Construction and application of cross-linkable phototherapy anti-tumor system based on up-conversion nano material

Abstract

The invention discloses construction and application of a cross-linkable phototherapy anti-tumor system based on an up-conversion nano material, and belongs to the technical field of biological medicines. A nano-drug is designed and synthesized, the drug can be retained in cells for a long time under photostimulation, the phototherapy (photothermal and photodynamic) anticancer effect is improved, and the administration frequency and dosage are reduced. The cancer treatment effect can be enhanced by combining a photo-thermal reagent with internal peroxide and combining a photodynamic photosensitizer with a singlet oxygen carrier. The core mechanism of the long-acting nano-drug is that near-infrared light irradiates an up-conversion nano-material, ultraviolet light released in the up-conversion process activates photoreactive groups such as diaziridine to generate an active intermediate, and the nano-drug is fixed to cells and focus parts through biomolecules or organelles such as covalent cross-linked protein of the intermediate. The constructed near-infrared light activated nano long-acting drug system provides a new thought for efficient and safe anti-cancer therapy, and is expected to reduce the dosage and frequency of administration.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and is the construction and application of a cross-linkable phototherapy anti-tumor system based on up-conversion nanomaterials. Background Art

[0002] In recent years, phototherapy has been widely used in cancer treatment due to its precision, safety, and non-invasiveness. For example, photothermal therapy is a popular non-invasive and local tumor treatment method that has attracted widespread research attention in the past few decades. Photothermal therapy usually relies on photothermal agents (PTAs) to generate sufficient heat under near-infrared (NIR) light to inhibit or even ablate tumors. Photothermal agents absorb the energy of photons under NIR irradiation and activate from the ground singlet state to the excited singlet state. Subsequently, the energy of the excited electron undergoes a non-radiative form of decay-vibrational relaxation, and returns to the ground state through collisions between PTA and surrounding molecules. The increased kinetic energy heats up the surrounding microenvironment, thereby producing a thermal effect. Photodynamic therapy is another emerging therapy for treating tumors. Its three elements are photosensitizers, light sources, and oxygen. The action process is that the photosensitizer is excited by light of a specific wavelength from the ground state to the singlet excited state, and then to the triplet excited state through intersystem crossing. The photosensitizer in the triplet excited state can effectively sensitize oxygen molecules to produce 1 O2 can not only directly kill tumor cells, but also damage the tumor's vascular system, causing tumor ischemia and hypoxia. 1 O2 has a very short half-life, and photosensitizers can only be produced in the presence of light. 1 O2, but no anti-tumor effect under dark conditions. In order to enhance the anti-cancer effect, we continue to add a 1 After the drug reaches the tumor tissue, the light source stimulates the photosensitizer to produce 1 O2 kills tumor cells and stores peroxide precursor compounds 1 O2, to achieve continuous release under dark conditions 1 The purpose of O2 is to kill tumors.

[0003] In order to enhance the uptake and retention of drugs in tumor cells and promote the enrichment of therapeutic drugs in tumors, photoreactive groups are introduced, which can be activated by ultraviolet rays of a specific wavelength to form active substances, and then can be covalently cross-linked with neighboring molecules. The main photoreactive groups are benzophenone, aromatic nitrogen compounds and diazirine. Lanthanide-doped upconversion nanoparticles (UCNPs) can convert low-energy radiation (usually near-infrared light) into high-energy emission (such as visible light, ultraviolet light and near-infrared light). Upconversion luminescence has a large anti-Stokes shift and is the process in which two or more photons combine in a medium to produce high-energy photons. Summary of the invention

[0004] By combining upconversion nanomaterials with photoreactive groups, the ultraviolet light released during the upconversion process activates photoreactive groups such as diazirine to produce active intermediates, and the nanomedicines are fixed to cells and lesions through covalently labeled proteins with intermediates. The near-infrared light-activated nano-long-acting drugs constructed in this way provide new ideas for efficient and safe anti-cancer therapy, and are expected to reduce the dosage and frequency of administration.

[0005] The technical solution adopted by the present invention is: a type of cross-linkable phototherapy material based on up-conversion nanomaterials, having the following structure:

[0006]

[0007] Among them, m1, m2, and m3 are each independently an integer not less than 1.

[0008] R2 is

[0009] R3 is ICG or methylene blue.

[0010] Among them, X - NO3 - 、F - , Cl - Br - ,I - PF6 - 、NO3 - 、SO4 2- CF3SO3 - 、CH3SO3 - , one of the organic carboxyl groups having 1 to 3 carbon atoms.

[0011] When R3 is a photothermal reagent, R1 is or

[0012] When R3 is a photosensitizer, R1 is or

[0013] Among them, R 1 -R 3 , R 5 -R 7 , R 9 -R 12 , R 14 -R 16 Each is independently selected from hydrogen, trimethylsilyl, C1-C10 alkyl, C1-C10 alkoxy, C5-C10 aryl, amino, and C1-C10 amino-substituted alkyl.

[0014] R 4, R 8 , R 13 , R 17 Each independently or The sum of n1 and n2 is independently an integer of 0-6, m is an integer of 0-6, and n is an integer of 1-2000.

[0015] It is upconversion nanoparticle UCNP.

[0016] For some specific materials, R2 is

[0017] R3 is or

[0018] R 1 -R 3 , R 5 -R 7 , R 9 -R 12 , R 14 -R 16 Each is independently selected from hydrogen, trimethylsilyl, C1-C5 alkyl, C1-C5 alkoxy, C6-C10 aryl, amino, and C1-C5 amino-substituted alkyl.

[0019] R 4 , R 8 , R 13 , R 17 Each independently or The sum of n1 and n2 is independently an integer of 0-6, m is an integer of 0-6, and n is an integer of 1-50.

[0020] m1, m2, and m3 are each independently an integer not less than 1.

[0021] For some specific materials, when R3 is When R1 is or

[0022] when When R1 is or

[0023]

[0024] R 1 -R 3 , R 5 -R 7 , R 14 -R 16Each is independently selected from hydrogen, trimethylsilyl, C1-C5 alkyl, C1-C5 alkoxy, phenyl, amino, and C1-C5 amino-substituted alkyl.

[0025] R 4 , R 8 , R 17 Each independently or The sum of n1 and n2 is independently an integer of 0-3, m is an integer of 0-3, and n is an integer of 1-15.

[0026] m1, m2, and m3 are each independently an integer not less than 1.

[0027] Some specific materials, R1 is When R3 is

[0028] R1 is or When R3 is

[0029] R 1 -R 3 , R 5 -R 7 , R 14 -R 16 , R 14 -R 16 Each is independently selected from hydrogen, trimethylsilyl, C1-C5 alkyl, C1-C5 alkoxy, and phenyl.

[0030] R 4 , R 8 , R 17 Each independently or

[0031] The sum of n1 and n2 is independently an integer of 0-3, m is an integer of 0-3, and n is an integer of 1-10.

[0032] For some specific materials, the total amount of R1, R2 and R3 loaded on the material is 1-10 μmol / g.

[0033] For some specific materials, the total amount of R1, R2 and R3 loaded on the material is 2-5 μmol / g.

[0034] The preparation method of the material comprises the following steps:

[0035] Stir R1-COOH, R2-COOH, R3-COOH and NHS in a solvent at room temperature for 1 to 5 hours, then dropwise add the nanomaterials dispersed in the solvent. The dispersion is stirred for 5 to 20 hours, centrifuged and washed to obtain the target material TM.

[0036] The molar ratio of R1-COOH, R2-COOH, R3-COOH and NHS is: 0.1~15:1:0.1~15:20~100.

[0037] R1--COOH and The mass ratio is 1:1~10.0.

[0038] In the preparation method, the molar ratio of R1-COOH, R2-COOH, R3-COOH and NHS is 5-15:1:5-15:40-80.

[0039] R1-COOH The mass ratio is 1:1~3.

[0040] In the preparation method, the molar ratio of R1-COOH, R2-COOH, R3-COOH and NHS is 5-12:1:5-12:50-70.

[0041] R1-COOH The mass ratio is 1:1~1.5.

[0042] The solvents are independently selected from at least one of DMF, DMSO, methanol and ethanol.

[0043] The material is used in preparing photothermal therapy synergistic with singlet oxygen anti-tumor drugs or in preparing continuous photodynamic therapy anti-tumor drugs under light and dark conditions.

[0044] The material is used in preparing long-retention drugs.

[0045] A specific nanomaterial TM-1 of the present invention has the following structure:

[0046]

[0047] A specific nanomaterial TM-2 of the present invention has the following structure:

[0048]

[0049] Nanomaterials Prepared using the following steps:

[0050] Step i: a mixture of YCl3, YbCl3, and TmCl3 in a mass ratio of 1:0.47:0.0056 with 1-octadecene and oleic acid is first reacted at 120-140°C under vacuum for 0.5-2h, then cooled to 40-60°C, and an alcohol solution containing NaOH and NH4F is added under an inert gas atmosphere, and maintained at 40-60°C for 0.5-1h, and then maintained under vacuum for 20-40min, and then the temperature is raised to 70°C and maintained for 30-90min, and then converted to 300-350°C under an inert gas atmosphere for 0.5-2h, centrifuged, and washed to obtain nanomaterial A.

[0051] Step .j: Add the nanomaterial A dispersed in the organic solvent in a mass ratio of 1:10 dropwise to the organic solvent dispersion of octylphenoxy poly(ethyleneoxy), ultrasonicate for 3 to 10 minutes, stir for 10 to 30 minutes, add 1.9 times the concentration of the nanomaterial A in 10 to 35% ammonia water, stir for 20 to 40 minutes after the solution becomes clear, then add 1.9 times the concentration of the nanomaterial A alkyl silicate and stir for 15 to 30 hours to obtain the nanomaterial

[0052] Step k: Add H2NCH2CH2CH2Si(OC2H5)3 dropwise to the nanomaterial at a mass ratio of 31.5:1. The nanomaterial B is obtained by stirring and reacting in an organic solvent dispersion;

[0053] Step 1: Nanomaterial B reacts with Bis-PEG6-NHS and DIPEA at a mass ratio of 1:1.5:1 in an organic solvent at room temperature to obtain nanomaterial C;

[0054] Step m: the nanomaterial C reacts with N-Boc-1,6-hexanediamine and DIPEA in a mass ratio of 1:1.1:2 in an organic solvent at room temperature to obtain the nanomaterial D;

[0055] Step n: Nanomaterial D and TFA react at a mass ratio of 1:1.5 in an organic solvent at room temperature to obtain nanomaterial

[0056] The organic solvents are independently selected from chloroform, dichloromethane, DMF, n-hexane, methanol and ethanol.

[0057] Upconversion Nanoparticles References: Recent Progress of Rare-Earth Doped Upconversion Nanoparticles: Synthesis, Optimization, and Applications[J].(Zhu X, Zhang J, Liu J, et al. Advanced science. 2019, 6(22): 1901358.) Prepare or purchase existing products.

[0058] The beneficial effects of the present invention are as follows: 980nm light energy is converted by UCNP into 365nm light to generate active carbene from diazirine to generate carbene intermediates, and then covalent bonds are formed with adjacent biological molecules through CC, CH, OH and XH (X = heteroatom) insertion reactions, so as to achieve the purpose of fixing the drug in the tumor and reducing drug loss, and then photothermal therapy is performed by irradiating 808nm light three times to synergistically release singlet oxygen to kill tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the photothermal effect of different concentrations of nanomaterial TM-1 under the excitation of 808nm light.

[0060] Figure 2 It is a test of the ability of nanomaterial TM-1 to capture singlet oxygen under 808nm laser irradiation.

[0061] Figure 3 is the cell survival rate after nanomaterial TM-1 was treated with different light.

[0062] Figure 4 This is a graph showing the anticancer efficacy of nanomaterial TM-1 at different compound loading ratios.

[0063] Figure 5 It is the toxicity of the nanomaterial TM-1 to cells when it is irradiated with 808nm laser.

[0064] Figure 6 This is a fluorescent imaging diagram of the fixation effect of nanomaterial TM-1 in cells.

[0065] Figure 7 This is the fluorescence imaging of intracellular ROS levels after the nanomaterial TM-1 was treated with different light.

[0066] Figure 8 This is the live / dead cell staining imaging of the nanomaterial TM-1 irradiated with different 808nm near-infrared light times.

[0067] Fig. 9 It is the toxicity of the nanomaterial TM-2 to cells when irradiated with 655nm laser. DETAILED DESCRIPTION

[0068] Preparation Example

[0069]

[0070] Step a: Under nitrogen protection, compound 1-1 levulinic acid (1.17 g, 10.07 mmol) and 10 mL of ammonia in 7N MeOH were mixed in a round-bottom flask and stirred in an ice-water bath for 3 h. Hydroxylamine-O-sulfonic acid (1.31 g, 11.59 mmol) was then dissolved in 10 mL MeOH and dripped into the reaction solution at a rate of 1 s / drop. After the dripping was completed, the reaction solution was stirred at 20-25 ° C for 16-17 h. After the reaction was completed, the ammonia in the reaction flask was pumped out for 1 h with a pump to remove the ammonia as much as possible, and vacuum filtered with a Buchner funnel and diatomaceous earth to obtain a light yellow filtrate. The liquid was transferred to a 250 mL round-bottom flask and concentrated by rotary evaporation. A light yellow to white oil should be left. Cover the reaction flask with aluminum foil to minimize the light exposure to the next reaction step. 10 mL MeOH was added to the flask to redissolve the above oil. The reaction flask was placed on an ice-water bath and covered with a rubber septum. After stirring the mixture for 5 min, 2.1 mL of TEA was added. The mixture was stirred for 5 min, and then iodine fragments were slowly added until the solution continued to maintain a reddish brown color. Theoretically, 2.56 g of iodine was required, but the actual amount depended on the yield of the previous step. The solution was then transferred to a separatory funnel and 50 mL of ethyl acetate was added. The solution was washed with 50 mL of 1 M HCl aqueous solution and 50 mL of 10% sodium thiosulfate in turn. The washing was repeated until the orange-red layer was nearly transparent and colorless because the iodine was reduced to iodide. A small amount of anhydrous sodium sulfate (enough to cover the bottom of the beaker) was added to dry the ethyl acetate layer in the glass beaker. The ethyl acetate was evaporated by rotary evaporation to obtain a crude product, which was finally purified by column chromatography (DCM: MeOH = 100: 1, v / v) to obtain compound 1-2 (yield 17.5%). 1 HNMR (400MHz, CDCl3) δ11.56 (s, 1H), 2.15-2.08 (m, 2H), 1.61-1.54 (m, 2H), 0.90 (s, 3H).

[0071] Step b: A mixture of compound 2-1 (993 mg, 4.9 mmol) and pyridine (400 μL, 4.95 mmol) was refluxed in acetone for 30 h. After the reaction was completed, the mixture was cooled and the solvent was removed by rotary evaporation under reduced pressure. The solid was then washed several times with n-hexane and filtered to obtain a white powder compound 2-2 (yield: 69.5%). 1H NMR (400MHz, CD3OD) δ9.96 (d, J=5.8Hz, 2H), 9.67 (d, J=2.4Hz, 1H), 9.53 (t, J=7 .9Hz, 1H), 9.40 (d, J=8.6Hz, 1H), 8.96 (t, J=7.0Hz, 2H), 8.91 (d, J=8.7Hz, 1H).

[0072] Step c: Dissolve aniline (320 μL, 3.47 mmol) and compound 2-2 (488 mg, 1.74 mmol) in 3 mL of ethanol respectively, add the aniline solution dropwise to the solution of compound 2-2, stir the mixture at room temperature for 30 min after the addition, remove the solvent by rotary evaporation under reduced pressure, wash the solid three times with ethanol and filter to obtain red powder compound 2-3 (yield: 55.6%). 1 H NMR (400MHz, CD3OD) δ8.98 (d, 1H), 8.43 (d, J=10.1Hz, 2H), 8.17 (m, 1H), 7.91 (t, J=12.9Hz, 1H), 7. 47-7.42 (m, 4H), 7.34 (m, 3H), 7.25 (t, J=7.4Hz, 2H), 7.07 (d, J=9.4Hz, 1H), 6.31 (t, J=12.2Hz, 2H).

[0073] Step d: Compound 3-1 (2.0 g, 9.00 mmol) and 3-bromopropionic acid (2.0 g, 13.07 mmol) were mixed in o-dichlorobenzene (20 mL), and the mixture was stirred and heated at 110 ° C for 18 h. After the reaction was completed, the mixture was cooled and filtered to collect the precipitate, and then repeatedly ground in DCM to remove unreacted substances until the solid turned into an off-white solid, and finally dried to obtain compound 3-2 (yield: 91.2%). 1 H NMR (400MHz, CD3OD) δ8.33 (d, J=8.4Hz, 1H), 8.24 (d, J=9.0Hz, 1H), 8.16 (d, J=8.2Hz, 1H), 8.04 (d, J=9.0Hz, 1H), 7.84-7.77 (t, 1H), 7.72 (t, J=8.0Hz, 1H), 3.17 (t, J=6.5Hz, 2H), 1.84 (s, 6H).

[0074] Step e: Compound 2-3 (142 mg, 0.5 mmol) and DIPEA (130 mg, 1.0 mmol) were dissolved in DCM (1 mL), Ac2O (60 mg, 0.59 mmol) was dissolved in DCM (0.25 mL) and added dropwise to the above reaction mixture at 0°C, and reacted at room temperature for 1 h to obtain reaction solution A. Compound 3-2 (410 mg, 1.13 mmol) and sodium acetate (160 mg, 1.95 mmol) were added to a mixed solution of acetonitrile and water (v / v, 19:1) and stirred at 85°C for 1 h to obtain reaction solution B, and then reaction solution A was added dropwise to reaction solution B, and the mixture was stirred at 85°C for 16 h. After the reaction was completed, it was cooled to room temperature, and then washed several times with acetonitrile, 5% hydrochloric acid solution, and ether, and filtered to obtain green solid compound 2-4 (yield: 56.3%). 1 H NMR (400MHz, DMSO) δ8.24 (d, J=8.6Hz, 1H), 8.10-7.93 (m, 3H), 7.76 (m, J=27.1, 11.0Hz, 2H), 7.64 (t, J=8.2Hz, 1H), 7.53 -7.46 (t, 1H), 6.60 (t, J = 12.6Hz, 1H), 6.46 (d, J = 13.7Hz, 1H), 4.43 (t, J = 7.1Hz, 2H), 2.77 (t, J = 7.2Hz, 2H), 1.91 (s, 6H).

[0075] Step f: Compound 3-1 (452 ​​mg, 1.35 mmol), phenylboronic acid (244 mg, 1.35 mmol), Pd(PPh3)4 (156 mg, 0.14 mmol) and Na2CO3 (6.96 g, 65.66 mmol) were mixed in toluene (18 mL), ethanol (9 mL) and water (6 mL). The reactants were heated at 100°C for 3 h. The mixture was extracted with ethyl acetate, the organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated by rotary evaporation, and purified by silica gel column chromatography to give compound 3-2 as a white solid. (Yield: 33.9%). 1 HNMR (400MHz, CDCl3) δ8.33 (d, 2H), 7.72 (m, J=10.1Hz, 2H), 7.66-7.54 (m, 7H), 7.49 (m, J=9.4Hz, 2H), 7.39-7.30 (m, 4H), 4.04 (s, 3H).

[0076] Step g: Compound 3-2 (80 mg, 0.21 mmol) was dissolved in 5 mL MeOH. 1 mL of 2M LiOH was added to the solution. The mixture was stirred at 60 ° C for 3 hours, and then the solution was acidified to pH 2.0, and then the mixture was extracted with ethyl acetate, the organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain compound 3-3 as a white solid. (Yield: 70.5%) 1 H NMR (400MHz, CDCl3) δ8.37 (d, J=7.7Hz, 2H), 7.71 (m, J=4.4Hz, 2H), 7.67-7.55 (m, 7H), 7.49 (m, J=7.0Hz, 2H), 7.35 (m, J=7.1Hz, 4H).

[0077] Step h: Compound 3-3 (50 mg, 0.13 mmol) was dissolved in DCM-THF (10 mL-5 mL), and then a catalytic amount of methylene blue was added and the solid was completely dissolved by ultrasound. The low temperature environment of 0°C was maintained throughout the process, and the reaction solution was irradiated with 630 nm light. The reaction process lasted for about 3 hours, and the reaction was monitored by TLC during the reaction. After the reaction was completed, the methylene blue was adsorbed by activated carbon, and the methylene blue was removed by filtration. Finally, the white solid compound 3-4 was obtained by vacuum concentration. (Yield: 90.2%) 1 H NMR (400MHz, CDCl3) δ8.37 (d, J=7.9Hz, 2H), 7.83 (d, J=8.0Hz, 2H), 7.70 (d, J=7.7 Hz, 2H), 7.64 (m, J=7.6Hz, 2H), 7.55 (m, J=7.1Hz, 1H), 7.15 (m, J=20.1, 8.2Hz, 8H).

[0078]

[0079] Step i: Add YCl3 (146.5 mg, 0.75 mmol), YbCl3 (69.0 mg, 0.247 mmol) and TmCl3 (0.83 mg, 0.003 mmol) to a 100 mL three-necked flask, then add 15 mL of 1-octadecene and 6 mL of oleic acid, place the reaction flask in a programmed temperature riser, first react at 130 ° C under vacuum for 1 h, and when the solution cools to 50 ° C, switch to nitrogen protection and drop 5 mL NaOH (100 mg, 2.5 mmol) and NH4F (146.5 mg, 4.0 mmol) dissolved in MeOH were maintained at 50°C for 40 min, then maintained under vacuum for 30 min, and then the temperature was raised to 70°C and maintained for 60 min, and then converted to 320°C under nitrogen protection for 1 h. After cooling, 30 mL of acetone was added, and then the centrifuge speed was adjusted to 12000 rpm for 6 min to obtain a solid precipitate. The precipitate was washed three times with n-hexane to obtain nanomaterial 1, and finally the nanomaterial 1 was stored in n-hexane.

[0080] Step j: Nanomaterial 1 (160 mg) was dispersed in 8 mL of n-hexane, octylphenoxy poly (ethyleneoxy) ethanol lgepal (1.6 mL) was dispersed in 30 mL of cyclohexane, and stirred at 750 rpm for 5 min. Then, the nanomaterial 1 suspended in n-hexane was added dropwise, ultrasonicated for 5 min, and 25% ammonia water (0.33 mL) was added after stirring for 15 min. At this time, the solution became clear. After stirring for another 30 min, ethyl silicate TEOS (0.33 mL) was added and stirred overnight. After the reaction was completed, 50 mL of ethanol was added to precipitate the solid, and then the centrifuge speed was adjusted to 12000 rpm for 6 min to obtain a solid precipitate. The precipitate was washed 3 times with ethanol to obtain nanomaterial 2. Finally, nanomaterial 2 was stored in ethanol.

[0081] Step k: Nanomaterial 2 (180 mg) was evenly dispersed in anhydrous ethanol (60 mL), and then H2NCH2CH2CH2Si(OC2H5)3APTES (6.0 mL) was added dropwise and stirred for 18 h. After the reaction was completed, the centrifuge was set at 12000 rpm for 6 min to obtain a solid precipitate. The precipitate was washed three times with ethanol to obtain nanomaterial 3, and finally the nanomaterial 3 was stored in ethanol.

[0082] Step 1: Nanomaterial 3 (160 mg) was evenly dispersed in DMF (2 mL), and Bis-PEG6-NHS (236 mg, 0.41 mmol) and DIPEA (0.22 mL, 1.23 mL) were added. After reacting overnight, the whole centrifuge speed was 12000 rpm for 10 min to obtain a solid precipitate. The precipitate was washed three times with DMF to obtain nanomaterial 4, and finally the nanomaterial 4 was stored in DMF.

[0083] Step m: Nanomaterial 4 was evenly dispersed in DMF (2 mL), and N-Boc-1,6-hexanediamine (177.4 mg, 0.82 mmol) and DIPEA (0.44 mL, 2.46 mmol) were added. After reacting overnight, the whole centrifuge speed was 12000 rpm for 10 min to obtain a solid precipitate. The precipitate was washed three times with DMF and then washed once with DCM to obtain nanomaterial 5. Finally, nanomaterial 5 was stored in DCM.

[0084] Step n: Nanomaterial 5 was evenly dispersed in DCM (1 mL), TFA (0.2 mL) was added dropwise at 0°C, and then reacted at room temperature for 6 h. The centrifuge speed was adjusted to 12000 rpm for 6 min to obtain a solid precipitate. The precipitate was washed three times with DCM and once with DMF to obtain nanomaterial 6. Finally, nanomaterial 6 was stored in DMF.

[0085] Step o: Compound 2-4 (64.1 mg, 0.10 mmol), compound 1-2 (1.3 mg, 0.01 mmol), compound 3-4 (40.6 mg, 0.10 mmol), EDC (97.1 mg, 0.63 mmol) and NHS (71.9 mg, 0.63 mmol) were stirred in DMF (2 mL) at room temperature for 2 h, and then nanomaterial 6 (48.72 mg) suspended in DMF (the amount of Bis-PEG6-NHS added from nanomaterial 3 to nanomaterial 4 was 0.42 mmol) was added dropwise, and stirred for 12 h. After the reaction, the centrifuge speed was adjusted to 12000 rpm for 10 min to obtain a solid precipitate. The precipitate was washed three times with DMF, and then washed once with DCM to obtain nanomaterial TM-1. Finally, the nanomaterial TM-1 was stored in DCM.

[0086] Referring to step o, the amount of compound 3-4, compound 1-2, and compound 2-4 is changed to 1:1:1 to obtain nanomaterial TM-1a. Referring to step 0, the amount of compound 3-4, compound 1-2, and compound 2-4 is changed to 1:10:1 to obtain nanomaterial TM-1b.

[0087] TM-2 was prepared by referring to the steps of preparing TM-1, wherein R1 is R2 is R3 is Will The molar ratio of NHS is 10:1:10:5. It is added with nano material 6 at a mass ratio of 1:1.2.

[0088] Example 1

[0089] In order to explore the photothermal effect of nanomaterial TM-1, suspensions with concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL were prepared using solvent DMF, and the optical density was 3 W / cm 2 The solution system was continuously irradiated with 808nm light, and the temperature change of the solution was recorded with a temperature measuring thermal imager. Figure 1 .like Figure 1 As shown, the nanomaterial TM-1 can successfully release heat under the excitation of 808nm light, proving that the nanomaterial TM-1 has a good photothermal effect.

[0090] Example 2

[0091] In order to investigate the ability of nanomaterial TM-1 to release singlet oxygen in vitro, the DPBF of nanomaterial TM-1 after multiple irradiation was measured. A solution with a concentration of 1 mg / mL of nanomaterial TM-1, a concentration of 50 μM of DPBF, and a solvent of DMF was prepared. The solution was irradiated with an optical density of 1 W / cm 2 The solution system was continuously irradiated with 808 nm light, and the UV-visible absorption spectrum of the solution system was measured once every 10 seconds. Figure 2 .like Figure 2 As shown, with the increase of 808nm laser irradiation time, the absorption intensity at 410nm gradually decreases, proving that singlet oxygen is generated in the nanomaterial TM-1 under 808nm laser irradiation.

[0092] Example 3

[0093] The MTT method was used to determine the anti-tumor efficacy of nanomaterial TM-1 after different light treatments. First, 4T1 cells were plated at 6×10 3 The cells were inoculated into two 96-well plates at a density of 100 μL and covered with PBS buffer solution on all sides. After 24 hours of incubation, the culture medium in the 96-well plates was discarded, and 100 μL of complete 1640 culture medium with the concentration of nanomaterial TM-1 of 0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL was added to the wells, respectively. The plates were incubated for 24 hours, and then the wells were washed with PBS buffer solution to remove free nanomaterials. This was repeated 3 times, and 100 μL of complete 1640 culture medium was added to each well. The 980- group did not receive any treatment, and the 980+ group was first treated with an optical density of 2 W / cm 2 , near-infrared light with a wavelength of 980nm was used for 2 minutes at intervals, and then both groups were exposed to light with an optical density of 2W / cm 2, 808nm near-infrared light was continuously irradiated for 5 minutes, and the two groups repeated the 5-minute 808nm light irradiation after an interval of 12 hours. After the treatment, incubate for 24 hours, add 10μL MTT solution (0.5mg / mL) to each well, and after culturing at 37℃ for 4 hours, carefully remove the culture medium, add 150μL DMSO dissolved in purple formazan to each well, and use a microplate reader to measure the absorbance at 570nm to calculate the cell survival rate. Figure 3 .like Figure 3 As shown, after irradiation with 980nm light, the photothermal anti-tumor effect of nanomaterial TM-1 was better, proving that 980nm laser successfully enhanced the anti-tumor efficacy of nanomaterial TM-1.

[0094] Example 4

[0095] Referring to Example 3, the anticancer efficacy diagrams of nanomaterial TM, nanomaterial TM-1a, and nanomaterial TM-1b with different compound loading ratios of nanomaterial TM-1 were investigated, and the results were obtained. Figure 4 .like Figure 4 As shown, since compound 2-4 has photothermal effect and compound 3-4 can release singlet oxygen to achieve anti-cancer effect, when the mass ratio of compound 1-2: compound 2-4: compound 3-4 is 1:10:10, the anti-cancer effect of the nanomaterial is the best. Therefore, this ratio is selected to synthesize TM-1 to complete subsequent experiments.

[0096] Example 5

[0097] Referring to Example 3, the toxicity of the nanomaterial TM-1 irradiated with 808 nm laser on cells was investigated. Figure 5 .like Figure 5 As shown, it can be seen that within 3 times, the more times the 808nm light is irradiated, the stronger the anti-tumor effect of the nanomaterial TM-1 prepared in the preparation example, indicating that each of the three 808nm light irradiations can stimulate the anti-cancer effect of the nanomaterial TM-1.

[0098] Example 6

[0099] 4T1 cells were plated at 2 × 10 per well. 3The cells were inoculated into 96-well plates at a density of 100 cells / well and covered with PBS buffer solution on all sides. After 24 hours of incubation, the culture medium in the 96-well plates was discarded, and 100 μL of complete 1640 culture medium containing nanomaterial TM-1 at a concentration of 100 μg / mL was added to the wells. The wells were irradiated with 980 nm laser at 3 W, 1.5 W, and no light treatment, respectively. Then, subsequent treatments were performed at intervals of 0 h, 8 h, 24 h, and 48 h: the well plates were washed with PBS buffer solution to remove free nanomaterials, and the process was repeated 3 times. Then, 100 μL of the nuclear dye Hoechst 33342 prepared according to the instructions was added to each well. After incubation at 37°C in the dark for 20 min, the wells were washed 3 times with PBS buffer solution, and finally, 100 μL of PBS buffer solution was added and the wells were placed in a high-content imager for photographing. Figure 6 .like Figure 6 As shown, it can be seen that at 8 hours, the fluorescence of the non-irradiated 980nm group was not as strong as that of the 980nm light irradiated group. At 24 hours, it can be clearly seen that there was almost no drug retention in the non-irradiated 980nm light group, while there was drug retention in the 980nm light irradiated group. At 48 hours, there was still a small amount of drug retention in the 980nm light irradiated group, proving that 980nm light can successfully excite the fixation effect of diaziridine to achieve the long retention effect of the nanomaterial TM-1.

[0100] Example 7

[0101] 4T1 cells were plated at 6 × 10 per well. 3 The cells were inoculated into a 96-well plate at a density of 100 cells / well and covered with PBS buffer solution on all sides. After incubation for 24 hours, the culture medium in the 96-well plate was discarded, and 100 μL of complete 1640 culture medium containing 100 μg / mL of nanomaterial TM-1 was added to the wells. 100 μL of complete 1640 culture medium was added to the control group and incubated for 24 hours. The well plate was then washed with PBS buffer solution to remove free nanomaterials. This was repeated three times, and 100 μL of complete 1640 culture medium was added to each well. The subjects were divided into four groups for different light treatments, namely, 980(-)+808(-) group (no light irradiation), 980(+)+808(+) group (one 980nm light irradiation and one 808nm light irradiation), 980(+)+808(++) group (one 980nm light irradiation and two 808nm light irradiations), and 980(+)+808(+++) group (one 980nm light irradiation and three 808nm light irradiations). Each 808nm laser irradiation was performed with an interval of 12 h, and the light density of the 980nm near-infrared light source was adjusted to 2 W / cm 2 The irradiation time was 2 min, and the light density was adjusted to 3 W / cm with 808 nm near-infrared light source. 2Each continuous irradiation lasted for 5 minutes. After treatment, 100 μL of DCFH-DA dye prepared according to the instructions was added to each well, incubated at 37°C in the dark for 30 minutes, washed three times with PBS buffer solution, and finally added 100 μL of PBS buffer solution and placed in the high-content imager to obtain Figure 7 .

[0102] like Figure 7 As shown, as the number of 808nm laser irradiation increases, the fluorescence intensity in the DCFH-DA graph becomes stronger, indicating that the ability of the nanomaterial TM-1 to generate singlet oxygen is stronger, proving that within 3 times, the more times the 808nm light is irradiated, the higher the level of ROS generated by the nanomaterial TM-1.

[0103] Example 8

[0104] 4T1 cells were plated at 6 × 10 per well. 3 The cells were inoculated into a 96-well plate at a density of 100 cells / well, and the PBS buffer solution was spread all around. After 24 hours of incubation, the medium in the 96-well plate was discarded, and 100 μL of complete 1640 medium containing 100 μg / mL of nanomaterial TM-1 was added to the wells. The control group was added with 100 μL of complete 1640 medium and incubated for 24 hours. The wells were then washed with PBS buffer solution to remove free nanomaterials. This was repeated 3 times, and 100 μL of complete 1640 medium was added to each well. The three groups of drug-added components were treated with different light treatments, namely, the group without any light treatment, the 980(+)808(+) group (1 time of 980nm light and 1 time of 808nm light), and the 980(+)808(+++) group (1 time of 980nm light and 3 times of 808nm light). The interval between each irradiation of 808nm laser was 12 hours, and the optical density of 980nm near-infrared light source was adjusted to 2W / cm 2 The irradiation time was 2 min, and the light density was adjusted to 3 W / cm with 808 nm near-infrared light source. 2 Each continuous irradiation lasted for 5 minutes. After treatment, 100 μL of Calcein AM / PI dye prepared according to the instructions was added to each well, incubated at 37°C in the dark for 30 minutes, washed three times with PBS buffer solution, and finally added 100 μL of PBS buffer solution and placed in the high-content imager to obtain Figure 8 .

[0105] like Figure 8As shown, there was no apoptosis in the control group, and a small amount of apoptosis in the drug-only group without light treatment, which may be due to the loading of compound 3-4 enabling the nanomaterial TM-1 to release singlet oxygen to kill tumor cells. The 980(+)808(+) group had more apoptosis than the group without any light treatment, proving that 808nm laser irradiation can make the nanomaterial TM-1 have a stronger ability to kill tumors. The 980(+)808(+++) group had more apoptosis than the 980(+)808(+) group, which also proved that the treatment regimen of 3 times of 808nm light irradiation has a stronger anti-tumor effect than the treatment regimen of 1 time of 808nm light irradiation.

[0106] Example 9

[0107] The MTT method was used to determine the anti-tumor efficacy of nanomaterial TM-2 after different times of 655 nm light irradiation. First, 4T1 cells were plated at 6×10 3 The cells were inoculated into two 96-well plates at a density of 100 μg / mL, and the 96-well plates were covered with PBS buffer solution. After 24 hours of incubation, the culture medium in the 96-well plates was discarded, and 100 μL of complete 1640 culture medium with the concentration of nanomaterial TM-1 of 0 μg / mL, 12.5 μeg / mL, 50 μgg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL was added to the wells, respectively, and incubated for 24 hours. The well plates were then washed with PBS buffer solution to remove free nanomaterials. This was repeated 3 times, and 100 μL of complete 1640 culture medium was added to each well. The three groups were first incubated with an optical density of 2 W / cm 2 , near-infrared light with a wavelength of 980 nm was used for 2 min at intervals, and then all three groups were exposed to light with an optical density of 20 mW / cm 2 , irradiate with near-infrared light of wavelength 655nm for 10min continuously, after 12h interval, the 655(++) group and 655(+++) group repeat the 10min irradiation of 655nm light, after another 12h interval, the 655(+++) group repeats the 10min irradiation of 655nm light. After treatment, incubate for 24h, then add 10μL MTT solution (0.5mg / mL) to each well, incubate at 37℃ for 4h, carefully remove the culture medium, add 150μL DMSO dissolved in purple formazan to each well, and use a microplate reader to measure the absorbance at 570nm to calculate the cell survival rate, and obtain Fig. 9 .like Fig. 9 As shown, it can be seen that the more times the 655nm light is irradiated within 3 times, the stronger the anti-tumor effect of the nanomaterial TM-2 prepared in the preparation example, indicating that each of the three 655nm light irradiations can stimulate the anti-cancer effect of the nanomaterial TM-2.

Claims

1. A class of cross-linkable phototherapy materials based on upconversion nanomaterials, characterized in that: It has the following structure: Wherein, m1, m2, and m3 are each independently an integer not less than 1; R2 is R3 is ICG or methylene blue; Among them, X - NO3 - 、F - , Cl - Br - ,I - PF6 - 、NO3 - 、SO4 2- CF3SO3 - 、CH3SO3 - , one of organic carboxylates having 1 to 3 carbon atoms; When R3 is a photothermal reagent, R1 is or When R3 is a photosensitizer, R1 is or Among them, R 1 -R 3 , R 5 -R 7 , R 9 -R 12 , R 14 -R 16 Each independently selected from hydrogen, trimethylsilyl, C1-C10 alkyl, C1-C10 alkoxy, C5-C10 aryl, amino, C1-C10 amino-substituted alkyl; R 4 , R 8 , R 13 , R 17 Each independently or The sum of n1 and n2 is independently an integer from 0 to 6, m is an integer from 0 to 6, and n is an integer from 1 to 2000; It is upconversion nanoparticle UCNP.

2. The material according to claim 1, characterized in that R2 is R3 is or R 1 -R 3 , R 5 -R 7 , R 9 -R 12 , R 14 -R 16 Each independently selected from hydrogen, trimethylsilyl, C1-C5 alkyl, C1-C5 alkoxy, C6-C10 aryl, amino, C1-C5 amino-substituted alkyl; R 4 , R 8 , R 13 , R 17 Each independently or The sum of n1 and n2 is independently an integer from 0 to 6, m is an integer from 0 to 6, and n is an integer from 1 to 50; m1, m2, and m3 are each independently an integer not less than 1.

3. The material according to claim 2, characterized in that When R3 is hour, R1 is or When R3 When R1 is or R 1 -R 3 , R 5 -R 7 , R 14 -R 16 Each independently selected from hydrogen, trimethylsilyl, C1-C5 alkyl, C1-C5 alkoxy, phenyl, amino, C1-C5 amino-substituted alkyl; R 4 , R 8 , R 17 Each independently or The sum of n1 and n2 is independently an integer from 0 to 3, m is an integer from 0 to 3, and n is an integer from 1 to 10; m1, m2, and m3 are each independently an integer not less than 1.

4. The material according to claim 3, characterized in that R1 is When R3 is R1 is or When R3 is R 1 -R 3 , R 5 -R 7 , R 14 -R 16 , R 14 -R 16 Each independently selected from hydrogen, trimethylsilyl, C1-C5 alkyl, C1-C5 alkoxy, and phenyl; R 4 , R 8 , R 17 Each is independent or The sum of n1 and n2 is independently an integer of 0-3, m is an integer of 0-3, and n is an integer of 1-5.

5. The material according to any one of claims 1 to 4, characterized in that The total amount of R1, R2 and R3 loaded on the material is 1-10 μmol / g.

6. The material according to claim 5, characterized in that The total amount of R1, R2 and R3 loaded on the material is 2-5 μmol / g.

7. The method for preparing the material according to any one of claims 1 to 4, characterized in that: The following steps are adopted: R1-COOH, R2-COOH, R3-COOH, and NHS are stirred in a solvent at room temperature for 1 to 5 hours, and then the nanomaterials dispersed in the solvent are added dropwise. The dispersion is stirred for 5 to 20 hours, centrifuged and washed to obtain the target material TM. The molar ratio of R1-COOH, R2-COOH, and R3-COOH is: 0.1-15:1:0.1-15; R1-COOH The mass ratio is 1:1~10.

0.

8. The preparation method according to claim 7, characterized in that: The molar ratio of R1--COOH, R2-COOH, and R3-COOH is: 5-15:1:5-15; R1--COOH and The mass ratio is 1:1~3.

0. The solvents are independently selected from at least one of DMF, DMSO, methanol and ethanol.

9. Use of the material according to any one of claims 1 to 4 in the preparation of photothermal therapy synergistic singlet oxygen anti-tumor drugs or in the preparation of continuous photodynamic therapy anti-tumor drugs under light and dark conditions.

10. Use of the material according to any one of claims 1 to 4 in the preparation of long-retention drugs.

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