxide solution. Sodium borohydride solution was added, and the reaction was terminated by adding sodium hydroxide solution. After purification, it was reacted with diphenylcyclooctyn-active ester to obtain diphenylcyclooctyn-modified gold nanoclusters after purification. (2) Synthesis of polypeptide molecules: The polypeptide sequence Val-Cit-Cys(StBu)-Lys(NH2)-Gly-Gly-Lys(N3) containing a CTSB-specific cleavage substrate was synthesized by solid-phase synthesis. Biotin was grafted onto the side chain of the polypeptide and combined with the amino acid Gly containing a 2-cyanobenzothiazole group to obtain the polypeptide molecule Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT. (3) Preparation of enzyme-responsive dual-modal imaging probes based on gold nanoclusters: The polypeptide molecule Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT described in step (2) was added to the gold nanocluster solution modified with diphenylcyclooctyne described in step (1), stirred evenly, and then purified.

[0012] The present invention also provides the application of the enzyme-responsive near-infrared II fluorescence / computed tomography dual-modal imaging probe based on gold nanoclusters in the imaging of liver cancer cells.

[0013] Among them, liver cancer cells highly express cathepsin B (CTSB).

[0014] This probe can be efficiently delivered to liver tumor sites under the action of the targeting group. After entering liver cancer cells, the highly expressed active enzyme cleaves the polypeptide, triggering the CBT-Cys click reaction, thereby self-assembling into larger nanoparticles, which enhances their NIR-II fluorescence and CT imaging signals, thus achieving accurate detection of liver cancer.

[0015] The active enzymes include one or more of cathepsin B, aminopeptidase N, cysteine-aspartic protease, serine protease, gamma-glutamyl transferase, and furin protease.

[0016] The targeting groups include one or more of biotin, folic acid, RGD peptide, and galactose / lactose.

[0017] The present invention also provides the application of the gold nanocluster-based enzyme-responsive near-infrared II fluorescence / computed tomography dual-modal imaging probe in near-infrared II fluorescence / computed tomography dual-modal imaging.

[0018] Principle of this invention: Figure 1As shown, this invention discloses a CTSB-specific responsive NIR-II fluorescence and CT imaging dual-modal imaging probe, comprising a gold nanocluster with NIR-II fluorescence and CT imaging functions; a polypeptide molecule grafted onto the surface of the gold nanocluster; wherein the probe is delivered to the liver tumor site under the action of a targeting group, and self-assembles under the action of an active enzyme highly expressed in the liver cancer cells to form larger nanoparticles.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. The probe of this invention has targeting capabilities; 2. The probe of this invention is responsive to cancer biomarkers, greatly reducing the possibility of false positives; 3. The probe of this invention has assembly and aggregation capabilities at the lesion site, which can greatly improve the signal intensity of dual-modal imaging. Through a CTSB-triggered "intelligent" self-assembly strategy, the probe can assemble from small particles into large particles at the tumor site. Combined with biotin-mediated efficient targeted delivery, this not only prolongs the probe's residence time in the tumor but also significantly enhances the imaging signal. In animal experiments, the probe exhibits simultaneous enhancement of NIR-II fluorescence and CT dual-modal signals, achieving accurate detection of liver cancer lesions. 4. The probe of this invention can detect extremely small lesions (a few millimeters in diameter). 5. The probe of this invention has good biocompatibility and a relatively fast in vivo metabolic rate, showing high potential for clinical application. 6. Compared with single-modal imaging, the advantage of dual-modal imaging lies in combining the real-time performance of NIR-II with the high resolution and high penetration depth of CT. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the enzyme-responsive self-assembly and imaging enhancement mechanism of the dual-modal imaging nanoprobe at the tumor site in an embodiment of the present invention. Figure 2 These are the mass spectrometry characterization results of polypeptide molecules in the embodiments of the present invention; Figure 3 This is a transmission electron microscope image of the dual-modal imaging nanoprobe in an embodiment of the present invention; Figure 4 This is a transmission electron microscope image of the dual-modal imaging nanoprobe after enzymatic digestion in an embodiment of the present invention; Figure 5 This is the cell viability detection result after co-incubation of HCCLM3 cells with the dual-modal imaging nanoprobe in this embodiment of the invention; Figure 6 The above are the NIR-II fluorescence and CT imaging results of HCCLM3 cells after taking up the nanoprobes in this embodiment of the invention. Figure 7 The images show the NIR-II fluorescence and CT imaging results of the tumor site in an orthotopic liver cancer mouse model after the nanoprobe was injected via the tail vein, as described in this embodiment of the invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1: Synthesis of a Dual-Modal Imaging Probe 1. Synthesis and purification of gold nanoclusters 1.5 mL of mercaptohexanoic acid solution (5 mM) and 0.9 mL of cysteine ​​solution (5 mM) were added to 2.35 mL of ultrapure water and mixed thoroughly. Then, under continuous stirring, 250 μL of chloroauric acid solution (20 mM) was added dropwise to the ligand solution. Next, 50 μL of sodium hydroxide solution (1 M) was added to the mixture. After stirring for 2 hours, 50 μL of sodium borohydride solution (11.4 mM) was added to the reaction system at 40°C. After reacting for 15 minutes, 150 μL of sodium hydroxide solution (1 M) was added to terminate the reaction. Finally, the obtained gold nanoclusters were purified and concentrated by ultrafiltration centrifugation (50 kDa).

[0023] 2. Synthesis and purification of diphenylcyclooctyne-modified gold nanoclusters 200 μL of diphenylcyclooctyn-active ester solution (100 μM) was added to 200 μL of gold nanocluster solution (1 mM) synthesized in the previous step. After stirring uniformly at room temperature for 12 hours, the sample was purified by ultrafiltration centrifugation (50 kDa) to remove excess unreacted diphenylcyclooctyn-active ester solution.

[0024] 3. Synthesis and purification of polypeptide molecules (1) A polypeptide molecule with the sequence FmocVal-Cit-Cys(StBu)-Lys(NH2)-Gly-Gly-Lys(N3) was synthesized using a solid-phase peptide synthesizer: First, 2-chlorotriphenylmethyl chloride resin (2 g) was swollen in 20 mL of N,N-dimethylformamide (DMF) for 30 minutes; then, the first amino acid, Fmoc-Lys(N3)-OH, was grafted onto the resin in a DMF solution containing N,N-diisopropylethylamine (DIPEA, 800 μL) for 1 hour. After washing three times with DMF to remove ungrafted amino acids from the reaction solution, a capping solution (DMF:methanol = 19:1, v / v) was added, and the reaction was carried out for 30 minutes to cap the resin; then, the Fmoc protecting group was removed with 20% piperidine-DMF solution for 30 minutes, and then washed three times with DMF to remove residual piperidine from the reaction solution. Next, the next amino acid, Fmoc-Gly-OH (3.2 mmol), was coupled to the free amino group using 457 mg 1-hydroxybenzotriazole (HOBt) / 1282 mg O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) / 1.3 mL DIPEA as coupling agents. Peptide growth was achieved by repeating these coupling and deprotection steps; finally, after washing with DMF (5 times), isopropanol (5 times), and n-hexane (5 times), the synthesized peptide was cleaved from the resin with 95% trifluoroacetic acid-dichloromethane solution over 30 minutes. The cleaved product was purified using a semi-preparative high-performance liquid chromatograph (Agilent Technologies, model: 1290 Infinity II, flow rate 10 mL / min, detection wavelengths 220 nm and 254 nm, injection volume: 4 mL). Specifically, the peptide molecules containing impurities were purified using a water-acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase eluent (from 60:40 to 0:100).

[0025] (2) Synthesis of Gly-CBT: Isobutyl chloroformate (IBCF) (168.6 μL, 1.3 mM) was added to 5 mL of a tetrahydrofuran (THF) mixture containing Fmoc-Gly-OH (297.3 mg, 1 mM) and 4-methylmorpholine (MMP) (285.8 μL, 2.6 mM) at 0°C. The mixture was stirred at 0°C for 1 hour. Then, 2-cyano-6-aminobenzothiazole (CBT, 192.7 mg, 1.1 mM) dissolved in 5 mL of THF was added, and stirring was continued at 0°C for 2 hours. Finally, the reaction mixture was stirred overnight at room temperature. The cleaved product was purified using semi-preparative high-performance liquid chromatography (HPLC). Specifically, the polypeptide molecules containing impurities were purified using a water-acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase eluent (from 20:80 to 0:100).

[0026] (3) Synthesis of FmocVal-Cit-Cys(StBu)-Lys(NH2)-Gly-Gly-Lys(N3)-Gly-CBT: Weigh 58 mg Gly-CBT (0.25 mM), 236 mg FmocVal-Cit-Cys(StBu)-Lys(NH2)-Gly-Gly-Lys(N3) (0.25 mM), 41 mg 1-hydroxy-7-azobenzotriazole (HOAt) (0.3 mM), and 114 mg 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (0.3 mM) and dissolve them in 2 mL DMF. Then, measure 153 μL LIPEA and add it to the reaction solution. React in a 40°C water bath for 12 hours. The cleaved product was purified using a semi-preparative high-performance liquid chromatography (HPLC) system. Specifically, the impurity-containing polypeptide molecules were purified using a water-acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase eluent (from 60:40 to 0:100).

[0027] (4) Synthesis of FmocVal-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT: Weigh 61 mg Biotin (0.25 mM), 259 mg FmocVal-Cit-Cys(StBu)-Lys(NH2)-Gly-Gly-Lys(N3)-Gly-CBT (0.25 mM), 41 mg 1-hydroxy-7-azobenzotriazole (HOAt) (0.3 mM), and 114 mg 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (0.3 mM) and dissolve them in 2 mL DMF. Then, measure 153 μL of DIPEA and add it to the reaction solution. React in a 40°C water bath for 12 hours. The cleaved product was purified using a semi-preparative high-performance liquid chromatography (HPLC) system. Specifically, the impurity-containing polypeptide molecules were purified using a water-acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase eluent (from 60:40 to 0:100).

[0028] (5) Synthesis of Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT: The peptide molecule FmocVal-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT was dissolved in 3 mL of DMF. A 12% piperidine solution was added at 0°C and reacted for 7 minutes to remove the Fmoc protecting group. Then, 360 μL of trifluoroacetic acid (TFA) was added to neutralize the alkaline solution. The cleaved product was purified using a semi-preparative high-performance liquid chromatography (HPLC). Specifically, a water-acetonitrile solution containing 0.1% trifluoroacetic acid was used as the mobile phase eluent (from 60:40 to 0:100) to purify the peptide molecule containing impurities. The structural characterization results of the peptide molecule are as follows: Figure 2 As shown, it is in line with expectations.

[0029] 4. Probe synthesis and purification A 100 μM solution of a peptide molecule with the sequence Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT was added to a 100 μM solution of gold nanoclusters modified with diphenylcyclooctyne. After stirring uniformly at room temperature for 12 hours, the sample was purified by ultrafiltration centrifugation (50 kDa) to remove excess unreacted peptide molecules, thus obtaining the final dual-modal imaging probe.

[0030] Example 2: Observation of micro-nanostructures of dual-modal imaging probes The probe was diluted tenfold and dropped onto a carbon-coated copper grid, then air-dried overnight. The image was then observed using a JEM-2100 transmission electron microscope. Figure 3 As shown, the synthesized probe has a size of approximately 2-3 nanometers, and exhibits good dispersibility and uniform size.

[0031] Example 3: Enzyme response characteristics test of dual-modal imaging probe Tris(2-carbonylethyl) phosphate hydrochloride (TECP) solution (8 mM) was added to 1 mL of 0.25 mg / mL dual-modal imaging probe solution. After sonication for one hour, 100 μL of 10 U / mL cathepsin B solution was added. The mixture was reacted in a shaker at 37°C for 24 hours. The reaction solution was then diluted tenfold and dropped onto a carbon-coated copper grid, and air-dried overnight. TEM images were then obtained using a JEM-2100 transmission electron microscope. Figure 4 As shown, gold clusters aggregate into nanoparticles with a size of about 30 nm.

[0032] Example 4: CCK-8 assay for HCCLM3 cell viability HCCLM3 cells were cultured in DMEM medium containing 10% FBS. HCCLM3 cells were seeded in 96-well plates (5000 cells / well). After 24 hours of culture, different concentrations of dual-modal imaging probe solutions (0, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2 mg / mL) were added to the plates. After another 24 hours, 100 µL of DMEM medium containing 10 µL / mL LCK-8 was used to replace the medium in each well. After incubation in a cell culture incubator for 2 hours, the absorbance density (OD) at 450 nm was measured using a microplate reader. Each experiment was performed at least three times, and the results are shown below. Figure 5 As shown, even with a probe concentration as high as 2 mg / mL, cell viability can still be maintained at around 80%.

[0033] Example 5: Dual-modal imaging probes for HCCLM3 cell imaging experiments HCCLM3 cells were cultured in DMEM medium containing 10% FBS. HCCLM3 cells were seeded in cell culture dishes (8,000,000 cells per dish) and cultured for 24 hours. Then, 1 mg / mL of dual-modality imaging probe solution was added to the cell culture dishes. After 24 hours, the medium was removed, and the cells obtained from digestion and centrifugation were dispersed in PBS solution containing 1.5 wt% agarose for NIR-II fluorescence and CT imaging observation. The results are as follows: Figure 6 As shown, both near-infrared II fluorescence and CT signals are enhanced.

[0034] Example 6: Establishment of an animal model Four-week-old female BALB / c nude mice (16±2 g) were selected; all experimental procedures complied with the provisions of the "Regulations on the Management of Laboratory Animals of the People's Republic of China"; to construct a tumor-bearing mouse model, 5×10 7 HCCLM3 cells (suspended in 100 μL PBS) were subcutaneously injected into the back of each mouse. After 10 days, when the tumor volume reached approximately 100 mm... 3 At that time, the tumor was removed and divided into sections of approximately 1 mm. 3 Tumor tissue blocks were extracted. These tissue blocks were transplanted into the xiphoid region of the mouse liver, the wound was sutured, and the mice were returned to the SPF environment for continued rearing.

[0035] Example 7: Evaluation of the in vivo tumor imaging effect of the probe Seven days after orthotopic liver tumor transplantation in mice, 100 µL of a probe at a concentration of 2 mg / mL was injected via the tail vein. NIR-II fluorescence and CT imaging results were collected at 2, 4, 6, 12, 24, 48, 72, and 144 hours post-injection. The results are as follows: Figure 7 As shown, in the near-infrared II fluorescence imaging results, it can be clearly seen that the tumor site was illuminated 3 hours after probe injection, while in the CT imaging results, the boundary of the lesion site is clearer, and the diameter of the lesion area is approximately 2 millimeters.

Claims

1. A gold nanocluster-based enzyme-responsive near-infrared two-region fluorescence / computed tomography bimodal imaging probe, characterized in that, The probe comprises: a gold nanocluster with 25 gold atoms or more and a polypeptide molecule grafted on the surface of the gold nanocluster; the polypeptide molecule has an azide group and a targeting group biotin. 2.The enzyme-responsive near-infrared two-region fluorescence / computed tomography bimodal imaging probe based on gold nanoclusters according to claim 1, wherein, The gold nanocluster is a ligand-protected gold nanocluster with a size of less than 2 nm. 3.The enzyme-responsive near-infrared two-region fluorescence / computed tomography bimodal imaging probe based on gold nanoclusters of claim 1, wherein, The gold nanocluster is a diphenyl cyclooctyne-modified gold nanocluster. 4.The enzyme-responsive near-infrared two-region fluorescence / computed tomography bimodal imaging probe based on gold nanoclusters of claim 1, wherein, The amino acid sequence of the polypeptide molecule is Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT.

5. A method for preparing the gold nanoclusters-based enzyme-responsive near-infrared two-region fluorescence / computed tomography bimodal imaging probe according to claim 3, characterized in that, The method comprises the following steps: adding the polypeptide molecule Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT to a solution of the diphenyl cyclooctyne-modified gold nanocluster, stirring until uniform, and then purifying.

6. The method of claim 5, wherein, The preparation of the diphenyl cyclooctyne-modified gold nanocluster comprises the following steps: mixing a mercaptohexanoic acid solution and a cysteamine solution in ultrapure water, adding a chloroauric acid solution dropwise under stirring, and then adding a sodium hydroxide solution; adding a sodium borohydride solution, stopping the reaction by adding a sodium hydroxide solution, and then reacting with a diphenyl cyclooctyne-active ester or an alkynyl-active ester after purification to obtain the diphenyl cyclooctyne-modified gold nanocluster.

7. The method of claim 5, wherein, The polypeptide molecule synthesis comprises the following steps: synthesizing a polypeptide sequence Val-Cit-Cys(StBu)-Lys(NH2)-Gly-Gly-Lys(N3) containing a CTSB-specific cleavage substrate by a solid-phase synthesis method; grafting biotin to a side chain of the polypeptide to obtain a polypeptide molecule Val-Cit-Cys(StBu)-Lys(Biotin)-Gly-Gly-Lys(N3)-Gly-CBT by combining an amino acid Gly containing a 2-cyanobenzothiazole group.

8. Use of the gold nanocluster-based enzyme-responsive near-infrared second region fluorescence / computed tomography bimodal imaging probe according to any one of claims 1 to 4 in imaging of liver cancer cells.

9. Use of the gold nanocluster-based enzyme-responsive near-infrared second region fluorescence / computed tomography bimodal imaging probe according to any one of claims 1 to 4 in near-infrared second region fluorescence / computed tomography bimodal imaging.

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