Lipid droplet targeted two-photon absorption material with aggregation-induced emission property, preparation method of lipid droplet targeted two-photon absorption material and application of lipid droplet targeted two-photon absorption material in osteosarcoma resistance

Abstract

The invention provides a lipid droplet targeted two-photon absorption material with aggregation-induced emission property, a preparation method thereof and application of the lipid droplet targeted two-photon absorption material in osteosarcoma resistance, and relates to the technical field of bioluminescent materials. According to the invention, the application of the material BMQMM in biological fluorescence imaging and photodynamic therapy is researched. The BMQMM provided by the invention has excellent light stability, remarkable aggregation-induced emission characteristic and I-type ROS generation capability, and can be used as an ideal candidate photosensitizer for efficient PDT of tumors. In addition, the BMQMM has good specific targeting property and fluorescence imaging capability on the lipid droplets, and can be used as a lipid droplet targeting fluorescence probe. Meanwhile, the BMQMM has excellent two-photon fluorescence imaging capability on living bodies, cells and fat-rich tissue slices, and can be developed as a fluorescence indicator for biological fluorescence imaging. Finally, the BMQMM has good biocompatibility, and the PDT mediated by the BMQMM shows excellent anti-tumor ability in vivo and in vitro. The BMQMM provided by the invention has a huge application prospect in biological fluorescence imaging and photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of bioluminescent materials, and in particular to a lipid droplet-targeted two-photon absorption material with aggregation-induced luminescence properties, a preparation method thereof, and an application thereof in anti-osteosarcoma. Background Art

[0002] Photodynamic therapy (PDT) is a non-destructive, reliable cancer treatment method with high spatiotemporal precision. It uses light sources to activate photosensitizers (PSs) to release cytotoxic reactive oxygen species (ROS) through type I (electron transfer) or type II (energy transfer) reactions. ROS rapidly interact with biological substances and exert anti-tumor effects by inducing cell apoptosis or necrosis, vascular damage, and immune response.

[0003] To date, many types of photosensitizers have been reported, including inorganic nanomaterials, metal-organic frameworks, semiconductor polymer nanoparticles, and pure organic molecules. However, the potential cytotoxicity and complex synthesis of inorganic and metal materials have largely limited their applications. In contrast, pure organic molecules have irreplaceable advantages such as good biocompatibility, structural certainty, and functional modifiability. In addition, traditional organic photosensitizers have aggregation-caused quenching (ACQ) characteristics, weak photostability, and narrow Stokes shift, which limit their clinical applications. Different from the ACQ characteristics of conventional organic photosensitizers, the aggregation-induced emission (AIE) characteristics endow PS with more stable fluorescence properties, achieving excellent properties such as good high-brightness fluorescence imaging, high stability, high ROS generation efficiency, good biocompatibility, tumor targeting, and efficient tumor cell killing effects.

[0004] Lipid droplets are organelles present in most cells of organisms, consisting of a neutral lipid (glyceride, sterol) core and a phospholipid monolayer inlaid with surface proteins. Studies have found that lipid droplets are highly dynamic, and their number, size and composition sometimes vary greatly in the same cell line, which usually reflects the metabolic status of the cell. In fact, lipid droplets play a key role in maintaining cellular energy balance, lipid homeostasis and signal transduction. Evidence has shown that lipid droplets play a vital role in the control of energy, metabolism and signal transduction in tumor cells. One of the hallmark changes of tumor cells is increased glucose catabolism, which leads to a hypoxic microenvironment in the tumor when blood supply is insufficient. In order to survive in this special environment, tumor cells have acquired adaptability, such as initiating a self-degradation and recycling mechanism called autophagy, as well as the accumulation of lipid droplets. Monitoring the dynamic changes of lipid droplets in cells and tissues is very important for the pathological study and clinical treatment of diseases related to lipid droplets.

[0005] Therefore, it is necessary to develop materials that can target lipid droplets and possess AIE properties and two-photon absorption properties for photodynamic therapy against osteosarcoma and bioluminescence imaging. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies of the prior art, the present invention provides a lipid droplet-targeted two-photon absorption material with aggregation-induced luminescence properties, a preparation method thereof, and an application thereof in anti-osteosarcoma.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] In the first aspect, the present invention provides a lipid droplet-targeted two-photon absorption material with aggregation-induced luminescence properties, the material is referred to as BMQMM, and the structural formula of BMQMM is shown in formula (I):

[0011]

[0012] In a second aspect, the present invention provides a method for preparing BMQMM, which specifically comprises the following steps:

[0013] (1) 1,2-bis(4-methoxyphenyl)ethane-1,2-dione and 3,4-diaminobenzophenone are mixed in a molar ratio of 1:1-2 and dissolved in acetic acid, and stirred and refluxed at 110°C for more than 3 hours; then cooled to room temperature, filtered, and washed with acetic acid to obtain an orange-red solid, which is dried in a vacuum oven for more than 12 hours to obtain 2,3-bis(4-methoxyphenyl)quinolin-6-yl(phenyl)methanone, referred to as BMQM. The structural formula of BMQM is shown in formula (II):

[0014]

[0015] (2) BMQM and malononitrile were mixed in a molar ratio of 1:18-30 and dissolved in dry chlorobenzene. Pyridine and TiCl were added under nitrogen atmosphere. 4 , then heated to reflux overnight; after the reaction was completed, chlorobenzene was removed by vacuum and the reactants were treated with deionized water; then the reactants were extracted with dichloromethane, and after the extraction was completed, anhydrous Na 2 SO 4 The organic layer was dried; the organic phase was then separated and concentrated to dryness, and purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate as eluent to obtain BMQMM.

[0016] In a third aspect, the present invention provides the use of the above-mentioned material in preparing a fluorescent probe targeting lipid droplets.

[0017] In a fourth aspect, the present invention provides the use of the above-mentioned material as a fluorescent agent in biological fluorescence imaging, wherein the biological fluorescence imaging includes cell imaging, living body imaging, and slice imaging.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned material in the preparation of drugs for photodynamic therapy.

[0019] (III) Beneficial effects

[0020] The BMQMM provided by the present invention has excellent optical stability, which is conducive to the implementation and in situ detection of cell morphology; at the same time, BMQMM has significant aggregation-induced luminescence characteristics and the ability to generate type I ROS, and can be used as an ideal candidate photosensitizer for efficient PDT of tumors.

[0021] The BMQMM provided by the present invention has good specific targeting and fluorescence imaging capabilities for lipid droplets, and BMQMM can be used as a fluorescent probe for lipid droplet targeting.

[0022] The BMQMM provided by the present invention has excellent two-photon fluorescence imaging capability for living bodies, cells and fat-rich tissue slices, and can be developed as a fluorescence indicator for biological fluorescence imaging.

[0023] The BMQMM provided by the present invention has good biocompatibility, and the PDT mediated by BMQMM shows excellent anti-tumor ability both in vivo and in vitro.

[0024] The lipid droplet-targeted two-photon absorption material BMQMM with aggregation-induced luminescence characteristics provided by the present invention has great application prospects in bioluminescence imaging and photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis route of BMQMM.

[0026] Figure 2 Results of characterization of optical properties of BMQMM; (A) Solid-state luminescence spectrum of BMQMM. Illustration: Images of BMQMM under natural light and ultraviolet light; (B) Luminescence spectrum analysis of BMQMM in H2O / EtOH mixed solutions with different ratios; (C) Luminescence power of BMQMM at 540nm wavelength varies with the change of fw parameters; BMQMM immersed in H 2 (D) BMQMM in H O and EtOH, while exposed to ultraviolet (UV) light; (D) BMQMM in H 2 Absorption spectra in H O and EtOH. The figure shows that BMQMM 2 Absorption spectrum in O.

[0027] Figure 3 The results of ROS detection in aqueous solution of compound BMQMM are shown below: (A) DCFH (5 μM) was used as an indicator to detect ROS under white light irradiation (30 mW cm -2 ) under the total ROS generated, I 0 and I are the fluorescence intensities of the indicator before and after irradiation, respectively; (B) DHR123 (5 μM) was used as an indicator to detect the fluorescence intensity of the indicator under white light irradiation (30 mW cm -2 ) 2 O 2 I 0 and I are the indicator fluorescence intensities before and after irradiation, respectively.

[0028] Figure 4 These are the photostability experimental results of BMQMM, BODIPY 493 / 503 and Nile Red; the laser excitation wavelength of BMQMM is 405nm, the laser excitation wavelength of BODIPY 493 / 503 is 488nm, and the laser excitation wavelength of Nile Red is 543nm; the corresponding emission range of BMQMM is 433-577nm, the corresponding emission range of BODIPY 493 / 503 is 500-600nm, and the corresponding emission range of Nile Red is 550-690nm.

[0029] Figure 5 MG-63 cells labeled for BMQMM co-localized with various commercially available organelle probes, including BODIPY 493 / 503, Nile Red, Cell Mask Deep Red, Lyso-Tracker Red, and Mito-Tracker Red. Before staining, cells were treated with 50 μmol / L oleic acid for 6 hours. Scale bar: 20 μm.

[0030] Figure 6 Application of BMQMM in bioluminescence imaging; A and B are single-photon CLSM images of zebrafish in vivo and zebrafish embryos treated with BMQMM; C and D are single-photon and two-photon CLSM images of multicellular spheroids of MG-63 cells treated with BMQMM at different depths along the z-axis under excitation of 405 and 880 nm, respectively, to evaluate penetration, scale bar: 50 μm; E and F are single-photon CLSM images of multicellular spheroids of MG-63 cells treated with BMQMM at 405 nm; G and H are two-photon CLSM images of multicellular spheroids of MG-63 cells treated with BMQMM at 880 nm; I and J are three-dimensional reconstructions of frozen sections of mouse liver and kidney labeled with BMQMM 880 nm; scale bar in A: 300 μm; scale bar in B, C, D: 50 μm.

[0031] Figure 7 In vivo fluorescence imaging of subcutaneous K7M2 osteosarcoma cells at different time points; A: Time-dependent fluorescence imaging. Excitation filter length: 420nm; emission wavelength: 570nm; B: Fluorescence imaging of different tissues such as mouse heart, liver, spleen, lung, kidney, intestine, and tumor after 24 hours of BMQMM staining.

[0032] Figure 8 A is the detection of ROS generation of MG-63 cells by DCFH-DA and DHR 123 in different groups under white light irradiation; Figure 8 B shows the changes of mitochondrial membrane potential in each group before and after white light irradiation; Figure 8 C is the Calcein-AM / PI detection of MG-63 cells and K7M2 cells under white light irradiation.

[0033] Fig. 9 Two-photon DCFH-DA detection of BMQMM-induced time-dependent ROS generation in MG-63 cells; scale bar: 50 μm.

[0034] Fig.10 Calcein AM / PI test in MG-63 and K7M2 cells induced by BMQMM after irradiation with low-power (30 mW cm-2) white light for 30 min; scale bar: 50 μm.

[0035] Fig.11 Time-dependent test of Calcein-AM / PI induction in MG-63 cells by BMQMM under two-photon irradiation; scale bar: 50 μm.

[0036] Fig.12 Effects of BMQMM-mediated PDT on tumor model mice; A: Changes in mouse body weight during the entire treatment process (mean±SD, n=4), ns: not significant; B: Tumor volume change curves of tumor-bearing mice under different treatment regimens (mean±SD, n=4), ****p<0.0001, ns: not statistically significant, T test; C: Changes in tumor weight after the entire treatment process (mean±SD, n=4), ****p<0.0001, ns: not statistically significant, T test; D: Photos of subcutaneous K7M2 tumors in mice before and after 16 days of BMQMM+Light treatment; F: Photos of tumors removed from mice in each group on the 16th day.

[0037] Fig.13 H&E staining analysis of tissues of mice in each group after different treatments. Scale bar: 100 μm. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] Preparation and characterization of lipid droplet-targeted two-photon absorption material BMQMM with aggregation-induced emission properties

[0041] 1 Preparation of BMQMM

[0042] The synthetic route of BMQMM is as follows Figure 1 As shown, the preparation is carried out according to the following steps:

[0043] (1) 1,2-bis(4-methoxyphenyl)ethane-1,2-dione (5 mmol) and 3,4-diaminobenzophenone (5 mmol) were dissolved in 10 mL of acetic acid; the mixed solution was stirred and refluxed at 110°C for 3 hours. Then it was cooled to room temperature, filtered, and washed with 10 mL of acetic acid to obtain an orange-red solid, which was dried in a vacuum oven for more than 12 hours to obtain 2,3-bis(4-methoxyphenyl)quinolin-6-yl(phenyl)methanone (BMQM);

[0044] (2) BMQM (1 mmol) and malononitrile (19.60 mmol) were dissolved in 50 mL of dry chlorobenzene. Under nitrogen, 0.75 mL of pyridine and 0.50 mL of TiCl4 were added to the solution, followed by heating and reflux overnight. After the reaction was completed, the chlorobenzene was removed by vacuum and the reactants were treated with deionized water. The reactants were then extracted with dichloromethane and anhydrous Na 2 SO 4 The organic layer was dried; the organic phase was then separated and concentrated to dryness, and purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate as eluent to obtain BMQMM.

[0045] BMQMM's H NMR, C NMR and MS results: 1 H NMR (600 MHz, CDCl 3 )δ(ppm):8.20(d,J=6Hz,2H),7.75(d,J=6Hz, 1 H),7.61(s,1H),7.51(m,8H),6.88(t,J=12Hz,4H),3.84(s,6H). 13 C NMR (600MHz, CDCl 3 )δ(ppm):173.74,160.79,155.28,154.68,142.53,140.12,136.68,135.88,132.90,132.45 ,131.42,131.26,130.43,129.83,129.08,113.93,82.82,55.35.HRMS(ESI-TOF)m / z:([M+H] + )Calcd for C 34 H 28 N 6 ,495.1743;Found:495.1951.

[0046] Optical properties characterization of 2BMQMM

[0047] 2.1 Test items

[0048] (1) UV-visible absorption and photoluminescence spectrum test of BMQMM

[0049] BMQMM was dissolved in EtOH to a concentration of 10 μM, 2 ml was taken into a quartz cuvette, and placed in a UV spectrophotometer and a fluorescence spectrometer for testing, and its absorption and emission spectra were recorded.

[0050] (2) AIE property test of BMQMM

[0051] BMQMM was dissolved in EtOH / water mixed solvents with different water contents (10 μM), excited at its maximum absorption wavelength, and its emission spectrum was recorded.

[0052] (3) Dynamic light scattering to detect the particle size of nanoparticles

[0053] BMQMM was dissolved in an aqueous solution containing 1 vol% EtOH to a concentration of 10 μM, and the concentration was measured at room temperature using a nanoparticle size potential analyzer (Zetasizer Nano-S90).

[0054] 2.2 Results

[0055] BMQMM emits weak fluorescence in a dispersed state in pure EtOH solution, but emits bright yellow-green fluorescence in a solid state ( Figure 2 A), indicating that BMQMM has AIE activity. To further confirm the AIE properties, we tested the photoluminescence (PL) changes of BMQMM in EtOH / water combinations. In water mixing systems with water fractions (fw) ranging from 0 to 60%, the emission intensity hardly increased. At 80% fw, the emission of the system increased significantly, with an intensity increase of 9 times compared to that of pure EtOH solution at 540nm ( Figure 2 B and Figure 2 C). Since BMQMM is insoluble in water, it may aggregate in high fw mixtures. In water-based solutions, obvious aggregation is observed in the visible region ( Figure 2 D). The average hydrodynamic diameter of these particles was approximately 21.2 nm as determined by dynamic light scattering (DLS).

[0056] Example 2

[0057] BMQMM detection of ROS generation in aqueous solution

[0058] 1. Detection method

[0059] 1.1 Determination of ROS generation efficiency

[0060] After DCFH-DA interacts with ROS, the fluorescence intensity increases. Therefore, we use 2',7'-dichlorodihydrofluorescein (DCFH-DA) as an indicator to measure the generation efficiency of ROS in extracellular ROS detection. The specific method is:

[0061] (1) Solution preparation: Using ethanol as solvent, prepare DCFH-DA into a 1 mM mother solution, and add 2 mL of 0.01 M NaOH to obtain a mixed solution, and then activate the mixed solution for 30 minutes without light; then add 10 mL of pre-cooled saline to make the pH value reach 7.4 to obtain an indicator solution, and then activate the indicator solution on ice without light for later use. Dissolve BMQMM in DMSO for later use to make the mother solution concentration 1 mM.

[0062] (2) The activated DCFH-DA and BMQMM were dissolved in physiological saline to a final concentration of 5 μM for DCFH-DA and 10 μM for BMQMM. -2 ROS were detected under white light. Using 488 nm as the excitation light wavelength and 500-600 nm as the emission spectrum range, the fluorescence signal of DCFH-DA was observed, and the generation efficiency of ROS was evaluated by recording the change of fluorescence intensity at 525 nm.

[0063] 1.2O 2 ·- Determination of generation efficiency

[0064] DHR 123 was used as an indicator to detect the -2 ) in aqueous solution 2 ·- DHR123 and BMQMM were dissolved in physiological saline to make the concentration of DHR123 5μM and the concentration of BMQMM 10μM; under white light irradiation (30mW cm -2 ) 2 ·- Using 495 nm as the excitation light source wavelength and 500-600 nm as the emission spectrum range, the fluorescence signal of DHR 123 was observed, and the changes in fluorescence intensity at 525 nm were recorded to evaluate O 2 ·- The generation efficiency

[0065] 2 Results

[0066] We used the indicator as the control group and the indicator + BMQMM as the experimental group to evaluate the ability of BMQMM to generate reactive oxygen species. Figure 3 As shown in A, compared with the DCFH-DA group, in the BMQMM+DCFH-DA group, after white light irradiation, the fluorescence of DCFH-DA was significantly enhanced, and the fluorescence intensity of DCFH-DA remained stable within 90s, producing an emission enhancement of up to 194 times in the absorption spectrum of 525nm, and BMQMM showed a very high ROS generation efficiency. Figure 3As shown in Figure B, compared with the DHR 123 group, in the BMQMM+DHR 123 group, after white light irradiation, the fluorescence of DHR 123 was significantly enhanced, and the fluorescence intensity increased by more than 95 times. The above results show that the BMQMM provided by the present invention has a significant ability to generate type I ROS, making it an ideal candidate photosensitizer for efficient PDT of tumors.

[0067] Example 3

[0068] Photostability determination of BMQMM

[0069] Photostability is an important indicator for long-term tracing imaging. Photobleaching is common in traditional photosensitizers. In order to study the anti-photobleaching ability of BMQMM, two commercial lipid droplet dyes BODIPY493 / 503 and Nile Red were used as controls to perform time-series continuous CLSM scanning. Figure 4 As shown in the figure, after 150 consecutive scans (187.85s), the signal intensity of BMQMM decayed by less than 2% (98.44%), while the signal intensity decay of BODIPY493 / 503 was 40% and that of Nile Red was 77%. The above results show that BMQMM has excellent photostability, which is conducive to real-time and in situ monitoring of cell morphology.

[0070] Example 4

[0071] Application of BMQMM as a lipid droplet-targeted fluorescent probe

[0072] We used laser confocal microscopy to verify the feasibility of BMQMM as a fluorescent probe specifically targeting lipid droplets. MG-63 cells treated with oleic acid for 6 hours were exposed to BMQMM at a concentration of 5 μmol / L for 1 hour, and then co-localized with BODIPY 493 / 503 and Nile Red, respectively.

[0073] The results are as follows Figure 5 As shown in the figure, the fluorescence of BMQMM and BODIPY 493 / 503 was almost complete, with Mander coefficients (MCs) as high as 0.929. In addition, a similar trend was found in the co-staining of BMQMM and Nile Red. Subsequently, several organelle probes, such as Cell Mask Deep Red, LysoTracker Red, and Mito-Tracker Red, were co-stained with BMQMM, and the MCs were 0.329, 0.354, and 0.483, respectively. The above results indicate that BMQMM has good specific targeting and fluorescence imaging capabilities for lipid droplets, and BMQMM can be used as a fluorescent probe for lipid droplet targeting.

[0074] Example 5

[0075] Application of BMQMM in bioluminescence imaging

[0076] To further investigate the potential of BMQMM in bioluminescence imaging, we selected live zebrafish eggs, zebrafish cell spheroids, and mouse organ cryosections as models. Zebrafish have a short reproductive cycle and fast growth rate, making them an ideal model for studying lipid metabolism. BMQMM was used to perform single-photon imaging of zebrafish eggs (3 days old) and zebrafish. Figure 6 As shown in AB, the changes in their outlines, lipid distribution, and lipid droplets at different developmental stages are clearly shown.

[0077] Three-dimensional (3D) spheroids serve as a bridge between cells and in vivo imaging. Unlike traditional cell culture, 3D cell culture reproduces the in vivo environment of cells, makes up for the shortcomings of monolayer culture, and makes the cell response to photosensitizers closer to the in vivo environment. Therefore, we used the spheroidization ability of MG-63 cells to culture MG-63 spheroids. After incubating MG-63 spheroids with BMQMM (5 μmol / L) for 2 hours, single-photon and two-photon CLSM imaging of the spheroids was performed using Zeiss LSM 980, as shown in Figure 2. Figure 6 Then, we selected single photon scanning depths of 50 μm and 100 μm ( Figure 6 EF) and two-photon ( Figure 6 GH) Confocal images. Compared with single-photon imaging of spheroids, two-photon imaging has greater penetration power, making it possible to study lipid droplets in tissues and organs.

[0078] We used BMQMM to perform two-photon imaging of lipid-rich mouse liver and kidney tissues ( Figure 6 IJ). Three-dimensional reconstruction of liver and kidney tissue clearly shows the microstructure of liver sinusoids, renal tubules, and glomeruli.

[0079] The above results indicate that BMQMM has excellent two-photon fluorescence imaging capabilities for living organisms, cells, and fat-rich tissue sections, which makes it possible to apply BMQMM to biological fluorescence imaging.

[0080] Example 6

[0081] Application of BMQMM in in vivo imaging of animals

[0082] In vivo fluorescence imaging provides an experimental basis for the practical application of tumor navigation drugs. Fluorescence imaging technology was used to verify the imaging ability of BMQMM in animals at different time intervals and for a long time using the Balb / c mouse K7M2 cell subcutaneous tumor model. With 420nm as the excitation wavelength and 570nm as the emission wavelength, the changes in fluorescence intensity in mice within 24 hours were observed. Figure 7As shown in A, the fluorescence intensity at the tumor location hardly changed within 24 hours after BMQMM staining. Figure 7 B) During imaging, it was found that only the tumor site retained the fluorescent signal. This may be due to the abundant lipid droplets in the tumor site, the specific targeting of BMQMM to lipid droplets, and the slow metabolism of BMQMM in the tumor site, which provides a basis for the application of BMQMM as a photosensitizer in PDT.

[0083] Example 7

[0084] Study on the Photodynamic Effect of BMQMM in Vitro

[0085] 1. Reactive oxygen production

[0086] Using DCFH-DA as an indicator, we studied the ROS generation of BMQMM in MG63 cells under white light irradiation. We divided the cells into four groups: Saline, Saline+light (L), BMQMM, and BMQMM+L. Figure 8 As shown in A, the CLSM image shows that only the cells treated with BMQMM+L produced bright green fluorescence, while this signal was not detected in other control groups, proving that BMQMM in MG63 cells produced significant ROS after light activation. To further clarify the type of ROS, DHR123 was used as an indicator. According to the same grouping treatment, the MG63 cells in the experimental group treated with BMQMM+L produced bright green fluorescence signals, while the other three groups produced almost no green signals, indicating that BMQMM can produce type I reactive oxygen species after light activation.

[0087] In order to investigate the ROS production of BMQMM under two-photon excitation, MG63 cells were irradiated with DCFH-DA as an indicator and 880 nm two-photon as an excitation light source. Fig. 9 As shown, with the continuous increase of irradiation time, the green signal intensity in MG63 cells also continued to increase, verifying the ROS-producing ability of BMQMM under two-photon excitation and providing a basis for two-photon photodynamic therapy.

[0088] 2. Mitochondrial membrane potential detection

[0089] BMQMM produces a large amount of ROS under light conditions, which will destroy the cell redox homeostasis and induce mitochondrial stress response. We use the JC-1 mitochondrial membrane potential staining kit for early apoptosis detection. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential (ΔΨm). It can detect the mitochondrial membrane potential of cells, tissues or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form polymers (J-aggregates), which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria. At this time, JC-1 is a monomer and can produce green fluorescence.

[0090] Figure 8 As shown in B, MG63 cells in four different treatment groups were stained with JC-1. Only in the BMQMM+L experimental group was the mitochondrial red fluorescence signal intensity reduced and the green fluorescence signal slightly increased, indicating that BMQMM can reduce mitochondrial membrane potential and induce cell apoptosis under light conditions.

[0091] 3. Tumor cell killing

[0092] To observe the phototherapy effect of BMQMM, MG63 cells and K7M2 cells in the four different treatment groups were stained with Calcein AM / PI to distinguish between dead and live cells. As expected, Calcein green fluorescence was observed in the three control groups, while in the BMQMM+L experimental group, the green fluorescence was not observed under low power (30 mW cm -2 ) After irradiation with white light for 30 minutes, about half of the cells showed PI red fluorescence. Fig.10 As shown in Figure 2, when the illumination time reached 1 h, almost all cells showed obvious PI red fluorescence ( Figure 8 C) The above results show that PDT mediated by BMQMM has a high killing efficiency on tumor cells.

[0093] We further studied the tumor killing ability of BMQMM under two-photon excitation by irradiating MG-63 cells induced by BMQMM stained with calcein / propidium iodide with 880nm two-photon laser, and performed live-death imaging at different time points (0min, 10min, 20min, 30min, and 40min time intervals). Fig.11As shown in the figure, with the increase of two-photon laser irradiation time, the green fluorescence signal intensity of chlorophyll decreases continuously, while the red fluorescence signal intensity of PI increases continuously. The above results show that BMQMM has excellent two-photon photodynamic properties, which provides conditions for the application of BMQMM in anti-tumor photodynamic therapy.

[0094] Example 8

[0095] Application of BMQMM in photodynamic therapy for osteosarcoma

[0096] The Balb / c mouse K7M2 cell subcutaneous tumor model was used as the research object to evaluate the tumoricidal activity of BMQMM against subcutaneous K7M2 osteosarcoma under white light irradiation.

[0097] The subcutaneous tumor model was randomly divided into Saline, Saline + light (L), BMQMM and BMQMM + L, with 4 mice in each group. The Saline and Saline + light (L) groups were given local administration of normal saline, and the BMQMM and BMQMM + L groups were given local administration of BMQMM (5 mg / Kg). 4 hours after administration, the Saline + light (L) and BMQMM + L groups were irradiated with white light (200 mW cm -2 ,10min).

[0098] During the experiment, there was no significant difference in body weight between the groups of mice (P>0.05). Fig.12 A). Fig.12 B is the tumor growth curve of mice in each group. As time went on, the tumor volume of mice in the Saline, Saline+light (L), and BMQMM groups increased rapidly. After 16 days of treatment, the tumor volume increased by about 4 times compared with the original value. There was no significant difference in tumor volume among the three groups (P>0.05). Compared with the three control groups, the tumor volume of mice in the BMQMM+L group was significantly reduced (P<0.05), and the tumor of mice in the BMQMM+L group was completely eradicated on the 10th day ( Fig.12 C. Fig.12 E), Importantly, no tumor recurrence was found in mice in the BMQMM+L group during the experiment, and only scars were left at the treatment site after 16 days of treatment ( Fig.12 D). The above results indicate that BMQMM-mediated PDT has significant anti-tumor properties.

[0099] The mice in the four groups did not show abnormal weight changes, and H&E results of the main organs showed no obvious damage ( Fig.13 ), confirming that BMQMM has good biosafety and negligible systemic side effects

[0100] In summary, the BMQMM provided by the present invention has good biocompatibility, and the PDT mediated by BMQMM shows excellent anti-tumor performance in the treatment of osteosarcoma.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid droplet-targeted two-photon absorption material with aggregation-induced luminescence properties, the material is referred to as BMQMM, and the structural formula of BMQMM is shown in formula (I):

2. The method for preparing the material BMQMM according to claim 1, characterized in that: The specific steps include: (1) 1,2-bis(4-methoxyphenyl)ethane-1,2-dione and 3,4-diaminobenzophenone are mixed in a molar ratio of 1:1-2 and dissolved in acetic acid, and stirred and refluxed at 110°C for more than 3 hours; then cooled to room temperature, filtered, and washed with acetic acid to obtain an orange-red solid, which is dried in a vacuum oven for more than 12 hours to obtain 2,3-bis(4-methoxyphenyl)quinolin-6-yl(phenyl)methanone, referred to as BMQM; (2) BMQM and malononitrile were mixed in a molar ratio of 1:18-30 and dissolved in dry chlorobenzene. Pyridine and TiCl4 were added under nitrogen atmosphere, followed by heating and reflux overnight. After the reaction was completed, chlorobenzene was removed by vacuum and the reactants were treated with deionized water. The reactants were then extracted with dichloromethane. After the extraction was completed, the organic layer was dried with anhydrous Na2SO4. The organic phase is then separated and concentrated to dryness, and purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate as eluent to obtain BMQMM.

3. Use of the material BMQMM as claimed in claim 1 in preparing a fluorescent probe targeting lipid droplets.

4. The use of the material BMQMM as claimed in claim 1 as a photosensitizer in bioluminescence imaging, wherein the bioluminescence imaging includes cell imaging, in vivo imaging, and slice imaging.

5. Use of the material BMQMM as claimed in claim 1 in the preparation of drugs for photodynamic therapy.

Images