A method for preparing alloy catalytic clusters capable of targeting lysosomes

Abstract

The application belongs to the technical field of functional biological nanomaterials, and particularly relates to preparation and function illustration of alloy catalytic clusters (KCK-AuCdNCs) capable of targeting lysosomes and having high enzyme-like activity. The KCK-AuCdNCs are synthesized by designing a short peptide sequence, doping a cadmium element and using a water phase synthesis method. The KCK-AuCdNCs have an average particle size of 1.46+0.05 nm, an absorption peak at 580 nm, an emission peak at 650 nm under 365 nm laser excitation, non-flashing fluorescence, and are suitable for fluorescence labeling imaging. The KCK-AuCdNCs have peroxidase activity and antioxidant capacity, cell experiments prove that the KCK-AuCdNCs are quickly taken up by cells and target lysosomes, the co-localization Pearson coefficient reaches 0.921, and the KCK-AuCdNCs can efficiently remove active oxygen free radicals. The preparation method is simple, the cost is low, the cadmium element doping improves the activity, and is beneficial to lysosome imaging and active oxygen removal.

Description

Technical Field

[0001] This invention belongs to the field of functional bionanomaterials technology, specifically relating to the preparation and functional description of an alloy catalytic cluster capable of targeting lysosomes. A simple and green aqueous-phase synthesis method is used to prepare a red fluorescent alloy cluster with high enzyme-like activity and the ability to target cellular lysosomes. Background Technology

[0002] Peroxidases are a class of enzymes that catalyze the decomposition of hydrogen peroxide and other peroxides. They are widely present in organisms and participate in many important physiological and pathological processes. Currently, many non-biological materials possess peroxidase activity, such as metal nanoparticles, metal oxides, and organic molecules. However, clusters with peroxidase activity also have some drawbacks: (1) their catalytic efficiency is usually low, requiring high reaction temperatures and long reaction times; (2) their catalytic specificity is poor, and they may react with non-target substrates, leading to false positive or false negative results; (3) they may produce toxic side effects on cells and tissues, such as inducing oxidative stress, inflammatory responses, and apoptosis. Therefore, it is urgent to develop a nanomaterial with peroxidase activity that has high catalytic efficiency, good specificity, good biocompatibility, high stability, and low toxicity for use in fields such as biosensing, drug detection, and antibacterial and anti-inflammatory applications.

[0003] Lysosomes are vesicle structures within cells, primarily responsible for degrading and recycling cellular waste and foreign substances. They play crucial roles in maintaining cellular homeostasis, regulating signal transduction, and inducing apoptosis. However, most lysosome-targeting clusters suffer from limited selectivity and affinity, leading to off-target effects, toxicity, instability, and poor permeability, making it difficult to reach the target site. Furthermore, many fluorescent probes targeting lysosomes are weakly basic; their retention within lysosomes can cause an increase in pH, affecting normal lysosome function. Compared to traditional fluorescent materials, fluorescent nanomaterials exhibit stronger affinity, higher stability, and surface modification potential, making them more suitable for targeting lysosomes.

[0004] Based on the above discussion, designing a reasonable and effective approach to develop and synthesize a nanocluster with high peroxidase activity that can target lysosomes has significant theoretical and practical value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and to seek a method for preparing alloy nanoclusters with peroxidase activity and targeted labeling of cell lysosomes, so as to avoid the problems of nanoclusters not having enzyme-like activity and reducing the background interference of existing cell lysosome labeling materials on cell imaging, as well as the problems of high production cost, high toxicity and complex preparation process.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] (1) Reagents should be prepared and used immediately. All glass bottles should be soaked in aqua regia for 2 hours to ensure thorough cleaning. After soaking, rinse thoroughly with ultrapure water and dry in a 65°C oven with the bottle opening facing down for later use.

[0008] (2) Mix the newly prepared KCK solution (20 mM, 500 μL) and TCEP (20 mM, 500 μL) and react in a 70 °C water bath in the dark for 15 min. Then quickly add HAuCl4·4H2O solution (20 mM, 480 μL) and CdCl2 (20 mM, 20 μL), and react in a 70 °C water bath in the dark for 10 min.

[0009] (3) Then, add NaOH solution (1.5M, 50μL) and NaBH4 solution (0.1M, 8μL) to the glass bottle in sequence. Wait until the color of the solution in the glass bottle quickly changes from pale yellow to brown, then add 3.442mL of ultrapure water to the glass bottle to adjust the Au concentration. + The final concentration was 2 mmol / L. The glass bottle was placed in a constant temperature water bath and reacted at 70°C for 10 h. The initial product KCK-AuCdNCs was obtained.

[0010] (4) Purification: The product was continuously ultrafiltered by centrifugation at 10,000 rpm at low temperature using 3kDa and 30kDa ultrafiltration tubes to finally obtain KCK-AuCdNCs alloy clusters with molecular weight in the range of 3kDa to 30kDa.

[0011] (5) Freeze-drying: A portion of the sample was freeze-dried in advance to obtain solid powdered alloy catalytic clusters.

[0012] The method of the present invention has the following advantages:

[0013] (1) The alloy catalytic clusters involved in this invention have an average particle size of 1.46±0.05nm and a significant absorption peak at 580nm; under 365nm laser excitation, they exhibit a broad fluorescence emission spectrum with an emission peak at 650nm; their single-particle fluorescence is strong, they are not resistant to bleaching, have high brightness, and do not have scintillation characteristics; they have peroxidase activity and have a targeting effect on lysosomes in cells;

[0014] (2) Compared with existing methods and materials, the alloy catalytic cluster preparation method of the present invention is simple, green, highly operable and low cost; the doping of cadmium element improves the activity of the cluster. Compared with traditional nanoprobes with enzyme-like activity or relatively conventional aromatic ring-rich nanoprobes, this is a brand-new nanocluster, which provides new ideas and methods for the scavenging of oxygen free radicals in living cells and the study of lysosomes. Attached Figure Description

[0015] Figure 1The images shown are transmission electron microscope images (a) and particle size distribution diagrams (b) of the alloy catalytic clusters involved in this invention.

[0016] Figure 2 This invention relates to the ultraviolet-visible absorption spectrum of the alloy catalytic clusters;

[0017] Figure 3 This invention relates to the three-dimensional fluorescence spectrum of alloy catalytic clusters;

[0018] Figure 4 This invention relates to the trajectory of fluorescence intensity change over time of the alloy catalytic clusters;

[0019] Figure 5 The present invention relates to the peroxidase activity of alloy catalytic clusters with air as a substrate (a) and hydrogen peroxide as a substrate (b);

[0020] Figure 6 The present invention relates to the absorption (a) and rate change (b) of peroxidase substrate TMB products catalyzed by alloy catalytic clusters;

[0021] Figure 7 The images show the fluorescence images of the scavenging level of the alloy catalytic clusters involved in this invention on intracellular reactive oxygen species (a) blank group, before (b) and after (c) H2O2 treatment with the addition of the alloy catalytic clusters.

[0022] Figure 8 This is a colocalization imaging image (c) of the alloy catalytic cluster (a) and the green lysosomal fluorescent probe Lyso-TrackerGreen (b) involved in this invention, and the colocalization Pearson coefficient (d) and fluorescence intensity (e, f) of the two. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0024] Example 1

[0025] I. Preparation method of alloy catalytic clusters:

[0026] (1) Reagents should be prepared and used immediately. All glass bottles should be soaked in aqua regia for 2 hours to ensure thorough cleaning. After soaking, rinse thoroughly with ultrapure water and dry in a 65°C oven with the bottle opening facing down for later use.

[0027] (2) Mix the newly prepared KCK solution (20mM, 500μL) and TCEP solution (20mM, 500μL) and react in a 70℃ water bath in the dark for 15 min. Then quickly add HAuCl4·4H2O solution (20mM, 480μL) and CdCl2 (20mM, 20μL) and react in a 70℃ water bath in the dark for 10 min.

[0028] (3) Then, add NaOH solution (1.5M, 50μL) and NaBH4 solution (0.1M, 8μL) to the glass bottle in sequence. Wait until the color of the solution in the glass bottle quickly changes from pale yellow to brown, then add 3.442mL of ultrapure water to the glass bottle to adjust the Au concentration. + The final concentration was 2 mmol / L. The glass bottle was placed in a constant temperature water bath and reacted at 70°C for 10 h. The initial product KCK-AuCdNCs was obtained.

[0029] (4) Purification: The product was continuously ultrafiltered by centrifugation at 10,000 rpm at low temperature using 3kDa and 30kDa ultrafiltration tubes to finally obtain KCK-AuCdNCs alloy clusters with molecular weight in the range of 3kDa to 30kDa.

[0030] (5) Freeze-drying: A portion of the sample was freeze-dried in advance to obtain solid powdered alloy catalytic clusters.

[0031] like Figure 1-4 As shown, the alloy catalytic cluster particles prepared in this embodiment are uniformly dispersed with an average particle size of 1.46±0.05nm and a significant absorption peak at 580nm. Under 365nm laser excitation, it exhibits a broad fluorescence emission spectrum with an emission peak at 650nm. Its single-particle fluorescence is strong, it is not resistant to bleaching, has high brightness, and does not have flickering characteristics. It has peroxidase activity and a targeting effect on lysosomes in cells.

[0032] Application Example 1

[0033] The alloy catalytic cluster (KCK-AuCdNCs, 100 mg / L) prepared in step 1 was added to the chromogenic substrate of Amplite™ IR (100 μM, 1×PBS) peroxidase and H2O2 system (total system 100 μL). After incubation at room temperature for 0-30 minutes, the fluorescence intensity at Ex / Em = 640 / 680 nm was monitored.

[0034] Figure 5 For Amplite TM IR fluorescence method for detecting peroxidase activity of KCK-AuCdNCs alloy catalytic clusters. The blank contains only Amplite. TM The fluorescence intensity is measured with IR dye and hydrogen peroxide; the other two values ​​represent the presence or absence of alloy catalytic clusters. No hydrogen peroxide solution was added at this time, and the reaction occurring was alloy cluster-catalyzed Amplite. TMThe IR dye reacts with oxygen in the air for oxidation. The fluorescence intensity after 10 min of reaction is shown in the figure, with a fluorescence enhancement of 2.11 times. The peroxidase activity of KCK-AuCdNCs with hydrogen peroxide as a substrate increased by 3.83 times after adding KCK-AuCdNCs and reacting for 10 min, indicating that this alloy catalytic cluster possesses excellent peroxidase activity.

[0035] Application Example 2

[0036] 20 μL of the alloy catalytic cluster (KCK-AuCdNCs, 100 mg / L) prepared in step 1 was added to 100 μL of TMB single-component substrate solution. The reaction was carried out at 37 °C, and the OD350 absorbance of the product was measured every 10 min using a Nanodrop ultra-micro UV-Vis spectrophotometer to obtain the change in catalytic activity of the alloy catalytic cluster over time.

[0037] In Figure 6, the absorbance at 350°C in (a) represents the content of blue formazan produced by the peroxidase substrate TMB. As time increases, the amount of formazan produced increases, and the growth trend in Figure (b) gradually slows down. The enzyme activity is strongest within the first 40 minutes of the reaction when the substrate and enzyme content are sufficient. It can be seen that the alloy catalytic cluster KCK-AuCdNCs has peroxidase activity.

[0038] Application Example 3

[0039] Commercially available reactive oxygen species (ROS) scavenging probe DCFH-DA (10 μM / L) was co-incubated with human cervical cancer cells (HeLa) at 37°C and 5% CO2 for 20 min. After stimulation with H2O2 (1%, diluted 1×PBS) for 20 min, the alloy catalytic cluster (KCK-AuCdNCs, 0.1 mM) prepared in Example 1 was incubated for 1 h. Imaging was performed using confocal fluorescence microscopy under the following conditions: 100× oil immersion, 405 nm laser excitation, and fluorescence signals were collected between 662 and 737 nm.

[0040] Figure 7The images show fluorescence imaging of intracellular reactive oxygen species (ROS) levels in the KCK-AuCdNCs alloy catalytic cluster and the DCFH-D reactive oxygen species scavenging probe. In the blank group (a), the green fluorescence in normal HeLa cells without H2O2 is minimal, indicating a low level of ROS. After H2O2 treatment (b), the green fluorescence significantly increases, indicating a substantial rise in intracellular ROS levels. After treatment with the KCK-AuCdNCs alloy cluster, the green fluorescence significantly weakens, as shown in (c), reflecting a reduction in intracellular ROS. Under the same conditions, cells stained with the alloy catalytic cluster in the 662–737 nm channel show a bright red color. This experiment demonstrates that the presence of the alloy catalytic cluster significantly reduces ROS levels, proving that the alloy catalytic cluster has the ability to scavenge ROS and protect normal cells in living cells, with a significant staining effect.

[0041] Application Example 4

[0042] The alloy catalytic clusters (KCK-AuCdNCs, 100 mg / L) prepared in step 1 were co-incubated with human cervical cancer cells (HeLa) at 37 °C and 5% CO2 for 2 h. After fixation with 4% paraformaldehyde, the cells were imaged using a confocal fluorescence microscope under the following conditions: 100× oil immersion, 405 nm laser excitation, and fluorescence signals between 662 and 737 nm were collected.

[0043] Commercially available green lysosomal fluorescent probe (Lyso-Tracker Green, 10 nM) was co-incubated with human cervical cancer cells (HeLa) at 37°C and 5% CO2 for 2 h. After fixation with 4% paraformaldehyde, the cells were imaged using a confocal fluorescence microscope under the following conditions: 100× oil immersion, 488 nm laser excitation, and fluorescence signals between 500 and 530 nm were collected.

[0044] Figure 8 The image shows the co-localization imaging of the alloy catalytic cluster KCK-AuCdNCs (b) and the green lysosomal fluorescent probe Lyso-Tracker Green (a). The image shows the intracellular localization overlap of KCK-AuCdNCs and Lyso-Tracker Green (c). A portion of the co-localization was selected for Pearson coefficient verification; the Recolor (d) result, close to the diagonal, is 0.921. Furthermore, the fluorescence intensity maps (e, f) of the two labeling methods are very similar, demonstrating that the alloy catalytic cluster KCK-AuCdNCs can target and label lysosomes in cells.

[0045] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing alloy catalytic clusters targeting lysosomes, characterized in that, The steps are as follows: (1) Prepare reagents immediately before use. Soak all glass bottles in aqua regia for 2 hours to ensure thorough cleaning. After soaking, rinse thoroughly with ultrapure water and dry in a 65°C oven with the bottle opening facing down for later use. (2) Mix 500 μL of newly prepared 20 mM KCK solution and 500 μL of 20 mM TCEP solution, and react in a 70°C water bath in the dark for 15 min; then quickly add 480 μL of 20 mM HAuCl4·4H2O and 20 μL of 20 mM CdCl2, and then react in a 70°C water bath in the dark for 10 min. (3) Then, 50 μL of 1.5 M NaOH solution and 8 μL of 0.1 M NaBH4 solution were added to the glass bottle in sequence. The color of the solution in the glass bottle quickly changed from pale yellow to brown. 3.442 mL of ultrapure water was added to the glass bottle to adjust the Au concentration in the solution. + The final concentration was 2 mmol / L; the glass bottle was placed in a constant temperature water bath and reacted at 70℃ for 10 h; the initial product KCK-AuCdNCs was obtained. (4) Purification: The KCK-AuCdNCs primary product was continuously ultrafiltered by centrifugation at 10,000 rpm at low temperature using 3kDa and 30kDa ultrafiltration tubes to finally obtain KCK-AuCdNCs alloy clusters with molecular weight in the range of 3kDa to 30kDa. (5) Freeze-drying: A portion of the sample was freeze-dried in advance to obtain solid powder KCK-AuCdNCs alloy clusters; The KCK-AuCdNCs alloy clusters have an average particle size of 1.46±0.05nm and a significant absorption peak at 580nm. Under 365nm laser excitation, they exhibit a broad fluorescence emission spectrum with an emission peak at 650nm. Their single-particle fluorescence is strong, they are not resistant to bleaching, have high brightness, and do not exhibit flickering characteristics. They also possess peroxidase activity and have a targeting effect on lysosomes in cells.

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