PPAB-derived near-infrared ultra-small fluorescent silicon dioxide nanoparticles as well as synthesis method and application thereof

Abstract

The invention relates to PPAB-derived near-infrared ultra-small fluorescent silicon dioxide nanoparticles as well as a synthesis method and application thereof, and the fluorescent silicon dioxide nanoparticles prepared by the invention show stronger near-infrared fluorescence and good water solubility, have strong fluorescence emission peaks near the wavelength of 650-700 nm, show good fluorescence spectrum performance, and can be applied to the field of near-infrared fluorescence detection, near-infrared fluorescence detection, near-infrared fluorescence detection, near-infrared fluorescence detection, near-infrared fluorescence detection, near-infrared fluorescence detection, near-infrared fluorescence detection and near-infrared detection. Therefore, the silicon quantum dot has relatively good cell tissue penetrability. Due to the unique optical property and relatively good cell tissue penetrability, living body fluorescence imaging can be effectively carried out. The fluorescent silicon dioxide nanoparticles prepared by the invention have excellent near-infrared optical properties and good biocompatibility, also have excellent singlet oxygen generation performance and peroxidase performance, can be used as a photodynamic / chemical dynamic synergistic treatment reagent for antibacterial and anti-tumor application, and have no toxic or side effect on organisms.

Description

Technical Field

[0001] The invention belongs to the technical field of silicon quantum dots and relates to PPAB-derived near-infrared ultra-small fluorescent silicon dioxide nanoparticles and a synthesis method and application thereof. Background Art

[0002] Ultrasmall inorganic nanoparticles are rapidly gaining interest as nanomedicines for cancer treatment. Some organic nanodrugs are already more competitive than traditional chemotherapeutics due to their multifunctional and multivalent effects. Inorganic nanoparticles further diversify the building blocks of nanomedicines and are likely to offer advantages related to their intrinsic physical properties and lower manufacturing costs. Safely translating nanoparticles from the laboratory to the clinic requires overcoming many significant scientific and regulatory barriers. The most important issue is the favorable biodistribution and its temporal evolution profile (pharmacokinetics).

[0003] To date, only a small number of composite inorganic nanoparticles with a size less than 10nm have been synthesized. Among them, polyethylene glycol-coated (PEGylated) fluorescent silica nanoparticles (SNPs) with a size less than 10nm, known as Cornell dots or simply C-dots, have been approved by the US Food and Drug Administration (FDA) as an Investigational New Drug (IND) for the first human clinical trial. Although the results of the first clinical trial in melanoma patients are encouraging, there are still synthesis problems for this sub-10nm size fluorescent organic-inorganic hybrid SNP. All previous C-dot-type SNP synthesis work followed a modified process, in which alcohol is used as a solvent. However, for materials intended for biological or clinical applications, water is preferred as the reaction medium. It will greatly simplify the synthesis and clean-up procedures and reduce volatile waste, thereby greatly improving the speed and cost-effectiveness of particle production.

[0004] We attempted to prepare small nano-sized fluorescent organic and inorganic hybrid SNPs using water as the reaction medium by controlling the hydrolysis and relatively slow condensation during the synthesis process and using efficient polyethylene glycol silane to induce particle growth termination. We synthesized ultra-small fluorescent silica nanoparticles with a diameter of 8-10nm by condensing the silane-conjugated fluorophore PPAB generated by the reaction of azide silane with pyrrolopyrrole aza-BODIPY (PPAB) modified by alkynyl into a silica matrix for near-infrared fluorescence-guided photodynamic anti-tumor therapy and biosafety assessment. The product was compared with PBS and free silane-conjugated fluorophore PPAB, showing a more significant tumor treatment effect. Summary of the invention

[0005] Technical problem to be solved: The present invention provides a PPAB-derived near-infrared ultra-small fluorescent silica nanoparticle, a synthesis method and application thereof.

[0006] Technical solution: A PPAB-derived near-infrared ultra-small fluorescent silica nanoparticle, the structure of which is shown in compound 1:

[0007]

[0008] The synthesis method of silica nanoparticles comprises the following steps: according to the following ratio, step 1, 1 mmol DPP and 10 mmol 2-amino-5-trimethylsilylethylpyridine are added into 75 mL dry toluene, refluxed at 110° C. under N2 protection, and after being fully dissolved, 15 mmol TiCl4, continue to reflux and stir for 10 minutes, then add 43mmol triethylamine, continue to reflux and stir for about 1-2h, monitor the formation of the intermediate product imine through thin layer chromatography silica gel plate, add 20.5mmol boron trifluoride etherate, then continue to reflux for 4h, cool to room temperature, pour the reaction mixture into water, and extract with CH2Cl2; add anhydrous sodium sulfate to the organic layer and dry it, filter, and remove CH2Cl2 by rotary evaporation. The crude product is separated by column chromatography to obtain compound 2; step 2, add 0.04mmol compound 2 to 0.15mL dry TBAF, reflux for 3-4h at room temperature and N2 protection in anhydrous and oxygen-free environment, cool to room temperature, and extract the reaction mixture with water; the organic layer is dried with anhydrous sodium sulfate and filtered, and the filtrate is free of solvent and separated by column chromatography to obtain compound 3; step 3, 20mg compound 3, 5μL azidosilane, 34mgKOAc, 2.6mg CuI, 2.4μL acetic acid and 4.9μL N,N-diisopropylethylamine was added to 1.0 mL of dry dichloromethane, and the mixture was refluxed for 6 to 8 h under N2 protection in a dark environment. The mixture was cooled to room temperature, and the solvent was removed by distillation under reduced pressure. A green solid, i.e., compound 1, was obtained after column chromatography separation.

[0009] Preferably, the molar ratio of DPP to 2-amino-5-trimethylsilylethylpyridine in step 1 is 1:10.

[0010] The above-mentioned near-infrared ultrasmall fluorescent silica nanoparticles are used in biomarkers and imaging.

[0011] The application of the above-mentioned near-infrared ultrasmall fluorescent silica nanoparticles in the preparation of tumor photodynamic therapy drugs.

[0012] An anti-tumor drug, the effective ingredient of which contains the near-infrared ultra-small fluorescent silicon dioxide nanoparticles.

[0013] A cell tracking reagent, the active ingredient of which comprises the near-infrared ultra-small fluorescent silica nanoparticles.

[0014] The application of the above-mentioned PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticles in the preparation of fluorescent silica nanoparticles PPAB@SNP.

[0015] The preparation method of PPAB@SNP is as follows: 100 μL of 2.0 M ammonia water is mixed with 10 mL of deionized water to prepare 0.02 M ammonia water, 1 mL of which is added to 9 mL of deionized water, and stirred at room temperature for 10 minutes. Under stirring, 0.43 mmol of tetramethyl orthosilicate (TMOS) and 100 μL of compound 1 are added, and the solution is stirred at room temperature; then, 0.21 mmol of polyethylene glycol silane is added and stirred at room temperature; the temperature is then raised to 80°C, stirring is stopped, and the solution is allowed to stand at 80°C; then, the solution is cooled to room temperature and transferred to a dialysis membrane tube (Da =10000); the solution in the dialysis tube was dialyzed in 2000mL of deionized water, with the water changed once a day for 3 days to wash away any residual reagent; the particles were then filtered through a 200nm filter to remove any aggregates or dust present in the particle solution; the resulting particle solution was stored for a long time at room temperature; the molar ratio of TMOS, ammonia, polyethylene glycol silane, and water reaction was 1:0.093:0.49:1292, i.e., near-infrared ultrasmall fluorescent silica nanoparticles (PPAB@SNPs).

[0016] The reaction formula is as follows:

[0017]

[0018] Beneficial effects: 1. The present invention directly uses PPAB aqueous solution and silane coupling reagent to prepare fluorescent silica nanoparticles through a one-step hydrothermal method. Since PPAB itself has a certain fluorescence intensity and high fluorescence intensity, high quantum yield, relatively uniform morphology, and good fluorescence stability, it is conducive to biomedical applications, and the preparation method is simple and convenient, has good biocompatibility and safety, and is low in cost.

[0019] 2. The fluorescent silica nanoparticles prepared by the present invention exhibit strong near-infrared fluorescence and good water solubility, have a strong fluorescence emission peak near the wavelength of 650nm-700nm, and exhibit good fluorescence spectrum performance, so silicon quantum dots have relatively good cell tissue penetration. Due to its unique optical properties and relatively good cell tissue penetration, in vivo fluorescence imaging can be effectively performed.

[0020] 3. In addition to excellent near-infrared optical properties and good biocompatibility, the fluorescent silica nanoparticles prepared by the present invention also have excellent singlet oxygen generation performance and peroxidase performance, and can be used as photodynamic / chemodynamic synergistic therapeutic agents for antibacterial and anti-tumor applications, and have no toxic side effects on the organism itself. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HRMS spectrum of the near-infrared ultra-small fluorescent molecule PPAB (Compound 2) prepared in Example 1;

[0022] Figure 2 HRMS spectrum of the near-infrared ultra-small fluorescent PPAB (Compound 1) prepared in Example 1;

[0023] Figure 3 TEM spectrum of fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2;

[0024] Figure 4 FTIR spectrum of the fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2;

[0025] Figure 5 is the XRD pattern of the fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2;

[0026] Figure 6 The ultraviolet absorption spectra of the fluorescent molecules PPAB and nanoparticles PPAB@SNPs prepared in Examples 1 and 2;

[0027] Figure 7 The in vivo anti-tumor efficacy and biosafety evaluation of the fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2: Figure 7-1 This is the fluorescence imaging of tumors in living animals after the fluorescent silica nanoparticles were injected into the tail vein at different time points. Figure 7-2 This is the in vitro fluorescence imaging of the main organs and tumors of mice 12 hours after injection of fluorescent silica nanomaterials PPAB@SNPs. Figure 7-3 These are representative images of tumor-bearing mice in the control group and different treatment groups on day 12 in the in vivo experiment. Figure 7-4 Figure 2 is a graph showing the weight changes of tumor-bearing mice during in vivo experimental treatment. Figure 7-5 The photos are of xenograft tumors dissected from mice after treatment with different agents in the in vivo experiment on day 12. The scale bar is 2 cm. Figure 7-6Tumor weights of xenograft tumors dissected from mice after treatment with different preparations in the in vivo experiment on day 12, P1: PBS, P2: PPAB, P3: PPAB@SNPs, P4: PPAB+Laser, P5: PPAB@SNPs+Laser. Figure 7-7 It is a dot-line graph of tumor growth in tumor-bearing mice after administration of different preparations during the in vivo experiment; DETAILED DESCRIPTION

[0028] The following examples are given to illustrate the present invention in more detail. It is necessary to point out that the following examples cannot be interpreted as limiting the scope of protection of the invention. Some non-essential improvements and adjustments made to the present invention by technical personnel skilled in the art based on the above invention content should still fall within the scope of protection of the present invention.

[0029] The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the experiments can be obtained from commercial sources unless otherwise specified. All the following reagents used in the examples are commercially available analytically pure or chemically pure.

[0030] Example 1

[0031] Preparation of near-infrared ultra-small fluorescent PPAB (compound 1)

[0032] DPP (305 mg, 1 mmol) and 2-amino-5-trimethylsilylethylpyridine (1.9 g, 10 mmol) were added to dry toluene (75 mL), and refluxed at 110 ° C under N2 protection. After fully dissolved, TiCl4 (1.65 mL, 15 mmol) was added to the mixed solution, and refluxed and stirred for 10 min before adding triethylamine (6.0 mL, 43 mmol). Reflux and stir for about 1 to 2 h. Boron trifluoride ether (2.5 mL, 20.5 mmol) was added after the formation of the intermediate product imine was monitored by thin layer chromatography silica gel plate, and then refluxed for 4 h. The reaction mixture was cooled to room temperature, poured into water, and extracted with CH2Cl2; anhydrous sodium sulfate was added to the organic layer to dry it, filtered, and CH2Cl2 was removed by rotary evaporation. The crude product was separated by column chromatography to obtain compound 2; compound 2 (29.6 mg, 0.04 mmol) was added to dry TBAF (0.15 mL). Reflux for 3 h in anhydrous and oxygen-free environment at room temperature under N2 protection. Cool to room temperature, extract the reaction mixture with water; add anhydrous sodium sulfate to the organic layer, dry it and filter it, and separate the residue by column chromatography to obtain compound 3; add compound 3 (20 mg), azidosilane (5 μL), KOAc (34 mg, 0.336 mmol), CuI (2.6 mg), acetic acid (2.4 μL) and N, N-diisopropylethylamine (4.9 μL) to dry dichloromethane (1.0 mL). Reflux for 6 h under N2 protection in the dark, cool to room temperature, distill under reduced pressure, remove the solvent and separate by column chromatography to obtain a green solid, namely compound 1.

[0033] Example 2

[0034] Preparation of fluorescent silica nanoparticles PPAB@SNP

[0035] 100 μL of 2.0M ammonia water was mixed with 10 mL of deionized water to prepare 0.02M ammonia water, and 1 mL was added to 9 mL of deionized water (pH = 8). Stir at room temperature for 10 minutes, and 0.43 mmol of tetramethyl orthosilicate (TMOS) and 100 μL of PPAB (compound 1) with silane azide added (the molar ratio of PPAB to TMOS was 1:1000) were added under vigorous stirring. The solution was then stirred at room temperature overnight. Then, 0.21 mmol of polyethylene glycol silane was added and stirred at room temperature overnight. The temperature was then raised to 80°C, stirring was stopped, and the solution was allowed to stand at 80°C overnight. Then, the solution was cooled to room temperature and transferred to a dialysis membrane tube (Da = 10000). The solution in the dialysis tube was dialyzed in 2000 mL of deionized water, with the water changed once a day for 3 days to wash away any residual reagents. The particles were then filtered through a 200 nm filter to remove any aggregates or dust present in the particle solution. The obtained particle solution was stored for a long time at room temperature. The molar ratio of TMOS, ammonia, polyethylene glycol silane and water was 1:0.093:0.49:1292, namely near-infrared ultrasmall fluorescent silica nanoparticles (PPAB@SNPs).

[0036] Example 3

[0037] The near-infrared ultrasmall fluorescent silica nanoparticles (PPAB@SNPs) prepared in Example 2 of the present invention were tested, including UV absorption, XRD, TEM, FTIR, etc.

[0038] Using TMOS as the silicon source, PPAB with azidosilane as the raw material, and polyethylene glycol silane to provide spatial stability, silica nanoparticles were synthesized by a one-step hydrothermal method at 80°C for 12 hours overnight. Figure 3 This is the TEM spectrum of fluorescent silica nanoparticles PPAB@SNPs. The particle size is mainly determined by TMOS concentration, pH value and reaction temperature. The size of the final prepared product PPAB@SNPs is within 5-10nm. Figure 4 The infrared spectrum of fluorescent silica nanoparticles PPAB@SNPs is shown in Figure 1. The characteristic peaks of each are marked, indicating the functional groups on the silica nanoparticles. -1 The azido group at 800 cm -1 With 1080cm -1 The absorption peak at is the peak position of the silicon oxygen group, which proves that the silicon source TMOS is hydrolyzed and condensed in the water environment condition of pH=8 and successfully reacts to generate silicon dioxide nanoparticles. Figure 5This is the XRD powder diffraction pattern of fluorescent silica nanoparticles PPAB@SNPs. As shown in the figure, the X-ray diffraction pattern shows the characteristic peaks of standard card PDF SiO2 at around 20° to 30°, further verifying the successful synthesis of silica nanoparticles. Figure 6 This is the ultraviolet absorption spectrum of PPAB and fluorescent silica nanoparticles PPAB@SNPs. It can be seen from the figure that the prepared fluorescent silica nanoparticles have a strong absorption peak in the range of 600-700nm.

[0039] Example 4

[0040] The anti-tumor efficacy of PPAB@SNPs was further evaluated. 4T1 tumor-bearing mice were used for in vivo anti-tumor experiments. First, BALB / c mice (6 weeks old, weighing about 20 g) were selected and divided into five groups, with 4 mice in each group. The five groups were Control group (PBS group) (P1), PPAB group (P2), PPAB@SNPs group (P3), PPAB+Laser group (P4) and PPAB@SNPs+Laser group (P5). About 1×10 6 4T1 cells were injected subcutaneously into the right leg of mice. 3 100 μL of 50 μg / mL aqueous solution of fluorescent silica nanoparticles was injected into the PPAB group and PPAB@SNPs+Laser group through the tail vein. The amount of free silicon-coupled fluorophore PPAB added was kept the same as that of PPAB contained in the fluorescent silica nanoparticles. The control group (PBS group) was injected with an equal amount of PBS. For the PPAB@SNPs+Laser group, after the injection of the material, the power was 0.5 W / cm 2 The tumor site was irradiated with a 660nm laser for 5 minutes (5 seconds for each irradiation, 5 seconds for rest, and a total of 5 minutes). For all groups, the tumor volume and mouse weight were recorded every two days, and the tail vein was injected every three days for a total of 12 days. After that, the mice were dissected, the solid tumors were removed for tumor size comparison, the heart, liver, spleen, lung, and kidney were removed for slice analysis, and the tumor was analyzed by H&E. Prior to the anti-tumor study, the in vivo transmission of nanomedicines was directly visualized by time-dependent fluorescence imaging. Figure 7-1 Fluorescence imaging of live mice by tail vein injection of fluorescent silica nanoparticles. Due to the fluorescent properties of PPAB, fluorescent imaging of mouse tumor sites can be performed using the FOBI live animal imager. Due to the EPR effect of the nanoparticles, PPAB@SNPs accumulate in the tumor. The mice were killed 72 hours after administration, and in vitro fluorescence imaging (removal of major organs and tumors) was performed. Figure 7-2As shown, after administration of PPAB@SNPs, bright fluorescence was observed in the liver and tumor, while the fluorescence in the heart, spleen, lung, and kidney was relatively weak.

[0041] Figure 7-3 The living mice in the PBS group, PPAB group, PPAB@SNPs group, PPAB+Laser group and PPAB@SNPs+Laser group were compared. The tumor size of the treatment group (PPAB@SNPs+Laser group) was significantly smaller than that of the other four groups, indicating that near-infrared fluorescent silica nanoparticles have a significant therapeutic effect on mouse tumors under laser irradiation conditions, and the PDT photodynamic therapy effect is obvious. Figure 7-4 This is a graph showing the weight changes of mice. There was no obvious weight change in all mice within 12 days, indicating that the fluorescent silica nanoparticles have no toxic side effects on mice. Figure 7-5 The tumor size of the five groups was compared after the mice were dissected. The tumor size of the PPAB@SNPs+Laser group was significantly smaller than that of the Control group (PBS group) and the PPAB group, indicating that PPAB@SNPs had a significant therapeutic effect on mouse tumors under laser irradiation. The tumor blocks were removed after the mice were dissected, and the weight of each mouse tumor was recorded. The PPAB group, PPAB@SNPs group, PPAB+Laser group and PPAB@SNPs+Laser group were compared with the Control group (PBS group), and the tumor inhibition rates of the PPAB group, PPAB@SNPs group and PPAB+Laser group were calculated to be 22.15%, 45.01% and 60.48%, respectively. The PPAB@SNPs+Laser group had the highest tumor inhibition rate of 87.66%. Figure 7-6 This is a comparison of the tumor inhibition rate of photodynamic therapy, which shows that the fluorescent silica nanoparticles have an outstanding PDT treatment effect on the tumor area of ​​mice. Figure 7-7 As shown in the graph of tumor volume changes in mice at 12 days, the tumor size of mice in the PPAB@SNPs+Laser group changed very little, almost unchanged, while the tumor size of mice in the other four groups increased significantly, indicating that the PDT treatment effect of fluorescent silica nanoparticles on the tumor area of ​​mice was significant.

[0042] The above is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A PPAB-derived near-infrared ultra-small fluorescent silica nanoparticle, characterized in that: The structure is shown in compound 1:

2. The method for synthesizing the PPAB-derived near-infrared ultra-small fluorescent silica nanoparticles according to claim 1, characterized in that: The steps are as follows: according to the following ratio, step 1, add 1mmol DPP and 10mmol 2-amino-5-trimethylsilylethylpyridine into 75mL dry toluene, reflux at 110°C under N2 protection, and add 15mmol TiCl4, continue to reflux and stir for 10 minutes, then add 43mmol triethylamine, continue to reflux and stir for about 1-2h, monitor the formation of the intermediate product imine through thin layer chromatography silica gel plate, add 20.5mmol boron trifluoride etherate, then continue to reflux for 4h, cool to room temperature, pour the reaction mixture into water, and extract with CH2Cl2; add anhydrous sodium sulfate to the organic layer and dry it, filter, and remove CH2Cl2 by rotary evaporation. The crude product is separated by column chromatography to obtain compound 2; step 2, add 0.04mmol compound 2 to 0.15mL dry TBAF, reflux for 3-4h at room temperature and N2 protection in anhydrous and oxygen-free environment, cool to room temperature, and extract the reaction mixture with water; the organic layer is dried with anhydrous sodium sulfate and filtered, and the filtrate is free of solvent and separated by column chromatography to obtain compound 3; step 3, 20mg compound 3, 5μL azidosilane, 34mg KOAc, 2.6mg CuI, 2.4 μL acetic acid and 4.9 μL N,N-diisopropylethylamine were added to 1.0 mL dry dichloromethane, refluxed for 6 to 8 h under N2 protection in a dark environment, cooled to room temperature, and distilled under reduced pressure to remove the solvent. After column chromatography separation, a green solid, namely compound 1, was obtained.

3. The method for synthesizing near-infrared ultra-small fluorescent silica nanoparticles derived from PPAB according to claim 2, characterized in that: The molar ratio of DPP to 2-amino-5-trimethylsilylethylpyridine in step 1 is 1:

10.

4. Use of the near-infrared ultrasmall fluorescent silica nanoparticles according to claim 1 in biomarkers and imaging.

5. Use of the near-infrared ultrasmall fluorescent silica nanoparticles according to claim 1 in the preparation of drugs for tumor photodynamic therapy.

6. An anti-tumor drug, characterized in that: The effective ingredient contains the near-infrared ultra-small fluorescent silica nanoparticles according to claim 1.

7. A cell tracking reagent, characterized in that The effective ingredients include the near-infrared ultra-small fluorescent silica nanoparticles according to claim 1.

8. Use of the PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticles according to claim 1 in the preparation of fluorescent silica nanoparticles PPAB@SNP.

9. The use according to claim 8, characterized in that: The preparation method is as follows: 100 μL of 2.0M ammonia water is mixed with 10 mL of deionized water to prepare 0.02M ammonia water, 1 mL of ammonia water is added to 9 mL of deionized water, and stirred at room temperature for 10 minutes. Under stirring, 0.43 mmol of tetramethyl orthosilicate (TMOS) and 100 μL of compound 1 are added, and the solution is stirred at room temperature; then, 0.21 mmol of polyethylene glycol silane is added, and stirred at room temperature; the temperature is then raised to 80°C, stirring is stopped, and the solution is allowed to stand at 80°C; then, the solution is cooled to room temperature and transferred to a 10000 Da column. The solution in the dialysis tube was dialyzed in 2000 mL of deionized water, with the water changed once a day for 3 days to wash away any residual reagents; the particles were then filtered through a 200 nm filter to remove any aggregates or dust present in the particle solution; the resulting particle solution was stored for a long time at room temperature; the molar ratio of TMOS, ammonia, polyethylene glycol silane, and water for the reaction was 1:0.093:0.49:1292, namely, near-infrared ultrasmall fluorescent silica nanoparticles (PPAB@SNPs).

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