CN113855808A - 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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- CN113855808A CN113855808A CN202111053714.5A CN202111053714A CN113855808A CN 113855808 A CN113855808 A CN 113855808A CN 202111053714 A CN202111053714 A CN 202111053714A CN 113855808 A CN113855808 A CN 113855808A
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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 dense structure capable of effectively penetrating through the surface layer of the cartilage tissues and entering cells in the deep layer of the cartilage tissues does not exist in the research of cartilage diseases at present. Dissolving o-phenylenediamine in deionized water, and magnetically stirring at room temperature to form a clear transparent solution; heating the solution in a high-pressure reaction kettle to 180 ℃ for reaction for 12 hours, and naturally cooling to room temperature to obtain carbon quantum dots; filtering with a 0.22 mu m carbon quantum dot filter membrane, and dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500 Da; freeze drying, and dissolving in deionized water to obtain the nitrogen-doped carbon quantum dot delivery system. The kit has good biocompatibility, high targeting property, high transfection efficiency and small volume, can smoothly pass through a compact structure on the surface of the cartilage tissue, and can become an excellent diagnosis method for the diseases of the cartilage tissue; smoothly enters cartilage cells through a shallow compact structure of cartilage tissues, and has great clinical treatment significance.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues.
Background
Articular cartilage diseases are common diseases and frequently encountered diseases seriously harming human health, and the main pathological manifestations are joint swelling, pain, dysfunction and the like, and are one of the main causes of disability of the middle-aged and elderly people [1 ]. The world health organization ranks articular cartilage diseases as the most widely harmful problem to human bodies besides three killers of cardiovascular and cerebrovascular diseases, cancer and diabetes [2 ]. Therefore, it is very necessary to attach importance to and reinforce the research on the articular cartilage diseases, which has positive effects and important realistic significance in improving the quality of life of people, controlling medical expenses, and accelerating the achievement of health care objectives.
Osteoarthritis (OA) is a joint cartilage disease with a high prevalence rate, and the clinically most common OA at present mainly includes 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 articular cartilage damage due to acute trauma directly or progressive degenerative articular cartilage damage due to post-traumatic knee joint tone abnormalities. With the increasing aging degree of the population and the popularization of the national fitness exercise in recent years, the incidence of OA is increasing year by year and the tendency of gradual rejuvenation is appearing [3 ].
The major pathological change in OA, progressive degeneration of cartilage tissue, is classified into 4 levels according to the arthroscopic scoring (Outerbridge score): stage I: the cartilage surface was slightly softened and swollen, but the surface was intact; II stage: superficial ulcer and fibrosis of rough nucleus with diameter of 1 cm; grade III: deep ulcers with a lesion diameter greater than 1cm, but lesions that do not reach the subchondral bone; stage IV: the full-thickness cartilage is stripped and torn, and subchondral bone is exposed [4 ].
The results of the existing studies show that: once the articular cartilage structure is destroyed, both drug therapy and surgical therapy are difficult to repair [5, 6], so early treatment before the cartilage structure is destroyed (I, II grade damage) may become an effective method for treating articular cartilage damage. However, knee cartilage has its unique structural features: mature cartilage tissue is usually divided into superficial, intermediate and deep layers, where the superficial collagen is finely and densely arranged and it is difficult for drugs to penetrate through this layer to achieve the desired therapeutic effect [7 ]. Because early cartilage tissue is less damaged and the compact structure of the surface layer still exists, how to effectively deliver the medicine into the cartilage tissue becomes a key problem to be solved urgently for treating OA in early stage.
The delivery system can accurately, targetedly and effectively deliver substances such as target genes, proteins or medicines to a target part by adopting a multidisciplinary means so as to achieve the aim of improving the bioavailability of the target substances at the target part. Various types of delivery systems have been developed: such as viral-based delivery systems and some non-viral vector delivery systems: such as plasmids, inorganic nanoparticles, cationic polymers, liposomes, and the like. These vectors are easy to be packaged and modified by chemical groups, but the above delivery photosystems all have the disadvantages of poor biocompatibility, single action, poor targeting, low transfection efficiency, large delivery system volume and incapability of tracing, so that good therapeutic effects cannot be achieved [8, 9 ].
The carbon quantum dot is a novel carbon nano material firstly discovered by Xu et al in 2004 when preparing single-walled carbon nanotubes [10 ]. With the continuous and intensive research on the carbon nanodots in recent years, researchers find that the carbon nanodots are zero-dimensional materials with the diameter of less than 10 nm, have excellent biocompatibility, are rich in surface groups, are easy to chemically modify, and have photoexcitation, electrochemiluminescence characteristics and stability, the excellent characteristics enable the carbon nanodots to integrate multiple functions, realize the functions of targeting, imaging, tracing, photothermal therapy, drug loading (gene/protein) or controlled drug release (gene/protein) and the like, are important novel multifunctional delivery systems, and have great potential application values in various aspects such as biology, medicine, environment, optics, analytical chemistry and the like [11-14 ].
Carbon element is one of the most important elements in living bodies, and forms a plurality of nano carbon materials, and in the nano particles, carbon quantum dots attract attention of the subject group through unique physicochemical properties of a sphere-like 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, fussy, expensive and inefficient 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 and pesticides by a simple, low-cost and mature and complete synthesis method. More importantly, compared with the traditional metal quantum dot, the carbon quantum dot serving as the fluorescent nano material has multiple unique properties of extremely high fluorescent quantum yield, multicolor photoluminescence, easily-modified surface, good light stability, outstanding biocompatibility and the like.
At present, the research of carbon quantum dots in the medical field mainly focuses on the diagnosis and treatment of tumors [15], and relevant reports in the research of cartilage diseases have not been searched yet.
The existing delivery systems have the disadvantages that: poor biocompatibility and large toxic and side effects; the effect is single; poor targeting; the transfection efficiency is low; the delivery system has large volume and cannot smoothly pass through the compact structure on the surface of the cartilage tissue; and the tracing cannot be carried out.
Disclosure of Invention
The invention provides an application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues in order to solve the key problem that no delivery system capable of penetrating a compact structure on the surface layer of cartilage exists at present, and researches the transfection efficiency, biocompatibility, targeting property, fluorescence property and the capability of penetrating the surface layer of the cartilage. The nitrogen-doped carbon quantum dot has the advantages of good biocompatibility, high targeting property, high transfection efficiency, small volume, smooth and compact structure passing through the surface of a cartilage tissue, and capability of tracing the cartilage tissue through a stable fluorescence signal.
The invention is realized by the following technical scheme: the application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues, wherein m-CQDs of the nitrogen-doped carbon quantum dot delivery system are as follows: 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 o-phenylenediamine is dissolved in 10ml deionized water, and the solution is magnetically stirred at room temperature to form clear and transparent solution; (2) adding the solution into a polytetrafluoroethylene high-pressure reaction kettle, heating the solution to 180 ℃ in a muffle furnace, reacting for 12 hours, and naturally cooling the solution to room temperature to obtain carbon quantum dots; (3) filtering the obtained carbon quantum dots by using a 0.22 mu m filter membrane, and then dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500 Da; (4) freeze-drying the dialyzed carbon quantum dots, and dissolving the carbon quantum dots in deionized water to obtain a nitrogen-doped carbon quantum dot delivery system; the nitrogen-doped carbon quantum dot can penetrate a compact structure on the surface layer of the cartilage tissue and effectively deliver biological factors or drugs to cells in the deep layer of the cartilage tissue.
Furthermore, the nitrogen-doped carbon quantum dot is applied to a fluorescence kit for labeling chondrocytes and living tissues.
The nitrogen-doped carbon quantum dot is applied to a fluorescence kit for labeling the cell nucleus of the chondrocyte.
The concentration of the carbon quantum dots in the deionized water is 0.05 mu g/ml or 0.025 mu g/ml.
The nitrogen-doped carbon quantum dots with the cell nucleus targeting function, prepared by the invention, are researched for the delivery effect of the nitrogen-doped carbon quantum dots in chondrocytes and cartilage tissues, and the research results show that: the carbon quantum dot in vitro transfection process is simple, the transfection speed is high (30 minutes), the transfection efficiency is high (close to 100 percent), the biocompatibility is high, the carbon quantum dot can enter cell nucleuses in a targeted mode, fluorescent signals are clear and stable, and the carbon quantum dot is small in size (4-5 nm) and can smoothly enter cartilage cells through a compact structure on the surface layer of cartilage tissues. The nitrogen-doped carbon quantum dots have great application value in mechanism and clinical diagnosis and treatment of cartilage diseases.
The nitrogen-doped carbon quantum dot m-CQDs prepared by the invention have the advantages of simple transfection process, high transfection speed, high transfection efficiency, small cytotoxicity, metabolizability, targeted entry into cell nucleus, clear and stable fluorescence signals 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 fluorescence labeling on chondrocytes, and dynamically recording biological processes such as proliferation, apoptosis, necrosis and the like of the chondrocytes, and the m-CQDs of the nitrogen-doped carbon quantum dots (m-CQDs) carrying detection targets can become an excellent cartilage tissue disease diagnosis method; nitrogen-doped carbon quantum dots (m-CQDs) can smoothly enter cartilage cells through a cartilage tissue shallow compact structure, so that the method can become a new, effective and visual drug delivery method suitable for cartilage tissue diseases such as cartilage injury and the like, 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 dots; 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 absorption spectra of m-CQDs; b, fluorescence excitation spectrum; c, fluorescence emission spectrum; b is fluorescence emission spectrum of m-CQDs under different excitation wavelengths;
FIG. 3 is a view showing dynamic observation of transfection of chondrocytes from 0 to 30 minutes after transfection of m-CQDs transfected group using a live cell workstation; taking pictures under a fluorescence microscope with the picture being 20 times and 10 times;
FIG. 4 is a view showing dynamic observation of transfection of adenovirus-GFP transfected chondrocytes at 0 to 24 hours after transfection using a live cell workstation; taking pictures under a fluorescence microscope with the picture being 20 times and 10 times;
FIG. 5 is a diagram showing dynamic observation of transfection of plasmid-GFP transfected chondrocytes at 0-24 hours after transfection using a live cell workstation; the results of FIGS. 3-5 show: compared with plasmid-GFP and adenovirus-GFP, m-CQDs have high transfection speed, and obvious nuclear imaging can be seen only in 30 minutes (the microscope multiples in the group images are respectively 20 times and 10 times);
FIG. 6 is a graph showing the transfection efficiency of m-CQDs, plasmid-GFP and adenovirus-GFP transfection groups; in the figure: a is a graph of the results of the transfection efficiency of each group detected by a flow cytometer; b is a statistical analysis graph of the flow-type results, the statistical analysis graph takes an m-CQDs group as a control group, and represents that P is less than 0.05 and P is less than 0.001;
FIG. 7 is a graph showing the cell viability of the m-CQDs, plasmid-GFP and adenovirus-GFP transfected groups; in the figure: a is a result graph of the survival rate of cells of each group detected by a flow cytometer; graph B is the statistical analysis graph of the flow result, the statistical analysis graph takes blank control group Con as the control group, which indicates P <0.05, and indicates P < 0.01;
FIG. 8 is a graph showing the cell proliferation potency of the m-CQDs transfected group at different concentrations; in the figure: a is a result graph of cell proliferation capacity of each group detected by an RTCA cell proliferation detector; b, a statistical analysis chart of the RTCA detection result;
FIG. 9 shows the morphological changes of 4d cells after transfection of different concentrations of m-CQDs transfection group under a fluorescence microscope: panel A shows that 4 days after transfection of the high concentration transfection group (passage and liquid change after 24 hours of transfection), the cell morphology of the two high concentration CQDs transfection groups (0.5 mu g/ml and 0.25 mu g/ml) is remarkably abnormal under a fluorescence microscope; b is the cell morphology under the fluorescence microscope of CODs (CODs are completely metabolized) and the cell morphology and growth are normal but no fluorescence signal is generated 4 days after the transfection of low concentration transfection groups (0.05. mu.g/ml and 0.025. mu.g/ml) (passage and liquid change are carried out 24 hours after the transfection); the microscope multiples in the group images are respectively 20 times and 10 times; A. in the diagram B, bright field, green fluorescence and superposition are sequentially performed from left to right;
FIG. 10 is nuclear targeting assay of m-CQDs under a fluorescence microscope at a live cell workstation, showing that obvious nuclear imaging can be seen after transfection of chondrocytes with m-CQDs; the microscope magnification in the group picture is 20 times;
FIG. 11 is a human cartilage groupTissue block culture process and cartilage tissue block selection standard; in the figure: panel a is a source of human cartilage tissue pieces: the relative normal cartilage remained after the joint replacement, and the tissue block treated to 4mm in the in vitro culture3Left and right size; c, performing safranin-fast green staining after paraffin section of a human cartilage tissue block, and selecting cartilage tissues suitable for the experiment through Mankin's scoring (the Mankin's scoring is 0-2 points);
FIG. 12 shows the distribution of m-CQDs in human cartilage tissue blocks under a fluorescence microscope after 48 hours of culture (without changing the fluid) in paraffin-embedded sections: a is the distribution of m-CQDs in the cartilage tissue blocks of the control group and the m-CQDs (0.025 mu g/ml) culture group, wherein green fluorescence represents the m-CQDs, blue fluorescence represents the position of cell nucleus by DAPI staining, a, b and c respectively represent the surface layer, the middle layer and the deep layer of the cartilage tissue, and the result shows that: green fluorescent signals representing the m-CQDs can be observed in chondrocytes at the surface layer, the middle layer and the deep layer of the cartilage tissue of the m-CQDs group, and the m-CQDs are mainly present in cell nuclei; b, performing statistical analysis on green fluorescence signals in the surface layer, the middle layer and the deep layer of the cartilage tissue of the m-CQDs group, and displaying the result: the transfection rate of m-CQDs at each layer is close to 100%, and the result indicates that the m-CQDs can effectively penetrate through compact tissues on the surface layer of the cartilage tissue and enter cartilage cells of deep cartilage tissue;
fig. 13 shows the mechanical properties of each group of cartilage tissues measured by a biological nanoindenter after culturing human cartilage tissue blocks for 48 hours (without changing the liquid), and the results show that: the m-CQDs group had no significant change in mechanical properties compared to the control group (Con.), suggesting: the m-CQDs have no obvious toxic effect on human cartilage tissues;
FIG. 14 is a small animal fluorescence in vivo imaging detection of m-CQDs in vivo fluorescence signal, and the results show that in vivo fluorescence signal can be detected after joint cavity injection of m-CQDs, and the signal gradually decays along with the time; the fluorescence signals in the picture are as follows from outside to inside in sequence: blue-yellow-red respectively represents the intensity of the fluorescence signal, the blue is weakest, the yellow is second, and the red represents the strongest fluorescence signal;
FIG. 15 is a micrograph of the distribution of m-CQDs in cartilage tissue examined by fluorescence microscopy after cryosectioning the cartilage tissue, from left to right, the first column being a micrograph; the second column and the third column are fluorescence micrographs, and the results show that the m-CQDs can penetrate through the compact structure on the surface layer of the cartilage tissue and enter cartilage cells in the deep layer of the cartilage tissue; the m-CQDs fluorescence signal is green and the picture is taken under a 20-fold fluorescence microscope.
FIG. 16 shows that m-CQDs are injected into the knee joint cavity of a rat for 48 hours, paraffin sections of cartilage tissues on the injection side are taken and stained with safranin-fast green, the content of glycosaminoglycan in the cartilage tissues is detected, the red part in the cartilage tissues represents the content of glycosaminoglycan, the darker the color shows that the content of glycosaminoglycan is higher, the result shows that the content of glycosaminoglycan in two groups of cartilage tissues is not obviously different, and the fact that the m-CQDs injected into the joint cavity have no obvious toxic reaction on the local part of the cartilage tissues is prompted.
FIG. 17 shows the results of the liver and kidney function index measurements in the serum of rats in the control group (Con.) and m-CQDs joint cavity injection group, where ALT is glutamic-pyruvic transaminase, AST is glutamic-oxaloacetic transaminase, AST/ALT is glutamic-oxaloacetic transaminase-glutamic-pyruvic transaminase ratio, TBIL is total bilirubin, DBIL is direct bilirubin, IBIL is indirect bilirubin, UREA is UREA, and Cr is creatinine; the results show that the indexes of the liver and kidney functions of two groups of rats have no obvious difference, and the joint cavity injection of the m-CQDs has no obvious systemic toxicity reaction.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Carbon element is one of the most important elements in living bodies, and forms a plurality of nano carbon materials, and in the nano particles, carbon quantum dots attract attention due to unique physicochemical properties such as a quasi-spherical structure with the 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, fussy, expensive and inefficient 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 and pesticides by a simple, low-cost and mature and complete synthesis method. More importantly, compared with the traditional metal quantum dot, the carbon quantum dot serving as the fluorescent nano material has multiple unique properties of extremely high fluorescent quantum yield, multicolor photoluminescence, easily-modified surface, good light stability, outstanding biocompatibility and the like. We therefore chose to build a carbon quantum dot delivery system.
Example 1: the nitrogen-doped carbon quantum dot delivery system is used for carrying out a high-temperature high-pressure reaction on o-phenylenediamine to generate nitrogen-doped carbon quantum dots;
the preparation method comprises the following steps: (1) 300mg o-phenylenediamine is dissolved in 10ml deionized water, and the solution is magnetically stirred at room temperature to form clear and transparent solution; (2) adding the solution into a polytetrafluoroethylene high-pressure reaction kettle, heating the solution to 180 ℃ in a muffle furnace, reacting for 12 hours, and naturally cooling the solution 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 (3) freeze-drying the dialyzed carbon quantum dots, and dissolving the carbon quantum dots in deionized water to obtain the nitrogen-doped carbon quantum dot delivery system (the concentration is 10 mu g/ml).
Example 2: detection of physical and chemical properties of nitrogen-doped carbon quantum dots
(1) Transmission Electron Microscope (TEM)
Diluting the prepared carbon quantum dots, dripping the diluted carbon quantum dots on an ultrathin copper mesh, drying the ultrathin copper mesh, and observing the size, the morphology and the distribution condition of the carbon quantum dots by using a TEM (transmission electron microscope).
The detection shows that: as shown in fig. 1, the m-CQDs prepared were spheroidal nanoparticles having good dispersibility and relatively uniform size, and a particle size distribution graph was obtained by measuring the particle size of 100 carbon quantum dots, and the average size of the carbon quantum dots was calculated to be 4nm and to exhibit a normal distribution.
(2) Fluorescence spectroscopy
The optical characteristics of the carbon quantum dots are analyzed by a fluorescence spectrophotometer, including an excitation spectrum in water, an emission spectrum under different excitation lights and an emission spectrum of the carbon dots under different pH values, and a scanning range of 200-800 nm.
(3) Absorption spectrum
And diluting the prepared carbon quantum dot solution to a proper concentration, detecting the absorption spectrum of the carbon quantum dot by using UV-8000A, and scanning the range of 190-1100 nm.
The UV-VIS absorption spectrum of m-CQDs is shown in FIG. 2A, from which it can be seen that: the carbon dots have very strong absorption bands in an ultraviolet region, an obvious absorption peak can be observed at 236 nm, meanwhile, the carbon quantum dots also have a wider absorption peak at 430 nm of visible light, a picture can be inserted to find that the light yellow carbon quantum dots emit very bright yellow-green fluorescence under the excitation of ultraviolet light, a curve B shows that the carbon quantum dots have an optimal excitation wavelength of 423 nm and correspond to an ultraviolet-visible spectrum, and the strongest fluorescence emission peak is positioned at 551 nm under the excitation of the wavelength.
Example 3: m-CQDs in vitro transfects chondrocytes, and detects the transfection efficiency, biocompatibility, targeting, fluorescent signal tracking and metabolic condition of the chondrocytes
1. And (3) detecting the transfection efficiency:
A. human chondrocyte extraction and culture process
1) After the joint replacement, knee joint tissues are selected from relatively normal parts and are cut into mud in a mixed solution of DMEM and mixed antibiotics (1%).
2) The crushed cartilage tissue was washed once with DMEM at 1200 rpm for 5 minutes and centrifuged, and the wash solution was discarded.
3) Pronase (2 mg/ml in HBSS formulation with 1% mixed antibiotic) was added to cartilage tissue at 37 ℃ for 30 minutes at 250 times/min.
4) The digestive juice is discarded, DMEM is added to wash the digestive juice once at 1200 rpm, the mixture is centrifuged for 5 minutes, and then the washing liquid is discarded.
5) Collagenase type II (0.1% -0.3%, prepared by 2% FBS culture medium and added with 1% mixed antibiotics) is added into the cartilage tissue and incubated for 6-8 hours at 37 ℃.
6) Blowing and beating the digested cartilage tissue by using a sterile pipette, filtering the cells by using a 75-100 micron filter sieve, centrifuging the filtrate at 1200 rpm for 5 minutes, and taking the cells.
7) DMEM was used to wash the cells 3 times as before.
8) Cells were cultured in 10% FBS medium (10% FBS formulation: 90ml DMEM/F12 was added to 10ml FBS).
B. Transfection of m-CQDs to chondrocytes cultured ex vivo
1) Cell inoculation (1X 10) in 33mm glass-bottom culture dish6) And the cells grow to 70% -80% for m-CQDs transfection, and the specific transfection concentrations are as follows:
0.5. mu.g/ml m-CQDs: 2ml 10% FBS, 100ul of m-CQDs (10. mu.g/ml);
0.25. mu.g/ml m-CQDs: 2ml 10% FBS, 50ul of m-CQDs (10. mu.g/ml) was added;
0.05. mu.g/ml m-CQDs: 2ml 10% FBS, 10ul of m-CQDs (10. mu.g/ml);
0.025. mu.g/ml m-CQDs: to 2ml of 10% FBS was added 5ul of m-CQDs (10. mu.g/ml).
2) Dynamically recording the m-CQDs transfection process by utilizing a living cell workstation 1-30 minutes after transfection;
3) and (3) observing the cell transfection condition and the cell morphology by using a fluorescence microscope 24 hours after transfection, and detecting the transfection efficiency and the cell survival rate by using a flow cytometer.
4) The transfection reagent was removed 24 hours after transfection, cells were cultured in 10% FBS medium, and fluorescence signals (m-CQDs metabolism) and cell morphology were observed at 48 hours and 4d after transfection.
C. Adenovirus transfection of chondrocytes cultured in vitro
1) Cell inoculation (1X 10) in 33mm glass-bottom culture dish6) Cells were grown to 70% -80% for adenovirus transfection (adenovirus was diluted to appropriate concentration with 10% FBS: 2ml of 10% FBS was added to 2ul of 1X 109Adenovirus of PFU).
2) Transfection reagents were removed 12 hours after transfection and cells were cultured by changing to 10% FBS medium.
3) Dynamically recording the adenovirus transfection process by utilizing a living cell workstation 1-24 hours after transfection;
4) and (3) observing the cell transfection condition and the cell morphology by using a fluorescence microscope 24 hours after transfection, and detecting the transfection efficiency and the cell survival rate by using a flow cytometer.
D. Plasmid transfected in vitro cultured chondrocytes
1) Cells were seeded one day in advance (33 mm glass-bottom culture dish seeded with cells 1X 10)6) Cells fused to 70-80% can be transfected with plasmids.
2) Mu.l of opti-MEM was added to 7.5. mu.l of lipofectamine 3000, and the mixture was vortexed briefly and allowed to stand at room temperature for 5 minutes.
3) Mu.g of plasmid DNA and 10. mu. l P-3000 (2. mu.l per. mu.g) were added to 250. mu.l of opti-MEM, and the mixture was mixed by pipetting and allowed to stand at room temperature for 5 minutes.
4) And (3) uniformly blowing the prepared liquid of 1) and 2), and standing at room temperature for 10-15 minutes.
5) Washing adherent cells once by the opti-MEM, adding the prepared liquid in the fourth step into 1.5ml of opti-MEM, blowing, uniformly mixing, and adding into a culture dish.
6) Transfection reagents were removed 12 hours after transfection and cells were cultured by changing to 10% FBS medium.
7) Dynamically recording the plasmid transfection process by utilizing a living cell workstation 1-24 hours after transfection;
8) the cell transfection condition and cell morphology were observed by fluorescence microscopy 24 hours after transfection, and the transfection efficiency and cell viability were examined by an elapsed cell analyzer.
E. Cell transfection efficiency and cell survival rate detection by using cell flow instrument
1) 24 hours after transfection, cells of each transfection group were digested with EDTA-free 0.25% trypsin and collected, washed twice with pre-cooled 1 XPBS, resuspended with 1 XPinding buffer, and diluted to 1 XP106Each group of cells/ml was tested for transfection efficiency and cell survival using PE Annexin V Apoptosis Detect Kit 1 (BD Pharmingen);
2) setting a blank control group, a fluorescence control group (the components are an m-CQDs fluorescence control group, a GFP-plasmid transfection group and a GFP-adenovirus transfection group), a cell single-staining PE-Annexin V group, a cell single-staining 7-AAD group and each detection group according to the instructions of the kit;
3) 100ul of cell suspension (1X 10) was taken per group5Each cell), the blank control group and the fluorescence control group are not added with dye, 5ul of PE-Annexin V dye is added into the PE-Annexin V group singly dyed by the cell, 5ul of 7-AAD dye is added into the 7-AAD group singly dyed by the cell, and 5ul of each PE-Annexin V dye and 7-AAD dye are added into each detection group;
4) blowing and beating the mixture, and incubating for 15 minutes at room temperature in a dark place;
5) binding buffer (400 ul, 1 Xis added to each group), and the transfection efficiency and cell survival rate of each group are detected by a flow cytometer within 1 hour.
F. Dynamic cell analysis technique (RTCA) cell proliferation assay
Cell suspension preparation:
1) absorbing the 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 of EDTA-containing trypsin solution (T75 flask) was added to the petri dish. Covering a cover, placing the cover in a 37 ℃ incubator for incubation for 2-6 minutes, observing the digestion condition of the cells under an inverted microscope, and if cytoplasm retracts, intermittently increasing the cells, and stopping digestion;
3) gently removing the digestive juice, adding the culture medium into a culture bottle with the volume of 10ml, and gently blowing the adherent cells by using a pipette repeatedly to form a cell suspension. The cell suspension was transferred to a 15ml centrifuge tube, centrifuged for 5 minutes at 1000 rpm, the supernatant removed, fresh medium added, and the cells pipetted evenly. Counting the cell suspension concentration with a counting plate;
G. E-Plate 96 preparation: adding 50 microliter of culture medium into a hole of the E-Plate 96; E-Plate 96 is put on RTCA Station; the RTCA system will automatically Scan ("Scan Plate") to see if the contact is good (Connection OK is shown on the "Message" page); starting to detect the baseline (Background), determining that the selected holes are in normal contact, and the Cell Index of all the holes is lower than 0.063; taking out the E-Plate 96, and adding 100 mul of uniformly mixed cell suspension into each hole to enable the number of cells in each hole to be 5,000 cells/100 mul; note: after the cells are added into the E-Plate 96, the cells do not need to be uniformly mixed with the original culture medium in the hole; the E-Plate 96 was placed in a clean bench at room temperature for 30 minutes; E-Plate 96 was placed on the RTCA Station in the incubator; after the system automatically scans for "Scan Plate", Step2 was started (overnight check cell proliferation curve).
The m-CQDs are subjected to sexual comparison with commonly-used chondrocyte in-vitro fluorescence transfection reagents, namely plasmid-GFP and adenovirus-GFP, and the results show that the m-CQDs are short in transfection time compared with the plasmid-GFP and the adenovirus-GFP, obvious nuclear imaging can be seen in only 30 minutes, a small amount of fluorescence signals can be detected only when plasmid-GFP and adenovirus-GFP transfection groups begin to detect 12 hours after transfection, and the maximum transfection rate is basically achieved 24 hours after transfection.
The transfection efficiency of each group was measured by a flow cytometer, and the results are shown in fig. 6, which shows that the transfection efficiency of the m-CQDs transfection group reaches about 91.76%, approaches 100%, and is significantly higher than that of the plasmid-GFP transfection group (about 30.34%) and that of the adenovirus transfection group (about 85.62%).
2. The result of biocompatibility test shows that m-CQDs have better biocompatibility compared with plasmid and adenovirus transfection groups, but when chondrocytes are transfected in vitro, a certain degree of cytotoxicity still exists even if the concentration is too high, and low concentration (0.05 mu g/ml and 0.025 mu g/ml) transfection is a better choice.
The cell survival rate of each group is detected by using a cell flow detection technology, and the result shows that: at 24h after transfection, the survival rate of the m-CQDs transfection group is about 89.37%, which is significantly higher than the transfection rates of the plasmid-GFP transfection group (about 77.3%) and the adenovirus transfection group (about 52.02%).
To further clarify the effect of m-CQDs on chondrocyte proliferation, ex vivo 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 chondrocyte proliferation was examined 0-48 hours after transfection using Real Time Cell Analysis (RTCA), and the results showed that: the cell proliferation ability gradually decreased with the increase of m-CQDs transfection concentration, and the cell proliferation condition of the low concentration group (0.05. mu.g/ml, 0.025. mu.g/ml) was close to that of the completely normal group.
To further clarify the effect of m-CQDs on chondrocytes, ex vivo 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 observed for morphology of 4d chondrocytes after transfection using a live cell workstation, and the results showed that: two high concentration groups (0.5. mu.g/ml, 0.25. mu.g/ml) showed significant abnormal changes in chondrocyte morphology, and the low concentration group (0.05. mu.g/ml, 0.025. mu.g/ml) showed normal chondrocyte morphology.
The results of the above experiments also demonstrate that m-CQDs can be metabolized in chondrocytes cultured ex vivo, the metabolic cycle is related to the transfection concentration, the higher the transfection concentration is, the longer the metabolic cycle is, and the two low concentration groups (0.05. mu.g/ml, 0.025. mu.g/ml) have a metabolic cycle of about 48 hours in the experiment.
3. Detection of targeting and fluorescence signals: the detection result of the living cell workstation shows that the m-CQDs with different concentrations can successfully transfect the chondrocytes within 30 minutes, and obvious nuclear imaging appears; and the m-CQDs can excite three fluorescent signals with different wavelengths of green, red and blue, and the fluorescent signals are stable.
Example 4: in order to detect whether the m-CQDs can pass through the compact structure of the articular cartilage surface layer, a human tissue block is cultured by respectively using a complete culture medium containing 10% FBS and the m-CQDs with the concentration of 0.025 mu g/ml, the section is embedded by paraffin after 48 hours, the distribution of the m-CQDs in the cartilage tissue block is observed by using a fluorescence microscope, and the biocompatibility of the m-CQDs is detected by detecting the mechanical property of the human cartilage tissue block by using a biological nano-indentor.
1. Culturing human cartilage tissue blocks: as shown in fig. 11
Human cartilage tissue block extraction and culture process
1) Knee joint tissue after joint replacement, selecting relatively normal part, and treating to about 4mm in DMEM and mixed antibiotic (1%) mixture3Left and right cartilage tissue mass.
2) The cartilage tissue block was washed once with DMEM and the wash solution was discarded.
3) Control groups human cartilage tissue blocks were cultured in six-well plates using 10% FBS medium (10% FBS formulation: 90ml DMEM/F12 was added to 10ml FBS).
4) m-CQDs group human cartilage tissue pieces were cultured in six-well plates using 0.025. mu.g/m lm-CQDs medium (0.025. mu.g/m lm-CQDs formulation: 2ml 10% FBS 5ul 10 ug/ml m-CQDs)
5) Culturing for 48 hours without changing liquid, and after 48 hours, reserving each group of cartilage tissue blocks for paraffin embedding and mechanical property detection respectively.
2. Human cartilage tissue block paraffin embedding, slicing and fluorescent microscope observation
1) After 48 hours of culture, the human cartilage tissue pieces were fixed with 10% formalin for 72 hours.
2) After fixation, the tissue is decalcified in a decalcifying machine for 2 months by using a Richman-Gelfand-Hill decalcifying liquid.
3) The tissue is then embedded in a separate embedding cassette using an embedding machine.
4) Using a paraffin microtome, 10 adjacent sections were collected at 0 μm, 100 μm and 200 μm intervals, and two consecutive 6 μm thick sections at each interval were used for safranin-O staining and DAPI nuclear staining, respectively.
The observation result under a fluorescence microscope shows that the m-CQDs can smoothly enter deep chondrocytes through a compact structure on the surface layer of the cartilage tissue (FIG. 12).
3. Detection of biomechanical properties of tissue mass by biological nanoindenter (Piuma)
1) Sample preparation: the sample is fixed on a glass culture dish by biological glue to ensure that the sample does not move when being pressed. The thickness of the sample is not required to be too thin, at least 5um is more than, and the indentation depth is not more than 10% of the thickness of the sample.
2) And (3) 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) Installing a probe, starting an interferometer, a controller and detection software required by detection, and calibrating an optical signal and the probe.
4) And (3) setting indentation parameters (displacement of 10 mu m, speed of 18 mu m/s and loading force of 80% of the maximum loading force), detecting the mechanical characteristics of the tissue by using a single-point test mode, and expressing the result in the form of Young's modulus (Yang's modulus) value.
The biological nanoindentation detection result is shown in fig. 13, and the result shows that compared with the control group, the Young modulus of the cartilage tissue has no significant change at low concentration of m-CQDs (0.025 mu g/ml), which indicates that the low concentration of m-CQDs (0.025 mu g/ml) has no obvious toxic effect on the cartilage tissue.
Example 5: in vivo experimental studies of m-CQDs: about 200g adult male SD rat is selected, 40ul m-CQDs (10 mu g/ml) are injected into the joint cavity, and the distribution, metabolism and biocompatibility of the m-CQDs in cartilage tissues are observed in vivo by utilizing a small animal fluorescence living body imaging system (FMT), a frozen section technology, safranin-fast green staining and liver and kidney function detection.
1. Rat articular cavity injection: selecting adult SD rat (180-220 g), and carrying out intraperitoneal injection anesthesia on 0.3% sodium pentobarbital at the dose of 1ml/100 g; sterilizing injection site with 2% iodine tincture and 75% alcohol (deiodination) in three times; injecting 40ul (10 μ g/ml) m-CQDs into the right hind limb of the anesthetized rat via joint cavity, injecting sterile 1 × PBS into the control group by the same method, finding the puncture point during injection, and puncturing the needle (avoiding main blood vessel and nerve); after injection, the needle opening is pressed by the sterilized cotton, and the needle is pulled out and covered by sterile gauze or hemostatic plaster; the knee joint is moved for several times after injection, so that the medicine is uniformly distributed in the joint cavity.
2. Small animal fluorescence Living body imaging System (FMT) in vivo detection of the m-CQDs fluorescence signal in the joint cavity: starting up a system; starting an FMT machine power supply and a computer; and (3) double-clicking TrueQuant software and starting a program about 30 seconds after the computer is started (a reconfiguration Queue background Reconstruction program is automatically started along with the TrueQuant software, an icon of the reconfiguration Queue background Reconstruction program is hidden in the lower right corner of a Windows system desktop, and if the reconfiguration Queue is not automatically started, the reconfiguration Queue background data needs to be manually added).
Animal preparation: anesthetizing the rat in advance, wherein the anesthetizing method is the same as the above, and removing hairs in the detection area for detection; taking an animal imaging box, synchronously rotating knobs at two sides 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 scanning and shooting areas; the knobs on two sides are synchronously rotated by two hands until the rat is just fixed, the thickness value of the rat can be obtained from the degree of the knobs, the imaging box is slowly pushed in, and the door of the internal imaging box and the door of the machine outer cabin are closed.
Setting photographing parameters: establishing Database under the Experiment function label, then establishing a new studio, and establishing a new Group under a studio path; in establishing Group, the Subjects values are input according to the number of experimental rats, and channels and fluorescent probe types of Agents are selected.
Scanning and image acquisition: after a name and a photographing parameter are established in an expert tag page, clicking to enter a Scan tag page; selecting Group and Subject in Select Subjects; carrying out fluorescence three-dimensional imaging scanning, reasonably selecting a scanning area according to a fluorescence signal and an experimental purpose during fluorescence scanning, wherein the scanning area at least comprises 35-75 scanning points, the number of the scanning points is not more than 120, adjusting the density of the scanning points after clicking Advanced to ensure that the numerical value of the Cassette Depth is consistent with the numerical values of knobs at two sides of a rat imaging box, selecting an option of Add to Reconstruction Queue, clicking Scan, and starting fluorescence scanning by an FMT system immediately; after the scanning is finished, the Reconstruction Queue software part automatically calculates and three-dimensionally reconstructs the scanning data in the background.
And (3) data analysis: entering an Analysis tab page, selecting scan Data to be analyzed in Data selection, and opening by double clicking; displaying the three-dimensional imaging result through 3D Subject; adjusting the transparency of the three-dimensional structure, probe concentration display, fluorescence volume display, projection of each section and color gradation by adjusting each threshold value in an Analysis label page; and (3) utilizing the ROI (region of interest) circle selection function at the upper left corner in the Analysis label page to circle the fluorescent signals needing quantification, and displaying accurate quantitative information such as the volume of the corresponding signals in a quantitative data window below the page.
3. Freezing tissue sections: killing rats 24 hours and 48 hours after injection with the joint cavity respectively by adopting a cervical dislocation method, separating complete knee joints, and reserving the distal femur, the proximal tibia and soft tissues around the joints; immersing the specimen in 50% sucrose, and transferring to a liquid nitrogen tank for quick freezing after bottom precipitation; embedding tissues by using an OCT frozen section embedding agent, transferring a sample into a cold chamber of a freezing microtome, 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 medium, placing the knee joint sample in a cold chamber for 30 minutes to reduce the temperature of the knee joint sample in the cold chamber; transferring the embedded sample to a sample fixing device, adjusting the position to enable the knee joint long shaft to be parallel to the blade long shaft, screwing a screw to firmly fix the sample, and avoiding loosening in the slicing process; and a disposable blade is used for slowly slicing, so that the speed is consistent in the slicing process. The blade slowly moves forwards and simultaneously lifts the film until the tissue is cut, and the film is completely lifted and placed on the glass slide; slicing, placing the slices at room temperature before observation, melting the embedding agent, and soaking the slices in 100% ethanol for 10 minutes before observation to remove bubbles; the sections were mounted on glass slides, mounted using coverslips, and the fluorescence signal of the sections was observed under a fluorescence microscope.
4. Safranin-fast green staining:
injecting the joint cavity for 48 hours, using a cervical dislocation method to sacrifice the rat, separating the complete knee joint, and reserving the distal end of the femur, the proximal end of the tibia and soft tissues around the joint; after the tissue was fixed with 10% formalin for 72 hours, the tissue was decalcified in a decalcifying machine using Richman-Gelfand-Hill decalcifying solution for 2 months, 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, and two consecutive 6 μm thick sections at each interval were used for safranin-fast green staining.
1) Slicing paraffin with thickness of 6 μm, placing on a baking sheet machine, baking at 60 deg.C for 30-60 min.
2) Dewaxing to water: placing the paraffin sections in dimethylbenzene I, dimethylbenzene II and 100% ethanol I in sequence, standing for 10 minutes, and then placing the paraffin sections in 100% ethanol II, 95% ethanol, 80% ethanol, 70% ethanol and dH in sequence2And standing in O for 5 minutes.
3) 50-100 mul of 0.02% fast green staining solution is sucked and dropped to the position of the paraffin section cartilage tissue, and the fast green staining solution is discarded after 1 minute.
4) And (3) sucking 50-100 mu l of 1% acetic acid solution and dripping the acetic acid solution to the position where the paraffin section cartilage tissue is positioned, carrying out color separation for 10-15 seconds, and discarding the acetic acid solution.
5) 50-100 mul of 0.2% safranin-O staining solution is sucked and dropped to the position of the paraffin section cartilage tissue, and the safranin-O staining solution is discarded after 2 minutes.
6) And (3) dehydrating: the paraffin sections are sequentially placed in 95% ethanol I, 95% ethanol II, 100% ethanol I, 100% ethanol II, xylene I and xylene II, and are kept stand for 5 minutes for dehydration.
7) The oily mounting medium was mounted for viewing under a light microscope.
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 all the items are scored respectively and then accumulated into a final score.
6. And (3) detecting the liver and kidney functions: after 48 hours of articular cavity injection, leaving about 2ml of venous blood, centrifuging at room temperature of 1200 r/min for 10 minutes, sucking upper serum, and detecting biological indexes reflecting liver functions in the serum by using a full-automatic biochemical detector: glutamate-pyruvate transaminase (ALT), aspartate-oxaloacetate transaminase (AST), glutamate-oxaloacetate transaminase-glutamate pyruvate transaminase ratio (AST/ALT), Total Bilirubin (TBIL), Direct Bilirubin (DBIL), Indirect Bilirubin (IBIL) and biological indicators reflecting renal function: UREA (UREA) and creatinine (Cr).
The FMT test results are shown in fig. 14, showing: after 24 hours of articular cavity injection, an obvious fluorescence signal can be detected, and 48 hours after injection, the fluorescence signal is obviously weakened after 24 hours.
After 24 hours and 48 hours after the joint cavity injection, the joint cartilage tissue is taken, the m-CQDs fluorescence signal in the joint cartilage tissue is observed under a fluorescence microscope after the frozen section is taken, and the result is shown in figure 15 and shows that: the m-CQDs can penetrate through compact tissues on the surface of the cartilage tissue to enter cartilage cells, the fluorescence signal is obvious 24 hours after injection, and the fluorescence signal is obviously weakened 48 hours after injection.
As shown in FIG. 16, 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 results of the liver and kidney function tests showed that: the low concentration of m-CQDs (0.025. mu.g/ml) had no significant systemic toxic effect on rats.
The nitrogen-doped carbon quantum dot has the advantages of simple in-vitro transfection process, high transfection speed (30 minutes), high transfection efficiency (close to 100 percent), high biocompatibility, capability of entering cell nucleus in a targeted mode, clear and stable fluorescence signals and convenience in tracing, and the carbon quantum dot has small volume (4-5 nm) and can smoothly enter chondrocytes through a compact structure on the surface layer of a cartilage tissue, so that the carbon quantum dot makes up the defects of the conventional delivery system, and has great application value in mechanism research and clinical diagnosis and treatment of cartilage diseases.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
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Claims (4)
1. The application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissues is characterized in that: the nitrogen-doped carbon quantum dot delivery system m-CQDs is as follows: 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 o-phenylenediamine is dissolved in 10ml deionized water, and the solution is magnetically stirred at room temperature to form clear and transparent solution; (2) adding the solution into a polytetrafluoroethylene high-pressure reaction kettle, heating the solution to 180 ℃ in a muffle furnace, reacting for 12 hours, and naturally cooling the solution to room temperature to obtain carbon quantum dots; (3) filtering the obtained carbon quantum dots by using a 0.22 mu m filter membrane, and then dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500 Da; (4) freeze-drying the dialyzed carbon quantum dots, and dissolving the carbon quantum dots in deionized water to obtain a nitrogen-doped carbon quantum dot delivery system; the nitrogen-doped carbon quantum dot can penetrate a compact structure on the surface layer of the cartilage tissue and effectively deliver biological factors or drugs to cells in the deep layer of the cartilage tissue.
2. The use of the nitrogen-doped carbon quantum dot delivery system of claim 1 in cartilage tissue, wherein: the nitrogen-doped carbon quantum dot is applied to a fluorescence kit for labeling chondrocytes and living tissues.
3. The use of the nitrogen-doped carbon quantum dot delivery system of claim 1 in cartilage tissue, wherein: the nitrogen-doped carbon quantum dot is applied to a fluorescence kit for labeling the cell nucleus of the chondrocyte.
4. The use of the nitrogen-doped carbon quantum dot delivery system of claim 1 in cartilage tissue, wherein: the concentration of the carbon quantum dots in the deionized water is 0.05 mu g/ml or 0.025 mu g/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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