CN113855808B - Application of nitrogen-doped carbon quantum dot delivery system in cartilage tissue - Google Patents
Application of nitrogen-doped carbon quantum dot delivery system in cartilage tissue Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Inorganic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and provides application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues, in order to solve the problem that a compact structure capable of effectively penetrating the surface layer of cartilage tissues and entering deep cells of the cartilage tissues does not exist in the research of cartilage diseases at present. O-phenylenediamine is dissolved in deionized water, and magnetic stirring is carried out at room temperature to form clear and transparent solution; heating the solution to 180 ℃ in a high-pressure reaction kettle to react for 12 hours, and naturally cooling to room temperature to obtain carbon quantum dots; filtering with a carbon quantum dot 0.22 mu m filter membrane, and dialyzing with a dialysis bag with molecular weight cutoff of 3500 Da to obtain 24 h; freeze drying and dissolving in deionized water to obtain the nitrogen doped carbon quantum dot delivery system. The method has the advantages of good biocompatibility, high targeting, high transfection efficiency and small volume, can smoothly pass through the compact structure of the cartilage tissue surface, and can become an excellent cartilage tissue disease diagnosis method; the cartilage cells smoothly enter the cartilage cells through the shallow compact structure of the cartilage tissue, and the method has great clinical treatment significance.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues.
Background
The articular cartilage disease is a common disease and frequently-occurring disease seriously endangering human health, and the main pathological manifestations are joint swelling, pain, dysfunction and the like, and is one of the main causes of disability of middle-aged and elderly people [1]. The world health organization lists the articular cartilage diseases as the most extensive hazard to the human body beyond the three killers of cardiovascular and cerebrovascular diseases, cancer and diabetes [2]. Therefore, it is extremely necessary to pay attention to and strengthen the study of articular cartilage diseases, which has positive effects and important practical significance for improving the quality of life of people, controlling medical costs, and accelerating the achievement of health care targets.
Osteoarthritis (OA) is a highly-priced articular cartilage disease, and the most common OA in the clinic at present is mainly composed of primary OA and traumatic OA. Primary OA is also known as senile OA, a progressive degenerative change of articular cartilage due to aging. Traumatic OA is a progressive degenerative joint cartilage injury resulting from either direct injury to the articular cartilage due to acute trauma or from abnormal stress in the knee joint following trauma. In recent years, along with the continuous increase of the aging degree of population and the popularization of body building exercises of the whole people, the incidence rate of OA is increased year by year, and the trend of gradual younger is seen [3].
OA major pathological changes, progressive degeneration of cartilage tissue, can be classified into 4 grades according to arthroscopic scoring (outpridge score): stage I: soft and swollen cartilage surface but intact surface; stage II: superficial ulcers and fibrosis of rough nuclei with a diameter of 1cm in small rain; class III: deep ulcers with lesion diameters greater than 1cm, but lesions that do not reach subchondral bone; grade IV: injury such as stripping and tearing of the full-thickness cartilage and exposure of subchondral bone [4].
The existing research results show that: once the cartilage structure is destroyed, it is difficult to repair both the drug therapy and the surgical therapy [5,6], so that early treatment (I, II class injury) before the cartilage structure is destroyed may become an effective method for treating the cartilage injury. However, knee cartilage has its unique structural features: mature cartilage tissue is generally divided into superficial, intermediate and deep layers, with superficial collagen being finely arranged and dense, and the drug hardly penetrating through the layers to achieve the desired therapeutic effect [7]. Since early cartilage tissue damage is small, the dense structure of the surface layer still exists, and therefore, how to effectively deliver drugs into cartilage tissue has become a critical issue for early treatment of OA.
The delivery system can accurately, target and effectively deliver substances such as target genes, proteins or medicines to a target position by adopting multidisciplinary means, so as to achieve the aim of improving the bioavailability of the target substances at the target position. Various types of delivery systems have been developed to establish: such as viral vector delivery systems and some non-viral vector delivery systems: such as plasmids, inorganic nanoparticles, cationic polymers, liposomes, and the like. These carriers are easily modified by chemical group packaging, but the above delivery optical systems have the disadvantages of poor biocompatibility, single action, poor targeting, low transfection efficiency, large delivery system volume, incapability of tracking, etc., so that good therapeutic effects cannot be achieved [8,9].
The carbon quantum dot is a novel carbon nanomaterial first discovered by Xu et al in 2004 when preparing single-wall carbon nanotubes [10]. With the continuous and intensive research of the carbon nanodots in recent years, researchers find that the carbon nanodots are zero-dimensional materials with diameters below 10 nm, have excellent biocompatibility, are rich in surface groups, are easy to chemically modify, have photoixcitation, electrochemiluminescence characteristics and stability, integrate multiple functions, realize targeting, imaging, tracing, photothermal therapy, drug (gene/protein) carrying or controlled drug release (gene/protein) and other functions, and are an important novel multifunctional delivery system, and have great potential application values in various aspects of biology, medicine, environment, optics, analytical chemistry and the like [11-14].
Carbon element is one of the most important elements in living bodies, and constitutes various nano carbon materials, and among these nano particles, carbon quantum dots attract attention of the subject group in terms of unique physicochemical properties such as a spheroid structure with a size of less than 10 nm, good dispersibility, high water solubility, and the like. And the construction of the carbon quantum dots does not need strict, complex, tedious, expensive and low-efficiency preparation steps, and the nontoxic green carbon quantum dots can be produced in a large scale from various common organic carbon sources such as glucose, wool, various fruits, pesticides and the like by a simple, low-cost and mature and perfect synthesis method. More importantly, compared with the traditional metal quantum dots, the carbon quantum dots have the unique properties of extremely high fluorescence quantum yield, multicolor photoluminescence, easy-to-modify surface, good light stability, excellent biocompatibility and the like.
At present, the research of the carbon quantum dots in the medical field is mainly focused on the diagnosis and treatment aspect of tumors [15], and related reports of the carbon quantum dots in the research of cartilage diseases are not searched yet.
The existing delivery systems suffer from the following disadvantages: poor biocompatibility and great toxic and side effects; the effect is single; the targeting is poor; the transfection efficiency is low; the delivery system has larger volume and can not smoothly pass through the compact structure of the cartilage tissue surface; the trace cannot be performed.
Disclosure of Invention
The invention provides an application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues, and researches on transfection efficiency, biocompatibility, targeting property, fluorescence performance and capacity of penetrating through cartilage surface layers in order to solve the key problem that no delivery system capable of penetrating through cartilage surface layers compact structures exists at present. The nitrogen-doped carbon quantum dot has good biocompatibility, high targeting property, high transfection efficiency and small volume, can smoothly pass through a compact structure of the cartilage tissue surface, and can trace the cartilage tissue surface through stable fluorescent signals.
The invention is realized by the following technical scheme: use of a nitrogen-doped carbon quantum dot delivery system in cartilage tissue, the nitrogen-doped carbon quantum dot delivery system m-CQDs being: o-phenylenediamine reacts at high temperature and high pressure to generate nitrogen-doped carbon quantum dots; the preparation method comprises the following steps: (1) 300mg of o-phenylenediamine is dissolved in 10ml of deionized water, and the solution is magnetically stirred at room temperature to form a clear and transparent solution; (2) Adding the solution into a polytetrafluoroethylene high-pressure reaction kettle, heating a muffle furnace to 180 ℃, reacting for 12 hours, and naturally cooling to room temperature to obtain carbon quantum dots; (3) Filtering the obtained carbon quantum dot 0.22 mu m filter membrane, and dialyzing 24h by using a dialysis bag with molecular weight cut-off of 3500 Da; (4) Freeze-drying the dialyzed carbon quantum dots, and dissolving the dialyzed carbon quantum dots in deionized water to obtain a nitrogen-doped carbon quantum dot delivery system; the nitrogen-doped carbon quantum dots are applied to penetrate through the compact structure of the surface layer of cartilage tissue and effectively deliver biological factors or medicines to deep cells of the cartilage tissue.
Further, the nitrogen-doped carbon quantum dots are applied to fluorescent kits for marking chondrocytes and living tissues.
The application of the nitrogen-doped carbon quantum dot in the fluorescent kit for marking the cell nucleus of the chondrocyte.
The concentration of the carbon quantum dots in deionized water is 0.05 mug/ml or 0.025 mug/ml.
The nitrogen doped carbon quantum dot with the cell nucleus targeting function prepared by the invention has the advantages that the delivery effect of the nitrogen doped carbon quantum dot in chondrocytes and cartilage tissues is researched, and the research result shows that: the carbon quantum dot in-vitro transfection flow is simple, the transfection speed is high (30 minutes), the transfection efficiency is high (approaching 100%), the biocompatibility is high, the targeting into cell nucleus can be realized, the fluorescent signal is clear and stable, and the carbon quantum dot can smoothly enter into the chondrocyte through the compact structure of the surface layer of the cartilage tissue due to the small volume (4-5 nm). The nitrogen-doped carbon quantum dots have great application value in the mechanism and clinical diagnosis and treatment of cartilage diseases.
The nitrogen-doped carbon quantum dots m-CQDs prepared by the invention have the advantages of simple transfection flow, high transfection speed, high transfection efficiency, low cytotoxicity, metabolizable property, targeted cell nucleus entering, clear and stable fluorescent signal and the like, and are an excellent biological delivery system; the above characteristics of the nitrogen-doped carbon quantum dots (m-CQDs) can be used for carrying out fluorescent marking on chondrocytes, and biological processes such as proliferation, apoptosis, necrosis and the like of the chondrocytes are dynamically recorded, and the nitrogen-doped carbon quantum dots (m-CQDs) carry detection targets and become an excellent cartilage tissue disease diagnosis method; the nitrogen-doped carbon quantum dots (m-CQDs) can smoothly enter chondrocytes through a cartilage tissue shallow layer compact structure, so that the method can be a novel, effective and visual drug delivery method suitable for cartilage tissue diseases such as cartilage injury, and has great clinical significance.
Drawings
FIG. 1 is a transmission electron microscope image of the prepared carbon quantum dots m-CQDs; in the figure: a is a transmission electron microscope image of the prepared carbon quantum dot; b is a particle size distribution diagram of the prepared carbon quantum dots;
in fig. 2: a is the ultraviolet-visible absorption spectrum of the prepared m-CQDs; in the figure: a: ultraviolet absorbance spectra of m-CQDs; b, fluorescence excitation spectrum; fluorescence emission spectrum; b is fluorescence emission spectrum of m-CQDs under different excitation wavelengths;
FIG. 3 is a dynamic observation of the transfection of chondrocytes from the m-CQDs transfection set using a living cell workstation at 0-30 minutes post-transfection; the pictures are taken under a 20-time and 10-time fluorescence microscope;
FIG. 4 is a dynamic observation of the transfection of chondrocytes from the adenovirus-GFP transfection group using a live cell workstation at 0-24 hours post-transfection; the pictures are taken under a 20-time and 10-time fluorescence microscope;
FIG. 5 is a dynamic observation of the transfection of chondrocytes from the plasmid-GFP transfection set 0-24 hours after transfection using a living cell workstation; the results of fig. 3-5 show that: compared with plasmid-GFP and adenovirus-GFP, the m-CQDs transfection speed is high, and obvious nuclear imaging can be seen only in 30 minutes (20 times and 10 times of microscope in the group chart respectively);
FIG. 6 is a graph showing the transfection efficiency of the m-CQDs, plasmid-GFP and adenovirus-GFP transfection sets; in the figure: panel A is a graph of results of flow cytometry detection of each set of transfection efficiencies; graph B is a statistical analysis graph of the flow results, wherein the statistical analysis graph uses m-CQDs as a control group, P <0.05 and P <0.001;
FIG. 7 is a graph showing cell viability assays of m-CQDs, plasmid-GFP and adenovirus-GFP transfected groups; in the figure: panel A is a graph of results of flow cytometry detection of cell viability of each group; b is a statistical analysis chart of the flow results, wherein the statistical analysis chart uses a blank control group con. Group as a control group, and P <0.05 and P <0.01 are respectively represented;
FIG. 8 is a graph showing the proliferation potency of transfected cells of different concentrations of m-CQDs; in the figure: FIG. A is a graph showing the results of the RTCA cell proliferation detector in detecting the proliferation capacity of each group of cells; the diagram B is a statistical analysis diagram of RTCA detection results;
FIG. 9 shows the morphological changes of 4d cells after transfection of m-CQDs transfected groups at different concentrations under a fluorescence microscope: panel A shows that the cell morphology appears remarkably abnormal under a fluorescence microscope in two high-concentration CQDs transfected groups (0.5 mug/ml and 0.25 mug/ml) 4 days after transfection (24 hours after transfection and liquid change); panel B shows the cell morphology under the CODs fluorescence microscope 4 days after transfection (24 hours passage and liquid change after transfection) of the low concentration transfection group (0.05. Mu.g/ml and 0.025. Mu.g/ml), the cell morphology and growth were normal, but no fluorescent signal was generated; the microscope magnification in the group chart is 20 times and 10 times respectively; A. in the diagram B, bright field, green fluorescence and superposition are sequentially carried out from left to right;
FIG. 10 is a graph showing nuclear targeting of m-CQDs under a fluorescence microscope at a living cell workstation, showing that significant nuclear imaging can be seen after transfection of chondrocytes with m-CQDs; the microscope magnification in the group chart is 20 times;
FIG. 11 is a human cartilage tissue block culture flow and cartilage tissue block selection criteria; in the figure: panel A shows the source of human cartilage tissue mass: relative normal cartilage left after joint replacement, panel B shows that tissue mass was treated to 4mm in ex vivo culture 3 Left and right size; panel C shows that after paraffin sections of human cartilage tissue blocks, safranin-fast green staining is performed, and cartilage tissue suitable for the experiment is selected by Mankin's scoring (Mankin's scoring 0-2 points);
FIG. 12 shows the distribution of m-CQDs in human cartilage tissue blocks under fluorescent microscopy after 48 hours of culture (without changing the liquid) in paraffin-embedded sections: panel A shows the distribution of m-CQDs in cartilage tissue blocks of control and m-CQDs (0.025. Mu.g/ml) cultures, where green fluorescence represents m-CQDs, blue fluorescence represents positions of nuclei by DAPI staining, and a, b, c represent the surface, middle and deep layers of cartilage tissue, respectively, and the results show that: green fluorescent signals representing m-CQDs are observed in all of the surface, middle and deep chondrocytes of the cartilage tissue of the m-CQDs group, and the m-CQDs are mainly present in the nucleus; and B, carrying out statistical analysis on green fluorescent signals in the surface layer, the middle layer and the deep layer of the cartilage tissue of the m-CQDs group, wherein the result shows that: the transfection efficiency of m-CQDs in each layer is close to 100%, and the result suggests that the m-CQDs can effectively penetrate through the surface compact tissue of the cartilage tissue into the chondrocytes of the deep cartilage tissue;
fig. 13 shows that after 48 hours of culture (without changing the liquid) of the human cartilage tissue blocks, the mechanical properties of the cartilage tissue of each group were detected by using a biological nano-indentation instrument, and the results show that: the mechanical properties of the m-CQDs group were not significantly changed compared to the control group (con.): m-CQDs have no obvious toxic effect on human cartilage tissue;
FIG. 14 is a graph of in vivo fluorescence signal from m-CQDs detected by fluorescence imaging of small animals, showing that in vivo fluorescence signal is detectable after injection of m-CQDs into the joint cavity and gradually decays with time; the fluorescent signals in the pictures are sequentially from outside to inside: blue-yellow-red, respectively, the intensity of the fluorescent signal, the weakest blue, the second yellow, the strongest red;
FIG. 15 shows the distribution of m-CQDs in cartilage tissue by fluorescence microscopy after frozen sections of cartilage tissue, from left to right, with the first column being a micrograph; the second and third columns are fluorescence micrographs, and the results show that m-CQDs can penetrate through the compact structure of the surface layer of cartilage tissue and enter into chondrocytes in the deep layer of cartilage tissue; the m-CQDs fluorescent signal was green and the picture was taken under a 20-fold fluorescence microscope.
FIG. 16 shows that m-CQDs are injected into knee joint cavities of rats for 48 hours, paraffin sections of cartilage tissues at the injection side are taken and subjected to safranin-fast green staining, the glycosaminoglycan content in the cartilage tissues is detected, the red part in the cartilage tissues represents the glycosaminoglycan content, the darker the color is to show that the glycosaminoglycan content is more, and the results show that the glycosaminoglycan content in the two groups of cartilage tissues is not significantly different, so that the m-CQDs injected into the knee joint cavities have no obvious toxic reaction to the local parts of the cartilage tissues.
FIG. 17 shows the results of liver and kidney function index measurements in serum of rats in the control group (Con.) and m-CQDs joint cavity injection group, wherein ALT is glutamic pyruvic transaminase, AST is glutamic oxaloacetic transaminase, AST/ALT is glutamic pyruvic transaminase ratio of glutamic pyruvic transaminase, TBIL is total bilirubin, DBIL is direct bilirubin, IBIL is indirect bilirubin, UREA is UREA, and Cr is creatinine; the results show that the liver and kidney function indexes of the two groups of rats have no obvious difference, which indicates that the joint cavity injection m-CQDs has no obvious systemic toxic reaction.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Carbon element is one of the most important elements in living body, and forms various nano carbon materials, and among the nano particles, carbon quantum dots attract attention in terms of unique physicochemical properties such as a spheroid structure with the size smaller than 10 nm, good dispersibility, high water solubility and the like. And the construction of the carbon quantum dots does not need strict, complex, tedious, expensive and low-efficiency preparation steps, and the nontoxic green carbon quantum dots can be produced in a large scale from various common organic carbon sources such as glucose, wool, various fruits, pesticides and the like by a simple, low-cost and mature and perfect synthesis method. More importantly, compared with the traditional metal quantum dots, the carbon quantum dots have the unique properties of extremely high fluorescence quantum yield, multicolor photoluminescence, easy-to-modify surface, good light stability, excellent biocompatibility and the like. We therefore opted to construct a carbon quantum dot delivery system.
Example 1: the method comprises the following steps of (1) reacting o-phenylenediamine at high temperature and high pressure to generate nitrogen-doped carbon quantum dots;
the preparation method comprises the following steps: (1) 300mg of o-phenylenediamine is dissolved in 10ml of deionized water, and the solution is magnetically stirred at room temperature to form a clear and transparent solution; (2) Adding the solution into a polytetrafluoroethylene high-pressure reaction kettle, heating a muffle furnace to 180 ℃, reacting for 12 hours, and naturally cooling to room temperature to obtain carbon quantum dots; (3) Filtering the obtained carbon quantum dot 0.22 mu m filter membrane to remove large-particle impurities, and then dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500 Da; (4) And freeze-drying the dialyzed carbon quantum dots, and dissolving the dialyzed carbon quantum dots in deionized water to obtain the nitrogen-doped carbon quantum dot delivery system (the concentration is 10 mug/ml).
Example 2: physical and chemical property detection of nitrogen-doped carbon quantum dots
(1) Transmission Electron Microscope (TEM)
And diluting the prepared carbon quantum dots, dripping the carbon quantum dots on an ultrathin copper net, drying the carbon quantum dots, and observing the size, the shape and the distribution of the carbon quantum dots by using a TEM.
The detection shows that: as shown in fig. 1, the prepared m-CQDs are spheroid-like nanoparticles having good dispersibility and relatively uniform size, a particle size distribution map is obtained by measuring the particle sizes of 100 carbon quantum dots, and the average size of the carbon quantum dots is calculated to be 4nm and to exhibit normal distribution.
(2) Fluorescence spectrum
The optical characteristics of the carbon quantum dots are analyzed by a fluorescence spectrophotometer, and the optical characteristics comprise excitation spectra in water, emission spectra under different excitation lights and emission spectra of the carbon dots under different pH values, and the scanning range is 200-800nm.
(3) Absorption spectrum
And diluting the prepared carbon quantum dot solution to a proper concentration, and detecting the absorption spectrum of the carbon quantum dot by using UV-8000A, wherein the scanning range is 190-1100nm.
The UV-visible absorption spectrum of m-CQDs is shown in FIG. 2A, which shows that: the carbon dots have very strong absorption bands in the ultraviolet region, obvious absorption peaks can be observed at 236 and nm, meanwhile, the carbon quantum dots have a wider absorption peak in the visible light 430 and nm, the carbon quantum dots with light yellow color can be found to emit very bright yellow-green fluorescence under ultraviolet excitation through an illustration, the carbon quantum dots can be found to have 423 and nm of optimal excitation wavelength corresponding to the ultraviolet-visible spectrum under the excitation of the wavelength, and the strongest fluorescence emission peak is positioned at 551 and nm.
Example 3: m-CQDs transfects chondrocytes in vitro, and detects transfection efficiency, biocompatibility, targeting property, fluorescent signal tracking and metabolism conditions
1. Transfection efficiency detection:
A. human chondrocyte extraction and culture process
1) Knee joint tissue after joint replacement surgery was minced into a paste in DMEM and mixed antibiotic (1%) mixed solution, selecting a relatively normal portion.
2) The crushed cartilage tissue DMEM was washed once at 1200 rpm, centrifuged for 5 minutes, and the washing solution was discarded.
3) The cartilage tissue was incubated with pronase (2 mg/ml, HBSS formulation, 1% antibiotic cocktail) at 37℃for 30 min at 250 times/min.
4) The digestion solution was discarded, washed once with DMEM, centrifuged for 5 minutes at 1200 rpm, and the wash solution was discarded.
5) Collagenase type II (0.1% -0.3%,2% FBS culture medium, 1% mixed antibiotic) was added to cartilage tissue, and incubated at 37deg.C for 6-8 hr.
6) The digested cartilage tissue is blown by a sterile straw, cells are filtered by a 75-100 micron filter screen, the filtrate is centrifuged at 1200 rpm for 5 minutes, and the cells are taken.
7) The cells were washed 3 times with DMEM, as before.
8) 10% FBS medium cells were cultured (10% FBS formulation: 90ml DMEM/F12 was added to 10ml FBS).
B. m-CQDs transfection of chondrocytes cultured ex vivo
1) Cell seeding in 33mm glass bottom Petri dishes (1X 10) 6 ) Cells were grown to 70% -80% for m-CQDs transfection at the following concentrations:
0.5 μg/ml m-CQDs:2ml of 10% FBS was added with 100ul of m-CQDs (10. Mu.g/ml);
0.25 μg/ml m-CQDs:2ml of 10% FBS was added with 50ul of m-CQDs (10. Mu.g/ml);
0.05 μg/ml m-CQDs:2ml of 10% FBS was added with 10ul of m-CQDs (10. Mu.g/ml);
0.025. Mu.g/ml m-CQDs:2ml of 10% FBS was added with 5ul of m-CQDs (10. Mu.g/ml).
2) Dynamically recording the m-CQDs transfection process by using a living cell workstation 1-30 minutes after transfection;
3) The transfection conditions and cell morphology were observed 24 hours after transfection using a fluorescence microscope, and the transfection efficiency and cell viability were also detected using a flow cytometer.
4) The transfection reagent was removed 24 hours after transfection, and the cells were cultured with 10% FBS medium, and fluorescent signals (m-CQDs metabolic status) and cell morphology were observed 48 hours and 4 days after transfection.
C. Adenovirus transfection of chondrocytes cultured in vitro
1) Cell seeding in 33mm glass bottom Petri dishes (1X 10) 6 ) Cells were grown to 70% -80% for adenovirus transfection (10% fbs diluted adenovirus to appropriate concentration: 2ul 1X 10 was added to 2ml 10% FBS 9 Adenovirus of PFU).
2) The transfection reagent was removed 12 hours after transfection and cells were cultured with 10% FBS medium.
3) Dynamically recording adenovirus transfection process by using a living cell workstation 1-24h after transfection;
4) The transfection conditions and cell morphology were observed 24 hours after transfection using a fluorescence microscope, and the transfection efficiency and cell viability were also detected using a flow cytometer.
D. Plasmid transfection of chondrocytes cultured in vitro
1) Cells were inoculated one day in advance (33 mm glass bottom dish 1X 10 cells) 6 ) Cells are fused to 70-80% and plasmid transfection can be performed.
2) Mu.l of opti-MEM was added to 7.5. Mu.l of lipofectamine 3000, and the mixture was stirred briefly and then allowed to stand at room temperature for 5 minutes.
3) Mu.l opti-MEM was added to 5. Mu.g of plasmid DNA and 10. Mu. l P-3000 (2. Mu.l per. Mu.g) and the mixture was stirred and stirred for 5 minutes at room temperature.
4) Blowing and mixing the liquid prepared in the steps 1) and 2), and standing for 10-15 minutes at room temperature.
5) The adherent cells were washed once with opti-MEM, and the liquid prepared in the fourth step was added to 1.5ml opti-MEM, and then added to a petri dish after being blown and mixed uniformly.
6) The transfection reagent was removed 12 hours after transfection and cells were cultured with 10% FBS medium.
7) Dynamically recording plasmid transfection process by using a living cell workstation 1-24 hours after transfection;
8) Cell transfection and cell morphology were observed 24 hours after transfection using a fluorescence microscope, while transfection efficiency and cell viability were measured using a lapse cytometer.
E. Cell flow meter for detecting cell transfection efficiency and cell survival rate
1) 24 hours after transfection, cells of each transfected group were digested with EDTA-free 0.25% trypsin and collected, washed twice with pre-chilled 1 XPBS, resuspended with 1 Xbinding buffer, and diluted to 1X 10 6 Each group was tested for transfection efficiency and cell viability using PE Annexin V Apoptosis Detect Kit (BD Pharmingen) per ml;
2) Setting a blank control group, a fluorescence control group (the m-CQDs fluorescence control group, a GFP-plasmid transfection group and a GFP-adenovirus transfection group) according to the using instruction of the kit, a cell single-transfection PE-Annexin V group, a cell single-transfection 7-AAD group and each detection group;
3) 100ul of cell suspension (1X 10) was taken per group 5 The cells) and the blank control group and the fluorescent control group are not added with dye, 5ul of PE-Annexin V dye is added into the cell single-dyeing PE-Annexin V group, 5ul of 7-AAD dye is added into the cell single-dyeing 7-AAD group, and 5ul of PE-Annexin V dye and 7-AAD dye are added into each detection group;
4) Mixing the mixture uniformly by a blowing pipe, and incubating the mixture at room temperature in a dark place for 15 minutes;
5) 400ul of 1 Xbinding buffer was added to each group, and each group was examined for transfection efficiency and cell viability by flow cytometry over 1 hour.
F. Dynamic cell analysis technique (RTCA) cell proliferation assay
Cell suspension preparation:
1) Sucking old culture solution in each group of cell culture dishes in an ultra-clean workbench under the aseptic condition;
2) The cells were washed 1-2 times with PBS, and 1ml (T75 flask) of trypsin solution containing EDTA was added to the dish. After the cover is covered and incubated in a temperature box at 37 ℃ for 2-6 minutes, observing the condition that the cells are digested under an inverted microscope, if the cytoplasm is retracted, the cells intermittently increase, and stopping the digestion;
3) The digestate was gently removed, added to a 10ml culture flask, and the adherent cells were gently swirled repeatedly with a pipette to form a cell suspension. The cell suspension was transferred to a 15ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant removed, fresh medium added and the cells were blown up with a pipette. Counting cell suspension concentrations with a counting plate;
G. E-Plate 96 preparation: 50 μl of medium was added to the wells of E-Plate 96; placing the E-Plate 96 onto the RTCA Station; the RTCA system automatically scans ("Scan Plate") to check if the contact is good (display Connection OK on the "Message" page); starting to detect baseline (Background), determining that selected wells are in normal contact, and Cell Index for all wells is below 0.063; taking out the E-Plate 96, adding 100 mu l of cell suspension which is uniformly mixed into the holes, and enabling the number of cells in each hole to be 5,000 cells/100 mu l; and (3) injection: after the cells are added into the E-Plate 96, the cells and the original culture medium in the holes are not required to be uniformly mixed; E-Plate 96 was placed in an ultra clean bench for 30 minutes at room temperature; placing the E-Plate 96 onto an RTCA Station in an incubator; step2 (overnight assay of cell proliferation curve) was started after the system automatically scanned "Scan Plate".
As a result of comparing the in-vitro fluorescence transfection reagent plasmid-GFP and adenovirus-GFP for m-CQDs with common chondrocytes, it was found that the m-CQDs were transfected for a short period of time compared with plasmid-GFP and adenovirus-GFP, and that significant nuclear imaging was seen in 30 minutes, whereas plasmid-GFP and adenovirus-GFP transfected groups were able to detect a small amount of fluorescence signal at 12 hours after transfection, until reaching the maximum transfection efficiency at substantially 24 hours after transfection.
The results of the transfection efficiency of each group were measured using a cell flow cytometer and are shown in FIG. 6, which shows that the m-CQDs transfection group was approximately 91.76%, approximately 100%, significantly higher than the plasmid-GFP transfection group (approximately 30.34%) and the adenovirus transfection group (approximately 85.62%).
2. The results of the biocompatibility test show that m-CQDs have better biocompatibility than plasmid and adenovirus transfection groups, but when chondrocytes are transfected in vitro, a certain degree of cytotoxicity exists due to overlarge concentration, and low-concentration (0.05 mug/ml, 0.025 mug/ml) transfection is a better choice.
Cell viability of each group was measured using cell flow detection techniques and the results showed that: 24h post-transfection, the m-CQDs transfected group had a cell viability of approximately 89.37% which was significantly higher than that of the plasmid-GFP transfected group (approximately 77.3%) and the adenovirus transfected group (approximately 52.02%).
To further clarify the effect of m-CQDs on chondrocyte proliferation, isolated cultured chondrocytes were transfected with different concentrations of m-CQDs (0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.05. Mu.g/ml, 0.025. Mu.g/ml), respectively, and the proliferation of chondrocytes was examined from 0 to 48 hours after transfection using real-time label-free dynamic cell analysis (Real time cell analysis, RTCA), which showed that: as the transfection concentration of m-CQDs increased, the cell proliferation capacity gradually decreased, and the low concentration group (0.05. Mu.g/ml, 0.025. Mu.g/ml) showed a cell proliferation close to that of the completely normal group.
To further clarify the effect of m-CQDs on chondrocytes, isolated cultured chondrocytes were transfected with different concentrations of m-CQDs (0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.05. Mu.g/ml, 0.025. Mu.g/ml), respectively, and the morphology of 4d chondrocytes after transfection was observed using a living cell workstation, which showed that: the morphology of chondrocytes was significantly abnormally altered in two high concentration groups (0.5. Mu.g/ml, 0.25. Mu.g/ml) and the morphology of chondrocytes was normal in the low concentration group (0.05. Mu.g/ml, 0.025. Mu.g/ml).
The results of the above experiments also demonstrate that m-CQDs are metabolizable in chondrocytes cultured ex vivo, with a metabolic cycle associated with transfection concentrations, the greater the transfection concentration the longer the metabolic cycle, and in this experiment the two low concentration groups (0.05. Mu.g/ml, 0.025. Mu.g/ml) were metabolised for about 48 hours.
3. Targeting and fluorescent signal detection: the detection result of the living cell workstation shows that the chondrocytes can be successfully transfected within 30 minutes by different concentrations of m-CQDs, and obvious nuclear imaging appears; and the m-CQDs can excite fluorescent signals of three different wavelengths of green, red and blue, and the fluorescent signals are stable.
Example 4: in order to detect whether the m-CQDs can penetrate through the compact structure of the articular cartilage surface layer, the m-CQDs are respectively cultured by using a complete culture medium containing 10% FBS and m-CQDs with the concentration of 0.025 mug/ml, paraffin embedding and slicing are carried out after 48 hours, the distribution of the m-CQDs in cartilage tissue blocks is observed by using a fluorescence microscope, and the mechanical properties of the cartilage tissue blocks are detected by using a biological nanoindenter to detect the biocompatibility of the m-CQDs.
1. Human cartilage tissue mass culture: as shown in fig. 11
Human cartilage tissue block extraction and culture process
1) Knee joint tissue after joint replacement, selected from relatively normal parts, was treated to about 4mm in a mixture of DMEM and mixed antibiotic (1%) 3 Left and right cartilage tissue pieces.
2) The cartilage tissue mass DMEM was washed once and the wash was discarded.
3) Control groups were cultured human cartilage tissue blocks in six well plates using 10% FBS medium (10% FBS formulation: 90ml DMEM/F12 was added to 10ml FBS).
4) The m-CQDs group cultures human cartilage tissue pieces in six well plates using 0.025. Mu.g/m lm-CQDs medium (0.025. Mu.g/m lm-CQDs formulation: 2ml of 10% FBS was added with 5ul of 10. Mu.g/ml m-CQDs
5) Culturing for 48 hours without changing liquid, and respectively taking each group of cartilage tissue blocks after 48 hours for paraffin embedding and mechanical property detection.
2. Paraffin embedding, slicing and fluorescent microscope observation of human cartilage tissue blocks
1) After 48 hours of incubation, the human cartilage tissue blocks were fixed with 10% formalin for 72 hours.
2) After fixation, tissues were decalcified in a decalcifier for 2 months using Richman-Gelfand-Hill decalcification solution.
3) The tissue is then embedded in a separate embedding cassette, which embeds the tissue with an embedder.
4) Using a paraffin microtome, 10 adjacent sections were collected at 0, 100 and 200 μm intervals, and two consecutive 6 μm thick sections per interval were used for safranin-O staining and DAPI nuclear staining, respectively.
The observation under the fluorescence microscope revealed that m-CQDs can smoothly enter deep chondrocytes through the dense structure of the surface layer of cartilage tissue (FIG. 12).
3. Biological nanometer indentation instrument (Piuma) for detecting biomechanical property of tissue block
1) Sample preparation: the sample is fixed on the glass culture dish by using the bio-glue to ensure that the sample cannot move during indentation. The thickness of the sample is not too thin, at least 5um or more, and the indentation depth is not more than 10% of the thickness of the sample.
2) Probe selection: and selecting a proper probe according to the elastic range of the sample, wherein the rigidity of the probe used in the research is 5.17N/m, and the diameter of the probe is 25 mu m.
3) And installing a probe, starting an interferometer, a controller and detection software required for detection, and calibrating the optical signal and the probe.
4) The indentation parameters (displacement 10 [ mu ] m, speed 18 [ mu ] m/s, loading force 80% of maximum loading force) are set, the single-point test mode is used for detecting the mechanical properties of the tissue, and the results are expressed in the form of Young's modulus values.
The biological nano-indentation detection results are shown in FIG. 13, and the results show that compared with the control group, the Young modulus of the cartilage tissue of the low-concentration m-CQDs (0.025 mug/ml) has no obvious change, and the low-concentration m-CQDs (0.025 mug/ml) has no obvious toxic effect on the cartilage tissue.
Example 5: m-CQDs in vivo experimental study: about 200g of adult male SD rats are selected, 40ul m-CQDs (10 mug/ml) are injected into joint cavities, and the distribution, metabolism and biocompatibility of m-CQDs in cartilage tissues are observed in vivo by using a small animal fluorescence living imaging system (FMT), a frozen section technology, safranin-fast green staining and liver and kidney function detection.
1. Rat joint cavity injection: adult SD rats (180-220 g) were selected and anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital at a dose of 1ml/100 g; conventional 2% iodine, 75% alcohol (deiodination) three injection site sterilization; taking 40ul (10 mug/ml) m-CQDs to inject joint cavity into right hind limb of anesthetized rat, injecting sterile 1 XPBS in the same way into control group, locating penetration point during injection, and penetrating needle (avoiding main blood vessel and nerve); after injection, the needle opening is pressed by sterilized cotton, and the needle is pulled out to cover sterilized gauze or hemostatic plaster; after injection, the knee joint is moved for several times, so that the medicine is uniformly distributed in the joint cavity.
2. Small animal fluorescence living imaging systems (FMT) detect m-CQDs fluorescent signals in the joint cavity: starting the system; starting an FMT machine power supply and a computer; about 30 seconds after the computer is started, the trueQuant software is double-clicked, and a starting program (Reconstruction Queue background rebuilding program is automatically started along with the trueQuant software, and an icon of the program is hidden at the right lower corner of a Windows system desktop, if the program is not automatically started, the program needs to be manually added when the background rebuilds data).
Animal preparation: the rats are anesthetized in advance, and the hair in the detection area is removed for detection in the same way; taking an animal imaging box, synchronously rotating two side knobs by two hands to open the imaging box, placing a rat in the imaging box, and reasonably adjusting the position of the rat according to a scanning and shooting area; the two hands synchronously rotate the two side knobs until the rats are just fixed, the thickness value of the rats can be obtained from the degrees of the knobs, the imaging box is slowly pushed in, and the inner imaging box cabin door and the machine outer cabin door are closed.
Photographing parameter setting: establishing a Database under the Experiment function label, then establishing a new Study, and establishing a new Group under the Study path; in establishing the Group, subjects values were input according to the number of experimental rats, while the channel of Agents and the type of fluorescent probe were selected.
Scanning and image acquisition: after establishing names and photographing parameters in the expert tab page, clicking to enter the Scan tab page; selecting a Group and a Subject from the Select Subjects; during fluorescence three-dimensional imaging scanning, reasonably selecting a scanning area according to fluorescence signals and experimental purposes, wherein the scanning area at least comprises 35-75 scanning points, the single scanning point is not more than 120, the density of the scanning points is adjusted after Advanced clicking, the value of a Cassette Depth is ensured to be consistent with the value of knobs on two sides of a rat imaging box, options Add toReconstruction Queue are checked, scan is clicked, and an FMT system immediately starts fluorescence scanning; after the scanning is finished, the Reconstruction Queue software part automatically calculates and reconstructs the scanning data in three dimensions in the background.
Data analysis: entering an Analysis tag page, selecting scan Data to be analyzed in Data selection, and opening by double-clicking; displaying the three-dimensional imaging result through the 3D Subject; the transparency, the probe concentration display, the fluorescence volume display, the projection of each section and the color level of the three-dimensional structure are adjusted by adjusting each threshold value in an Analysis tag page; and (3) performing circle selection on fluorescent signals to be quantified by utilizing the ROI circle selection function of the upper left corner in the Analysis label page, and displaying accurate quantitative information such as the volume of the corresponding signals in a quantitative data window below the page.
3. Frozen tissue sections: killing the rat by adopting a cervical dislocation method 24 hours and 48 hours after the joint cavity injection, separating the complete knee joint, and reserving the distal femur, proximal tibia and soft tissues around the joint; immersing the specimen in 50% sucrose, transferring to a liquid nitrogen tank port for quick freezing after sinking; embedding tissues by using an OCT frozen section embedding agent, transferring a specimen into a cold chamber of a frozen section machine, and pre-cooling the temperature in the cold chamber and the temperature of a blade to-25 to-20 ℃ in advance; after the knee joint sample is completely embedded by the embedding agent, the knee joint sample is placed in a cold room for 30 minutes, so that the temperature in the cold room is reduced; transferring the embedded sample to a sample fixing device, adjusting the position to enable the long axis of the knee joint to be parallel to the long axis of the blade, and screwing a screw to firmly fix the sample so as to avoid loosening in the slicing process; and the disposable blade is used for slowly slicing, so that speed consistency in the slicing process is ensured. The blade slowly moves forward and lifts the film at the same time until the tissue is cut, and the film is completely lifted and placed on the glass slide; slicing, namely transferring the slice to room temperature before observation, melting the embedding agent, and immersing the slice in 100% ethanol for 10 minutes before observation to remove bubbles; the sections were placed on slides and blocked with coverslips and the fluorescence signal of the sections was observed under a fluorescence microscope.
4. Safranin-fast green staining:
the rats are sacrificed by cervical dislocation after 48 hours of joint cavity injection, the complete knee joint is separated, and the distal femur, proximal tibia and soft tissues around the joint are reserved; after fixing the tissue with 10% formalin for 72 hours, the tissue was decalcified in a decalcifier for 2 months using Richman-gelfan-Hill decalcification solution, after which the tissue was embedded in a separate embedding cassette using an embedding machine. 10 adjacent sections were collected using a paraffin microtome at 0 μm, 100 μm and 200 μm intervals, with two consecutive 6 μm thick sections per interval being used for safranin-fast green staining.
1) Slicing 6 μm thick paraffin, and baking at 60deg.C for 30-60 min.
2) Dewaxing to water: sequentially placing paraffin sections in xylene I, xylene II and 100% ethanol I, standing for 10 min, sequentially placing paraffin sections in 100% ethanol II, 95% ethanol, 80% ethanol, 70% ethanol and dH 2 And standing in O for 5 minutes.
3) Sucking 50-100 μl of 0.02% fast green staining solution to the position of the cartilage tissue of paraffin section, and discarding the fast green staining solution after 1 min.
4) Sucking 50-100 μl of 1% acetic acid solution to the position of paraffin section cartilage tissue, separating color for 10-15 seconds, and discarding acetic acid solution.
5) Sucking 50-100 μl of 0.2% safranin-O staining solution to the position of cartilage tissue of paraffin section, and discarding safranin-O staining solution after 2 min.
6) Dehydrating: paraffin sections were placed in 95% ethanol I, 95% ethanol II, 100% ethanol I, 100% ethanol II, xylene I and xylene II in this order, and left to stand for 5 minutes for dehydration.
7) The oily tablet is sealed and observed under a lens.
5. Mankin's scoring criteria: as shown in table 1.
Table 1: mankin's scoring criteria
The Mankin's scores are four items in total, and the individual scores are accumulated to be the final score.
6. Liver and kidney function detection: after 48 hours of joint cavity injection, about 2ml of venous blood is reserved, the room temperature is 1200 rpm, the blood is centrifuged for 10 minutes, the upper serum is sucked, and the biological index reflecting the liver function in the serum is detected by using a full-automatic biochemical detector: glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), glutamic-oxaloacetic transaminase ratio (AST/ALT), total Bilirubin (TBIL), direct Bilirubin (DBIL), indirect Bilirubin (IBIL), and biological indicators reflecting kidney function: UREA (UREA) and creatinine (Cr).
The FMT test results are shown in fig. 14, which shows that: after 24 hours of joint cavity injection, a distinct fluorescent signal was detected, which was significantly attenuated at 48 hours after injection compared to 24 hours.
The fluorescence signals of m-CQDs in the articular cartilage tissue were observed under a fluorescence microscope after taking the articular cartilage tissue 24 hours and 48 hours after the articular cavity injection, and the results are shown in FIG. 15, and the results show that: the m-CQDs can penetrate through the cartilage tissue surface compact tissue to enter the cartilage cells, and the fluorescence signal is obvious at 24 hours after injection and is obviously weakened at 48 hours after injection.
As shown in FIG. 16, the safranin-fast green staining results showed that low concentrations of m-CQDs (0.025. Mu.g/ml) had no significant toxic effect on cartilage tissue.
As shown in fig. 17, the liver and kidney function detection result shows that: low concentrations of m-CQDs (0.025. Mu.g/ml) had no significant systemic toxic effects on rats.
The in-vitro transfection flow of the nitrogen-doped carbon quantum dot is simple, the transfection speed is high (30 minutes), the transfection efficiency is high (close to 100%), the biocompatibility is high, the targeting is achieved, the fluorescent signal is clear and stable and is convenient for tracing, and the carbon quantum dot can smoothly enter the chondrocyte through the compact structure of the surface layer of the cartilage tissue due to the small volume (4-5 nm), so that the carbon quantum dot overcomes the defects of the existing delivery system, and has great application value in the mechanism research and clinical diagnosis of the cartilage disease.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Reference is made to:
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3、Benjamin Abramoff, Franklin E Caldera. Osteoarthritis: Pathology, Diagnosis, andTreatment Options.Med Clin North Am. 2020 Mar;104(2):293-311.
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Claims (3)
1. An application of a nitrogen-doped carbon quantum dot delivery system in preparing a fluorescence kit for marking chondrocytes, which is characterized in that: the nitrogen-doped carbon quantum dot delivery system m-CQDs is: o-phenylenediamine reacts at high temperature and high pressure to generate nitrogen-doped carbon quantum dots; the preparation method comprises the following steps: (1) 300mg of o-phenylenediamine is dissolved in 10ml of deionized water, and the solution is magnetically stirred at room temperature to form a clear and transparent solution; (2) Adding the solution into a polytetrafluoroethylene high-pressure reaction kettle, heating a muffle furnace to 180 ℃, reacting for 12 hours, and naturally cooling to room temperature to obtain carbon quantum dots; (3) Filtering the obtained carbon quantum dot 0.22 mu m filter membrane, and dialyzing 24h by using a dialysis bag with molecular weight cut-off of 3500 Da; (4) And freeze-drying the dialyzed carbon quantum dots, and dissolving the dialyzed carbon quantum dots in deionized water to obtain the nitrogen-doped carbon quantum dot delivery system.
2. The use according to claim 1, characterized in that: the application of the nitrogen-doped carbon quantum dot in the fluorescent kit for marking the cell nucleus of the chondrocyte.
3. The use according to claim 1, characterized in that: the concentration of the carbon quantum dots in deionized water is 0.05 mug/ml or 0.025 mug/ml.
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| CN110791289A (en) * | 2019-10-21 | 2020-02-14 | 天津科技大学 | Nitrogen-phosphorus-doped biomass-based carbon quantum dot, application and method for cell imaging detection by using nitrogen-phosphorus-doped biomass-based carbon quantum dot |
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| CN110791289A (en) * | 2019-10-21 | 2020-02-14 | 天津科技大学 | Nitrogen-phosphorus-doped biomass-based carbon quantum dot, application and method for cell imaging detection by using nitrogen-phosphorus-doped biomass-based carbon quantum dot |
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