Near-infrared two-region imaging quantum dot material as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared two-region imaging quantum dot material and a preparation method and application thereof, the near-infrared two-region imaging quantum dot material comprises a quantum dot aggregate, and the quantum dot aggregate is formed by assembling CdTe quantum dots and carbon quantum dots; the maximum fluorescence emission wavelength of the near-infrared two-region imaging material is 1300 to 1800 nm when the excitation wavelength of the near-infrared two-region imaging material is 650 to 900 nm. The band gap of CdTe / CDs prepared by self-assembly of the carbon nanodots and the cadmium telluride quantum dots in the infrared two-region imaging material is reduced, the maximum fluorescence emission wavelength is red-shifted to 1700-1800 nm from original 855 nm, the whole near-infrared two-region window is covered, the application of the cadmium telluride quantum dots in the near-infrared two-region window is realized for the first time, and the application range of the cadmium telluride quantum dots in the near-infrared two-region window is widened. And an effective strategy is provided for the red shift of the fluorescence emission wavelength of the near-infrared quantum dots.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging, specifically to a near-infrared II imaging quantum dot material, its preparation method, and its applications. Background Technology

[0002] Fluorescence imaging is an important biomedical imaging method with very high detection sensitivity. By combining fluorescent probes for specific target recognition, it can achieve real-time, dynamic, and high spatiotemporal resolution imaging at the molecular and cellular levels, accurately acquiring dynamic biological information in vivo, making it one of the most promising methods for precision disease diagnosis and treatment. Therefore, fluorescence imaging technology shows significant clinical application value. However, fluorescence imaging is easily interfered with by tissue absorption, scattering, and autofluorescence, especially since traditional fluorescence imaging is mainly in the visible to near-infrared I region (NIR-I, 650-950nm), with short wavelengths (<1000nm), particularly visible light, resulting in poor photon penetration depth, which severely limits the clinical application of biomedical fluorescence imaging in vivo. Compared with visible light and NIR-I light, near-infrared II (NIR-II, 1000-2000nm) imaging, due to significantly reduced light scattering, low tissue autofluorescence, and improved water absorption, can not only achieve real-time monitoring of deep tissues but also utilize intraoperative navigation for lesion resection, showing broad clinical application prospects. Therefore, the NIR-II window is more suitable for fluorescence imaging.

[0003] In recent years, with the rapid development of NIR-II fluorescence imaging, single-walled carbon nanotubes (SWCNTs), quantum dots (QDs), rare-earth-doped nanoparticles (RENPs), and organic fluorophores (OFs) have been developed for NIR-II fluorescence imaging. However, SWCNTs are often plagued by low quantum yield (QY), strong hydrophobicity, and poor pharmacokinetics; OFs are often susceptible to photobleaching, resulting in poor stability and limiting their long-term applications. RENPs are limited by their small absorption cross-section and low QY. Compared with these NIR-II materials, quantum dots exhibit superior QY and stability, especially in aqueous solutions, and have attracted much attention due to their good biocompatibility and satisfactory pharmacokinetics. Currently, multifunctional quantum dots, such as lead sulfide (PbS), silver chalcogenides (Ag₂X; X = S, Se, Te), and copper indium selenide (CuInSe₂), emit fluorescence in the NIR II region. However, the aforementioned NIR II quantum dots suffer from poor fluorescence stability in air, a narrow tunable range in the near-infrared spectrum, weak exciton absorption, and unsatisfactory brightness in NIR-IIa (1300-1400nm) and NIR-IIb (1500-1700nm), which limit the practical application of near-infrared quantum dots. Summary of the Invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, one of the objectives of the present invention is to provide a near-infrared II imaging material.

[0005] The second objective of this invention is to provide a method for preparing near-infrared II imaging materials.

[0006] The third objective of this invention is to provide a near-infrared II region fluorescent molecular imaging probe.

[0007] The fourth objective of this invention is to provide a near-infrared II fluorescent contrast agent.

[0008] The fifth objective of this invention is to provide a detection system.

[0009] The sixth objective of this invention is to provide an application of near-infrared II imaging material in the field of fluorescence imaging.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of this invention provides a near-infrared II imaging material, comprising a quantum dot aggregate assembled from CdTe quantum dots and carbon quantum dots; the near-infrared II imaging material has a maximum fluorescence emission wavelength of 1300–1800 nm at an excitation wavelength of 650–900 nm. The maximum fluorescence emission wavelength of the near-infrared II imaging material of this invention is adjustable within the range of 1300–1800 nm at an excitation wavelength of 650–900 nm.

[0012] Preferably, the band gap of the near-infrared II imaging material is 0.01–1.15 eV. This invention utilizes the self-assembly of CdTe quantum dots and carbon quantum dots into quantum dot aggregates, which significantly reduces the band gap of the CdTe quantum dot material. This red-shifts the fluorescence emission wavelength of the CdTe quantum dot material to the near-infrared II bioimaging window, enabling its use for near-infrared II fluorescence imaging.

[0013] Preferably, the quantum dot aggregate is spherical, near-spherical, or rod-shaped.

[0014] Preferably, the particle size of the near-infrared II imaging material is 5–500 nm.

[0015] Preferably, the mass ratio of the CdTe quantum dots to the carbon quantum dots is 1:(0.001~1).

[0016] Preferably, the near-infrared II imaging material has a maximum fluorescence emission wavelength of 1300–1800 nm when the excitation wavelength is 808 nm; more preferably, the near-infrared II imaging material has a maximum fluorescence emission wavelength of 1500–1800 nm when the excitation wavelength is 808 nm; even more preferably, the near-infrared II imaging material has a maximum fluorescence emission wavelength of 1700–1800 nm when the excitation wavelength is 808 nm. When the excitation wavelength is 808 nm, the maximum fluorescence emission wavelength of the near-infrared II imaging material of the present invention is adjustable within the range of 1300–1800 nm, and its maximum excitation wavelength covers the entire near-infrared II bioimaging window, making it suitable for near-infrared II fluorescence imaging.

[0017] The CdTe quantum dots in this invention possess high water solubility, stability, and crystallinity, making them suitable for applications in biology and medicine. Their quantum yield in aqueous solution can reach over 80%. By combining carbon quantum dots and CdTe quantum dots, a new bandgap arrangement is achieved, shifting the fluorescence emission wavelength of CdTe quantum dots from the near-infrared I region to the near-infrared II region. This solves the technical problem in existing technologies where the fluorescence emission wavelength of CdTe quantum dots is below 900 nm, resulting in insufficient penetration depth for fluorescence imaging, limiting it to cellular imaging and preventing its use in in vivo deep tissue imaging. Under excitation light of 650–900 nm, the maximum fluorescence emission wavelength of the near-infrared II imaging material in this invention is tunable within the range of 1300–1800 nm. Near-infrared II vascular imaging, lymphatic imaging, and tumor imaging were successfully achieved in live mice, and a successful resection of the popliteal lymph nodes in the hind limbs of mice was performed under imaging guidance. This demonstrates its suitability as a high-resolution near-infrared II fluorescent contrast agent for in vitro and in vivo imaging. The near-infrared II imaging material in this invention improves the stability of the near-infrared II imaging material and reduces its biotoxicity by combining CdTe quantum dots with carbon quantum dots.

[0018] The second aspect of the present invention provides a method for preparing the near-infrared II imaging material provided in the first aspect of the present invention, comprising the following steps:

[0019] The near-infrared II imaging material was prepared by mixing carbon quantum dots, a cadmium source, and mercaptopropionic acid, adjusting the pH, and then reacting it with NaHTe.

[0020] This invention improves the quantum yield and brightness of near-infrared II imaging materials by optimizing synthesis conditions. The material is synthesized directly in an aqueous phase using a green synthesis method. The resulting near-infrared II imaging material exhibits good water solubility, excellent fluorescence stability in aqueous solution, and high brightness without further modification. This largely achieves fluorescence protection, ensuring no loss of fluorescence intensity in the near-infrared II imaging material. Enhancing the brightness of quantum dots can improve spatial and temporal resolution, but low quantum yield and fluorescence intensity loss due to quantum dot surface modification have been limiting factors for the further development of quantum dot materials. The near-infrared II imaging material in this invention exhibits no fluorescence intensity loss during preparation and high quantum yield, ultimately meeting the requirements for high spatiotemporal resolution fluorescence imaging in vivo. This invention prepares CdTe quantum dots in an aqueous phase using o-phenylenediamine as a precursor. Carbon quantum dots are synthesized via a solvothermal method, and CdTe quantum dots are grown in situ in an aqueous phase using carbon quantum dots as templates. This results in near-infrared II imaging materials. This not only overcomes the existing limitation that the emission wavelength of cadmium telluride quantum dots is only below 900 nm, but also, for the first time, this control strategy achieves continuous tunability of the fluorescence emission wavelength of cadmium telluride quantum dots in the range of 1300–1800 nm, covering the entire near-infrared II region.

[0021] Preferably, the step of adjusting the pH involves using an alkaline solution to adjust the pH value to be greater than 7.

[0022] Preferably, the reaction temperature is 80–160°C.

[0023] Preferably, the reaction time is 0.5 to 72 hours.

[0024] Preferably, the preparation method is carried out under the protection of an inert gas.

[0025] Preferably, the inert gas is selected from at least one of nitrogen, argon, and xenon.

[0026] Preferably, the alkaline solution is selected from sodium hydroxide, potassium hydroxide, or a combination thereof.

[0027] Preferably, the cadmium source is selected from cadmium chloride hemipentahydrate.

[0028] Preferably, the carbon quantum dots are prepared by reacting a carbon precursor and a solvent at 100–220°C for 2–72 h. More preferably, the carbon quantum dots are prepared by reacting a carbon precursor and a solvent at 100–220°C for 2–72 h, followed by dialysis purification and freeze-drying.

[0029] Preferably, the carbon precursor is selected from disubstituted aromatic compounds.

[0030] Preferably, the aromatic compound is selected from benzene, naphthalene, anthracene, or phenanthrene.

[0031] Preferably, the carbon precursor is selected from at least one of o-phenylenediamine, p-phenylenediamine, and m-phenylenediamine.

[0032] Preferably, the solvent is selected from at least one of ethanol, formamide, N,N-dimethylformamide, water, and dimethyl sulfoxide.

[0033] A third aspect of the present invention provides a near-infrared II fluorescent molecular imaging probe, comprising the near-infrared II imaging material provided in the first aspect of the present invention.

[0034] A fourth aspect of the present invention provides a near-infrared II fluorescent contrast agent, comprising the near-infrared II imaging material provided in the first aspect of the present invention.

[0035] A fifth aspect of the present invention provides a detection system comprising a near-infrared II fluorescent molecular imaging probe provided in the third aspect of the present invention or a near-infrared II fluorescent contrast agent provided in the fourth aspect of the present invention.

[0036] The sixth aspect of the present invention provides the application of the near-infrared II imaging material provided in the first aspect of the present invention in the field of fluorescence imaging.

[0037] The beneficial effects of this invention are as follows: The near-infrared II imaging material of this invention, CdTe / CDs, is prepared by self-assembly of carbon nanodots and cadmium telluride quantum dots. Due to the introduction of carbon dots, a new energy level structure is imparted. By controlling the band gap, the maximum fluorescence emission wavelength is redshifted from the original 855 nm to 1300–1800 nm, covering the entire near-infrared II window. This is the first time that cadmium telluride quantum dots have been applied in the near-infrared II window, providing an effective strategy for adjusting the redshift of the fluorescence emission wavelength of near-infrared quantum dots. The near-infrared II imaging material of this invention can be applied in the fields of fluorescent molecular imaging probes and near-infrared II fluorescent contrast agents. It can achieve fluorescence imaging of deep tissues in vivo in the near-infrared II region, such as vascular imaging, lymphatic imaging, and imaging of deeper tissues in living organisms, and can be used for surgical navigation guided by imaging. Attached Figure Description

[0038] Figure 1 The image shows a TEM image of the CdTe QDs prepared in Comparative Example 1.

[0039] Figure 2 This is a TEM image of the CDs prepared in Example 1.

[0040] Figure 3 This is a TEM image of the CdTe / CDs prepared in Example 1.

[0041] Figure 4This is a TEM image of the CdTe / CDs prepared in Example 4.

[0042] Figure 5 The image shows the XRD pattern of the CdTe QDs prepared in Comparative Example 1.

[0043] Figure 6 The image shows the XRD pattern of the CDs prepared in Example 1.

[0044] Figure 7 The image shows the EDS diagram of CdTe / CDs obtained in Example 1.

[0045] Figure 8 The image shows the EDS diagram of CdTe / CDs obtained in Example 4.

[0046] Figure 9 The image shows the fluorescence emission pattern of the CdTe QDs prepared in Comparative Example 1.

[0047] Figure 10 The image shows the fluorescence emission pattern of the CdTe / CDs prepared in Example 1.

[0048] Figure 11 The image shows the maximum fluorescence emission of the CdTe / CDs prepared in Example 1.

[0049] Figure 12 The image shows the bandgap test results for CdTe QDs and CdTe / CDs in Example 1.

[0050] Figure 13 The image shows the fluorescence of the CdTe / CDs aqueous solution prepared in Example 1.

[0051] Figure 14 This is a biosafety evaluation test diagram of the CdTe / CDs prepared in Example 1.

[0052] Figure 15 This is a graph showing the cell hemolysis rate of CdTe / CDs prepared in Example 1.

[0053] Figure 16 The image shows the fluorescence test results of CdTe / CDs in PBS, NaCl, and H2O in Example 1.

[0054] Figure 17 The image shows the fluorescence test results of CdTe / CDs in Example 1 in H2O and different concentrations of NaCl.

[0055] Figure 18 This is a near-infrared 2D vascular imaging image of CdTe / CDs in mice in Example 1.

[0056] Figure 19The images show tumor imaging, fluorescence intensity in the tumor region, and fluorescence intensity in the liver region of CdTe / CDs in Example 1 at different times.

[0057] Figure 20 This is a surgical image of the popliteal lymph node resection in the hind limb of a mouse under lymph node imaging guidance, using CdTe / CDs in Example 1. Detailed Implementation

[0058] The specific implementation of the present invention will be further described in detail below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0059] Example 1

[0060] This example provides a method for preparing a near-infrared II imaging material, including the following steps:

[0061] (1) Preparation of carbon quantum dots (CDs):

[0062] 0.05 g of o-phenylenediamine and 30 mL of formamide were placed in a high-pressure reactor and reacted at 160 °C for 10 h. After the reaction was completed, the carbon quantum dots were collected, purified by dialyzing, and then freeze-dried to obtain carbon quantum dot solid powder.

[0063] (2) Preparation of CdTe / CDs:

[0064] 0.05 g of CDs and 0.2284 g of cadmium chloride hemipentahydrate (CdCl2·5 / 2H2O) were placed in a beaker, and 100 mL of deionized water was added. The mixture was sonicated for 30 min to ensure complete dissolution. Then, 132 μL of mercaptopropionic acid (MPA) was added to the flask. While stirring, the pH of the reaction solution was adjusted to 11 with 1 mol / L NaOH solution. As NaOH solution was continuously added, the solution changed from clear to milky white and turbid. When the pH of the solution was greater than 7, the solution color became clear and transparent and no longer changed. The solution was then transferred to a 250 mL three-necked flask, stirred, and purged with nitrogen for 30 min to remove oxygen. The prepared NaHTe solution was then quickly added, and the mixture was refluxed at 100 °C for 18 h to obtain an aqueous solution of CdTe / CDs.

[0065] (3) Purification of CdTe / CDs:

[0066] The prepared CdTe / CDs solution was mixed with anhydrous ethanol at a volume ratio of 1:1 and centrifuged at 10,000 rpm for 10 min to remove excess mercaptopropionic acid and other ions. After centrifugation, the CdTe / CDs were redispersed in deionized water and stored in a brown bottle at 4°C in the dark to obtain the near-infrared II fluorescence imaging material in this example, denoted as CdTe / CDs.

[0067] Example 2

[0068] This example provides a near-infrared II fluorescent molecular imaging probe, which uses the near-infrared II fluorescent imaging material from Example 1.

[0069] Example 3

[0070] This example provides a near-infrared II fluorescent contrast agent, which uses the near-infrared II fluorescent imaging material from Example 1.

[0071] Example 4

[0072] This example provides a near-infrared II fluorescent contrast agent. The preparation method of this agent differs from that of the fluorescent imaging material in Example 1 in that the following step is added after step (2) in Example 1: the aqueous solution of CdTe / CDs is first treated by ultrasonication, then subjected to gradient centrifugation, and the precipitate is taken at 5000 rpm. The precipitate is then purified by the purification step in step (3) to obtain the final product.

[0073] Comparative Example 1

[0074] This example provides a method for preparing CdTe quantum dots, including the following steps:

[0075] (1) Synthesis of the precursor sodium telluride (NaHTe):

[0076] According to the reaction equation 4NaBH4 + 2Te + 7H2O → 2NaHTe + Na2B4O7 + 12H2↑, and based on the reaction ratio of each substance, Cd 2+ The ratio of MPA to HTe- is 1:1.5:0.2. Weigh 0.0255g of tellurium powder and 0.15g of sodium borohydride into 10mL vials, add 1.5mL of deionized water, seal the vial, insert a needle into the rubber stopper to release the hydrogen gas released during the reaction, stir overnight at room temperature. After the reaction is complete, a milky white suspension is obtained. After standing for 15 minutes, the colorless, transparent and clear solution in the upper layer of the vial is the sodium telluride solution.

[0077] (2) Preparation of water-soluble CdTe QDs by hot reflux method:

[0078] Weigh 0.2284 g of cadmium chloride hemipentahydrate (CdCl2·5 / 2H2O) into a beaker, add 100 mL of deionized water, stir thoroughly to dissolve, and then add 132 μL MPA. While stirring, adjust the pH of the reaction solution to 11 using 1 mol / L NaOH solution. As NaOH solution is continuously added, the solution changes from clear to milky white turbidity. When the pH of the solution is greater than 7, the solution becomes clear and transparent and no longer changes color. Transfer the solution to a 250 mL three-necked flask, stir, and purge with nitrogen for 30 min to remove oxygen. Then, quickly add the prepared NaHTe solution. Reflux at 100 °C for 36 h to obtain a water-soluble CdTeQDs solution.

[0079] Performance testing:

[0080] (1) Microscopic morphology test

[0081] The microstructure of the CdTe QDs in Comparative Example 1 was examined using transmission electron microscopy and high-resolution electron microscopy (HRTEM), specifically as follows: Figure 1 As shown, where, Figure 1 (a) and Figure 1 (b) Transmission electron microscopy images with scale bars of 20 nm and 5 nm, respectively. Figure 1 (a) It can be seen that the CdTe QDs particles are round in shape, uniform in size, and evenly distributed without agglomeration. Figure 1 (b) shows a high-resolution electron microscope image of a single CdTe QDs within the white box, clearly demonstrating that the CdTe QDs have a circular lattice structure.

[0082] The microstructure of the CDs in Example 1 was examined using transmission electron microscopy and high-resolution electron microscopy (HRTEM), specifically as follows: Figure 2 As shown, where, Figure 2 (a) and Figure 2 (b) Transmission electron microscopy images with scale bars of 20 nm and 5 nm, respectively. Figure 2 It can be seen that the prepared carbon quantum dots have a size of about 3 nm and a lattice spacing of about 0.21 nm. They have a spherical structure, are uniformly distributed, and do not exhibit agglomeration.

[0083] The microstructure of the near-infrared II fluorescence imaging materials in Examples 1 and 4 was tested using transmission electron microscopy, specifically as follows: Figure 3 and 4 As shown, by Figure 3 It can be seen that the CdTe / CDs in Example 1 are spherical aggregates, which are composite quantum dot aggregates formed by the self-assembly of individual CdTeQDs and CDsQDs. Figure 4It can be seen that the CdTe / CDs in Example 4 are rod-shaped aggregates, which are composite quantum dot aggregates formed by the self-assembly of individual CdTe QDs and CDs QDs.

[0084] (2) Crystal structure testing

[0085] The crystal structure of CdTe QDs was tested using X-ray diffraction, specifically as follows: Figure 5 As shown, by Figure 5 It can be seen that the XRD pattern of CdTeQDs shows three distinct diffraction peaks at 26.02°, 43.74°, and 51.98°, which correspond to the (111), (220), and (311) crystal planes of the cubic zincblende structure in the PDF standard comparison card (PDF#75-2086), thus conforming to the cubic crystal system of zincblende. Figure 5 This also proves that CdTe QDs were successfully prepared and that the prepared CdTe QDs had high crystallinity, complete crystal form, and high purity.

[0086] The crystal structure of CDs was tested using an X-ray diffractometer, specifically as follows: Figure 6 As shown, by Figure 6 As can be seen, carbon quantum dots were successfully prepared in Example 1, which have a broad peak centered at 26.9°, indicating that the carbon quantum dots prepared in Example 1 have a disordered structure.

[0087] (3) EDS test

[0088] The EDS spectra of CdTe / CDs prepared in Examples 1 and 4 are as follows: Figure 7 and 8 As shown, by Figure 7 and 8 It is known that CdTe / CDs aggregates contain elements such as C, N, O, Cd, and Te, which proves that CdTe QDs and CDs QDs have successfully composited.

[0089] (4) Fluorescence property test

[0090] The fluorescence emission spectra of the CdTe QDs synthesized in Comparative Example 1 were tested, as shown in the figure below. Figure 9 As shown, by Figure 9 It can be seen that CdTeQDs have a maximum emission wavelength of about 820 nm under the excitation condition of 365 nm excitation light.

[0091] Fluorescence emission spectra of the product were sampled and tested at different time points (5h, 6h, 7h, 8h, 9h, 10h, 12h, 15h, 18h) during the 100℃ reflux process in step (2) of Example 1, under excitation by an 808nm laser. The specific results are as follows: Figure 10As shown, the fluorescence emission spectrum of the purified CdTe / CDs from Example 1 was then tested, specifically as follows: Figure 11 As shown, by Figure 10 It can be seen that under the excitation conditions of an 808nm laser, the fluorescence emission wavelength of CdTe / CDs can be tunable in the range of 1300–1800nm; from Figure 11 It can be seen that CdTe / CDs, under the excitation condition of an 808nm laser, can achieve a maximum emission wavelength of 1730nm, which is 910nm redshifted compared to pure CdTe QDs (820nm).

[0092] (5) Bandgap test

[0093] The DRS of CdTe QDs and CdTe / CDs were recorded using an integrating sphere of a UV-Vis spectrophotometer, and the corresponding bandgap energies were calculated using Tauc plots. Specific test results are shown below. Figure 12 As shown, where, Figure 12 (a) and Figure 12 (c) UV-Vis DRS spectra of CdTe QDs and CdTe / CDs, respectively. Figure 12 (b) and Figure 12 (d) Bandgap energies (ahv) of CdTe QDs and CdTe / CDs, respectively. 1 / 2 A graph showing the relationship between photon energy (hv) and photon energy. Figure 12 It is known that the band gap of CdTe QDs is 1.18 eV, while the band gap of CdTe / CDs is reduced from 1.18 eV to 0.418 eV by assembling CdTe QDs and CDs into aggregates. This further indicates that this is an effective strategy to adjust the fluorescence emission wavelength of quantum dots by controlling the energy level band gap.

[0094] (6) Evaluation of luminescence performance

[0095] The luminescence properties of the CdTe / CDs in Example 1 were evaluated using 660nm, 808nm, and 980nm lasers from a near-infrared II small animal in vivo imager. The results are as follows: Figure 13 As shown, Figure 13 (a) Figure 13 (b) Figure 13 (c) are fluorescence images of CdTe QDs under the following conditions: 30ms exposure time with a 660nm laser, 30ms exposure time with an 808nm laser, and 30ms exposure time with an 808nm laser. Figure 13The samples in each centrifuge tube in each figure are CdTe / CDs samples obtained from different time points (sampling times from left to right are 5h, 3h, 6h, 9h, 12h, 15h, and 18h) during the 100℃ reflux process in step (2) of Example 1. Figure 13 It can be seen that CdTe / CDs can be used for in vitro fluorescence imaging under the excitation conditions of three different lasers: 660nm, 808nm, and 980nm, and all of them have good fluorescence performance. Among them, under the same conditions, the imaging effect is the best and the fluorescence intensity is the highest when using an 808nm laser.

[0096] (7) Biosafety Evaluation - MTT Test

[0097] The cytotoxicity of CdTe QDs comes not only from the release of Cd 2+ The toxicity of CdTe QDs also stems from their intracellular distribution and associated nanoscale properties. In some in vitro studies, the toxicity of CdTe QDs may originate from their physical and chemical properties (size, concentration, shape, charge, composition, surface functional groups, mechanical stability, surface charge, and photolytic stability) and the environment. These factors include properties such as the size, concentration, chemical composition, charge, and capped ligands of CdTe QDs, as well as their oxidative, mechanical, and photocatalytic stability. Studies on the toxic use patterns of CdTe QDs in the cell nucleus have revealed that CdTe QDs can induce DNA mutations, thereby preventing the proliferation of new cells that may carry the same disease. To ensure the safe use of CdTe / CDs as fluorescent probes in bioimaging, this invention conducted a biosafety evaluation—the MTT assay—using the following specific testing methods:

[0098] (a) Cell culture: Select L929 cells in good growth condition and in the logarithmic growth phase. Pipette culture medium, wash cells with 1 mL of sterile PBS, digest cells with 0.25% trypsin, add 1 mL of cell culture medium to stop digestion, centrifuge, resuspend cells, calculate cell density using a cell counting chamber, and then add cell culture medium to achieve a cell density of 1 × 10⁻⁶ cells / mL. 5 Then, take 200 μL of cell suspension and seed it into sterile 96-well plates, and set up a culture medium zeroing well.

[0099] (b) Sample addition: After culturing cells in a CO2 incubator for 24 hours, observe the cells under a microscope. When the cells are in good growth condition and reach a suitable cell density, discard the cell culture medium with a pipette and add CdTe / CDs (6 replicates per sample) at different concentrations (0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) to different 96-well plates and continue culturing.

[0100] (c) MTT staining: After culturing in 96-well plates for 24 hours, wash out the cell culture medium with a pipette, add 0.5 mg / mL, 200 μL MTT to each well. (Since the synthesized CdTe / CDs particles are dark in color, they may affect the absorbance in the solution. Therefore, the cells should be washed with sterile PBS to remove particles that may be non-specifically adsorbed on the cell surface.) Wrap the 96-well plate with aluminum foil to protect it from light, and continue incubation for 4 hours. After that, remove the 96-well plate, remove the culture medium with a pipette, add 200 μL DMSO to each well, and shake slowly on a shaker for 5 minutes to completely dissolve the purple-red crystals at the bottom of the 96-well plate.

[0101] (d) Cell viability assessment: The absorbance of each well in a 96-well plate at 490 nm was measured using a multi-functional microplate reader, and the cell viability of each well was calculated based on (sample group - zeroing well) / (control group - zeroing well).

[0102] The results obtained according to the above test method are as follows: Figure 14 As shown. By Figure 14 It is known that the CdTe / CDs prepared in this invention did not show obvious cytotoxicity to L929 cells, and the cell viability could still be maintained at more than 80% even at a concentration as high as 200 μg / mL.

[0103] (8) Cell hemolysis experiment:

[0104] Considering that the prepared CdTe / CDs probes need to be intravenously injected into animal blood vessels in the later stages, it is essential to ensure the blood compatibility of the probes, such as low hemolytic activity. Therefore, this invention conducted a cell hemolysis test on CdTe / CDs, and the specific test method is as follows:

[0105] (a) Take 500 μL of fresh blood from the mouse eyeball and add an anticoagulant to prevent blood clotting;

[0106] (b) Add 5 mL of sterile PBS to the blood, centrifuge at 10,000 rpm for 10 min, discard the supernatant, repeat the centrifugation several times until the supernatant no longer appears red;

[0107] (c) The centrifuged and purified red blood cells were resuspended in 10 mL of PBS. Positive control group: 200 μL red blood cell suspension + 800 μL water; negative control: 200 μL red blood cell suspension + 800 μL PBS; experimental group: 200 μL red blood cell suspension + 800 μL PBS solution of different concentrations of probe (CdTe / CDs concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL).

[0108] (d) After incubation in a 37℃ water bath for 4 hours, centrifuge at 10000 rpm for 10 minutes. Transfer 800 μL of the supernatant to a cuvette and measure the UV absorbance at 541 nm using a UV-8000 microscope. Calculate the hemolysis rate. Hemolysis rate (%) = (Sample absorbance - Negative control absorbance) / (Positive control absorbance - Negative control) × 100%.

[0109] The results obtained according to the above test method are as follows: Figure 15 As shown. By Figure 15 It was found that CdTe / CDs nanoparticles at different concentrations did not exhibit significant hemolytic activity. The exact hemolytic rate of erythrocytes was determined by measuring the absorbance of the supernatant (hemoglobin) at 541 nm using UV-Vis spectroscopy. At the highest probe concentration of 200 μg / mL, only 4.38% hemolytic activity was observed, and the hemolytic rate decreased with decreasing probe concentration. A hemolytic rate exceeding 5% was considered hemolysis. The low hemolytic activity indicates that the CdTe / CDs probe synthesized in this invention causes negligible damage to erythrocytes and holds promise for applications in the biomedical field.

[0110] (9) In vitro near-infrared two-zone fluorescence imaging

[0111] Near-infrared II fluorescence imaging of the CdTe / CDs from Example 1 was tested in PBS, different concentrations of NaCl, and H2O. The test conditions were: 900nm LP filter, 1ms exposure time, and NaCl concentrations of 50mmol / L, 100mmol / L, 150mmol / L, 200mmol / L, 300mmol / L, 400mmol / L, and 500mmol / L. Specific test results are as follows: Figure 16 As shown and Figure 17 As shown, where, Figure 16 The concentration of NaCl in the solution is 0.9% NaCl. Figure 17 (a) is a photograph of CdTe / CDs in H2O and NaCl of different concentrations. Figure 17 (b) Fluorescence spectra of CdTe / CDs in H2O and NaCl of different concentrations. Figure 16 and Figure 17It can be seen that CdTe / CDs exhibit strong second-region fluorescence signals in PBS, NaCl, and H2O. Furthermore, CdTe / CDs show bright second-region fluorescence signals in NaCl solutions of different concentrations. Even when the NaCl concentration is higher than 500 mmol / L, the fluorescence signal of CdTe / CDs does not decrease, indicating that the prepared CdTe / CDs have excellent fluorescence stability and can withstand excessively high ion concentration environments.

[0112] (10) Near-infrared two-zone vascular imaging

[0113] Kunming mice were selected for near-infrared II (NIR-II) vascular imaging. Prior to imaging, all mice were treated with hair removal cream (such as Shuke depilatory cream) and anesthetized with chloral hydrate or isoflurane. They were then placed supine on the imaging stage, and their limbs were secured with tape. At least three mice were used as parallel controls in each imaging experiment. Images were collected on a two-dimensional InGaAs array using an 808 nm laser and a power density of 6000 mA. By adjusting the instrument's exposure time, the InGaAs camera captured images in the NIR-II window. Specific test results are shown below. Figure 18 As shown, where, Figure 18 (a) Figure 18 (b) Figure 18 (c) and Figure 18 (d) are images of the whole-body blood vessels, head blood vessels, hind limb blood vessels, and lymphatic vessels of the mice after injection. Figure 18 As shown in (a), after CdTe / CDs were injected into the tail vein of mice, the entire vascular network of the mouse immediately lit up under 808 nm laser and 1400 nm LP conditions. Figure 18 As shown in (b), due to the high brightness of CdTe / CDs within the transparent window of biological tissue, the spatial resolution and tissue penetration are improved. Furthermore, the pericerebral veins, superior sagittal sinus, and transverse sinus, located approximately 1-2 mm deep beneath the scalp, can be clearly and non-invasively imaged, demonstrating the potential of CdTe / CD nanomaterials in the visualization and monitoring of cerebrovascular diseases. Moreover, even minute capillaries in the higher branches of the vascular network can be illuminated with high spatial resolution. Figure 18 As shown in (c), the femoral artery and vein in the mouse hind limb can be clearly distinguished. Figure 18 As shown in (d), CdTe / CDs can be used for high-brightness lymph node imaging.

[0114] Figure 19 (a) Tumor images recorded at different times using a near-infrared 2D in vivo imaging system after CdTe / CDs were injected into mice via the tail vein. Figure 19 (b) is a fluorescence intensity map of the tumor region. Figure 19(c) is a fluorescence intensity map of the liver region. (From...) Figure 19 The results show that CdTe / CDs can enter the tumor site through the EPR effect and successfully perform tumor imaging, further demonstrating that CdTe / CDs have the ability to perform high-resolution imaging in the near-infrared II region, and further illustrating that the prepared CdTe / CDs nanomaterials have the potential to be applied in the biomedical field.

[0115] (11) Surgical navigation under near-infrared II imaging of CdTe / CDs

[0116] Given that CdTe / CDs can be used for lymph node imaging, Kunming mice were first anesthetized with chloral hydrate. Then, a PBS solution containing CdTe / CDs was subcutaneously injected into the paw pads of the mice's hind limbs, and gentle pressure was applied for 1 minute to promote migration towards lymphatic vessels and nodes, ensuring clear observation of these structures during imaging. Subsequently, a circular incision was made on one hind limb, both internally and externally, to remove the lymph nodes. Finally, the wound was sutured for further experimental applications. Specific test results are as follows: Figure 20 As shown, where, Figure 20 (a) Figure 20 (b) and Figure 20 (c) are actual images of mice before surgery, mice after surgery, and lymph nodes removed after surgery; Figure 20 (d) Figure 20 (e) and Figure 20 (f) Fluorescence imaging images of lymph nodes removed from mice before surgery, after surgery, and after surgery, respectively. Figure 20 It can be seen that CdTe / CDs can successfully perform popliteal lymph node resection surgery on the hind limb of mice under the guidance of near-infrared II imaging.

[0117] In summary, this invention reduces the band gap of CdTe / CDs fabricated by combining carbon nanodots and cadmium telluride quantum dots, redshifting the maximum fluorescence emission wavelength from 855 nm to 1730 nm, covering the entire near-infrared II window. This is the first time that cadmium telluride quantum dots have been applied in the near-infrared II window, providing an effective strategy for redshifting the fluorescence emission wavelength of near-infrared quantum dots. The CdTe / CDs of this invention can be applied in fields such as fluorescent molecular imaging probes and near-infrared II fluorescent contrast agents. Furthermore, the CdTe / CDs of this invention enable deep in vivo fluorescence imaging in the near-infrared II region, such as high-resolution vascular imaging, lymphatic imaging, and imaging of deeper tissues within the body. In contrast, the maximum fluorescence emission wavelength of CdTe quantum dots is below 900 nm, limiting their imaging capabilities to the visible light band and preventing their application in near-infrared II imaging. Because the short fluorescence emission wavelength of CdTe quantum dots affects their penetrating power, the application of CdTe quantum dots is mainly limited to fluorescence imaging at the cellular level, and they cannot be used for imaging at deeper levels in living organisms, such as vascular imaging, lymphatic imaging, and imaging of deeper tissues in living organisms.

[0118] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A near-infrared II imaging material, characterized in that: It includes quantum dot aggregates, which are assembled from CdTe quantum dots and carbon quantum dots; the near-infrared II imaging material has a maximum fluorescence emission wavelength of 1300-1800 nm when the excitation wavelength is 650-900 nm.

2. The near-infrared II imaging material according to claim 1, characterized in that: The band gap of the near-infrared II imaging material is 0.01–1.15 eV; And / or, the quantum dot aggregates are spherical, near-spherical, or rod-shaped; And / or, the particle size of the near-infrared II imaging material is 5–500 nm.

3. The near-infrared II imaging material according to claim 1, characterized in that: The mass ratio of the CdTe quantum dots to the carbon quantum dots is 1:(0.001~1).

4. The near-infrared II imaging material according to claim 1, characterized in that: The near-infrared II imaging material has a maximum fluorescence emission wavelength of 1300–1800 nm when the excitation wavelength is 808 nm.

5. The method for preparing the near-infrared II imaging material according to any one of claims 1 to 4, characterized in that: Includes the following steps: The near-infrared II imaging material was prepared by mixing carbon quantum dots, a cadmium source, and mercaptopropionic acid, adjusting the pH, and then reacting it with NaHTe.

6. The method for preparing near-infrared II imaging material according to claim 5, characterized in that: The step of adjusting pH is to use an alkaline solution to adjust the pH value to be greater than 7. And / or, the reaction temperature is 80–160°C; And / or, the reaction time is 0.5 to 72 h.

7. A near-infrared II region fluorescent molecular imaging probe, characterized in that: Includes the near-infrared II imaging material as described in any one of claims 1 to 4.

8. A near-infrared II fluorescent contrast agent, characterized in that: Includes the near-infrared II imaging material as described in any one of claims 1 to 4.

9. A detection system, characterized in that: It includes the near-infrared II fluorescent molecular imaging probe of claim 7 or the near-infrared II fluorescent contrast agent of claim 8.

10. The application of the near-infrared II imaging material according to any one of claims 1 to 4 in the field of fluorescence imaging.

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