Preparation method and application of aggregation-induced emission nanoparticles

Abstract

The application provides a preparation method of an aggregation-induced emission nanoparticle, which comprises the following steps: adopting triphenylamine as an electron donor (D), pyridine bromide as an electron acceptor (A), constructing a D-π-A structure, and synthesizing a nanoscale AIE photosensitizer nanoparticle by acting with an albumin stock solution; the nanoparticle synthesis method is easy to operate, has high yield and low cost, can simultaneously generate type I and type II reactive oxygen species (ROS), including ·OH (type I) and O2 (type II), and has good mitochondrial targeting function, and a Pearson correlation coefficient is as high as 0.97. 1 In addition, due to the modification of the terminal carboxyl group of TTPA, the nanoparticle has a pH response effect, can increase the accumulation or endocytosis of the nanoparticle in tumors, and can enhance the in-vivo imaging and treatment effect, and has potential clinical application value of intelligent response cancer diagnosis and treatment function.

Description

Technical Field

[0001] This invention belongs to the field of luminescent bionanomaterials technology, specifically relating to a method for preparing aggregation-induced luminescent nanoparticles and their applications. Background Technology

[0002] Photodynamic therapy (PDT) is a method that involves injecting photosensitizers (PSs) followed by laser irradiation to generate highly cytotoxic reactive oxygen species (ROS), causing tumor cells to degenerate and die. Compared to traditional surgery or chemotherapy, PDT has advantages such as being non-invasive, well-controllable, and having fewer systemic side effects, making it an important clinical treatment for tumors. ROS mainly includes singlet oxygen (…). 1 O2), hydroxyl radicals (·OH), hydrogen peroxide (H2O2), superoxide (O 2- Based on their photoreaction mechanism, ROS can be divided into Type I and Type II. Type I ROS mainly refers to the formation of ·OH and H2O2, while Type II ROS includes... 1 O2 and O 2- Type I ROS, in addition to generating additional oxygen in situ, typically exhibit higher cytotoxicity than type II ROS, thus significantly modulating the hypoxic environment of tumors. Therefore, most type II photosensitizers face the fundamental paradox between the hypoxic microenvironment of solid tumors and the severe oxygen consumption associated with photodynamic therapy (PDT). Consequently, there is an urgent need to develop photosensitizers with high PDT and low O2 dependence. Therefore, the use of organic fluorescent photosensitizers for bioluminescence imaging diagnosis and PDT applications has received widespread attention in order to achieve better tumor diagnosis and treatment outcomes.

[0003] However, traditional organic photosensitive polymers (PSs) such as porphyrins and phthalocyanines generally suffer from some inherent defects. The main drawbacks are a lack of binding capacity for efficient fluorescence emission and, in the aggregated state, π-π stacking leading to weakened fluorescence and reduced ROS generation—the so-called aggregation quenching (ACQ) effect—which significantly inhibits their therapeutic effects in bioimaging, diagnosis, and phototherapy (PDT). Due to the defects of ACQ, a novel type of PS with aggregation-induced emission (AIE) properties has gradually attracted widespread attention in phototherapy. AIE properties involve the consumption of excited-state energy in solution through non-radiative internal conversion from the lowest excited state (singlet state) to the ground state, thus exhibiting weak fluorescence. In the aggregated state, non-radiative internal conversion restricts intramolecular motion, causing the singlet state to release energy through radiative transitions or intersystem cross-channeling, which enhances the relative fluorescence intensity and provides sufficient ROS generation, effectively overcoming the limitations of the traditional ACQ effect and making it more suitable for biomedical applications with higher working concentrations and higher sensitivity. Meanwhile, to improve biocompatibility and targeting, nanostructures have been found to have significant advantages, including size-dependent physical properties, long period times, and the ability to be used for active targeting.

[0004] Many existing photosensitizers suffer from drawbacks such as poor biocompatibility, low tissue penetration, low tumor-specific targeting, and inability to overcome the hypoxic environment of tumors, limiting their clinical application in in vivo imaging and photodynamic therapy. Therefore, there is a need for a photosensitizer that can efficiently generate ROS under both aerobic and hypoxic conditions, and possesses mitochondrial targeting, good tissue penetration, and biocompatibility. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for preparing aggregation-induced emission nanoparticles and their applications. This method synthesizes a photosensitizer TTPA molecule with AIE characteristics through a simple three-step process. Under white light excitation, the TTPA molecule can simultaneously generate hydroxyl radicals (·OH) and singlet oxygen (·OH). 1 The nano-assemblies TTPA-NPs, made from O2, release photosensitizer TTPA molecules under weakly acidic conditions after entering tumor cells. They then target mitochondria and induce the production of superoxide, thereby enabling in vivo imaging and photodynamic therapy in animals.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing aggregation-induced emission nanoparticles, the method comprising the following steps:

[0007] S1. Methylpyridine and 9-bromononanoic acid are mixed in acetone and refluxed overnight. After cooling to room temperature, the mixture is filtered under reduced pressure to obtain a solid. The solid is washed with acetone several times to obtain an intermediate product, which is methylpyridine bromide.

[0008] S2. The intermediate product obtained in S1 is dissolved in ethanol with 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde to obtain a mixture. A few drops of piperidine are added to the mixture, and the mixture is refluxed overnight. After cooling to room temperature, it is evaporated under reduced pressure to obtain a crude product. The crude product is purified by column chromatography to obtain the aggregation-induced emission photosensitizer TTPA molecule.

[0009] S3. Dissolve the aggregation-induced emission photosensitizer TTPA molecule obtained in S2 in dimethyl sulfoxide (DMSO) to obtain TTPA stock solution; mix the TTPA stock solution with albumin stock solution and dilute with phosphate buffer solution (PBS) to obtain aggregation-induced emission nanoparticles, i.e., nano-assemblies TTPA-NPs.

[0010] Preferably, the solid-liquid ratio of methylpyridine, 9-bromononanoic acid and acetone in S1 is (0.5-2.8) g: (1.2-7) g: (60-150) mL; and the number of washes is 3.

[0011] Preferably, the overnight reflux reaction in S1 and the overnight reflux reaction in S2 are both 10-14 hours.

[0012] Preferably, the molar ratio of the intermediate product and 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde in S2 is (4.0-10):(3.2-10); the concentration of the intermediate product in the mixture is 0.02-0.2 mol / L.

[0013] Preferably, the adsorbent for column chromatography in S2 is silica gel, and the eluent is dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 8:1.

[0014] Preferably, the molecular formula of the aggregation-induced emission photosensitizer TTPA molecule in S2 is C 38 H 39 BrN2O2S, the molecular structural formula is:

[0015]

[0016] Preferably, the number of piperidine drops in S2 is 3 to 6 drops; the vacuum degree of the reduced pressure evaporation is -0.08 MPa, the temperature is 40°C, and the time is 30 min.

[0017] Preferably, in S3, the solid-liquid ratio of the aggregation-induced emission photosensitizer TTPA molecule and dimethyl sulfoxide is 0.0067 g: 10 mL; the concentration of the TTPA stock solution is 1 mM; and the volume ratio of the TTPA stock solution, albumin stock solution, and phosphate buffer solution is 1:1:98.

[0018] Preferably, the albumin stock solution in S3 is prepared by dissolving albumin in a phosphate buffer solution, and the solid-liquid ratio of albumin to the phosphate buffer solution is 0.6645 g: 10 mL; the concentration of the albumin stock solution is 1 mM, and the albumin is bovine serum albumin.

[0019] The application of the above-prepared aggregation-induced emission nanoparticles in photodynamic therapy is also provided.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. This invention uses triphenylamine as an electron donor (D) and pyridine bromide as an electron acceptor (A) to construct a D-π-A structure. The loading and release of the photosensitizer TTPA molecule by the terminal carboxyl group of the alkyl chain attached to the pyridine ring is regulated by the alkyl group. The AIE photosensitizer TTPA molecule provided by this invention can simultaneously generate hydroxyl radicals (·OH) and singlet oxygen (·OH) under white light excitation. 1 O2), so it can kill tumor cells in aerobic / hypoxic environments.

[0022] 2. This invention utilizes characterization techniques such as ultraviolet and fluorescence spectroscopy, DLS dynamic light scattering particle size analyzer, and transmission electron microscopy to explore the relationship between the emission properties of AIE nanoassemblies TTPA-NPs. Furthermore, theoretical calculations were used to study the interaction between the photosensitizer TTPA molecules and albumin, in order to gain a deeper understanding of the mechanism of nanoassembly and provide unique design ideas for the design of AIE photosensitizer nanomaterials.

[0023] 3. This invention synthesizes a D-π-A photosensitizer molecule (TTPA molecule) by design. Experimental results show that the TTPA molecule can simultaneously and efficiently generate type I and type II ROS. Therefore, it has high PDT efficiency under both normoxic and hypoxic environments. Furthermore, after forming aggregation-induced luminescent nanoparticles with bovine serum albumin (BSA), it can effectively target mitochondria and efficiently generate ROS in mitochondria, exhibiting excellent cell-killing ability.

[0024] 4. The aggregation-induced emission nanoparticles prepared by this invention have excellent mitochondrial targeting function, with a Pearson correlation coefficient as high as 0.97. In addition, due to the modification of the terminal carboxyl group of TTPA, the nanoparticles have a pH-responsive effect, which can increase their accumulation or endocytosis in tumors, enhance in vivo imaging and therapeutic effects, and have potential clinical application value for intelligent response cancer diagnosis and treatment. Moreover, the preparation method of this invention has the characteristics of low cost, simple synthesis, easy operation, and high PDT efficiency.

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] Figure 1 (a) is the 1H NMR spectrum of the TTPA molecule obtained in Example 1 of this invention. Figure 1 (b) is the carbon NMR spectrum of the TTPA molecule.

[0027] Figure 2 This is the mass spectrum of the TTPA molecule obtained in Example 1 of this invention.

[0028] Figure 3 This is a fluorescence emission variation diagram of TTPA molecules obtained in Example 1 of the present invention in methanol solution with different water contents.

[0029] Figure 4 This is a graph showing the total ROS generation efficiency of TTPA molecules obtained in Example 1 of this invention.

[0030] Figure 5 This is a graph showing the efficiency of hydroxyl radical generation in TTPA molecules obtained in Example 1 of this invention.

[0031] Figure 6 This is a graph showing the singlet oxygen generation efficiency of TTPA molecules obtained in Example 1 of this invention.

[0032] Figure 7 (a) is a dynamic light scattering diagram of the nano-assemblies TTPA-NPs prepared in Example 1 of this invention. Figure 7 (b) is a transmission electron microscope image of the nano-assemblies TTPA-NPs.

[0033] Figure 8 The fluorescence spectra of the nano-assemblies TTPA-NPs and free TTPA molecules prepared in Example 1 of this invention are shown.

[0034] Figure 9 The fluorescence spectra of TTPA molecules obtained in Example 1 of this invention, mixed with culture medium and serum, are based on free TTPA molecules.

[0035] Figure 10 This is a molecular docking simulation diagram of the nano-assemblies TTPA-NPs prepared in Example 1 of this invention.

[0036] Figure 11 This is a fluorescence change diagram of the nano-assemblies TTPA-NPs prepared in Example 1 of this invention in different pH buffer solutions.

[0037] Figure 12 This is a mitochondrial targeting diagram of the nano-assemblies TTPA-NPs prepared in Example 1 of this invention.

[0038] Figure 13This is a diagram showing the reactive oxygen species generation effect of the nano-assemblies TTPA-NPs prepared in Example 1 of this invention in tumor cells.

[0039] Figure 14 This is a schematic diagram illustrating the preparation of the nano-assemblies TTPA-NPs in Example 1 of this invention.

[0040] Figure 15 The images show the phototoxicity of the nano-assemblies TTPA-NPs prepared in Example 1 of this invention to B16F10, CT26 and 4T1 tumor cells under white light irradiation (15(a)-15(c)), the dark toxicity of the cells under light-shielded conditions (15(d)), and the fluorescence images of 4T1 cells incubated with the nano-assemblies TTPA-NPs under white light irradiation (15(e)). Detailed Implementation

[0041] Example 1

[0042] This embodiment provides a method for preparing aggregation-induced emission nanoparticles, which includes the following steps:

[0043] S1. 0.94 g of methylpyridine and 2.37 g of 9-bromononanoic acid were mixed in 80 mL of acetone and refluxed overnight for 10 h. After cooling to room temperature (25 °C), a large amount of white solid precipitated. The solvent was removed by vacuum filtration to obtain a solid substance. The solid substance was washed three times with acetone to obtain an intermediate product, which was methylpyridine bromide.

[0044] S2, 1.2 mmol of the intermediate obtained in S1 is reacted with 1.0 mmol of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde. Dissolved in 60 mL of ethanol, then 3 drops of piperidine were added as a catalyst, and the mixture was refluxed overnight for 10 h. After cooling to room temperature (25 °C), the solvent was removed by vacuum evaporation for 30 min at a vacuum of -0.08 MPa and a temperature of 40 °C to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 8:1 to obtain the red aggregation-induced emission photosensitizer TTPA molecule. The molecular formula of the aggregation-induced emission photosensitizer TTPA molecule is C0. 38 H 39 BrN2O2S, the molecular structural formula is:

[0045]

[0046] S3. Dissolve 0.0067g of the aggregation-induced emission photosensitizer TTPA molecule obtained in S2 in 10mL of DMSO to prepare a TTPA stock solution with a concentration of 1mM; then, take 100μL of the stock solution and mix it with 100μL of bovine serum albumin (BSA) stock solution, and dilute it with PBS to a final volume of 10mL to obtain aggregation-induced emission nanoparticles, i.e., nano-assemblies TTPA-NPs;

[0047] The bovine serum albumin stock solution was prepared by dissolving 0.6645 g of bovine serum albumin in 10 mL of PBS, and the concentration of the albumin stock solution was 1 mM. The synthesis principle of the nano-assemblies TTPA-NPs is as follows: Figure 14 As shown;

[0048] The synthetic route for the aggregation-induced emission photosensitizer TTPA molecule (hereinafter referred to as TTPA molecule) described in S2 is as follows:

[0049]

[0050] Figure 1 The NMR spectra of the TTPA molecules obtained in this embodiment are shown, where (a) is the 1H NMR spectrum and (b) is the 1C NMR spectrum; the NMR characterization data of the TTPA molecules are as follows: 1 HNMR(600MHz,d6-DMSO)δ:8.90(d,J=6.6Hz,2H),8.26(d,J=16.2.0Hz,1H),8. 19(d,J=6.6Hz,2H),7.63(d,J=9.0Hz,2H),7.51(m,2H),7.36(t,J=7.8Hz,4H) ,7.15-7.11(m,3H),7.09(d,J=7.8Hz,4H),6.99(d,J=9.0Hz,2H),4.45(t,J=7 .8Hz,2H),2.15(t,J=7.2Hz,2H),1.89(m,2H),1.47(m,2H),1.28-1.25(m,8H). 13 CNMR (150MHz, d6-DMSO) δ: 175.1, 153.0, 148.3, 147.7, 147.0, 144.4, 139.1, 134.5, 134.3, 130.2, 127.3, 126.7, 125.2, 124.7, 124.4, 123.7, 122.6, 121.7, 60.0, 34.4, 30.9, 29.0, 28.9, 28.7, 25.8, 25.0; These 1H and 1C NMR spectra preliminarily confirm the successful synthesis of the target product TTPA molecule.

[0051] Figure 2This is the mass spectrum of the TTPA molecule prepared in this embodiment, where the peak with a mass-to-charge ratio of 587.27311 corresponds to the TTPA molecule with the molecular formula C. 38 H 39 N2O2S + [M-Br] + Its theoretical value C 38 H 39 N2O2S + [M-Br] + The result matches 587.27268, further proving the successful synthesis of the target product TTPA molecule.

[0052] Experiment 1: Detection of fluorescence emission changes of TTPA molecules obtained in Example 1 under different water contents:

[0053] To investigate the aggregation-induced emission properties of the photosensitizer TTPA, 0.0067 g of TTPA molecules were dissolved in 10 mL of methanol to prepare a solution with a concentration of 1 × 10⁻⁶ g / mL. -3 A stock solution of mol / L was prepared; the above stock solution was used to prepare solutions with a concentration of 1×10⁻⁶. -5 Fluorescence intensity was measured in solutions with water contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% under excitation conditions (excitation wavelength 498 nm). The results are as follows: Figure 3 As shown.

[0054] Figure 3 This is a fluorescence emission graph of TTPA molecules in methanol solution with different water contents (excitation wavelength 498 nm). Figure 3 (a) shows the fluorescence emission of TTPA molecules in methanol solution with different water contents. Figure 3 (b) is a fluorescence change graph at an emission wavelength of 640 nm. The graph shows that the photosensitizer TTPA molecule exhibits fluorescence changes at a water content (f...). w There is almost no fluorescence at 0% and 60%, and it increases with water content (f) w From 60% to 95%, the emission intensity of the photosensitizer TTPA molecule increased by approximately 20-fold. Figure 3 (cd) It can be seen that due to the continuous increase in water content, the formation of TTPA molecular aggregates leads to a significant increase in fluorescence intensity, indicating that the compound has obvious aggregation-induced emission (AIE) characteristics.

[0055] Experiment 2: Determination of total reactive oxygen species (ROS) in the TTPA molecules obtained in Example 1:

[0056] 2',7'-Dichlorodihydrofluorescein (DCFH) was used as a common commercial indicator of ROS generation to measure the TTPA molecule's performance under white light (50 mW·cm⁻¹). -2 The total ROS efficiency generated under irradiation. Considering the role of ROS in photodynamic therapy, the experiment utilized DCFH (1 μM PBS solution) and TTPA molecules (final concentration 10 μM, stock solution 1 × 10⁻⁶) to measure the total ROS efficiency. -3 Mix 1 mol / L DMSO solution or the same concentration of the conventional photosensitizer dihydroporphyrin E6 (Ce6) in PBS. Then use white light (50 mW·cm⁻¹). -2 After irradiation for different times, the fluorescence spectrum of 2',7'-dichlorofluorescein (DCF) was measured at an excitation wavelength of 480 nm. Figure 4 This is a comparison chart of the total reactive oxygen species (ROS) measurements of TTPA molecules and the traditional photosensitizer dihydroporphyrin e6.

[0057] Figure 4 This is a curve showing the change in relative emission intensity (I / I0⁻¹) at an emission wavelength of 523 nm as a function of irradiation time. The results show that within a short irradiation time (within 60 seconds), TTPA molecules ( Figure 4 a) Under white light irradiation (50mW·cm) -2 When ), the fluorescence intensity is higher than that of the equivalent concentration of Ce6 ( Figure 4 b) is significantly enhanced, while DCFH alone ( Figure 4 c) Almost no change. Therefore, this research data indicates that TTPA molecules have a higher ROS generation efficiency than the traditional photosensitizer Ce6; Figure 4 (d) is a comparison of the emission intensities of TTPA, Ce6, and DCFH molecules. The reason why AIE photosensitizers generate ROS efficiently may be that the stronger donor-acceptor effect leads to a smaller singlet and three-state band gap, which is conducive to the crossover process from singlet to three-state and greatly improves the yield of three-state excited states.

[0058] Experiment 3: Determination of the generation efficiency of hydroxyl radicals (·OH) from the TTPA molecules obtained in Example 1:

[0059] To investigate the types of ROS generated by TTPA molecules, fluorescein hydrochloride (HPF) (5 mM in 2 μL of DMF) was used as a commercial ·OH indicator, and TTPA molecules (final concentration 10 μM, stock solution 1 × 10⁻⁶) were compared with those (OH-). -3 1 mL of a mixture of DMSO solution (1 mol / L) or Ce6 (10 μM, activity control group) and PBS. Light was applied using white light (50 mW·cm⁻¹). -2 The changes in the fluorescence spectrum of HPF were measured at 480 nm (excitation wavelength) after irradiation for different times.

[0060] Figure 5 This is a graph and curve showing the change in relative emission intensity (I / I0⁻¹) at 512 nm as a function of illumination time. Under the same experimental conditions as the control group, TTPA molecules (…) were excited by white light. Figure 5 (a) resulted in a significant increase in the fluorescence intensity of HPF, while the control group Ce6 ( Figure 5 (b) and HPF ( Figure 5 (c) The generation of ·OH is almost invisible. Unlike type II ROS, ·OH, a type I ROS, can produce significant cytotoxicity through superoxide dismutase-mediated dismutation reactions. More importantly, the cascade of biological reactions initiated by ·OH can supplement additional oxygen in situ, further improving the hypoxic environment at the tumor site and increasing the efficiency of PDT applications.

[0061] Experiment 4: Singlet oxygen in TTPA molecules obtained in Example 1 ( 1 O2) generation efficiency determination:

[0062] 1 The efficiency of type II ROS in O2 is beneficial for PDT treatment. This study used the commonly used singlet oxygen indicator 9,10-anthrayl-bis(methylene)dimalonic acid (ABDA) to evaluate this. 1 O2 is generated because the oxidation of ABDA leads to a decrease in its UV-vis absorbance at 378 nm. Experiment 1 was prepared by reacting 200 μM ABDA with TTPA molecules (final concentration 10 μM, stock solution 1 × 10⁻⁶). -3 A PBS mixture was prepared using 10 μM DMSO solution. Ce6 (10 μM) and blank ABDA were used as control groups. The solution was irradiated with white light (50 mW·cm⁻¹). -2 The absorbance of ABDA under white light with an excitation wavelength of 378 nm for 0, 1, 2, 3, 4 and 5 min was measured using a UV spectrophotometer at different times.

[0063] Figure 6 For TTPA molecules and Ce6 at 50 mW·cm -2 The graph shows the changes in the ultraviolet absorption spectrum of ABDA at 378 nm after white light irradiation for different times. It can be seen that ABDA alone... Figure 6 (c) The absorption peaks under white light excitation at different times showed almost no change, while the absorption rate of TTPA molecules decreased significantly under the same amount of light. Figure 6 (a)), with equivalent concentrations of Ce6 ( Figure 6 (b) Compared to TTPA molecules ( Figure 6 (a) The decrease in absorbance of ABDA is more pronounced. This indicates that, at the same dosage, TTPA molecules have a higher absorbance than the widely used photosensitizer Ce6.1 O2 generation efficiency.

[0064] Experiment 5 Figure 7 The dynamic light scattering particle size distribution of the nano-assemblies TTPA-NPs prepared in Example 1 is shown. Figure 7 (a) and transmission electron microscopy (TEM) images Figure 7 (b) Dynamic light scattering particle size distribution and transmission electron microscopy results both show that the particle size of the nano-assemblies TTPA-NPs is around 160 nm, and they are uniform spherical nanoparticles with a good Tyndall effect. Figure 7 (b) The inset in the upper left corner shows the Tyndall effect in the nanomaterial solution. The results of this experiment provide sufficient evidence for the further application of TTPA molecules in in-situ self-assembly into nanoparticles in blood.

[0065] Experiment 6: Detection of fluorescence spectra of the nano-assemblies TTPA-NPs and free TTPA molecules prepared in Example 1:

[0066] In this experiment, 10 μM PBS solutions of TTPA molecules and TTPA-NPs nanoassemblies were prepared. The fluorescence intensity at an excitation wavelength of 498 nm was measured using a fluorescence spectrophotometer, and the fluorescence emission from 500 to 850 nm was recorded.

[0067] To investigate whether the assembly of TTPA molecules with BSA has a positive impact on the aggregation-induced emission effect of TTPA molecules, the fluorescence intensity of the nano-assembled TTPA-NPs and free TTPA molecules was measured using fluorescence spectroscopy. The results are as follows: Figure 8 As shown, the fluorescence intensity of the nano-assemblies TTPA-NPs is significantly enhanced, laying the foundation for effective in vitro and in vivo photodynamic therapy of tumors.

[0068] Experiment 7: Fluorescence spectral changes of the TTPA molecules obtained in Example 1 in serum (Plasma) and culture medium (CCM).

[0069] Based on the abundance of albumin in complete culture medium and serum, the fluorescence spectra of TTPA molecules mixed with plasma and blood were measured. 100 μL of TTPA molecules (1 mM MSO stock solution) was mixed with PBS solutions of plasma and culture medium, respectively, and diluted to 10 mL with PBS. The final concentrations of TTPA and BSA were consistent with those in Example 7. The fluorescence changes before and after mixing were measured.

[0070] Figure 9 The image shows the fluorescence spectra of TTPA molecules in serum and culture medium. The results indicate that TTPA molecules exhibit strong red emission around 640 nm in both serum and culture medium, with an intensity almost equal to that in serum. Figure 8Similarly, this clearly demonstrates that the assembly of TTPA molecules with albumin can rapidly and stably generate nanoassemblies TTPA-NPs in situ in complete culture medium or blood.

[0071] Experiment 8: Theoretical molecular docking study of the interaction between TTPA molecules and BSA in the nano-assemblies TTPA-NPs prepared in Example 1:

[0072] To investigate in more detail how TTPA molecules bind to BSA, molecular docking simulations were performed. The autodockvina software was used to dock TTPA molecules with BSA to estimate potential interactions between the protein and TTPA. The crystal structure of albumin was obtained from the RCSB Protein Data Bank (PDBID: 3V03). The structure of the TTPA molecule was modeled using the ChemDraw program. The predicted assemblies were then optimized and ranked using the empirical scoring function ScreenScore. Each docking was performed twice, with each operation selecting 100 conformations favorable for docking of the TTPA molecule.

[0073] The results are as follows Figure 10 As shown, the carboxyl group is located on the surface of the protein molecule. However, the triphenylamine is close to the hydrophobic binding bag of the domain, resulting in BSA encapsulating the entire TTPA molecule. The TTPA molecule can form multiple hydrogen bonds with the amino acid residues of Tyr137, Phe133, and Tyr160, with hydrogen bond strengths of [missing values]. and Furthermore, multiple CH···π interactions exist between the phenyl and thiophene groups, and these multiple intermolecular forces promote the self-assembly of TTPA molecules with BSA, thereby forming TTPA-NPs nanoparticles.

[0074] Experiment 9: Fluorescence changes of the nanoassemblies TTPA-NPs prepared in Example 1 at different pH values.

[0075] Given that the weakly acidic microenvironment of tumor tissue (pH 6.5-6.8) is lower than that of normal tissue (pH ~7.4), we further investigated the photoluminescence intensity changes of the nano-assemblies TTPA-NPs under pH conditions of 2-12, and used free TTPA molecules as a control group in the experiment. Figure 11 This indicates that free TTPA molecules in DMSO emit almost no emission at pH values ​​between 2 and 12. Figure 11 (a)). Figure 11(b) At pH below 7, the TTPA-NP nanoassemblies also exhibited weak photoluminescence intensity, indicating that the TTPA-NP nanoassemblies may be disrupted under acidic conditions, releasing TTPA molecules, which is beneficial for in vivo applications. However, at pH above 7, the fluorescence intensity was significant, possibly due to the recombination of TTPA molecules with albumin. Therefore, the TTPA-NP nanoassemblies possess strong pH responsiveness, providing a basis for in vivo photodynamic therapy and imaging.

[0076] Experiment 10: Mitochondrial targeting assay of the nanoassemblies TTPA-NPs prepared in Example 1 within cells:

[0077] To observe the mitochondrial targeting effect of TTPA molecules, 5 × 10 4 4T1 cells were seeded into 24-well plates containing 1 mL of culture medium and incubated for 24 h. The culture medium containing 10 μM TTPA stock solution was discarded. After incubation at 37°C for 0.5 h, the cells were gently washed twice with PBS. Then, 1 mL of fresh culture medium containing 0.05 μM Mito-Tracker green solution was added to each well, and incubation continued for 30 min. Next, the cells were washed three times with PBS and fixed with 4% PFA for 15 min. The nuclei were stained with DAPI and then washed with PBS. Finally, the distribution of TTPA in 4T1 cells was imaged using CLSM.

[0078] The results showed that as the nanoassemblies TTPA-NPs entered the mitochondria, the red fluorescence emitted by the molecules fused with the green markers of the mitochondria, thus forming a yellow color. Figure 12 It can be seen that the PCC values ​​of the mitochondrial green fluorescent probe and the TTPA molecule are 0.97, indicating that the TTPA molecule can effectively target mitochondria.

[0079] Experiment 11: Determination of reactive oxygen species generation in cells using the TTPA-NPs nanoassemblies prepared in Example 1.

[0080] The experiment used 2',7'-dichlorodihydrofluorescein-acetoacetate (DCFH-DA) as a cellular ROS detection method. Therefore, we used DCFH-DA to assess ROS produced by cells under white light irradiation with TTPA molecules. 4T1 cells were irradiated with 5 × 10⁻⁶ cells / day. 4 Cells were cultured at high density in 24-well plates containing coverslips and incubated overnight. The old medium was replaced with medium containing 10 μM TPA stock solution, and then incubated under white light (approximately 50 mW·cm⁻¹). -2Irradiate for 2 min. After 12 h, discard the cells and incubate with 10 μM DCFH-DA in culture medium at 37°C for 20 min. Finally, label the cell nuclei with DAPI and observe using a confocal laser scanning microscope (CLSM). Additionally, quantitatively measure green fluorescence using flow cytometry. Figure 13 As shown, both the PBS and PBS+Light groups were relatively dark, indicating that ROS generation was negligible. In the light-irradiated group, TTPA molecules exhibited strong fluorescence and necrosis. This is because TTPA molecules generate smaller nanoparticles in situ in the culture medium, facilitating their entry into cells and releasing TTPA molecules in the acidic environment of the tumor. Simultaneously, flow cytometry quantitative analysis of green fluorescence showed that when cells were only exposed to white light (PBS+light), the percentage of green fluorescence changed very little from 0.57% (PBS group) to 0.79%. However, the fluorescence percentage of TTPA molecules reached 89.6%, indicating that TTPA molecules can efficiently generate ROS within cells.

[0081] Experiment 12: Determination of cytotoxicity of the nanoassemblies TTPA-NPs prepared in Example 1:

[0082] The photodynamic killing effect on cells was evaluated using the CCK8 assay and a live / dead cell double staining kit (CalceinAM / PI). For the CCK8 assay, 4T1, CT26, and B16F10 cells were seeded in 96-well plates at a seeding density of 2 × 10⁶ cells / well. 3 24 hours later, new medium containing different final concentrations of TTPA was added to complete medium (3.125, 6.25, 12.5, 25, 50, 100 μM) to replace the old medium. 4 hours later, the culture dishes containing 4T1 cells were immersed in white light (approximately 50 mW·cm²). -2 After irradiation for 5 min, another group of culture dishes containing 4T1 cells was placed in the dark as a control. 48 h later, 10 μM LCCK8 was added to each well and incubated for 2 h. Absorbance was read at 450 nm using a microplate reader, and cell viability was calculated compared to the control group. To more directly observe the cell-killing effect of TTPA, cells were treated with 10 μM TTPA, and after 4 h, placed under white light for 2 min for Calcein AM / PI assay. Cells were then stained and observed using CLSM.

[0083] Dark toxicity of TTPA molecules was evaluated using 4T1 cells in the absence of white light irradiation. 4T1 cells were used at 2 × 10⁶ cells per well. 3Cells were seeded in 96-well plates and incubated with intact culture medium for 24 h. Subsequently, the original medium was replaced with fresh medium containing a gradient of TTPA compounds (3.125, 6.25, 12.5, 25, 50, 100 μM). After 48 h, 10 μL CCK8 was added to each well, and the plates were incubated for 2 h. Finally, the absorbance was read at 450 nm on the microplate. Cytotoxicity was calculated using the same method as described above.

[0084] The photodynamic therapy efficacy of TTPA molecules was evaluated using Calcein AM / PI staining and the CCK8 assay. Results were as follows: Figure 15 As shown. From Figure 15 (ac) It can be seen that when the TTPA molecule dose is greater than 50 μM, the proliferation inhibition rate of almost all cells is higher than 90%. Furthermore, even at relatively low concentrations (3.125, 6.25 μM), cell viability is still less than 20%. Among the three tumor cell types tested (B16F10, CT26, 4T1), 4T1 cells had the highest mortality rate. The dark toxicity of TTPA molecules under no-light conditions is shown in the results... Figure 15 (d) shows the opposite of the phototoxicity results: in the absence of light, even 4T1 cells treated with 25 μM TTPA showed no significant cytotoxicity, while under the same concentration of light, almost all cells died. Therefore, the high tumor cell killing efficacy of TTPA molecules is attributed to the generation of large amounts of ROS.

[0085] Meanwhile, Calcein AM / PI staining results ( Figure 15 (e) shows that almost all 4T1 cells in the TTPA molecule were stained red by PI, indicating that almost all cells were killed. However, the PBS and PBS+Light groups showed near-complete green fluorescence, indicating no phototoxicity.

[0086] In summary, this invention synthesizes a series of aggregation-induced emission photosensitizer TTPA molecules, which exhibit strong fluorescence in the aggregated state and can simultaneously generate type I (·OH) and type II (·OH) upon white light excitation. 1 O2)ROS. When rapidly internalized by cancer cells, they can induce photodynamic therapy (PDT) under both aerobic and anaerobic conditions. Furthermore, molecular docking, DLS, and TEM results showed that TTPA molecules can assemble with albumin in culture medium or serum to form TTPA-NP nanoparticles. The assembled nanoassemblies, TTPA-NPs, can be targeted and delivered to intracellular mitochondria, further generating large amounts of ROS, even more effectively than Ce6, leading to widespread tumor cell damage. Therefore, this work reveals that TTPA molecules can produce a prominent PDT effect and are ideal candidate photosensitizers for near-infrared imaging-guided tumor therapy.

[0087] Example 2

[0088] This embodiment provides a method for preparing aggregation-induced emission nanoparticles, which includes the following steps:

[0089] S1. 0.5 g of methylpyridine and 1.2 g of 9-bromononanoic acid were mixed in 60 mL of acetone and refluxed overnight for 12 h. After cooling to room temperature (25 °C), a large amount of white solid precipitated. The solvent was removed by vacuum filtration to obtain a solid substance. The solid substance was washed three times with acetone to obtain an intermediate product, which was methylpyridine bromide.

[0090] S2, 4 mmol of the intermediate obtained in S1 is reacted with 10 mmol of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde. Dissolved in 200 mL of ethanol, then 4 drops of piperidine were added as a catalyst, and the mixture was refluxed overnight for 12 h. After cooling to room temperature (25 °C), the solvent was removed by vacuum evaporation for 30 min at a vacuum of -0.08 MPa and a temperature of 40 °C to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 8:1 to obtain the red aggregation-induced emission photosensitizer TTPA molecule. The molecular formula of the aggregation-induced emission photosensitizer TTPA molecule is C 38 H 39 BrN2O2S, the molecular structural formula is:

[0091]

[0092] S3. Dissolve 0.0067g of the aggregation-induced emission photosensitizer TTPA molecule obtained in S2 in 10mL of DMSO to prepare a TTPA stock solution with a concentration of 1mM; then, take 100μL of the stock solution and mix it with 100μL of bovine serum albumin (BSA) stock solution, and dilute it with PBS to a final volume of 10mL to obtain aggregation-induced emission nanoparticles, i.e., nano-assemblies TTPA-NPs;

[0093] The bovine serum albumin stock solution was prepared by dissolving 0.6645 g of bovine serum albumin in 10 mL of PBS, and the concentration of the albumin stock solution was 1 mM. The synthesis principle of the nano-assemblies TTPA-NPs is as follows: Figure 14 As shown;

[0094] The synthetic route for the aggregation-induced emission photosensitizer TTPA molecule described in S2 is as follows:

[0095]

[0096] Example 3

[0097] This embodiment provides a method for preparing aggregation-induced emission nanoparticles, which includes the following steps:

[0098] S1. 2.8 g of methylpyridine and 7 g of 9-bromononanoic acid were mixed in 150 mL of acetone and refluxed overnight for 14 h. After cooling to room temperature (25 °C), a large amount of white solid precipitated. The solvent was removed by vacuum filtration to obtain a solid substance. The solid substance was washed three times with acetone to obtain an intermediate product, which was methylpyridine bromide.

[0099] S2, 10 mmol of the intermediate obtained in S1 is reacted with 3.2 mmol of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde. Dissolved in 50 mL of ethanol, then 6 drops of piperidine were added as a catalyst, and the mixture was refluxed overnight for 14 h. After cooling to room temperature (25 °C), the solvent was removed by vacuum evaporation for 30 min at a vacuum of -0.08 MPa and a temperature of 40 °C to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 8:1 to obtain the red aggregation-induced emission photosensitizer TTPA molecule. The molecular formula of the aggregation-induced emission photosensitizer TTPA molecule is C0. 38 H 39 BrN2O2S, the molecular structural formula is:

[0100]

[0101] S3. Dissolve 0.0067g of the aggregation-induced emission photosensitizer TTPA molecule obtained in S2 in 10mL of DMSO to prepare a TTPA stock solution with a concentration of 1mM; then, take 100μL of the stock solution and mix it with 100μL of bovine serum albumin (BSA) stock solution, and dilute it with PBS to a final volume of 10mL to obtain aggregation-induced emission nanoparticles, i.e., nano-assemblies TTPA-NPs;

[0102] The bovine serum albumin stock solution was prepared by dissolving 0.6645 g of bovine serum albumin in 10 mL of PBS, and the concentration of the albumin stock solution was 1 mM. The synthesis principle of the nano-assemblies TTPA-NPs is as follows: Figure 14 As shown;

[0103] The synthetic route for the aggregation-induced emission photosensitizer TTPA molecule described in S2 is as follows:

[0104]

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing aggregation-induced emission nanoparticles, characterized in that, The method includes the following steps: S1. Methylpyridine and 9-bromononanoic acid are mixed in acetone and refluxed overnight. After cooling to room temperature, the mixture is filtered under reduced pressure to obtain a solid. The solid is washed with acetone several times to obtain an intermediate product, which is methylpyridine bromide. S2. The intermediate product obtained in S1 is dissolved in ethanol with 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde to obtain a mixture. A few drops of piperidine are added to the mixture, and the mixture is refluxed overnight. After cooling to room temperature, it is evaporated under reduced pressure to obtain a crude product. The crude product is purified by column chromatography to obtain the aggregation-induced emission photosensitizer TTPA molecule. The molecular formula of the aggregation-induced emission photosensitizer TTPA molecule is C38H39BrN2O2S, and the molecular structure is as follows: ； S3. Dissolve the aggregation-induced emission photosensitizer TTPA molecules obtained in S2 in dimethyl sulfoxide to obtain TTPA stock solution; mix the TTPA stock solution with albumin stock solution and dilute with phosphate buffer solution to obtain aggregation-induced emission nanoparticles.

2. The preparation method according to claim 1, characterized in that, The solid-liquid ratio of methylpyridine, 9-bromononanoic acid and acetone in S1 is (0.5~2.8) g : (1.2~7) g : (60~150) mL; the number of washes is 3.

3. The preparation method according to claim 1, characterized in that, The overnight reflux reaction time described in S1 and the overnight reflux reaction time described in S2 are both 10~14h.

4. The preparation method according to claim 1, characterized in that, The molar ratio of the intermediate product and 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde in S2 is (4.0~10):(3.2~10); the concentration of the intermediate product in the mixture is 0.02~0.2 mol / L.

5. The preparation method according to claim 1, characterized in that, The adsorbent for column chromatography described in S2 is silica gel, and the eluent is dichloromethane and methanol, with a volume ratio of 8:1 between the dichloromethane and methanol.

6. The preparation method according to claim 1, characterized in that, The number of piperidine drops in S2 is 3 to 6 drops; the vacuum degree of the reduced pressure evaporation is -0.08 MPa, the temperature is 40°C, and the time is 30 min.

7. The preparation method according to claim 1, characterized in that, The solid-liquid ratio of the aggregation-induced emission photosensitizer TTPA molecule and dimethyl sulfoxide in S3 is 0.0067 g: 10 mL; the concentration of the TTPA stock solution is 1 mM; and the volume ratio of the TTPA stock solution, albumin stock solution and phosphate buffer solution is 1:1:

98.

8. The preparation method according to claim 1, characterized in that, The albumin stock solution described in S3 is prepared by dissolving albumin in a phosphate buffer solution, wherein the solid-liquid ratio of albumin to the phosphate buffer solution is 0.6645 g: 10 mL; and the concentration of the albumin stock solution is 1 mM.

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