CN118542940A - AIE photodynamic nanoparticle targeting lysosomes and its preparation method and application - Google Patents

Abstract

The invention discloses a lysosome-targeted AIE photodynamic nanoparticle and a preparation method and application thereof, wherein the preparation method comprises the following steps: s1, mixing AIE photosensitive material A1 and F127 polyether in tetrahydrofuran to obtain a mixed solution, drying to obtain a glue layer, and adding an acidic aqueous solution with the concentration of 0.77-0.93M into the glue layer to form F127 micelles coated with AIE photosensitive material A1; s2, adding tetraethoxysilane, stirring for 0.5-3 hours, and taking the F127 micelle as a template and tetraethoxysilane as a silicon source to generate silicon dioxide nano particles coated with AIE photosensitive material A1; s3, dropwise adding aminopropyl triethoxysilane, stirring for 22-26 hours, filtering, ultrafiltering and centrifuging for 9-11 minutes to obtain silica nanoparticles with amino groups on the surfaces; s4, adding 3- (4-morpholinyl) propyl isothiocyanate, stirring for 22-26 hours, and obtaining the AIE photodynamic nanoparticle through ultrafiltration and centrifugation. The preparation method provided by the invention is simple and low in cost, and the AIE photodynamic nanoparticle A1@SiO ₂ -MP has good stability and biocompatibility, can be better enriched in a cell lysosome, can generate active oxygen to perform oxidative damage on the lysosome after excitation, thereby inducing apoptosis and having good photodynamic therapy application potential.

Description

AIE photodynamic nanoparticle targeting lysosomes and its preparation method and application

Technical Field

The present invention relates to the field of biomedicine, and in particular to an AIE photodynamic nanoparticle targeting lysosomes, and a preparation method and application thereof.

Background Art

Tumors are diseases in which autologous cells abnormally proliferate and form lumps in local parts of the body. They are highly harmful and difficult to treat, and pose a serious threat to the physical and mental health of patients and the happiness and stability of their families and society. Photodynamic therapy is an emerging technology that uses the photodynamic effect to treat tumors. It mainly uses excitation light of a specific wavelength to induce photosensitizers located in the tumor site to produce reactive oxygen species (ROS), causing oxidative damage to the tumor site and inducing tumor cell apoptosis. Compared with traditional tumor chemotherapy strategies, photodynamic therapy can only irradiate the tumor site, with the advantages of precise treatment and fewer toxic side effects.

Photosensitive materials that generate reactive oxygen species when stimulated play a vital role in photodynamic therapy, and their development has always attracted much attention. At present, traditional small-molecule photosensitive materials have been used in clinical photodynamic therapy. However, their hydrophobicity and large conjugation make them easy to form aggregates in an aqueous environment, and then aggregation leads to the luminescence quenching effect (ACQ), which reduces the generation of ROS, and the therapeutic effect is often restricted. On the other hand, aggregation-induced emission (AIE) is an emerging fluorescence generation technology in recent years. The characteristic is that materials with AIE characteristics emit weak light at low concentrations or in a dispersed state, and significantly enhance the light emission at high concentrations or in a solid aggregate state. Current research on the AIE phenomenon believes that the unique propeller structure of AIE molecules in the aggregated state restricts the intramolecular motion, which can greatly avoid non-radiative decay, thereby having enhanced fluorescence and ROS generation performance compared with traditional luminescent materials limited by the ACQ effect.

In the field of photodynamic therapy, AIE photosensitive materials are often encapsulated with amphiphilic materials such as polyether F127 and phosphoethanolamine phospholipids to form water-soluble AIE photodynamic nanoparticles, which have the advantages of high ROS generation efficiency and strong resistance to photobleaching. However, the above-mentioned micellar nanoparticles are prone to dissociation when the concentration in the biological environment is lower than the critical micelle concentration, resulting in the leakage of AIE photosensitive materials. On the other hand, the current AIE photodynamic nanoparticles generally do not have subcellular structure targeting. After entering the cell, they are mainly distributed in the cytoplasm. The reactive oxygen species generated by the stimulation are easily removed in large quantities by the cell's reactive oxygen scavenging mechanism (such as superoxide dismutase SOD, catalase CAT, ascorbic acid Vc, etc.), thereby reducing the photodynamic killing effect on tumor cells. If the photodynamic killing effect is simply improved by increasing the amount of nanoparticles, toxic side effects may occur. Therefore, how to improve the stability and tumor killing efficiency of photodynamic nanoparticles is an important issue facing the field of photodynamic therapy.

Summary of the invention

The present invention designs and synthesizes an AIE photodynamic nanoparticle targeting lysosomes, wherein the AIE photosensitive material core is wrapped by a silicon dioxide layer and has good stability and biocompatibility; the surface of the nanoparticle is modified with a morpholine group that can target lysosomes, so that it can be better enriched in the lysosomes, and when stimulated, it can produce reactive oxygen to cause oxidative damage to the lysosomes, thereby inducing tumor cell apoptosis, and has a good photodynamic therapy effect.

The method for preparing the AIE photodynamic nanoparticle A1@SiO ₂ -MP that can target lysosomes provided by the present invention comprises the following steps:

S1. AIE photosensitive material A1 and polyether F127 are mixed in tetrahydrofuran to obtain a mixed solution, wherein the concentration of the AIE photosensitive material A1 in the mixed solution is 0.30-0.36 mM, and the concentration of the polyether F127 is 60-72 mg/mL, and then dried to obtain a glue layer, and an acidic aqueous solution with a concentration of 0.77-0.93 M is added to the glue layer, and the volume ratio of the acidic aqueous solution to the mixed solution is (0.9-1.1): 1, to form F127 micelles with a hydrophobic core encapsulating the AIE photosensitive material A1;

S2. adding tetraethoxysilane, wherein the volume ratio of tetraethoxysilane to the mixed solution is (0.9-1.1): 20, stirring for 0.5-3 hours, using the hydrolyzed tetraethoxysilane as a silicon source and the polyether F127 micelle as a template to generate silica nanoparticles encapsulated with the AIE photosensitive material A1;

S3. aminopropyl triethoxysilane is added dropwise, wherein the volume ratio of the aminopropyl triethoxysilane to the tetraethoxysilane is (0.9-1.1): 1, stirred for 22-26 hours, filtered, and ultrafiltered by centrifugation for 9-11 minutes to obtain silica nanoparticles A1@SiO ₂ -NH ₂ with amino groups on the surface;

S4. dissolving the nanoparticles A1@SiO ₂ -NH ₂ with amino groups on the surface in ultrapure water, adding 3-(4-morpholinyl)propyl isothiocyanate, wherein the volume ratio of the 3-(4-morpholinyl)propyl isothiocyanate to the aminopropyltriethoxysilane is (0.9-1.1):3, stirring for 22-26 hours, and obtaining the AIE photodynamic nanoparticles A1@SiO ₂ -MP by ultrafiltration and centrifugation;

Wherein, the structural formula of the AIE photosensitive material A1 is: .

Furthermore, the gas used for drying in step S1 is nitrogen.

Furthermore, the stirring time in step S2 is 2 hours.

Furthermore, the filtering process in step S3 is filtering using filter paper and 0.22µm syringe filter membrane in sequence.

Furthermore, the ultrafiltration centrifugation conditions in steps S3 and S4 are both 100 kDa, 5000 rcf for 10 minutes.

Furthermore, the preparation method of the AIE photosensitive material A1 comprises the following steps:

(1) dissolving compound A (4-bromo-4',4'-dimethoxytriphenylamine), 5-formyl-2-thiopheneboronic acid and potassium carbonate in a molar ratio of (1.8-2.2):(2.7-3.3):10 in a mixed solution of toluene and methanol to obtain a first mixed solution, wherein the volume ratio of toluene to methanol is (0.9-1.1):1, evacuating the solution and replacing with nitrogen;

(2) dissolving the catalyst [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride in toluene, wherein the molar concentration of the catalyst after dissolution is 0.45-0.55 mol/L, and adding the dissolved catalyst to the first mixed solution;

(3) stirring at 68-83° C. for 16-20 hours, cooling to room temperature, and removing the solvent using a rotary evaporator to obtain a second mixed solution;

(4) Purifying the second mixed liquid by silica gel column chromatography with dichloromethane as the eluent to obtain an intermediate product compound B, the structural formula of the compound B being: ;

(5) dissolving compound B and malononitrile in a molar ratio of (0.9-1.1):2 in dichloromethane, and adding triethylamine dropwise, wherein the volume ratio of the triethylamine to the toluene in step (2) is (0.9-1.1):2;

(6) stirring at 27-33° C. for 16-20 hours, cooling to room temperature, and removing the solvent by rotary evaporation to obtain a mixture;

(7) Purifying the mixture by silica gel column chromatography using n-hexane and ethyl acetate in a volume ratio of (4.5-5.5):1 as eluent to obtain the AIE photosensitive material A1.

The present invention also provides an AIE photodynamic nanoparticle that can target lysosomes, which is prepared using the above-mentioned preparation method.

The present invention also provides the use of the above-mentioned AIE photodynamic nanoparticles in tumor treatment.

Compared with the prior art, the present invention has the following beneficial effects:

1. The preparation method of AIE photodynamic nanoparticles provided by the present invention uses silica materials with high mechanical strength and biochemical inertness, which can effectively encapsulate AIE materials to avoid leakage and loss and enhance biocompatibility. The obtained AIE photodynamic nanoparticles A1@SiO ₂ -MP have no obvious cell and biological toxicity in the unexcited state, can stay in the cells for more than 6 hours, and are then normally excreted by the cells, and have no obvious effect on the morphology and survival rate of the cells during this period;

2. The present invention provides a method for preparing AIE photodynamic nanoparticles targeting lysosomes. The obtained nanoparticles A1@SiO ₂ -MP have weakly alkaline morpholine groups on their surfaces, so that the nanoparticles can specifically target and enrich lysosomes after entering cells. Under the irradiation of excitation light, the nanoparticles located in the lysosomes produce reactive oxygen species, causing oxidative damage to the lysosomes and inducing cell apoptosis. Through in vitro cell and tumor-bearing mouse photodynamic therapy experiments, it was confirmed that the AIE photodynamic nanoparticles A1@SiO ₂ -MP have good photodynamic therapy effects;

3. The nanoparticle preparation method provided by the present invention has the advantages of low cost and simple preparation. Based on the inspiration provided by the present invention, different fluorescent materials or subcellular structure targeting groups can be introduced to prepare a variety of functional nanoparticles for fluorescent labeling of subcellular structures or tumor photodynamic therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is a chemical synthesis route of AIE photosensitive material A1;

Figure 2 shows the preparation route of nanoparticles A1@SiO ₂ -MP;

FIG3 shows the particle size distribution and TEM morphology of the nanoparticles A1@SiO ₂ -MP in Example 3;

FIG4 shows the fluorescence characteristics of the nanoparticles A1@SiO ₂ -MP in Example 3;

FIG5 is a comparison of the photodynamic generation of active oxygen by the nanoparticles A1@SiO ₂ -MP in Example 4 and the commercial photosensitive material Ce6;

FIG6 is a cytotoxicity experiment of nanoparticles A1@SiO ₂ -MP in Example 5;

FIG. 7 is a hemolysis experiment of nanoparticles A1@SiO ₂ -MP in Example 5;

FIG8 is a cell uptake-efflux experiment of nanoparticles A1@SiO ₂ -MP in Example 6;

FIG9 shows the fluorescence intensity changes of the nanoparticles A1@SiO ₂ -MP in Example 7 mixed in an acidic solution of pH=5.0 at different mixing times;

FIG. 10 shows the particle size changes of the nanoparticles A1@SiO ₂ -MP in Example 7 after mixing in an acidic solution of pH=5.0 for different time periods;

FIG. 11 is a co-localization experiment of the nanoparticles A1@SiO ₂ -MP and the commercial lysosomal probe LTG in Example 8;

FIG. 12 is a diagram showing the use of active oxygen probe DCFH to test the active oxygen generated by nanoparticles A1@SiO ₂ -MP in lysosomes in Example 9, and a co-localization test;

FIG. 13 shows cells containing the active oxygen probe DCFH treated with nanoparticles A1@SiO ₂ -MP at different concentrations in Example 10, and then treated in the dark or in the light, and then flow cytometry was performed to detect the generation of active oxygen;

FIG. 14 shows cells treated with nanoparticles A1@SiO ₂ -MP at different concentrations in Example 11, treated in the dark or in the light, and stained for cell death and viability using Calcein-AM/PI;

FIG. 15 shows cells treated with nanoparticles A1@SiO ₂ -MP at different concentrations in Example 11, and then treated in the dark or in the light, and CCK8 cell activity was detected;

FIG. 16 shows the cells treated with nanoparticles A1@SiO ₂ -MP and illuminated for different time periods in Example 12, and the cell apoptosis was examined by Annexin V-FITC/PI staining and flow cytometry experiments were performed;

FIG. 17 shows the weight changes of tumor-bearing mice during 15 days of photodynamic therapy using PBS or nanoparticles A1@SiO ₂ -MP injected intratumorally in Example 13;

FIG. 18 shows the changes in tumor volume during 15 days of photodynamic therapy using PBS or nanoparticles A1@SiO ₂ -MP injected intratumorally into tumor-bearing mice in Example 13;

FIG. 19 shows that PBS or nanoparticles A1@SiO ₂ -MP were injected intratumorally into tumor-bearing mice in Example 13, and the mice were killed and the tumors were removed on the 15th day after photodynamic therapy to confirm the therapeutic effect;

FIG. 20 is a diagram showing the results of TUNEL staining analysis of mouse tumor tissue sections in Example 13.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, 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 intended to limit the present invention. The present invention is specifically introduced below in conjunction with the specific embodiments.

The present invention provides a method for preparing AIE photodynamic nanoparticles that can target lysosomes, comprising the following steps:

S1. AIE photosensitive material A1 and F127 polyether are mixed in tetrahydrofuran to obtain a mixed solution, wherein the concentration of the AIE photosensitive material A1 in the mixed solution is 0.30-0.36 mM, and the concentration of the F127 polyether is 60-72 mg/mL, and then dried to obtain a glue layer, and an acidic aqueous solution with a concentration of 0.77-0.93 M is added to the glue layer, and the volume ratio of the acidic aqueous solution to the mixed solution is (0.9-1.1): 1, to form F127 micelles with a hydrophobic core encapsulating the AIE photosensitive material A1;

S2. adding tetraethoxysilane, wherein the volume ratio of the tetraethoxysilane to the mixed solution is (0.9-1.1): 20, stirring for 0.5-3 hours, the tetraethoxysilane is hydrolyzed and a silica layer is formed on the outer edge of the core of the F127 micelles encapsulating the AIE photosensitive material A1, thereby generating silica nanoparticles encapsulating the AIE photosensitive material A1;

S3. aminopropyl triethoxysilane is added dropwise, wherein the volume ratio of the aminopropyl triethoxysilane to the tetraethoxysilane is (0.9-1.1): 1, stirred for 22-26 hours, filtered, and ultrafiltered by centrifugation for 9-11 minutes to obtain silica nanoparticles A1@SiO ₂ -NH ₂ with amino groups on the surface;

S4. dissolving the nanoparticles A1@SiO ₂ -NH ₂ with amino groups on the surface in ultrapure water, adding 3-(4-morpholinyl)propyl isothiocyanate, wherein the volume ratio of the 3-(4-morpholinyl)propyl isothiocyanate to the aminopropyltriethoxysilane is (0.9-1.1):3, stirring for 22-26 hours, and obtaining the AIE photodynamic nanoparticles A1@SiO ₂ -MP by ultrafiltration and centrifugation;

The structural formula of the AIE photosensitive material A1 is: .

Specifically, the chemical synthesis route of the AIE photodynamic nanoparticles is shown in FIG2 .

Specifically, the gas used for drying in step S1 is nitrogen.

Specifically, the acidic aqueous solution used in step S1 is hydrochloric acid.

Preferably, the stirring time in step S2 is 2 hours.

Specifically, the filtering process in step S3 is filtering using filter paper and 0.22µm syringe filter membrane in sequence.

Preferably, the ultrafiltration centrifugation conditions in steps S3 and S4 are both to use an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 kDa and centrifuge at 5000 rcf for 10 minutes.

Specifically, the chemical synthesis route of the AIE photosensitive material A1 is as follows:

Specifically, the preparation method of the AIE photosensitive material A1 comprises the following steps:

(1) dissolving compound A (4-bromo-4',4'-dimethoxytriphenylamine), 5-formyl-2-thiopheneboronic acid and potassium carbonate in a molar ratio of (1.8-2.2):(2.7-3.3):10 in a mixed solution of toluene and methanol to obtain a first mixed solution, wherein the volume ratio of toluene to methanol is (0.9-1.1):1, evacuating the solution and replacing with nitrogen;

(2) dissolving the catalyst [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride in toluene, wherein the molar concentration of the catalyst after dissolution is 0.45-0.55 mol/L, and adding the dissolved catalyst to the first mixed solution;

(3) stirring at 68-83° C. for 16-20 hours, cooling to room temperature, and removing the solvent using a rotary evaporator to obtain a second mixed solution;

(4) Purifying the second mixed liquid by silica gel column chromatography with dichloromethane as the eluent to obtain an intermediate product, compound B, wherein the structural formula of compound B is:

;

(5) dissolving compound B and malononitrile in a molar ratio of (0.9-1.1):2 in dichloromethane, and adding triethylamine dropwise, wherein the volume ratio of the triethylamine to the toluene in step (2) is (0.9-1.1):2;

(6) stirring at 27-33° C. for 16-20 hours, cooling to room temperature, and removing the solvent by rotary evaporation to obtain a mixture;

(7) Purifying the mixture by silica gel column chromatography using n-hexane and ethyl acetate in a volume ratio of (4.5-5.5):1 as eluent to obtain the AIE photosensitive material A1.

The present invention also provides an AIE photodynamic nanoparticle that can target lysosomes, which is prepared using the above-mentioned preparation method. The structure of the AIE photodynamic nanoparticle can be expressed as follows:

.

The embodiments of the present invention also provide the use of the AIE photodynamic nanoparticles as described above in photodynamic therapy of tumors.

The following is described in conjunction with specific embodiments:

Example 1: Preparation and characterization of AIE photosensitive material A1

The chemical structure and preparation method of AIE photosensitive material A1 are shown in FIG1 . Specifically, compound A (4-bromo-4',4'-dimethoxytriphenylamine, 1920 mg, 5 mmol), 5-formyl-2-thiopheneboronic acid (2340 mg, 7.5 mmol) and potassium carbonate (3450 mg, 25 mmol) are dissolved in a mixture of toluene and methanol (1:1, v/v). The mixture is transferred to a three-necked flask, evacuated and replaced with nitrogen. The catalyst [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (360 mg, 0.5 mmol) is dissolved in 1 mL toluene and injected into the three-necked flask via a syringe. The mixture is stirred at 75°C for 18 hours, cooled to room temperature, and the solvent is removed as much as possible using a rotary evaporator. The resulting mixture is purified by silica gel column chromatography, with dichloromethane as the eluent, to obtain the intermediate product compound B as an orange-red solid. Compound B (415 mg, 0.5 mmol) and malononitrile (60 mg, 1 mmol) were dissolved in 5 mL of dichloromethane, and 0.5 mL of triethylamine was added dropwise. The mixture was stirred at 27-33°C for 18 hours, and then the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography using n-hexane:ethyl acetate (5:1, v/v) as the eluent to obtain compound A1 as a black solid. A sample of the obtained compound A1 was dissolved in a deuterated reagent and characterized by NMR (hydrogen spectrum and carbon spectrum) using a Bruker AVANCE Ⅲ 500M superconducting NMR spectrometer: ¹ H NMR (500MHz, Chloroform-d) δ 7.72 (s, 1H), 7.66 (d, 1H), 7.49 (d, 2H), 7.27 (d, 1H),7.08 (d, 4H), 6.89 (d, 6H), 3.83 (s, 6H). ¹³ C NMR (126 MHz, DMSO-d6) δ 157.10, 156.81 , 152.71 , 150.80 , 143.44 , 139.24 , 132.87 , 128.16 , 124.11 ,122.87 , 118.15 , 115.63 , 114.72 , 72.91 , 55.76 . Mass spectrometry was performed using a Waters Xevo G2-XS Qtof time-of-flight tandem high-resolution mass spectrometer: ESI-MS calcd for C ₂₈ H ₂₁ N ₃ O ₂ S, [M + H] ⁺ 464.1432, found 464.1498.

Example 2: Preparation of Nanoparticles A1@SiO ₂ -MP

The preparation method of nanoparticles A1@SiO ₂ -MP is shown in Figure 2. Specifically, 1 mL of THF solution (1 mM) of AIE photosensitive material A1 is prepared, and it is fully mixed with 2 mL of THF solution containing F127 (100 mg/mL). After stirring for 30 minutes, it is gently blown dry with nitrogen to obtain a glue layer. 3 mL of hydrochloric acid solution (0.85 M) is added to the glue layer, and ultrasonication is performed for 5 minutes to obtain F127 micelles encapsulating the AIE photosensitive material A1. 0.15 mL of tetraethoxysilane (TEOS) is added dropwise during stirring and stirred for 2 hours to generate silica nanoparticles encapsulating A1. 0.15 mL of aminopropyltriethoxysilane (APTES) is added dropwise and stirred for 24 hours. The resulting solution is filtered using filter paper and 0.22 μm syringe filter membrane in turn, and centrifuged using an ultrafiltration centrifuge tube (100 kDa, 5000 rcf) for 10 minutes to obtain nanoparticles A1@SiO ₂ -NH ₂ with amino groups on the surface. The obtained A1@SiO ₂ -NH ₂ was dissolved in 4 mL of ultrapure water, and 50 μL of 3-(4-morpholino)propyl isothiocyanate (MP-NCS) was added and stirred for 24 h. The nanoparticles A1@SiO ₂ -MP were obtained by ultrafiltration centrifugation (100 kDa, 5000 rcf, 10 min).

Keeping the preparation conditions unchanged, only changing the stirring time after adding 0.15 mL of ethoxysilane (TEOS) can adjust the particle size of the nanoparticles A1@SiO ₂ -MP. Through single factor change analysis, the average particle size of the obtained nanoparticles was 22±6.7 nm, 54±8.1 nm, 91±8.4 nm, 103±2.7 nm, 126±4.3 nm, and 165±5.7 nm when the stirring time was 0.5, 1, 1.5, 2, 2.5, and 3 h, respectively. Nanoparticles that just reached the submicron level (103±2.7 nm) were selected for further study.

Example 3: Characterization of Nanoparticles A1@SiO ₂ -MP

The THF solution containing the AIE photosensitive material A1 was diluted with deionized water to obtain aqueous solutions with concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 mM. The absorbance at 512 nm was detected by a UV-visible spectrophotometer, and a concentration-absorbance standard curve was drawn. The concentration of the nanoparticle A1@SiO ₂ -MP was calculated by substituting its absorbance at 512 nm into the concentration-absorbance standard curve. As shown in Figure 3, A1@SiO ₂ -MP solution (0.1 mM) was prepared and the average particle size of the nanoparticles was measured to be 103±2.7 nm using a nanoparticle tracking analyzer; the morphology of the nanoparticles was observed using a transmission electron microscope, and a clear AIE core and a silica outer layer structure were observed; the Zeta potential was measured to be 10.13 mV using a nanoparticle size potentiometer. As shown in Figure 4, 1 mL of nanoparticle A1@SiO ₂ -MP solution (0.1 mM) was taken to plot the absorption curve using a UV-visible spectrophotometer, and the absorption peak was measured to be 512 nm; the emission curve was plotted using a fluorescence spectrophotometer, and the emission peak was measured to be 675 nm. It was calculated that the nanoparticle A1@SiO ₂ -MP has a large Stokes shift (163 nm), which is helpful in avoiding the background signal from the excitation light during imaging, and has great potential in biological applications.

Example 4: Detection of the reactive oxygen generation capability of nanoparticles A1@SiO ₂ -MP

Prepare the reactive oxygen indicator DCFH-DA solution (1 mM, 0.5 mL), then add NaOH solution (10 mM, 2 mL) to activate DCFH-DA into a probe DCFH that can directly detect reactive oxygen. Use pre-cooled PBS to dilute and obtain DCFH solution (40 μM). Take DCFH solution (40 μM), A1@SiO ₂ -MP solution (2 μM) and commercial photosensitizer Ce6 solution (2 μM) to make samples, transfer them to quartz cuvettes, and then irradiate with white light (120 mW/cm ² ). Place the cuvette in a fluorescence spectrophotometer at 0, 2, 4, 6, 8, and 10 minutes, use 488 nm as the excitation light, and detect the fluorescence intensity of the oxidation product DCF at 525 nm. The results are shown in Figure 5. When there is no photosensitizer in the solution, continuous illumination fails to cause the reactive oxygen probe DCFH to oxidize to DCF and emit a fluorescent signal. When the solution contains A1@SiO ₂ -MP or photosensitizer Ce6, continuous illumination also does not produce fluorescence at 525 nm. When the solution contains both photosensitizer and reactive oxygen probe DCFH, illumination causes the photosensitizer to produce reactive oxygen, causing the reactive oxygen probe DCFH to be oxidized to the fluorescent substance DCF with a fluorescence emission peak at 525 nm. Based on the positive correlation between the fluorescence intensity of DCF and the reactive oxygen concentration, it can be seen that the nanoparticles A1@SiO ₂ -MP exhibit a stronger reactive oxygen generation ability than the commercial photosensitizer Ce6. At the 10-minute time point, the reactive oxygen generated by the former is 7.5 times that of the latter.

Example 5: Cytotoxicity study of nanoparticles A1@SiO ₂ -MP

HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37°C and 5% _CO2 . Two days before the experiment, trypsin was used for digestion and the cells were transferred from the culture flask to a 96-well plate. During the experiment, DMEM medium containing different concentrations of A1@ _SiO2 -MP (0, 0.5, 1, 2, 5, 10, 20, 50, 100 μM) was added, and 4 replicates were set for each concentration, and the cells were cultured for 24 hours. The cells were washed with PBS, and 100 μL of serum-free medium containing 10% CCK8 was added to each well. After 1 hour of culture, the 96-well plate was placed in an ELISA reader to read the absorbance at 450 nm. The data were normalized to the absorbance value of the control group (0 μM A1@ _SiO2 -MP) at 450 nm. As shown in Figure 6, the cells treated with A1@SiO ₂ -MP at different concentrations (0-100 μM) did not significantly inhibit cell activity, and the cell activity of the high concentration group (100 μM) was not significantly different from that of the control group (P>0.05). As shown in Figure 7, fresh mouse blood was obtained to prepare 2% mouse blood-normal saline suspension 2.0 ml, and different concentrations of A1@SiO ₂ -MP (0, 1, 2, 5, 10, 20, 50, 100 μM) were added, shaken, and placed at 37°C for 1 hour. It can be seen that the color of the solution gradually deepened with the increase of the concentration of A1@SiO ₂ -MP. Compared with the positive (+) hemolysis control group, no hemolysis was observed in the experimental group containing A1@SiO ₂ -MP. The results suggest that the nanoparticles A1@SiO ₂ -MP have good biocompatibility and no obvious cytotoxicity was observed.

Example 6: Cellular uptake experiment of nanoparticles A1@SiO ₂ -MP

HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37°C and 5% CO _2. Two days before the experiment, trypsin was used for digestion and the cells were transferred from the culture flask to a 96-well plate. DMEM medium containing A1@SiO ₂ -MP (5 μM) was added during the experiment. Four replicate wells were selected at 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours, the old medium was discarded, and 4% paraformaldehyde solution was added for fixation for 15 minutes. After washing with PBS, DAPI (200 μM) was added and incubated for 15 minutes to mark the cell nucleus. After washing with PBS three times, microscopic imaging was performed. The DAPI imaging settings were: excitation/emission = 405/410-473 nm; the A1@SiO ₂ -MP imaging settings were: excitation/emission = 525/600-730 nm. The results are shown in Figure 8. Nanoparticles A1@SiO ₂ -MP can be taken up by cells. The uptake gradually increases from 0 to 4 hours. The nanoparticles A1@SiO ₂ -MP in the cells reach a peak at 4 hours. Then, from 24 hours on, the nanoparticles A1@SiO ₂ -MP in the cells gradually decrease. During the entire 0-24h process, the cells remain active and there is no obvious change in morphology, which further suggests that the nanoparticles A1@SiO ₂ -MP have good biocompatibility.

Example 7: Stability of Nanoparticles A1@SiO ₂ -MP

Nanoparticles A1@SiO ₂ -MP (100 μM) were dissolved in an acidic solution of pH=5.0 to simulate the environment of intracellular lysosomes. The fluorescence intensity and particle size were detected at 0, 1, 2, 4, 6, 8, 10, and 12 hours. As shown in Figures 9 and 10, after treatment with acidic solution, the fluorescence intensity and particle size of nanoparticles A1@SiO ₂ -MP did not change significantly, indicating that nanoparticles A1@SiO ₂ -MP can still maintain good stability in the acidic environment of cell lysosomes.

Example 8: Lysosomal targeting of nanoparticles A1@SiO ₂ -MP

HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37℃, 5% CO2 environment. Two days before the experiment, the cells were digested with trypsin and transferred to a glass-bottomed culture dish with a diameter of 35 mm. During the experiment, serum-free DMEM medium containing nanoparticles A1@SiO ₂ -MP (5 μM) and commercial lysosomal probe Lysotracker Green (LTG, 1 μM) was prepared. After incubating the cells for 1 hour, they were washed three times with PBS and imaged using a confocal microscope after adding fresh serum-free DMEM medium. The imaging parameters were set as follows: the imaging settings of nanoparticles A1@SiO ₂ -MP were: excitation/emission = 525 nm/560-730 nm; the imaging settings of commercial dye LTG were: excitation/emission = 488 nm/500-550 nm. The results are shown in Figure 11. The nanoparticles A1@SiO ₂ -MP that entered the cells were locally distributed in the cells, and their locations overlapped well with the commercial probe LTG. The calculated Pearson colocalization coefficient was 0.92, indicating that A1@SiO ₂ -MP can be targeted and enriched in cell lysosomes. This result creates conditions for the subsequent realization of photodynamic therapy targeting lysosomes.

Example 9: Ability of Nanoparticle A1@SiO ₂ -MP to Generate Reactive Oxygen Species in Lysosomes

After confirming that the nanoparticles A1@SiO ₂ -MP can be targeted and aggregated in lysosomes, their ability to generate reactive oxygen species in lysosomes was further studied. HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37°C and 5% CO ₂ environment. Two days before the experiment, the cells were digested with trypsin and transferred to a glass-bottomed culture dish with a diameter of 35 mm. The cells were co-cultured with A1@SiO ₂ -MP (5 μM) for 1 hour, washed 3 times with PBS, and then the ROS indicator DCFH (10 μM) was added and incubated in the dark for 0.5 hours. After washing with PBS, the cell culture dish was placed under a white light source (120 mW/cm ² ) for 5 minutes, and then confocal microscopy imaging was performed immediately. The imaging parameters were set as follows: the imaging setting of nanoparticles A1@SiO ₂ -MP was: excitation/emission = 525 nm/560-730 nm; the imaging setting of ROS indicator DCFH was: excitation/emission = 488 nm/500-550 nm. As shown in Figure 12, nanoparticles A1@SiO ₂ -MP were enriched in lysosomes; ROS indicator DCFH had no subcellular structure targeting group and diffused freely in the cytoplasm after passing through the cell membrane. After the cells were irradiated, green fluorescence was observed in the lysosomes, indicating that DCFH in the lysosomes generated fluorescent substance DCF after contacting reactive oxygen. The Pearson colocalization coefficient between nanoparticles A1@SiO ₂ -MP and the oxidation product DCF was 0.88, indicating that nanoparticles A1@SiO ₂ -MP enriched in lysosomes could generate reactive oxygen in situ in the lysosomes after being excited.

Example 10: Factors affecting the generation of reactive oxygen species by nanoparticles A1@SiO ₂ -MP

HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37°C and 5% CO _2. Two days before the experiment, cells were digested with trypsin and transferred to 6-well plates. ROS indicator DCFH (10 μM) was added and incubated in the dark for 0.5 hours, and then the cells were treated with 0, 0.1, 0.5, 1, and 5 μM nanoparticles A1@SiO ₂ -MP, followed by dark or light treatment (120mW/cm ² , 5 minutes). The detection settings of ROS indicator DCFH in flow cytometry experiments were: excitation/emission = 488 nm/500-550 nm. The results are shown in Figure 13. Under no-light conditions, regardless of whether the cells contain A1@SiO ₂ -MP, the reactive oxygen concentration in the cells is extremely low; simple illumination also does not cause an increase in the reactive oxygen content; when the cells are treated with nanoparticles A1@SiO ₂ -MP and illuminated at the same time, it can be seen that the reactive oxygen content in the cells is significantly increased, and the content of reactive oxygen generated is positively correlated with the amount of nanoparticles introduced. It can be seen that changing the amount of nanoparticles A1@SiO ₂ -MP introduced and the illumination time can affect the amount of reactive oxygen generated in the cells.

Example 11: Photodynamic Tumor Cell Killing by Nanoparticles A1@SiO ₂ -MP

HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37°C and 5% CO _2. Two days before the experiment, cells were digested with trypsin and transferred to 96-well plates. Serum-free DMEM medium containing nanoparticles A1@SiO ₂ -MP (0, 0.1, 0.2, 0.5, 1, 2, 5, 10 μM) was added to the cells for 1 hour, washed three times with PBS, and fresh serum-free DMEM medium was added. The cells were irradiated with a white light source (120 mW/cm ² ) for 5 minutes. After irradiation, the cells were washed with PBS and fresh DMEM medium was added, and returned to the cell culture incubator for 3 hours. Then, Calcein-AM (5 μM) and dye PI (10 μM) were used to stain the cells for live or dead. After washing with PBS, the cells were observed under a fluorescence microscope. The imaging settings of the dye Calcein-AM were: excitation/emission = 488 nm/500-550 nm; the imaging settings of the dye PI were: excitation/emission = 535 nm/580-630 nm. As shown in Figure 14, after Calcein-AM entered the cells, the esterase hydrolyzed it into the green fluorescent product Calcein and it remained in the cells. When the nanoparticles A1@SiO ₂ -MP were treated and irradiated at the same time, the green fluorescence of Calcein-AM was weakened and the red fluorescence of PI was gradually enhanced in the cells, indicating that after A1@SiO ₂ -MP entered the cells and targeted the lysosomes, it was stimulated to produce reactive oxygen species in situ, inducing cell apoptosis. After washing with PBS, serum-free culture medium containing 10% CCK8 was added. After culturing for 2 hours, the 96-well plate was placed in an ELISA reader to read the absorbance at 450 nm. As shown in FIG. 15 , the CCK8 experiment indicates that the introduction of nanoparticles A1@SiO ₂ -MP does not affect cell activity without illumination; once illuminated, nanoparticles A1@SiO ₂ -MP can achieve photodynamic therapy to effectively kill cells; the effect of photodynamic therapy is positively correlated with the amount of nanoparticles A1@SiO ₂ -MP introduced.

Example 12: Photodynamic apoptosis detection using nanoparticles A1@SiO ₂ -MP

HeLa cells were cultured in DMEM medium (containing 10% FBS, 1% double antibody) at 37℃, 5% _CO2 . Two days before the experiment, cells were trypsinized and transferred to 96-well plates. Cells were treated with A1@ _SiO2 -MP (5 μM) for 2 hours, irradiated with white light source (120 mW/ ^cm2 ) for 0, 1, 3, and 5 minutes, and then cultured in the dark for 12 hours. 200 μL of PBS solution containing Annexin V-FITC (2.5%) and PI (5%) was added. After 20 minutes, the cells were washed with PBS and fluorescent imaging was performed. The imaging settings of the dye Annexin V-FITC were: excitation/emission = 488 nm/500-530 nm; the imaging settings of the dye PI were: excitation/emission = 543 nm/550-650 nm. As shown in FIG16 , as the irradiation time increases, the green fluorescence of the cell membrane and the red fluorescence of the cell nucleus PI gradually increase. The flow cytometry experiment confirms the results of the microscope experiment, indicating that the photodynamic treatment of the nanoparticles A1@SiO ₂ -MP on the cells induces the occurrence of cell apoptosis.

Example 13: Photodynamic therapy of mouse tumors using nanoparticles A1@SiO ₂ -MP

Six-week-old female nude mice were fed for one week and injected with 0.1 mL of cervical cancer HeLa cells (cell number: 3×10 ⁶ ) into the right hind leg. The experiment was performed when the tumor grew to 40-60 mm ^3. The tumor-bearing mice were randomly divided into 4 groups, with 5 mice in each group. On day 0, mice in groups A and B were injected with 0.02 mL of PBS intratumorally; mice in groups C and D were injected with 0.2 mL of PBS containing A1@SiO ₂ -MP (1 mM) intratumorally. In the following 14 days, the mice in each group were weighed and the tumor volume was measured every 3 days. The mice in groups B and D were anesthetized with isoflurane gas every day and the tumor site was irradiated with white light (120 mW/cm ² ) for 10 minutes. The mice were killed on day 15, and the tumor volume was measured and sliced for TUNEL staining to observe cell apoptosis.

As shown in FIG17 , the weight change of mice injected with nanoparticles A1@SiO ₂ -MP and irradiated was not significantly different from that of mice in the PBS control group. The same situation also occurred in the group injected with nanoparticles A1@SiO ₂ -MP alone and the group injected with PBS and irradiated. The results indicate that nanoparticles A1@SiO ₂ -MP have good biocompatibility, and photodynamic therapy using them has no significant effect on the overall physiological condition of mice. As shown in FIG18 and FIG19 , the tumor volume of mice in the PBS control group gradually increased over time over 15 days. This phenomenon also occurred in the group injected with nanoparticles A1@SiO ₂ -MP alone and the group injected with PBS and irradiated, indicating that the simple introduction of nanoparticles A1@SiO ₂ -MP or the simple irradiation of tumors cannot inhibit the development of tumors. However, when the mouse tumor was injected with nanoparticles A1@SiO ₂ -MP and irradiated, the growth of tumor volume was significantly inhibited. As shown in Figure 20, in the TUNEL apoptosis experiment of tumor tissue sections, no green fluorescence indicating apoptosis was observed in the samples of groups A, B, and C, but obvious green fluorescence was observed in group D, indicating that apoptosis occurred in the tumor cells of this group. The above experimental results show that the nanoparticles A1@SiO ₂ -MP injected into the tumor generate reactive oxygen species in the lysosomes of tumor cells after being stimulated, oxidatively damaging the lysosomes and inducing apoptosis, thereby inhibiting tumor development and achieving photodynamic therapy.

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 (8)

1. A method for preparing AIE photodynamic nanoparticles that can target lysosomes, characterized in that it comprises the following steps: S1. AIE photosensitive material A1 and F127 polyether are mixed in tetrahydrofuran to obtain a mixed solution, wherein the concentration of the AIE photosensitive material A1 in the mixed solution is 0.30-0.36 mM, and the concentration of the F127 polyether is 60-72 mg/mL, and then blow-dried to obtain a glue layer, and an acidic aqueous solution with a concentration of 0.77-0.93 M is added to the glue layer, and the volume ratio of the acidic aqueous solution to the mixed solution is (0.9-1.1):1, to form F127 micelles encapsulating the AIE photosensitive material A1; S2. adding tetraethoxysilane, wherein the volume ratio of the tetraethoxysilane to the mixed solution is (0.9-1.1): 20, stirring for 0.5-3 hours, using F127 micelles as a template and tetraethoxysilane as a silicon source to generate silica nanoparticles coated with the AIE photosensitive material A1; S3. adding aminopropyl triethoxysilane dropwise, wherein the volume ratio of the aminopropyl triethoxysilane to the tetraethoxysilane is (0.9-1.1): 1, stirring for 22-26 hours, filtering, and ultrafiltration centrifugation for 9-11 minutes to obtain nanoparticles with amino groups on the surface; S4. dissolving the nanoparticles with amino groups on the surface in ultrapure water, adding 3-(4-morpholinyl)propyl isothiocyanate, wherein the volume ratio of the 3-(4-morpholinyl)propyl isothiocyanate to the aminopropyltriethoxysilane is (0.9-1.1):3, stirring for 22-26 hours, and obtaining the AIE photodynamic nanoparticles by ultrafiltration and centrifugation; Wherein, the structural formula of the AIE photosensitive material A1 is: . 2. The method for preparing lysosome-targetable AIE photodynamic nanoparticles according to claim 1, wherein the gas used for drying in step S1 is nitrogen. 3. The method for preparing lysosome-targetable AIE photodynamic nanoparticles according to claim 1, wherein the stirring time in step S2 is 2 hours. 4. The method for preparing lysosome-targetable AIE photodynamic nanoparticles according to claim 1, characterized in that the filtration process in step S3 is performed by filtering using filter paper and a 0.22 µm syringe filter membrane in sequence. 5. The method for preparing lysosome-targetable AIE photodynamic nanoparticles according to claim 1, characterized in that the ultrafiltration centrifugation conditions in steps S3 and S4 are both centrifugation at 100 kDa and 5000 rcf for 10 minutes. 6. The method for preparing the AIE photodynamic nanoparticles that can target lysosomes according to claim 1, characterized in that the method for preparing the AIE photosensitive material A1 comprises the following steps: (1) dissolving 4-bromo-4',4'-dimethoxytriphenylamine, 5-formyl-2-thiopheneboronic acid and potassium carbonate in a molar ratio of (1.8-2.2):(2.7-3.3):10 in a mixed solution of toluene and methanol to obtain a first mixed solution, wherein the volume ratio of toluene to methanol is (0.9-1.1):1, evacuating and replacing with nitrogen; (2) dissolving the catalyst [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride in toluene, wherein the molar concentration of the catalyst after dissolution is 0.45-0.55 mol/L, and adding the dissolved catalyst to the first mixed solution; (3) stirring at 68-83° C. for 16-20 hours, cooling to room temperature, and removing the solvent using a rotary evaporator to obtain a second mixed solution; (4) Purifying the second mixed liquid by silica gel column chromatography with dichloromethane as the eluent to obtain an intermediate product, compound B, wherein the structural formula of compound B is: ; (5) dissolving compound B and malononitrile in a molar ratio of (0.9-1.1):2 in dichloromethane, and adding triethylamine dropwise, wherein the volume ratio of the triethylamine to the toluene in step (2) is (0.9-1.1):2; (6) stirring at 27-33° C. for 16-20 hours, cooling to room temperature, and removing the solvent by rotary evaporation to obtain a mixture; (7) Purifying the mixture by silica gel column chromatography using n-hexane and ethyl acetate in a volume ratio of (4.5-5.5):1 as eluent to obtain the AIE photosensitive material A1. 7. An AIE photodynamic nanoparticle that can target lysosomes, characterized in that it is prepared using the preparation method described in any one of claims 1 to 6. 8. Use of the lysosome-targeting AIE photodynamic nanoparticles as claimed in claim 7 in photodynamic tumor therapy.