Red fluorescent silicon nanodots, their preparation methods and applications

Abstract

This invention discloses a red fluorescent silicon nanodot, its preparation method, and its application. The method includes the following steps: S1, dissolving 2-nitro-4-aminodiphenylamine in anhydrous ethanol, then adding 3-aminopropyltriethoxysilane and stirring until homogeneous; S2, transferring the mixture obtained in step S1 to a reaction vessel and reacting under heating; S3, after the reactants have cooled to room temperature, eluting and purifying them using a silica gel chromatography column; S4, lyophilizing the purified product to obtain red fluorescent silicon nanodots. The red fluorescent silicon nanodots provided by this invention not only possess photostability and high quantum yield but also contain lipophilic cations, enabling them to target mitochondria in living cells; furthermore, these silicon nanodots also have the ability to penetrate the blood-brain barrier; the invented red fluorescent silicon nanodots can be applied to mitochondrial dynamic tracking imaging and blood-brain barrier penetration imaging, and are expected to provide a potentially powerful tool for diagnosing mitochondrial-related brain diseases.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials, and in particular to a red fluorescent silicon nanodot, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the primary site of aerobic respiration in eukaryotic cells, not only provide energy but are also closely related to various cellular activities, such as signal transduction, calcium storage, and regulation of the intracellular environment. Furthermore, mitochondria participate in the exchange of substances with other organelles, such as endoplasmic reticulum-mitochondrial contact regulating lipid flux and mitochondrial-lysosomal contact clearing damaged mitochondria. Mitochondrial dysfunction can directly lead to cell degeneration and even death. More severe mitochondrial disorders can also cause various diseases such as diabetes, arrhythmia, and Alzheimer's disease. Therefore, real-time monitoring of the dynamic changes in mitochondria is of great significance for studying mitochondrial function and diagnosing diseases.

[0003] Fluorescent probes are considered the preferred materials for mitochondrial imaging due to their excellent targeting and real-time feedback capabilities. Several commercial fluorescent dyes have been developed, such as Rhodamine 123 (Rho 123) and Mito-Tracker Green (MTG). However, these organic dyes typically have poor photobleaching resistance, and their fluorescence intensity gradually decreases under continuous excitation, making it impossible to continuously observe the dynamic changes in mitochondria. Although these commercial fluorescent probes can target mitochondria, there are few reports of them penetrating the blood-brain barrier (BBB). Given the association between brain diseases such as Alzheimer's disease and mitochondria, the inability to penetrate the BBB cannot further meet the needs of imaging mitochondrial-related brain diseases.

[0004] Silicon nanodots (SiNDs) are an emerging nanomaterial that has attracted widespread attention due to their non-toxic and environmentally friendly properties. Simultaneously, the excellent optical properties of SiNDs (good photostability, high quantum yield, and tunable emission wavelength) have also spurred their rapid development in the field of fluorescent probes. It is well known that fluorescent materials with red light emission possess unique advantages in imaging, such as low phototoxicity, high tissue permeability, and low interference from biological autofluorescence. Although SiNDs have made progress in detection and imaging, most of their fluorescence emission is in the blue to yellow range. Reports on red-emitting SiNDs are scarce, indicating the importance of designing novel SiNDs with excellent optical properties. Furthermore, small-diameter SiNDs with positively charged surfaces and amphiphilic properties can meet the basic requirements for penetrating the blood-brain barrier, but reports on their direct application in blood-brain barrier penetration imaging are limited. Therefore, developing novel fluorescent probes with blood-brain barrier penetration capability and excellent optical properties is of great significance for long-term imaging. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a red fluorescent silicon nanodot, its preparation method and application, in order to address the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing red fluorescent silicon nanodots, comprising the following steps:

[0007] S1. Dissolve 2-nitro-4-aminodiphenylamine in anhydrous ethanol, then add 3-aminopropyltriethoxysilane and stir until homogeneous;

[0008] S2. Transfer the mixture obtained in step S1 to a reaction vessel, and then react it under heating;

[0009] S3. After the reactants have cooled to room temperature, they are purified by elution using a silica gel chromatography column.

[0010] S4. The purified product obtained in step S3 is freeze-dried to obtain red fluorescent silicon nanodots.

[0011] Preferably, step S1 specifically includes:

[0012] Weigh 0.1-0.4 g of 2-nitro-4-aminodiphenylamine, dissolve it in 10-40 mL of anhydrous ethanol, then add 0.5-2.0 mL of 3-aminopropyltriethoxysilane and stir until homogeneous.

[0013] Preferably, step S2 specifically includes:

[0014] Transfer the mixture obtained in step S1 to a reaction vessel, place the reaction vessel in a drying oven, and heat it at 160-200℃ for 6-24 hours.

[0015] Preferably, step S3 specifically includes:

[0016] S3. After the reactants have cooled to room temperature, they are purified by elution using a silica gel chromatography column. The eluent consists of dichloromethane and methanol.

[0017] Preferably, the volume ratio of dichloromethane to methanol in the eluent is 5:1 to 15:1.

[0018] Preferably, the volume ratio of dichloromethane to methanol in the eluent is 10:1.

[0019] Preferably, the method for preparing the red fluorescent silicon nanodots includes the following steps:

[0020] S1. Weigh 0.2g of 2-nitro-4-aminodiphenylamine and place it in a beaker. Add 20mL of anhydrous ethanol to dissolve it, then add 1.0mL of 3-aminopropyltriethoxysilane and stir until well mixed.

[0021] S2. Transfer the mixture obtained in step S1 to a reaction vessel, place the reaction vessel in a drying oven, and heat it at 180°C for 12 hours.

[0022] S3. After the reactants have cooled to room temperature, they are purified by elution using a silica gel chromatography column. The eluent consists of dichloromethane and methanol in a volume ratio of 10:1.

[0023] S4. The purified product obtained in step S3 is freeze-dried to obtain red fluorescent silicon nanodots, which are then stored at 4°C.

[0024] The present invention also provides a red fluorescent silicon nanodot, which is prepared by the method described above.

[0025] The present invention also provides an application of the red fluorescent silicon nanodots described above in mitochondrial dynamic tracking imaging.

[0026] The present invention also provides an application of the red fluorescent silicon nanodots as described above in blood-brain barrier penetration imaging.

[0027] The beneficial effects of this invention are:

[0028] This invention provides a novel silicon nanodot with red fluorescence emission. This silicon nanodot not only has photostability and high quantum yield (22.86%), but also contains lipophilic cations, enabling it to target mitochondria in living cells. Furthermore, this silicon nanodot also has the ability to penetrate the blood-brain barrier.

[0029] The R-SiNDs prepared in this invention have good targeting ability for mitochondria. In addition, zebrafish imaging shows that the R-SiNDs can penetrate the blood-brain barrier without any ligands. Therefore, the red fluorescent silicon nanodots of this invention can be applied to mitochondrial dynamic tracking imaging and blood-brain barrier penetration imaging. They are expected to provide a potentially powerful tool for diagnosing mitochondrial-related brain diseases and have important biomedical application value. Attached Figure Description

[0030] Figure 1 The results show the structural and fluorescence properties of the red fluorescent silicon nanodots (R-SiNDs) prepared in Example 1. Figure 1 In the middle: (a) FT-IR spectrum of R-SiNDs, (b) XPS spectrum of R-SiNDs, (c) Zeta potential of R-SiNDs, (d) UV absorption spectrum, optimal excitation and emission spectrum of R-SiNDs, (e) Fluorescence emission spectrum of R-SiNDs under different excitations, (f) CIE color coordinates of R-SiNDs;

[0031] Figure 2This is a schematic diagram showing the distribution of R-SiNDs in PANC-1 cells after staining with R-SiNDs following the addition of CCCP and 4% PFA, respectively. Figure 2 (a) Confocal microscopy image of cells incubated with CCCP, (b) Confocal microscopy image of PANC-1 cells pretreated with R-SiNDs, (c) Confocal microscopy image of cells incubated with 4% PFA; Scale bar: 10 μm;

[0032] Figure 3 The results of testing R-SiNDs applied to mitochondrial dynamic monitoring. Figure 3 (a) Confocal microscopy image of mitochondria after staining PANC-1 cells with R-SiNDs; (b) R-SiNDs-labeled mitochondrial fusion process; (c) R-SiNDs-labeled mitochondrial fission process; (d) Schematic diagram of mitochondrial fusion and fission; Scale bar: 10 μm;

[0033] Figure 4 These are confocal microscope images of wild-type zebrafish cultured using R-SiNDs. Figure 4 In the middle: (a) the area above the white dashed line is the central nervous system region, and the area below is the notochord region; (b) the white arrow above is the central canal of the zebrafish spinal cord, and the white arrow below is the notochord region; scale bar: 20μm. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0035] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0037] Example 1

[0038] A red fluorescent silicon nanodot is prepared by the following steps:

[0039] S1. Weigh 0.2g of 2-nitro-4-aminodiphenylamine and place it in a beaker. Add 20mL of anhydrous ethanol to dissolve it, then add 1.0mL of 3-aminopropyltriethoxysilane and stir until well mixed.

[0040] S2. Transfer the mixture obtained in step S1 to a reaction vessel, place the reaction vessel in a drying oven, and heat it at 180°C for 12 hours.

[0041] S3. After the reactants have cooled to room temperature, they are purified by elution using a silica gel chromatography column. The eluent consists of dichloromethane and methanol in a volume ratio of 10:1.

[0042] S4. The purified product obtained in step S3 is freeze-dried to obtain red fluorescent silicon nanodots, denoted as R-SiNDs, which are stored at 4°C for later use.

[0043] Sample characterization and performance testing

[0044] (1) Structure and fluorescence properties

[0045] The FT-IR and XPS characterization results of R-SiNDs are as follows: Figure 1 As shown in Figures a and 1b, the characteristic peaks of silicon and the presence of silicon-based functional groups prove the successful synthesis of silicon nanodots; the measured potential of R-SiNDs is 12.8 ± 1.1 mV. Figure 1 c) This indicates that the R-SiNDs surface is positively charged; from Figure 1 As can be seen from d, the maximum excitation wavelength of silicon nanodots is 470nm, and the maximum emission peak is 614nm. Figure 1 The "e" indicates that the fluorescence emission of silicon nanodots is excitation-independent; its red fluorescence emission is also consistent with the analysis results of the International Commission on Illumination (CIE). Figure 1 f).

[0046] (2) Targeting mechanism and dynamic tracking of silicon nanodots to mitochondria

[0047] Mitochondrial membrane potential is fundamental to maintaining normal mitochondrial function. In this invention, CCCP and 4% PFA were selected to verify the mechanism by which R-SiNDs target mitochondria. Figure 2 (a-2c) In the control group, the mitochondrial membrane potential was normal, and the R-SiNDs-labeled mitochondria appeared as clear filaments. When CCCP was added to lower the membrane potential, the R-SiNDs in the cytoplasm became blurred, but did not completely disappear. Under 4% PFA fixation treatment, the mitochondrial membrane potential decreased, and the permeability of the nuclear membrane increased after disruption, leading to the appearance of R-SiNDs in the cytoplasm and nucleus. Combined with the positive potential of R-SiNDs in the characterization results, it indicates that the targeting effect of R-SiNDs on mitochondria mainly depends on electrostatic interactions.

[0048] Furthermore, lipophilicity is an essential characteristic of mitochondrial-targeting dyes. The log P values ​​of the two precursors, APTES and NAP, used in the experiment were 1.37 and 2.42, respectively, indicating that they both possess good lipophilicity. Introducing these two structures into R-SiNDs allows the R-SiNDs to maintain lipophilicity similar to that of the precursors. In summary, the mitochondrial-targeting properties of the R-SiNDs prepared in this invention, like most mitochondrial-targeting dyes, benefit from their lipophilic cations.

[0049] Based on their excellent resistance to photobleaching, R-SiNDs can be applied to the dynamic monitoring of mitochondria. Figure 3 a represents the long-term tracking imaging results of mitochondria using R-SiNDs as a fluorescent probe; Figure 3 As shown in the white circle in b, the fusion process of mitochondria can be seen, with several close branches gradually merging into a complete curve; Figure 3 c records the fission process occurring in another region, where a complete filamentous mitochondria eventually splits into three fragments, a fact confirmed by the relative fluorescence intensity image; a schematic diagram of this fusion and fission process is shown below. Figure 3 As shown in d.

[0050] (3) Silicon nanodots for zebrafish blood-brain barrier penetration imaging

[0051] After soaking zebrafish in a culture medium solution containing R-SiNDs for 5 hours, the areas imaged by R-SiNDs were observed under a confocal microscope. The distribution of R-SiNDs in the central nervous system (CNS) was clearly visible. Figure 4 a). Simultaneously, R-SiNDs were also observed in the central canal of the spinal cord ( Figure 4 b) indicates that R-SiNDs have the ability to penetrate the blood-brain barrier. Since R-SiNDs do not require any ligand coupling to achieve blood-brain barrier penetration, it is speculated that this process involves passive diffusion.

[0052] The permeation mechanism is related to the properties of the blood-brain barrier itself. First, although the endothelial cells forming the blood-brain barrier are tightly connected, there is still a 4-6 nm gap between them, while the particle size of R-SiNDs is 2.74 nm, smaller than the gap between the epithelial cells, which facilitates passive diffusion. Second, the negatively charged endothelial cells of the blood-brain barrier will attract positively charged R-SiNDs near the barrier through electrostatic attraction, and then increase the permeability of the blood-brain barrier through charge neutralization. Third, amphiphilic substances tend to penetrate the blood-brain barrier, and the amino and hydroxyl functional groups on the surface of R-SiNDs ensure their hydrophilicity. At the same time, the APTES structure used in this experiment contains -(CH2)3-, and the introduction of the weakly polar n-propyl group enhances the lipophilicity of R-SiNDs. Another precursor, NAP (log P = 2.42), also has suitable lipophilicity. The introduction of these two precursors gives R-SiNDs good hydrophilicity and lipophilicity, making it an amphiphilic substance. In summary, the small particle size, positive charge, and amphiphilicity contribute to the ability of R-SiNDs to penetrate the blood-brain barrier, which lays the foundation for the diagnosis of brain-related diseases.

[0053] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing red fluorescent silicon nanodots, characterized in that, The method comprises the following steps: S1, 2-nitro-4-amino diphenylamine is dissolved in anhydrous ethanol, and 3-aminopropyl triethoxysilane is added and stirred uniformly; S2, the mixture obtained in step S1 is transferred to a reaction kettle, and then reacted under heating; S3, after the reactant is cooled to room temperature, it is purified by elution with a silica gel chromatographic column; S4, the purified product obtained in step S3 is freeze-dried to obtain red fluorescent silicon nanodots.

2. The method for preparing red fluorescent silicon nanodots according to claim 1, characterized in that, Step S1 is specifically: 0.1-0.4g of 2-nitro-4-amino diphenylamine is weighed, dissolved in 10-40mL of anhydrous ethanol, and then 0.5-2.0ml of 3-aminopropyl triethoxysilane is added and stirred uniformly.

3. The method for preparing red fluorescent silicon nanodots according to claim 1, characterized in that, Step S2 is specifically: The mixture obtained in step S1 is transferred to a reaction kettle, the reaction kettle is placed in a drying box, and the reaction is heated at 160-200°C for 6-24 hours.

4. The method for preparing red fluorescent silicon nanodots according to claim 1, characterized in that, Step S3 is specifically: S3, after the reactant is cooled to room temperature, it is purified by elution with a silica gel chromatographic column; 5. The method for preparing red fluorescent silicon nanodots according to claim 4, characterized in that, The volume ratio of dichloromethane to methanol in the eluent is 5:1-15:

1.

6. The method for preparing red fluorescent silicon nanodots according to claim 5, characterized in that, The volume ratio of dichloromethane to methanol in the eluent is 10:

1.

7. The method for preparing red fluorescent silicon nanodots according to claim 1, characterized in that, The method comprises the following steps: S1, 2-nitro-4-amino diphenylamine is dissolved in anhydrous ethanol, and 3-aminopropyl triethoxysilane is added and stirred uniformly; S2, the mixture obtained in step S1 is transferred to a reaction kettle, and then reacted under heating; S3, after the reactant is cooled to room temperature, it is purified by elution with a silica gel chromatographic column; S4, the purified product obtained in step S3 is freeze-dried to obtain red fluorescent silicon nanodots, which are stored at 4°C.

8. A red fluorescent silicon nanodot, characterized in that, It is prepared by the method of any one of claims 1-7.

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