CN114129739A - Water-soluble ultrathin carbon nitride two-dimensional nanosheet, and preparation method and application thereof - Google Patents
Water-soluble ultrathin carbon nitride two-dimensional nanosheet, and preparation method and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A—HUMAN NECESSITIES
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Abstract
The invention discloses a preparation method and application of a water-soluble ultrathin carbon nitride two-dimensional nanosheet, wherein the preparation process comprises the following steps: calcining the nitrogen-containing precursor at 550 ℃ to obtain initial blocky carbon nitride; stripping the initial massive carbon nitride in a mixed acid solution of concentrated sulfuric acid and concentrated nitric acid to obtain porous carbon nitride; and carrying out long-term ultrasonic stripping reaction on the porous carbon nitride to obtain a final product. The invention has simple preparation process, low cost and good repeatability. The nanosheet provided by the invention has a good fluorescence imaging function, and can dye cells; the nano-sheet provided by the invention can generate a large amount of active oxygen under the illumination condition, thereby realizing the function of photodynamic therapy; the nanosheet provided by the invention can be used as a photoresponse non-viral gene vector, so that the delivery of DNA is promoted and the cell nucleus can be targeted. The nanosheet provided by the invention can load a P53 gene, and can realize gene and photodynamic combined treatment on tumors.
Description
Technical Field
The invention relates to a non-viral gene vector, in particular to a water-soluble ultrathin carbon nitride two-dimensional nanosheet, a preparation method and application thereof.
Background
Gene therapy refers to the delivery of nucleic acids (e.g., DNA, mRNA, and siRNA) with therapeutic functions into target cells to regulate the expression of proteins, further effecting disease therapy. Gene therapy has received much attention from researchers as a novel therapeutic approach. However, once the exogenous genetic material enters cells through endocytosis, the exogenous genetic material is degraded by lysosomes, and the treatment effect is influenced. Therefore, the construction of a safe and efficient gene delivery system is a decisive factor for the success of gene therapy.
Carbon nitride as a novel graphene-like material gradually draws great interest in the field of biomedicine due to its excellent physicochemical properties such as easy preparation, stable fluorescence, appropriate energy level, wide excitation wavelength and the like. The carbon nitride surface is rich in electrons and can be modified by various functional units. Compared with other biological materials, the carbon nitride also has the advantages of adjustable solubility and photoelectric property, high biocompatibility and the like, and is beneficial to the application of the carbon nitride in the field of biomedicine. The fluorescent properties of carbon nitride are applicable to biological imaging. In addition, the proper energy level (2.7 eV) can promote the deposition of electrons, promote carbon nitride to generate active oxygen under the condition of illumination, and therefore lysosome escape of the material is achieved and the material has a photodynamic therapy function. These properties can overcome the problems that the traditional non-viral gene delivery system is easy to be captured by lysosomes, has high toxicity, poor single gene treatment effect and the like, and has great potential for clinical application.
Therefore, the construction of the non-viral gene vector with multiple functions, photostimulation response, high transfection efficiency and tumor combined treatment effect by taking the water-soluble ultrathin carbon nitride two-dimensional nanosheet as the core has great research value.
Disclosure of Invention
The invention aims to provide a water-soluble ultrathin carbon nitride two-dimensional nanosheet, a preparation method and an application thereof, wherein the water-soluble ultrathin carbon nitride two-dimensional nanosheet is prepared by taking carbon nitride as a core and constructing the ultrathin water-soluble two-dimensional nanosheet through mixed acid treatment, long-term ultrasound and centrifugation; the nano-sheet can be self-assembled with DNA into nano-particles in water; after the nanosheet is combined with DNA, the gene delivery process can be monitored in real time; after the nano-sheet is self-assembled with DNA, the gene can be delivered into cell nucleus, and the characteristic can be used as a non-viral gene vector; the nano-sheet can generate active oxygen under the illumination condition, and the nano-sheet can stimulate and respond to light by utilizing the characteristic, so that lysosome escape is facilitated, and photodynamic therapy can be realized; the nano-sheet can transfect a P53 gene, so that gene and photodynamic combined treatment of tumors is realized; solves at least the above problems and/or disadvantages and provides at least the advantages described hereinafter.
To achieve these objects and other advantages in accordance with the present invention, the technical solutions adopted by the technical solutions of the present invention are as follows:
the embodiment of the invention provides a water-soluble ultrathin carbon nitride two-dimensional nanosheet, which is characterized in that the water-soluble ultrathin carbon nitride two-dimensional nanosheet is in a nanosheet shape, the size of the nanosheet is 100-170 nm, and the thickness of the nanosheet is 20-50 nm; the nanosheets are water-soluble and have endocytosis; the nanoplatelets can be payload DNA with which nanoparticles are formed.
The embodiment of the invention provides a preparation method of a water-soluble ultrathin carbon nitride two-dimensional nanosheet, which is characterized by comprising the following steps:
(1) calcining the nitrogen-containing precursor under the nitrogen protection atmosphere to obtain initial massive carbon nitride;
(2) placing the blocky carbon nitride obtained in the step (1) in a mixed acid solution of 98% concentrated sulfuric acid and 68% concentrated nitric acid for reaction to obtain porous carbon nitride;
(3) and (3) ultrasonically stripping the porous carbon nitride obtained in the step (2) in water, centrifuging, and taking supernatant to obtain the water-soluble ultrathin carbon nitride vitamin nanosheet.
Further, the preparation of the initial bulk carbon nitride in the step (1) specifically includes: under the condition of nitrogen or argon, the nitrogen-containing precursor is melamine, and the calcining temperature is 500-550 ℃ in a tubular furnace; calcining for 1-3 h; the temperature rising speed is 2-8 ℃/min, and the initial blocky carbon nitride is obtained.
Further, the step (2) of preparing porous carbon nitride specifically includes: and stirring the obtained initial blocky carbon nitride in 40 mL of mixed acid solution of 98% concentrated sulfuric acid and 68% concentrated nitric acid for reaction for 2 hours, wherein the ratio of the concentrated sulfuric acid to the concentrated nitric acid is 1: 1. And collecting and filtering the white floccule after the reaction is finished to obtain the porous carbon nitride.
Further, the preparation of the water-soluble ultrathin carbon nitride two-dimensional nanosheet in the step (3) specifically comprises: and (3) dispersing the porous carbon nitride obtained in the step (2) in 100 mL of ultrapure water, and carrying out ultrasonic treatment for 6-14 h, preferably 12 h.
Further, in the step (3), the centrifugal speed is 3000-7000 rpm, and the centrifugal time is 30 min.
The water-soluble ultrathin carbon nitride two-dimensional nanosheet prepared by the method is also within the protection range. The carbon nitride obtained in the step (3) is water-soluble ultrathin two-dimensional nano flaky structure which is nano flaky, the size of the nano sheet is 100-170 nm, and the thickness of the nano sheet is 20-50 nm.
The embodiment of the invention also provides application of the water-soluble ultrathin carbon nitride two-dimensional nanosheet as a non-viral gene vector in gene therapy.
The embodiment of the invention also provides application of the water-soluble ultrathin carbon nitride two-dimensional nanosheet as a photosensitizer in photodynamic therapy.
The embodiment of the invention also provides application of the water-soluble ultrathin carbon nitride two-dimensional nanosheet in tumor gene and photodynamic combined therapy.
The embodiment of the invention also provides application of the water-soluble ultrathin carbon nitride two-dimensional nanosheet as a DNA marker.
The invention at least comprises the following beneficial effects:
1. the invention constructs the water-soluble ultrathin carbon nitride two-dimensional nanosheet, has low toxicity, is easy to apply in the field of biomedicine, can respond to light stimulation, can generate active oxygen under the illumination condition, promotes lysosomes of the compound to escape, and realizes photodynamic therapy.
2. The nanosheet of the present invention can effectively support DNA, forming nanoparticles therewith.
3. The binary compound formed by self-assembling the nanosheets and the DNA can enter a cell nucleus to realize gene transfection, and gene therapy can be realized after the P53 gene is loaded.
4. The nano-sheet can realize gene and photodynamic combined therapy of tumors, and has potential of practical application.
5. The nano-sheet can carry out fluorescence imaging and real-time tracing on the gene transfection process, is favorable for researching the transfection mechanism, and lays a foundation for the research and development of novel gene vectors.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
Figure 1 is an X-ray diffraction (XRD) pattern of a nanoplate of the present invention.
FIG. 2 Transmission Electron Microscopy (TEM) image of nanoplatelets of the invention.
Fig. 3 is an Atomic Force Microscope (AFM) spectrum of a nanoplate of the present invention.
FIG. 4 shows the cell survival rate of the nanosheet of the present invention under illumination at a wavelength of 400 nm for various periods of time.
FIG. 5 is an agarose gel retardation experiment of pUC18 DNA by the nanosheet of the present invention.
FIG. 6 is a confocal diagram of the green fluorescent protein transfection expression of EGFP gene by the nanosheet of the present invention.
FIG. 7 shows the cell survival rate of the nanosheet-loaded P53 gene under non-illumination and illumination conditions.
Fig. 8 is a picture of co-localization of nanoplatelets of the present invention with lysosomal probes under non-lighting and lighting conditions.
Fig. 9 is a confocal map of uptake of the positive nanoplate and FAM-DNA complexes of the invention by cells at different time periods.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It is to be understood that the terms "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
< example 1>
A water-soluble ultrathin carbon nitride two-dimensional nanosheet takes carbon nitride as a carrier, and the carbon nitride is further constructed into the water-soluble ultrathin two-dimensional nanosheet.
The preparation method of the water-soluble ultrathin carbon nitride two-dimensional nanosheet of the embodiment includes the following steps:
(1) placing 5 g of melamine in a crucible, covering the crucible with a crucible cover, placing the crucible in a tubular furnace under the protection of nitrogen for calcination, controlling the heating rate of the tubular furnace to be 5 ℃/min, keeping the temperature at 550 ℃ for 2 h, and cooling and grinding the calcined product to obtain the blocky carbon nitride.
(2) Adding 1 g of blocky carbon nitride into 40 mL of mixed acid solution of 98% concentrated sulfuric acid and 68% concentrated nitric acid, and stirring for reaction for 2 hours, wherein the ratio of the concentrated sulfuric acid to the concentrated nitric acid is 1: 1. And collecting and filtering the white floccule after the reaction is finished to obtain the porous carbon nitride.
(3) Dispersing 100 mg of porous carbon nitride in 100 mL of ultrapure water, performing ultrasonic treatment for 12 h, centrifuging, and taking supernatant to obtain the water-soluble ultrathin carbon nitride two-dimensional nanosheet.
As shown in fig. 1, all samples contained the characteristic peak (002) of carbon nitride, indicating that the bulk of all samples was carbon nitride and no excess impurities.
As can be seen from FIGS. 2 and 3, the carbon nitride nanosheets prepared by the method have the morphology of a typical thermopolymer and have a sheet-like structure. The carbon nitride nanosheet prepared by the method has an ultrathin sheet structure, which indicates that the water-soluble ultrathin carbon nitride two-dimensional nanosheet is successfully prepared.
< example 2>
Preparing 50 mu g/mL solution of ultrathin carbon nitride nanosheets, incubating the solution with Hela cells for 12 h, and respectively using an LED light source (450 mW/cm) with the wavelength of 400 nm2) Different durations of illumination were used without light as a control. The medium was then replaced, incubated for an additional 12 h, and 20. mu.L of MTT solution (5 mg/mL) was added to the cells. After 4 hours of incubation, the medium was changed to 200 μ L DMSO. The resulting solution was measured at a wavelength of 570 nm in a Bio Tech Synergy H4 to obtain cell activity.
FIG. 4 shows the survival rate of Hela cells of the carbon nitride nanosheets of the present invention under light irradiation; as can be seen from fig. 4, the survival rate of Hela cells is lower and lower with the increase of time under the illumination condition of the carbon nitride nanosheets. The carbon nitride nanosheet prepared by the method can promote cancer cell apoptosis under the illumination condition. From the example 2, the water-soluble ultrathin carbon nitride nanosheet prepared by the method has the photodynamic therapy effect.
< example 3>
Placing carbon nitride nanosheets and pUC18 plasmid DNA (9 mug/mL) with different concentrations in a water bath at 37 ℃, incubating for 1 h, and then performing a DNA agarose gel retardation experiment to obtain a gel retardation result of compounds with different concentrations on the pUC18 DNA.
FIG. 5 shows the result of agarose gel retardation experiment of carbon nitride nanosheets of the present invention against pUC18 DNA; the values noted above in FIG. 5 are the test concentrations (. mu.g/mL); as can be seen from FIG. 5, the carbon nitride nanosheets prepared by the present invention can completely block DNA migration in agarose at a lower concentration. From the example 3, the water-soluble ultrathin carbon nitride nanosheet prepared by the method can effectively coagulate DNA to form nanoparticles, and can be used as a non-viral gene vector.
< example 4>
50 mu g/mL of carbon nitride nanosheets and EGFP-DNA are incubated for 30 min, added to Hela cells for further culture, and then the complex is illuminated for 5 min by an LED with the wavelength of 400 nm, and an unilluminated and commercial transfection reagent lipofectamine 2000 is used as a control group. Finally, the medium was aspirated, incubated for 24 h with complete medium containing FBS, and photographed by confocal laser microscopy.
FIG. 6 shows the effect of carbon nitride nanosheets + light conditions, carbon nitride nanosheets + no light conditions, and commercial transfection reagent lipofectamine 2000 on the expression of EGFP; as can be seen from FIG. 6, the water-soluble ultrathin carbon nitride nanosheet prepared by the method disclosed by the invention can be used for effectively carrying out gene transfection, and the gene transfection is facilitated by illumination for a proper time.
< example 5>
And (3) incubating the carbon nitride nanosheets and the P53 gene for 0.5 h, adding the carbon nitride nanosheets into Hela cells, culturing for 4 h, illuminating for 20 min by using an LED with the wavelength of 400 nm, culturing for 24 h, and testing the survival rate of the cells by an MTT (maximum temperature test) experiment according to the method in the embodiment 2, wherein the unirradiated group is used as a control.
FIG. 7 is a graph showing the apoptosis effect of the carbon nitride nanosheet-loaded P53 gene on Hela cells under light conditions. As can be seen from fig. 7 and example 5, the carbon nitride nanosheets enable gene and photodynamic combination therapy of cancer cells.
< example 6>
Incubating carbon nitride nanosheets and DNA which is not fluorescently labeled for 30 min, adding the carbon nitride nanosheets and lysosome dye LysoTracker into Hela cells for culturing for 30 min, and illuminating for 5 min by using an LED with the wavelength of 400 nm, wherein the unilluminated group is used as a control. Then the culture medium is sucked out, washed for 3-5 times by PBS, imaged by a laser confocal microscope, and the escape condition of lysosomes is observed.
FIG. 8 shows that carbon nitride nanosheets aggregated and fluorescently-unlabeled DNA was added to Hela cells to perform a lysosome escape experiment. As can be derived from fig. 8, lysosomal escape of the vector was accelerated after light irradiation.
< example 7>
And incubating the carbon nitride nanosheets and the FAM-DNA for 30 min, adding Hela cells to culture for different time, sucking out the culture medium, washing the culture medium for 3-5 times by PBS (phosphate buffer solution), photographing by a laser confocal microscope, and tracing the gene transfection process.
FIG. 9 is an image of cells obtained by incubating FAM-DNA aggregated by carbon nitride nanosheets with Hela cells for different durations. As can be seen from example 7, the present invention can trace the process of gene delivery in real time, and 1 h can deliver DNA to the nucleus.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Claims (10)
1. The water-soluble ultrathin carbon nitride two-dimensional nanosheet is characterized in that the water-soluble ultrathin carbon nitride two-dimensional nanosheet is in a nanosheet shape, the size of the nanosheet is 100-170 nm, and the thickness of the nanosheet is 20-50 nm; the nanosheets are water-soluble and have endocytosis; the nanoplatelets can be payload DNA and form nanoparticles therewith.
2. A preparation method of water-soluble ultrathin carbon nitride two-dimensional nanosheets is characterized by comprising the following steps:
(1) calcining the nitrogen-containing precursor under the nitrogen protection atmosphere to obtain initial massive carbon nitride;
(2) placing the blocky carbon nitride obtained in the step (1) in a mixed acid solution of 98% concentrated sulfuric acid and 68% concentrated nitric acid for reaction to obtain porous carbon nitride;
(3) ultrasonically stripping the porous carbon nitride obtained in the step (2) in water, centrifuging and taking supernatant liquid to obtain the water-soluble ultrathin carbon nitride vitamin nanosheet.
3. The preparation method of the water-soluble ultrathin carbon nitride two-dimensional nanosheet according to claim 1, characterized in that: in the step (1), the nitrogen-containing precursor is melamine; the calcining temperature is 500-550 ℃; the calcination time is 1-3 h; the temperature rising speed is 2-8 ℃/min.
4. The preparation method of the water-soluble ultrathin carbon nitride two-dimensional nanosheet according to claim 1, characterized in that: in the step (2), the ratio of concentrated sulfuric acid to concentrated nitric acid is 1: 1.
5. The preparation method of the water-soluble ultrathin carbon nitride two-dimensional nanosheet according to claim 1, characterized in that: in the step (3), the ultrasonic stripping time is 6-14 h; the centrifugal speed is 3000-7000 rpm, and the centrifugal time is 30 min.
6. The preparation method of water-soluble ultrathin carbon nitride two-dimensional nanosheets of any one of claims 2-5, wherein: the carbon nitride obtained in the step (3) is of a water-soluble ultrathin two-dimensional nano flaky structure; the size of the nano-chip is 100-170 nm, and the thickness is 20-50 nm.
7. Use of the water-soluble ultrathin carbon nitride two-dimensional nanoplates of claim 1 as photosensitizers in photodynamic therapy.
8. Use of the water-soluble ultrathin carbon nitride nanosheets of claim 1 as a non-viral gene carrier in gene therapy.
9. An application of the water-soluble ultrathin carbon nitride two-dimensional nanosheet of claim 1 in tumor gene and photodynamic combination therapy.
10. Use of the water-soluble ultrathin carbon nitride two-dimensional nanosheets of claim 1 as a DNA marker.
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| CN105417507A (en) * | 2015-12-10 | 2016-03-23 | 济南大学 | Preparing method for carbon nitride nano particles and obtained product |
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| RU2690810C1 (en) * | 2018-10-05 | 2019-06-05 | Федеральное государственное бюджетное учреждение науки Научно-технологический центр уникального приборостроения Российской академии наук (НТЦ УП РАН) | Method of producing carbon nitride having an abnormally high level of fluorescence under the action of visible laser radiation |
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