CN116426275B

CN116426275B - Fluorescent silicon quantum dot and preparation method and application thereof

Info

Publication number: CN116426275B
Application number: CN202310236502.3A
Authority: CN
Inventors: 葛介超; 孔琳; 刘卫敏; 汪鹏飞
Original assignee: Technical Institute of Physics and Chemistry of CAS
Current assignee: Technical Institute of Physics and Chemistry of CAS
Priority date: 2023-03-13
Filing date: 2023-03-13
Publication date: 2024-05-28
Anticipated expiration: 2043-03-13
Also published as: CN116426275A

Abstract

The invention discloses a fluorescent silicon quantum dot which is dot-shaped nano particles with the size of 5-10 nm. The fluorescence of the fluorescent silicon quantum dot has excitation dependence, can target a lysosome of a cancer cell, can generate singlet oxygen under laser irradiation, and can be effectively used in fluorescence imaging-mediated photodynamic therapy. The invention also discloses a preparation method and application of the fluorescent silicon quantum dot.

Description

Fluorescent silicon quantum dot and preparation method and application thereof

Technical Field

The invention relates to the technical field of silicon quantum dots. More particularly, relates to a fluorescent silicon quantum dot and a preparation method and application thereof.

Background

Photodynamic therapy (Photodynamic Therapy, abbreviated PDT) is a highly effective treatment technique for malignant tumors that has been newly developed in recent years. PDT has the characteristics of strong selectivity, small side effect, repeated treatment and the like, and is another novel phototherapy technology besides photothermal treatment (Photothermal Therapy, abbreviated as PTT). The basic principle of PDT is that when a photosensitizer is accumulated in tumor tissue, it is irradiated with light of an appropriate wavelength, and the photosensitizer generates active oxygen by destroying genetic material of cancer cells, etc., thereby killing the cancer cells. One is that a photosensitizer in a singlet excited state transitions to a triplet state through intersystem crossing, directly transferring energy to oxygen, generating singlet oxygen, a process known as type II PDT. Another form is that the photosensitizer in the triplet state and oxygen undergo electron transfer to generate hydroxyl radicals, or superoxide anions, or active oxygen such as hydrogen peroxide, which is type I PDT [ chem. Soc. Rev.,2016,45,6488-6519 ]. In addition, photosensitizers with fluorescent properties, when entering tumor tissue, can be imaged by fluorescence. The photosensitizer integrating diagnosis and treatment is a hotspot of current attention of researchers, and can lay a solid foundation for accurate treatment of cancers.

Photodynamic therapy (PDT), while having some of the advantages described above, has some drawbacks that have prevented its clinical use. For example, active oxygen generated during PDT has a lifetime of less than 5 microseconds and a diffusion distance of less than 30nm (Angew. Chem.2016,128, 10101-10105). In addition, the cell membrane and cytoplasm of cancer cells also have a loss of active oxygen produced. Therefore, the preparation of the photosensitizer which can not only perform fluorescence imaging on cancer cells and generate active oxygen, but also target subcellular organelles is an important method for improving the PDT curative effect. In all subcellular organelles of cancer cells, lysosomes play an important role in cell proliferation differentiation and steady-state regulation, so that the preparation of a photosensitizer targeting lysosomes will greatly improve the efficiency of PDT.

Silicon quantum dots are a kind of silicon nanomaterial that has recently been closely focused on by researchers. The preparation method has the characteristics of abundant raw materials, simple preparation, excellent electric/optical properties, good biocompatibility, low toxicity, easy surface modification and the like, so that the preparation method is widely applied to the fields of microelectronic materials, clean energy sources, biomedicine and the like (chem. Rev.2016,116, 215-257). Although silicon quantum dots have also made a series of advances in cancer diagnostics, silicon quantum dot photosensitizers that can be used to target lysosomes and photodynamic diagnostics simultaneously have not been reported.

Disclosure of Invention

Based on the facts, the invention aims to provide a fluorescent silicon quantum dot and a preparation method and application thereof. The fluorescence of the fluorescent silicon quantum dot has excitation dependence, can target a lysosome of a cancer cell, can generate singlet oxygen under laser irradiation, and can be effectively used in fluorescence imaging-mediated photodynamic therapy.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a fluorescent silicon quantum dot which is a dot-shaped nanoparticle with a size of 5-10 nm.

Further, using Cu-ka radiation, the X-ray powder diffraction expressed in terms of 2θ angle has characteristic diffraction peaks at about 27.8 °, 39.8 °, 58.8 °.

The research of the invention shows that the selection of raw materials influences the performance of the finally prepared silicon quantum dots. Further, the fluorescent silicon quantum dots are obtained by reducing a cross-linked substance of the reducing agent and silicon tetrachloride by the reducing agent.

Further, the reducing agent is selected from diethylene glycol.

Further, the fluorescent silicon quantum dot is obtained by reducing diethylene glycol-silicon tetrachloride cross-linked matter through diethylene glycol.

Further, the ratio of the reducing agent to the silicon tetrachloride is (200-400): 1, preferably (200-250): 1. According to the invention, diethylene glycol and silicon tetrachloride are mixed to obtain a cross-linked product, and the excessive diethylene glycol is further used as a reducing agent to reduce the cross-linked product, so that the fluorescent silicon quantum dot is obtained.

In yet another aspect, the present invention provides a method for preparing the fluorescent silicon quantum dot as described above, comprising the steps of:

and uniformly mixing silicon tetrachloride with a reducing agent, heating to perform a reduction reaction, and dialyzing, filtering and freeze-drying after the reaction is finished to obtain the fluorescent silicon quantum dot.

Since silicon tetrachloride is stored in a closed state, and in particular, contact with water is avoided, the silicon tetrachloride and the reducing agent are preferably mixed by injecting the silicon tetrachloride into the reducing agent. And the reduction reaction is preferably carried out in an oil bath environment. The oil bath is adopted to heat more conveniently, the reaction is more uniform, the reduction degree is easy to control, and the preparation cost is reduced.

Further, the mixing mode is stirring, and the stirring speed is preferably 600r/min.

Further, the temperature of the reduction reaction is 140-170 ℃ and the time is 5-6h. Researches show that if the reduction reaction temperature is too low, the obtained product has no fluorescence effect; if the reduction reaction temperature is too high, blue shift of fluorescence occurs.

Further, the reducing agent is diethylene glycol.

Further, the volume ratio of the reducing agent to the silicon tetrachloride is (200-400): 1. preferably, the volume ratio of the reducing agent to the silicon tetrachloride is (200-250): 1. at this time, the prepared fluorescent silicon quantum dot has high purity and no impurity.

In the preparation method, diethylene glycol and silicon tetrachloride are uniformly mixed to obtain a cross-linked product, and the excessive diethylene glycol is further used as a reducing agent to reduce the cross-linked product to obtain the fluorescent silicon quantum dot.

Further, the dialysis has a molecular weight cut-off of 3000Da for 24-36 hours.

Further, the pore size of the filter membrane for filtering is 0.4-0.8um.

Further, the freeze-drying temperature is-50 ℃ to-55 ℃ and the time is 24-48h.

In yet another aspect, the invention provides an application of the fluorescent silicon quantum dot in preparing a cancer cell lysosome targeting drug or photodynamic therapy drug.

The fluorescence of the fluorescent silicon quantum dot has excitation dependence under excitation of different wavelengths, can target a lysosome of a cancer cell, can generate singlet oxygen under laser irradiation, and can be effectively used in photodynamic therapy mediated by fluorescence imaging.

Further, the fluorescent silicon quantum dots may generate singlet oxygen under laser irradiation including, but not limited to, 635nm wavelength, 532nm wavelength, 577nm wavelength. Preferably, the singlet oxygen generation effect under the irradiation of laser light with a wavelength of 635nm is better.

The beneficial effects of the invention are as follows:

The fluorescent silicon quantum dot provided by the invention fills the blank in the technical field of silicon quantum dots which can be simultaneously used for targeting lysosomes and photodynamic diagnosis and treatment. The fluorescence of the fluorescent silicon quantum dot has excitation dependence, and singlet oxygen can be generated after laser irradiation. The preparation raw materials are abundant in reserves, simple, convenient and easy to obtain. The preparation of the fluorescent silicon quantum dot provided by the invention can obtain excellent optical performance without manually doping other fluorescent groups in the process of synthesizing the silicon quantum dot, and has high biosafety. The fluorescent silicon quantum dot provided by the invention can be well applied to the targeting cancer cell lysosome, and can be used as a fluorescent probe. Can generate singlet oxygen under the irradiation of laser, thereby killing cancer cells and being used as a photodiagnosis and treatment integrated reagent.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the drawings.

Fig. 1 shows a transmission electron microscope image showing the fluorescent silicon quantum dots prepared in example 1.

Fig. 2 shows an XRD spectrum of the fluorescent silicon quantum dots prepared in example 1.

Fig. 3 shows an absorption spectrum of the fluorescent silicon quantum dot prepared in example 1.

Fig. 4 shows a fluorescence spectrum of the fluorescent silicon quantum dot prepared in example 1.

Fig. 5 shows the singlet oxygen generating capacity of the fluorescent silicon quantum dot prepared in example 1, whose absorption is reduced by degradation of ABDA.

Fig. 6 shows that the fluorescent silicon quantum dots prepared in example 1 can generate fluorescence under laser irradiation after being incubated with Hela cells for different times.

Figure 7 shows the ability of fluorescent silicon quantum dots prepared in example 1 to target cancer cell lysosomes.

Fig. 8 shows the dark toxicity and the ability to kill Hela cells under laser irradiation of the fluorescent silicon quantum dots prepared in example 1.

Fig. 9 shows a transmission electron microscope image of the silicon quantum dots obtained in example 6.

Fig. 10 shows a transmission electron microscope image of the solid powder obtained in example 7.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

Example 1

A preparation method of fluorescent silicon quantum dots comprises the following steps:

40mL of the diethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 200uL of silicon tetrachloride solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 160℃in an oil bath and reacted for 6 hours. After the solution cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove the unreacted complete diethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. Then, the powder was filtered using a filter membrane having a pore size of 0.80um and dried for 24 hours using a vacuum freeze dryer to obtain fluorescent silicon quantum dot solid powder.

Fig. 1 is a transmission electron microscope image of the fluorescent silicon quantum dot corresponding to example 1. The results show that fluorescent silicon quantum dots prepared by reducing silicon tetrachloride by diethylene glycol are uniformly dispersed dot-shaped particles, and the size is between 5 and 10 nm.

Fig. 2 is an XRD spectrum of the fluorescent silicon quantum dot prepared in example 1. From the graph, the fluorescent silicon quantum dot has street incidence peaks at the positions of 27.8 degrees, 39.8 degrees and 58.8 degrees, corresponding to the (111), (200) and (311) crystal faces of dry silicon, and the characterization result proves that the fluorescent silicon quantum dot is successfully prepared.

Fig. 3 is an absorption spectrum of the fluorescent silicon quantum dot according to example 1. The result shows that fluorescent silicon quantum dots prepared by reducing silicon tetrachloride by diethylene glycol have absorption at 450-700 nm.

Fig. 4 is a graph of excitation spectra of the fluorescent silicon quantum dots corresponding to example 1 at different wavelengths, and the result shows that the fluorescence of the silicon quantum dots has excitation dependence.

Example 2

An application of fluorescent silicon quantum in preparing photodynamic reagent.

The fluorescent silicon quantum dots prepared in example 1 above were dispersed in water to prepare a 400ug/mL solution. 1mL of the solution was placed in a cuvette, and background absorption was subtracted. Then 60uL of 1mg/mL ABDA solution was added to the cuvette and its absorbance spectrum was measured. This was then irradiated with a 635nm laser of 0.1W/cm ², and the absorption spectrum was measured every one minute. Judging whether the silicon quantum dot can generate singlet oxygen or not by judging the change of the absorption spectrum of the ABDA.

Fig. 5 shows that the fluorescence silicon quantum dot corresponding to example 1 uses the change of the absorption spectrum of ABDA to monitor singlet oxygen, and the absorption spectrum is gradually decreased after 635nm laser irradiation, so that singlet oxygen can be generated, and the fluorescence silicon quantum dot has potential for photodynamic therapy of cancer cells.

Example 3

An application of fluorescent silicon quantum dots in fluorescent imaging. The fluorescent silicon quantum dots prepared in example 1 were prepared as a 400ug/mL solution and added to a HeLa cell confocal dish. And (3) observing whether the material can smoothly enter cells and emit fluorescence under a confocal fluorescence microscope when incubating the 3 rd, the 6 th, the 9 th and the 12 th, and performing fluorescence imaging on the cells.

Fig. 6 shows that the fluorescent silicon quantum dots corresponding to example 1 successfully enter cytoplasm after incubation with Hela cells for 3, 6, 9, 12h, and the fluorescence intensity gradually increases under laser irradiation. Silicon quantum dots are illustrated as being useful for fluorescence imaging.

Example 4

An application of fluorescent silicon quantum dots in targeting lysosomes. The fluorescent silicon quantum dots prepared in example 1 were prepared as a 400ug/mL solution, added to a HeLa cell confocal dish, and incubated for 9h. Then 10uL of lysosome targeted red fluorescent probe was added and fluorescence coincidence and co-localization coefficient were observed under confocal fluorescence microscope.

FIG. 7 is a co-localization map of fluorescent silicon quantum dots and lysosome targeted red fluorescent probes corresponding to example 1. The green fluorescence of the silicon quantum dot and the red fluorescence of the probe can be well overlapped, the co-localization coefficient reaches 0.92, and the lysosome can be well targeted.

Example 5

Dark toxicity and phototoxicity experiments of cells:

Approximately 1 x 10 ⁴ Hela cells per well were seeded in 96-well plates in 6 rows and 10 columns and incubated at 37 ℃ in 5% co ₂ for 48h. Then, a new culture medium is replaced by a 96-well plate, and the fluorescent silicon quantum dot material prepared in the embodiment 1 is added, so that the concentration of each row of the wells is respectively 0, 50, 100, 200 and 300ug/mL, and the incubation is continued for 9 hours. This was irradiated with a 635nm laser at 0.1W/cm ² and incubation continued for 15h. Thereafter, relative viability of the cells was examined using MTT colorimetry.

Fig. 8 shows the dark toxicity and phototoxicity of the fluorescent silicon quantum dots corresponding to example 1 to Hela cells. The fluorescent silicon quantum dot nano material has low dark toxicity to Hela cells and high phototoxicity, and can generate singlet oxygen after laser irradiation, so that proliferation of cancer cells can be obviously inhibited.

Example 6

40mL of the diethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 100uL of silicon tetrachloride solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 160℃in an oil bath and reacted for 6 hours. After the solution cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove the unreacted complete diethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. Then, the powder was filtered using a filter membrane having a pore size of 0.80um and dried for 24 hours using a vacuum freeze dryer to obtain fluorescent silicon quantum dot solid powder.

Fig. 9 is a transmission electron microscope of the obtained material, and it is observed that impurities having non-uniform sizes exist in the prepared silicon quantum dots.

Example 7

30mL of the diethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 100uL of silicon tetrachloride solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 160℃in an oil bath and reacted for 6 hours. After the solution cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove the unreacted complete diethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. This was then filtered using a filter membrane with a pore size of 0.80um and dried using a vacuum freeze dryer for 24h to give a solid powder.

Fig. 10 is a transmission electron microscope of the obtained material, and it is observed that the prepared material does not have the morphology of quantum dots.

Comparative example 1

A preparation method of silicon quantum dots comprises the following steps:

40mL of the diethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 200uL of silicon tetrachloride solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 80℃in an oil bath and reacted for 6 hours. After the solution cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove the unreacted complete diethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. It was then filtered using a filter membrane with a pore size of 0.80um and dried using a vacuum freeze dryer for 24 hours to obtain silicon quantum dot solid powder.

The silicon quantum dots did not fluoresce upon irradiation with an ultraviolet lamp.

Comparative example 2

A preparation method of silicon quantum dots comprises the following steps:

40mL of the diethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 200uL of silicon tetrachloride solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 180 ℃ in an oil bath and reacted for 6 hours. After the solution cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove the unreacted complete diethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. It was then filtered using a filter membrane with a pore size of 0.80um and dried using a vacuum freeze dryer for 24 hours to obtain silicon quantum dot solid powder.

Through detection, the fluorescence of the silicon quantum dot is blue shifted.

Comparative example 3

A preparation method of silicon quantum dots comprises the following steps:

40mL of the ethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 200uL of silicon tetrachloride solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 160℃in an oil bath and reacted for 6 hours. After the solution was cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove unreacted complete ethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. It was then filtered using a filter membrane with a pore size of 0.80um and dried using a vacuum freeze dryer for 24 hours to obtain silicon nanorods instead of silicon quantum dots. And the silicon nanorods cannot target lysosomes.

Comparative example 4

A preparation method of silicon quantum dots comprises the following steps:

40mL of the diethylene glycol solution was weighed into a 250mL two-necked flask and sealed with a rubber stopper. 200uL of gamma-aminopropyl triethoxysilane solution was injected into the flask using a syringe and magnetically stirred at 600r/min. Then heated to 160℃in an oil bath and reacted for 6 hours. After the solution cooled, the solution was dialyzed with a semipermeable membrane (molecular weight 3000 Da) for 24 hours to remove the unreacted complete diethylene glycol solution, to obtain an aqueous solution of silicon quantum dots. It was then filtered using a filter membrane with a pore size of 0.80um and dried using a vacuum freeze dryer for 24 hours to obtain silicon quantum dot solid powder.

It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. The fluorescent silicon quantum dot is characterized in that the fluorescent silicon quantum dot is dot-shaped nano particles, and the size of the fluorescent silicon quantum dot is between 5 and 10 nm;

the fluorescent silicon quantum dots are obtained by reducing a cross-linked product of the reducing agent and silicon tetrachloride by the reducing agent;

The reducing agent is selected from diethylene glycol;

The temperature of the reduction is 140-170 ℃ and the time is 5-6h;

The volume ratio of the reducing agent to the silicon tetrachloride is 200-400:1.

2. The fluorescent silicon quantum dot of claim 1, wherein using Cu-ka radiation, X-ray powder diffraction expressed in terms of 2Θ angles has characteristic diffraction peaks at 27.8 °, 39.8 °, 58.8 °.

3. The fluorescent silicon quantum dot according to claim 1, wherein the fluorescent silicon quantum dot is obtained by reducing diethylene glycol-silicon tetrachloride cross-linked product by diethylene glycol.

4. A method of preparing a fluorescent silicon quantum dot according to any one of claims 1 to 3, comprising the steps of:

5. The process according to claim 4, wherein the ratio of reducing agent to silicon tetrachloride is 200-250:1 by volume.

6. The method according to claim 4, wherein the dialysis has a molecular weight cut-off of 3000Da for a period of 24-36 hours; and/or

The aperture of the filter membrane for filtering is 0.4-0.8um; and/or

The freeze-drying temperature is-50 to-55 ℃ and the time is 24-48h.

7. Use of the fluorescent silicon quantum dot according to any one of claims 1-3 for the preparation of cancer cell lysosome targeting drugs or photodynamic therapy drugs.