Method for marking in situ and synchronously promoting secretion of extracellular vesicles

Abstract

The invention discloses a method for in-situ labeling and synchronous promotion of extracellular vesicle secretion, and a preparation method of a glucose modified photosensitizer comprises the following steps: carrying out an amidation reaction on glucosamine, a photosensitizer, benzotriazole-N, N, N ', N'-tetramethylurea hexafluorophosphate, 4-(dimethylamino) pyridine and N, N-diisopropylethylamine in a solvent, cooling to room temperature, filtering, washing, and drying to obtain the glucose modified photosensitizer. According to the glucose-modified photosensitizer and the preparation method thereof disclosed by the invention, the glucose is modified by the photosensitizer to be loaded into the EV by utilizing the metabolic process of the glucose, so that the drug loading rate of a disease treatment drug can be improved while the integrity of an EV membrane is maintained; and negative side effects of physical or chemical intervention are avoided, so that the functional EV with higher purity is obtained.

Description

Technical Field

[0001] This invention belongs to the field of chemical and biomolecular engineering technology, specifically relating to a glucose-modified photosensitizer, its preparation method, and its application. Background Technology

[0002] With the development of medical technology, various therapeutic drugs have been widely used in cancer treatment. However, these drugs currently face many challenges, such as limited water solubility, poor targeting ability, and non-specific distribution. Therefore, many synthetic nanoscale drug delivery systems, such as liposomes, metal nanoparticles, and polymer micelles, have been developed to overcome these obstacles. These nanoformulations, due to their high drug loading capacity, surface functionalization with targeting ligands, and high permeability and retention effects, can be used to promote drug accumulation at the lesion site and minimize off-target effects. However, drawbacks such as poor biocompatibility, high immunogenicity, and rapid in vivo clearance still need to be addressed.

[0003] In recent years, extracellular vesicles (EVs) have been widely used as drug delivery vehicles for cancer treatment due to their low immunogenicity, high biocompatibility, and excellent homologous targeting capabilities. Generally, the application of naturally occurring EVs in cancer treatment is limited by low secretion efficiency and unsatisfactory therapeutic effects. Therefore, it is necessary to intervene in the secretion process of EVs and to functionalize and modify them.

[0004] Currently, common strategies for loading extracellular vesicles (EVs) with drugs typically employ physical or chemical methods, such as post-loading methods like electroporation, extrusion, and chemiprecipitation, followed by EV repurification. However, these post-loading methods can impair the biological function of EVs due to the negative side effects of physical or chemical interventions. For example, the pulses used during electroporation often lead to EV aggregation, and extrusion can disrupt the EV membrane structure.

[0005] Preloading is an emerging approach that enables the simple and continuous production of functional EVs through gene transfection or co-culturing cells with drugs without compromising membrane integrity. However, current preloading strategies are still in their infancy, being inefficient and limiting the selection of loadable biomolecules. Therefore, there is an urgent need to develop a simple and efficient EV drug loading strategy. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a glucose-modified drug that enables the large-scale and in-situ production of functionalized drug-loaded EVs through glucose metabolism.

[0007] Another object of the present invention is to provide a method for preparing glucose-modified drugs.

[0008] Another object of the present invention is to provide a glucose-modified photosensitizer.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned glucose-modified photosensitizer, wherein the photosensitizer is prepared by amidation reaction of glucosamine and TB.

[0010] Another object of the present invention is to provide the use of glucose-modified drugs in cancer treatment drugs.

[0011] Another object of the present invention is to provide the use of glucose-modified photosensitizers in cancer treatment drugs.

[0012] The objective of this invention is achieved through the following technical solution.

[0013] A glucose-modified drug has the following structural formula:

[0014]

[0015] Wherein, R is a group that has cancer treatment effects.

[0016] In the above technical solution, R has the effect of inhibiting or killing cancer cells.

[0017] In the above technical solution, R has AIE characteristics.

[0018] In the above technical solution, the cancer treatment effect is achieved by generating reactive oxygen species (ROS) under light irradiation.

[0019] A method for preparing the above-mentioned glucose-modified drug includes: subjecting glucosamine to an amidation reaction to obtain the glucose-modified drug, wherein the reactant has the structural formula: R-OH.

[0020] A glucose-modified photosensitizer has the following structural formula:

[0021]

[0022] A method for preparing a glucose-modified photosensitizer (TBG), comprising:

[0023] Glucosamine, a photosensitizer, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 4-(dimethylamino)pyridine, and N,N-diisopropylethylamine (DIPEA) were subjected to an amide reaction in a solvent. The reaction was cooled to room temperature, extracted, and purified by column chromatography to obtain a glucose-modified photosensitizer, wherein the photosensitizer is TB, and the structural formula of TB is [insert structural formula here].

[0024] In the above technical solution, the temperature for the amide reaction is 35-40℃, and the reaction time is 24-36h.

[0025] In the above technical solution, the solvent is dimethylformamide (DMF).

[0026] In the above technical solution, the ratio of the photosensitizer, glucosamine, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 4-(dimethylamino)pyridine and N,N-diisopropylethylamine (DIPEA) is 1:(1-3):(5-15):(2-4):(6-8) by molar amount.

[0027] In the above technical solution, the ratio of the molar amount of the photosensitizer to the volume fraction of the solvent is 3:(1000-1500), the unit of the molar amount is mmol, and the unit of the volume fraction is mL.

[0028] In the above technical solution, the dimethylformamide (DMF) is ultra-dry DMF.

[0029] In the above technical solution, the extraction uses a mixture of saturated brine and dichloromethane, wherein the volume ratio of saturated brine to dichloromethane is 1:1.

[0030] The above-mentioned glucose-modified drugs are used in cancer treatment drugs.

[0031] The use of the above-mentioned glucose-modified photosensitizers in cancer treatment drugs.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) The glucose-modified drug / glucose-modified photosensitizer of the present invention is based on the glucose on it being transported to the cell via the glucose transporter protein (GLUT-1) to produce EV, thereby obtaining EV loaded with glucose-modified drug / TBG.

[0034] This invention utilizes the metabolic process of glucose to load therapeutic drugs (reactants / TB) into EVs by modifying glucose. This can improve the loading rate of therapeutic drugs while maintaining the integrity of the EV membrane, and avoid the negative side effects of physical or chemical interventions, thereby obtaining EVs with higher purity.

[0035] TB has a highly efficient ability to generate reactive oxygen species (ROS) under light irradiation. ROS kill cancer cells by damaging their proteins, RNA, and DNA.

[0036] (2) By co-incubating cells with glucose-modified drugs / glucose-modified photosensitizers, cells generated TBG-rich EVs (TBG-EVs) at a higher yield. TBG has excellent solubility and aggregation-induced emission (AIE) properties, eliminating the need for a washing step in imaging the formation and secretion of EVs, thus simplifying the imaging process and enabling imaging of in situ EV generation.

[0037] (3) Co-culturing the TBG prepared in this invention with the mother cell can increase the yield of TBG-EV.

[0038] (4) The TBG-treated cells obtained in this invention showed high EV labeling efficiency (77.4%). Attached Figure Description

[0039] Figure 1 The synthetic route for TBG;

[0040] Figure 2 TB in deuterated methanol 1 H NMR;

[0041] Figure 3 TB in deuterated methanol 13 C NMR;

[0042] Figure 4 ESI-mass spectrometry for TB;

[0043] Figure 5 TBG in deuterated methanol 1 H NMR;

[0044] Figure 6 TBG in deuterated methanol 13 C NMR;

[0045] Figure 7 ESI-mass spectrometry for TBG;

[0046] Figure 8 The UV / Vis absorption and emission spectra of TBG;

[0047] Figure 9 'a' represents the emission spectrum of TBG in TBG mixed solutions with different toluene ratios. Figure 9 b is Figure 9 The relationship between the relative emission intensity (I / I0) at 720 nm wavelength in a and the proportion of toluene in the TBG mixed solution;

[0048] Figure 10 'a' represents the fluorescence spectrum of the DCFH control group solution obtained at different irradiation time points. Figure 10 b represents the fluorescence spectrum of the TBG test solution obtained at different irradiation time points. Figure 10c is Figure 10 a and Figure 10 The change of peak intensity at 525 nm in b with illumination time;

[0049] Figure 11 The cell survival rate of HeLa cells;

[0050] Figure 12 TEM images of HeLa cells;

[0051] Figure 13 Fluorescence confocal images of HeLa cells after co-incubation with TBG and TB for 30 mins (scale line = 10 μm);

[0052] Figure 14 Fluorescence confocal images of HeLa cells after co-incubation with TBG for different times (scale line = 10 μm);

[0053] Figure 15 A schematic diagram of collecting EVs using differential centrifugation;

[0054] Figure 16 a) Images of EV, TBG, and TBG-EV under natural light (NL) and ultraviolet light (UV). Figure 16 b) is a fluorescence confocal image of TBG-EV;

[0055] Figure 17 a) represents the percentage of markers used in the negative control group. Figure 17 b) represents the percentage of TBG-EV labeled in the test solution;

[0056] Figure 18 a) represents the Zeta potential of TBG-EV and EV. Figure 18 b) is the average hydrodynamic diameter of TBG-EV and EV at different times. Inset: Transmission electron microscope image of TBG-EV, scale line = 200 nm.

[0057] Figure 19 Western blot analysis of HeLa cells, EVs, and TBG-EVs;

[0058] Figure 20 A comparison chart of TBG-EV and EV production volumes;

[0059] Figure 21 Fluorescence confocal images of MDA-MB-231 cells and A549 cells (scale line = 10 μm);

[0060] Figure 22 a) is a volcano diagram. Figure 22 b) is a bar chart of functional enrichment analysis for GO items;

[0061] Figure 23 a) is for genome enrichment analysis (transmembrane transporter activity pathways). Figure 23 b) is the genome enrichment analysis (ATP synthesis pathway), (NES refers to the normalized enrichment score, FDR refers to the false discovery rate, FDR < 0.05). Figure 23 c) is an immunofluorescence confocal laser scanning microscopy image of GLUT-1 in HeLa cells. Figure 23 d) is a Western blot analysis of HeLa cells. Figure 23 e) is the ImageJ analysis graph. Detailed Implementation

[0062] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0063] The raw materials and their manufacturers involved in the following embodiments are as follows:

[0064] Glucosamine chloride 4-(dimethylamino)pyridine, N,N-diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), DMF, 2',7'-dichlorodihydrofluorescein (DCFH) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich.

[0065] Reference for the preparation method of TA:

[0066] 1% (v / v) penicillin / streptomycin (1 unit: 1 ug, Pen Strep Glutamine (100×)) was purchased from Gibco.

[0067] Dulbecco's Modified Eagle Medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Gibco, with the PBS pH set at 7.4.

[0068] HeLa (human cervical cancer cell line), A549 (lung cancer epithelial cell line), and MDA-MB-231 (triple-negative breast cancer cell line) were purchased from the American Type Culture Collection (ATCC, USA).

[0069] The instruments and their models involved in the following embodiments are as follows:

[0070] Shimadzu UV-1700 UV-Vis detector;

[0071] Waters 2545 high performance liquid chromatograph;

[0072] Bruker ARX 400 nuclear magnetic resonance imaging (MRI) instrument;

[0073] Proteome X-LTQ electrospray ionization mass spectrometer;

[0074] Edinburgh FS5 fluorescence emission spectrometer.

[0075] The Leica SP8 laser scanning confocal microscope (CLSM) was excited at a wavelength of 493 nm and observed at a wavelength of 720 nm.

[0076] Example 1

[0077]

[0078] like Figure 1 As shown, the synthesis method of TB is as follows:

[0079] TA (191 mg, 0.42 mol) and 5-bromopentanoic acid (152 mg, 0.84 mol) were dissolved in 50 mL of acetonitrile and placed in a 100 mL round-bottom flask. The mixture was refluxed at 85 °C for 24 h. After cooling to room temperature, the solvent was evaporated to dryness, and the mixture was purified by preparative thin-layer chromatography (using a mixture of methanol and dichloromethane as eluent, with a volume ratio of 1:20) to obtain a dark red solid powder, TB (56 mg, 21% yield). The structural formula of TA is [insert structural formula here].

[0080] The structural formula of TB is

[0081] like Figure 2 and Figure 3 As shown, the NMR data for TB are: 1H NMR (400MHz, MeOD) δ9.07(d,J=5.8Hz,2H),8.99(d,J=5.8Hz,2H),8.47(d,J=7.4Hz,1H),8.06(d,J=7.4 Hz,3H),7.36(t,J=7.3Hz,4H),7.21-7.09(m,8H),4.70(s,2H),2.46(s,2H),2.15(s,2H),1.75(s,2H). 13 C NMR(101MHz,MeOD)δ175.23,153.84,153.10,153.01,149.25,147.18,144.06,137.78,132.07,130 .36,129.26,129.19,126.47,126.29,125.03,123.87,123.72,121.54,60.51,30.31,29.37,21.07.

[0082] like Figure 4 As shown, the mass spectrometry data for TB are HR ESI-MS, m / z: [M-Br]. + calcd557.2006,found557.2084.

[0083] like Figure 1 As shown, a method for preparing a glucose-modified photosensitizer (TBG) includes the following steps:

[0084] TB (20 mg, 0.03 mmol), glucosamine chloride (13.2 mg, 0.06 mmol), 4-(dimethylamino)pyridine (10 mg, 0.08 mmol), N,N-diisopropylethylamine (DIPEA) (36 μL, 0.20 mmol), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (130 mg, 0.34 mmol) were dissolved in 10 mL of ultra-dry DMF, and the mixture was stirred at 38 °C for 36 h to carry out the amide reaction. After the reaction was completed, the mixture was cooled to room temperature and extracted with a mixture of saturated brine (room temperature) and dichloromethane (1:1 volume ratio) to obtain the crude product. The crude product was dissolved in methanol and purified by high performance liquid chromatography (HPLC) to obtain a dark red solid as TBG (10.27 mg, 41% yield). HPLC conditions were as follows: column: Agilent ZORBAX SB-C18 (9.4 × 150 mm); gradient: 0-23-24-26-27-30 min; eluent was a mixture of solution A and solution B, with the volume of solution B varying with the gradient as follows: 70%-30%-0%-0%-70%-70% (solution A was ultrapure water containing 0.1% (v / v) trifluoroacetic acid (TFA), and solution B was acetonitrile containing 0.1% (v / v) TFA); flow rate was 2.0 mL / min. -1 UV-Vis detector: 480nm.

[0085] like Figure 5 and Figure 6 As shown, the NMR data for TBG are as follows: 1 H NMR (400MHz, CD3OD) δ9.02(d,J=5.8Hz,2H),8.96(d,J=5.9Hz,2H),8.44(d,J=7. 5Hz,1H),8.04(t,J=6.7Hz,3H),7.34(t,J=7.4Hz,4H),7.34-7.14(m,8H),4.67( t,J=7.0Hz,2H),3.86(d,J=11.3Hz,1H),3.79(d,J=10.8Hz,1H),3.76-3.47(m,3 H),3.47-3.31(m,2H),2.38(d,J=5.4Hz,2H),2.13(d,J=7.2Hz,2H),1.75(s,2H). 13C NMR (101MHz, CD3OD) δ174.08,153.86,153.10,152.99,149.27,147.20,144.07,137.81,132.05,130.37,129.26,129.20,126 .48,126.29,125.04,123.90,123.73,121.55,91.19,71.73,71.29,71.19,61.33,60.38,54.40,47.60,30.17,29.39,21.69.

[0086] like Figure 7 As shown, the mass spectrometry data for TBG are: HR ESI-MS, m / z: [M-Br] + calcd718.2694,found718.2904.

[0087] like Figures 2-7 As shown, the structure of the above molecules was confirmed using nuclear magnetic resonance (NMR) and ESI mass spectrometry. The TBG solid powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. 10 μL of this stock solution was added to 990 μL of phosphate buffered saline (PBS) to characterize its photophysical properties, as shown below. Figure 8 As shown. Figure 8 The UV / Vis absorption and emission spectra of TBG are shown. TBG has a broad absorption spectrum (400-600 nm) and emits red fluorescence centered at 720 nm.

[0088] A 10 μM TBG mixed solution (molecular state) was prepared, comprising TBG, toluene, and methanol. Toluene, as a poor solvent for TBG, was mixed with methanol, as a good solvent, in different proportions (toluene volume ratios in the TBG mixed solution were 0%, 20%, 40%, 60%, 80%, 90%, and 100%). The aggregation-induced emission (AIE) characteristics of TBG were detected for TBG mixed solutions with different toluene ratios. Figure 9 As shown, Figure 9 The emission spectra of TBG mixed solutions with different toluene ratios are shown in Figure 'a'. It can be seen that as the toluene ratio increases, the emission intensity increases accordingly. Figure 9 b is Figure 9 The relative emission intensity (I / I0) at 720 nm in TBG mixed solutions with different toluene ratios shows that the fluorescence gradually increases with the increase of toluene component (aggregated state), which proves the aggregation-induced emission (AIE) characteristics of TBG. Therefore, TBG has a high signal-to-noise ratio during real-time observation, thus eliminating the need for water washing when fluorescently labeling and tracking EVs.

[0089] 2',7'-Dichlorodihydrofluorescein (DCFH) emits strong fluorescence upon oxidation by ROS, thus it can be used as a ROS indicator to assess the ROS-generating ability of TBG. 10 μL of the above stock solution was added to 990 μL of PBS containing 10 μM DCFH as the TBG test solution, with a final TBG concentration of 10 μM. A mixture of DCFH and PBS was used as the DCFH control solution, with a DCFH concentration of 10 μM. The photophysical properties of the TBG test solution and the DCFH control solution were characterized. The TBG test solution and the DCFH control solution were heated to 20 mW cm⁻¹. -2 The sample was irradiated with light, and the fluorescence emission spectra at 400–700 nm were measured using a fluorescence spectrometer at different time points: 0 min, 1 min, 2 min, 3 min, 4 min, and 5 min. Figure 10 'a' represents the fluorescence spectrum of the DCFH control group solution obtained at different irradiation time points. Figure 10 b represents the fluorescence spectrum of the TBG test solution obtained at different irradiation time points, derived from the above. Figure 10 a and Figure 10 As shown in b, the fluorescence intensity of DCFH after TBG treatment increases significantly with prolonged irradiation time. Figure 10 c is Figure 10 a (DCFH control group solution) and Figure 10 The change in peak intensity at 525 nm in b (TBG test solution) with illumination time, combined with Figure 10 The c further reveals TBG's efficient ROS generation capability.

[0090] The method for obtaining EV-free FBS in the following examples is as follows: Fetal bovine serum (FBS) is ultracentrifuged in an ultracentrifuge (Beckman Coulter, CA) at 110,000×g and 4°C for 2h, and then filtered through a 0.22μm filter (Millipore, Merck, Germany) to obtain EV-free FBS.

[0091] Furthermore, the ability of TBG to promote EV production from cancer cells was investigated. HeLa cells (human cervical cancer cells) were used as a model cancer cell line, and EVs were generated through co-incubation with TBG. To ensure efficient EV production, HeLa cells must remain viable during the process. Since ROS generated by TBG can damage HeLa cells under light, the entire experimental procedure was conducted under dark conditions. The viability of TBG-treated HeLa cells was investigated using the MTT assay: 10,000 HeLa cells (human cervical cancer cells) were seeded in 100 μL of culture medium and incubated at 37°C for 20 h under light-protected conditions (5% (v / v) CO2 environment). Different amounts of TBG were then added to the medium to achieve TBG concentrations of 0 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. The culture medium consisted of Dulbecco's Modified Eagle Medium (DMEM), 10% (v / v) EV-free FBS, and 1% (v / v) penicillin / streptomycin (1 unit: 1 μg, Pen Strep Glutamine (100×)). After another 24 h of incubation, the cell viability of HeLa cells was as follows: Figure 11 As shown. From Figure 11 It can be seen that the cell survival rate is above 90%, which indicates that TBG has high biocompatibility in the process of secreting TBG-EV under light-free conditions.

[0092] To investigate the changes in TBG-EV production after TBG culture, 10,000 HeLa cells (human cervical cancer cells) were seeded in 100 μL of culture medium and incubated at 37°C for 20 h in the dark (5% (v / v) CO2 environment). TBG was then added to the medium to achieve a TBG concentration of 10 μM, and the cells were cultured for another 30 min. Transmission electron microscopy (TEM) images were obtained as follows: Figure 12 As shown in the first row. 10,000 HeLa cells (human cervical cancer cells) were seeded in 100 μL of culture medium and incubated at 37°C for 20 h under light-protected conditions (5% (v / v) carbon dioxide). Then, an equal volume of PBS (the amount of TBG added when the TBG concentration in the culture medium was 10 μM) was added to the medium, and the cells were incubated for another 30 min. Transmission electron microscopy (TEM) images were obtained as shown below. Figure 12 As shown in the second row, observations revealed that TBG-EVs were more abundant around HeLa cells treated with TBG, with an average size of approximately 200 nm, indicating that TBG-EV production was increased after TBG culture.

[0093] Cell culture method: 10,000 HeLa cells (human cervical cancer cells) were seeded in 100 μL of culture medium and incubated at 37°C for 20 h in the dark (5% (v / v) CO2 environment). TBG was then added to the culture medium to a concentration of 10 μM, and the cells were cultured at 37°C for another 30 min in the dark. The cells were excited at 493 nm using a laser scanning confocal microscope (CLSM) and observed at 720 nm. Figure 13 As shown in the first row. 10,000 HeLa cells (human cervical cancer cells) were seeded in 100 μL of culture medium and incubated at 37°C for 20 h in the dark (5% (v / v) CO2 environment). Then, TB was added to the culture medium to bring the concentration of TB to 10 μM, and the HeLa cells were cultured at 37°C for another 30 min in the dark. The cells were excited at 493 nm using a laser scanning confocal microscope (CLSM) and observed at 720 nm. Figure 13 As shown in the second row. Figure 13 As shown, TBG-treated cells have more TBG-EVs (EVs loaded with TBG) around them, while TB-treated cells have very few TB-EVs (EVs loaded with TB).

[0094] 10,000 HeLa cells (human cervical cancer cells) were seeded in 100 μL of culture medium and incubated at 37°C for 20 h in the dark (5% (v / v) CO2 environment). TBG was then added to the culture medium to a concentration of 10 μM, and the cells were cultured at 37°C for another 24 h in the dark. At 8 h and 24 h after TBG addition, the cells were observed using a laser scanning confocal microscope (CLSM) at 493 nm and 720 nm, respectively. Figure 14 As shown, CLSM revealed that HeLa cells continued to produce TBG-EVs and release them into the extracellular space after long-term co-culture with TBG.

[0095] HeLa cells (human cervical cancer cells) were placed in T175 culture flasks (using the same culture medium as described above) and incubated at 37°C for 20 hours in the dark (with 5% (v / v) carbon dioxide). Then, TBG was added to the culture medium to bring the TBG concentration to 10 μM, and the cells were cultured for another 48 hours to obtain the first experimental group. HeLa cells (human cervical cancer cells) were placed in T175 culture flasks (using the same culture medium as described above) and incubated at 37°C for 20 hours in the dark (with 5% (v / v) carbon dioxide). Then, PBS of the same volume as the TBG added to the culture medium (to achieve the 10 μM TBG concentration in the first experimental group) was added, and the cells were cultured for another 48 hours to obtain the second experimental group (the volumes of the first and second experimental groups were the same). Figure 15 As shown, to extract EVs, the released EVs were collected by differential centrifugation: the first and second experimental groups were centrifuged at 4°C for 10 min (2000×g) and 30 min (10000×g), respectively, to remove dead cells and cell debris. The supernatant was then collected and centrifuged at 110,000×g for 2 h at 4°C. The supernatant cell culture medium was discarded, and the lower layer was washed twice with PBS, then ultracentrifuged at 110,000×g for 1 h at 4°C. The lower layer was collected to obtain TBG-EVs (first experimental group) and EVs (second experimental group), which were stored at -80°C.

[0096] EV and TBG-EV were separately dispersed in PBS to obtain EV solution and TBG-EV solution, respectively; TBG was dissolved in PBS to obtain TBG solution. The EV solution, TBG solution, and TBG-EV solution were observed under natural light (NL) and ultraviolet light (UV = 365 nm). Figure 16 As shown in "a", "EV", "TBG", and "TBG-EV" (EV solution, TBG solution, and TBG-EV solution have the same volume; the mass of TBG in TBG-EV solution is the same as the mass of TBG in TBG solution; the mass of EV in TBG-EV solution is the same as the mass of EV in EV solution). Figure 16 As shown in a, obvious fluorescence can be observed in TBG-EV solution, which is due to the AIE effect of TBG molecules.

[0097] To observe the fluorescence of TBG-EV in a CLSM, the TBG-EV solution was first fixed with 4% (w / v) paraformaldehyde for 30 min. After washing twice with PBS, 10 μL of the solution was dropped onto a microscope slide, excited with a CLSM at a wavelength of 493 nm, and images were acquired at a wavelength of 720 nm. Figure 16 As shown in b, significant fluorescence was also observed, demonstrating the successful loading of TBG in EV.

[0098] The EV solution and TBG-EV solution were diluted 1000-fold with PBS, serving as the negative control and TBG-EV test solution, respectively. Analysis was performed using a NanoAnalyzer (NanoFCM). A relatively small threshold setting (68-155S16M-Exo) was used to detect the TBG-EV test solution and the negative control, and the results were analyzed in the PC5 channel (λ). ex =488nm / λ em =670nm) to detect the group of markers. Figure 17 'a' represents the percentage of the negative control labeled, with a labeling efficiency of approximately 1.25%. It is worth noting that... Figure 17 b represents the percentage of TBG-EV labeled in the test solution. The labeling efficiency of TBG for EV is approximately 77.4%, significantly exceeding the 17.4% labeling efficiency reported in the literature (C Zhou, SJ Cox-Vázquez, GWN Chia, RJ Vázquez, HY Lai, SJW Chan, J Limwongyut, GC Bazan, Water-soluble extracellular vesicle probes based on conjugatedoligoelectrolytes[J], Science Advances, 11 Jan 2023, Vol 9, Issue 2). This may be because TBG is a metabolism-directed active labeling agent, rather than a passive labeling agent as described in the literature.

[0099] The zeta potential of EV solution and TBG-EV solution was measured separately, and the results are as follows: Figure 18 As shown in Figure 18a, the surface charge of TBG-EV is -7.69±0.7mV, which is slightly higher than that of EV (-9.42±0.8mV).

[0100] To investigate the importance of stability for the practical application of EVs, dynamic light scattering (DLS) was used to measure the stability of EV solutions and TBG-EV solutions at 4°C on days 1, 2, 3, and 7. The results are as follows: Figure 18 As shown in b of Figure 18, the average hydrodynamic diameter of TBG-EV is 145.5 ± 4.8 nm, which is comparable to that of EV (134.3 ± 1.7 nm). Furthermore, both EV and TBG-EV exhibit good stability over time.

[0101] TBG-EV solution was fixed with 4% (v / v) paraformaldehyde for 30 min. After washing twice with PBS, 10 μL of the solution was dropped onto a copper grid. Then, 10 μL of 2 wt% phosphotungstic acid aqueous solution was used to wet the surface of the copper grid for 10 s, followed by blotting with absorbent paper. This process was repeated three times, and the grid was then left to dry at room temperature. The morphology of the negatively stained TBG-EV was observed using transmission electron microscopy (TEM), as shown in the inset of Figure 18(b). The results showed that TBG-EV was spherical, and the particle size was consistent with the results of DLS determination.

[0102] TBG-EV, EV, and HeLa cells (for control) were analyzed using Western blotting (with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a protein reference) to detect EV biomarkers. The results are as follows: Figure 19 As shown, Figure 19 The images show specific enrichment of tumor susceptibility gene 101 (TSG101), ALG-2-interacting protein X (Alix), and transmembrane protein (CD9) in EVs and TBG-EVs. (TSG101 and Alix are involved in the biogeneration and secretion of EVs and are widely used as marker proteins for EVs. Furthermore, Alix and CD9 are known to mediate EV internalization and uptake.) This indicates that the membrane structure of TBG-EVs is undamaged and their composition and function remain largely unchanged. Neither EVs nor TBG-EVs contain cadherin (cadherin is a marker of the endoplasmic reticulum (ER) and is mainly found in cells, but not in EVs), suggesting that the collected product is high-purity EVs, not cell debris.

[0103] Furthermore, quantification of EVs was performed using NanoFCM: TBG-EV and EV were diluted 100-fold with PBS before testing. The results are as follows: Figure 20 As shown, the amount of EV collected in the TBG-treated group (TBG-EV) was almost 4.3 times that in the untreated group (EV), indicating that this method can effectively promote EV secretion and thus increase EV production.

[0104] Furthermore, to observe whether other cell types also exhibited increased EV production after TBG treatment, HeLa cells (human cervical cancer cells) were replaced with MDA-MB-231 cells (triple-negative breast cancer cells) according to the aforementioned "cell culture method." These cells were excited at 493 nm using a laser scanning confocal microscope (CLSM) and observed at 720 nm. Figure 21As shown in the first and second rows. Following the aforementioned "cell culture method," HeLa cells (human cervical cancer cells) were replaced with A549 (lung cancer epithelial cells), excited at 493 nm wavelength using a laser scanning confocal microscope (CLSM), and observed at 720 nm wavelength, as shown... Figure 21 As shown in the third and fourth lines of the document. From Figure 21 It was found that TBG-treated A549 and MDA-MB-231 cells showed more EVs than TB-treated A549 and MDA-MB-231 cells. Compared with TB-treated cells, TBG could elicit more EVs in all cell lines. This observation further demonstrates the potential of TBG, a photosensitizer that binds TB to glucose, to obtain relatively high TBG-EV yields.

[0105] Further investigation was conducted into the mechanism by which TBG promotes EV production through glucose metabolism: HeLa cells (human cervical cancer cells) were placed in T175 culture flasks (using the same culture medium as described above) and incubated at 37°C for 20 hours in the dark (within a 5% (v / v) carbon dioxide environment). TBG was then added to the culture medium to achieve a concentration of 10 μM, and the cells were cultured for another 0.5 hours to obtain the third experimental group. RNA sequencing analysis was performed on the HeLa cells obtained from the third experimental group and on untreated HeLa cells. Figure 22 As shown in the volcano plot in Figure a, compared with the control group (HeLa cells without any treatment), TBG-treated HeLa cells (HeLa cells obtained from the third experimental group) showed upregulated expression of 19 genes and downregulated expression of 394 genes. To better understand the relevant signaling pathways, GO term functional enrichment analysis was performed on differentially expressed genes using the Metascape database, such as... Figure 22 As shown in b, the results indicate that differentially expressed genes are significantly enriched in positively regulated pathways of stimulus response, metabolic processes, and biological processes. Specifically, these pathways involve glucose uptake and energy supply, which play crucial roles in the biogeneration and secretion of cancer-derived EVs. Therefore, it is hypothesized that the addition of TBG stimulates HeLa cells to upregulate glucose metabolism and energy supply to generate TBG-EVs.

[0106] To verify the hypothesis, genomic enrichment analysis (GSEA) was performed on differentially expressed genes using the Metascape database, such as... Figure 23 a (transmembrane transporter active pathway) and Figure 23 As shown in b (ATP synthesis pathway), from Figure 23 a and Figure 23As can be seen from b, TBG-treated HeLa cells (HeLa cells obtained from the third experimental group) exhibited enhanced transmembrane transporter activity and significantly increased ATP synthesis levels within 30 min (given that the entry of glucose-containing molecules into cells is an active transport process mediated by the glucose facilitated diffusion transporter GLUT-1, and glucose is subsequently loaded into EVs under the GLUT-1 mediation of EVs, the activity of active transmembrane transporters is of primary concern).

[0107] Furthermore, immunocytochemical staining of GLUT-1 in HeLa cells treated with TBG and PBS was performed. The specific method was as follows: HeLa cells were cultured in medium containing 20 μM TBG for 6 h, and then washed three times with PBS as the "TBG-treated" group. At room temperature (RT), both the "TBG-treated" group and untreated HeLa cells ("w / o TBG-treated") were fixed with 4% (v / v) paraformaldehyde for 15 min, and then washed three times with PBS. Next, HeLa cells were incubated with PBS containing 1 wt% skim milk at room temperature for 30 min, then incubated with anti-glucose transporter primary antibody (anti-GLUT-1) at 4°C for 12 h, then incubated with Alexa Fluor488 fluorescent secondary antibody at room temperature in the dark for 2 h, and finally stained with 1 μg / mL Hoechst (PBS as solvent) for 5 min, followed by CLSM imaging. Figure 23 c is shown and Figure 23 The left image in figure e is shown (MFI: mean fluorescence intensity). (By...) Figure 23 As can be seen from c, the GLUT-1 transporter activity of HeLa cells treated with TBG was significantly increased, and the average fluorescence intensity of the "TBG-treated" group was about 1.7 times that of the untreated HeLa cells ("w / o TBG-treated").

[0108] HeLa cells were cultured in a medium containing 20 μM TBG for 6 hours as... Figure 23 d and Figure 23 The “TBG-treated” group in e used untreated HeLa cells as… Figure 23 d and Figure 23 The expression levels of GLUT-1 in the "w / o TBG-treated" group were tested using Western blot analysis. Figure 23 d and Figure 23 The image on the right in the image is shown in section e (ID: grayscale value). Figure 23 d and Figure 23 The results in the right figure of Figure e show that the band integration density in the "TBG-treated" group was approximately 1.6 times that of the "w / o TBG-treated" group. This result indicates that once TBG is absorbed by HeLa cells, it stimulates higher GLUT-1 activity, leading to increased glucose uptake. Glucose is one of the most widely utilized metabolic substrates for ATP supply and plays a crucial role in promoting EV secretion.

[0109] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A glucose-modified medicament, characterized in that, The structural formula is as follows: wherein R is a group having a cancer treatment effect.

2. The glucose-modifying medicament of claim 1, wherein, The R has AIE characteristics.

3. The glucose-modifying drug according to claim 1 or 2, characterized in that, The cancer treatment effect is to generate active oxygen under light irradiation.

4. A method of preparing the glucose modified medicament of claim 1, wherein, The method comprises the following steps: The glucosamine is subjected to amidation reaction with a reactant to obtain a glucose modified drug, wherein the structural formula of the reactant is R-OH.

5. A photosensitizer modified with glucose, characterized in that, The structural formula is as follows:

6. A method for preparing a photosensitizer modified with glucose, characterized by, The method comprises the following steps: The glucosamine, a photosensitizer, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(dimethylamino)pyridine and N,N-diisopropylethylamine are subjected to amidation reaction in a solvent, cooled to room temperature, extracted, and subjected to column chromatography purification to obtain a glucose modified photosensitizer, wherein the structural formula of the photosensitizer is 7. The production method according to claim 6, wherein The temperature of the amidation reaction is 35-40 DEG C, and the time of the amidation reaction is 24-36 h.

8. The preparation method according to claim 6, characterized in that, The solvent is dimethylformamide.

9. The use of the glucose modified drug in claim 1 in a cancer treatment drug.

10. The use of the glucose modified photosensitizer in claim 5 in a cancer treatment drug.

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