Fluorescent nano carbon dot for tumor photodynamic therapy based on ribosome affinity, preparation and application

Abstract

The invention discloses a ribosome affinity-based fluorescent nano carbon dot for tumor photodynamic therapy as well as preparation and application thereof, and relates to the field of subcellular organelle imaging and photodynamic therapy. Levofloxacin, toluidine blue and polyethyleneimine are used as raw materials, and the carbon dots with red fluorescence and photodynamic therapy effects are synthesized through heating. Levofloxacin can be modified to the surfaces of the carbon dots and is used for being combined with ribosome positioned on a rough surface endoplasmic reticulum; toluidine blue serves as a photosensitizer to serve as a carbon core in carbon dot synthesis, and formation of the carbon dots is facilitated; the polyethyleneimine is used as a cationic polymer, so that the surfaces of the carbon dots have proper positive charges, and rapid uptake of the carbon dots by cells is facilitated. The nano carbon dots can specifically mark cellular ribosome, so that imaging of the ribosome is realized, and in addition, photodynamic therapy can be realized under laser irradiation. The carbon dots are high in specificity and rapid in marking, and can generate singlet oxygen for photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to the field of subcellular organelle imaging and photodynamic therapy, and more specifically, to a ribosome-affinity-based fluorescent carbon nanodot for tumor photodynamic therapy, its preparation and application, and particularly to the preparation and application of a carbon dot with photodynamic function based on the specific affinity ability of levofloxacin to ribosomes. Background Art

[0002] As the site for protein translation and transportation, the damage, repair and regulation of ribosome functions directly affect the cell phenotype and physiological state, and further affect various diseases including tumors. The synthesis of proteins in cells all starts with the ribosome binding to mRNA through its large and small subunits. After the N-terminal signal peptide is translated by the ribosome in the initial stage, it is recognized by the signal recognition particle (SRP) in the cell matrix together with the large and small subunits of the ribosome. Subsequently, it is actively transported to the vicinity of the rough endoplasmic reticulum, and the SRP receptor on the rough endoplasmic reticulum membrane is recognized by SRP, so as to stay in the endoplasmic reticulum. After the translation is completed, the ribosome leaves the rough endoplasmic reticulum. Ribosomes have a highly complex three-dimensional structure, which provides binding sites for mRNA, tRNA, peptide chains, etc., and also provides targets for a variety of specific affinity molecules.

[0003] Traditional ribosome active targeting particles are designed based on the principle of pairing targeting of nucleic acid sequences to ribosomal RNA or receptor binding. Limited by the multiple organelles and complex transport environment in cells and the low specificity of receptors, the targeting accuracy is insufficient. Therefore, designing a new ribosome targeting mechanism for precise imaging and efficient photodynamic therapy is still a huge challenge. Summary of the Invention

[0004] Aiming at the problem of insufficient precise delivery of existing endoplasmic reticulum drugs, the present invention provides a cell ribosome carbon nanodot based on the specific affinity ability of levofloxacin to ribosomes. Using levofloxacin, toluidine blue and polyethyleneimine as raw materials, fluorescent carbon dots are synthesized by heating. In the present invention, levofloxacin modifies the surface of the carbon dots to bind to ribosomes located on the rough endoplasmic reticulum; toluidine blue is used as a photosensitizer and serves as a carbon core in the synthesis of carbon dots, which helps the formation of carbon dots. Part of the toluidine blue is distributed on the surface of the carbon dots to provide a photodynamic therapy effect, and it has a conjugated structure, endowing the carbon dots with good fluorescence characteristics; polyethyleneimine is used as a cationic polymer, making the surface of the carbon dots carry appropriate positive charges, which helps the cells to rapidly uptake the carbon dots.

[0005] According to the first aspect of the present invention, a preparation method of a ribosome-affinity-based fluorescent carbon nanodot is provided, including the following steps: (1) Heat the mixed solution of levofloxacin, toluidine blue, and polyethyleneimine to form carbon nanodots; the toluidine blue serves as the carbon core for the formation of carbon dots; the polyethyleneimine serves as a cationic polymer to make the surface of the carbon dots carry a positive charge; the levofloxacin is modified on the surface of the carbon dots, and the levofloxacin is used to target ribosomes on the rough endoplasmic reticulum; (2) Filter the solution obtained in step (1) through an aqueous filter to remove insoluble substances and retain the filtrate; then perform dialysis using a dialysis membrane to obtain fluorescent carbon nanodots based on ribosome affinity for tumor photodynamic therapy.

[0006] Preferably, in the mixed solution, the mass ratio of levofloxacin, toluidine blue, and polyethyleneimine is (2 - 4):(10 - 15):(1 - 2).

[0007] Preferably, the heating method is microwave heating, the heating power is 500 W - 700 W, and the heating time is 5 min - 10 min.

[0008] According to another aspect of the present invention, there is provided fluorescent carbon nanodots based on ribosome affinity prepared by any one of the methods.

[0009] Preferably, the particle size range of the fluorescent carbon nanodots is 1 nm - 10 nm.

[0010] Preferably, the surface potential range of the fluorescent carbon nanodots is 20 mV - 40 mV.

[0011] According to another aspect of the present invention, there is provided the application of the fluorescent carbon nanodots based on ribosome affinity as described in any one of the above in the preparation of tumor photodynamic therapy reagents.

[0012] According to another aspect of the present invention, there is provided the application of the fluorescent carbon nanodots based on ribosome affinity as described in any one of the above in the preparation of imaging reagents.

[0013] Generally speaking, compared with the existing photodynamic therapy probes, the above technical solutions designed in the present invention have the following principles and technical advantages: (1) Levofloxacin kills bacteria by binding to the functional site of ribosomes to interfere with protein synthesis in bacteria. Similarly, levofloxacin can also bind to eukaryotic ribosomes, but eukaryotic cells have significantly higher tolerance to it. The main reason is that its binding site to eukaryotic ribosomes is a non-functional site, and the interference with protein synthesis is limited. Therefore, in the present invention, levofloxacin is used as a ribosome-targeting molecule, and levofloxacin-modified carbon nanodots are synthesized by a one-step method, which can bind to ribosomes and emit red fluorescence and generate reactive oxygen species under light excitation for photodynamic therapy.

[0014] (2) The ribosomal nanocarbodots in the present invention have precise and highly specific targeting capabilities with strong specificity. The high affinity of levofloxacin for eukaryotic ribosomes and the cellular behavior of ribosomes concentrated and enriched in the rough endoplasmic reticulum are utilized to accurately locate the endoplasmic reticulum. Different from the principle of traditional ribosomal fluorescent probes targeting ribosomal proteins and RNA with antibody molecules or nucleic acid sequences, the target of the nanocarbodots in the present invention is the tertiary structure of ribosomal rRNA, thereby achieving the localization of ribosomes.

[0015] (3) In the present invention, due to the affinity of levofloxacin for ribosomes, it has low toxicity to eukaryotes. By utilizing the differences in the structures and conformations of bacterial and mammalian ribosomes, precise affinity for eukaryotic ribosomes is achieved, while interference with the functions of normal cell ribosomes is avoided, and the physiological toxicity of the probe is minimized to the greatest extent.

[0016] (4) The present invention proposes a new targeting technology for eukaryotic endoplasmic reticulum. Levofloxacin is combined with ribosomes enriched in the rough endoplasmic reticulum to target the endoplasmic reticulum, and it has the ability of tracing.

[0017] (5) The preparation method of the present invention is simple, which is a one-step microwave method or a one-step hydrothermal synthesis, and the synthesis conditions are simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the synthesis process of the nanocarbodots described in the present invention.

[0019] Figure 2 It is a flow chart of the binding of ribosomes and targeting of the endoplasmic reticulum in cells by the nanocarbodots based on ribosome affinity for tumor photodynamic therapy described in the present invention.

[0020] Figure 3 It is the transmission electron microscope (TEM) image and high-resolution transmission electron microscope (HRTEM) image of the fluorescent carbodots described in the present invention.

[0021] Figure 4 It is the X-ray diffraction spectrum (XRD) of the fluorescent carbodots described in the present invention.

[0022] Figure 5 It is the particle size distribution diagram of the fluorescent carbodots described in the present invention.

[0023] Figure 6 It is the ultraviolet-visible light absorption spectrum diagram of the fluorescent carbodots described in the present invention.

[0024] Figure 7 It is the fluorescence spectrum diagram of the fluorescent carbodots described in the present invention.

[0025] Figure 8 It is the Fourier transform infrared spectrum diagram of the fluorescent carbodots described in the present invention.

[0026] Figure 9 1H NMR spectrum of the fluorescent carbon dots of the present invention.

[0027] Figure 10 ICP-MS mass spectrum of the fluorescent carbon dots of the present invention.

[0028] Figure 11 After the fluorescent carbon dots of the present invention are irradiated with a 660 nm laser, 1 O2 is generated and causes 1 A graph showing a significant decrease in the absorption of the O2 capture reagent DPBF at 420 nm.

[0029] Figure 12 Fluorescence co-localization map of the nanocarbon dots of the present invention with the endoplasmic reticulum, lysosomes, cell membrane, and mitochondria in cells. The hemolytic properties of the detection probe are detected after the nanocarbon dots of the present invention are incubated with red blood cells.

[0030] Figure 13 Co-localization map of the nanocarbon dots of the present invention with the endoplasmic reticulum in different cells (Hela, SKBR-3, MDA-MB-231, BT549).

[0031] Figure 14 Fluorescence co-localization map of the fluorescent carbon dots of the present invention with the endoplasmic reticulum, lysosomes, cell membrane, and mitochondria in cells.

[0032] Figure 15 Verification of ROS generation by the fluorescent nanocarbon dots of the present invention in cells.

[0033] Figure 16 Statistical chart of the cell therapy effect of the fluorescent nanocarbon dots of the present invention. Detailed implementation manners

[0034] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] The present invention relates to a kind of nano-carbon dots for tumor photodynamic therapy based on ribosome affinity. Using levofloxacin, toluidine blue and polyethyleneimine as raw materials, carbon dots with red fluorescence and photodynamic therapy effect are synthesized by microwave method or hydrothermal method; the levofloxacin can be modified on the surface of the carbon dots for binding to ribosomes located on the rough endoplasmic reticulum; the toluidine blue is used as a photosensitizer and serves as a carbon core in the synthesis of carbon dots, which helps the formation of carbon dots. Part of the toluidine blue is distributed on the surface of the carbon dots, which can provide the photodynamic therapy effect, and it has a conjugated structure, endowing the carbon dots with good fluorescence characteristics; the polyethyleneimine is used as a cationic polymer, making the surface of the carbon dots carry appropriate positive charges, which helps the cells to rapidly uptake the carbon dots.

[0036] The targeting site of the nano-carbon dots of the present invention is specific to ribosomes on the rough endoplasmic reticulum in cells, so as to target ribosomes in cells and perform photodynamic therapy near the endoplasmic reticulum.

[0037] The present invention provides nano-carbon dots for tumor photodynamic therapy based on ribosome affinity and explores their synthesis steps. The carbon dots are simply referred to as LT-CD, and their synthesis steps are as Figure 1 shown.

[0038] The present invention utilizes the affinity of levofloxacin for ribosomes to realize a ribosome affinity strategy with ribosomal ribonucleic acid as the target on ribosomes. Levofloxacin is modified on the surface of the carbon dots to bind ribosomal RNA, and then the carbon dots are anchored to ribosomes to achieve precise fluorescence imaging and targeted therapy.

[0039] The preparation method of the nano-carbon dots for tumor photodynamic therapy based on ribosome affinity of the present invention includes the following steps: (1) Dissolve levofloxacin in hydrochloric acid aqueous solution in advance and stir until the solution is transparent, which is called solution A; (2) Add toluidine blue aqueous solution and polyethyleneimine aqueous solution to the above solution A, mix well, and then transfer it to a round-bottom flask, which is called solution B.

[0040] (3) Place the solution B in step (2) in a household microwave oven to heat, take it out and cool it to room temperature, then add a large amount of ultrapure water and disperse it by ultrasonic wave.

[0041] (4) Filter the insoluble substances from the aqueous solution in step (3) through a water-based filter and retain the filtrate. Finally, perform dialysis for one week using a dialysis membrane. And freeze-dry to obtain the final carbon dots, which are called LT-CD.

[0042] The ultraviolet absorption peaks of the ribosome nano-carbon dots of the present invention are respectively located at 280 nm, 600 nm and 660 nm.

[0043] Under the irradiation of excitation light at 580 nm, the main fluorescence peak emitted by the ribosomal nanocarbodots of the present invention is located at 690 nm.

[0044] The fluorescent nanocarbodots based on ribosome affinity for tumor photodynamic therapy of the present invention can generate singlet oxygen under the irradiation of 660 nm laser and can be used for photodynamic therapy.

[0045] The fluorescent nanocarbodots based on ribosome affinity for tumor photodynamic therapy of the present invention can emit red fluorescence after being irradiated by excitation light, so as to trace inside cells and be used for the application of intracellular imaging.

[0046] In some embodiments, the particle size range of the ribosomal nanocarbodots of the present invention is 1 nm - 10 nm.

[0047] In some embodiments, the surface potential range of the ribosomal nanocarbodots of the present invention is 20 mV - 40 mV.

[0048] The principle of the present invention is as follows: First, levofloxacin can be modified to the surface of the carbodots for binding to ribosomal RNA; second, toluidine blue is used as a photosensitizer and serves as a carbon core in the synthesis of carbodots, which helps the formation of carbodots, endows the carbodots with photodynamic therapy effects, and has a conjugated structure, endowing the carbodots with good fluorescence properties; third, polyethyleneimine is used as a cationic polymer, making the surface of the carbodots carry appropriate positive charges, which helps the cells to rapidly uptake the carbodots.

[0049] As Figure 2 shown, the embodiment of the present invention provides a flow chart of a nanocarbodots based on ribosome affinity for tumor photodynamic therapy binding to ribosomes and targeting the endoplasmic reticulum inside cells. It is mainly divided into two stages. First, LT-CDs bind to ribosomes in the cytoplasmic matrix and then target to the endoplasmic reticulum together with the ribosomes. And because LT-CDs have the ability to produce reactive oxygen species, photodynamic therapy can be carried out on the endoplasmic reticulum membrane.

[0050] Example 1 The preparation method of the nanocarbodots based on ribosome affinity for tumor photodynamic therapy of the present invention includes the following steps: Step 1: Dissolve 0.5 g of levofloxacin in 10 mL of 5% hydrochloric acid aqueous solution in advance, stir at 50 °C until the solution is transparent, and it is called solution A; Step 2: Add 10 mL of toluidine blue aqueous solution and 5 mL of polyethyleneimine aqueous solution to the above solution A, mix well, and then transfer it to a 50 mL round-bottom flask, which is called solution B; the mass ratio of levofloxacin, toluidine blue, and polyethyleneimine has been optimized, and the optimized mass ratio is 2:10:1; Step 3: Place the solution B in Step 2 in a household microwave oven with a microwave power of 700 W and a heating time of 5 min. After taking it out and cooling it to room temperature, add a large amount of ultrapure water and disperse it by ultrasonic waves. Step 4: Filter the insoluble substances from the aqueous solution in Step 3 through a 0.22 μm water-based filter and retain the filtrate. Finally, perform dialysis for one week using a dialysis membrane with a molecular weight cut-off of 3500 MWCO. And freeze-dry to obtain the final carbon dots, called LT-CD.

[0051] As Figure 3 shown are the transmission electron microscope (TEM) images and high-resolution transmission electron microscope (HRTEM) images of the fluorescent carbon dots of the present invention. The carbon dots are evenly dispersed and the particle size is below 5 nm. The lattice of the carbon dots can be significantly seen in the HRTEM image.

[0052] As Figure 4 shown is the X-ray diffraction spectrum (XRD) of the fluorescent carbon dots of the present invention. A broad peak appears at a 2θ angle of about 25.5, which is the typical XRD peak position of the carbon dot lattice. It indicates the formation of the carbon dot lattice.

[0053] As Figure 5 shown is the particle size distribution diagram of the fluorescent carbon dots of the present invention. The hydrated particle size of most carbon dots is between 1.5 nm and 7.5 nm.

[0054] As Figure 6 shown is the ultraviolet-visible light absorption spectrum diagram of the fluorescent carbon dots of the present invention. The carbon dots LT-CD have obvious absorption peaks of toluidine blue and levofloxacin. It indicates that levofloxacin has been modified on the surface of the carbon dots.

[0055] As Figure 7 shown is the fluorescence spectrum diagram of the fluorescent carbon dots of the present invention. When the carbon dots LT-CD use 580 nm as the excitation light source, they have the strongest fluorescence. And the fluorescence emission is located at 690 nm, which helps to avoid fluorescence interference during imaging.

[0056] As Figure 8 shown is the Fourier transform infrared spectrum diagram of the fluorescent carbon dots of the present invention. There is a typical -CO-NH- infrared peak in the spectrum, which is attributed to toluidine blue having -NH2. During the carbonization process, part of the -NH2 is on the surface of the carbon dots and couples with the -COOH of levofloxacin to form -CO-NH-. Thus, levofloxacin is coupled to the surface of the carbon dots.

[0057] As Figure 9 shown is the nuclear magnetic resonance hydrogen spectrum diagram of the fluorescent carbon dots of the present invention. The nuclear magnetic resonance hydrogen spectrum shows the chemical shift of levofloxacin on the surface of the carbon dots, further proving that levofloxacin is not carbonized but remains on the surface of the carbon dots.

[0058] As shown Figure 10 in the ICP-MS mass spectrum of the fluorescent carbon dots of the present invention; it can be seen from the mass spectrum that the positions of m / z of levofloxacin fragments are at 330.34 and 348.35, further proving that levofloxacin has been modified to the surface of the carbon dots.

[0059] As shown Figure 11 in the figure, after the fluorescent carbon dots of the present invention are irradiated with a 660 nm laser, 1 O2 is generated and causes 1 the absorption of the O2 capture reagent DPBF at 420 nm to decrease significantly, proving that the carbon dots can effectively generate 1 O2.

[0060] As shown Figure 12 in the figure is the statistical result of red blood cell hemolysis after the fluorescent carbon dots of the present invention are incubated with red blood cells for different times. After incubation for more than 6 h, almost no hemolysis of red blood cells occurs, indicating that the carbon dots have good biocompatibility.

[0061] As shown Figure 13 in the figure is the co-localization map of the nano-carbon dots of the present invention with the endoplasmic reticulum in different cells (Hela, SKBR-3, MDA-MB-231, BT549). It shows that the carbon dots have no cell specificity and have excellent endoplasmic reticulum tracing effects in different cells.

[0062] Figure 14 This is the fluorescence co-localization map of the fluorescent carbon dots of the present invention with the endoplasmic reticulum, lysosome, cell membrane, and mitochondria in cells. Since ribosomes are located on the rough endoplasmic reticulum, the co-localization with the endoplasmic reticulum in the results indicates that the carbon dots have been successfully bound to ribosomes. The fluorescent carbon dots have no co-localization effects with the cell membrane, mitochondria, and lysosome in cells, proving that the carbon dots can avoid non-specific binding to other organelles.

[0063] Figure 15 This is the verification of the generation of ROS by the fluorescent nano-carbon dots of the present invention in cells. DCFH-DA is used to detect ROS in cells, and obvious green fluorescence appears after DCFH-DA reacts with ROS.

[0064] Figure 16 This is the statistical chart of the cell treatment effect of the fluorescent nano-carbon dots of the present invention. After LT-CDs are incubated with cells and then irradiated with a laser, the cell viability is detected by the MTT method. After laser irradiation, a significant decrease in cell viability can be detected.

[0065] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of fluorescent carbon nanodots based on ribosome affinity, characterized in that, Comprising the following steps: (1) Heating a mixed solution of levofloxacin, toluidine blue, and polyethyleneimine to form carbon nanodots; the toluidine blue serves as the carbon core for the formation of carbon dots; the polyethyleneimine serves as a cationic polymer such that the surface of the carbon dots carries a positive charge; the levofloxacin is modified on the surface of the carbon dots, and the levofloxacin is used to target ribosomes on the rough endoplasmic reticulum; (2) Filtering the solution obtained in step (1) through an aqueous filter to remove insoluble substances and retaining the filtrate; then performing dialysis using a dialysis membrane, thereby obtaining fluorescence carbon nanodots for tumor photodynamic therapy based on ribosome affinity.

2. The preparation method of the fluorescence nanocarbons dots based on ribosome affinity according to claim 1, wherein, In the mixed solution, the mass ratio of levofloxacin, toluidine blue, and polyethyleneimine is (2 - 4):(10 - 15):(1 - 2).

3. The preparation method of the ribosome affinity-based fluorescent carbon nanodots according to claim 1, characterized in that, The heating method is microwave heating, the heating power is 500 W - 700 W, and the heating time is 5 min - 10 min.

4. Fluorescence carbon nanodots based on ribosome affinity prepared by the method according to any one of claims 1 - 3.

5. The ribosome affinity-based fluorescent carbon nanodots according to claim 4, wherein The particle size range of the fluorescence carbon nanodots is 1 nm - 10 nm.

6. The ribosome affinity-based fluorescent carbon nanodots according to claim 4, wherein The surface potential range of the fluorescence carbon nanodots is 20 mV - 40 mV.

7. Use of the fluorescence carbon nanodots based on ribosome affinity according to any one of claims 4 - 6 for the preparation of tumor photodynamic therapy reagents.

8. Use of the fluorescence carbon nanodots based on ribosome affinity according to any one of claims 4 - 6 for the preparation of imaging reagents.

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