CN111484840B - Conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot and preparation method and application thereof - Google Patents
Conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot and a preparation method and application thereof, and belongs to the technical field of quantum dots. The quantum dot is a sulfur-nitrogen double-doped graphene quantum dot modified by polypeptide c (RGDFC). The synthetic method is simple and convenient to operate. The synthesized quantum dots not only have good biocompatibility, but also retain the advantage of high efficiency and stability of the sulfur-nitrogen double-doped graphene quantum dots; the compound has tumor cell selectivity, can enter lysosomes and mitochondria of cells independently, can overcome photodynamic therapy resistance generated by the cells in the repeated treatment process of sulfur-nitrogen double-doped graphene quantum dots, continuously generates active oxygen to kill the tumor cells in the repeated treatment process, and can be used as a high-efficiency anti-tumor photosensitizer; the quantum dot prepared by the invention solves the technical problem which is difficult to solve in the prior art, and has wide clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of quantum dots, and particularly relates to a conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot and a preparation method and application thereof.
Background
The photodynamic therapy is a new treatment means, and has the advantages of small wound, wide tumor treatment types, repeated treatment, short treatment period, no influence on other treatments and the like. Organic photosensitizers have been studied for a long time, and several organic photosensitizers have been used clinically, but the organic photosensitizers have poor water solubility, cannot be transported in vivo alone, have weak light stability, and need to be stored for a long time in a dark place. And most of the organic photosensitizers have poor targeting property and are not easy to enrich in tumor tissues. In addition, many experimental and clinical cases prove that the cells of the organic photosensitizer are easy to generate photodynamic therapy resistance in repeated photodynamic therapy, and the curative effect of the photodynamic therapy is greatly reduced. The nano particles have good biocompatibility, proper particle size and shape and excellent surface physicochemical property, occupy a certain position in the research and development of photosensitizers, wherein the graphene quantum dots are simple and convenient in synthesis process, easy to modify surfaces, good in water solubility, low in toxicity and excellent in photophysical performance, and are attracted attention in the research and development of photodynamic therapy.
The sulfur-nitrogen double-doped graphene quantum dot prepared by the literature (Jiechao Ge et al. A graphene quantum dot photo-dynamic thermal with high single oxygen generation, nat. Commun.2014, 5, 4596) has the advantages of good water solubility, excellent biocompatibility, high singlet oxygen yield and the like, can mediate high-efficiency photodynamic therapy, and can be used as a good nano photosensitizer. However, the pure sulfur and nitrogen double-doped graphene quantum dots do not have tumor targeting and cannot actively target tumor cells; meanwhile, a certain amount of accumulation can be generated in normal tissue cells, and a certain side effect can be generated in the process of photodynamic therapy; in addition, the problem that the cells generate photodynamic therapy resistance in the repeated photodynamic therapy process cannot be solved by the pure sulfur-nitrogen double-doped graphene quantum dots.
Modification on the surface of the nano photosensitizer is a method for solving the lack of targeting, for example, RGD polypeptide is modified on graphene quantum dots, so that the quantum dots have targeting; however, no research has proved that the cell can solve the problem of generating photodynamic therapy resistance in the process of repeated photodynamic therapy. Therefore, the search for a simple and feasible synthetic method, the endowment of the nano photosensitizer to the tumor cell targeting and the overcoming of the resistance problem of photodynamic therapy have important significance.
Disclosure of Invention
The invention aims to provide a conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot, and a preparation method and application thereof.
The invention provides a conjugated c (RGDfC) sulfur and nitrogen double-doped graphene quantum dot, which is a sulfur and nitrogen double-doped graphene quantum dot modified by polypeptide c (RGDfC).
Further, the conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dot is prepared from sulfur-nitrogen double-doped graphene quantum dots and polypeptide c (rgdfc) as raw materials;
the molar ratio of the sulfur-nitrogen double-doped graphene quantum dots to the polypeptide c (RGDFC) is 1 (80-120);
preferably, the molar ratio of the sulfur-nitrogen double-doped graphene quantum dot to the polypeptide c (rgdfc) is 1: 100.
Further, the sequence of the polypeptide c (RGDFC) is cyclo (Arg-Gly-Asp-d-Phe-Cys), and the structural formula is shown as the formula I:
further, the preparation method of the sulfur-nitrogen double-doped graphene quantum dot comprises the following steps:
1)
dissolving 4-bromobenzyl bromide in CH2Cl2/CH3Adding N, N-dimethyldodecane into the OH mixed solutionAmine reacts at room temperature, and a reactant is concentrated, filtered, washed and dried to obtain a compound 1; reacting compound 1 with Na2CO3、Pd(PPh3)4Dissolving thiophene-3-boric acid in ethanol, adding double distilled water under the protection of nitrogen, refluxing at 90 ℃ for 6 hours, decompressing the reactant to remove ethanol, extracting, drying, and purifying by a chromatographic column to obtain a compound 2; under the protection of nitrogen, FeCl is contained3Anhydrous CHCl3Adding the solution into the compound 2, reacting at room temperature, collecting precipitate, washing and drying to obtain a compound PT 2;
2) dissolving a compound PT2 in a NaOH solution, carrying out ultrasonic treatment, heating to 170 ℃, reacting for 24h, cooling to room temperature, filtering, and dialyzing to obtain the compound PT 2.
Further, the air conditioner is provided with a fan,
in the step 1), the molar ratio of the 4-bromobenzyl bromide to the N, N-dimethyldodecylamine is 1: 1.3;
and/or, in step 1), the CH2Cl2/CH3In OH mixed solution, CH2Cl2And CH3OH volume ratio is 3: 2;
and/or, in step 1), the compound 1, Na2CO3、Pd(PPh3)4The mass ratio of the thiophene-3-boric acid is 0.4:0.5:0.2: 0.128;
and/or, in step 1), the extraction is carried out by using CH2Cl2Extracting;
and/or, in step 1), the FeCl3And compound 2 in an equivalent ratio of 4: 1;
and/or, in the step 2), the concentration of the NaOH solution is 0.5 mM;
and/or, in the step 2), the filtration is the filtration by using a 0.22 μm filter membrane;
and/or, in step 2), the dialysis is dialysis in distilled water.
The invention also provides a preparation method of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot, which comprises the following steps:
dissolving sulfur-nitrogen double-doped graphene quantum dots in a solvent, and adding polypeptide c (RGDFC) for reaction;
ultrafiltering the reaction liquid obtained in the first step to obtain the product;
preferably, in the step (i), the solvent is double distilled water;
and/or in the step (i), the reaction temperature is 4-25 ℃; the reaction time is 2-6 h;
and/or in the second step, during the ultrafiltration, the molecular weight cut-off of the ultrafiltration tube is 10 kDa;
and/or, in the second step, the centrifugal temperature is 0-4 ℃ during ultrafiltration; the centrifugal force is 5000-10000 g; centrifuging for 5-10 min;
more preferably still, the first and second liquid crystal compositions are,
in the step I, the reaction temperature is 4 ℃; the reaction time is 4 h;
and/or, in the second step, the centrifugal temperature is 4 ℃ during ultrafiltration; the centrifugal force is 10000 g; the centrifugation time was 6 min.
The invention also provides a preparation method of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot, which comprises the following steps:
(1) dissolving the sulfur-nitrogen double-doped graphene quantum dots in a solvent, and adding an EDC/NHS mixture for activation;
(2) adding polypeptide c (RGDFC) into the activated sulfur-nitrogen double-doped graphene quantum dots for reaction;
(3) and (3) carrying out ultrafiltration on the reaction liquid obtained in the step (2).
Further, the EDC/NHS mixture consists of EDC and NHS; the molar ratio of the sulfur-nitrogen double-doped graphene quantum dots to the EDC/NHS mixture is 1 (2000-4000);
preferably, the molar ratio of the sulfur-nitrogen double-doped graphene quantum dot to the EDC/NHS mixture is 1: 4000; and/or the mass ratio of EDC to NHS is 2: 1.
Further, the air conditioner is provided with a fan,
in the step (1), the solvent is double distilled water;
and/or in the step (1), the activation temperature is 4-25 ℃; the activation time is 10-60 min;
and/or in the step (2), the reaction temperature is 4-25 ℃; the reaction time is 2-6 h;
and/or, in the step (3), during the ultrafiltration, the molecular weight cut-off of the ultrafiltration tube is 10 kDa;
and/or in the step (3), the centrifugal temperature is 0-4 ℃ during ultrafiltration; the centrifugal force is 5000-10000 g; centrifuging for 5-10 min;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the activation temperature is 25 ℃; the activation time is 20 min;
and/or, in the step (2), the reaction temperature is 4 ℃; the reaction time is 4 h;
and/or, in the step (3), the centrifugal temperature is 4 ℃ during the ultrafiltration; the centrifugal force is 80000 g; the centrifugation time was 6 min.
The invention also provides application of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot in preparation of antitumor drugs;
preferably, the anti-tumor drug is a photosensitizer;
more preferably, the photosensitizer is a photosensitizer that targets a tumor and/or overcomes resistance to photodynamic therapy.
In the invention, the room temperature is 25 +/-5 ℃; the overnight period was 12. + -.2 h.
By adopting the method, the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dots are successfully synthesized, and the synthesis method is simple and convenient to operate. The synthesized conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot not only has good biocompatibility, but also retains the advantage of high efficiency and stability of the sulfur-nitrogen double-doped graphene quantum dot; the tumor cell selectivity is realized, the alpha v beta 3 integrin positive tumor cells can be targeted, and the cells can not enter the alpha v beta 3 integrin negative normal tissue cells; more importantly, the conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dot can overcome the photodynamic therapy resistance generated by cells in the repeated treatment process of the sulfur and nitrogen double-doped graphene quantum dot, can enter lysosomes and mitochondria of the cells automatically, continuously generates active oxygen to kill tumor cells in the repeated treatment process, and can be used as a high-efficiency anti-tumor photosensitizer; the quantum dot prepared by the invention solves the technical problem which is difficult to solve in the prior art, and has wide clinical application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Fig. 1 is an XPS representation of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots of the present invention.
Fig. 2 is a Zeta potential distribution diagram of the conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dot of the invention.
Fig. 3 shows the transmission electron microscope and particle size measurement results of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots of the present invention: a is a transmission electron microscope test result; and B is the result of hydrated particle size test.
Fig. 4 shows flow detection results and fluorescence images of tumor cells a375 and U87 and mouse fibroblast L929 after uptake of sulfur and nitrogen double-doped graphene quantum dots or conjugated c (rgdfc) sulfur and nitrogen double-doped graphene quantum dots, wherein red in the fluorescence images is autofluorescence of the material, and green is cytoskeletal staining: a is tumor cell A375; b is tumor cell U87; c is mouse fibroblast L929.
Fig. 5 shows cytotoxicity results of conjugated c (rgdfc) sulfur and nitrogen double-doped graphene quantum dots and sulfur and nitrogen double-doped graphene quantum dots at different concentrations on tumor cells a375, U87 and mouse fibroblast L929: a is the cytotoxicity result of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots to the tumor cells A375 under the condition of no illumination; b is the cytotoxicity result of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots to the tumor cells U87 under the condition of no illumination; c is the cytotoxicity result of conjugated C (RGDfC) sulfur-nitrogen double-doped graphene quantum dots and sulfur-nitrogen double-doped graphene quantum dots with different concentrations to tumor cells A375 under the illumination condition; d is the cytotoxicity result of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots and sulfur-nitrogen double-doped graphene quantum dots with different concentrations on the tumor cells U87 under the illumination condition; and E is the cytotoxicity result of the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dots and sulfur-nitrogen double-doped graphene quantum dots with different concentrations to mouse fibroblast L929 under the illumination condition.
Fig. 6 is a fluorescent image of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots positioned in tumor cells a375 and U87, wherein red is autofluorescence of the material, and Green is labeled by a lysosome probe (Lyso-Tracker Green) or a mitochondrial probe (Mito-Tracker Green): a is tumor cell A375; b is tumor cell U87.
Fig. 7 shows the killing ability of the sulfur-nitrogen double-doped graphene quantum dots or the conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots to the normal tumor cell a375 and the tumor cell a375 resistant to the photodynamic therapy.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products. Wherein:
EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS for N-hydroxy succinimide.
(rgdfc) is a polypeptide having the sequence: cyclo (Arg-Gly-Asp-d-Phe-Cys), of the formula:
in the present invention, the preparation method of the sulfur-nitrogen double-doped graphene quantum dot is described in reference to the preparation method of the sulfur-nitrogen double-doped graphene quantum dot (Jiechao Ge, Minhuan Lan, Bingjiang Zhou, et al. A graphene quantum dot and photo dynamic thermal gene with high sensitivity generation. Nat. Comm,2014,5,1-8.DOI: 10.1038/ncoms 5596; Minhuan Lan, Jiansheng Wu, Weimin Liuet. Copolythiophene-Derived Colorimetric and fluoro Sensor for visual superior sensitivity Determination of Lipocalization polypeptide. J. am. chem. Soc. 134, 6685-6694.).
The specific method comprises the following steps:
(1) preparation of polythiophene analogue PT 2:
the preparation route is as follows:
4-Bromobenzylbromide (0.25g, 1mmol) was dissolved in 20mL CH2Cl2/CH3To the OH (v/v ═ 3/2) mixed solution was then added N, N-dimethyldodecylamine (0.4mL, 1.3mmol), and stirred at room temperature for 12 h. The reaction solution was concentrated to 5mL, poured into 200mL of anhydrous ether, filtered, washed and dried to give compound 1. Compound 1(0.4g), Na2CO3(0.5g)，Pd(PPh3)4(200mg) and thiophene-3-boronic acid (0.128g) were dissolved in ethanol (20mL), 10mL of double distilled water was added under nitrogen, reflux was carried out at 90 ℃ for 6 hours, and ethanol was removed under reduced pressure. CH (CH)2Cl2The residue was extracted (3X 20mL), and the organic layer was collected over anhydrous MgSO4Drying and purifying by a chromatographic column to obtain the compound 2. Under the protection of nitrogen, 4 equivalents of FeCl3Dissolved in 30mL of anhydrous CHCl3In (1), dissolved in 20mL of CHCl was added dropwise31 equivalent of compound 2 in (1) is stirred at room temperature for 2 days. The precipitate was collected, washed with methanol and dried in vacuo to give PT2 powder, gpc.mn 6.655 × 104(PDI＝1.161)。
(2) Preparing sulfur-nitrogen double-doped graphene quantum dots:
dissolving 30mg of PT2 powder in 0.5mM NaOH solution, carrying out ultrasonic treatment for 30min, transferring the solution into an autoclave, and heating to 170 ℃ for reaction for 24 h; and cooling to room temperature, filtering larger particles by using a 0.22-micron membrane, and dialyzing in distilled water to remove residual sodium hydroxide to obtain the sulfur-nitrogen double-doped graphene quantum dot solution.
The conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot can be linked with c (RGDFC) after being activated by using an EDC/NHS mixture; c (RGDFC) may also be linked directly.
Example 1 preparation of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
(1) 50 mul of sulfur and nitrogen double-doped graphene quantum dot aqueous solution (prepared by double distilled water) with the concentration of 20 mul is taken and placed in 750 mul of double distilled water, then 100 mul of EDC/NHS mixture aqueous solution with the concentration of 40mM (the mass ratio of EDC to NHS is 2:1, prepared by double distilled water) is added for reaction, and the reaction is carried out for 20min at the temperature of 25 ℃.
(2) Mu.l of a 1mM aqueous solution of c (RGDFC) (prepared in double distilled water) was added to the reaction mixture, and the mixture was kept at 4 ℃ for 2 hours.
(3) Transferring the reactant to a 10kDa ultrafiltration tube, centrifuging for 6min at 4 ℃ under the centrifugal force condition of 8000g, and removing filtrate to obtain the sulfur-nitrogen double-doped graphene quantum dot conjugated with c (RGDFC).
Embodiment 2, preparation of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
(1) 50 mul of sulfur and nitrogen double-doped graphene quantum dot aqueous solution (prepared by double distilled water) with the concentration of 20 mul is taken and placed in 750 mul of double distilled water, then 100 mul of EDC/NHS mixture aqueous solution with the concentration of 40mM (the mass ratio of EDC to NHS is 2:1, prepared by double distilled water) is added for reaction, and the reaction is carried out for 10min at the temperature of 25 ℃.
(2) Mu.l of a 1mM aqueous solution of c (RGDFC) (prepared in double distilled water) was added to the reaction mixture, and the mixture was kept at 4 ℃ for 4 hours.
(3) Transferring the reactant to a 10kDa ultrafiltration tube, centrifuging for 6min at 4 ℃ under the centrifugal force condition of 8000g, and removing filtrate to obtain the sulfur-nitrogen double-doped graphene quantum dot conjugated with c (RGDFC).
Embodiment 3 preparation of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
(1) 50 mul of sulfur and nitrogen double-doped graphene quantum dot aqueous solution (prepared by double distilled water) with the concentration of 20 mul is taken and placed in 750 mul of double distilled water, then 100 mul of EDC/NHS mixture aqueous solution with the concentration of 40mM (the mass ratio of EDC to NHS is 2:1, prepared by double distilled water) is added for reaction, and the reaction is carried out for 60min at the temperature of 25 ℃.
(2) Mu.l of a 1mM aqueous solution of c (RGDFC) (prepared in double distilled water) was added to the reaction mixture, and the mixture was kept at 4 ℃ for 6 hours.
(3) Transferring the reactant to a 10kDa ultrafiltration tube, centrifuging for 6min at 4 ℃ under the centrifugal force condition of 6000g, and removing filtrate to obtain the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots.
Embodiment 4 preparation of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
(1) 50 mul of sulfur and nitrogen double-doped graphene quantum dot aqueous solution (prepared by double distilled water) with the concentration of 20 mul is taken and placed in 840 mul of double distilled water, then 100 mul of EDC/NHS mixture aqueous solution with the concentration of 40mM (the mass ratio of EDC to NHS is 2:1, prepared by double distilled water) is added for reaction, and the reaction time is 20min at the temperature of 20 ℃.
(2) To the reaction mixture was added 8. mu.l of a 10mM aqueous solution of c (RGDFC) (prepared in double distilled water), and the mixture was reacted at 4 ℃ for 2 hours.
(3) Transferring the reactant to a 10kDa ultrafiltration tube, centrifuging for 6min at 4 ℃ under the centrifugal force condition of 7000g, and removing filtrate to obtain the sulfur-nitrogen double-doped graphene quantum dot conjugated with c (RGDFC).
Embodiment 5 preparation of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
(1) 100 mul of sulfur and nitrogen double-doped graphene quantum dot aqueous solution (prepared by double distilled water) with the concentration of 20 mul is taken and placed in 680 mul of double distilled water, and then 200 mul of EDC/NHS mixture aqueous solution with the concentration of 20mM (prepared by double distilled water with the mass ratio of EDC to NHS being 2: 1) is added for reaction, and the reaction time is 20min at 4 ℃.
(2) Mu.l of a 10mM aqueous solution of c (RGDFC) (prepared in double distilled water) was added to the reaction mixture, and the mixture was kept at 4 ℃ for 2 hours.
(3) Transferring the reactant to a 10kDa ultrafiltration tube, centrifuging for 6min at 4 ℃ under the centrifugal force condition of 10000g, and removing filtrate to obtain the sulfur-nitrogen double-doped graphene quantum dot conjugated with c (RGDFC).
Embodiment 6 preparation of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
(1) 50 mul of sulfur and nitrogen double-doped graphene quantum dot aqueous solution (prepared by double distilled water) with the concentration of 20 mul is put into 850 mul of double distilled water.
(2) To the above solution, 100. mu.l of a 1mM aqueous solution of c (RGDFC) (prepared in double distilled water) was added, and the reaction was carried out at 4 ℃ for 4 hours.
(3) Transferring the reactant to a 10kDa ultrafiltration tube, centrifuging for 6min at 4 ℃ under the centrifugal force condition of 10000g, and removing filtrate to obtain the sulfur-nitrogen double-doped graphene quantum dot conjugated with c (RGDFC).
The advantageous effects of the present invention are demonstrated by specific test examples below.
Test example 1 XPS test of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
Test samples: the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot prepared in embodiment 1 of the invention.
XPS test: diluting the sample prepared in the embodiment 1 of the invention to 1 mu M with filtered double distilled water, dripping a drop of conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dot solution on a 3mm multiplied by 3mm glass sheet, adhering the solution on double-sided adhesive, airing the solution at room temperature, detecting the sample according to an X-ray photoelectron spectroscopy operation method, wherein the detection result is shown in figure 1.
And (3) testing results: the element composition of the synthesized nano particles is tested by utilizing X-ray photoelectron spectroscopy, and characteristic peaks of carbon, sulfur, nitrogen and oxygen elements can be seen in figure 1 and are consistent with the accepted absorption peaks of four elements. The synthesized conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots contain carbon, sulfur and nitrogen elements in the sulfur-nitrogen double-doped graphene quantum dots and carbon, nitrogen and oxygen elements in the polypeptide, so that the successful synthesis of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots is proved.
Test example 2 Zeta potential test of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots
Test samples: the conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots (denoted as cRGD @ GQDs in fig. 2), the raw material sulfur-nitrogen double-doped graphene quantum dots (denoted as GQDs in fig. 2) and the raw material c (rgdfc) (denoted as cRGD in fig. 2) prepared in embodiment 1 of the present invention.
Zeta potential test: the measurement is carried out by a dynamic light scattering method, wherein the measuring cell is cleaned three times by absolute ethyl alcohol, and then cleaned three times by filtered double distilled water. Respectively taking 1mL of 10nM conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot solution, sulfur-nitrogen double-doped graphene quantum dot solution and c (RGDfC) solution diluted by double distilled water after filtration, enabling the height of a sample in a measuring pool to be higher than a metal sheet of the measuring pool, opening a sample pool cover according to the instruction of an instrument, putting the sample pool into the measuring pool, starting to measure, measuring for three times, and enabling the measuring result to be shown in figure 2.
And (3) testing results: the dynamic light scattering method is used for measuring the surface charge of the quantum dot, and the result shows that c (RGDfC) is negative charge and the sulfur and nitrogen double-doped graphene quantum dot is positive charge.
Test example 3 particle size detection of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots of the present invention
Test samples: the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot prepared in embodiment 1 of the invention.
And (3) transmission electron microscope testing: diluting the prepared sample to 10nM by using filtered double distilled water, dripping a drop of conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dot solution into a copper mesh, drying the copper mesh under an infrared lamp, and finally detecting the sample according to a transmission electron microscope operation method, wherein the detection result is shown in a picture 3 (A).
Hydrated particle size testing: the measurement is carried out by a dynamic light scattering method, wherein the measuring cell is cleaned three times by absolute ethyl alcohol, and then cleaned three times by filtered double distilled water. Taking 1mL of 10nM conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dot solution diluted by double distilled water after filtration, enabling the height of a sample in a measuring cell to be higher than that of a metal sheet in the measuring cell, opening a sample cell cover according to the instruction of an instrument, placing the sample cell into the measuring cell, starting to measure for three times, and obtaining a measuring result shown in a picture 3 (B).
And (3) testing results: and detecting and analyzing the particle size of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot by using a transmission electron microscope and dynamic light scattering. The result shows that the particle size of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot prepared by the invention is about 40 nm.
Test example 4 test for cell uptake of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots
Test samples: the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot prepared in embodiment 1 of the invention and the raw material sulfur-nitrogen double-doped graphene quantum dot are prepared.
Human malignant melanoma cell A375, human malignant glioma cell U87(U87-MG) and mouse fibroblast L929 were used as experimental cells, and digested with EDTA-containing 0.25% trypsin to give cells with a concentration of 5X 104Suspension per mL.
Flow cytometry testing: seeding 2ml of 5X 10 density in 6-well plates4Overnight, removing the culture medium after the cells adhere to the wall, then adding 2mL of culture medium into each pore plate, and adding 1.25 mul of sulfur-nitrogen double-doped graphene quantum dot solution with the concentration of 20 muM or conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot solution (the quantum dots adopt ddH)2O is prepared as a solvent), after 24 hours of culture, is digested by 0.25 percent trypsin containing EDTA, centrifuged for 5 minutes under the condition of 3000g centrifugal force, and washed by PBS for 3 times to finally prepare 1 × 106The cell concentration per mL of the suspension was examined on the machine according to the flow cytometer method of operation, and the results are shown in FIG. 4.
Laser confocal testing: the confocal dish was inoculated with 1ml of a seed having a density of 5X 104Adding 1mL of culture medium into each small dish after the cells are adhered overnight and then adding 1.25 mul of sulfur-nitrogen double-doped graphene quantum dot solution with the concentration of 20 MuM or conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot solution of the invention into each 1mL of culture medium for culturing (the quantum dots adopt ddH)2O formulated as a solvent). After 24h of cell culture, 4% paraformaldehyde was fixed for 30min, the cells were washed 3 times with PBS, F-actin of the cells was stained with FITC-labeled phalloidin, incubated at 37 ℃ for 30min, and finally fluorescence images were taken using a laser scanning confocal microscope (AIR-MP, Nikon, Japan), and the results are shown in FIG. 4.
And (3) testing results: after incubating sulfur-nitrogen double-doped graphene quantum dots (shown as GQDs in figure 4) and conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots (shown as cRGD @ GQDs in figure 4) for 24h, compared with the sulfur-nitrogen double-doped graphene quantum dots, cells A375 and U87 with high expression of integrin alpha v beta 3 take up more conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots; and the normal tissue cell L929 takes up less conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots. The conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dots have targeting property, and can target tumor cells with high integrin alpha v beta 3 expression compared with normal cells.
Test example 5 test of cell compatibility of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots of the present invention
Test samples: the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot prepared in embodiment 1 of the invention and the raw material sulfur-nitrogen double-doped graphene quantum dot are prepared.
Cell types: human malignant melanoma cell A375, human malignant glioma cell U87 and mouse fibroblast L929 were used as experimental cells.
CCK-8 cell activity assay: the cells were digested with 0.25% trypsin containing EDTA to give a cell concentration of 5X 104 Inoculating 100 mu l of cell suspension into each hole of a 96-hole plate, culturing for one day, removing the upper culture medium, adding new culture medium containing different concentrations of sulfur-nitrogen double-doped graphene quantum dots (25nM,50nM) or conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots (0nM,10nM,25nM,50nM,100nM,250nM,500nM) in the same volume, culturing for 24h, removing the culture medium, slightly washing with PBS, adding the culture medium without the quantum dots to the illumination group, and using an LED lamp with the wavelength of 420nM to 25J-cm-2The light intensity was kept for 5min and washed gently with PBS. The illuminated and non-illuminated plates were incubated at 37 ℃ for 1h with 100. mu.l of serum-free medium containing 10% CCK-8 reagent per well. After the reaction, the absorbance at a wavelength of 450nm per well was measured with a microplate reader (VariOskanFlas 3001, Thermo, u.s.).
And (3) testing results: the CCK-8 detection result is shown in fig. 5, and fig. 5A and 5B show the influence of the conjugated c (rgdfc) sulfur and nitrogen double-doped graphene quantum dots on cells a375 and U87 under the condition of no light, and the result shows that the conjugated c (rgdfc) sulfur and nitrogen double-doped graphene quantum dots have no influence on the cell viability under the condition of no light, which shows that the conjugated c (rgdfc) sulfur and nitrogen double-doped graphene quantum dots have good biocompatibility. Fig. 5C to E show the effect of different quantum dots on cells under the illumination condition, and it can be known that under the illumination condition of 420nm, the sulfur-nitrogen double-doped graphene quantum dots can not only kill tumor cells, but also have great lethality to normal tissue cells; the capability of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot of the invention for killing tumor cells is obviously better than that of the sulfur-nitrogen double-doped graphene quantum dot, but the capability of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot of the invention for killing normal cells is less. Therefore, the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot can kill tumor cells and protect normal tissue cells at the same time.
The conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dot has strong killing power on tumor cells, has targeting performance on the tumor cells, and cannot damage normal cells when inhibiting the growth of the tumor cells.
Experimental example 6 positioning of nanoparticles of conjugated c (RGDFC) S/N double-doped graphene quantum dots
Test samples: the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot prepared in embodiment 1 of the invention.
Intracellular localization test: the confocal dish was seeded with 1ml of cells at a cell density of 5X 104A375 or U87 cell suspension/mL, after the cells attached overnight, 1mL of culture medium was added to each dish, and 1.25. mu.l of conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot solution with 20. mu.M concentration was added to each 1mL of culture medium for culture. After 24h of cell culture, lysosome dye (Lyso-Tracker Green, Thermo Fisher Scientific) and mitochondrial dye (Mito-Tracker Green, Beyotime Biotechnology) were added in the state of viable cells, incubated at 37 ℃ for 30min, cells were washed 3 times with PBS, and finally fluorescence images were taken using a laser scanning confocal microscope (AIR-MP, Nikon, Japan), and the results are shown in FIG. 6.
And (3) testing results: the relation between the quantum dot and the lysosome has important significance for the research of a drug carrying system, and the photosensitizer is simultaneously positioned in mitochondria and the lysosome and can activate mitochondrial-mediated apoptosis so as to mediate more efficient photodynamic therapy. In the invention, the lysosome fluorescent probe and the mitochondrial fluorescent probe are adopted to observe the relationship among lysosome, mitochondria and prepared nanoparticles. After 24h of culture, most of the conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dots are positioned in lysosomes, and a part of the conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dots are also positioned in mitochondria, and the detection result is shown in figure 6, which illustrates that the conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dots prepared by the invention can be positioned in mitochondria and lysosomes, so that more efficient photodynamic therapy is mediated.
Test example 7. conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots of the invention for overcoming photodynamic therapy resistance
Test samples: the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots (cRGD @ GQDs) and the raw material sulfur-nitrogen double-doped Graphene Quantum Dots (GQDs) prepared in embodiment 1 of the invention.
(1) Detecting cell resistance after sulfur-nitrogen double-doped Graphene Quantum Dots (GQDs) photodynamic therapy: the tumor cells A375 were digested with EDTA-containing 0.25% trypsin to give a cell concentration of 5X 104Inoculating 2mL of cell suspension into each 6-well plate, culturing for one day, removing the upper culture medium, adding 2mL of new culture medium containing 50nM sulfur-nitrogen double-doped Graphene Quantum Dots (GQDs), culturing for 24h, removing the culture medium, washing with PBS, adding culture medium without quantum dots, and illuminating with LED lamp with wavelength of 420nM at 25J-cm-2The light intensity of (c) was illuminated for 3min, and the illumination was repeated 1 time after every 2 hours (2, 4,6 or 8 times total illumination, respectively, representing 2, 4,6 or 8 times of repeated photodynamic therapy). After the completion of the light irradiation in each test group (2 times total, 4 times total, 6 times total or 8 times total), the cells were gently washed with PBS, the fresh medium was replaced, and after 24 hours of culture, the tumor cells A375 were digested with 0.25% trypsin containing EDTA to give a cell concentration of 5X 104Each well of the suspension is inoculated with 100 mu L of cell suspension in a 96-well plate, after 24 hours of culture, the upper medium layer is discarded, and 100 mu L of sulfur and nitrogen double-doped graphene quantum dots (GQDs: 5nM,10nM,20nM,50nM,100nM) with different concentrations are added. Culturing for 24 hr, removing culture medium, washing with PBS, adding quantum dot-free culture medium, and culturing with LED lamp with wavelength of 420nm at 25J cm-2The cells were irradiated with light for 5min, gently washed with PBS, and 100. mu.l of a serum-free medium containing 10% CCK-8 reagent was added to each well, followed by incubation at 37 ℃ for 1 hour. After the reaction, each well was measured at a wavelength of 450nm using a microplate reader (VarioSkanFlas 3001, Thermo, U.S.)Absorbance of (d) in (d). From the results, IC50 and the resistance coefficient were calculated. Resistance coefficient-IC 50 for n repeated photodynamic therapy/IC 50 for a single photodynamic therapy. The results are shown in Table 1.
TABLE 1 resistance coefficient and IC (50) of Sulfur-Nitrogen double doped Graphene Quantum Dots (GQDs)
Note: in table 1, pdtx 2 represents IC50 and the resistance coefficient after 2 repetitions of photodynamic therapy; pdtx 4 represents the IC50 and resistance coefficient after 4 repetitions of photodynamic therapy; pdtx 6 represents the IC50 and resistance coefficient after 6 repetitions of photodynamic therapy; PDT x 8 represents the IC50 and the resistance coefficient after 8 repetitions of photodynamic therapy.
And (3) detection results: in photodynamic therapy, a resistance coefficient greater than 1.5 indicates that the cell is resistant to the treatment. After 2 times of repeated light irradiation treatment, the resistance coefficient of the sulfur-nitrogen double-doped graphene quantum dot mediated photodynamic treatment is 1.55; after repeated illumination for 4 times, the resistance coefficient is 1.81; after repeated illumination for 6 times, the resistance coefficient is 2.02; after 8 times of repeated light irradiation, the resistance coefficient was 2.24. The results show that the tumor cell A375 can generate photodynamic therapy resistance after being subjected to the photodynamic therapy by the sulfur-nitrogen double-doped graphene quantum dot for 2 times, and the sulfur-nitrogen double-doped graphene quantum dot is used for performing the photodynamic therapy repeatedly, so that the killing efficiency of the cell is obviously reduced, and the treatment efficacy is obviously reduced.
(2) Killing capacity of conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dots to cells generating photodynamic therapy resistance: normal tumor cells A375 (without photodynamic therapy) and tumor cells A375 with photodynamic therapy resistance after 2 times, 4 times, 6 times or 8 times of repeated photodynamic therapy are treated at 5 × 104The cells were inoculated into 96-well plates at a cell concentration of one/mL, and 100. mu.L of the cell suspension was inoculated into each well, and after one day of culture, the upper medium was discarded. Then respectively adding 100 μ L of new culture medium containing 50nM sulfur-nitrogen double-doped graphene quantum dots or 50nM conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dots, culturing for 24 hr, discarding the culture medium, and lightly adding PBSWashing, adding quantum dot-free culture medium, and irradiating with LED lamp with wavelength of 420nm at 25J cm-2The cells were irradiated with light for 5min, gently washed with PBS, and 100. mu.L of a serum-free medium containing 10% CCK-8 reagent was added to each well, followed by incubation at 37 ℃ for 1 hour. After the reaction, the absorbance at a wavelength of 450nm per well was measured with a microplate reader (VariOskanFlas 3001, Thermo, u.s.). The results are shown in FIG. 7.
And (3) testing results: the killing power of the sulfur-nitrogen double-doped graphene quantum on normal tumor cells is more than 60% (the cell activity is less than 40%), the killing efficiency on the tumor cells generating the photodynamic therapy resistance is obviously weakened, the killing power is as low as 40% after 8 times of photodynamic therapy, and the killing efficiency is weakened by 33.4%. The killing power of the conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dots to normal tumor cells is more than 90%, the killing efficiency to the tumor cells generating photodynamic therapy resistance can also reach more than 80% after 8 times of photodynamic therapy, and the killing efficiency is only reduced by 11.1% at most. The result shows that the conjugated c (RGDfC) sulfur and nitrogen double-doped graphene quantum dots have higher killing efficiency on common tumor cells and tumor cells with resistance, and the conjugated c (RGDfC) sulfur and nitrogen double-doped graphene quantum dots can overcome photodynamic therapy resistance generated in the repeated treatment process of the sulfur and nitrogen double-doped graphene quantum dots and can maintain higher killing efficiency on the tumor cells.
In conclusion, the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot is successfully synthesized by the method, and the method is simple and convenient to operate. The synthesized conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot not only has good biocompatibility, but also retains the advantage of high efficiency and stability of the sulfur-nitrogen double-doped graphene quantum dot; the tumor cell selectivity is realized, the alpha v beta 3 integrin positive tumor cells can be targeted, and the cells can not enter the alpha v beta 3 integrin negative normal tissue cells; more importantly, the conjugated c (RGDFC) sulfur and nitrogen double-doped graphene quantum dot can overcome the photodynamic therapy resistance generated by cells in the repeated treatment process of the sulfur and nitrogen double-doped graphene quantum dot, can enter lysosomes and mitochondria of the cells automatically, continuously generates active oxygen to kill tumor cells in the repeated treatment process, and can be used as a high-efficiency anti-tumor photosensitizer; the quantum dot prepared by the invention solves the technical problem which is difficult to solve in the prior art, and has wide clinical application prospect.
Claims (15)
1. A conjugated c (RGDFC) sulfur-nitrogen double-doped graphene quantum dot is characterized in that: the graphene quantum dot is a sulfur-nitrogen double-doped graphene quantum dot modified by polypeptide c (RGDFC);
the preparation method of the sulfur-nitrogen double-doped graphene quantum dot comprises the following steps:
1)
dissolving 4-bromobenzyl bromide in CH2Cl2/CH3Adding N, N-dimethyl dodecyl amine into the OH mixed solution, reacting at room temperature, concentrating, filtering, washing and drying reactants to obtain a compound 1; reacting compound 1 with Na2CO3、Pd(PPh3)4Dissolving thiophene-3-boric acid in ethanol, adding double distilled water under the protection of nitrogen, refluxing at 90 ℃ for 6 hours, decompressing the reactant to remove ethanol, extracting, drying, and purifying by a chromatographic column to obtain a compound 2; under the protection of nitrogen, FeCl is contained3Anhydrous CHCl3Adding the solution into the compound 2, reacting at room temperature, collecting precipitate, washing and drying to obtain a compound PT 2;
2) dissolving a compound PT2 in a NaOH solution, carrying out ultrasonic treatment, heating to 170 ℃, reacting for 24h, cooling to room temperature, filtering, and dialyzing to obtain the compound PT 2.
2. The conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dot according to claim 1, wherein: the molar ratio of the sulfur-nitrogen double-doped graphene quantum dots to the polypeptide c (RGDFC) is 1 (80-120).
3. The conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dot according to claim 2, wherein: the molar ratio of the sulfur-nitrogen double-doped graphene quantum dots to the polypeptide c (RGDFC) is 1: 100.
4. The conjugated c (rgdfc) sulfur-nitrogen double-doped graphene quantum dot according to claim 1, wherein:
in the step 1), the molar ratio of the 4-bromobenzyl bromide to the N, N-dimethyldodecylamine is 1: 1.3;
and/or, in step 1), the CH2Cl2/CH3In OH mixed solution, CH2Cl2And CH3OH volume ratio is 3: 2;
and/or, in step 1), the compound 1, Na2CO3、Pd(PPh3)4The mass ratio of the thiophene-3-boric acid is 0.4:0.5:0.2: 0.128;
and/or, in step 1), the extraction is carried out by using CH2Cl2Extracting;
and/or, in step 1), the FeCl3And compound 2 in an equivalent ratio of 4: 1;
and/or, in the step 2), the concentration of the NaOH solution is 0.5 mM;
and/or, in the step 2), the filtration is the filtration by using a 0.22 μm filter membrane;
and/or, in step 2), the dialysis is dialysis in distilled water.
5. The method for preparing the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot according to any one of claims 1 to 4, wherein the method comprises the following steps: it comprises the following steps:
dissolving sulfur-nitrogen double-doped graphene quantum dots in a solvent, and adding polypeptide c (RGDFC) for reaction;
and (II) ultrafiltering the reaction liquid obtained in the step (I) to obtain the product.
6. The method of claim 5, wherein: in the step I, the solvent is double distilled water;
and/or in the step (i), the reaction temperature is 4-25 ℃; the reaction time is 2-6 h;
and/or in the second step, during the ultrafiltration, the molecular weight cut-off of the ultrafiltration tube is 10 kDa;
and/or, in the second step, the centrifugal temperature is 0-4 ℃ during ultrafiltration; the centrifugal force is 5000-10000 g; the centrifugation time is 5-10 min.
7. The method of claim 6, wherein:
in the step I, the reaction temperature is 4 ℃; the reaction time is 4 h;
and/or, in the second step, the centrifugal temperature is 4 ℃ during ultrafiltration; the centrifugal force is 10000 g; the centrifugation time was 6 min.
8. The method for preparing the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot according to any one of claims 1 to 4, wherein the method comprises the following steps: it comprises the following steps:
(1) dissolving the sulfur-nitrogen double-doped graphene quantum dots in a solvent, and adding an EDC/NHS mixture for activation;
(2) adding polypeptide c (RGDFC) into the activated sulfur-nitrogen double-doped graphene quantum dots for reaction;
(3) and (3) carrying out ultrafiltration on the reaction liquid obtained in the step (2).
9. The method of claim 8, wherein: the EDC/NHS mixture consists of EDC and NHS; the molar ratio of the sulfur-nitrogen double-doped graphene quantum dot to the EDC/NHS mixture is 1 (2000-4000).
10. The method of claim 9, wherein: the molar ratio of the sulfur-nitrogen double-doped graphene quantum dots to the EDC/NHS mixture is 1: 4000; and/or the mass ratio of EDC to NHS is 2: 1.
11. The method of claim 8, wherein:
in the step (1), the solvent is double distilled water;
and/or in the step (1), the activation temperature is 4-25 ℃; the activation time is 10-60 min;
and/or in the step (2), the reaction temperature is 4-25 ℃; the reaction time is 2-6 h;
and/or, in the step (3), during the ultrafiltration, the molecular weight cut-off of the ultrafiltration tube is 10 kDa;
and/or in the step (3), the centrifugal temperature is 0-4 ℃ during ultrafiltration; the centrifugal force is 5000-10000 g; the centrifugation time is 5-10 min.
12. The method of claim 11, wherein:
in the step (1), the activation temperature is 25 ℃; the activation time is 20 min;
and/or, in the step (2), the reaction temperature is 4 ℃; the reaction time is 4 h;
and/or, in the step (3), the centrifugal temperature is 4 ℃ during the ultrafiltration; the centrifugal force is 80000 g; the centrifugation time was 6 min.
13. Use of the conjugated c (RGDfC) sulfur-nitrogen double-doped graphene quantum dot according to any one of claims 1 to 4 in preparation of antitumor drugs.
14. Use according to claim 13, characterized in that: the anti-tumor drug is a photosensitizer.
15. Use according to claim 14, characterized in that: the photosensitizer is a photosensitizer that targets a tumor and/or overcomes resistance to photodynamic therapy.
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