Preparation method of acid-activated aggregation-induced emission sound-sensitive agent, light-emitting sound-sensitive agent and application of light-emitting sound-sensitive agent

Abstract

The invention relates to the technical field of medicine, in particular to an aggregation-induced emission sonosensitizer and application of the aggregation-induced emission sonosensitizer to treatment of in-situ breast cancer through sonodynamic therapy. The preparation method of the luminescent sound-sensitive agent comprises the following steps: taking a TTh derivative or a pharmaceutically acceptable salt of the TTh derivative, adding the TTh derivative or the pharmaceutically acceptable salt of the TTh derivative and a tetrahydrofuran solution of PAE-PEG into ultrapure water to obtain a first solution, and the chemical formula of the TTh derivative is # imgabs0 #; stirring the first solution, and then performing ultrafiltration to obtain nanoparticles; and dispersing the nano-particles in a buffer solution to obtain the luminescent sound-sensitive agent. The sonosensitizer can effectively overcome the pi-pi accumulation effect, still maintains a certain free radical yield in an aggregation state, and after PAE-PEG modification, the sonosensitizer can target a tumor area with an acidic microenvironment, increase the uptake of tumor cells, reduce the influence on normal tissues and reduce side effects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing an aggregation-induced luminescence sonosensitizer, a luminescent sonosensitizer and an application of the sonosensitizer in preparing a drug for treating in situ breast cancer through sonodynamic therapy. Background Art

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

[0003] Sonodynamic therapy (SDT) is a new treatment for tumors that can produce reactive oxygen species (ROS) and is non-invasive, tissue penetrating, and highly controllable. Sonosensitizers can induce the separation of electron-hole pairs under ultrasound (US) irradiation. The released energy, electrons, and holes can react with the surrounding oxygen or water to produce reactive oxygen species (ROS) with strong oxidizing and cytotoxicity, which can destroy lipids, proteins, DNA and other biological macromolecules, thereby achieving the purpose of killing tumor cells. At the same time, sonodynamic therapy uses ultrasound to have a strong penetration ability in biological tissues, and can focus the sound energy on deep tissues without trauma. It has broad prospects in the treatment of deep or large solid tumors.

[0004] Organic sonosensitizers have attracted 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 sonosensitizers, and the densely packed nanoparticles naturally limit the energy and electron exchange between sonosensitizers and environmental oxygen. These two factors greatly limit the free radical yield of sonosensitizers in the nanoparticle state. Therefore, the key to achieving efficient sonodynamic therapy based on organic sonosensitizers lies in the reasonable molecular structure design and the effective control of the molecular aggregation state. Summary of the invention

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

[0006] The first aspect of the present invention provides a method for preparing an acid-activated aggregation-induced luminescence sonosensitizer, comprising the following steps: S1. Take a TTh derivative or a pharmaceutically acceptable salt of the TTh derivative, and add it to ultrapure water together with a tetrahydrofuran (THF) solution of PAE-PEG to obtain a first solution, wherein the chemical formula of the TTh derivative is: , wherein R1 and R2 are the same or different, R1 and R2 are phenyl, 4-methylphenyl, tetraphenylvinyl, 4-tert-butylbenzene or 4-methoxybenzene, and R3 is hydrogen; S2. stirring the first solution, and then ultrafiltering to obtain nanoparticles; S3. Dispersing the nanoparticles in a buffer solution to obtain a luminescent sonosensitizer.

[0007] Furthermore, in step S1, the mass ratio of the TTh derivative or the pharmaceutically acceptable salt of the TTh derivative to PAE-PEG is (0.1-0.3):1; the mass volume ratio of the TTh derivative or the pharmaceutically acceptable salt of the TTh derivative to THF is 0.8-1.3 mg / mL; and the volume ratio of ultrapure water to THF solution is (19-21):1.

[0008] Furthermore, in step S2, the stirring speed is 700-800 rpm, which lasts for 1.8-2.2 minutes; and the ultrafiltration speed is 4000-4400 rpm, which lasts for 8-12 minutes.

[0009] Furthermore, the buffer in step S3 is PBS buffer with a pH of 7.0-7.4.

[0010] The second aspect of the present invention provides a luminescent sonosensitizer prepared by the above preparation method.

[0011] Preferably, the luminescent sonosensitizer exerts its effect after being induced by acid.

[0012] Furthermore, the acid-induced condition is pH=5.0±0.2.

[0013] Preferably, the luminescent sonosensitizer is a nanoparticle.

[0014] The third aspect of the present invention provides the use of the luminescent sonosensitizer as described above in the preparation of a drug for treating in situ breast cancer.

[0015] Furthermore, the drug for treating in situ breast cancer is a sonodynamic therapy drug.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The TTh derivative luminescent sonosensitizer provided by the present invention is a sonosensitizer with aggregation-induced luminescence properties, which can effectively overcome the π-π stacking effect and maintain a certain free radical yield in the aggregated state.

[0017] (2) The present invention uses PAE-PEG to modify TTh derivatives, and the resulting nanoparticles can remove the surface polymer shell in an acidic environment, exposing the positively charged core, promoting the cellular uptake of the nanoparticles, and inducing the release of drugs.

[0018] (3) The PAE-PEG-modified TTh derivative provided by the present invention is an acid-activated nanoparticle that can target tumor areas with acidic microenvironment, increase the uptake of tumor cells, reduce the impact on normal tissues, and reduce side effects.

[0019] (4) The acid-activated nanoparticles provided by the present invention have a high drug encapsulation rate and an adjustable drug loading ratio, which can improve the drug delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0021] FIG1 (a) is a hydrogen nuclear magnetic resonance spectrum of MeTTh provided in an embodiment of the present invention; (b) is a carbon nuclear magnetic resonance spectrum of MeTTh provided in an embodiment of the present invention; (c) is a time-of-flight mass spectrum of MeTTh provided in an embodiment of the present invention; Figure 2 The ultraviolet absorption spectrum and fluorescence emission spectrum of MeTTh provided in the embodiments of the present invention; Figure 3 The AIE curve of MeTTh provided by the embodiment of the present invention; Figure 4 The absorption and emission spectra of MeTTh nNPs and MeTTh NPs provided in the embodiments of the present invention; Figure 5 DLS images of MeTTh nNPs and MeTTh NPs provided in the embodiments of the present invention; Figure 6 The size changes of MeTTh nNPs and MeTTh NPs after incubation under neutral or acidic conditions; Figure 7 Zeta potential diagrams of MeTTh nNPs and MeTTh NPs after incubation under neutral or acidic conditions; Figure 8 The release rate and fluorescence intensity change diagram of MeTTh nNPs and MeTTh NPs; Fig. 9is the ESR spectrum; Fig.10 for the sonodynamic effects of MeTTh nNPs and MeTTh NPs; Fig.11 Cellular uptake results of MeTTh nNPs and MeTTh NPs; Figure 12 shows the intracellular ROS production results of MeTTh nNPs and MeTTh NPs: (a) Confocal imaging 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; Figure 13 shows the results of cell sonodynamic therapy of MeTTh nNPs and MeTTh NPs: (a) live and dead cell staining method (scale bar: 100 μm); (b) cck8 kit detection; Fig.14 (a) Cytocompatibility experiments of MeTTh nNPs and (b) MeTTh NPs. Figure 15 (a) is the whole-body fluorescence images of mice at different time points after tail vein injection of MeTTh nNPs and MeTTh NPs, (b) is the result of fluorescence normalization of the MeTTh nNPs group; (c) is the result of fluorescence normalization of the MeTTh NPs group; Fig.16 Body weight changes of mice in different groups during 14 days of ultrasound treatment; Fig.17 are the tumor growth curves of mice in each group during the 14-day treatment (I: PBS, II: + US, III: MeTThNPs, IV: MeTTh NPs + US - 12 h, V: MeTTh NPs + US - 24 h); Fig.18 is the weight of the tumor 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); Fig.19 The pictures are of the tumors of mice in each group dissected out after 14 days of treatment; Fig. 20 The results of H&E, TUNEL, CD31 and Ki67 staining of tumor tissues (scale bar: H&E is 100 μm, and the rest are 200 μm); Fig.21 These are the results of H&E staining of the heart, liver, spleen, lung, and kidney of mice in the PBS group and the MeTTh NPs group. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and 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.

[0023] Some abbreviations used in this example are shown in Table 1.

[0024] Table 1 English abbreviation comparison table The present invention provides a method for preparing an acid-activated aggregation-induced luminescence sonosensitizer, comprising the following steps: S1: Take a TTh derivative or a pharmaceutically acceptable salt of the TTh derivative and a tetrahydrofuran (THF) solution of PAE-PEG and add them to ultrapure water to obtain a first solution. The chemical formula of the TTh derivative is: , wherein R1 and R2 are the same or different, R1 and R2 are phenyl, 4-methylphenyl, tetraphenylvinyl, 4-tert-butylbenzene or 4-methoxybenzene, and R3 is hydrogen; S2: stirring the first solution, and then ultrafiltering to obtain nanoparticles; S3: Dispersing the nanoparticles in a buffer solution to obtain a luminescent sonosensitizer.

[0025] The TTh derivative or the pharmaceutically acceptable salt of the TTh derivative provided in the embodiment 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. The novel sonosensitizer with aggregation-induced emission characteristics constructed in this way can effectively overcome the π-π stacking effect and still maintain a certain free radical yield in the aggregated state.

[0026] 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 ease of description, the TTh derivative is represented by the English abbreviation MeTTh.

[0027] Through step S1, MeTTh or a pharmaceutically acceptable salt of MeTTh is modified by PAE-PEG (poly(β-amino lipid)-polyethylene glycol) to obtain corresponding nanoparticles (MeTTh Nano Particles, MeTTh NPs). The nanoparticles are a type of acid-activated nanoparticles, in which PAE is protonated and converted into a hydrophilic structure in an acidic environment, and its charge state is converted from negative to positive. This charge conversion causes the nanoparticles composed of PAE-PEG to remove the polymer shell on the surface due to charge repulsion in an acidic environment, exposing the positively charged core, thereby promoting the cellular uptake of the nanoparticles and inducing the release of drugs.

[0028] Modified acid-activated MeTTh nanoparticles MeTTh NPs can utilize the acidic characteristics of the tumor microenvironment to improve the targeting efficiency of drugs. After the drug is activated at the tumor site, the positively charged nanoparticles bind to the negatively charged cell membrane and biomacromolecules in the cell, reducing spillover. As the uptake of nanoparticles by cancer cells increases, the total amount of enrichment increases and the therapeutic effect is enhanced. In addition, since the acid-activated nanoparticles specifically release drugs in the tumor microenvironment, they can reduce the impact on normal tissues and reduce side effects.

[0029] Acid-activated nanoparticles also have high drug encapsulation efficiency and adjustable drug loading ratio, which can improve the drug delivery efficiency.

[0030] Specifically, in step S1, the mass ratio of TTh derivative to PAE-PEG is (0.1-0.3):1; the mass volume ratio of TTh derivative or pharmaceutically acceptable salt of TTh derivative to THF is 0.8-1.3 mg / mL; and the volume ratio of ultrapure water to THF solution is (19-21):1.

[0031] Preferably, the mass volume ratio of the TTh derivative or the pharmaceutically acceptable salt of the TTh derivative to THF is 1.0 mg / mL, and the volume ratio of ultrapure water to the THF solution is 20:1.

[0032] Specifically, the molecular weight of PAE in PAE-PEG is 10 KDa, and the molecular weight of PEG is 5 KDa.

[0033] Specifically, in step S2, the stirring speed is 700-800 rpm, which lasts for 1.8-2.2 minutes; the ultrafiltration speed is 4000-4400 rpm, which lasts for 8-12 minutes.

[0034] Specifically, the buffer in step S3 can be any buffer, preferably PBS buffer with a pH of 7.0-7.4.

[0035] Specifically, the amount of buffer used in step S3 is such that the concentration of the nanoparticles therein is 50 μM.

[0036] The embodiment of the present invention also provides a luminescent sonosensitizer prepared by the above preparation method.

[0037] Specifically, the luminescent sonosensitizer exerts its effect after being induced by acid.

[0038] Specifically, the luminescent sonosensitizer is a nanoparticle.

[0039] Specifically, the particle size of the nanoparticles is 16-140 nm, preferably 70 nm.

[0040] Specifically, the acid-induced condition is pH=5.0±0.2.

[0041] 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.

[0042] The embodiment of the present invention also provides the use of the luminescent sonosensitizer as described above in the preparation of a drug, and the drug is used to treat in situ breast cancer through sonodynamic therapy.

[0043] The following is explained in conjunction with specific embodiments: The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified, and the techniques not described in detail are all performed according to standard methods well known to those skilled in the art.

[0044] Example 1 Preparation of compound MeTTh Formula (I) is a flow chart for preparing compound MeTTh. According to formula (I), compound MeTTh can be prepared.

[0045] Formula (I) In a 25 mL two-necked round-bottom flask, 190 mg, 0.5 mmol of 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carboxaldehyde (CAS No.: 654067-66-0), 64 mg, 0.4 mmol of 3-ethyl-2-thioxo-4-thiazolidinedione (CAS No.: 7648-01-3) and 125 mg, 1.5 mmol of sodium acetate (CAS No.: 127-09-3) were added. The two-necked round-bottom flask was evacuated and purged with dry nitrogen three times. Then 10 mL of glacial acetic acid was added, the temperature was raised to 90 ° C for reaction for 24 h, and then cooled to room temperature. 10 mL of water was added to 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 over 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 as a dark red solid with a yield of 86%.

[0046] Its structure was identified by H NMR spectrum, C NMR spectrum and matrix-assisted laser desorption time-of-flight mass spectrometry, as shown in Figure 1. Figure 1 (a) is its H NMR 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 carbon NMR 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 MW 526.121, measured MW 526.255.

[0047] Example 2 Preparation of PAE-PEG modified sonosensitizer nanoparticles S1: a tetrahydrofuran (THF) solution containing MeTTh (prepared in Example 1) and PAE-PEG (poly(β-amino ester)-polyethylene glycol) in a mass ratio of 0.1:1 (based on MeTTh, the mass volume concentration is 1.0 mg / mL) is added to ultrapure water, and the volume ratio of ultrapure water to THF solution is 19:1; S2: After vigorous stirring at 750 rpm for 1.8 min, ultrafiltration was performed at 4000 rpm for 12 min; S3: The nanoparticles obtained by ultrafiltration are dispersed in a PBS buffer solution with a pH of 7.4 to obtain the sonosensitizer (MeTThNPs).

[0048] Example 3 Preparation of PAE-PEG modified sonosensitizer nanoparticles S1: a tetrahydrofuran (THF) solution containing MeTTh (prepared in Example 1) and PAE-PEG (poly(β-amino ester)-polyethylene glycol) in a mass ratio of 0.3:1 (based on MeTTh, the mass volume concentration is 1.0 mg / mL) is added to ultrapure water, and the volume ratio of ultrapure water to THF solution is 21:1; S2: After vigorous stirring at 750 rpm for 2.2 minutes, ultrafiltration was performed at a rotation speed of 4400 rpm for 8 minutes; S3: The nanoparticles obtained by ultrafiltration are dispersed in a PBS buffer solution with a pH of 7.4 to obtain the sonosensitizer (MeTThNPs).

[0049] Comparative Example 1 Preparation of DSPE-PEG2000 modified sonosensitizer nanoparticles S1: A THF solution containing MeTTh (prepared in Example 1) and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoacetamide-N-amino(polyethylene glycol)-2000) in a mass ratio of 0.4:1 is added to ultrapure water, and the volume ratio of ultrapure water to THF solution is 5:1; S2: After vigorous stirring for 1.8 minutes, ultrafiltration was performed at a rotation speed of 4000 rpm for 12 minutes; S3: The nanoparticles obtained by ultrafiltration are dispersed in a PBS buffer with a pH of 7.4 to obtain control nanoparticles (MeTTh nNPs).

[0050] Example 4 Characterization of MeTTh A solution of MeTTh prepared in Example 1 with a concentration of 0.1 M was prepared using acetonitrile, and the absorption spectrum and emission spectrum of MeTTh were detected by UV-vis-NIR spectrophotometer and fluorescence spectrophotometer. Figure 2 The maximum absorption wavelength and maximum emission wavelength of MeTTh are 495 nm and 700 nm, respectively.

[0051] By changing the ratio of water to acetonitrile (based on water volume concentration), the AIE characteristic curve of MeTTh was detected, such as Figure 3 As shown. It can be seen that the maximum emission wavelength remains basically unchanged, but the emission intensity changes with the proportion of water in the mixed solvent. This is because as the proportion of water increases, the polarity of the mixed solvent gradually increases, and the increase in solvent polarity will cause small organic 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 of the groups (heat energy), and ultimately radiates the energy in the form of light. Therefore, the higher the solvent polarity, the higher the degree of molecular aggregation, and the greater the emission intensity.

[0052] Example 5 Characterization of MeTTh nNPs and MeTTh NPs 1. Absorption and emission spectra Sample solutions of MeTTh nNPs prepared in Comparative Example 1 and MeTTh NPs prepared in Example 2 were prepared according to the method in Example 4. The absorption spectra and emission spectra of MeTThnNPs prepared in Comparative Example 1 and MeTTh NPs prepared in Example 1 were detected by UV-vis-NIR spectrophotometer and fluorescence spectrophotometer. Figure 4 As shown, it can be seen that the two nanoparticles have the same emission curve, which is the same as the MeTTh compound; however, the absorption curve of the MeTTh NPs in Example 2 has a certain red-shift trend, while the absorption curve of the MeTTh nNPs in the comparative example remains unchanged.

[0053] 2. Particle size Dynamic light scattering (DLS) was used to detect the diameters of MeTTh nNPs and MeTTh NPs. Figure 5 As shown in Figure 2, the hydrodynamic size of MeTTh nNPs and MeTTh NPs was ~48 nm as measured by dynamic light scattering. After incubation in an acidic environment (PBS buffer at pH = 5.0) at room temperature and in the dark for 24 h, the particle size was measured again. Figure 6 It was detected that the size of MeTTh NPs became larger, while the size of MeTTh nNPs remained basically unchanged.

[0054] 3. Zeta potential The Zeta potential of MeTTh nNPs and MeTTh NPs was detected. Under neutral conditions (PBS solution with pH = 7.4), both were negative potentials. However, MeTTh NPs underwent charge reversal after being incubated in an acidic environment (PBS solution with pH = 5.0) at room temperature in the dark for 24 h. Figure 7 shown.

[0055] 4. Release rate and fluorescence intensity The release rate and fluorescence intensity of MeTTh nNPs and MeTTh NPs at different time points after incubation in neutral and acidic environments were detected. Figure 8 As shown in the figure, 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, which may be due to the increase in size, loose structure of nanoparticles, and decreased aggregation of molecules encapsulated therein, resulting in weakened fluorescence.

[0056] 5. ESR signal like Fig. 9 As shown, using 2,2,6,6-tetramethylpiperidine (TEMP) as a spin trap, different ESR signals indicate the type I and type II nature of ROS. When ultrasound irradiation is added, the presence of three free radicals can be detected; when ultrasound irradiation is removed, the free radical signal disappears.

[0057] The ROS production of MeTTh nNPs and MeTTh NPs under ultrasound incubation for 10 min in neutral and acidic environments was further tested. Fig.10As shown in the figure, I-I0 represents the difference in fluorescence intensity between each group at different time points and the initial state (0 min). It can be seen that the fluorescence intensity of MeTTh NPs increased rapidly after incubation in an acidic environment, which was higher than that of the other groups. This result well demonstrates the potential of MeTTh NPs to produce ROS in an acidic environment.

[0058] Example 6 Cell Sonodynamic Therapy (1) Cells and cell culture Mouse breast cancer cell line (4T1 cells) purchased from the American Type Culture Collection (ATCC) was selected. The cells were added with 10% (v / v) fetal bovine serum (Hyclone), 100 UI mL -1 Penicillin and 100 UI mL -1 The cells were cultured in RPMI-1640 medium containing streptomycin at 37°C in an incubator containing 5% carbon dioxide until the cell density reached 70%-80% of the culture flask, thereby obtaining pre-cultured 4T1 cells.

[0059] (2) Cellular uptake The 4T1 cells pre-cultured in (1) were cultured at 5×10 4 The cells were seeded into a 24-well plate at a density of 10 cells / well and cultured for 12 h as described in (1) to allow them to attach. MeTTh NPs were added to 12 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 three times with PBS (pH 7.4), and the uptake efficiency of each group was evaluated by flow cytometry (CytoFLEX, Beckman) (excitation at 488 nm and emission collected with a 690 / 50 nm bandpass filter). Fig.11 As shown. It can be seen that compared with ordinary nanoparticles, the cell uptake efficiency of acid-activated nanoparticles is much higher. Acid-activated nanoparticles achieved 99.5% uptake effect in one hour. Ordinary nanoparticles need to achieve 99.2% uptake effect in 12 hours.

[0060] (3) Cellular reactive oxygen species detection The cells were 5×10 4The cells were seeded on the confocal dish at a density of 10 cells / well and cultured for 12 h as described in (1). When the 4T1 cells grew to about 80%, 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 the same concentration of nanoparticles (10 uM) for 4 h, and 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).

[0061] like Fig.12a As shown, with a duty cycle of 50%, the power density is 1.5W / cm 2 After 10 minutes of ultrasound irradiation at a frequency of 1 MHz (i.e., the experimental group), an obvious green fluorescence signal was observed in the 4T1 cells of the MeTTh NPs + US group, indicating that MeTThNPs effectively produced ROS under ultrasound irradiation. In contrast, no green fluorescence signal was observed in the control group and the material group (without ultrasound irradiation). Figure 12b , c is the distribution of fluorescence intensity inside cells after different treatments of cells reacted by flow cytometry, where Figure 12b is the original data graph, Fig.12c It is a bar graph after quantification. As shown in the figure, after ultrasound irradiation, the average fluorescence intensity of the experimental group was significantly enhanced. This result well demonstrates the ability of MeTTh NPs to generate ROS under ultrasound irradiation and its potential for cancer treatment.

[0062] (4) The killing effect of sonodynamic therapy on cancer cells In this example, the killing effect on 4T1 cells was investigated through live / dead cell staining experiments and CCK-8 experiments.

[0063] Live / dead cell staining experiment: 4T1 cells were cultured at 5×10 4 The cells were inoculated at a density of 10 cells / well on the confocal dish and cultured for 12 h. 300 µL of 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 treatment group. The control group (Blank) directly replaced the waste liquid with 300 µL of culture medium. After incubation for 4 hours, ultrasonication was performed for 10 minutes and the incubation continued. The waste liquid was then 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 using a confocal microscope. The results are shown in Fig.13a Only the MeTTh NPs group could kill cells under ultrasound irradiation, while the other groups had almost no cytotoxic effect.

[0064] CCK-8 assay: 4T1 cells were cultured at 5×10 3 The cells were seeded at a density of 10 cells / well on a 96-well plate and cultured for 12 h. The waste liquid was replaced with culture medium containing different concentrations of MeTTh nNPs and MeTTh NPs. After incubation for 4 hours, the cells were ultrasonically irradiated for 5 minutes and incubated for another 24 hours. Then 10 µL of CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole 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 (%) = (absolute mean value of the treatment group / absolute mean value of the control group) × 100%. The results are shown in Fig.13b As shown in Figure 3, with the increase in the concentration of MeTTh nNPs and MeTTh NPs, the cell survival rate decreased. In the MeTTh NPs+US treatment group, the cell survival rate decreased most significantly, which was similar to the results of the live / dead cell staining experiment.

[0065] (5) Cytotoxicity test 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 Beina Biotechnology were cultured at 5 × 10 cells per well. 3 Cells were seeded in a 96-well plate (100 µL) and cultured for 12 h. The original culture medium was replaced with 100 µL of culture medium 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-disulfonylbenzene)-2H-tetrazole monosodium salt) solution was added to each well to determine the OD value, which indirectly reflects the number of living 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 (%) = (absolute mean value of the treatment group / absolute mean value of the control group) × 100%. Fig.14 As shown, Fig.14 a is the result of cell compatibility of MeTTh nNPs. Fig.14 b is the cell compatibility result diagram of MeTTh NPs. It can be seen that the killing ability of MeTTh nNPs on various tumor cells is very limited regardless of the concentration, while the killing ability of MeTTh NPs in the experimental group on tumor cells increases with the increase of its concentration.

[0066] Example 7 In vivo sonodynamic therapy (1) In vivo fluorescence imaging distribution study of MeTTh nNPs and MeTTh NPs MeTTh nNPs and MeTTh NPs materials (100 µL, 10 mg / kg) were injected through the tail vein. The fluorescence distribution of MeTThnNPs and MeTTh NPs in mice was observed using a small animal fluorescence imaging system (IVIS Spectrum, Perkelmer, US) at 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h, as shown in Figure 15. The material was enriched in the tumor of mice in the MeTTh nNPs group at 24 h, while the fluorescence of mice in the MeTTh NPs group was the strongest at 12 h and decreased at 24 h. Since it was not certain whether the fluorescence at 24 h was weakened due to the decomposition of nanoparticles under acidic conditions or due to the clearance of nanoparticles by mice, two time points of 12 h and 24 h were set for treatment during sonodynamic therapy.

[0067] (2) Intracorporeal sonodynamic therapy Female Balb / c mice were randomly divided into 5 groups, with 5 mice in each group. 4T1 cells (5×10 5 The tumor volume reached about 80-100mm 3 When the mice were primed for different treatments: Blank group I: normal saline was injected into the tail vein; ultrasound group II: normal saline was injected into the tail vein and ultrasound was irradiated for 10 min; MeTTh NPs group III: MeTTh NPs were injected into the tail vein; MeTTh NPs + US – 12 h group IV: MeTTh NPs were injected into the tail vein and ultrasound was irradiated for 10 min 12 h later; MeTTh NPs + US – 24 h group V: MeTTh NPs were injected into the tail vein and ultrasound was irradiated for 10 min 24 h later.

[0068] The body weight of mice was recorded every 2 days. Fig.16 The length and width of the tumor were measured every 2 days. Fig.17 As shown. It can be seen that the tumor volume 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 group. Specifically, there was a significant difference between Group IV (MeTTh NPs + US - 12 h) and the three control groups (p < 0.05), and there was a more significant difference between Group V (MeTTh NPs + US - 24h) and the three control groups (p < 0.01).

[0069] After 14 days, the tumors of mice in each group were removed, weighed and photographed. Fig.18 , 19 As shown. The tumor mass of the control groups (PBS group, ultrasound group, and MeTTh NPs group) increased dramatically during the treatment, and there was no significant difference among the three groups, while there was a significant difference between the experimental groups and the control group. Specifically, there was a significant difference between group IV (MeTTh NPs + US - 12 h) and the three control groups (p<0.05), while there was a more significant difference between group V (MeTTh NPs + US - 24 h) and the three control groups (p<0.01). Therefore, it can be concluded that after ultrasound treatment, MeTTh NPs showed a significant inhibitory effect on tumor growth. In addition, tumor weight also indicated that MeTTh NPs-mediated sonodynamic therapy had a strong therapeutic effect on tumors in tumor-bearing mice.

[0070] 24 h after ultrasound treatment, the tumor tissues of the mice were collected for hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), anti-Ki67 monoclonal antibody immunofluorescence staining (Ki67 staining), and platelet endothelial cell adhesion molecule immunofluorescence staining (CD31 staining). Fig. 20 shown.

[0071] The results of hematoxylin-eosin (HE) staining of tumor sections in each group showed that a large number of nuclear loss and obvious nuclear condensation were only observed in the tumor sections treated with MeTTh NPs + US, indicating that SDT successfully destroyed the tumor tissue.

[0072] In addition, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was performed on tumor tissues to further verify the MeTTh NPs-mediated sonodynamic-induced apoptosis of tumor tissue cells. A large number of TUNEL-positive red fluorescence signals were observed in ultrasound-treated tumor tissues, while no red fluorescence signals were detected in the control group, indicating that ultrasound induced severe cell apoptosis.

[0073] 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 their mechanism of action. The number of green-positive proliferating cells in the MeTTh NPs + US group was greatly reduced. In sharp contrast, densely arranged 4T1 cells with strong proliferation activity were seen in the tumor tissues of the control group. These results strongly demonstrate that ultrasound-activated aggregation-induced luminescence sonosensitizers can effectively inhibit tumor growth by inducing tumor cell apoptosis and inhibiting tumor cell proliferation.

[0074] In addition, we evaluated whether MeTTh NPs would cause damage to major organs. At the end of the treatment, we injected pentobarbital into the mice at a dose of 100 mg / kg and euthanized the mice. The heart, liver, spleen, lung, and kidney organs of the mice were sliced ​​and stained with H&E, and then observed under a bright field microscope. Fig.21 The results showed that there was no significant difference between the mice in the MeTTh NPs injection group and the PBS control group, indicating that MeTTh NPs did not cause lesions in major organs and that MeTTh NPs did not have acute toxicity.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an acid-activated aggregation-induced luminescence sonosensitizer, comprising: S1: Take a TTh derivative or a pharmaceutically acceptable salt of the TTh derivative and a tetrahydrofuran solution of PAE-PEG and add them to ultrapure water to obtain a first solution. The chemical formula of the TTh derivative is: , wherein R1 and R2 are the same or different, R1 and R2 are phenyl, 4-methylphenyl, tetraphenylvinyl, 4-tert-butylbenzene or 4-methoxybenzene, and R3 is hydrogen; S2: stirring the first solution, and then ultrafiltering to obtain nanoparticles; S3: Dispersing the nanoparticles in a buffer solution to obtain a luminescent sonosensitizer.

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

1.

3. The method for preparing the luminescent sonosensitizer according to claim 1, characterized in that: 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.

4. The method for preparing the luminescent sonosensitizer according to claim 1, characterized in that: The buffer in step S3 is PBS buffer with a pH of 7.0-7.

4.

5. A luminescent sonosensitizer prepared by the method for preparing a luminescent sonosensitizer according to any one of claims 1 to 4.

6. The luminescent sonosensitizer according to claim 5, characterized in that The luminescent sonosensitizer takes effect after being induced by acid.

7. The luminescent sonosensitizer according to claim 6, characterized in that The acid-induced condition is pH=5.0±0.

2.

8. The luminescent sonosensitizer according to claim 5, characterized in that The luminescent sonosensitizer is a nanoparticle.

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

10. The use according to claim 9, characterized in that The drug for treating in situ breast cancer is a sonodynamic therapy drug.

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