Quantum dot synthesized by cell in-situ programmed synthesis as well as preparation method and application of quantum dot

Abstract

The invention relates to the technical field of biological materials, in particular to a cell in-situ programmed synthesis quantum dot as well as a preparation method and application thereof. The core innovation of the invention lies in that a multi-level nano synthesizer with responsiveness is designed, and the bottleneck that in-situ synthesis of functional nano materials is difficult to realize at living body and cell levels is broken through. The technology promotes the development of nano material biosynthesis, especially in-situ programmed synthesis of quantum dots. After an original nano synthesizer is internalized by cells, diselenide bonds are split by glutathione to generate active selenium species, and then a silicon shell skeleton collapses, a silver precursor is released and reacts with a selenium precursor to generate the quantum dots. The quantum dot provided by the invention can be synthesized in situ at a tumor site, the photothermal effect of oxidative stress combined near-infrared quantum dots is induced to finally cause apoptosis or necrosis of tumor cells, the development of tumors of tumor-bearing mice is effectively delayed, and meanwhile, the lifetime of the tumors is prolonged.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to in-situ programmed synthesis of quantum dots in cells and a preparation method and application thereof. BACKGROUND

[0002] As a kind of nanomaterials with fluorescence and light conversion ability, Ag2Se quantum dots show excellent potential for deep tissue bioimaging. However, traditional synthesis methods often rely on harsh chemical reaction conditions, and the prepared quantum dots have poor biocompatibility. Although surface modification strategies can improve their water dispersibility and biocompatibility, there are still problems such as non-specific distribution and rapid clearance during in vivo delivery.

[0003] Currently, by artificially regulating, calling and coupling different biochemical reaction pathways in living cells, only by feeding related reactants to cells, the controllable synthesis of quantum dots with different emission wavelengths can be realized in fungi, bacteria and mammalian cells. By regulating metabolic pathways, the close relationship between quantum dots and glutathione, thioredoxin and other systems is revealed, and the synthesis process is mechanically replicated in earthworms. However, in complex living organisms such as mammals, due to the wide distribution of exogenous precursors along with the circulatory system and the element-specific tissue tropism, it is difficult to achieve the required spatiotemporal coupling. In addition, the existing strategy is limited by the selectivity of a single precursor and endogenous molecules, and can only direct the synthesis of sulfur / oxides, which severely limits the diversity of products.

[0004] Therefore, there is an urgent need for a new quantum dot synthesis method. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the related art. To this end, the first object of the present application is to provide a preparation method of quantum dots synthesized in-situ in cells; the second object of the present application is to provide quantum dots synthesized in-situ in cells; and the third object of the present application is to provide applications of quantum dots synthesized in-situ in cells.

[0006] To achieve the first object, the technical solution adopted by the present application is as follows: The preparation method of quantum dots synthesized in-situ in cells comprises the following steps: S100, preparing nanoparticles I by using a soluble silver salt and a protein; S200, preparing nanoparticles II by using nanoparticles I, an ester silicate compound and a disulfide-containing bisalkoxysilane compound; S300, modifying nanoparticles II by using a coupling agent to obtain nanoparticles III; S400, activating the coupling group of the diselenium bond compound with activator I, and then coupling with nanoparticles III to prepare nanoparticles IV; S500, activating the targeting molecule with activator II, and then coupling with nanoparticles IV to prepare nanoparticles V; S600, using nanoparticles IV or nanoparticles V to release selenium precursors and silver precursors in cells to obtain in-situ programmed synthesis of Ag2Se quantum dots.

[0007] Preferably, in step S100, the protein is selected from at least one of ferritin, apoferritin and albumin, and the soluble silver salt is selected from silver nitrate or silver acetate; The molar ratio of the protein to the soluble silver salt is 1:100-1:400.

[0008] Preferably, in step S200, the silicate compound is selected from any one of methyl orthosilicate, ethyl orthosilicate and propyl orthosilicate; The disulfide-containing bisalkoxysilane compound is selected from any one of bis(triethoxysilylpropyl) disulfide, bis[3-(trimethoxysilyl)propyl] disulfide and bis[3-(triethoxysilyl)propyl] disulfide.

[0009] Preferably, in step S300, the coupling agent is selected from any one of an amino silane coupling agent and a carboxyl silane coupling agent; The amino silane coupling agent is selected from any one of 3-aminopropyl triethoxysilane, 3-aminopropyl methyldiethoxysilane and γ-aminopropyl triethoxysilane; The carboxyl silane coupling agent is selected from any one of 3-carboxypropyl trimethoxysilane, 3-carboxypropyl triethoxysilane and carboxyethyl silane triol sodium salt.

[0010] Preferably, in step S400, the activator I is selected from a carboxylic acid activator, and the carboxylic acid activator is selected from EDC and NHS; EDC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide.

[0011] Preferably, in step S400, the diselenium bond compound is selected from HOOC–(CH2) n –Se–Se–(CH2) m –COOH, n and m are each independently one of an integer from 1 to 10; The mass ratio of the diselenium bond compound to nanoparticles III is 1:1-3.5:1.

[0012] Preferably, in step S500, the activating agent II is selected from a carboxylic acid activating agent selected from EDC and NHS. wherein EDC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide. The targeting molecule is selected from a tumor targeting molecule, and the tumor targeting molecule is selected from a folate targeting conjugate derivative. The folate targeting conjugate derivative is selected from FA-(C2H4O) k -COOH, k is 1000, 1500, 2000. wherein FA is folate.

[0013] Preferably, in step S600, the cell is selected from any one of 4T1 cells, MCF-7 cells, A549 cells and HeLa cells.

[0014] To achieve the second object, the technical scheme adopted by the present application is: The cell in-situ programmed synthesized quantum dots are prepared by using the preparation method of the cell in-situ programmed synthesized quantum dots according to any one of the above.

[0015] To achieve the third object, the technical scheme adopted by the present application is: The application of the cell in-situ programmed synthesized quantum dots, as described above, includes any one or several of the following: For preparing a pharmaceutical preparation for treating and / or preventing tumors; For preparing a medical device for treating and / or preventing tumors; For preparing a medical imaging product; For preparing a tumor diagnosis product.

[0016] The above one or more technical solutions in the embodiments of the present application have at least one of the following technical effects: The preparation method of the cell in-situ programmed synthesized quantum dots provided by the present application has the core innovation that a multi-level nanosynthesizer with glutathione responsiveness is designed, which breaks through the bottleneck that the spatiotemporal regulation strategy for cell synthesis of quantum dots is difficult to realize at the mouse level. This technology promotes the development of quantum dot biosynthesis technology and further promotes the progress of in-situ programmed synthesis of quantum dots.

[0017] The experimental results show that: after the nanoparticles provided by the application are internalized by cells, the Se-Se bond thereof is cleaved by glutathione, and then active selenium species are generated, at the same time, the silicon shell skeleton collapses, so that the silver precursor is released, and finally the synthesis of quantum dots is completed in the cell. The cascade reaction achieves the spatiotemporal coupling of selenium and silver precursor, and creates a key condition for the synthesis of quantum dots in the tumor in vivo.

[0018] The in vivo experimental results of tumor-bearing mice show that: after the tail vein injection of the nanoparticles V modified by folic acid, the near-infrared fluorescence signal is observed at the tumor site after 2h, and the signal remains relatively stable over time. When the unmodified nanoparticles IV are injected intratumorally, clear fluorescence appears within 2h and increases over time, while the buffer control group still has no significant signal after 84h; 36h after the second injection, the fluorescence intensity in the tumor area is 3x10 4 times that of the adjacent normal tissue; quantitative analysis shows that the fluorescence intensity of the intratumoral injection group is 5.7 times that of the intravenous injection group at the same concentration; The above results show that: the nanoparticles IV and the nanoparticles V modified by the targeting molecule can synthesize quantum dots in situ in vivo.

[0019] The cell viability assay and the intracellular reactive oxygen species (ROS) detection results show that: the nanoparticles III can significantly increase the ROS level. This result shows that the cleavage of S-S and Se-Se bonds induces oxidative stress, which eventually leads to the apoptosis or necrosis of tumor cells.

[0020] The in vivo pharmacodynamics experimental results of tumor-bearing mice show that: after the unmodified nanoparticles IV are injected intratumorally, the quantum dots are synthesized in the tumor microenvironment, and then the local temperature can rapidly increase to 56℃ after laser irradiation, which is significantly higher than 30℃ of the buffer control group, which fully embodies the excellent photothermal conversion performance; after the unmodified nanoparticles IV are injected intratumorally, the tumor growth inhibition rate reaches 51.2% after 30 days, which can effectively delay the progression of tumor and prolong the survival period of tumor-bearing mice; the body weight of mice in all groups remains stable during the entire experimental period, indicating that the mice have good tolerance to the nanoparticles.

[0021] Based on the above cell experiment and in vivo experiment results, it can be known that the quantum dots synthesized in situ by cells based on the preparation method provided by the application have application potential in the preparation field of drug preparations for treating and / or preventing tumors, medical devices for treating and / or preventing tumors, medical imaging products and tumor diagnosis products.

[0022] Additional aspects and advantages of the application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 This is the X-ray photoelectron energy (XPS) spectrum of Ft-Ag(I) provided in Embodiment 1 of the present invention.

[0024] Figure 2 This is the result of multi-dimensional characterization of FtAg@SS / SiO2 nanoparticles and detection of the influence of GSH on them provided in Example 1 of this invention.

[0025] Figure 3 This is a Zeta potential histogram of FtAg@SS / SiO2 and FtAg@SS / SiO2-NH2 nanoparticles provided in Example 1 of the present invention.

[0026] Figure 4 This is a high-resolution mass spectrum of the target product 5,5-diselenodivalerate (DSeBPA) provided in Example 1 of the present invention.

[0027] Figure 5 This is a diagram illustrating the detection of H2Se generation using lead acetate test paper, provided in Embodiment 1 of the present invention.

[0028] Figure 6 This is the characterization of the FtAg@SS / SiO2-Se nanoparticles provided in Example 1 of this invention.

[0029] Figure 7 This shows the fluorescence of FtAg@SS / SiO2-Se nanoparticles provided in Example 1 of this invention after co-incubation with 4T1 cells.

[0030] Figure 8 This describes the particle size distribution of FtAg@SS / SiO2-Se nanoparticles and FtAg@SS / SiO2-Se-FA nanoparticles provided in Example 1 of this invention.

[0031] Figure 9 This is an imaging diagram showing the change of fluorescence distribution signal over time when tumor-bearing mice are intravenously injected with different concentrations and different injection times, as provided in Example 2 of the present invention.

[0032] Figure 10 This is an imaging diagram showing the change of fluorescence distribution signal over time when different concentrations and different injection times are injected into the tumor of a tumor-bearing mouse, as provided in Example 2 of the present invention.

[0033] Figure 11 This is a line graph showing the relative level of fluorescence signal at the tumor site over time under different administration methods (injection concentration of 20 mg / mL) provided in Example 2 of the present invention.

[0034] Figure 12This is a bar chart showing the change of mean fluorescence intensity (MFI) at the tumor site over time under different injection methods and different injection times (injection concentration of 20 mg / mL) provided in Example 2 of the present invention.

[0035] Figure 13 This is a bar chart showing the effect of different nanoparticles on cell activity provided in Example 3 of the present invention.

[0036] Figure 14 This is a fluorescence staining image of reactive oxygen species after co-culturing different nanoparticles with 4T1 cells, as provided in Example 4 of this invention.

[0037] Figure 15 This is a line graph showing the photothermal heating effect after intratumoral injection of FtAg@SS / SiO2-Se nanoparticles, as provided in Example 5 of this invention.

[0038] Figure 16 This describes the effect of intratumoral injection of FtAg@SS / SiO2-Se nanoparticles provided in Example 5 of the present invention on tumor-bearing mice. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0040] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0041] In the following embodiments: The silver source was selected from ferritin-encapsulated silver source, denoted as Ft-Ag(I); The selenium source was selected from 5,5'-diselenodivalerate (DSeBPA). The structure contains a Se–Se bond, which can be cleaved by glutathione (GSH) to generate active selenium species. Silica: Doped with disulfide bonds, it undergoes GSH-responsive degradation, providing a time difference for the coupling of silver selenium precursors.

[0042] Example 1 I. Preparation of Nanoparticle I (a protein-encapsulated silver source nanocomposite), the preparation process of which is described below: 10 mL of 2 mg / mL ferritin solution (in BR buffer) was heated to 60 °C and the pH was adjusted to 8.0. Then, 400 μL of 3.77 mg / mL silver nitrate aqueous solution was added dropwise. After reacting at 60 °C in the dark for 1 h, the unbound Ag(I) ions were purified and removed by passing them through a PD-10 desalting column to obtain nanoparticle I, denoted as Ft-Ag(I). Among them, BR buffer is Britton-Robinson universal buffer.

[0043] XPS spectra of Ft-Ag(I), such as Figure 1 As shown in the figure, Ft-Ag(I) and silver nitrate have the same 3d characteristic peak of Ag, which indicates the successful incorporation of silver(I).

[0044] II. Preparation of Nanoparticles II (FtAg@SS / SiO2 Nanoparticles), the process is as follows: FtAg@SS / SiO2 nanoparticles were synthesized using the sol-gel method disclosed in the following references: Yuan, P. et al. Intracellular delivery of functional nativeantibodies under hypoxic conditions by using a biodegradable silicananoquencher. Angew. Chem. Int. Edit. 129, 12655–12659 (2017); The process is as follows: Triton X-100 (1.06 mL) and n-hexanol (1.08 mL) were dissolved in cyclohexane (4.5 mL) and stirred at room temperature to form a homogeneous solution. Subsequently, 180 μL of 1 mg / mL Ft-Ag(I) solution (in BR buffer), 24 μL of tetraethyl orthosilicate (TEOS), and 45 μL of bis[3-(triethoxysilyl)propyl]disulfide (BTEPDS) were mixed and added to the aforementioned solution containing Triton X-100 and n-hexanol. Then, 36 μL of ammonia was added to the reaction system to initiate a hydrolysis-condensation reaction. After stirring the reaction continuously at room temperature for 12 h, the nanoparticles were precipitated with acetone and washed three times each with ethanol and ultrapure water to obtain FtAg@SS / SiO2 nanoparticles. The product was freeze-dried and stored at 4 °C.

[0045] Compared to FtAg@SiO2 nanoparticles: BTEPDS were not introduced, but TEOS (60 μL) was added. The rest of the preparation process was the same as that of FtAg@SS / SiO2 nanoparticles.

[0046] Replace 1 mg / mL Ft-Ag(I) solution (180 μL in BR buffer) with BR buffer (pH 7.4, 180 μL), denoted as SS / SiO2; FtAg@SS / SiO2+GSH refers to FtAg@SS / SiO2 nanoparticles treated with 5mM GSH for 24 hours.

[0047] Characterization of FtAg@SS / SiO2 nanoparticles and detection results of the effect of GSH on them, such as Figure 2 As shown; Figure A shows the thermogravimetric analysis results of FtAg@SS / SiO2 nanoparticles and their control SS / SiO2. From this figure, it can be seen that FtAg@SS / SiO2 has a faster mass loss and a lower residual mass, indicating that it successfully introduced Ft-Ag(I). Figure B shows the infrared spectra of FtAg@SS / SiO2 nanoparticles, Ft-Ag(I), and SS / SiO2. From this figure, it can be seen that FtAg@SS / SiO2 simultaneously contains amide groups (corresponding to 1538 cm⁻¹). -1 (The peak at 794 cm⁻¹ is a characteristic peak for amide bonds) and silicon-oxygen bonds. -1 The characteristic structure of the peak at the Si–O–Si stretching vibration point indicates that SiO2 has successfully coated Ft-Ag(I); Figure C shows the Raman spectra of different substances. From this figure, it can be seen that FtAg@SS / SiO2 and SS / SiO2 exhibit the following Raman spectra at 490 cm⁻¹. -1 S–S bond vibration signal was detected at FtAg@SiO2, which was not detected in FtAg@SiO2. The S–S bond vibration signal disappeared after FtAg@SS / SiO2 was treated with GSH, indicating that the S–S bond was embedded in the silicon skeleton and could be cut by GSH. Figure D shows the morphology of FtAg@SS / SiO2 nanoparticles and FtAg@SiO2 nanoparticles before and after treatment with 5mM GSH for 12 hours. As can be seen from the figure, the structure of FtAg@SS / SiO2 completely collapsed after GSH treatment; while the morphology and size of the control group FtAg@SiO2 remained stable under the same conditions, indicating that FtAg@SS / SiO2 nanoparticles have a clear GSH response function.

[0048] III. Amination modification of FtAg@SS / SiO2 nanoparticles to prepare amination-modified nanoparticles III (FtAg@SS / SiO2-NH2 nanoparticles), the process is as follows: 100 mg of FtAg@SS / SiO2 nanoparticles were dispersed in 25 mL of ethanol. 1 mL of 3-aminopropyltriethoxysilane was added under continuous stirring. After reacting for 10 h, a precipitate was obtained. The precipitate was washed three times with ethanol and ultrapure water to obtain the aminated product FtAg@SS / SiO2-NH2 nanoparticles.

[0049] Zeta potential analysis results of FtAg@SS / SiO2 nanoparticles and FtAg@SS / SiO2-NH2 nanoparticles, as follows: Figure 3 As shown in the figure, the Zeta potential of FtAg@SS / SiO2 is -19.0 mV, and the Zeta potential of FtAg@SS / SiO2-NH2 is +28.8 mV, indicating that FtAg@SS / SiO2 was successfully amination.

[0050] IV. Synthesize DSeBPA according to the following references: Pan, S., Li, T., Tan, Y.&Xu, H. Selenium-containing nanoparticles synergistically enhance Pemetrexed&NK cell-based chemoimmunotherapy. Biomater ials 280, 121321 (2022).

[0051] The process is as follows: Selenium powder (884.8 mg) and sodium borohydride (414.4 mg) were placed in a three-necked round-bottom flask, with two of the necks sealed with stoppers. The middle neck was connected to a condenser equipped with a three-way valve, which was connected to an argon protection system. After completing three vacuum-argon replacement cycles, the condenser circulation system was turned on, and ultrapure water (20 mL) was slowly injected through a syringe. The reaction was carried out at 50 °C for 30 min to obtain the reaction solution. Separately, 5-bromopentanoic acid (2001.0 mg) was dissolved in tetrahydrofuran (40 mL), and the aforementioned reaction solution was added. The reaction was continued at 50 °C for 6 h. After that, the tetrahydrofuran was removed by rotary evaporation under reduced pressure at 40 °C. The residue was extracted with dichloromethane, and the organic phase was collected. The organic phase was washed with 1% hydrochloric acid aqueous solution, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to remove dichloromethane. The resulting residue was dissolved in dichloromethane, recrystallized 2-3 times with petroleum ether, and then filtered under vacuum to obtain the target product DSeBPA. Its characterization data are as follows: 1 ¹H NMR (400MHz, deuterated chloroform): δ=2.94(t, J=7.2Hz, 4H), 2.41(t, J =6.8Hz, 4H), 1.82–1.74(m, 8H); 13 C NMR (100MHz, deuterated chloroform): δ=180.1, 33.6, 30.4, 29.4, 24.6.

[0052] A 50 wt% methanol aqueous solution containing glutathione (5 mM, 0.2 mL) and DSeBPA (15.6 mM, 1.8 mL) was incubated at 37 °C for 30 min, followed immediately by high-resolution mass spectrometry analysis. The results are as follows: Figure 4 As shown.

[0053] The formation of H2Se was detected using lead acetate test paper. The procedure was as follows: DSeBPA (23.8 mM) or Na2SeO3 (47.6 mM) was reacted with 150 μL of GSH (190.4 mM) ultrapure aqueous solution at 37 °C with shaking for 20 min. Then, 40 μL of GSH (47.6 mM) ultrapure aqueous solution was added, and immediately 150 μL of the reaction solution was transferred to one well of a 96-well plate. The well opening was quickly covered with moistened lead acetate test paper and sealed. After reacting for 12 h, the test paper was removed for image acquisition. The results are shown below. Figure 5 As shown.

[0054] Depend on Figure 4 and Figure 5 It can be seen that DSeBPA, under the action of glutathione, can generate active selenium species such as 5-selenopentanoic acid, GSSePA, and hydrogen selenide.

[0055] V. Preparation of unmodified nanoparticles IV (FtAg@SS / SiO2-Se nanoparticles), the process is as follows: Different masses (100 mg, 150 mg, 200 mg, 250 mg, 300 mg, and 350 mg) of DSeBPA were mixed with EDC (515 mg) and NHS (345 mg) in N,N-dimethylformamide (DMF) (10 mL). After activation for 2 h, the reaction solution was dispersed in DMF (5 mL) containing 100 mg of FtAg@SS / SiO2-NH2 nanoparticles. After continuous stirring for 24 h, the product was washed with ethanol and ultrapure water to obtain FtAg@SS / SiO2-Se nanoparticles. The characterization results are as follows: Figure 6 As shown; Figure A shows a transmission electron microscope (TEM) image of DSeBPA with a mass of 300 mg. The image shows a regular spherical nanoparticle structure with good particle dispersion. Figure B shows the particle size distribution of DSeBPA at a mass of 300 mg. As can be seen from the figure, the average particle size of the nanoparticles is 74.6 ± 0.4 nm, and the particle size distribution is concentrated with narrow peaks, indicating that the nanoparticles have good monodispersity (highly uniform particle size). Figure C shows the results of the Zeta potential analysis. From this figure, we can see that by grafting DSeBPA onto the material surface through the amidation reaction, the Zeta potential is restored to a negative value. System optimization determines that the optimal loading mass ratio of DSeBPA is 3.0. Figure D is an X-ray photoelectron spectroscopy (XPS) spectrum. From this spectrum, we can see the 3d characteristic peak of Se (55.8 eV) and the enhanced N 1s and C 1s signals, indicating that the selenium element was successfully introduced and formed a new bonding structure with the original component. Figure E shows the Raman spectrum, from which the S–S bond vibration peak (485.15 cm⁻¹) can be seen. -1 ) and the characteristic peak of Se–Se bonds in grafted DSeBPA (252.20 cm⁻¹) -1 (A redshift compared to the free state) This result indicates that DSeBPA was successfully introduced into nanoparticles through covalent modification.

[0056] VI. In-situ programmed synthesis of quantum dots in cells, the process is as follows: In cell culture dishes / plates, an appropriate amount of 4T1 cells were seeded and cultured for 24 hours. Once the cell density reached approximately 90%, the cell culture medium was aspirated, and serum-free DMEM medium containing FtAg@SS / SiO2-Se nanoparticles was added to a final concentration of 10.4 mg / mL. After incubation for different times, near-infrared fluorescent Ag2Se quantum dots were synthesized intracellularly, i.e., quantum dots synthesized in situ, denoted as Ag2Se@cells. Ag2Se@cells were uniformly dispersed in Tris-HCl buffer solution (1 mL), and 600 μL was added to a quartz cuvette. The emission spectra of the intracellular quantum dots were collected using a fluorescence spectrometer (excitation source and detector: 808 nm laser and PMT1700). The results are as follows: Figure 7 As shown; Figure A shows the fluorescence of FtAg@SS / SiO2-Se and 4T1 cells after co-incubation for different times. As can be seen from the figure, a near-infrared fluorescence signal appears at 1025nm, and the intensity gradually increases with the extension of incubation time, reaching the peak at 12h of co-incubation. Figure B is a fluorescence microscope image, which shows that the fluorescence signal is mainly located in the cell nucleus.

[0057] VII. Preparation of folic acid (FA) modified nanoparticles V (FtAg@SS / SiO2-Se-FA nanoparticles), the process is as follows: FA-(C2H4O) 2000 -COOH (25 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (19 mg) and N-hydroxysuccinimide (NHS) (12 mg) were dissolved in a mixed solvent (6 mL) of dimethyl sulfoxide (DMSO) and N,N-dimethylformamide in a volume ratio of 1:1. After carboxyl activation for 3 h, FtAg@SS / SiO2-Se nanoparticles (30 mg) dispersed in DMSO (4 mL) were added dropwise to the activation solution. The reaction was continued at room temperature for 24 h. The product was washed three times with DMSO and ultrapure water, and then freeze-dried to obtain FtAg@SS / SiO2-Se-FA nanoparticles. The particle size distribution of FtAg@SS / SiO2-Se nanoparticles and FtAg@SS / SiO2-Se-FA nanoparticles, as follows: Figure 8 As shown; Figure A shows the particle size distribution of FtAg@SS / SiO2-Se nanoparticles. From this figure, we can see that the particle size distribution range of FtAg@SS / SiO2-Se is 92.1±1.0 nm. B is a statistical diagram of the particle size distribution of FtAg@SS / SiO2-Se-FA nanoparticles. From this diagram, it can be seen that the hydrated particle size of the nanoparticles after folic acid modification is 105.8±1.3nm.

[0058] Example 2 Quantum dots were synthesized in vivo using FtAg@SS / SiO2-Se-FA nanoparticles, as follows: FtAg@SS / SiO2-Se-FA nanoparticles at concentrations of 4 mg / mL, 8 mg / mL, 12 mg / mL, 16 mg / mL and 20 mg / mL were injected intravenously twice into tumor-bearing mice. FtAg@SS / SiO2-Se-FA nanoparticles at concentrations of 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL and 60 mg / mL were injected into the tumor twice. The control group was injected with an equal volume of BR buffer (pH 7.4). Imaging images showing the changes in fluorescence distribution signals over time at different concentrations and injection numbers, obtained using a near-infrared II imaging system, such as... Figure 9 and Figure 10 As shown; from Figure 9It can be seen that: tumor-bearing mice injected intravenously with FtAg@SS / SiO2-Se-FA nanoparticles at concentrations of 12 mg / mL, 16 mg / mL and 20 mg / mL showed near-infrared fluorescence signals at the tumor site, and the fluorescence signals remained relatively stable over time and number of injections, while the control group showed no obvious fluorescence signals from 2 h to 36 h. from Figure 10 It can be seen that tumor-bearing mice injected with FtAg@SS / SiO2-Se-FA nanoparticles at concentrations of 20 mg / mL, 30 mg / mL, and 40 mg / mL showed clear fluorescence signals within 2 hours of injection, and the fluorescence signals increased with time and number of injections. The control group showed no obvious fluorescence signals from 2 hours to 36 hours.

[0059] Line graphs showing the relative levels of fluorescence signal at the tumor site over time under different administration routes (injection concentration of 20 mg / mL), as shown. Figure 11 As shown in the figure, MFI represents the average fluorescence intensity; from Figure 11 It can be seen that the fluorescence signal ratio of the intratumoral injection group is much higher than that of the intravenous injection group, indicating that intratumoral injection can achieve effective enrichment of nanoparticles at the tumor site.

[0060] A bar chart showing the change in mean fluorescence intensity (MFI) at the tumor site over time under different injection methods and injection frequencies (injection concentration of 20 mg / mL), as shown below. Figure 12 As shown in the figure, the MFI in the intratumoral injection group is much higher than that in the intravenous injection group (the MFI in the intravenous group is generally lower than 1×10). 7 The highest value in the intratumoral group was close to 4.5 × 10⁻⁶. 7 This indicates that intratumoral injection allows nanoparticles to accumulate more concentratedly at the tumor site; under the same injection method, the MFI at each time point of the second intratumoral injection is generally higher than that of the first injection; while the MFI difference between the two intravenous injections is very small.

[0061] The above results indicate that FtAg@SS / SiO2-Se-FA nanoparticles can efficiently synthesize quantum dots in situ in tumor-bearing mice.

[0062] Example 3 After culturing 4T1 cells in 96-well plates for 24 h, the medium was replaced with fresh medium containing different concentrations (based on the final concentration of silver) of FtAg@SS / SiO2-Se, FtAg@SS / SiO2, and Ft-Ag(I) for 24 h. Then, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C for 30 min. The absorbance at 450 nm was then measured using a microplate reader. Calculate relative cell viability using the following formula: Cell viability (%) = (sample absorbance - background absorbance) / (control group absorbance - background absorbance) × 100%; The PBS-treated group served as a negative control.

[0063] Bar chart showing the effects of different nanoparticles on cell activity, such as... Figure 13 As shown in the figure, it can be seen that FtAg@SS / SiO2-Se, FtAg@SS / SiO2 and Ft-Ag(I) exhibit concentration-dependent cytotoxicity, with FtAg@SS / SiO2-Se nanoparticles having the most significant effect on cell activity.

[0064] Example 4 After culturing 4T1 cells in 12-well plates for 24 h, they were incubated for 12 h in serum-free medium containing FtAg@SS / SiO2-Se, FtAg@SS / SiO2, or Ft-Ag(I) (based on the final concentration of silver). The medium was then replaced with fresh serum-free medium containing 5 μM fluorescein diacetate (2',7'-dichlorodihydrofluorescein diacetate) and incubated at 37°C in the dark for 30 min. After washing three times with PBS, fluorescence imaging was performed using a confocal microscope equipped with a 488 nm laser and 525 / 50 nm filters. The results are as follows: Figure 14 As shown in the figure, both FtAg@SS / SiO2-Se and FtAg@SS / SiO2 can significantly increase ROS levels in a concentration-dependent manner. This result indicates that the breaking of S–S and Se–Se bonds induces oxidative stress, ultimately leading to tumor cell apoptosis or necrosis.

[0065] Example 5 FtAg@SS / SiO2-Se (30 mg / mL) or BR buffer (pH 7.4) as a control was injected twice into the tumor of 4T1 tumor-bearing BALB / c mice. 36 hours after the second injection, the tumor area was irradiated with an 808 nm laser (power density 1.5 W / cm²). 2 Irradiation was performed for 9 minutes or no irradiation was performed. During the irradiation period, temperature changes and thermal images were recorded using an infrared thermal imager. Tumor volume and mouse weight were monitored every two days for 30 days, and survival status was recorded throughout the process.

[0066] Linear graph showing the photothermal heating effect after intratumoral injection of FtAg@SS / SiO2-Se nanoparticles and synthesis of quantum dots in the tumor microenvironment, as shown. Figure 15 As shown in the figure, laser irradiation can rapidly raise the local temperature to 56°C, which is significantly higher than the 30°C of the control group. This result indicates that quantum dots synthesized using living cells have excellent photothermal conversion performance.

[0067] The effects of intratumoral injection of FtAg@SS / SiO2-Se nanoparticles on tumor-bearing mice, such as Figure 16 As shown; Figure A shows the curve of tumor volume change over time. The figure shows that the tumor growth inhibition rate reached 51.2% after 30 days. This result indicates that intratumoral injection of FtAg@SS / SiO2-Se has significant antitumor activity. Figure B shows the survival rate over time. It can be seen from the figure that intratumoral injection of FtAg@SS / SiO2-Se can effectively delay tumor progression and prolong survival.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for the preparation of quantum dots synthesized in situ in cells, characterized in that, The method comprises the following steps: S100, preparing nanoparticles I by using a soluble silver salt and a protein; S200, preparing nanoparticles II by using nanoparticles I, a silicate compound and a double alkoxysilane compound containing a disulfide bond; S300, modifying nanoparticles II by using a coupling agent to obtain nanoparticles III; S400, activating the coupling group of a diselenide compound by using an activator I, and then coupling the activated group with nanoparticles III to prepare nanoparticles IV; S500, activating a targeting molecule by using an activator II, and then coupling the activated molecule with nanoparticles IV to prepare nanoparticles V; S600, releasing selenium precursors and silver precursors in cells by using nanoparticles IV or nanoparticles V to obtain in-situ programmed synthesis Ag2Se quantum dots.

2. The method of claim 1, wherein the cell is a human cell. In step S100, the protein is selected from at least one of ferritin, apoferritin and albumin, and the soluble silver salt is selected from silver nitrate or silver acetate. The molar ratio of the protein to the soluble silver salt is 1:100-1:

400.

3. The method of claim 1, wherein the cell is a human cell. In step S200, the silicate compound is selected from any one of methyl orthosilicate, ethyl orthosilicate and propyl orthosilicate. The double alkoxysilane compound containing a disulfide bond is selected from any one of bis(triethoxysilylpropyl) disulfide, bis[3-(trimethoxysilyl)propyl] disulfide and bis[3-(triethoxysilyl)propyl] disulfide.

4. The method of claim 1, wherein the cell is a human cell. In step S300, the coupling agent is selected from any one of an amino silane coupling agent and a carboxyl silane coupling agent. The amino silane coupling agent is selected from any one of 3-aminopropyl triethoxysilane, 3-aminopropyl methyldiethoxysilane and γ-aminopropyl triethoxysilane. The carboxyl silane coupling agent is selected from any one of 3-carboxypropyl trimethoxysilane, 3-carboxypropyl triethoxysilane and carboxyethyl silane triol sodium salt.

5. The method of claim 1, wherein the cell is a human cell. In step S400, the activator I is selected from a carboxylic acid activator, and the carboxylic acid activator is selected from EDC and NHS. EDC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide.

6. The method of claim 1, wherein the cell is a human cell. In step S400, the diselenide compound is selected from the group consisting of HOOC - (CH2) n - Se - Se - (CH2) m - COOH, n and m are each independently one of an integer from 1 to 10; The mass ratio of the diselenide compound to nanoparticles III is 1:1-3.5:

1.

7. The method of claim 1, wherein the cell is a human cell. In step S500, the activator II is selected from a carboxylic acid activator, and the carboxylic acid activator is selected from EDC and NHS. EDC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide. The targeting molecule is selected from a tumor targeting molecule, and the tumor targeting molecule is selected from a folic acid targeting coupling derivative. said folate targeted conjugated derivative is selected from the group consisting of FA-(C2H4O) k -COOH, k is 1000, 1500, 2000; FA is folic acid.

8. The method of claim 1, wherein the cell is a human cell. In step S600, the cells are selected from any one of 4T1 cells, MCF-7 cells, A549 cells and HeLa cells.

9. Cell in situ procedurally synthesized quantum dots, characterized in that, The in-situ programmed synthesis quantum dots are prepared by using the preparation method of the in-situ programmed synthesis quantum dots according to any one of claims 1 to 8.

10. Use of in situ procedures for the synthesis of quantum dots in cells, characterized in that, The in-situ programmed synthesis quantum dots according to claim 9 are used in any one or more of the following applications: for preparing a pharmaceutical preparation for treating and / or preventing tumors; for preparing a medical device for treating and / or preventing tumors; For the preparation of a medical imaging product; For the preparation of a tumor diagnostic product.

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