Mof-based high-order multi-photon excitation fluorescent material and preparation method and application thereof

Abstract

The application discloses a MOF-based high-order multi-photon excitation fluorescent material and a preparation method and application thereof, relates to the technical field of multi-photon excitation fluorescent materials, and the preparation method comprises the following steps: 1) preparation of ZrTc; 2) preparation of Au NRs; 3) preparation of Au NR / ZrTc; and 4) preparation of Au NR / ZrTc@FA. The MOF-based high-order multi-photon excitation fluorescent material prepared by the application has good biocompatibility and low dark toxicity, can realize enhanced two / three-photon fluorescence imaging under near-infrared two-region laser irradiation, and can be applied to cancer cell specific targeting and deep tissue fluorescence imaging.

Description

Technical fields:

[0001] This invention relates to the field of multiphoton-excited fluorescent materials technology, specifically to a MOF-based high-order multiphoton-excited fluorescent material, its preparation method, and its application. Background technology:

[0002] In recent years, nonlinear optical materials have attracted widespread attention for their unique strong penetration depth, high spatial resolution, and 3D reconstruction capabilities for biological samples, particularly in various non-invasive biological applications such as in vitro and in vivo imaging. In this field, multiphoton-excited fluorescence materials are a research hotspot for many chemistry researchers, especially high-order multiphoton-excited (n≥3) fluorescence (H-MPEF) materials in the near-infrared II (NIR-II) region. These materials offer stronger spatial confinement, deeper tissue penetration, less Rayleigh scattering, and less tissue damage, which is extremely beneficial for practical applications in bioimaging. However, due to the lack of suitable construction strategies and complex structural modulation of traditional multiphoton materials, there are currently few reports on H-MPEF-responsive nanomaterials. Therefore, researchers are dedicated to developing novel H-MPEF-responsive nanomaterials to overcome these challenges.

[0003] Nanoscale metal-organic frameworks (nMOFs) not only possess the advantages of both organic and inorganic nanomaterials but also exhibit inherent porosity, high design flexibility, and general modifiability. These properties enable precise integration from structural design to controlled fabrication, providing unique solutions for developing H-MPEF-responsive materials and achieving efficient bioimaging. The Vittal research group reported the first multiphoton-excitation-sensitive MOF, exhibiting large two-, three-, and four-photon absorption cross-sections by incorporating nonlinear optics (NLO)-active organic chromophores as ligands. Fisher et al. investigated a series of H-MPEF-responsive MOFs by varying parameters such as ligand deformation or chromophore spatial arrangement, providing valuable insights for designing H-MPEF-active MOFs. Our group reports an H-MPEF-responsive MOF (ZrTc) for bioimaging, utilizing a thiazothiazolium-based unit as an organic linker for higher-order multiphoton excitation. However, current strategies for improving the H-MPEF performance of MOFs typically focus on structural modifications such as using ligands with optimized structures and adjusting the degree of conjugation in the system, which makes MOF synthesis challenging. Therefore, a simple and effective general approach is needed to enhance the activity of H-MPEFs. Previous studies have shown that combining localized surface plasmon resonance (LSPR) with nonlinear optical (NLO) materials can significantly improve their NLO activity, including second-harmonic and third-order nonlinear two-photon absorption effects. However, to date, there are no reports on LSPR-driven enhancements to the performance of H-MPEFs. Summary of the Invention:

[0004] The technical problem to be solved by this invention is to provide a MOF-based high-order multiphoton excitation fluorescent material (AuNR / ZrTc@FA), whose molecular structure includes Au NRs units as plasmonic exciters and ZrTc units with multiphoton response, thereby achieving enhanced two-photon excitation fluorescence (TPEF) and H-MPEF performance. The degree of enhancement strongly depends on the overlap between the multiphoton excitation wavelength of MOFs and the local surface plasmon resonance absorbance of Au NRs. Through the encapsulation effect of folic acid (FA), its application in cancer cell-specific targeting and deep tissue fluorescence imaging is realized.

[0005] The technical problem to be solved by this invention is achieved by the following technical solution:

[0006] The first objective of this invention is to provide a method for preparing MOF-based higher-order multiphoton-excited fluorescent materials, comprising the following steps:

[0007] 1) Preparation of ZrTc:

[0008] 1a. Using methyl 4-formylbenzoate and dithiooxazone as raw materials, ligand Tc was obtained through condensation and hydrolysis reactions.

[0009] The reaction equation is as follows:

[0010]

[0011] 1b. ZrCl4 undergoes a hydrothermal reaction with ligand Tc and benzoic acid to yield ZrTc;

[0012] 2) Preparation of Au NRs:

[0013] 2a. Prepare seed solution from HAuCl4, CTAB, and sodium borohydride;

[0014] 2b. Prepare a growth solution from HAuCl4, CTAB, sodium oleate, silver nitrate and ascorbic acid;

[0015] 2c. Add seed solution to growth solution, let stand to react, and obtain Au NRs;

[0016] 3) Preparation of Au NR / ZrTc:

[0017] Mixing ZrTc and Au NRs yields Au NR / ZrTc;

[0018] 4) Preparation of Au NR / ZrTc@FA:

[0019] Au NR / ZrTc was encapsulated with folic acid to obtain Au NR / ZrTc@FA.

[0020] The second objective of this invention is to provide a MOF-based high-order multiphoton excited fluorescent material obtained by the aforementioned preparation method.

[0021] A third objective of this invention is to provide the application of the aforementioned MOF-based higher-order multiphoton-excited fluorescent materials in fluorescence imaging of deep tissues.

[0022] A fourth objective of this invention is to provide the application of the aforementioned MOF-based higher-order multiphoton-excited fluorescent materials in cancer cell-specific targeting.

[0023] The beneficial effects of this invention are:

[0024] 1. The method for preparing MOF-based high-order multiphoton excitation fluorescent materials provided by the present invention is simple and easy to operate, with low cost, and the obtained MOF-based high-order multiphoton excitation fluorescent materials have good biocompatibility and low dark toxicity.

[0025] 2. The MOF-based high-order multiphoton excitation fluorescent material prepared by this invention can achieve enhanced two / three-photon fluorescence imaging under near-infrared II laser irradiation, and can be applied to cancer cell-specific targeting and deep tissue fluorescence imaging. Attached image description:

[0026] Figure 1 Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of AZF prepared in Example 1;

[0027] Figure 2 Two-photon excitation fluorescence spectra of AZF prepared in Example 1 and ZF prepared in Comparative Example 1;

[0028] Figure 3 The three-photon excitation fluorescence spectra of AZF prepared in Example 1 and ZF prepared in Comparative Example 1 are shown.

[0029] Figure 4 Cell viability of AZF prepared in Example 1 under dark conditions, as determined by the MTT assay;

[0030] Figure 5 The targeting of AZF prepared in Example 1 to normal cells HEK 293T and cancer cells Hep G2;

[0031] Figure 6 Two / three-photon fluorescence imaging of AZF prepared in Example 1 and ZF prepared in Comparative Example 1;

[0032] Figure 7 Confocal tissue imaging images of AZF prepared in Example 1 under laser irradiation at 458nm, 950nm, and 1150nm. Detailed implementation method:

[0033] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments and illustrations.

[0034] This invention provides a method for preparing MOF-based higher-order multiphoton-excited fluorescent materials, comprising the following steps:

[0035] 1) Preparation of ZrTc:

[0036] 1a. Using methyl 4-formylbenzoate and dithiooxazone as raw materials, ligand Tc was obtained through condensation and hydrolysis reactions.

[0037] The reaction equation is as follows:

[0038]

[0039] 1b. ZrCl4 undergoes a hydrothermal reaction with ligand Tc and benzoic acid to yield ZrTc;

[0040] 2) Preparation of Au NRs:

[0041] 2a. Prepare seed solution from HAuCl4, CTAB, and sodium borohydride;

[0042] 2b. Prepare a growth solution from HAuCl4, CTAB, sodium oleate, silver nitrate and ascorbic acid;

[0043] 2c. Add seed solution to growth solution, let stand to react, and obtain Au NRs;

[0044] 3) Preparation of Au NR / ZrTc:

[0045] Mixing ZrTc and Au NRs yields Au NR / ZrTc;

[0046] 4) Preparation of Au NR / ZrTc@FA:

[0047] Au NR / ZrTc was encapsulated with folic acid to obtain Au NR / ZrTc@FA.

[0048] In a further technical solution, the molar ratio of methyl 4-formylbenzoate to dithiooxazone is (2.1-2.5):1.

[0049] In a further technical solution, the molar ratio of ZrCl4 to ligand Tc and benzoic acid is 1:(1~2):(3~4).

[0050] In a further technical solution, the temperature of the hydrothermal reaction is 120°C, and the time is 18–72 h.

[0051] In a further technical solution, the molar ratio of HAuCl4 to CTAB and sodium borohydride is 0.01:(300-400):(2-3).

[0052] In a further technical solution, the molar ratio of HAuCl4 to CTAB, sodium oleate, silver nitrate, and ascorbic acid is 0.01:(100~200):(10~30):(0.2~0.3):(1.5~2).

[0053] In a further technical solution, the temperature of the static reaction is 28-30°C, and the time is 12-18 hours.

[0054] In a further technical solution, the mass ratio of ZrTc to Au NRs is 1:(1.25~2).

[0055] In a further technical solution, the mass ratio of Au NR / ZrTc to folic acid is (1-3):1.

[0056] The present invention also provides a MOF-based high-order multiphoton excitation fluorescent material obtained by the aforementioned preparation method. The size of the MOF-based high-order multiphoton excitation fluorescent material is 150–200 nm.

[0057] This invention also provides the application of the aforementioned MOF-based higher-order multiphoton-excited fluorescent materials in deep tissue fluorescence imaging.

[0058] This invention also provides the application of the aforementioned MOF-based higher-order multiphoton-excited fluorescent materials in cancer cell-specific targeting.

[0059] Explanation of abbreviations in this invention:

[0060] AZF: Au NR / ZrTc@FA;

[0061] ZF: ZrTc@FA;

[0062] ZrTc: Zr-MOF nanometal-organic framework;

[0063] Au NRs: gold nanorods;

[0064] DMF: N,N-dimethylformamide;

[0065] KOH: Potassium hydroxide;

[0066] ZrCl4: Zirconium chloride;

[0067] HAuCl4: Tetrachloroauric acid;

[0068] CTAB: Hexadecyltrimethylammonium bromide;

[0069] NaBH4: Sodium borohydride.

[0070] Example 1: Preparation of AZF

[0071] 1) Preparation of ZrTc

[0072] Preparation of ligand Tc: 30 mL of DMF was added to a flask and bubbled under nitrogen for 1 h. Then, methyl 4-formylbenzoate (1.96 g, 12 mmol) and dithiooxazone (0.6 g, 5 mmol) were added, and the mixture was refluxed for 6 h. After cooling to room temperature, the mixture was filtered, washed with DMF, washed with water, and dried under vacuum to obtain a pale yellow solid. 0.2 g (0.48 mmol) of the pale yellow solid was dissolved in a mixed solvent of tetrahydrofuran (8 mL) and methanol (8 mL), and an aqueous solution of KOH (0.08 g, 14.4 mmol) was added. The mixture was refluxed for 12 h. After cooling to room temperature, the solvent was evaporated at 70 °C, dissolved in water, and the pH was adjusted to 2–3 with hydrochloric acid. The mixture was filtered and dried under vacuum to obtain ligand Tc (0.18 g).

[0073] Preparation of ZrTc: A mixed solution containing the prepared ligand Tc (0.014 mol / L), ZrCl4 (0.01 mol / L), benzoic acid (0.14 g), and DMF (30 mL) was added to a hydrothermal reactor, sealed, and maintained at 120 °C for 72 h. After cooling to room temperature, the solid was collected by centrifugation, washed with DMF and ethanol, and dried under vacuum to obtain ZrTc.

[0074] 2) Preparation of Au NRs

[0075] Take 10 mL of CTAB aqueous solution (0.10 M) and 0.1 mL of HAuCl4 aqueous solution (2.5 × 10⁻⁶ M). -4 Mix and stir, then add ice-cold NaBH4 aqueous solution (0.6 mL, 0.01 M), stir vigorously, and let stand at 29 °C for 2 h. The solution turns brownish-yellow, and the seed solution is obtained.

[0076] Take 50 mL of an aqueous solution of CTAB (0.1 M) and sodium oleate (0.01 M), then add 1 mL of HAuCl4 aqueous solution (2.5 × 10⁻⁶). -4 The mixture of ascorbic acid (M) and AgNO3 aqueous solution (0.72 mL, 0.01 M) was stirred at 28 °C. After the color of the mixture changed from yellow to colorless, hydrochloric acid (0.3 mL, 12 M) was added dropwise to adjust the pH. Then, ascorbic acid aqueous solution (0.6 mL, 0.078 M) was added to obtain the growth solution.

[0077] Add 0.1 mL of seed solution to the above growth solution, let stand at 28 °C for 12 h, centrifuge, wash and collect the product to obtain Au NRs solution.

[0078] 3) Preparation of Au NR / ZrTc

[0079] 1 mg ZrTc was added to 4 mL of ethanol and ultrasonically dispersed. 48 mL of the Au NRs solution with absorbance AR = 1.8 prepared above was added dropwise while stirring. After the addition was complete, stirring was continued for 24 h. The product was collected by centrifugation, washed with water, and freeze-dried to obtain Au NR / ZrTc.

[0080] 4) Preparation of Au NR / ZrTc@FA

[0081] Dissolve 3 mg of folic acid in 3 mL of DMF by sonication, then add 3 mg of the Au NR / ZrTc prepared above, disperse by sonication, stir at room temperature in the dark for 12 h, centrifuge, wash with DMF, wash with water, freeze dry to obtain AZF.

[0082] from Figure 1 It can be seen that the size of the AZF prepared in Example 1 is about 150 nm, wherein ZrTc has a spherical structure and AuNR has a rod-like structure.

[0083] Comparative Example 1: Preparation of ZF

[0084] The difference from Example 1 is that Au NRs are not prepared; ZrTc is directly encapsulated with FA.

[0085] Dissolve 3 mg of folic acid in 3 mL of DMF by sonication, then add 3 mg of the ZrTc prepared above, disperse by sonication, stir at room temperature in the dark for 12 h, centrifuge, wash with DMF, wash with water, freeze dry to obtain ZF.

[0086] Example 2: Two-photon excitation fluorescence (2PEF) performance detection

[0087] Two PEF spectra were obtained using a femtosecond laser pulse and a Ti:sapphire system (680–1080 nm, 80 MHz, 140 fs) as the light source, employing the two-photon excitation fluorescence (2PEF) method. The reference sample was Rhodamine B with an ethanol concentration of 1.0 × 10⁻⁶. -3 The concentrations of M, ZF, and AZF were both 200 μg / mL. The formula for calculating the 2PA cross section is as follows:

[0088]

[0089] Where ref is the reference sample, δ is the two-photon absorption cross section, Φ is the fluorescence quantum yield, c is the sample concentration, n is the refractive index, and F is the two-photon fluorescence integral area.

[0090] from Figure 2 It can be seen that, compared with the ZF prepared in Comparative Example 1, the AZF prepared in Example 1 has stronger two-photon excitation fluorescence performance.

[0091] Example 3: Detection of Multiphoton Excited Fluorescence (3PEF) Performance

[0092] MPEF spectra were obtained using multiphoton excitation fluorescence method with a coherent +TOPAS Prime (1050–1350 nm, 1 kHz, 120 fs) as the light source. The reference sample was Rhodamine 6G (1.0 × 10⁻⁶). -3 M). The concentrations of ZF and AZF are 200 μg / mL. The formula for calculating the multiphoton absorption cross section is as follows:

[0093]

[0094] Where γ is the absorption coefficient of the three photons, λ is the wavelength of the incident light, and N A d is Avogadro's constant, d0 is the sample concentration (200 μg / mL), and n is the number of absorbed photons (n ​​= 3).

[0095] from Figure 3 It can be seen that, compared with the ZF prepared in Comparative Example 1, the AZF prepared in Example 1 has stronger three-photon excitation fluorescence performance.

[0096] Example 4: Biocompatibility Test

[0097] The cytotoxicity of AZF was studied using the MTT assay. AZF was diluted with fresh DMEM to obtain different concentrations (0, 10, 30, 50, 70, 90, 110, 130 μg / mL). Before the experiment, HeLa cells were cultured in 96-well plates for 24 h, then the medium was exchanged with different concentrations of AZF solution, and incubated at 37°C and 5% CO2 for 12 h. Cell viability was then assessed using the MTT assay. The medium was exchanged with 100 μL of fresh DMEM, and then 20 μL of MTT aqueous solution (5 mg / mL) was added to each well. The cell plates were incubated at 37°C and 5% CO2 for 4 h, the MTT and medium were removed, and 100 μL of formazan in DMSO solution was added to each well. Cell viability was calculated by measuring the absorbance at 490 nm using a microplate reader.

[0098] from Figure 4 It can be seen that the AZF prepared in Example 1 has good biocompatibility and can be further used for biological tissue imaging.

[0099] Example 5: Cancer Cell Specific Targeting

[0100] To verify the tumor-specific targeting of AZF, HEK 293T cells (cd44-negative cells) and HepG2 cells (cd44-positive cells) were seeded into their respective cell culture dishes and cultured to approximately 70% confluence before use. HEK 293T cells and HepG2 cells were treated with AZF (30 μg / mL), respectively. After 12 h of incubation, the cellular uptake capacity of AZF was analyzed using an Olympus FVMPE-RS confocal microscopy system with a 458 nm excitation channel.

[0101] from Figure 5 It can be seen that, compared with normal cells HEK 293T, the AZF prepared in Example 1 can specifically target tumor cells HepG2.

[0102] Example 6: Two / Three Photon Fluorescence Imaging

[0103] Two / three-photon fluorescence imaging was achieved using an Olympus FVMPE-RS system equipped with a femtosecond laser (output wavelength 690–1300 nm). Tissue sections were obtained from Balb / c mouse myocardial tissue, cut to a thickness of 300 μm, and incubated with ZF / AZF for 60 min at 950 nm and 1150 nm (0.4 W / cm²). 2 Two-photon and three-photon three-dimensional fluorescence imaging was observed under excitation. Hep G2 cells treated with ZF and AZF (30 μg / mL) were selected for cell fluorescence imaging.

[0104] from Figure 6 It can be seen that, compared with the ZF prepared in Comparative Example 1, the AZF prepared in Example 1 has higher fluorescence imaging intensity and resolution under 950nm and 1150nm laser irradiation.

[0105] from Figure 7 It can be seen that the AZF prepared in Example 1 can achieve deep tissue fluorescence imaging under near-infrared II laser irradiation.

[0106] Example 7: Preparation of AZF

[0107] The preparation method of AZF in this embodiment is the same as in Example 1, except that the hydrothermal reaction time is adjusted to 24 hours.

[0108] Example 8: Preparation of AZF

[0109] The preparation method of AZF in this embodiment is the same as that in Example 1, except that the molar ratio of ZrCl4 to ligand Tc is adjusted to 1:1.8.

[0110] Example 9: Preparation of AZF

[0111] The preparation method of AZF in this embodiment is the same as in Example 1, except that the mass ratio of ZrTc to Au NRs is adjusted to 1:2.

[0112] Example 10: Preparation of AZF

[0113] The preparation method of AZF in this embodiment is the same as in Example 1, except that the mass ratio of Au NR / ZrTc to folic acid is adjusted to 2:1.

[0114] Example 11: Preparation of AZF

[0115] The preparation method of AZF in this embodiment is the same as that in Example 1, except that the molar ratio of HAuCl4 to CTAB and sodium borohydride in the seed solution preparation is adjusted to 0.01:300:2.

[0116] Example 12: Preparation of AZF

[0117] The preparation method of AZF in this embodiment is the same as that in Example 1, except that the molar ratio of HAuCl4 to CTAB, sodium oleate, silver nitrate and ascorbic acid in the preparation of the growth solution is adjusted to 0.01:150:15:0.3:1.5.

[0118] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing MOF-based high-order multiphoton-excited fluorescent materials, characterized in that, Includes the following steps: 1) Preparation of ZrTc: 1a. Using methyl 4-formylbenzoate and dithiooxazone as raw materials, ligand Tc was obtained through condensation and hydrolysis reactions. ； 1b. ZrCl4 undergoes a hydrothermal reaction with ligand Tc and benzoic acid to obtain ZrTc; the hydrothermal reaction is carried out at a temperature of 120℃ for a time of 18~72 h. 2) Preparation of Au NRs: 2a. Prepare seed solution from HAuCl4, CTAB, and sodium borohydride; 2b. Prepare a growth solution from HAuCl4, CTAB, sodium oleate, silver nitrate and ascorbic acid; 2c. Add seed solution to growth solution, let stand to react, and obtain Au NRs; 3) Preparation of Au NR / ZrTc: Mixing ZrTc and Au NRs yields Au NR / ZrTc; 4) Preparation of Au NR / ZrTc@FA: Au NR / ZrTc was encapsulated with folic acid to obtain Au NR / ZrTc@FA.

2. The preparation method according to claim 1, characterized in that: The molar ratio of methyl 4-formylbenzoate to dithiooxazone is (2.1~2.5):1; the molar ratio of ZrCl4 to ligand Tc and benzoic acid is 1:(1~2):(3~4).

3. The preparation method according to claim 1, characterized in that: The molar ratio of HAuCl4 to CTAB and sodium borohydride is 0.01 : (300~400) : (2~3); the molar ratio of HAuCl4 to CTAB, sodium oleate, silver nitrate and ascorbic acid is 0.01 : (100~200) : (10~30) : (0.2~0.3) : (1.5~2).

4. The preparation method according to claim 1, characterized in that: The static reaction is carried out at a temperature of 28-30°C for 12-18 hours.

5. The preparation method according to claim 1, characterized in that: The mass ratio of ZrTc to Au NRs is 1:(1.25~2).

6. The preparation method according to claim 1, characterized in that: The mass ratio of Au NR / ZrTc to folic acid is (1~3):

1.

7. MOF-based high-order multiphoton excitation fluorescent materials obtained by the preparation method according to any one of claims 1-6.

8. The application of the MOF-based high-order multiphoton-excited fluorescent material of claim 7 in fluorescence imaging of deep tissues, wherein the application is for non-therapeutic and non-diagnostic purposes.

9. The application of the MOF-based high-order multiphoton-excited fluorescent material of claim 7 in cancer cell-specific targeting, wherein the application is for non-therapeutic and non-diagnostic purposes.

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