Preparation Method of Acid-Activated Aggregation-Induced Emission Photosensitizer, the Photosensitizer and Its Application

Abstract

The present invention relates to the field of medical technology, and particularly relates to an aggregation-induced emission photosensitizer and its application for treating in-situ breast cancer by sonodynamic therapy. The preparation method of the photosensitizer includes: taking a TTh derivative or a pharmaceutically acceptable salt of the TTh derivative, and adding the tetrahydrofuran solution of PAE-PEG to ultrapure water to obtain a first solution, and the chemical formula of the TTh derivative is: ; stirring the first solution, and then performing ultrafiltration to obtain nanoparticles; dispersing the nanoparticles in a buffer solution to obtain the photosensitizer. The photosensitizer of the present invention can effectively overcome the π-π stacking effect, maintain a certain free radical yield in the aggregated state, and after being modified by PAE-PEG, can target tumor regions with an acidic microenvironment, increase the uptake of tumor cells, reduce the impact on normal tissues, and reduce side effects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly relates to a preparation method of an aggregation-induced emission photosensitizer, a luminescent photosensitizer, and their application in the preparation of a drug for treating in-situ breast cancer by sonodynamic therapy. Background Art

[0002] In recent years, the incidence and mortality rates of breast cancer have both shown an upward trend, seriously threatening human health. Traditional treatment methods such as chemotherapy, radiotherapy, and surgical treatment cause greater trauma to the human body and have problems such as incomplete treatment and serious side effects.

[0003] Sonodynamic Therapy (SDT) is a new therapy for treating tumors, which can generate reactive oxygen species (ROS) and has the characteristics of non-invasiveness, tissue penetration, and high controllability. Under ultrasonic (US) irradiation, a photosensitizer can induce the separation of electron-hole pairs, and the released energy, electrons, and holes can react with surrounding oxygen or water to generate reactive oxygen species (ROS) with strong oxidizing and cytotoxic properties, which can destroy biological macromolecules such as lipids, proteins, and DNA, thereby achieving the purpose of killing tumor cells. At the same time, sonodynamic therapy uses the strong penetration ability of ultrasonic waves on biological tissues to focus acoustic energy on deep tissues without trauma, and has broad prospects in the treatment of deep or large solid tumors.

[0004] Organic photosensitizers have received extensive attention due to their clear molecular structure, easy modification, high biocompatibility, and biodegradability. However, their therapeutic effects are often limited by two factors. The π-π stacking effect of traditional organic small molecules greatly inhibits the triplet exciton yield of the photosensitizer, and the nanoparticles in a dense packaging state naturally limit the energy and electron exchange between the photosensitizer and environmental oxygen. These two factors greatly limit the radical yield of the photosensitizer in the nanoparticle state. Therefore, the key to realizing efficient sonodynamic therapy based on organic photosensitizers lies in reasonable molecular structure design and effective control of the molecular aggregation state. Summary of the Invention

[0005] To solve the above problems, the present invention provides a preparation method of an acid-activated aggregation-induced emission (AIE) photosensitizer, a luminescent photosensitizer, and their application in the preparation of a drug for treating in-situ breast cancer by sonodynamic therapy.

[0006] In the first aspect of the present invention, a preparation method of an acid-activated aggregation-induced emission photosensitizer is provided, including the following steps:

[0007] S1. Take the TTh derivative or a pharmaceutically acceptable salt thereof, and add it together with a tetrahydrofuran (THF) solution of PAE-PEG to ultrapure water to obtain a first solution. The chemical formula of the TTh derivative is: , where R1 and R2 are the same or different, and R1 and R2 are each phenyl, 4-methylphenyl, tetraphenylethylene, 4-tert-butylbenzene, or 4-methoxybenzene, and R3 is hydrogen;

[0008] S2. Stir the first solution, and then perform ultrafiltration to obtain nanoparticles;

[0009] S3. Disperse the nanoparticles in a buffer solution to obtain a luminescent photosensitizer.

[0010] Furthermore, in step S1, the mass ratio of the TTh derivative or a pharmaceutically acceptable salt thereof to PAE-PEG is (0.1 - 0.3):1; the mass-volume ratio of the TTh derivative or a pharmaceutically acceptable salt thereof to THF is 0.8 - 1.3 mg / mL; the volume ratio of ultrapure water to the THF solution is (19 - 21):1.

[0011] Furthermore, in step S2, the stirring speed is 700 - 800 rpm and lasts for 1.8 - 2.2 minutes; the ultrafiltration speed is 4000 - 4400 rpm and lasts for 8 - 12 minutes.

[0012] Furthermore, in step S3, the buffer solution is a PBS buffer solution with a pH of 7.0 - 7.4.

[0013] The second aspect of the present invention provides a luminescent photosensitizer prepared by the above preparation method.

[0014] Preferably, the luminescent photosensitizer exerts its effect after acid induction.

[0015] Furthermore, the conditions for acid induction are pH = 5.0 ± 0.2.

[0016] Preferably, the luminescent photosensitizer is nanoparticles.

[0017] The third aspect of the present invention provides the use of the above-mentioned luminescent photosensitizer in the preparation of a drug for treating in situ breast cancer.

[0018] Furthermore, the drug for treating in situ breast cancer is a sonodynamic therapy drug.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1)The TTh derivative-based luminescent photosensitizer provided by the present invention is a photosensitizer with aggregation-induced emission characteristics, which can effectively overcome π-π stacking and maintain a certain free radical yield in the aggregated state.

[0021] (2)The present invention uses PAE-PEG to modify the TTh derivative, and the obtained nanoparticles can remove the surface polymer shell in an acidic environment, expose the positively charged core, promote the cellular uptake of the nanoparticles, and induce the release of the drug.

[0022] (3)The PAE-PEG modified TTh derivative provided by the present invention is an acid-activated nanoparticle, which can target tumor regions with an acidic microenvironment, increase the uptake of tumor cells, reduce the impact on normal tissues, and reduce side effects.

[0023] (4)The acid-activated nanoparticles provided by the present invention have a high drug encapsulation efficiency and an adjustable drug loading ratio, which can improve the drug delivery efficiency. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 (a) is the 1H NMR spectrum of MeTTh provided by the embodiment of the present invention; (b) is the 13C NMR spectrum of MeTTh provided by the embodiment of the present invention; (c) is the time-of-flight mass spectrum of MeTTh provided by the embodiment of the present invention;

[0026] Figure 2 is the UV absorption spectrum and fluorescence emission spectrum of MeTTh provided by the embodiment of the present invention;

[0027] Figure 3 is the AIE curve of MeTTh provided by the embodiment of the present invention;

[0028] Figure 4 is the absorption spectrum and emission spectrum of MeTTh nNPs and MeTTh NPs provided by the embodiment of the present invention;

[0029] Figure 5 is the DLS diagram of MeTTh nNPs and MeTTh NPs provided by the embodiment of the present invention;

[0030] Figure 6 is the size change diagram of MeTTh nNPs and MeTTh NPs after incubation under neutral or acidic conditions;

[0031] Figure 7 Zeta potential diagrams of MeTTh nNPs and MeTTh NPs after incubation under neutral or acidic conditions;

[0032] Figure 8 Graphs of the release rates and fluorescence intensity changes of MeTTh nNPs and MeTTh NPs;

[0033] Figure 9 ESR spectrum;

[0034] Figure 10 Sonodynamic effects of MeTTh nNPs and MeTTh NPs;

[0035] Figure 11 Cell uptake results of MeTTh nNPs and MeTTh NPs;

[0036] Figure 12 shows the intracellular ROS generation results of MeTTh nNPs and MeTTh NPs: (a) Confocal microscopy images of DCF fluorescence (scale bar: 10 μm); (b) Raw data of DCF fluorescence intensity detected by flow cytometry; (c) Histogram of DCF fluorescence intensity detected by flow cytometry;

[0037] Figure 13 shows the cell sonodynamic therapy results of MeTTh nNPs and MeTTh NPs: (a) Live / dead cell staining (scale bar: 100 μm); (b) Detection by cck8 kit;

[0038] Figure 14 Cell compatibility experiments of (a) MeTTh nNPs and (b) MeTTh NPs;

[0039] Figure 15 (a) shows the whole-body fluorescence images of mice at different time points after tail vein injection of MeTTh nNPs and MeTTh NPs, (b) shows the fluorescence normalization results of the MeTTh nNPs group; (c) shows the fluorescence normalization results of the MeTTh NPs group;

[0040] Figure 16 Weight changes of mice in different groups during 14 days of ultrasound treatment;

[0041] Figure 17 Tumor growth curves of mice in each group during 14 days of treatment (I: PBS, II: + US, III: MeTTh NPs, IV: MeTTh NPs + US - 12 h, V: MeTTh NPs + US - 24 h);

[0042] Figure 18Tumor weights of mice in each group after 14 days of treatment (I: PBS, II: +US, III: MeTTh NPs, IV: MeTTh NPs + US - 12 h, V: MeTTh NPs + US - 24 h);

[0043] Figure 19 Pictures of tumors dissected from mice in each group after 14 days of treatment;

[0044] Figure 20 Results of H&E, TUNEL, CD31, and Ki67 staining of tumor tissues; (Scale bar: 100 μm for H&E, 200 μm for the rest);

[0045] Figure 21 Results of H&E staining of the heart, liver, spleen, lungs, and kidneys of mice in the PBS group and the MeTTh NPs group. Detailed implementation manners

[0046] In order to make the technical problems, technical solutions, and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] Some abbreviations used in this embodiment are shown in Table 1.

[0048] Table 1 English abbreviation comparison table

[0049]

[0050] The embodiment of the present invention provides a preparation method of an acid-activated aggregation-induced emission photosensitizer, including the following steps:

[0051] S1: Take a TTh derivative or a pharmaceutically acceptable salt of the TTh derivative, and add it together with a tetrahydrofuran (THF) solution of PAE-PEG to ultrapure water to obtain a first solution. The chemical formula of the TTh derivative is: , where R1 and R2 are the same or different, R1 and R2 are respectively phenyl, 4-methylphenyl, tetraphenylethylene, 4-tert-butylbenzene, or 4-methoxybenzene, and R3 is hydrogen;

[0052] S2: Stir the first solution, and then perform ultrafiltration to obtain nanoparticles;

[0053] S3: Disperse the nanoparticles in a buffer solution to obtain a luminescent photosensitizer.

[0054] The TTh derivative or a pharmaceutically acceptable salt thereof provided by the embodiments of the present invention uses a phenyl-containing aromatic group as an electron donor, a thiophene structure as a π-bridge, and a rhodanine structure as an electron acceptor, thereby constructing a novel sonosensitizer with aggregation-induced emission characteristics, which can effectively overcome the π-π stacking effect and still maintain a certain free radical yield in the aggregated state.

[0055] Preferably, in some embodiments of the present invention, in the chemical formula of the TTh derivative, R1 and R2 are 4-methylphenyl, and R3 is hydrogen. For the sake of convenience of description, the TTh derivative at this time is represented by the English abbreviation MeTTh.

[0056] Through step S1, MeTTh or a pharmaceutically acceptable salt thereof is modified with PAE-PEG (poly(β-amino ester)-polyethylene glycol) to obtain corresponding nanoparticles (MeTTh Nano Particles, MeTTh NPs). This type of nanoparticle is an acid-activated nanoparticle. In an acidic environment, PAE undergoes protonation to transform into a hydrophilic structure, and at the same time its charge state changes from negative to positive. This charge transformation causes the nanoparticle composed of PAE-PEG to remove the surface polymer shell due to charge repulsion in an acidic environment, exposing the positively charged core, which can thus promote the cellular uptake of the nanoparticle and induce the release of the drug.

[0057] The acid-activated nanoparticles of modified MeTTh, MeTTh NPs, can utilize the acidic characteristics of the tumor microenvironment to improve the targeting efficiency of the drug. After the drug is activated at the tumor site, the positively charged nanoparticles bind to the negatively charged cell membranes and biomacromolecules inside the cell, reducing spillage. As the uptake of the nanoparticles by cancer cells increases, the total enrichment amount increases, and the therapeutic effect is enhanced. In addition, since this type of acid-activated nanoparticle specifically releases the drug in the tumor microenvironment, it can reduce the impact on normal tissues and reduce side effects.

[0058] The acid-activated nanoparticles also have a high drug encapsulation rate and an adjustable drug loading ratio, which can improve the drug delivery efficiency.

[0059] Specifically, in step S1, the mass ratio of the TTh derivative to PAE-PEG is (0.1-0.3):1; the mass-volume ratio of the TTh derivative or a pharmaceutically acceptable salt thereof to THF is 0.8-1.3 mg / mL; the volume ratio of ultrapure water to the THF solution is (19-21):1.

[0060] Preferably, the mass-volume ratio of the TTh derivative or a pharmaceutically acceptable salt thereof to THF is 1.0 mg / mL, and the volume ratio of ultrapure water to the THF solution is 20:1.

[0061] Specifically, the molecular weight of PAE in PAE-PEG is 10 KDa, and the molecular weight of PEG is 5 KDa.

[0062] Specifically, in step S2, the stirring speed is 700 - 800 rpm and lasts for 1.8 - 2.2 minutes; the ultrafiltration speed is 4000 - 4400 rpm and lasts for 8 - 12 minutes.

[0063] Specifically, in step S3, the buffer solution can be any buffer solution, preferably PBS buffer solution with a pH of 7.0 - 7.4.

[0064] Specifically, the amount of the buffer solution in step S3 is such that the concentration of the nanoparticles in it is 50 μM.

[0065] The embodiment of the present invention also provides a luminescent sonosensitizer prepared by the above preparation method.

[0066] Specifically, the luminescent sonosensitizer exerts its effect after acid induction.

[0067] Specifically, the luminescent sonosensitizer is nanoparticles.

[0068] Specifically, the particle size of the nanoparticles is 16 - 140 nm, preferably 70 nm.

[0069] Specifically, the conditions for acid induction are pH = 5.0 ± 0.2.

[0070] Specifically, the acid used for acid induction can be any organic acid and / or inorganic acid such as acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, etc.

[0071] The embodiment of the present invention also provides the application of the luminescent sonosensitizer as described above in the preparation of a drug, and the drug is used for the treatment of in situ breast cancer by sonodynamic therapy.

[0072] The following is illustrated with specific examples:

[0073] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified, and the technologies not described in detail are all carried out according to the standard methods well-known to those skilled in the art.

[0074] Example 1 Preparation of Compound MeTTh

[0075] Formula (I) is the flow chart for preparing Compound MeTTh. As shown in Formula (I), Compound MeTTh can be prepared.

[0076]

[0077] Formula (I)

[0078] 190 mg (0.5 mmol) of 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carbaldehyde (CAS No.: 654067-66-0), 64 mg (0.4 mmol) of 3-ethyl-2-thioxo-4-thiazolidinone (CAS No.: 7648-01-3) and 125 mg (1.5 mmol) of sodium acetate (CAS No.: 127-09-3) were added into a 25 mL two-necked round-bottom flask. The two-necked round-bottom flask was evacuated and purged with dry nitrogen three times. Then 10 mL of glacial acetic acid was added, and the temperature was raised to 90 °C for reaction for 24 h. Subsequently, it was cooled to room temperature. 10 mL of water was added into the two-necked round-bottom flask, and the mixture was washed three times with dichloromethane, 20 mL each time. The dichloromethane layer was collected, dried with anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain 210 mg of pure MeTTh, which was a dark red solid with a yield of 86%.

[0079] Its structure was identified by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, as shown in Figure 1. Among them, Figure 1 (a) is its nuclear magnetic resonance hydrogen spectrum, 1 H NMR(400 MHz, chloroform- d ) δ 7.84 (s, 1H),7.45 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 4.0 Hz, 1H), 7.25 (d, J = 3.3 Hz, 1H), 7.10(d, J = 8.4 Hz, 4H), 7.03 (d, J = 8.3 Hz, 4H), 6.99 (d, J = 8.7 Hz, 2H), 4.18 (q, J =7.1 Hz, 2H), 2.33 (s, 6H), 1.28 (t, J = 7.1 Hz, 3H); Figure 1 (b) is its nuclear magnetic resonance carbon spectrum, 13 CNMR(126 MHz, chloroform- d) δ 192.22, 182.59, 167.38, 153.28, 144.42, 135.85, 133.67, 130.12, 127.16, 126.77, 125.69, 125.37, 123.38, 122.58, 121.30, 121.09, 119.45, 39.91, 20.90, 12.31; Figure 1(c) is its time-of-flight mass spectrum, MALDI-TOF-MS: expected M.W. 526.121, measured M.W. 526.255.

[0080] Example 2 Preparation of PAE-PEG Modified Sonosensitizer Nanoparticles

[0081] S1: Add a tetrahydrofuran (THF) solution containing MeTTh (prepared in Example 1) and PAE-PEG (poly(β-amino ester)-polyethylene glycol) with a mass ratio of 0.1:1 (with a mass-volume concentration of 1.0 mg / mL based on MeTTh) to ultrapure water. The volume ratio of ultrapure water to the THF solution is 19:1;

[0082] S2: Stir vigorously at 750 rpm for 1.8 minutes and then perform ultrafiltration at 4000 rpm for 12 minutes;

[0083] S3: Disperse the nanoparticles obtained by ultrafiltration in PBS buffer with a pH of 7.4 to obtain the sonosensitizer (MeTThNPs).

[0084] Example 3 Preparation of PAE-PEG Modified Sonosensitizer Nanoparticles

[0085] S1: Add a tetrahydrofuran (THF) solution containing MeTTh (prepared in Example 1) and PAE-PEG (poly(β-amino ester)-polyethylene glycol) with a mass ratio of 0.3:1 (with a mass-volume concentration of 1.0 mg / mL based on MeTTh) to ultrapure water. The volume ratio of ultrapure water to the THF solution is 21:1;

[0086] S2: Stir vigorously at 750 rpm for 2.2 minutes and then perform ultrafiltration at 4400 rpm for 8 minutes;

[0087] S3: Disperse the nanoparticles obtained by ultrafiltration in PBS buffer with a pH of 7.4 to obtain the sonosensitizer (MeTThNPs).

[0088] Comparative Example 1 Preparation of DSPE-PEG2000 Modified Sonosensitizer Nanoparticles

[0089] S1: Add the THF solution containing MeTTh (prepared in Example 1) and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000) with a mass ratio of 0.4:1 to ultrapure water. The volume ratio of ultrapure water to the THF solution is 5:1.

[0090] S2: After vigorously stirring for 1.8 minutes, perform ultrafiltration at a rotational speed of 4000 rpm for 12 minutes.

[0091] S3: Disperse the nanoparticles obtained by ultrafiltration in a PBS buffer solution with a pH of 7.4 to obtain the control nanoparticles (MeTTh nNPs).

[0092] Example 4 Characterization of MeTTh

[0093] Prepare a solution of MeTTh prepared in Example 1 with a concentration of 0.1 M using acetonitrile, and detect the absorption spectrum and emission spectrum of MeTTh using a UV-vis-NIR spectrophotometer and a fluorescence spectrophotometer, as Figure 2 shown. The maximum absorption wavelength and maximum emission wavelength of MeTTh are 495 nm and 700 nm, respectively.

[0094] Change the ratio of water to acetonitrile (in terms of the volume concentration of water), and detect the AIE characteristic curve of MeTTh, as Figure 3 shown. It can be seen that its maximum emission wavelength remains basically unchanged, but the emission intensity changes with the ratio of water in the mixed solvent. This is because as the ratio of water increases, the polarity of the mixed solvent gradually increases, and the increase in solvent polarity causes the organic small molecules to aggregate. The aggregation of molecules restricts the movement of molecular groups, and the energy absorbed by the molecules cannot be dissipated through the movement (thermal energy) of the groups. Finally, the energy is radiated in the form of luminescence. Therefore, the higher the solvent polarity, the higher the degree of molecular aggregation and the greater the emission intensity.

[0095] Example 5 Characterization of MeTTh nNPs and MeTTh NPs

[0096] 1. Absorption and Emission Spectra

[0097] Prepare the sample solutions of MeTTh nNPs prepared in Comparative Example 1 and MeTTh NPs prepared in Example 2 according to the method in Example 4, and detect the absorption spectra and emission spectra of MeTTh nNPs prepared in Comparative Example 1 and MeTTh NPs prepared in Example 1 using a UV-vis-NIR spectrophotometer and a fluorescence spectrophotometer, as Figure 4As shown, it can be seen that the two kinds of nanoparticles have the same emission curve, which is the same as that of the MeTTh compound; however, the absorption curve of MeTTh NPs in Example 2 has a certain red-shift trend, while the absorption curve of MeTTh nNPs in the comparative example remains unchanged.

[0098] 2. Particle size

[0099] The diameters of MeTTh nNPs and MeTTh NPs were detected using dynamic light scattering (DLS). As Figure 5 shown, the hydrodynamic sizes of MeTTh nNPs and MeTTh NPs measured by dynamic light scattering were ~48 nm. After incubation for 24 h in an acidic environment (PBS buffer at pH = 5.0) under dark conditions at room temperature, the particle size was detected again, and the results are as Figure 6 shown. It was detected that the size of MeTTh NPs increased, while the size of MeTTh nNPs remained basically unchanged.

[0100] 3. Zeta potential

[0101] The Zeta potentials of MeTTh nNPs and MeTTh NPs were detected. Under neutral conditions (PBS solution at pH = 7.4), both of them had negative potentials. However, after incubation for 24 h in an acidic environment (PBS solution at pH = 5.0) under dark conditions at room temperature, MeTTh NPs underwent charge reversal, as Figure 7 shown.

[0102] 4. Release rate and fluorescence intensity

[0103] The release rates and fluorescence intensities of MeTTh nNPs and MeTTh NPs at each time point during incubation in neutral and acidic environments were detected. As Figure 8 shown, the release rate of MeTTh NPs reached 80% after incubation for 8 h in an acidic environment, which was significantly higher than that of other groups; the fluorescence intensity of MeTTh NPs decreased after incubation in an acidic environment, probably because the size increased, the structure of the nanoparticles became looser, and the aggregation degree of the molecules encapsulated inside decreased, resulting in weakened fluorescence.

[0104] 5. ESR signal

[0105] As Figure 9 shown, using 2,2,6,6-tetramethylpiperidine (TEMP) as a spin trap, different ESR signals indicate the type I and type II properties of ROS. When ultrasonic irradiation was added, three kinds of free radicals could be detected; while when the ultrasonic irradiation was removed, the free radical signal disappeared.

[0106] Further detect the ROS production of MeTTh nNPs and MeTTh NPs under ultrasonic action within 10 min of incubation in neutral and acidic environments. As Figure 10 shown, I-I0 represents the difference in fluorescence intensity of each group at different time points and in the initial state (0 min). It can be seen that the fluorescence intensity of MeTTh NPs increases rapidly after incubation in acidic environment, higher than that of the other groups. This result well demonstrates the potential of MeTTh NPs to produce ROS in acidic environment.

[0107] Example 6 Cell sonodynamic therapy

[0108] (1) Cells and cell culture

[0109] Select the mouse breast cancer cell line (4T1 cells) purchased from the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 medium supplemented with 10% (v / v) fetal bovine serum (Hyclone), 100 UI / mL -1 penicillin and 100 UI / mL -1 streptomycin in an incubator containing 5% carbon dioxide at 37°C until the cell density reached 70%-80% of the culture flask, and the pre-cultured 4T1 cells were obtained.

[0110] (2) Cell uptake

[0111] The pre-cultured 4T1 cells in (1) were seeded into a 24-well plate at a density of 5×10 4 cells / well. After culturing for 12 h in the same manner as in (1), they were allowed to adhere. MeTTh NPs were added to 12 of the wells as the experimental group, and MeTThnNPs were added to the other 12 wells as the control group. After incubation at each time point, the 4T1 cells were washed 3 times with PBS (pH 7.4), and the uptake efficiency of each group was evaluated using a flow cytometer (CytoFLEX, Beckman) (excited at 488 nm and the emission was collected using a 690 / 50 nm bandpass filter), as Figure 11 shown. It can be seen that compared with ordinary nanoparticles, the cell uptake efficiency of acid-activated nanoparticles is much higher. The acid-activated nanoparticles achieved a 99.5% uptake effect at one hour. Ordinary nanoparticles required 12 hours to achieve a 99.2% uptake effect.

[0112] (3) Detection of intracellular reactive oxygen species

[0113] The cells were seeded at 5×10 4Cells were seeded onto confocal dishes at a density of cells / well and cultured for 12 h in the manner described in (1). When the 4T1 cells reached approximately 80% confluence, they were divided into a control group (PBS), a material group (MeTTh nNPs and MeTTh NPs), and an experimental group (MeTThnNPs + US and MeTTh NPs + US). The material group and the experimental group were incubated with nanoparticles at the same concentration (10 μM) for 4 h, then 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was added and incubated for 30 minutes, and then the 4T1 cells were washed 3 times with PBS (pH 7.4).

[0114] As Figure 12a shown, after 10 minutes of ultrasound irradiation at a duty cycle of 50%, a power density of 1.5 W / cm 2 , and a frequency of 1 MHz (i.e., the experimental group), obvious green fluorescence signals were observed in the 4T1 cells of the MeTTh NPs + US group, indicating that MeTThNPs effectively generated ROS under ultrasound irradiation. In contrast, no green fluorescence signals were observed in the control group and the material group (without ultrasound irradiation). As Figure 12b , c shows the distribution of the fluorescence intensity inside the cells after different treatments of the cells detected by flow cytometry, where Figure 12b is the original data graph, Figure 12c is the bar graph after quantification. As shown in the figure, after ultrasound irradiation, the average fluorescence intensity of the experimental group increased significantly, and this result well demonstrated the ability of MeTTh NPs to generate ROS under ultrasound irradiation and its potential for cancer treatment.

[0115] (4) Killing effect of sonodynamic therapy on cancer cells

[0116] In this example, the killing effect on 4T1 cells was explored through live / dead cell staining experiments and CCK-8 experiments.

[0117] Live / dead cell staining experiment: 4T1 cells were seeded onto confocal dishes at a density of 5×10 4 cells / well and cultured for 12 h. 300 μL of the culture medium containing MeTTh nNPs and MeTTh NPs at a concentration of 50 μM was used to replace the waste liquid as the material group and the treatment group, while the control group (Blank) directly replaced the waste liquid with 300 μL of the culture medium. After incubation for 4 h, ultrasound treatment was applied for 10 minutes, and then incubation continued. Then, the waste liquid was aspirated, and the staining solution (300 μL, containing 2 μM Calcein AM and 8 μM PI) was mixed. After 15 minutes, fluorescence imaging was taken with a confocal microscope. The results are as Figure 13a shown. Only the MeTTh NPs group could exert a cell killing effect under ultrasound irradiation, and the other groups had almost no cytotoxic effect.

[0118] CCK-8 assay: 4T1 cells were seeded into 96-well plates at a density of 5×10 3 cells / well and cultured for 12 h. The culture media containing different concentrations of MeTTh nNPs and MeTTh NPs were used to replace the waste liquid. After incubation for 4 h, ultrasound irradiation was performed for 5 minutes, and then incubation was continued for 24 h. Then, 10 µL of CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) was added to each well. After incubation for 30 minutes, the absorbance at 450 nm of each well was measured on a Microplate Reader (BioTek). The cell viability was calculated using the following formula: Cell viability (%) = (mean absolute value of the treatment group / mean absolute value of the control group) × 100%. The results are as Figure 13b shown. With the increase in the concentrations of MeTTh nNPs and MeTTh NPs, the cell viability decreased. In the MeTTh NPs + US treatment group, the cell viability decreased most significantly, which was similar to the results of the live / dead cell staining experiment.

[0119] (5) Cytotoxicity test

[0120] To measure the cytotoxicity of MeTTh nNPs and MeTTh NPs, 4T1 cells purchased from ATCC and MCF-10A, Hela, and Bend.3 cells purchased from Beijing Na Biotechnology were seeded into 96-well plates at a density of 5×10 3 cells per well (100 µL) and cultured for 12 h. The original culture media were replaced with 100 µL of culture media containing different concentrations of MeTTh nNPs and MeTTh NPs, and then incubated for 24 h. 10 µL of CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) solution was added to each well to measure the OD value, indirectly reflecting the number of live cells. After incubation with CCK-8 for 30 minutes, the absorbance at 450 nm of each well was measured on a Microplate Reader (BioTek). The cell viability was calculated using the following formula: Cell viability (%) = (mean absolute value of the treatment group / mean absolute value of the control group) × 100%. As Figure 14 shown, Figure 14 a is the cell compatibility result graph of MeTTh nNPs, Figure 14 b is the cell compatibility result graph of MeTTh NPs. It can be seen that the killing effect of MeTTh nNPs on various types of tumor cells is very limited regardless of the concentration, while the killing effect of MeTTh NPs in the experimental group increases with the increase in its concentration.

[0121] Example 7 In Vivo Sonodynamic Therapy

[0122] (1)In Vivo Fluorescence Imaging Distribution Study of MeTTh nNPs and MeTTh NPs

[0123] Inject MeTTh nNPs and MeTTh NPs materials (100 µL, 10 mg / kg) via the tail vein. At 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h, observe the fluorescence distribution of MeTTh nNPs and MeTTh NPs in mice using a small animal fluorescence imaging system (IVIS Spectrum, Perkelmer, US), as shown in Figure 15. In the MeTTh nNPs group of mice, the material had the maximum enrichment in the tumor at 24 h, while in the MeTTh NPs group of mice, the fluorescence was the strongest at 12 h and decreased at 24 h. Since it was not certain whether the fluorescence attenuation at 24 h was due to the decomposition of the nanoparticles under acidic conditions or the clearance of the nanoparticles by the mice, two time points, 12 h and 24 h, were set for treatment during sonodynamic therapy.

[0124] (2)In Vivo Sonodynamic Therapy

[0125] Randomly divide female Balb / c mice into 5 groups, with 5 mice in each group. Inject 4T1 cells (5×10 5 cells / mouse) to establish a tumor-bearing mouse model. When the tumor volume reaches approximately 80 - 100 mm 3 , the mice are ready to receive different treatments:

[0126] Blank group I: Inject normal saline via the tail vein; Ultrasound group II: Inject normal saline via the tail vein and irradiate with ultrasound for 10 min; MeTTh NPs group III: Inject MeTTh NPs via the tail vein; MeTTh NPs + US – 12 h group IV: Inject MeTTh NPs via the tail vein and irradiate with ultrasound for 10 min after 12 h; MeTTh NPs + US – 24 h group V: Inject MeTTh NPs via the tail vein and irradiate with ultrasound for 10 min after 24 h.

[0127] Record the body weight of the mice every 2 days, as Figure 16 shown. Measure the length and width of the tumor every 2 days, as Figure 17As shown, it can be seen that the tumor volumes of the control groups (PBS group, ultrasound group, MeTTh NPs group) increased sharply during the treatment, and there was no significant difference among the three groups, while there was a significant difference between the experimental group and the control groups. Specifically, there was a significant difference between the fourth group (MeTTh NPs + US - 12 h) and the three control groups (p < 0.05), and there was a more significant difference between the fifth group (MeTTh NPs + US - 24 h) and the three control groups (p < 0.01).

[0128] After 14 days, the tumors of the mice in each group were dissected, weighed and photographed, as Figure 18 、 19 shown. The tumor masses of the control groups (PBS group, ultrasound group, MeTTh NPs group) increased sharply during the treatment, and there was no significant difference among the three groups, while there was a significant difference between the experimental group and the control groups. Specifically, there was a significant difference between the fourth group (MeTTh NPs + US - 12 h) and the three control groups (p < 0.05), and there was a more significant difference between the fifth group (MeTTh NPs + US - 24 h) and the three control groups (p < 0.01). Thus, it can be concluded that after ultrasound treatment, MeTTh NPs showed an obvious inhibitory effect on tumor growth. In addition, the tumor weight also indicated that the sonodynamic therapy mediated by MeTTh NPs had a strong therapeutic effect on the tumors of tumor-bearing mice.

[0129] After 24 h of ultrasound treatment, the tumor tissues of the mice were collected for hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), anti-Ki67 monoclonal antibody immunofluorescence staining (Ki67 staining), and platelet endothelial cell adhesion molecule immunofluorescence staining (CD31 staining). As Figure 20 shown.

[0130] The hematoxylin-eosin (HE) staining results of the tumor sections of each group showed that a large number of karyopyknosis and obvious karyopyknosis could only be observed on the tumor sections treated with MeTTh NPs + US, indicating that SDT successfully destroyed the tumor tissue.

[0131] In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of the tumor tissue was performed to further verify the apoptosis of tumor tissue cells induced by sonodynamic therapy mediated by MeTTh NPs. A large number of TUNEL-positive red fluorescence signals were observed in the ultrasound-treated tumor tissue, while no red fluorescence signals were detected in the control group, indicating that ultrasound induced severe apoptosis.

[0132] Anti-Ki67 monoclonal antibody immunofluorescence staining (Ki67 staining) and platelet endothelial cell adhesion molecule immunofluorescence staining (CD31 staining) were performed on tumor tissues to further explore its mechanism of action. A large number of green positive proliferating cells in the MeTTh NPs + US group were significantly reduced. In sharp contrast, densely arranged 4T1 cells with strong proliferative activity were visible in the tumor tissues of the control group. These results strongly demonstrated that ultrasound-activated aggregation-induced emission photosensitizers could effectively inhibit tumor growth by inducing tumor cell apoptosis and inhibiting tumor cell proliferation.

[0133] In addition, we evaluated whether MeTTh NPs would cause damage to major organs. At the end of the treatment period, mice were euthanized by injecting pentobarbital at a dose of 100 mg / kg. The heart, liver, spleen, lung, and kidney organs of the mice were taken for sectioning and H&E staining, and then observed under a bright-field microscope. Figure 21 The results showed that there was no significant difference between the mice in the MeTTh NPs injection group and the PBS control group. This indicated that MeTTh NPs did not cause lesions in major organs, suggesting that MeTTh NPs had no acute toxicity.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of an acid-activated aggregation-induced emission photosensitizer, comprising: S1: Taking a TTh derivative or a pharmaceutically acceptable salt thereof, and adding a tetrahydrofuran solution of poly(β-amino ester)-polyethylene glycol PAE-PEG to ultrapure water to obtain a first solution, and the chemical formula of the TTh derivative is: ； S2: Stirring the first solution, and then performing ultrafiltration to obtain nanoparticles; S3: Dispersing the nanoparticles in a buffer solution to obtain a photosensitizer.

2. The preparation method of the luminescent photosensitizer according to claim 1, characterized in that, In the step S1, the mass ratio of the TTh derivative or the pharmaceutically acceptable salt thereof to PAE-PEG is (0.1-0.3):1; the mass-volume ratio of the TTh derivative or the pharmaceutically acceptable salt thereof to tetrahydrofuran is 0.8-1.3 mg / mL; the volume ratio of ultrapure water to the tetrahydrofuran solution is (19-21):

1.

3. The preparation method of the luminescent photosensitizer according to claim 1, characterized in that, In the step S2, the stirring speed is 700-800 rpm and lasts for 1.8-2.2 minutes; the ultrafiltration speed is 4000-4400 rpm and lasts for 8-12 minutes.

4. The preparation method of the luminescent photosensitizer according to claim 1, wherein, In the step S3, the buffer solution is PBS buffer solution with a pH of 7.0-7.

4.

5. A photosensitizer prepared by the preparation method of the photosensitizer according to any one of claims 1-4.

6. The luminescent photosensitizer according to claim 5, wherein The photosensitizer exerts its function after acid induction.

7. The luminescent photosensitizer according to claim 6, wherein The conditions for acid induction are pH = 5.0 ± 0.

2.

8. The luminescent photosensitizer according to claim 5, wherein, The photosensitizer is nanoparticles.

9. Use of the photosensitizer according to any one of claims 5-8 in the preparation of a drug for treating in situ breast cancer.

10. The application according to claim 9, wherein The drug for treating in situ breast cancer is a sonodynamic therapy drug.

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