Ppab-derived near-infrared ultrasmall fluorescent silica nanoparticles, methods of synthesis and applications thereof

Abstract

PPAB derived near-infrared super-small fluorescent silica nanoparticles and synthesis method and application thereof, the fluorescent silica nanoparticles prepared by the present application exhibit strong near-infrared fluorescence and good water solubility, have a strong fluorescence emission peak near 650 nm-700 nm wavelength, exhibit good fluorescence spectrum performance, and therefore the silicon quantum dots have relatively good cell tissue penetration. Due to the unique optical properties and relatively good cell tissue penetration, the in vivo fluorescence imaging can be effectively carried out. The fluorescent silica nanoparticles prepared by the present application have excellent singlet oxygen generation performance and peroxidase performance in addition to excellent near-infrared optical performance and good biocompatibility, can be used as a photodynamic / chemodynamic synergistic treatment reagent, and are used for antibacterial and antitumor applications, and have no toxic side effects on the organism itself.

Description

Technical Field

[0001] This invention belongs to the field of silicon quantum dot technology, and relates to PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticles, their synthesis methods, and applications. Background Technology

[0002] Currently, ultrasmall inorganic nanoparticles are rapidly attracting interest as nanomedicines for cancer treatment. Due to their multifunctionality and multivalent effects, some organic nanomedicines are already more competitive than traditional chemotherapy drugs. Inorganic nanoparticles further diversify the building blocks of nanomedicines and are highly likely to offer advantages related to their intrinsic physical properties and lower manufacturing costs. Safely translating nanoparticles from the laboratory into clinical applications requires overcoming many significant scientific and regulatory hurdles. The most important issue is achieving favorable biodistribution and its temporal evolution profile (pharmacokinetics).

[0003] To date, only a small number of composite inorganic nanoparticles with sizes smaller than 10 nm have been synthesized. Among them, polyethylene glycol-coated (PEGylated) fluorescent silica nanoparticles (SNPs) with a size smaller than 10 nm, known as Cornell points or simply C-points, have been approved by the U.S. Food and Drug Administration (FDA) for the first human clinical trial as an Investigational New Drug (IND). Although the results of the first clinical trial in melanoma patients were encouraging, synthetic challenges remain for this sub-10 nm fluorescent organic-inorganic hybrid SNP. All previous synthesis efforts for C-point SNPs followed an improved method... The process uses alcohol as a solvent. However, for materials intended for biological or clinical applications, water is preferred as the reaction medium. This significantly simplifies synthesis and cleaning procedures, reduces volatile waste, and thus greatly increases the speed and cost-effectiveness of pellet production.

[0004] We attempted to synthesize small-sized fluorescent organic-inorganic hybrid SNPs using water as the reaction medium by controlling hydrolysis, relatively slow condensation, and employing efficient polyethylene glycol silane-induced particle growth termination during the synthesis process. We synthesized ultrasmall fluorescent silica nanoparticles with diameters of 8-10 nm by condensing the silane conjugated fluorophore PPAB generated from the reaction of azidosilane with alkyne-modified pyrrolopyrroloaza-BODIPY (PPAB) into a silica matrix. These nanoparticles were then used for near-infrared fluorescence-guided photodynamic antitumor therapy, and their biocompatibility was evaluated. The effects of the product were compared with those of PBS and free silane conjugated fluorophores PPAB, showing a more significant tumor therapeutic effect. Summary of the Invention

[0005] Technical problem solved: This invention provides a PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticle, its synthesis method, and its application.

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

[0007]

[0008] The method for synthesizing silica nanoparticles includes the following steps: In step 1, 1 mmol DPP and 10 mmol 2-amino-5-trimethylsilaneethylpyridine are added to 75 mL of dry toluene. The mixture is refluxed at 110 °C under N2 protection until fully dissolved. Then, 15 mmol DPP is added to the mixed solution. TiCl4 was added and refluxed for 10 min, followed by stirring. 43 mmol of triethylamine was added, and the mixture was refluxed and stirred for approximately 1–2 h. After the formation of the intermediate imine was detected by thin-layer chromatography on silica gel plates, 20.5 mmol of boron trifluoride diethyl ether was added, and the mixture was refluxed for another 4 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with CH2Cl2. The organic layer was dried with anhydrous sodium sulfate, filtered, and CH2Cl2 was removed by rotary evaporation. The crude product was separated by column chromatography to obtain compound 2. In step 2, 0.04 mmol of compound 2 was added to 0.15 mL of dry TBAF and refluxed for 3–4 h under anhydrous and oxygen-free conditions at room temperature and under N2 protection. After cooling to room temperature, the reaction mixture was extracted with water. The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was separated by column chromatography to obtain compound 3. In step 3, 20 mg of compound 3, 5 μL of azidosilane, 34 mg of KOAc, 2.6 mg of CuI, 2.4 μL of acetic acid, and 4.9 μL of... N,N-diisopropylethylamine was added to 1.0 mL of dry dichloromethane and refluxed for 6–8 h under light protection and N2 protection. After cooling to room temperature, the solvent was removed by vacuum distillation, and the product was separated by column chromatography to obtain a green solid, namely compound 1.

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

[0010] Applications of the aforementioned near-infrared ultrasmall fluorescent silica nanoparticles in biolabeling 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 antitumor drug, the active ingredient of which contains the aforementioned near-infrared ultra-small fluorescent silica nanoparticles.

[0013] A cell tracking reagent, the active ingredient of which comprises the aforementioned 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 solution is mixed with 10 mL of deionized water to prepare 0.02 M ammonia solution. 1 mL of this solution is added to 9 mL of deionized water and stirred at room temperature for 10 minutes. While 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 increased to 80 °C, stirring is stopped, and the solution is allowed to stand at 80 °C. Finally, the solution is cooled to room temperature and transferred to a dialysis membrane tube (Da). =10000); Dialyze the solution in the dialysis tube in 2000 mL of deionized water, changing the water once a day for 3 days to wash away any residual reagents; then filter the particles through a 200 nm filter to remove any aggregates or dust present in the particle solution; the resulting particle solution is stored at room temperature for a long time; the molar ratio of TMOS, ammonia, polyethylene glycol silane, and water is 1:0.093:0.49:1292, i.e., near-infrared ultra-small fluorescent silica nanoparticles (PPAB@SNPs).

[0016] The reaction formula is shown below:

[0017]

[0018] Beneficial effects: 1. This invention directly utilizes PPAB aqueous solution and silane coupling reagent to prepare fluorescent silica nanoparticles via a one-step hydrothermal method. Since PPAB itself possesses a certain fluorescence intensity and high fluorescence intensity, a relatively uniform morphology, and good fluorescence stability, it is beneficial for biomedical applications. Furthermore, the preparation method is simple and convenient, exhibits good biocompatibility and safety, and is low in cost.

[0019] 2. The fluorescent silica nanoparticles prepared in this invention exhibit strong near-infrared fluorescence and good water solubility, with a strong fluorescence emission peak in the 650nm-700nm wavelength range, demonstrating excellent fluorescence spectral performance. Therefore, silicon quantum dots have relatively good cell and tissue penetration. Due to their unique optical properties and relatively good cell and tissue penetration, they can be effectively used for in vivo fluorescence imaging.

[0020] 3. In addition to excellent near-infrared optical properties and good biocompatibility, the fluorescent silica nanoparticles prepared by this invention also have excellent singlet oxygen generation performance and peroxidase performance. They can be used as photodynamic / chemodynamic synergistic therapeutic agents for antibacterial and antitumor applications, and have no toxic side effects on organisms. Attached Figure Description

[0021] Figure 1 The mass spectrometry (HRMS) spectrum of PPAB (compound 2), a near-infrared ultrasmall fluorescent molecule, prepared in Example 1;

[0022] Figure 2 The mass spectrometry (HRMS) spectrum of the near-infrared ultrasmall fluorescent PPAB (compound 1) prepared in Example 1 is shown.

[0023] Figure 3 The TEM image of the fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2 is shown below.

[0024] Figure 4 The FTIR spectrum of the fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2 is shown below.

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

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

[0027] Figure 7 Evaluation of the in vivo antitumor efficacy and biosafety of the fluorescent silica nanoparticles PPAB@SNPs prepared in Example 2: Figure 7-1 These are fluorescence images of tumors in live animals after tail vein injection of fluorescent silica nanoparticles at different time points. Figure 7-2 In vitro fluorescence imaging of major organs and tumors in mice 12 hours after injection of fluorescent silica nanomaterials PPAB@SNPs. Figure 7-3 These are representative images of tumor-bearing mice on day 12 after treatment in the control group and different treatment groups in in vivo experiments. Figure 7-4 This is a graph showing the changes in body weight of tumor-bearing mice during in vivo experimental treatment. Figure 7-5 These are photographs of xenograft tumors dissected from mice on day 12 after treatment with different agents in an in vivo experiment. The scale bar is 2 cm. Figure 7-6The tumor weights of xenografted tumors dissected from mice on day 12 after treatment with different formulations in the in vivo experiment were: P1: PBS, P2: PPAB, P3: PPAB@SNPs, P4: PPAB+Laser, P5: PPAB@SNPs+Laser. Figure 7-7 Line graphs showing tumor growth in tumor-bearing mice after administration of different formulations during in vivo experiments; Detailed Implementation

[0028] The following examples illustrate the invention in more detail. It should be noted that the following embodiments should not be construed as limiting the scope of protection of the invention. Any non-essential improvements and adjustments made to the invention by those skilled in the art based on the above-described invention should still fall within the scope of protection of the invention.

[0029] Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, all materials and reagents used in the experiments are commercially available. All reagents used in the examples are commercially available analytical grade or chemically pure.

[0030] Example 1

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

[0032] DPP (305 mg, 1 mmol) and 2-amino-5-trimethylsilaneethylpyridine (1.9 g, 10 mmol) were added to dry toluene (75 mL) and refluxed at 110 °C under N2 protection. After complete dissolution, TiCl4 (1.65 mL, 15 mmol) was added to the mixture, and the mixture was refluxed and stirred for another 10 min. Then, triethylamine (6.0 mL, 43 mmol) was added. The mixture was refluxed and stirred for another 1–2 h. After the formation of the intermediate imine was detected by thin-layer chromatography on silica gel plates, boron trifluoride diethyl ether (2.5 mL, 20.5 mmol) was added, and the mixture was refluxed for another 4 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with CH2Cl2. The organic layer was dried with anhydrous sodium sulfate, 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). The mixture was refluxed for 3 h under anhydrous and oxygen-free conditions at room temperature and under N2 protection. After cooling to room temperature, the reaction mixture was extracted with water; the organic layer was dried with anhydrous sodium sulfate and filtered. The residue was separated by column chromatography to obtain compound 3. 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) were added to dry dichloromethane (1.0 mL). The mixture was refluxed for 6 h under N2 protection in the dark, cooled to room temperature, and distilled under reduced pressure to remove the solvent. The residue was then separated by column chromatography to obtain a green solid, namely compound 1.

[0033] Example 2

[0034] Preparation of fluorescent silica nanoparticles PPAB@SNP

[0035] Prepare a 0.02M ammonia solution by mixing 100 μL of 2.0M ammonia solution with 10 mL of deionized water. Take 1 mL of this solution and add it to 9 mL of deionized water (pH = 8). Stir at room temperature for 10 minutes. Under vigorous stirring, add 0.43 mmol of tetramethyl orthosilicate (TMOS) and 100 μL of PPAB (compound 1) with added azidosilane (PPAB to TMOS molar ratio 1:1000). Then, stir the solution at room temperature overnight. Next, add 0.21 mmol of polyethylene glycol silane and stir at room temperature overnight. Raise the temperature to 80°C, stop stirring, and let the solution stand at 80°C overnight. Then, cool the solution to room temperature and transfer it to a dialysis membrane tube (Da = 10000). Dialyze the solution in the dialysis tube to 2000 mL of deionized water, changing the water daily for 3 days to wash away any residual reagents. Then filter the particles through a 200 nm filter to remove any aggregates or dust present in the particle solution. The resulting particulate solution was stored at room temperature for an extended period. The molar ratio of TMOS, ammonia, polyethylene glycol silane, and water was 1:0.093:0.49:1292, which yielded near-infrared ultra-small fluorescent silica nanoparticles (PPAB@SNPs).

[0036] Example 3

[0037] The near-infrared ultra-small fluorescent silica nanoparticles (PPAB@SNPs) prepared in Example 2 of this invention were tested using methods including ultraviolet absorption, XRD, TEM, and FTIR.

[0038] Using TMOS as the silicon source and PPAB with azidosilane as the raw material, with polyethylene glycol silane providing steric stability, silica nanoparticles were synthesized overnight at 80°C for 12 hours using a one-step hydrothermal method. Figure 3 The image shows the TEM spectrum of fluorescent silica nanoparticles PPAB@SNPs. The particle size is mainly determined by the TMOS concentration, pH value and reaction temperature. The final product PPAB@SNPs has a size within 5-10 nm. Figure 4 The image shows the infrared spectrum of fluorescent silica nanoparticles PPAB@SNPs. The characteristic peaks of each nanoparticle are marked, indicating the functional groups present on the silica nanoparticles. PPAB@SNPs show a peak density of 1500 cm⁻¹. -1 The presence of azido groups at the α-terminus demonstrates the successful reaction of PPAB with azidosilane, and at 800 cm⁻¹ -1 With 1080cm -1 The absorption peak at the point is the peak position of the siloxane group, proving that the silicon-sourced TMOS successfully reacted and generated silicon dioxide nanoparticles through hydrolysis and condensation under aqueous conditions of pH=8. Figure 5The XRD powder diffraction pattern of fluorescent silica nanoparticles PPAB@SNPs is shown in the figure. The X-ray diffraction pattern shows the characteristic peaks of the standard card PDF SiO2 at around 20° to 30°, further verifying the successful synthesis of silica nanoparticles. Figure 6 The image shows the UV absorption spectra of PPAB and fluorescent silica nanoparticles PPAB@SNPs. As can be seen from the image, the prepared fluorescent silica nanoparticles have strong absorption peaks in the 600-700 nm range.

[0039] Example 4

[0040] Further evaluation of the antitumor efficacy of PPAB@SNPs was conducted. In vivo antitumor experiments were performed using 4T1 tumor-bearing mice. First, all mice were BALB / c mice (6 weeks old, approximately 20g in weight) and divided into five groups of four mice each. 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). Approximately 1×10⁻⁶ PPAB@SNPs were suspended in 20μL PBS. 6 4T1 cells were injected subcutaneously into the right leg of mice. When the tumor volume reached approximately 50 mm... 3 The PPAB group and the PPAB@SNPs+Laser group were injected intravenously with 100 μL of a fluorescent silica nanoparticle aqueous solution at a concentration of 50 μg / mL. The amount of free silica-coupled fluorophores PPAB added was kept the same as that contained in the fluorescent silica nanoparticles. The control group (PBS group) was injected with an equal volume of PBS. For the PPAB@SNPs+Laser group, after the material was injected, a 0.5 W / cm² solution was used. 2 The tumor site was irradiated with a 660nm laser for 5 minutes (5 seconds of irradiation followed by a 5-second pause, for a total of 5 minutes). For all groups, tumor volume and mouse weight were recorded every two days, and the tumor was injected via the tail vein every three days for a total of 12 days. Afterwards, the mice were dissected, solid tumors were removed for size comparison, and the heart, liver, spleen, lung, and kidney were extracted for section analysis. Hematologic and immunological analysis (H&E) of the tumors was also performed. Prior to anti-tumor research, the in vivo delivery of nanomedicines was directly visualized using time-dependent fluorescence imaging. Figure 7-1 Fluorescence imaging of live mice was performed using a tail vein injection of fluorescent silica nanoparticles (PPAB). Due to the fluorescent properties of PPAB, fluorescence imaging of tumor sites in mice was achieved using a FOBI in vivo animal imaging system. PPAB@SNPs aggregated in the tumor due to the EPR effect of the nanoparticles. Mice were sacrificed 72 hours after administration, and in vitro fluorescence imaging (of the resected major organs and tumors) was performed. Figure 7-2As shown, after administration of PPAB@SNPs, bright fluorescence was observed on the liver and tumors, while the fluorescence on the heart, spleen, lungs, and kidneys was relatively weak.

[0041] Figure 7-3 Compared with live mice in the PBS group, PPAB group, PPAB@SNPs group, PPAB+Laser group and PPAB@SNPs+Laser group, the tumor size in the treatment group (PPAB@SNPs+Laser group) was significantly smaller than that in the other four groups, indicating that near-infrared fluorescent silica nanoparticles have a significant therapeutic effect on mouse tumors under laser irradiation, and PDT photodynamic therapy has a significant effect. Figure 7-4 The graph shows the change in mouse body weight. The change in body weight of all mice was not significant within 12 days, indicating that the fluorescent silica nanoparticles have no toxic side effects on mice. Figure 7-5 To compare the tumor sizes of the five groups after dissecting the mice, the tumor size in the PPAB@SNPs+Laser group was significantly smaller than that in the Control group (PBS group) and the PPAB group, indicating that PPAB@SNPs has a significant therapeutic effect on mouse tumors under laser irradiation. After dissecting the mice, the tumor masses were removed, and the weight of the tumor in each mouse was recorded. The PPAB group, PPAB@SNPs group, PPAB+Laser group, and PPAB@SNPs+Laser group were compared with the Control group (PBS group). 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, with the PPAB@SNPs+Laser group showing the highest tumor inhibition rate of 87.66%. Figure 7-6 The comparison of tumor inhibition rates in photodynamic therapy (PDT) demonstrates the outstanding therapeutic effect of fluorescent silica nanoparticles on tumor regions in mice. Figure 7-7 The tumor volume change in mice over 12 days is shown in the figure. The tumor size of the mice in the PPAB@SNPs+Laser group changed very little and hardly changed, while the tumor size of the other four groups of mice increased significantly, indicating that fluorescent silica nanoparticles have a significant PDT therapeutic effect on the tumor area of ​​mice.

[0042] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticle, characterized in that, The structure is shown in compound 1: 。 2. The method for synthesizing PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticles according to claim 1, characterized in that, The steps are as follows: Step 1, according to the following proportions, 1 mmol DPP and 10 mmol 2-amino-5-trimethylsilane ethylpyridine were added to 75 mL of dry toluene and refluxed at 110 °C under N2 protection. After complete dissolution, 15 mmol TiCl4 was added to the mixture, and reflux and stirring were continued for 10 min. Then, 43 mmol triethylamine was added, and reflux and stirring were continued for about 1-2 h. After the formation of the intermediate imine was detected by thin-layer chromatography on silica gel plates, 20.5 mmol boron trifluoride diethyl ether was added, and reflux was continued for 4 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with CH2Cl2. The organic layer was dried with anhydrous sodium sulfate, filtered, and CH2Cl2 was removed by rotary evaporation. The crude product was separated by column chromatography to obtain compound 2. Step 2, 0.04 mmol of compound 2 was added to 0.15 mL of dry TBAF and refluxed at room temperature under N2 protection for 3-4 hours in an anhydrous and oxygen-free environment. h, cool to room temperature, extract the reaction mixture with water; dry the organic layer with anhydrous sodium sulfate and filter, remove the solvent from the filtrate and separate by column chromatography to obtain compound 3; in step 3, add 20 mg of compound 3, 5 μL of azidosilane, 34 mg of KOAc, 2.6 mg of CuI, 2.4 μL of acetic acid and 4.9 μL of N,N-diisopropylethylamine to 1.0 mL of dry dichloromethane, reflux for 6-8 h under light protection and N2 protection, cool to room temperature, distill under reduced pressure to remove the solvent, and separate by column chromatography to obtain a green solid, namely compound 1.

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

10.

4. The application of the near-infrared ultrasmall fluorescent silica nanoparticles according to claim 1 in the preparation of biolabeling and imaging formulations.

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

6. An antitumor drug, characterized in that, The active ingredient contains the near-infrared ultra-small fluorescent silica nanoparticles as described in claim 1.

7. A cell tracking reagent, characterized in that, The active ingredient comprises the near-infrared ultra-small fluorescent silica nanoparticles as described in claim 1.

8. The application of the PPAB-derived near-infrared ultrasmall fluorescent silica nanoparticles of claim 1 in the preparation of fluorescent silica nanoparticles PPAB@SNP.

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

Images