Graphene base targeting DNA major groove and inhibiting topoisomerase and preparation method and application thereof
阅读说明：本技术 靶向dna大沟并抑制拓扑异构酶的石墨烯碱及其制法和用途 (Graphene base targeting DNA major groove and inhibiting topoisomerase and preparation method and application thereof ) 是由 潘登余 耿弼江 于 2020-07-06 设计创作，主要内容包括：本发明公开了一种靶向DNA大沟并抑制拓扑异构酶的石墨烯碱及其制法和用途,所述靶向DNA大沟并抑制拓扑异构酶的石墨烯碱作为抗癌活性成分,其包括仅几个纳米尺寸、具有平面结构的石墨烯骨架和位于所述石墨烯骨架周围的氮杂环簇。该生物活性纳米粒子的石墨烯碱,结构简单、易于合成,其不仅拥有对核DNA大沟的精准靶向功能,而且具有对拓扑异构酶I和II的高效抑制活性,尤其是在不含小分子药物成分且不含嫁接的特殊靶向分子的情况下纳米粒子本身就能显示高于化疗药物的抗癌活性。(The invention discloses a graphene base targeting a DNA major groove and inhibiting topoisomerase, a preparation method and application thereof. The graphene base of the bioactive nano particle has a simple structure, is easy to synthesize, has an accurate targeting function on a nuclear DNA major groove, has high-efficiency inhibition activity on topoisomerase I and topoisomerase II, and particularly can show anticancer activity higher than that of a chemotherapeutic drug under the condition that the graphene base does not contain small molecular drug components and grafted special targeting molecules.)
1. The graphene base is characterized by comprising a nano-scale graphene planar structure, wherein the size of the graphene planar structure is 1-10 nm, and the thickness of the graphene planar structure is 0.34-3.4 nm.
2. The graphene base targeting the DNA major groove and inhibiting topoisomerase according to claim 1, wherein the graphene base has a quasi-molecular weight of 2000-20000.
3. The graphene base for targeting the DNA major groove and inhibiting topoisomerase according to claim 1, further comprising a nitrogen heterocyclic cluster structure covalently bonded to the edges of the graphene planar structure.
4. The graphene base targeting the DNA major groove and inhibiting topoisomerase according to claim 3, wherein said nitrogen heterocyclic ring cluster structure comprises a pyridine ring, a pyrrole ring, a phenol ring, or a combination thereof.
5. The graphene base targeting the DNA major groove and inhibiting topoisomerase according to claim 4, wherein the nitrogen heterocyclic cluster structure comprises a combination of 10 to 50 heterocycles.
6. The graphene base targeting the DNA major groove and inhibiting topoisomerase according to claim 4, wherein the atomic ratio of N in the pyridine ring structure, N in the pyrrole ring structure and N in the graphite N ring structure is pyridine N: pyrrole N: graphite N ═ (2.0 to 5.0): (0.1-0.8): 1.
7. the graphene base targeting the DNA major groove and inhibiting topoisomerase according to claim 1, wherein N is present in the graphene base at 5 to 20 at%.
8. The DNA major groove-targeted topoisomerase inhibiting graphene base according to claim 1, wherein the graphene base has a Zeta potential of +33.2 to +72.3eV at physiological pH (pH 6.5 to 7.4) and/or at a concentration range of 0.01 to 1.0 mg/mL.
9. The graphene base targeting the DNA major groove and inhibiting topoisomerase according to claim 1, wherein the maximum absorption of the aqueous solution of graphene alkaloid is at 430-480 nm and/or the fluorescence emission is at 530-560 nm.
10. A method for preparing the graphene base targeting the DNA major groove and inhibiting topoisomerase according to any one of claims 1 to 9, comprising the steps of:
(a) one-step reaction: in the presence of a catalyst, in an alcohol solvent, carrying out a one-step reaction on a precursor to form the graphene base;
wherein the precursor is Julolidine (juliodine); the catalyst is organic acid and/or anhydride thereof, and/or a phosphorus-containing compound.
11. The preparation method according to claim 10, wherein in the step (a), the organic acid catalyst is selected from one or more of acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, adipic acid, maleic acid, tartaric acid, benzoic acid, phenylacetic acid and phthalic acid.
12. The preparation method according to claim 10, wherein in step (a), the phosphorus-containing compound is selected from one or more of phosphorus pentoxide, phosphorus oxychloride and phosphorus pentachloride.
13. The preparation method according to claim 10, wherein in the step (a), the alcoholic solvent is selected from one or more of ethanol, n-propanol, isopropanol, n-butanol, and isoamyl alcohol.
14. The preparation method according to claim 10, wherein in the step (a), the mass-to-volume ratio (mg: ml) of the precursor to the alcohol solvent is (2.0-3.0): 1.
15. The preparation method according to claim 10, wherein in the step (a), when the catalyst is a liquid, the volume ratio of the catalyst to the alcohol solvent is 1 (4-40).
16. The method according to claim 10, wherein in the step (a), when the catalyst is a solid, the ratio of the mass volume (mg: ml) of the catalyst to the mass volume (mg: ml) of the alcoholic solvent is 1: (40-400).
17. The method according to claim 10, wherein the reaction temperature in step (a) is 100 to 300 ℃ and the reaction time is 0.05 to 24 hours.
18. The method of claim 10, further comprising the steps of:
(b) and (3) post-treatment: separating and/or purifying the graphene base formed in the one-step reaction.
19. The method of claim 18, wherein the post-treating step is: (i) a post-treatment based on petroleum ether extraction, (ii) a post-treatment based on column chromatography separation, and/or (ii) a post-treatment based on salt precipitation centrifugation.
20. Use of a graphene base according to any one of claims 1 to 9 for targeting the DNA major groove and inhibiting topoisomerase, wherein (i) the graphene base is used for the preparation of a topoisomerase inhibitor; and/or (ii) for the manufacture of a medicament or composition thereof for the treatment and/or prevention of cancer.
21. The use of a graphene base to target the DNA major groove and inhibit topoisomerase according to claim 20 wherein the topoisomerase comprises both topoisomerase I and II types.
22. The use of graphene base for targeting the DNA major groove and inhibiting topoisomerase according to claim 20, wherein said pharmaceutical composition comprises said graphene base and a pharmaceutically acceptable carrier or a small molecule drug or an antibody or an immune drug.
23. The use of graphene base to target the DNA major groove and inhibit topoisomerase according to claim 20 wherein said cancer comprises: colon cancer, breast cancer, gastric cancer, lung cancer, carcinoma of large intestine, pancreatic cancer, ovarian cancer, prostatic cancer, renal cancer, hepatocarcinoma, brain cancer, melanoma, multiple myeloma, chronic myelogenous leukemia, blood tumor, and lymphoma.
24. The use of graphene base to target the DNA major groove and inhibit topoisomerase according to claim 20 wherein the treatment and/or prevention comprises: reversing drug resistance in resistant cells and/or inhibiting metastasis of cancer cells.
25. A method of inhibiting topoisomerase I and/or topoisomerase II comprising the steps of:
contacting the graphene base of any one of claims 1-9 with a subject to inhibit topoisomerase I and/or topoisomerase II.
26. The method of inhibiting topoisomerase I and/or topoisomerase II according to claim 25, wherein said subject comprises: a cell.
27. The method of inhibiting topoisomerase I and/or topoisomerase II according to claim 26, wherein said cell comprises: MCF-7 human breast cancer cells, human osteogenic sarcoma MG-63, human pancreatic cancer PANC-1, human liver cancer HepG2, human lung cancer A549, mouse breast cancer 4T1, gastric cancer Hgc-27, human colon cancer HCT-8, malignant melanoma A375, human cervical cancer Hela or a combination thereof.
28. A method of treating and/or preventing a disease, comprising the steps of:
administering the graphene base according to any one of claims 1 to 9 to a subject in need thereof.
29. The method of treating and/or preventing a disease according to claim 28, wherein the disease comprises: cancer, a disease associated with too high activity of topoisomerase I and/or topoisomerase II, or a combination thereof.
30. The application of graphene base is characterized in that the graphene base can target DNA major groove and comprises a nano-scale graphene planar structure, the size of the graphene planar structure is 1-10 nm, and the thickness of the graphene planar structure is 0.34-3.4 nm; the application is the application of the grapheme alkali in preparing a reversal agent for reversing the multidrug resistance of the tumor and/or the application in preparing an inhibitor for inhibiting the metastasis of cancer cells.
31. The use of the graphene base according to claim 30, wherein the tumor multidrug resistance comprises one or more of breast cancer doxorubicin resistance, colon cancer paclitaxel resistance and cervical cancer cisplatin resistance.
32. A fluorescent probe for labeling nucleic acids and/or cell nuclei, comprising a graphene base; the graphene base can target a DNA major groove and comprises a nano-scale graphene planar structure, wherein the size of the graphene planar structure is 1-10 nm, and the thickness of the graphene planar structure is 0.34-3.4 nm.
33. The fluorescent probe of claim 32, wherein the fluorescent probe can be used for labeling isolated DNA or nuclei in living cells.
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to graphene base which targets DNA major groove and inhibits topoisomerase, and a preparation method and application thereof.
Background
DNA and topoisomerase (Topo) are important targets for anticancer drugs. Topoisomerase inhibitors which have been widely used clinically and are under development are mainly DNA intercalators which indirectly interfere with the function of topoisomerase I or II mainly by means of DNA base intercalation, resulting in DNA damage and apoptosis. In recent years, in order to overcome the drug resistance limitation of single topoisomerase inhibitors, some dual inhibitors targeting Topo I and Topo II are discovered, such as indolyl quinoline derivative TAS-103, prodigiosin, curcumin and the like. However, the discovered small-molecule dual Topo inhibitor has large toxic and side effects and poor clinical test effect.
The development of novel anti-cancer drugs targeted to the DNA major groove is of particular interest because, in addition to the topoisomerase target binding to the DNA major groove, many other nuclear protein targets of interest, such as polymerases, transcription factors, and DNA repair proteins, can also specifically bind to the DNA major groove. In particular, the DNA major groove targeted drug can compete with a plurality of major groove binding proteins for DNA major groove sites to directly interfere the functions of the DNA major groove targeted drug, so that the multi-target anticancer effect can be realized, the metastasis of malignant tumors can be obviously inhibited, and the multi-drug resistance of the tumors can be overcome. However, small molecule drugs and dyes interact with DNA primarily by way of base insertion or minor groove binding, and only a few dyes have been found to have the property of binding to the major groove of DNA, but they have not been found to have anticancer activity. An anticancer drug with definite DNA major groove targeting characteristics has not been reported or clinically applied so far.
In summary, the DNA-embedded or minor groove-bound small molecule chemotherapeutic drugs have the defects of single action target, difficulty in inhibiting tumor metastasis, easy generation of multidrug resistance, large toxic and side effects, and the like. Therefore, the development of a multi-target anti-cancer drug which targets the DNA major groove, simultaneously inhibits topoisomerase I and topoisomerase II with high efficiency, has good safety, is not easy to generate drug resistance and particularly can obviously inhibit tumor metastasis is urgently needed.
Disclosure of Invention
The invention provides a graphene base which targets a DNA major groove and inhibits topoisomerase and a preparation method and application thereof to solve the problems in the prior art.
The invention provides a bioactive nano particle graphene base which is simple in structure and easy to synthesize, wherein the nano particle graphene base not only has a targeting function on nuclear DNA major groove, but also has high-efficiency inhibition activity on topoisomerase I and topoisomerase II, and particularly can show anticancer activity higher than that of chemotherapeutic drugs under the condition that the nano particle graphene base does not contain small molecular drug components and grafted special targeting molecules. In addition, the graphene base also has the activity of reversing tumor multidrug resistance (MDR) and inhibiting tumor metastasis. The graphene base can also be used as a DNA fluorescent probe for labeling nucleic acid and cell nucleus.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a graphene base for targeting a DNA major groove and inhibiting topoisomerase, which comprises a nano-scale graphene planar structure different from a planar structure of micromolecule alkaloid, wherein the size of the graphene planar structure is 1-10 nm, and the thickness of the graphene planar structure is 0.34-3.4 nm.
Further, the geometrical size of the graphene base is as follows: the average transverse dimension is 3.5 +/-0.5 nm, and the thickness range is 0.34-1.0 nm. Where the average lateral dimension is the TEM measured dimension and the thickness is the AFM measured dimension.
Further, the quasi-molecular weight of the graphene base is 2000-20000.
Further preferably, the quasi-molecular weight of the graphene base is 6000-10000.
Further, besides the graphene planar structure, the graphene base also contains a nitrogen heterocyclic ring cluster structure which is covalently bonded to the edge of the graphene planar structure.
Further preferably, the nitrogen heterocyclic ring cluster structure comprises a pyridine ring, a pyrrole ring, a phenol ring or a combination thereof.
More preferably, the nitrogen heterocyclic ring cluster structure comprises a combination of 10-50 heterocyclic rings.
More preferably, the nitrogen heterocyclic ring cluster structure comprises a combination of 15-30 heterocyclic rings.
More preferably, the ratio of N in the pyridine ring structure, N in the pyrrole ring structure and N in the graphite N ring structure is pyridine N: pyrrole N: graphite N (atomic ratio) ═ 2.0 to 5.0: (0.1-0.8): 1.
further, the content of N in the graphene base is 5-20 at%, and at% refers to atomic percentage.
More preferably, the content of N in the graphene base is 7.8 to 15.6 at%, and at% refers to atomic percentage.
More preferably, the content of N in the graphene base is 10.4 ± 0.5 at%, and at% refers to atomic%.
Further, the Zeta potential of the grapheme base is between +33.2 and +72.3eV at physiological pH (pH between 6.5 and 7.4) and/or in a concentration range of 0.01 and 1.0 mg/mL.
Further, the maximum absorption of the aqueous solution of the graphene alkaloid is 430-480 nm, and/or the fluorescence emission is 530-560 nm. The GA fluorescence emission peak red-shifted less than 5nm when the excitation wavelength was shifted from 400nm to 500 nm.
In a second aspect, the present invention provides a method for preparing a graphene base targeting a DNA major groove and inhibiting topoisomerase, comprising the steps of:
(a) one-step reaction: in the presence of a catalyst, in an alcohol solvent, carrying out a one-step reaction on a precursor to form the graphene base;
wherein the precursor is Julolidine (juliodine); the catalyst is organic acid and/or anhydride thereof, and/or a phosphorus-containing compound.
Further, in the step (a), the organic acid catalyst is selected from one or more of acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, adipic acid, maleic acid, tartaric acid, benzoic acid, phenylacetic acid and phthalic acid.
Further, in the step (a), the phosphorus-containing compound is one or a combination of several selected from phosphorus pentoxide, phosphorus oxychloride and phosphorus pentachloride.
Further, in the step (a), the alcohol solvent is selected from one or a combination of several of ethanol, n-propanol, isopropanol, n-butanol and isoamyl alcohol.
Further, in the step (a), the mass-to-volume ratio (mg: ml) of the precursor to the alcohol solvent is (2.0-3.0): 1.
Further preferably, in the step (a), the mass-to-volume ratio (mg: ml) of the precursor to the alcohol solvent is (2.0-2.5): 1.
Further, in the step (a), when the catalyst is a liquid, the volume ratio of the catalyst to the alcohol solvent is 1 (4-40).
Further preferably, in the step (a), when the catalyst is a liquid, the volume ratio of the catalyst to the alcohol solvent is 1 (10-40).
Further, in the step (a), when the catalyst is a solid, the ratio of the mass volume (mg: ml) of the catalyst to the mass volume (mg: ml) of the alcohol solvent is 1: (40-400).
Further preferably, in the step (a), when the catalyst is a solid, the ratio of the mass volume (mg: ml) of the catalyst to the mass volume (mg: ml) of the alcoholic solvent is 1: (100-400).
Further, in the step (a), the reaction temperature is 100-300 ℃, and the reaction time is 0.05-24 h.
Further, the preparation method of the graphene base which targets the DNA major groove and inhibits topoisomerase further comprises the following steps:
(b) and (3) post-treatment: separating and/or purifying the graphene base formed in the one-step reaction.
Further, the post-processing steps are as follows: (i) a post-treatment based on petroleum ether extraction, (ii) a post-treatment based on column chromatography separation, and/or (ii) a post-treatment based on salt precipitation centrifugation.
A third aspect of the invention provides a use of a graphene base as defined in any one of the above for targeting the DNA major groove and inhibiting topoisomerase, (i) for the preparation of an inhibitor for inhibiting topoisomerase; and/or (ii) for the manufacture of a medicament or composition thereof for the treatment and/or prevention of cancer.
Further, the topoisomerase includes two types of topoisomerase I and topoisomerase II.
Further, the pharmaceutical composition comprises the graphene base and a pharmaceutically acceptable carrier or a small molecule drug or an antibody or an immune drug.
Further, the cancer includes: colon cancer, breast cancer, gastric cancer, lung cancer, carcinoma of large intestine, pancreatic cancer, ovarian cancer, prostatic cancer, renal cancer, hepatocarcinoma, brain cancer, melanoma, multiple myeloma, chronic myelogenous leukemia, and malignant lymphoma.
Optionally, the treatment and/or prevention comprises: reversing drug resistance in resistant cells and/or inhibiting metastasis of cancer cells.
In a fourth aspect of the invention there is provided a method of inhibiting topoisomerase I and/or topoisomerase II comprising the steps of: contacting the graphene base with a subject as described above to inhibit topoisomerase I and/or topoisomerase II.
Further, the object includes: a cell. Preferably, the cell comprises: MCF-7 human breast cancer cells, human osteogenic sarcoma MG-63, human pancreatic cancer PANC-1, human liver cancer HepG2, human lung cancer A549, mouse breast cancer 4T1, gastric cancer Hgc-27, human colon cancer HCT-8, malignant melanoma A375, and one or more of human cervical cancer Hela.
A fifth aspect of the present invention provides a method for treating and/or preventing a disease, comprising the steps of: administering to a subject in need thereof a graphene base as described above.
Further, the disease is selected from: cancer, a disease associated with too high activity of topoisomerase I and/or topoisomerase II, or a combination thereof.
Further, the subject is an animal. Preferably, the subject is a mammal; more preferably, the subject is a human.
The sixth aspect of the invention provides an application of graphene base, in a specific embodiment, the application is an application of graphene base in preparation of a reversal agent for reversing tumor multidrug resistance and/or an application in preparation of an inhibitor for inhibiting cancer cell metastasis, wherein the graphene base can target a DNA major groove and comprises a nano-scale graphene planar structure, and the size of the graphene planar structure is 1-10 nm, and the thickness of the graphene planar structure is 0.34-3.4 nm.
Optionally, the subject to which the reversal agent or inhibitor is administered is an animal or a cell. Further optionally, the subject is a mammal.
Optionally, the tumor multidrug resistance comprises one or more of breast cancer adriamycin resistance, colon cancer paclitaxel resistance and cervical cancer cisplatin resistance.
Further optionally, the subject to which the reversal agent is administered comprises one or more of human breast cancer doxorubicin-resistant cells MCF-7/ADR, human colon cancer paclitaxel-resistant cells HCT-8/PTX, and human cervical cancer cisplatin-resistant cells Hela/DDP.
In a seventh aspect, the invention provides a fluorescent probe for labeling nucleic acids and/or cell nuclei. In one embodiment, the fluorescent probe comprises a graphene base; the graphene base can target a DNA major groove and comprises a nano-scale graphene planar structure, wherein the size of the graphene planar structure is 1-10 nm, and the thickness of the graphene planar structure is 0.34-3.4 nm. Wherein labeling the nucleus comprises nuclear imaging.
Further, the fluorescent probe can be used for labeling isolated DNA or nuclei in living cells.
Optionally, when the fluorescent probe is used for DNA labeling, the wavelength range of the exciting light is 350-500nm, and the wavelength of the emitting light is 530-560 nm; when the fluorescent probe is used for the nuclear marking of living cells, the excitation wavelength is selected from 405nm, 488nm and 543nm, and the corresponding detection wavelengths are respectively 410-500nm, 500-600nm and 550-620 nm.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
(a) the graphene base is not easy to cause drug resistance of a treatment object; (b) the graphene base can effectively inhibit cancer cell metastasis; (c) the graphene has less toxic and side effects; (d) the graphene base can directly target tumor tissues, and particularly can target nucleic acid or ribozyme; (e) the graphene base can overcome multiple biological barriers in cells; (f) the graphene alkali provided by the invention is stable in structure and easy to dissolve in water; (g) the graphene base is simple to prepare and easy to control; (h) the graphene base can be used as an ultra-stable fluorescent probe.
Drawings
Figure 1 shows a structural model of graphene base GA;
TEM picture of a) GA in fig. 2; b) HRTEM picture of GA; c) AFM pictures of GA.
In fig. 3a) X-ray diffraction pattern of graphene base GA; b) a raman spectrogram of graphene base GA;
figure 4 a) XPS summary of graphene base GA; b) c1s spectrum of graphene base GA; c) an N1s spectrum of graphene alkaloid; d) an O1s spectrum of graphene base GA;
in fig. 5a) Zeta potential of graphene base GA under different pH conditions; b) zeta potential of graphene base GA under different concentrations;
in fig. 6 a) a fluorescence picture, uv-vis absorption spectrum and fluorescence excitation and emission spectrum of graphene base GA ethanol solution; b) ultraviolet visible absorption spectrum and fluorescence excitation and emission spectrum of the graphene alkali GA aqueous solution; c) testing the fluorescence stability of the graphene alkali GA ethanol solution and the graphene alkali aqueous solution under continuous ultraviolet irradiation;
figure 7 a) effect of graphene oxide GA on cell survival of various types of cancer cell lines; b) graphene base GA pairSemi-lethal concentration (IC) of cancer cell lines of the same type50)；
FIG. 8 compares the semi-lethal concentration (IC) of graphene GA on Hela cells with the small molecule topoisomerase inhibitors Doxorubicin (DOX), hydroxycamptothecin (CPT-11) and etoposide (VP-16)50)；
Figure 9 shows experimental evidence of graphene base GA binding to the major groove of double stranded DNA: influence of different concentrations of graphene base GA on characteristic circular dichroism peaks generated by binding of major groove binding dye methyl green with ct-DNA;
figure 10 shows the results of an assessment of inhibition of topoisomerase I activity by graphene base GA;
figure 11 shows the results of an assessment of inhibition of topoisomerase II activity by graphene base GA;
figure 12 shows the therapeutic effect of graphene base GA on MCF-7 tumors by intratumoral injection in vivo; wherein, adriamycin DOX is used as a positive control;
FIG. 13 shows the therapeutic effect of graphene base GA on Hela, HepG-2, 4T1 and A549 tumors by intravenous injection in vivo (doxorubicin DOX as a positive control);
figure 14 shows the change in body weight of mice after treatment with graphenic base GA (doxorubicin as a positive control);
figure 15 shows the number of metastases of the tumor in the lung after treatment of 4T1 tumor with graphene base GA by intravenous injection in vivo (doxorubicin DOX as positive control);
fig. 16 shows the plasma concentration-time curve after a single tail vein injection of graphene base GA in mice;
figure 17 shows in vivo imaging of graphene base GA passively targeted to tumors by intravenous injection;
figure 18 shows the distribution of graphene base GA in organs in vivo following administration by in vivo imaging after intravenous injection;
figure 19 shows long-term toxicity assessment of graphene base GA, wherein major organs were taken at different time points for histological analysis after intravenous injection of graphene base GA;
fig. 20 shows long-term toxicity evaluation of graphene base GA, where whole blood was taken at different time points for routine blood analysis and biochemical blood analysis after intravenous injection of graphene base GA;
fig. 21 shows a hemolysis experiment of graphene base GA;
FIG. 22 shows cellular imaging of graphene base GA and doxorubicin DOX on drug-sensitive and drug-resistant cells;
FIG. 23 shows the semi-lethal concentrations of graphene base GA and the commonly used small molecule chemotherapeutic drugs on drug-sensitive cells (MCF-7) and drug-resistant cells (MCF-7/ADR), showing the change in the resistance index of the small molecule chemotherapeutic drugs in the presence or absence of graphene base.
FIG. 24 shows the semi-lethal concentrations of graphene base GA and the commonly used small molecule chemotherapeutic drugs on drug-sensitive cells (HCT-8) and drug-resistant cells (HCT-8/PTX), showing the change in the resistance index of the small molecule chemotherapeutic drugs in the presence or absence of graphene base.
FIG. 25 shows a scratch experiment of the ability of graphene base GA to inhibit cell migration of 4 cancer cells (A375, HepG2, 4T1, PANC-1).
Figure 26 shows a Transwell experiment of the ability of graphene base GA and small molecule chemotherapeutic drugs (doxorubicin DOX and paclitaxel PTX) to inhibit cell migration of 4T1 cells.
Figure 27 shows a Transwell experiment of the ability of graphene base GA and small molecule chemotherapeutic drugs (doxorubicin DOX and paclitaxel PTX) to inhibit cell invasion of 4T1 cells.
Figure 28 shows in vivo imaging of the ability of the graphenes GA and doxorubicin DOX to inhibit lung metastasis of 4T1-Luc cells in vivo.
Fig. 29 shows a gel electrophoresis of graphene base GA and commercial nucleic acid dye-labeled DNA.
Fig. 30 shows targeted nuclear imaging of graphene base GA and the commercial nucleic
Fig. 31 shows that the change in fluorescence imaging effect (stability) was observed when the excitation time was prolonged when the graphene base GA was subjected to nuclear imaging with the commercial nucleic
Detailed Description
The present invention, through extensive and intensive studies, has unexpectedly developed a graphene-based anticancer drug GA having a novel structure, which has excellent inhibitory activities of topoisomerase I and II for the first time. In addition, research also shows that the GA medicament provided by the invention has a planar structure of ultrathin nano graphene. The present invention has been completed based on this finding.
As used herein, "graphene alkaloid", "graphene alkaloid drug", "graphene base drug", "graphene base nano drug", "graphene base anti-cancer drug", "graphene quantum dot active drug", and "GA drug" are used interchangeably and refer to planar ultra-thin nano-graphene alkaloid, GA.
As used herein, the nanopharmaceutical or active ingredient of the present invention refers to the graphene base of the first aspect.
Graphene base medicine
The invention provides a non-small molecule type anticancer drug which is targeted to act on nuclear DNA major groove and simultaneously inhibits topoisomerase I and II.
The molecular structure of the graphene alkaloid of the present invention is substantially as shown in fig. 1.
In addition, compared with the traditional micromolecular drugs, the graphene alkali anticancer drug has obviously different structural characteristics, physicochemical characteristics and pharmacological characteristics.
In one embodiment, the graphenic base used as the active ingredient in the present invention has one or more of the following characteristics:
one of the characteristics is as follows: the graphene alkali is a derivative of graphene, can be regarded as an ultra-stable pi-conjugated planar macromolecule, is formed by fusing more than dozens of benzene rings and nitrogen heterocycles, and has the average molecular weight equivalent to that of a polypeptide molecule. The medicine can maintain chemical stability and biological activity even in special physiological environment (such as gastric acid, liver, cell lysosome), and will not be biodegraded to lose efficacy.
The second characteristic: graphene alkali belongs to an artificially synthesized alkaloid-like active medicine. Different from natural alkaloid drugs, the pharmacophore of graphene base is an alkaline nitrogen heterocyclic cluster which surrounds the perimeter of a graphene planar framework and is tightly fused with a graphene base surface, and mainly comprises a six-membered pyridine ring, a five-membered pyrrole ring and a phenol ring.
Thirdly, the characteristics are as follows: the graphene alkali has adjustable size, so that the drug can be targeted on tumor tissue cell nucleuses without influencing normal tissue cells. The lateral size of the graphene base can be limited within the range of 2-10 nanometers, and the upper limit of the size of the graphene base allows nanoparticles to smoothly pass through nuclear pores (the diameter is 10nm), so that the graphene base targets a DNA double chain acting on tumor cell nucleuses. The lower size limit is set to take into account the significant difference in microvascular structure and permeability between normal and tumor tissues. For normal tissues without pathological changes, the capillaries are complete, endothelial cells are closely arranged, the gaps are as small as 1-2 nanometers, small-molecule medicines are easy to permeate and generate toxic and side effects, and the nano medicines are not easy to permeate and can reduce the toxic and side effects. Solid tumor tissue is different: poor structural integrity of blood vessels, abnormal widening of endothelial cell gaps, and lymphatic return loss, resulting in high permeability and retention (EPR effect) to nano-drugs. By utilizing the EPR effect of the nanoparticles, the passive targeting of the graphene drug to the solid tumor can be realized.
The characteristics are as follows: the graphene base selectively targets a large groove region of nuclear DNA and is a dual inhibitor of topoisomerase I and II. The graphene base is irregular in shape, and the edges of the graphene base are provided with fine edges and corners, so that the graphene base can be selectively inserted into the bottom of a DNA major groove in a targeted manner. The graphene corner tip part contains a plurality of nitrogen heterocycles with positive charges, and generates strong electrostatic interaction with a DNA phosphodiester bond framework with negative charges, and meanwhile, the drug tip contains a hydrogen bond donor and an acceptor, and can generate hydrogen bond interaction with the acceptor or the donor of a base in a DNA major groove. The major groove region of DNA is the binding site of various DNA binding proteins such as topoisomerase I and II and protein transcription factors, and the graphene base plane inserted into the major groove of DNA can block the reconnection of topoisomerase to DNA single strand break points or double strand break points, resulting in irreversible damage to DNA. And the graphene alkaloid inserted into the DNA major groove further hinders the repair of DNA damage by cells, so that the cells are subjected to apoptosis. The unique graphene-DNA-protein nano interface effect forms the pharmacological basis of the graphene base planar drug as a dual topoisomerase I and II inhibitor.
The method is characterized by comprising the following steps: the graphene base has the optical characteristics of quantum dots, has extremely stable fluorescence property, and can be applied to DNA labeling and cell nucleus fluorescence imaging.
Preparation method
The second purpose of the invention is to provide a synthetic method of the graphene base.
The method for synthesizing the graphene base is based on an organic phase catalytic molecular fusion method, wherein a nitrogen heterocyclic aromatic compound (Julolidine) is used as a drug precursor, and a graphene base solution product (glucose injection or physiological saline injection thereof) which can be stably stored at room temperature for a long time without agglomeration is finally obtained through one-step solvent thermocatalytic reaction and subsequent separation and purification in an alcohol solvent. Compared with the synthesis of the traditional organic micromolecule chemotherapeutic drug, the synthesis method has the advantages of few synthesis steps (only single-step synthesis), mild and safe reaction (120 ℃), short synthesis time (1h), green and environment-friendly synthesis process (only using nontoxic ethanol and acetic acid), good repeatability, low synthesis cost, high total yield (55%), and the like.
In another preferred embodiment, the alcohol solvent can be replaced by ethanol, n-propanol, isopropanol, n-butanol, or isoamyl alcohol in carrying out the above reaction. The catalyst can be replaced by acetic acid, propionic acid, butyric acid, caprylic acid, adipic acid, oxalic acid, malonic acid, succinic acid, maleic acid, tartaric acid, benzoic acid, phenylacetic acid, phthalic acid or anhydride, or phosphorus oxychloride, phosphorus pentachloride and phosphorus pentoxide.
In carrying out the above synthesis, the solvothermal reaction is carried out in a reaction vessel (e.g., a teflon reaction vessel), or may be carried out under microwave-assisted conditions, or may be carried out under reflux in a high boiling point solvent at normal pressure. Preferably, a solvothermal synthesis method (performed in a taffon reaction kettle or an industrial reaction kettle) is adopted, and the method is easy to produce on a large scale and has lower cost.
In the process of separating and purifying the product, petroleum ether is used for extracting and removing fat-soluble small molecular impurities. The product separation can also adopt a chromatographic column separation method and a salt sedimentation centrifugal separation method.
In a specific embodiment, the invention provides a preparation method of a graphene base nano-drug, wherein the preparation method comprises the following steps:
a. julolidine (juliodine) is taken as a precursor, dissolved in an alcohol solvent, added with a catalyst and stirred together, and then transferred into a polytetrafluoroethylene high-pressure reaction kettle to be subjected to heat preservation reaction for 1-12 hours under the heating condition of 120-230 ℃;
b. after natural cooling, filtering with a 220nm filter membrane to remove insoluble impurities, and further separating and purifying to remove non-anticancer active organic small molecular impurities.
In another preferred example, in step a, the alcohol solvent can be one of ethanol, n-propanol, isopropanol, n-butanol and isoamyl alcohol. Preferably, ethanol is selected as the solvent.
In another preferred embodiment, in step a, the catalyst may be one of acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, adipic acid, maleic acid, tartaric acid, benzoic acid, phenylacetic acid, phthalic acid, organic acid, phosphorus pentoxide, phosphorus oxychloride and phosphorus pentachloride, and preferably, acetic acid is used as the catalyst.
In another preferred example, the concentration of the precursor in the alcohol solvent is 2.0-2.5 mg/mL, and the volume ratio of the solvent to the catalyst is 4-40.
In another preferred embodiment, in the step b, the purification is performed by an extraction purification method, which comprises the steps of: extracting with petroleum ether, and removing residual small molecular impurities for multiple times.
In another preferred embodiment, in step b, the separation is performed by a chromatographic column separation method, comprising the steps of: firstly dispersing graphene alkali in a trichloromethane solution, then adding the solution into a neutral aluminum oxide chromatographic column, using a mixed solution of trichloromethane, cyclohexane and ethanol as an eluent, and collecting the separated graphene alkali solution according to the characteristic that the graphene alkaloid medicament emits yellow fluorescence under an ultraviolet lamp.
In another preferred embodiment, in step b, the separation is performed by salt settling centrifugation comprising the steps of: removing the solvent in the graphene alkali ethanol solution by a rotary evaporation method, dispersing in 20mL of water, adding 0.2g of nitrate for dissolving, standing the solution for 10 minutes, separating out graphene alkali from the solution, and separating the separated graphene alkaloid by centrifugation.
In a specific embodiment, the invention also provides another preparation method of the graphene base nano-drug, which comprises the following steps: adding 0.1g of juliodine (juliodine) into 40mL of high boiling point solvent such as isoamyl alcohol, n-octanol or dodecanol, and then adding 1-3 mL of acetic acid, or 0.02-0.1 g of acetic anhydride, or 0.02-0.1 g of phosphorus pentoxide (or phosphorus oxychloride, phosphorus pentachloride and the like) into the solution. And transferring the mixture into a 100mL three-neck flask after the mixture is completely dissolved, heating the mixture in an oil bath kettle in a reflux manner at the temperature of 130-170 ℃, and preserving the heat for 40-120 min. After the reaction is finished, separating and purifying by a nitrate selective sedimentation method.
In a specific embodiment, the invention also provides another preparation method of the graphene base nano-drug, which comprises the following steps: julolidine (juliodine) (0.1g) is dissolved in 10mL of absolute ethanol, and 0.1-1 mL of acetic acid is added and stirred for 10 min. Then transferring the solution into a 35mL reaction tube of a microwave reactor, and reacting for 5-60 min under the microwave heating condition of 120-200 ℃. After the reaction is finished, the graphene alkali is selectively settled by nitrate, and then the graphene alkali is separated and purified.
Application of graphene base
The third purpose of the invention is to provide the anticancer application of the graphene alkaloid.
GA or an anticancer drug containing it combines the unique biological activity of graphene alkaloids with the unique pharmacological activity of alkaloid compounds. The research on the in vitro and in vivo anticancer activity shows that the medicine is superior to the traditional micromolecule anticancer medicines such as anthracycline antibiotics, cis-platinum, taxol and the like, can effectively inhibit the drug resistance mechanism regulated and controlled by transport protein P-gp, and has system toxicity obviously lower than that of micromolecule medicines (such as no cardiotoxicity, no immunosuppressive side effect and the like). The drug adopts a stable graphene structure and an alkaline nitrogen heterocycle design, overcomes the problems of structural instability, low water solubility and the like of small molecule drugs, and is suitable for systemic drug delivery such as intravenous injection or instillation, body cavity injection, oral administration and the like and interventional therapy modes such as hepatic artery infusion, bladder infusion, pulmonary artery infusion and the like. The ultrathin graphene alkaloid has large specific surface area and contains various in-plane and edge active sites, so that structural optimization and surface functionalization can be further performed, a small-molecule drug and a gene drug are allowed to be loaded, a targeting molecule, a PEG molecule and the like are connected, the ultrathin graphene alkaloid can also be loaded into a common nano-drug carrier, the pharmacokinetics and the therapeutic index of the drug are improved, and multiple choices of treatment schemes such as combined drug therapy, personalized therapy and the like are promoted.
The graphene alkaloid medicine can be conveniently prepared into high-concentration stably-dispersed water-soluble injection, such as glucose injection (about 3.2mg mL)-1) And no organic cosolvent is needed. The injection can be stored at room temperature for a long time without low temperature and light. The injection can also kill various bacteria and pathogenic protozoa with high efficiency. The injection is suitable for various tumor types, including but not limited to: colon cancer, breast cancer, gastric cancer, lung cancer, carcinoma of large intestine, pancreatic cancer, ovarian cancer, prostatic cancer, renal cancer, hepatocarcinoma, brain cancer, melanoma, multiple myeloma, chronic myelogenous leukemia, and lymphoma.
The injection will show specific efficacy in the treatment of tumors as follows, although a large body of clinical trial evidence is also required:
(1) primary tumor metastasis inhibition. The graphene alkaloid medicine can kill latent cancer cells such as tumor stem cells, and has a special effect on inhibiting the diffusion and migration of primary cancer cells. Is particularly suitable for tumor patients who need auxiliary chemotherapy to prevent primary tumor metastasis after surgical resection.
(2) The new adjuvant chemotherapy and interventional therapy of intratumoral injection. The nano-drug is applied to a systemic administration mode (intravenous injection and cavity injection), is also obviously superior to the traditional small molecular drug in the aspect of intratumoral injection treatment, can obviously reduce the volume of tumor mass, even completely eliminates the tumor, and is particularly suitable for interventional therapy or new auxiliary chemotherapy of the tumor (the volume of the tumor mass is reduced by using the chemotherapy in advance for surgical excision). The advantages of topical treatment benefit from the fact that graphene alkaloids can maintain high intratumoral concentrations for periods of up to 24-36 hours.
In a specific embodiment, the invention provides an anticancer application of graphene alkaloid, wherein the graphene alkaloid nanoparticles can be used as a high-efficiency dual topoisomerase I and II inhibitor to prepare a pharmacologically acceptable anticancer drug or dosage form.
In another preferred embodiment, the graphene alkaloid of the present invention has a broad anti-tumor spectrum, and can be used for the following diseases including but not limited to: colon cancer, breast cancer, gastric cancer, lung cancer, carcinoma of large intestine, pancreatic cancer, ovarian cancer, prostatic cancer, renal cancer, hepatocarcinoma, brain cancer, melanoma, multiple myeloma, chronic myelogenous leukemia, and lymphoma.
In another preferred example, the graphene alkaloid is suitable for systemic administration such as intravenous injection or instillation, body cavity injection, oral administration and interventional therapy modes such as hepatic artery perfusion, bladder perfusion and pulmonary artery perfusion.
The fourth purpose of the invention is to provide the application of the grapheme alkali in reversing tumor multidrug resistance and inhibiting tumor-resistant cell metastasis.
The graphene base can effectively reverse the drug resistance of at least MCF-7/ADR and HCT-8/PTX cells, and can more effectively inhibit the migration and invasion of cancer cells compared with the common traditional small-molecule anticancer drugs.
The fifth purpose of the invention is to provide the application of the graphene base as a fluorescent probe.
Pharmaceutical compositions and methods of administration
Since the compound of the present invention has excellent inhibitory activity against topoisomerase I and/or topoisomerase II, the compound of the present invention (i.e., graphene alkaloid) and the pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for the treatment, prevention and alleviation of diseases mediated by topoisomerase I and/or topoisomerase II. According to the prior art, the compounds of the invention are useful for the treatment of the following diseases: cancer. Such cancers include, but are not limited to: colon cancer, breast cancer, gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, renal cancer, liver cancer, brain cancer, melanoma, multiple myeloma, chronic myelogenous leukemia, lymphoma, and the like.
The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-1000mg of a compound of the invention per dose, more preferably, 10-500mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are liposomes, albumin, cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. vomit-ethanol, sodium lauryl sulfate
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): intravenous drip, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.
The sterile injection solution for intravenous drip includes a glucose aqueous solution or a physiological saline solution containing graphene base GA.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 1000mg, preferably 20 to 600 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
It is noted that nano-active drugs differ from small molecule cytotoxic drugs in the definition of drug structure. The latter has a limited molecular structural formula or a structural general formula to give strict protection instructions, while the nano toxic drug has no limited chemical structural formula or a structural general formula and can be protected only by describing the main composition, structural characteristics and physicochemical properties of the nano toxic drug. The graphene alkaloid drugs of the present invention are defined by the main structural features described in the summary of the invention and the main physicochemical properties described in the embodiments.