A surface-modified DNA nanoscale metal-organic framework and its preparation method and application

Abstract

The present invention relates to a surface-modified DNA-based nano metal-organic framework (Metal-Organic Framework, abbreviated as MOF) and its application. The organic framework serves as a universal platform for activatable photodynamic cancer therapy, that is, a DNA-functionalized nano metal-organic framework serves as a universal platform for activatable photodynamic cancer therapy. Specifically, it uses an iron-based MOF to load different types of photosensitizers, and utilizes the Fenton-like reaction participated by ferric iron in the tumor microenvironment to specifically release and activate the photosensitizers at the tumor site, improving the therapeutic effect. The surface-modified DNA can not only improve the biocompatibility of the nanoparticles but also effectively prevent drug leakage during blood circulation, thereby reducing the impact on normal tissue cells. It is a targeted therapeutic nano drug and belongs to the technical fields of material chemistry and nano medicine.

Description

Technical Field

[0001] The present invention relates to a surface-modified DNA nano metal-organic framework (Metal-Organic Framework, abbreviated as MOF), a preparation method and an application thereof. The organic framework serves as a general platform for activatable photodynamic cancer therapy, that is, a DNA-functionalized nano metal-organic framework serves as a general platform for activatable photodynamic cancer therapy. Specifically, it uses an iron-based MOF to load different types of photosensitizers, and utilizes the Fenton-like reaction participated by ferric iron in the tumor microenvironment to specifically release and activate the photosensitizers at the tumor site, improving the treatment effect. The surface-modified DNA can not only improve the biocompatibility of the nanoparticles but also effectively prevent drug leakage during blood circulation, thereby reducing the impact on normal tissue cells. It is a targeted therapeutic nano-drug and belongs to the technical fields of material chemistry and nanomedicine. Background Art

[0002] Photodynamic Therapy (PDT) is a tumor treatment technique that relies on photosensitizers (PS) to generate cytotoxic reactive oxygen species (ROS), including peroxides, superoxides, singlet oxygen ( 1 O2), etc. to kill cancer cells, and has the advantages of spatio-temporal precision, non-invasiveness and low side effects. However, most PS do not necessarily distribute in malignant tissues, and PS present in the skin and healthy tissues often exhibit non-specific off-target phototoxicity. Therefore, researchers have recently developed activatable PS that can be activated in tumor tissues to generate ROS while remaining inert in normal cells. However, these strategies are usually limited to a few PS that have undergone specific molecular modifications.

[0003] MOF has high porosity and structural diversity, which is convenient for the transport and biofunctionalization of guest molecules, and has great potential in biomedical applications as a nanocarrier. In recent years, researchers have developed an activatable PS system assisted by nano-hydrophobic MOF with Fe(III) or Cu(II) as nodes to achieve selective activation of PS in tumor tissues. However, this strategy is only applicable to hydrophobic PS because the hydrophilic photosensitizers transported by hydrophobic MOF are prone to leakage due to hydrophilic interaction during transportation, which not only reduces the treatment effect but also causes damage to healthy parts. Summary of the Invention

[0004] When the technology of the present invention solves problems: It overcomes the deficiencies of the prior art and provides a nano metal-organic framework with surface-modified DNA, its preparation method and application. This DNA-functionalized nano metal-organic framework serves as a general platform for activatable photodynamic cancer therapy, which is a general and highly efficient targeted therapy strategy. Aiming at the problems of poor targeting and easy leakage of hydrophilic PS in the nano drug delivery system in current PDT, it can greatly improve the accuracy of targeted therapy and the efficiency of activation, and has excellent versatility for various types of PS.

[0005] The object of the present invention is achieved by the following technical solutions.

[0006] A nano metal-organic framework with surface-modified DNA, where the nano metal-organic framework is MIL-101 modified with azide groups (MIL-101-N3), a photosensitizer is loaded in the pores of the nano metal-organic framework, and DNA is modified on the surface of the nano metal-organic framework;

[0007] The photosensitizer is a hydrophobic photosensitizer or a hydrophilic photosensitizer. The hydrophobic photosensitizer is 2-(4-(diphenylamino)phenyl)anthraquinone (TPAAQ), and the hydrophilic photosensitizer is methylene blue (MB);

[0008] The DNA is dibenzocyclooctyne-functionalized DNA (DBCO-DNA).

[0009] A preparation method of a nano metal-organic framework with surface-modified DNA. This method uses the solvothermal method to prepare nano-scale MOF particles with amino groups, converts the amino groups into azides, then loads the photosensitizer into the MOF pores, and finally modifies the surface of the MOF with DNA to obtain the final product. The steps of this method include:

[0010] The first step is to prepare MIL-101-NH2. The specific method is: Mix 2-aminoterephthalic acid (BDC-NH2), ferric chloride hexahydrate (FeCl3·6H2O), cetyltrimethylammonium bromide (CTAB) and N,N-dimethylformamide (DMF) solvent. The mixing temperature is 50 - 80 °C, and the mixing time is 12 - 24 h to obtain MIL-101-NH2, and then activate the obtained MIL-101-NH2;

[0011] The molar ratio of the 2-aminoterephthalic acid (BDC-NH2), ferric chloride hexahydrate (FeCl3·6H2O), cetyltrimethylammonium bromide (CTAB) and N,N-dimethylformamide (DMF) solvent is 100 - 150:200 - 300:1:15000 - 20000;

[0012] The activation method is as follows: extract with ethanol in a Soxhlet extractor for 2 - 4 h, then extract with DMF for more than 10 h, wash several times with methanol, and then store in absolute ethanol at room temperature;

[0013] In the second step, prepare MIL - 101 - N3. The specific method is as follows:

[0014] Mix azidotrimethylsilane (TMSN3), tert - butyl nitrite (tBuONO), MIL - 101 - NH2 obtained in the first step, and tetrahydrofuran (THF) solvent for more than 8 h to obtain MIL - 101 - N3;

[0015] The molar ratio of the azidotrimethylsilane (TMSN3), tert - butyl nitrite (tBuONO), MIL - 101 - NH2 obtained in the first step, and tetrahydrofuran (THF) solvent is 15000 - 20000:150:80 - 150:50 - 100;

[0016] In the third step, prepare PS@MIL - 101 - N3. The specific method is as follows: Disperse MIL - 101 - N3 obtained in the second step in ethanol and mix it with a PS (photosensitizer) solution by stirring. When mixing, the final concentrations of PS (photosensitizer) and MIL - 101 - N3 are 1 - 4 mg mL -1 and 0.5 - 1.0 mg mL -1 , then the PS (photosensitizer) is impregnated into the pores of MIL - 101 - N3 to obtain PS@MIL - 101 - N3; The solvent in the PS solution is tetrahydrofuran, and the concentration of the PS (photosensitizer) solution is 2 - 8 mg mL -1 ; The concentration ratio of MIL - 101 - N3 to the PS (photosensitizer) solution is 1:1 - 1:8;

[0017] The PS is 2 - (4 - (diphenylamino)phenyl) - anthraquinone (TPAAQ) with aggregation - induced emission properties and hydrophobicity or methylene blue (MB) with aggregation - induced quenching properties and hydrophilicity;

[0018] Among them, TPAAQ is obtained by the Suzuki coupling chemical method of 4 - (diphenylamino)phenylboronic acid pinacol ester and 2 - bromoanthraquinone;

[0019] The 2 - (4 - (diphenylamino)phenyl)anthraquinone (TPAAQ) is a hydrophobic photosensitizer, and the methylene blue (MB) is a hydrophilic photosensitizer.

[0020] The method for preparing TPAAQ is as follows: Degas and charge nitrogen into a mixture of 4-(diphenylamino)phenylboronic acid pinacol ester (0.30 - 0.80 mmol), 2-bromoanthraquinone (0.30 - 0.80 mmol), potassium carbonate (2 - 8 mmol), 10 - 20 mL of tetrahydrofuran, 3 - 7 mL of water, and tetrakis(triphenylphosphine)palladium (3 - 5 mol%). Stir the reaction mixture at 50 - 70 °C for more than 12 h. After cooling to ambient temperature, stop the reaction by adding water, extract with dichloromethane, and wash with brine to obtain TPAAQ;

[0021] The calculation method for the PS content loaded on the prepared MIL-101-N3 is as follows:

[0022] Add MIL-101-N3 (100 mg) to a 10 mL THF solution of TPAAQ (30 mg mL -1 ), stir at room temperature, and monitor the PS loading amount (wt%) in real-time through the ultraviolet-visible absorption spectrum of the supernatant obtained by centrifugation;

[0023] Add MIL-101-N3 (100 mg) to a 10 mL THF solution of MB (30 mg mL -1 ), stir at room temperature, and monitor the PS loading amount (wt%) in real-time through the ultraviolet-visible absorption spectrum of the supernatant obtained by centrifugation;

[0024]

[0025] Among them, A PS负载前 is the ultraviolet-visible absorption value of PS before loading; A PS负载后 is the ultraviolet-visible absorption value of PS after loading; C PS is the concentration of PS; C MIL-101-N3 is the concentration of MIL-101-N3;

[0026] In the fourth step, convert the PS@MIL-101-N3 prepared in the third step into DNA-PS@MIL-101-N3 by click chemistry. The specific method is as follows: Graft DNA to the PS@MIL-101-N3 prepared in the third step to obtain DNA-PS@MIL-101-N3, that is, react MIL-101-N3 with dibenzocyclooctyne (DBCO)-functionalized DNA to obtain a mixed solution and stir at 30 - 50 °C for 48 - 72 h. After centrifugation, wash the precipitate with PBS solution several times to remove unreacted nucleic acids to obtain DNA-PS@MIL-101-N3, that is, a nanoscale metal-organic framework with DNA surface modification and loaded with photosensitizer; For example: MIL-101-N3 (0.5 - 3.0 mg mL -1 , 2 mL) and DBCO-functionalized DNA (100×10-6 React with M (0.2 - 1.0 mL), and stir the resulting mixture solution at 40 °C for more than 24 h.

[0027] The DNA-functionalized nano metal-organic framework (Metal-Organic Framework, abbreviated as MOF) is used as a universal platform for activatable photodynamic cancer therapy. The MOF is formed by grafting DNA onto the surface of azide-modified iron terephthalate metal-organic framework material (MIL-101) through a click reaction, and a photosensitizer is loaded in the MOF;

[0028] The azide-modified MIL-101 (MIL-101-N3) is obtained by transforming amino-modified MIL-101 (MIL-101-NH2);

[0029] The DNA is dibenzocyclooctyne-functionalized DNA (DBCO-DNA);

[0030] An application of a nano metal-organic framework with surface-modified DNA, that is, as a universal platform of DNA-functionalized MOF for activating photodynamic therapy of tumors.

[0031] The present invention has the following beneficial effects:

[0032] (1) The nano metal-organic framework with surface-modified DNA of the present invention can load various photosensitizers, including PSs with hydrophilic and hydrophobic properties, greatly expanding the versatility of various PSs.

[0033] (2) The nano metal-organic framework with surface-modified DNA of the present invention can effectively increase the oxygen concentration at the tumor site, so it can activate the photosensitizer while releasing the photosensitizer, improving the effect of killing tumor cells.

[0034] (3) The nano metal-organic framework with surface-modified DNA of the present invention can improve the biocompatibility of the nanoparticles and prolong the blood circulation time, so as to make full use of the enhanced permeability and retention effect (EPR effect) to enrich at the tumor site.

[0035] (4) The nano metal-organic framework with surface-modified DNA of the present invention can greatly prevent the leakage of drugs during blood circulation and reduce the damage to normal tissue cells. Brief Description of the Drawings

[0036] Figure 1 For the preparation process and action principle of the nano drug delivery platform;

[0037] Figure 2 For the powder X-ray diffraction of MIL-101-N3 in Example 1;

[0038] Figure 3 Infrared spectra of MIL-101-N3 and MIL-101-NH2 in Example 1;

[0039] Figure 4 1H NMR spectrum of MIL-101-NH2 in Example 1;

[0040] Figure 5 1H NMR spectrum of MIL-101-N3 in Example 1;

[0041] Figure 6 Transmission electron microscope of MIL-101-N3 in Example 1;

[0042] Figure 7 1H NMR spectrum of TPAAQ in Example 2;

[0043] Figure 8 13C NMR spectrum of TPAAQ in Example 2;

[0044] Figure 9 UV-Vis absorption spectrum of MIL-101-N3 loaded with MB in Example 3;

[0045] Figure 10 Thermogravimetric curve of MIL-101-N3 loaded with MB in Example 3;

[0046] Figure 11 UV-Vis absorption spectrum of MIL-101-N3 loaded with TPAAQ in Example 3;

[0047] Figure 12 Thermogravimetric curve of MIL-101-N3 loaded with TPAAQ in Example 3;

[0048] Figure 13 N2 adsorption curve of MIL-101-N3 loaded with PS in Example 3;

[0049] Figure 14 Gel electrophoresis and transmission electron microscope images of DNA-MB@MIL-101-N3 in Example 4;

[0050] Figure 15 Gel electrophoresis and transmission electron microscope images of DNA-TPAAQ@MIL-101-N3 in Example 4;

[0051] Figure 16 Transmission electron microscope image of DNA-MB@MIL-101 treated with deoxyribonuclease I (DNase-I) in Example 4;

[0052] Figure 17Transmission electron microscopy image of DNA-TPAAQ@MIL-101 treated with deoxyribonuclease I (DNase-I) in Example 4;

[0053] Figure 18 Zeta potential diagrams of DNA-MB@MIL-101 and DNA-TPAAQ@MIL-101 in Example 5;

[0054] Figure 19 Dynamic light scattering particle size analyzer of DNA-MB@MIL-101 and DNA-TPAAQ@MIL-101 in Example 5;

[0055] Figure 20 Transmission electron microscopy image of DNA-MB@MIL-101 after treatment with H2O2 in Example 5;

[0056] Figure 21 Transmission electron microscopy image of DNA-TPAAQ@MIL-101 after treatment with H2O2 in Example 5;

[0057] Figure 22 Reactive oxygen species generation efficiency diagrams of various MB-containing nanosystems in Example 6;

[0058] Figure 23 Reactive oxygen species generation efficiency diagrams of various TPAAQ-containing nanosystems in Example 6;

[0059] Figure 24 Fluorescence confocal microscopy images of HeLa cells of various TPAAQ-containing nanosystems in Example 7;

[0060] Figure 25 Fluorescence confocal microscopy images of HeLa cells of various MB-containing nanosystems in Example 7;

[0061] Figure 26 Cell survival rate diagrams of various PS nanosystems under different conditions in Example 8. Detailed implementation manners

[0062] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but is not limited thereto.

[0063] In the following examples, the instruments used are as follows:

[0064] 1. Nuclear magnetic resonance spectrometer, model: Bruker Avance 400, manufacturer: Varian, USA. All nuclear magnetic operations involved in this experiment are liquid nuclear magnetic, 400M nuclear magnetic.

[0065] 2. Transmission electron microscope, model: JEOL-JEM 2010F, manufacturer: Hitachi, Ltd., Japan. The specific operation of this experiment was to disperse the obtained sample in methanol solution and place it on a copper mesh for scanning.

[0066] 3. Fourier transform infrared spectrometer, model: Bruker ALPHA, manufacturer: Bruker Corporation, Germany. The infrared scanning range was from 400 - 4000 cm -1 .

[0067] 4. Ultraviolet-visible spectrophotometer, model: Shimadzu UV-1700, manufacturer: Shimadzu Corporation, Japan.

[0068] 5. Photoluminescence spectrometer, model: Perkin-Elmer LS 55, manufacturer: PerkinElmer Inc.

[0069] 6. X-ray powder diffractometer, model: Bruker D8, manufacturer: Bruker Corporation, Germany. The X-ray powder sample scanning involved in this experiment was carried out at 298K, with a voltage of 40kV, a current of 50mA, and the X-ray radiation source was Cu-Kα.

[0070] 7. Thermogravimetric analyzer, model: Shimadzu DTG-60AH, manufacturer: Shimadzu Corporation, Japan.

[0071] 8. Physical adsorption analyzer, model: Micromeritics ASAP 2020, manufacturer: Micromeritics Instrument Corporation, USA.

[0072] Example 1: Synthesis of MIL-101-NH2 and MIL-101-N3.

[0073] (1) The synthesis method of MIL-101-NH2 is as follows:

[0074] Using 2 mM BDC-NH2, 2.5 mM FeCl3·6H2O and 20 μM cetyltrimethylammonium bromide (CTAB) as raw materials, and 30 mL of DMF as the solvent, react in a hydrothermal autoclave at 110 °C for 18 h to obtain MIL-101-NH2. Clean the pores in a Soxhlet extractor with DMF and ethanol for 12 h successively, wash several times with methanol, and then store in absolute ethanol.

[0075] (2) Synthesis of MIL-101-N3:

[0076] Disperse MIL-101-NH2 in 6 ml of THF. Then, add 1.8 mL of tert-butyl nitrite and 1.6 mL of azidotrimethylsilane respectively. Stir the mixture overnight at room temperature. After the reaction is completed, filter, wash successively with THF and dichloromethane, and finally dry under vacuum to obtain MIL-101-N3.

[0077] The characterization results of Example 1 are as follows:

[0078] (1) Test results of X-ray powder diffractometer:

[0079] As Figure 2 shown, the powder X-ray diffraction peaks of MIL-101-N3 match well with those of MIL-101, proving that it has the correct crystal type, and it can be seen from the figure that it has good crystallinity.

[0080] (2) Test results of infrared spectrometer:

[0081] As Figure 3 shown, from the curve, the bending vibration peak (1580 cm -1 ) and shear vibration peak (780 cm -1 ) of the amino group of MIL-101-NH2 can be seen, while the characteristic peak of the amino group cannot be observed from the curve representing MIL-101-N3, and the characteristic peak of azide (2103 cm -1 ) appears. This fully indicates that the amino group has been successfully converted into an azide group.

[0082] (3) Test results of nuclear magnetic resonance hydrogen spectrum:

[0083] After digesting the MOF with deuterated hydrochloric acid, the corresponding tests were carried out. Figure 4 is the nuclear magnetic resonance hydrogen spectrum of MIL-101-NH2, from which the chemical shifts of the three hydrogen atoms of the protonated amino group are located at 7.11 ppm, 7.43 ppm, and 7.73 ppm respectively. Figure 5 is the nuclear magnetic resonance hydrogen spectrum of MIL-101-N3. Only the chemical shifts of the hydrogen atoms on the benzene ring can be observed, and the chemical shift of the amino hydrogen cannot be observed. This also proves that the amino group has been successfully converted into an azide group.

[0084] (4) Test results of transmission electron microscope:

[0085] The test results of the transmission electron microscope of the MIL-101-N3 material are as Figure 6 shown. Its particle size is about 70 nm, and the crystal morphology is similar to that of nano-MIL-101(Fe) reported in the literature. Such nanoparticles below 200 nm will be very beneficial for cell absorption.

[0086] Example 2: The synthesis method of photosensitizer TPAAQ is as follows:

[0087] Add 4-(diphenylamino)phenylboronic acid pinacol ester (220 mg, 0.6 mmol), 2-bromoanthraquinone (144 mg, 0.5 mmol), potassium carbonate (680 mg), 15 mL of THF, 5 mL of water and tetrakis(triphenylphosphine)palladium (3 mol%) into a round-bottom flask, stir and react at 60 °C for 12 h under a nitrogen atmosphere. After cooling to ambient temperature, add 20 mL of water, extract with 20 mL of dichloromethane, and wash with saturated brine. The organic layer is dried with anhydrous magnesium sulfate and purified by column chromatography using n-hexane / dichloromethane (1 / 5, v / v) as the eluent to obtain red solid TPAAQ (183.1 mg, 81.2%).

[0088] The characterization results of Example 2 are as follows:

[0089] 1H NMR test results:

[0090] As Figure 7 shown, 1 H NMR (400 MHz, CDCl3, 298 K), δ (TMS, ppm): 8.49 (d, J = 2.0 Hz, 1H, ArH), 8.30 (m, 3H, ArH), 7.97 (m, 1H, ArH), 7.79 (m, 2H, ArH), 7.61 (d, J = 8.8 Hz, 2H, ArH), 7.31 (t, J = 7.2 Hz, 4H, ArH), 7.17 (d, J = 7.6 Hz, 6H, ArH), 7.09 (t, J = 7.2 Hz, 2H, ArH).

[0091] 13C NMR test results:

[0092] As Figure 8 shown, 13 C NMR (100 MHz, CDCl3, 298 K), δ (ppm): 183.3, 182.8, 148.8, 147.3, 146.3, 134.1, 133.9, 133.7, 133.6, 131.9, 131.5, 131.5, 129.5, 128.1, 128.0, 127.2, 127.2, 125.0, 124.7, 123.7, 122.9.

[0093] Example 3: MIL-101-N3 is respectively loaded with hydrophobic photosensitizer TPAAQ and hydrophilic photosensitizer MB to obtain PS@MIL-101-N3:

[0094] Dissolve PS in tetrahydrofuran, and then impregnate and load it with MIL-101-N3 dispersed in ethanol. The final concentrations of PS and MIL-101-N3 are 2 mg mL -1 and 1 mg mL -1 . Centrifuge the impregnated mixture to obtain the supernatant, and analyze the loading situation in real time by ultraviolet-visible absorption spectroscopy.

[0095] The characterization results of Example 3 are as follows:

[0096] (1) Thermogravimetric analysis and ultraviolet-visible absorption spectroscopy test results:

[0097] It can be seen from the ultraviolet-visible absorption spectroscopy and thermogravimetric analysis that the maximum loading weight percentage (wt%) of MB in MIL-101-N3 reaches about 26% within 16 h ( Figure 9 , 10), and the maximum loading amount of TPAAQ in MIL-101-N3 reaches about 48% within 8 h ( Figure 11 , 12).

[0098] (2) Physical adsorption test results:

[0099] As Figure 13 shown, the adsorption amount of N2 by MIL-101-N3 decreases significantly after loading PS, indicating that PS is indeed loaded into the pores rather than adsorbed on the particle surface.

[0100] (3) X-ray powder diffractometer test results:

[0101] The X-ray powder diffractometer test results of MIL-101-N3 after loading PS are as Figure 2 shown, and the results show that MIL-101-N3 still maintains good crystallinity after loading PS.

[0102] Example 4: Graft DNA onto MIL-101-N3 loaded with PS to obtain DNA-PS@MIL-101-N3:

[0103] React MIL-101-N3 (2 mg mL -1 , 2 mL) with dibenzocyclooctyne (DBCO)-functionalized DNA (100×10 -6 M, 0.8 mL), and stir the resulting mixed solution at 40 °C for 72 h. After centrifugation, the precipitate obtained is washed several times with PBS solution to remove unreacted nucleic acids. In addition, we encapsulated MB@MIL-101-N3 and TPAAQ@MIL-101-N3 by hydrophobic interaction using F-127 to obtain F127-MB@MIL101 and F127-TPAAQ@MIL-101 as controls.

[0104] The test results of Example 4 are as follows:

[0105] (1) Test results of gel electrophoresis:

[0106] As Figure 14 and 15 show, after the click reaction, the mobility of DBCO-DNA detected by gel electrophoresis decreased significantly, proving that DNA was successfully grafted onto MB@MIL-101 and TPAAQ@MIL-101;

[0107] (2) Test results of transmission electron microscopy (TEM):

[0108] The test results of transmission electron microscopy are as Figure 14 and 15 shown. The particle sizes of both DNA-MB@MIL-101 and DNA-TPAAQ@MIL-101 were 80 - 120 nm, and some coronas were captured outside PS@MIL-101. To further verify the composition of the coronas, DNA-MB@MIL-101 and DNA-TPAAQ@MIL-101 were treated with deoxyribonuclease I (DNase-I). From Figure 16 and 17 , it can be observed that the above-mentioned coronas became indistinguishable under TEM. Considering that DNase-I can specifically digest DNA after cleavage, the coronas outside PS@MIL-101 under TEM can prove to be DNA grafted onto the MOF surface.

[0109] Example 5: Stability of DNA-PS@MIL-101-N3 in physiological environment and degradation in response to H2O2 stimulation:

[0110] DNA-MB@MIL-101 and DNA-TPAAQ@MIL-10 were immersed in water, DMEM medium, and fetal bovine serum (FBS) to study their stability in the biological environment.

[0111] DNA-MB@MIL-101 and DNA-TPAAQ@MIL-10 were immersed in a 10% H2O2 dilute solution to study their degradation in response to stimulation.

[0112] The test results of Example 5 are as follows:

[0113] As Figure 18 and 19 shown, the hydrodynamic diameters and Zeta potentials of the two nanoparticles, DNA-MB@MIL-101 and DNA-TPAAQ@MIL-10, changed little, and the degradation degree in the biological medium was moderate, confirming their good stability in the biological environment before reacting with H2O2.

[0114] As Figure 20 and 21 shown, after treatment with H2O2, both DNA-MB@MIL-101 and DNA-TPAAQ@MIL-101 lost their crystalline morphology and became fragmented. This indicates that DNA-MB@MIL-101 and DNA-TPAAQ@MIL-101 are H2O2-triggered degradations.

[0115] Example 6: Test for Reactive Oxygen Species Generation Efficiency

[0116] 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used as a singlet oxygen indicator to test the singlet oxygen generation efficiency of different PSs.

[0117] The test results of Example 6 are as follows:

[0118] As Figure 22 and 23 shown, in the absence of H2O2 stimulation, DNA-MB@MIL-101 and DNA-TPAAQ@MIL-10 showed obvious reactive oxygen species generation quenching effects compared to free MB and TPAA, indicating that the surface-modified DNA strands can effectively prevent the leakage of PS; after treatment with reactive oxygen species, the two nanoparticles showed better reactive oxygen species generation efficiency than free MB and TPAA, indicating that H2O2 can not only activate the release of drugs, but also improve the reactive oxygen species generation efficiency due to the Fenton-like reaction with ferric ions to produce oxygen.

[0119] Example 7: Intracellular Experiment

[0120] After incubating cancerous HeLa cells with different PSs for 24 hours, they were labeled with a commercial nuclear dye Hoechst (showing blue fluorescence) and an intracellular reactive oxygen species probe 2,7-dichlorofluorescein diacetate (DCFDA shows green fluorescence when encountering reactive oxygen species). The specific operation was to inoculate HeLa cells in an 8-well glass culture chamber and culture for 12 h. When 80% confluence was reached, various PS-loaded reagents (5 μg mL based on PS) -1) Add it to the culture medium and incubate with cancer cells for 24 h. Then wash the cells with PBS, culture them with fresh culture medium containing DCFDA (20 μM) and Hoechst (10 μM) for 30 min respectively, and then irradiate with light for 5 min. Rinse the cells with PBS and perform confocal laser scanning imaging. The confocal laser scanning microscope collects the fluorescence signal of TPAAQ in the cells under excitation at 480 nm, and the collection wavelength is 550 - 800 nm. Capture the MB fluorescence signal under excitation at 650 nm and collect the signal in the range of 670 - 800 nm. Capture the DCFDA fluorescence signal under excitation at 488 nm and collect the signal at 505 - 525 nm. Capture the Hoechst fluorescence signal under excitation at 400 nm and collect the signal from 430 - 480 nm.

[0121] The test results of Example 7 are as follows:

[0122] As Figure 24 shown, no signals of PS and reactive oxygen species were observed in cells incubated without hydrophobic TPAAQ and only with H2O2. In sharp contrast, bright green fluorescence was observed in the cytoplasm after treatment with H2O2 for F127 - TPAAQ@MIL - 101 and DNA - TPAAQ@MIL - 101, indicating the generation of reactive oxygen species.

[0123] It is worth noting that for hydrophilic PS (MB), only DNA - MB@MIL - 101 can achieve the activation of H2O2, while F127 - MB@MIL - 101 cannot achieve this function ( Figure 25 ). It shows that DNA functionalization is crucial for the generality of this nanodrug delivery platform.

[0124] Example 8: Cell viability test

[0125] Seed HeLa cells in a 96 - well plate at a density of 3000 cells, 200 μL per well, and incubate for 12 h. Add various drugs at different concentrations to the cell culture medium respectively, incubate the cells for 24 h, with or without H2O2 treatment, and then wash with fresh culture medium. After light treatment or no light treatment for 5 min, add thiazolyl blue (MTT 80 μL, 1 mg mL -1 ) to the culture medium and keep it for 3 h. Take out the culture medium, add dimethyl sulfoxide (300 μL) to each well, and gently shake at room temperature for 10 minutes. The absorbance of MTT at 550 nm is measured with a SpectraMax M5 microplate reader. The cell viability is determined by the ratio of the absorbance of cells incubated with different nanoparticles to the absorbance of cells incubated with normal culture medium.

[0126] The test results of Example 8 are as follows:

[0127] AsFigure 26 As shown, in the presence of H2O2, the IC values of F127-TPAAQ@MIL-101 and DNA-TPAAQ@MIL-101 against HeLa cells under light irradiation are both around 10 μg mL 50 , which are much lower than the IC values of the two nanoparticles not activated by H2O2. On the contrary, F127-MB@MIL101 did not show the inhibitory ablation effect on cancer cells, while DNA-MB@MIL-101 was measured to successfully inhibit photodynamic ablation. This indicates that F127-MB@MIL101 cannot control the leakage of hydrophilic PS. Under dark conditions, all kinds of nanoparticles showed quite good biosafety. -1 As described above, the above is only a description of the specific implementation of the present invention and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. 50

[0128] ​​

Claims

1. A nano metal-organic framework with surface-modified DNA, characterized in that: The nano metal-organic framework is MIL-101 modified with azide groups. A photosensitizer is loaded in the pores of the nano metal-organic framework, and DNA is modified on the surface of the nano metal-organic framework; The photosensitizer is a hydrophobic photosensitizer or a hydrophilic photosensitizer. The hydrophobic photosensitizer is 2-(4-(diphenylamino)phenyl)anthraquinone, and the hydrophilic photosensitizer is methylene blue; The DNA is dibenzocyclooctyne-functionalized DNA; The preparation method of the nano metal-organic framework with surface-modified DNA includes the following steps: The first step is to prepare MIL-101-NH2 and then activate MIL-101-NH2; The second step is to prepare MIL-101-N3 using the MIL-101-NH2 activated in the first step; The third step is to prepare PS@MIL-101-N3 using the MIL-101-N3 prepared in the second step; The fourth step is to convert the PS@MIL-101-N3 prepared in the third step into DNA-PS@MIL-101-N3 by click chemical reaction, that is, to obtain a nano metal-organic framework with surface-modified DNA and loaded with a photosensitizer; In the first step, the specific method for preparing MIL-101-NH2 is as follows: Mix 2-aminoterephthalic acid, ferric chloride hexahydrate, cetyltrimethylammonium bromide, and N,N-dimethylformamide at a temperature of 50-80 °C for 12-24 h to obtain MIL-101-NH2. The molar ratio of 2-aminoterephthalic acid, ferric chloride hexahydrate, cetyltrimethylammonium bromide, and N,N-dimethylformamide is 100-150:200-300:1:15000-20000; In the first step, the method for activating MIL-101-NH2 is as follows: Extract with ethanol in a Soxhlet extractor for 2-4 h, then extract with DMF for more than 10 h, wash several times with methanol, and then store in absolute ethanol at room temperature; In the second step, the specific method for preparing MIL-101-N3 is as follows: Mix trimethylsilyl azide, tert-butyl nitrite, MIL-101-NH2, and tetrahydrofuran for more than 8 h to obtain MIL-101-N3; The molar ratio of trimethylsilyl azide, tert-butyl nitrite, MIL-101-NH2, and tetrahydrofuran is 15000-20000:150:80-150:50-100; In the third step, the specific method for preparing PS@MIL-101-N3 is as follows: Disperse MIL-101-N3 in ethanol and mix it with the photosensitizer solution by stirring. When mixing, the final concentrations of the photosensitizer and MIL-101-N3 are 1-4 mg mL -1 and 0.5-1.0 mg mL -1 , respectively. The photosensitizer is impregnated into the pores of MIL-101-N3 to obtain PS@MIL-101-N3; The solvent in the photosensitizer solution is tetrahydrofuran, and the concentration of the photosensitizer solution is 2 - 8 mg / mL -1 , and the concentration ratio of MIL-101-N3 to the photosensitizer is 1:1 - 1:8; In the fourth step, the specific method for converting PS@MIL-101-N3 into DNA-PS@MIL-101-N3 by click chemical reaction is as follows: PS@MIL-101-N3 was grafted with DNA to obtain DNA-PS@MIL-101-N3, that is, MIL-101-N3 was reacted with dibenzocyclooctyne-functionalized DNA to obtain a mixed solution, which was stirred at 30-50 °C for 48-72 h. After centrifugation, the precipitate was washed several times with PBS solution to obtain DNA-PS@MIL-101-N3, which is a nano metal-organic framework with surface-modified DNA and loaded with photosensitizer.

2. A nano metal-organic framework with surface-modified DNA according to claim 1, characterized in that: 2-(4-(Diphenylamino)phenyl)anthraquinone is obtained by the Suzuki coupling chemical method using 4-(diphenylamino)phenylboronic acid pinacol ester and 2-bromoanthraquinone; The method for preparing 2-(4-(diphenylamino)phenyl)anthraquinone is as follows: A mixture of 0.30-0.80 mmol of 4-(diphenylamino)phenylboronic acid pinacol ester, 2-bromoanthraquinone (0.30-0.80 mmol), potassium carbonate (2-8 mmol), 10-20 mL of tetrahydrofuran, 3-7 mL of water and tetrakis(triphenylphosphine)palladium (3-5 mol%) was degassed and filled with nitrogen. The reaction mixture was stirred at 50-70 °C for more than 12 h. After cooling to ambient temperature, the reaction was stopped by adding water, extracted with dichloromethane and washed with brine to obtain 2-(4-(diphenylamino)phenyl)anthraquinone; Among them, the ratio of 4-(diphenylamino)phenylboronic acid pinacol ester, 2-bromoanthraquinone, potassium carbonate, tetrahydrofuran, and water is: 0.30-0.80 mmol: 0.30-0.80 mmol: 2-8 mmol: 10-20 mL: 3-7 mL, and the concentration of tetrakis(triphenylphosphine)palladium is 3-5 mol%.

3. A nano metal-organic framework with surface-modified DNA according to claim 1, characterized in that: The calculation method for the PS content loaded on the prepared MIL-101-N3 is: 100 mg of MIL-101-N3 was added to 10 mL of a THF solution of 2-(4-(diphenylamino)phenyl)anthraquinone at 30 mg mL -1 , stirred at room temperature, and the wt% loading of PS was monitored in real time by the UV-visible absorption spectrum of the supernatant obtained by centrifugation; 100 mg of MIL-101-N3 was added to 10 mL of a THF solution of 30 mg mL -1 MB, and the mixture was stirred at room temperature. The wt% loading of PS was monitored in real time by the UV-vis absorption spectrum of the supernatant obtained by centrifugation; Among them, A PS负载前 is the UV-visible absorption value of PS before loading; A PS负载后 is the UV-visible absorption value of PS after loading; C PS is the concentration of PS; C MIL-101-N3 is the concentration of MIL-101-N3.

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