CN113855808B - Application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissue - Google Patents

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

Application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissue

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to the application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissue.

Background technique

Articular cartilage disease is a common and frequently-occurring disease that seriously endangers human health. Its main pathological manifestations are joint swelling, pain, dysfunction, etc. It is one of the main causes of disability in middle-aged and elderly people [1]. The World Health Organization lists articular cartilage disease as the most widespread problem that harms the human body after the "three major killers" of cardiovascular and cerebrovascular diseases, cancer, and diabetes [2]. Therefore, it is extremely necessary to pay attention to and strengthen research on articular cartilage diseases, which has a positive impact and important practical significance for improving people's quality of life, controlling medical expenses, and accelerating the realization of health care goals.

Osteoarthritis (OA) is an articular cartilage disease with a high prevalence. Currently, the most common clinical OA is primary OA and traumatic OA. Primary OA is also called senile OA, which is the progressive degenerative change of articular cartilage caused by aging. Traumatic OA is articular cartilage damage directly caused by acute trauma or progressive degenerative articular cartilage damage caused by abnormal force on the knee joint after trauma. In recent years, with the increasing aging of the population and the popularization of national fitness exercises, the incidence of OA has increased year by year, and there has been a trend of getting younger [3].

The main pathological change of OA is the progressive degeneration of cartilage tissue. According to the arthroscopic scoring method (Outerbridge score), the degree of cartilage damage can be divided into 4 levels: Level I: the cartilage surface is slightly softened and swollen, but the surface is intact; Level II : Superficial ulcers and fibrosis with rough core with a diameter of less than 1cm; Grade III: Deep ulcers with a diameter of more than 1cm, but the damage does not reach the subchondral bone; Grade IV: Full-thickness cartilage peeling, tearing and other injuries, and subchondral bone is exposed [4].

Existing research results show that once the articular cartilage structure is destroyed, it is difficult to repair regardless of drug treatment or surgical treatment [5, 6]. Therefore, early treatment before the cartilage structure is destroyed (level I, II injuries) may be Become an effective method to treat articular cartilage damage. However, knee joint cartilage has its own unique structural characteristics: mature cartilage tissue can usually be divided into superficial layer, middle layer and deep layer. The superficial layer of collagen is finer and denser, making it difficult for drugs to penetrate this layer to achieve the expected therapeutic effect [7] . Since early cartilage tissue is less damaged and its surface dense structure still exists, how to effectively deliver drugs into cartilage tissue has become a key issue that needs to be solved in the early treatment of OA.

The delivery system can use multidisciplinary means to deliver target genes, proteins, drugs and other substances to the target site accurately, targetedly and effectively, in order to achieve the purpose of improving the bioavailability of the target substance at the target site. Various types of delivery systems have been developed and established: such as virus-based delivery systems and some non-viral vector delivery systems: such as plasmids, inorganic nanoparticles, cationic polymers, liposomes, etc. These carriers are easy to be packaged and modified by chemical groups. However, the above delivery systems all have shortcomings such as poor biocompatibility, single action, poor targeting, low transfection efficiency, large volume of the delivery system and the inability to be traced. Therefore, they cannot achieve many goals. Good therapeutic effect[8,9].

Carbon quantum dots are a new type of carbon nanomaterial first discovered by Xu et al. in 2004 when preparing single-walled carbon nanotubes [10]. In recent years, as their research has continued to deepen, researchers have discovered that carbon nanodots are zero-dimensional materials with a diameter of less than 10 nm. They not only have excellent biocompatibility, are rich in surface groups and are easy to be chemically modified, but also have photoinduced properties. Excitation, electrochemiluminescence properties and stability, these excellent properties allow it to integrate multiple functions into one, while achieving targeting, imaging, tracing, photothermal therapy, drug loading (gene/protein) or controlled drug release (gene/protein) and other functions, it is an important new multifunctional delivery system with huge potential application value in many aspects such as biology, medicine, environment, optics and analytical chemistry [11-14].

Carbon is one of the most important elements in life and constitutes a variety of nanocarbon materials. Among these nanoparticles, carbon quantum dots have a spherical structure with a size less than 10 nm, good dispersion, high water solubility, etc. The unique physical and chemical properties attracted the attention of this research group. Moreover, the construction of carbon quantum dots does not require strict, complex, tedious, expensive and inefficient preparation steps. Through simple, low-cost and mature synthesis methods, it can be made from various common materials such as glucose, wool, various fruits and pesticides. Large-scale production of non-toxic green carbon quantum dots from organic carbon sources. More importantly, as a fluorescent nanomaterial, compared to traditional metal quantum dots, carbon quantum dots have extremely high fluorescence quantum yield, multi-color photoluminescence, easy-to-modify surface, good photostability and outstanding Biocompatibility and many other unique properties.

At present, research on carbon quantum dots in the medical field mainly focuses on the diagnosis and treatment of tumors [15], and no relevant reports on their use in cartilage disease research have been retrieved.

The shortcomings of the existing delivery system are: poor biocompatibility, large toxic and side effects; single effect; poor targeting; low transfection efficiency; the delivery system is large and cannot smoothly pass through the dense structure of the cartilage tissue surface; it cannot be traced .

Contents of the invention

In order to solve the key problem that there is currently no delivery system that can penetrate the dense structure of the cartilage surface, the present invention provides an application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissue, and compares its transfection efficiency, biological phase and The capacitance, targeting, fluorescence properties and ability to penetrate the cartilage surface were studied. The nitrogen-doped carbon quantum dots have good biocompatibility, high targeting, high transfection efficiency, small size, can smoothly pass through the dense structure of the cartilage tissue surface, and can be traced through stable fluorescence signals. A multifunctional chondrocyte/tissue delivery system.

The present invention is realized by the following technical solutions: the application of a nitrogen-doped carbon quantum dot delivery system in cartilage tissue. The nitrogen-doped carbon quantum dot delivery system m-CQDs is: o-phenylenediamine reacts at high temperature and high pressure, Generate nitrogen-doped carbon quantum dots; the specific preparation method is: (1) Dissolve 300mg o-phenylenediamine in 10ml deionized water, stir magnetically at room temperature to form a clear and transparent solution; (2) Add the solution to a polytetrafluoroethylene high-pressure reactor , heat the muffle furnace to 180°C, react for 12 hours, and naturally cool to room temperature to obtain carbon quantum dots; (3) Filter the obtained carbon quantum dots with a 0.22µm filter membrane, and then dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da. 24 h; (4) The dialyzed carbon quantum dots are freeze-dried and dissolved in deionized water to form a nitrogen-doped carbon quantum dot delivery system; the nitrogen-doped carbon quantum dots are effective in penetrating the dense structure of the surface layer of cartilage tissue. Application of delivering biological factors or drugs to cells deep in cartilage tissue.

Further, the nitrogen-doped carbon quantum dots are used in fluorescent kits for labeling chondrocytes and living tissues.

Application of the nitrogen-doped carbon quantum dots in a fluorescence kit for labeling the nucleus of chondrocytes.

The concentration of the carbon quantum dots in deionized water is 0.05 μg/ml or 0.025 μg/ml.

The delivery effect of the nitrogen-doped carbon quantum dots with cell nucleus targeting function prepared by the present invention in chondrocytes and cartilage tissues has been studied. The research results show that the carbon quantum dots have a simple in vitro transfection process and are efficient in transfection. The transfection speed is fast (30 minutes), the transfection efficiency is high (nearly 100%), the biocompatibility is high, it can be targeted into the cell nucleus, the fluorescence signal is clear and stable, and due to the small size of the carbon quantum dots (4-5nm), it can be smoothly Enters the chondrocytes through the dense structure of the cartilage tissue surface. 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 present invention have the advantages of simple transfection process, fast transfection speed, high transfection efficiency, low cytotoxicity, metabolizability, targeting into the cell nucleus, and clear and stable fluorescence signal. It is an excellent biological delivery system; the above characteristics of nitrogen-doped carbon quantum dots (m-CQDs) can fluorescently label chondrocytes and dynamically record biological processes such as chondrocyte proliferation, apoptosis, and necrosis. Nitrogen-doped carbon quantum dots (m-CQDs) m-CQDs carrying detection targets can become an excellent diagnostic method for cartilage tissue diseases; nitrogen-doped carbon quantum dots (m-CQDs) can smoothly enter chondrocytes through the shallow dense structure of cartilage tissue, which may It has become a new, effective and visual drug delivery method suitable for cartilage tissue diseases such as cartilage damage, which has great clinical significance.

Description of the drawings

Figure 1 is a transmission electron microscope image of the prepared carbon quantum dots m-CQDs; in the figure: A is the transmission electron microscope image of the prepared carbon quantum dots; B is the particle size distribution map of the prepared carbon quantum dots;

In Figure 2: A is the UV-visible absorption spectrum of the prepared m-CQDs; In the figure: a: UV absorption spectrum of m-CQDs; b: Fluorescence excitation spectrum; c: Fluorescence emission spectrum; B is under different excitation wavelengths Fluorescence emission spectrum of m-CQDs;

Figure 3 shows the dynamic observation of the transfection status of chondrocytes in the m-CQDs transfection group 0-30 minutes after transfection using a living cell workstation; the pictures are taken under a 20x and 10x fluorescence microscope;

Figure 4 shows the dynamic observation of the transfection status of chondrocytes in the adenovirus-GFP transfection group 0-24 hours after transfection using a live cell workstation; the pictures are taken under a 20x and 10x fluorescence microscope;

Figure 5 shows the dynamic observation of the transfection status of chondrocytes in the plasmid-GFP transfection group 0-24 hours after transfection using a live cell workstation; Figure 3-5 results show: compared with plasmid-GFP and adenovirus-GFP, m -CQDs transfection is fast, and obvious nuclear imaging can be seen in just 30 minutes (microscope magnifications in the group pictures are 20x and 10x respectively);

Figure 6 is a graph showing the transfection efficiency detection of m-CQDs, plasmid-GFP and adenovirus-GFP transfection groups; in the figure: Figure A is a flow cytometer test of the transfection efficiency results of each group; Figure B is a flow cytometry result. Statistical analysis diagram, the statistical analysis diagram uses the m-CQDs group as the control group, * indicates P<0.05, *** indicates P<0.001;

Figure 7 shows the detection of cell survival rate in m-CQDs, plasmid-GFP and adenovirus-GFP transfection groups; in the figure: Figure A shows the results of flow cytometry detection of cell survival rate in each group; Figure B shows the flow cytometry results Statistical analysis chart, the statistical analysis chart uses the blank control group Con. group as the control group, * means P<0.05, ** means P<0.01;

Figure 8 is a graph showing the detection of cell proliferation ability in different concentrations of m-CQDs transfection groups; in the figure: Figure A is a graph showing the results of RTCA cell proliferation detector testing the cell proliferation ability of each group; Figure B is a statistical analysis graph of RTCA detection results;

Figure 9 shows the changes in cell morphology observed under a fluorescence microscope 4 days after transfection in the m-CQDs transfection group with different concentrations: Picture A shows the high-concentration transfection group 4 days after transfection (passage and medium replacement 24 hours after transfection), two A high-concentration CQDs transfection group (0.5 μg/ml and 0.25 μg/ml) showed significant abnormal cell morphology under a fluorescence microscope; Picture B shows a low-concentration transfection group (0.05 μg/ml and 0.025 μg/ml) 4 days after transfection Day (24 hours after transfection and passage and medium change), the cell morphology under the CODs fluorescence microscope shows that the CODs have been completely metabolized, and the cell morphology and growth are normal, but there is no fluorescence signal; the microscope magnifications in the group pictures are 20 times and 10 times respectively; A , In picture B, from left to right are bright field, green fluorescence, and superposition;

Figure 10 shows the nuclear targeting detection of m-CQDs under the fluorescence microscope of the live cell workstation. The results show that obvious nuclear imaging can be seen after m-CQDs are transfected into chondrocytes; the microscope magnification in the group picture is 20 times;

Figure 11 shows the human cartilage tissue block culture process and cartilage tissue block selection criteria. In the figure: Picture A shows the source of the human cartilage tissue block: relatively normal cartilage collected after joint replacement. Picture B shows the tissue block processed into 4mm during in vitro culture. About ³ in size; Picture C shows a human cartilage tissue block paraffin sectioned and stained with safranin-fast green. The cartilage tissue suitable for this experiment was selected based on Mankin's score (Mankin's score 0-2 points);

Figure 12 shows the distribution of m-CQDs in human cartilage tissue blocks after culture for 48 hours (without changing the medium), paraffin-embedded sections, and observation under a fluorescence microscope: Picture A shows the control group and m-CQDs (0.025 μg /ml) The distribution of m-CQDs in the cartilage tissue blocks of the culture group. The green fluorescence in the figure represents m-CQDs, and the blue fluorescence represents the location of the cell nucleus stained by DAPI. a, b, and c represent the surface, middle and deep layers of the cartilage tissue, respectively. The results show that green fluorescence signals representing m-CQDs can be observed in the surface, middle and deep cartilage cells of the cartilage tissue in the m-CQDs group, and m-CQDs mainly exist in the nucleus; Picture B shows the cartilage tissue of the m-CQDs group Statistical analysis of green fluorescence signals in the surface, middle and deep layers showed that the transfection rate of m-CQDs in each layer was close to 100%. This result suggests that m-CQDs can effectively penetrate the surface dense tissue of cartilage tissue and enter the deep cartilage tissue. in chondrocytes;

Figure 13 shows that after culturing human cartilage tissue blocks for 48 hours (without changing the medium), the mechanical properties of the cartilage tissues in each group were tested using a biological nanoindentation instrument. The results showed that compared with the control group (Con.), the mechanical properties of the m-CQDs group were no different. Significant changes indicate that m-CQDs have no obvious toxic effects on human cartilage tissue;

Figure 14 shows the fluorescence in vivo imaging of small animals to detect the in vivo fluorescence signal of m-CQDs. The results show that the in vivo fluorescence signal can be detected after m-CQDs are injected into the joint cavity, and the signal gradually decays over time; the fluorescence signal in the picture From the outside to the inside, they are: blue-yellow-red, which respectively indicate the intensity of the fluorescence signal. Blue is the weakest, followed by yellow, and red indicates the strongest fluorescence signal;

Figure 15 shows the distribution of m-CQDs in cartilage tissue detected by fluorescence microscopy after frozen sectioning of cartilage tissue. From left to right, the first column is a micrograph; the second and third columns are fluorescence micrographs. The results show m-CQDs can penetrate the dense structure of the surface layer of cartilage tissue and enter the chondrocytes deep in the cartilage tissue; the fluorescence signal of m-CQDs is green, and this picture was taken under a 20x fluorescence microscope.

Figure 16 shows a rat knee joint injected with m-CQDs for 48 hours. Paraffin sections of the cartilage tissue on the injection side were taken and stained with safranin-fast green to detect the glycosaminoglycan content in the cartilage tissue. The red part in the cartilage tissue represents the glycosaminoglycan content. , the darker the color, the higher the glycosaminoglycan content. The results showed that there was no significant difference in the glycosaminoglycan content in the cartilage tissue between the two groups, suggesting that m-CQDs injected into the joint cavity had no obvious local toxic effects on the cartilage tissue.

Figure 17 shows the test results of liver function and kidney function indicators in the serum of rats in the control group (Con.) and the m-CQDs intra-articular injection group. In the figure, ALT is alanine aminotransferase, AST is aspartate aminotransferase, and AST/ALT is aspartate aminotransferase alanine aminotransferase. Transaminase ratio, TBIL is total bilirubin, DBIL is direct bilirubin, IBIL is indirect bilirubin, UREA is urea, Cr is creatinine; the results show that there is no significant difference in various indicators of liver and kidney function between the two groups of rats, suggesting Intra-articular injection of m-CQDs has no obvious systemic toxic effects.

Detailed ways

In order to make the purpose, 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. Obviously, the described embodiments are part of the embodiments of the present invention, not All embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

Carbon is one of the most important elements in life and constitutes a variety of nanocarbon materials. Among these nanoparticles, carbon quantum dots have a spherical structure with a size less than 10 nm, good dispersion, high water solubility, etc. The unique physical and chemical properties have attracted attention. Moreover, the construction of carbon quantum dots does not require strict, complex, tedious, expensive and inefficient preparation steps. Through simple, low-cost and mature synthesis methods, it can be made from various common materials such as glucose, wool, various fruits and pesticides. Large-scale production of non-toxic green carbon quantum dots from organic carbon sources. More importantly, as a fluorescent nanomaterial, compared to traditional metal quantum dots, carbon quantum dots have extremely high fluorescence quantum yield, multi-color photoluminescence, easy-to-modify surface, good photostability and outstanding Biocompatibility and many other unique properties. So we chose to build a carbon quantum dot delivery system.

Example 1: Nitrogen-doped carbon quantum dot delivery system, o-phenylenediamine reacts at high temperature and high pressure to generate nitrogen-doped carbon quantum dots;

The specific preparation method is: (1) Dissolve 300mg o-phenylenediamine in 10ml deionized water, stir magnetically at room temperature to form a clear and transparent solution; (2) Add the solution into a polytetrafluoroethylene high-pressure reactor, and heat the muffle furnace to 180 ℃, react for 12 hours, naturally cool to room temperature, and obtain carbon quantum dots; (3) Filter the obtained carbon quantum dots through a 0.22µm filter membrane to remove large particle impurities, and then dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours; (4) The dialyzed carbon quantum dots are freeze-dried and dissolved in deionized water to form a nitrogen-doped carbon quantum dot delivery system (concentration: 10 μg/ml).

Example 2: Detection of physical and chemical properties of nitrogen-doped carbon quantum dots

(1) Transmission electron microscope (TEM)

The prepared carbon quantum dots were diluted, dropped on an ultra-thin copper mesh and allowed to dry. The size, morphology and distribution of the carbon quantum dots were observed using TEM.

Testing showed that: as shown in Figure 1, the prepared m-CQDs were spherical nanoparticles with good dispersion and relatively uniform size. The particle size distribution diagram was obtained by measuring the particle size of 100 carbon quantum dots, and the calculation was The average size of carbon quantum dots is 4nm and exhibits a normal distribution.

(2) Fluorescence spectrum

The optical characteristics of carbon quantum dots were analyzed by fluorescence spectrophotometer, including excitation spectra in water, emission spectra under different excitation lights and emission spectra of carbon dots at different pH, with a scanning range of 200-800nm.

(3) Absorption spectrum

Dilute the prepared carbon quantum dot solution to a suitable concentration, and use UV-8000A to detect the absorption spectrum of the carbon quantum dots, with a scanning range of 190-1100nm.

The UV-visible absorption spectrum of m-CQDs is shown in Figure 2A. It can be seen from the figure that carbon dots have a very strong absorption band in the ultraviolet region, and an obvious absorption peak can be observed at 236 nm. At the same time, carbon quantum dots have There is also a broad absorption peak at 430 nm in visible light. Through the illustration, it can be found that the light yellow carbon quantum dots emit very bright yellow-green fluorescence under ultraviolet light excitation. From curve B, it can be seen that the optimal excitation wavelength of carbon quantum dots is 423 nm. , corresponding to the UV-visible spectrum, under excitation at this wavelength, the strongest fluorescence emission peak is located at 551 nm.

Example 3: m-CQDs were transfected into chondrocytes in vitro, and their transfection efficiency, biocompatibility, targeting, fluorescence signal tracking and metabolism were detected.

1. Transfection rate detection:

A. Human chondrocyte extraction and culture process

1) Select the relatively normal part of the knee joint tissue after joint replacement and chop it into a paste in a mixture of DMEM and antibiotics (1%).

2) Wash the crushed cartilage tissue once with DMEM, centrifuge at 1200 rpm for 5 minutes, and discard the washing solution.

3) Add pronase (2mg/ml, HBSS configuration, 1% mixed antibiotic) to the cartilage tissue at 37°C, 250 times/min, and incubate for 30 minutes.

4) Discard the digestive juice, add DMEM to wash once, centrifuge at 1200 rpm for 5 minutes, discard the washing liquid.

5) Add type II collagenase (0.1% - 0.3%, prepared in 2% FBS medium, add 1% mixed antibiotics) to the cartilage tissue, and incubate at 37°C for 6-8 hours.

6) Puff the digested cartilage tissue with a sterile pipette, filter the cells through a 75-100 micron sieve, centrifuge the filtrate at 1200 rpm for 5 minutes, and collect the cells.

7) Wash the cells 3 times with DMEM, using the same method as before.

8) Culture cells in 10% FBS medium (10% FBS formula: add 90ml DMEM/F12 to 10ml FBS).

B. m-CQDs transfected into cultured chondrocytes in vitro

1) Inoculate cells (1×10 ⁶ ) in a 33mm glass-bottomed culture dish. Cells will grow to 70% - 80% for transfection with m-CQDs. The specific transfection concentration is as follows:

0.5μg/ml m-CQDs: Add 100ul of m-CQDs (10μg/ml) to 2ml 10%FBS;

0.25μg/ml m-CQDs: Add 50ul of m-CQDs (10μg/ml) to 2ml 10%FBS;

0.05μg/ml m-CQDs: Add 10ul of m-CQDs (10μg/ml) to 2ml 10%FBS;

0.025μg/ml m-CQDs: Add 5ul of m-CQDs (10μg/ml) to 2ml 10%FBS.

2) Use a live cell workstation to dynamically record the m-CQDs transfection process 1-30 minutes after transfection;

3) 24 hours after transfection, use a fluorescence microscope to observe the cell transfection status and cell morphology, and use a flow cytometer to detect the transfection efficiency and cell survival rate.

4) Remove the transfection reagent 24 hours after transfection, replace with 10% FBS medium to culture the cells, and observe the fluorescence signal (m-CQDs metabolism) and cell morphology 48 hours and 4 days after transfection.

C. Adenovirus transfection of chondrocytes cultured in vitro

1) Inoculate cells (1×10 ⁶ ) in a 33mm glass bottom culture dish and grow the cells to 70% - 80% for adenovirus transfection (10% FBS dilutes the adenovirus to an appropriate concentration: add 2ul 1×10 to 2ml 10% FBS ⁹ PFU of adenovirus).

2) The transfection reagent was removed 12 hours after transfection and replaced with 10% FBS medium to culture the cells.

3) Use a live cell workstation to dynamically record the adenovirus transfection process 1-24 hours after transfection;

4) 24 hours after transfection, use a fluorescence microscope to observe the cell transfection status and cell morphology, and use a flow cytometer to detect the transfection efficiency and cell survival rate.

D. Plasmid transfection of chondrocytes cultured in vitro

1) Inoculate cells one day in advance (1 × 10 ⁶ cells in a 33mm glass-bottomed culture dish). Cell confluence reaches 70-80%, and plasmid transfection can be carried out.

2) Take 250μl opti-MEM, add 7.5μl lipofectamine 3000, vortex briefly to mix, and let stand at room temperature for 5 minutes.

3) Take 250μl opti-MEM, add 5μg plasmid DNA and 10μl P-3000 (2μl per μg), mix by pipetting and let stand at room temperature for 5 minutes.

4) Mix the liquids prepared in 1) and 2) by pipetting and let stand at room temperature for 10-15 minutes.

5) Wash the adherent cells once with opti-MEM. Add the liquid prepared in step 4 to 1.5ml of opti-MEM, mix by pipetting, and add it to the culture dish.

6) The transfection reagent was removed 12 hours after transfection and replaced with 10% FBS medium to culture the cells.

7) Use a live cell workstation to dynamically record the plasmid transfection process 1-24 hours after transfection;

8) 24 hours after transfection, use a fluorescence microscope to observe the cell transfection status and cell morphology, and use a flow cytometer to detect the transfection efficiency and cell survival rate.

E. Cell flow cytometry to detect cell transfection efficiency and cell survival rate

1) 24 hours after transfection, use EDTA-free 0.25% trypsin to digest and collect the cells in each transfection group, wash the cells twice with pre-cooled 1×PBS, resuspend the cells in 1× binding buffer, and dilute the concentration to 1×10 ⁶ cells/ml, and PE Annexin V Apoptosis Detect Kit 1 (BD Pharmingen) was used to detect the transfection efficiency and cell survival rate of each group;

2) Set the blank control group and fluorescence control group (this group is divided into m-CQDs fluorescence control group, GFP-plasmid transfection group and GFP-adenovirus transfection group) according to the instructions of the kit, and cells are single-stained with PE-Annexin V group, cell single-stained 7-AAD group, and each detection group;

3) Take 100ul of cell suspension (1×10 ⁵ cells) from each group. No dye is added to the blank control group and fluorescent control group. In the PE-Annexin V group, 5ul of PE-Annexin V dye is added to the single-stained cells. The cells are single-stained for 7- 5ul of 7-AAD dye was added to the AAD group, and 5ul of PE-Annexin V dye and 7-AAD dye were added to each detection group;

4) Mix the tube by pipetting, and incubate at room temperature in the dark for 15 minutes;

5) Add 400ul of 1× binding buffer to each group, and use flow cytometry to detect the transfection rate and cell survival rate of each group within 1 hour.

F. Dynamic cell analysis technology (RTCA) cell proliferation detection

Cell suspension preparation:

1) In the ultra-clean workbench, under sterile conditions, suck out the old culture medium in each group of cell culture dishes;

2) Wash 1-2 times with PBS solution, and add 1ml (T75 culture flask) trypsin solution containing EDTA to the culture dish. Cover the lid and incubate it in a 37˚C incubator for 2-6 minutes. Observe the digestion of the cells under an inverted microscope. If the cytoplasm retracts and the cells enlarge intermittently, the digestion is terminated;

3) Gently aspirate the digestion liquid, add 10ml of culture medium/culture flask, and gently pipet the adherent cells repeatedly with a pipette to form a cell suspension. Transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, add fresh culture medium, and pipette the cells evenly. Use a counting board to count the cell suspension concentration;

G. E-Plate 96 preparation: Add 50 µl culture medium to the well of E-Plate 96; place E-Plate 96 on the RTCA Station; the RTCA system will automatically scan (“Scan Plate”) to check whether the contact is good (Connection OK is displayed on the "Message" page); start to detect the baseline (Background), make sure the selected wells are in normal contact, and the Cell Index of all wells is lower than 0.063; take out the E-Plate 96, add 100 µl to the wells and mix evenly Cell suspension, so that the number of cells in each well is 5,000 cells/100 µl; Note: After adding cells to E-Plate 96, there is no need to mix the cells and the original culture medium in the well; place E-Plate 96 in a clean bench Leave it at room temperature for 30 minutes; place E-Plate 96 on the RTCA Station in the incubator; after the system automatically scans "Scan Plate", start Step 2 (overnight detection of cell proliferation curve).

The progress of m-CQDs was compared with that of commonly used chondrocyte in vitro fluorescent transfection reagents plasmid-GFP and adenovirus-GFP. The results showed that compared with plasmid-GFP and adenovirus-GFP, the transfection time of m-CQDs was shorter and only It takes 30 minutes to see obvious nuclear imaging. However, in the plasmid-GFP and adenovirus-GFP transfection groups, a small amount of fluorescent signal can only be detected 12 hours after transfection, and the maximum transfection rate is basically reached 24 hours after transfection. .

The transfection rate of each group was tested using a cell flow cytometer. The results are shown in Figure 6. The results showed that the transfection rate of the m-CQDs transfection group reached approximately 91.76%, close to 100%, which was significantly higher than the plasmid-GFP transfection group (approximately 30.34%) and adenovirus transfection group (approximately 85.62%) transfection rate.

2. Biocompatibility test results show that compared with plasmid and adenovirus transfection groups, m-CQDs have better biocompatibility, but when transfecting chondrocytes in vitro, the concentration is still too high to a certain extent. For the cytotoxic effect, low concentration (0.05μg/ml, 0.025μg/ml) transfection is a better choice.

Cell flow cytometry was used to detect the survival rate of cells in each group. The results showed that 24 hours after transfection, the survival rate of transfected cells in the m-CQDs transfection group was about 89.37%, which was significantly higher than the plasmid-GFP transfection group (about 77.3%) and Transfection rate of adenovirus transfection group (approximately 52.02%).

In order to further clarify the effect of m-CQDs on chondrocyte proliferation, different concentrations of m-CQDs (0.5 μg/ml, 0.25 μg/ml, 0.05 μg/ml, 0.025 μg/ml) were used to transfect in vitro cultured chondrocytes. Real-time label-free dynamic cell analysis (RTCA) was used to detect chondrocyte proliferation 0-48 hours after transfection. The results showed that as the m-CQDs transfection concentration increased, the cell proliferation ability gradually increased. Decreased, the cell proliferation of the low concentration group (0.05μg/ml, 0.025μg/ml) was close to that of the completely normal group.

In order to further clarify the effect of m-CQDs on chondrocytes, different concentrations of m-CQDs (0.5 μg/ml, 0.25 μg/ml, 0.05 μg/ml, 0.025 μg/ml) were used to transfect in vitro cultured chondrocytes, and The living cell workstation was used to observe the morphology of chondrocytes 4 days after transfection. The results showed that the morphology of chondrocytes in the two high-concentration groups (0.5μg/ml, 0.25μg/ml) had obvious abnormal changes, and the morphology of chondrocytes in the low-concentration group (0.05μg/ml ,0.025μg/ml) chondrocyte morphology was normal.

The above experimental results also prove that m-CQDs can be metabolized in in vitro cultured chondrocytes. The metabolic cycle is related to the transfection concentration. The greater the transfection concentration, the longer the metabolic cycle. In this experiment, the two lower The metabolic cycle of the concentration group (0.05μg/ml, 0.025μg/ml) is about 48 hours.

3. Targeting and fluorescence signal detection: Live cell workstation detection results show that m-CQDs of different concentrations can be successfully transfected into chondrocytes within 30 minutes, with obvious nuclear imaging; and m-CQDs can excite green, red, and blue It can produce fluorescence signals of three different wavelengths, and the fluorescence signals are stable.

Example 4: In order to detect whether m-CQDs can penetrate the dense structure of the surface layer of articular cartilage, human tissue blocks were cultured using complete culture medium containing 10% FBS and m-CQDs at a concentration of 0.025 μg/ml, and then embedded in paraffin after 48 hours. Sections were made, and a fluorescence microscope was used to observe the distribution of m-CQDs in the cartilage tissue block. A biological nanoindentation instrument was used to detect the mechanical properties of the human cartilage tissue block and detect the biocompatibility of m-CQDs.

1. Human cartilage tissue block culture: as shown in Figure 11

Human cartilage tissue block extraction and culture process

1) For knee joint tissue after joint replacement, select a relatively normal part and process it into a cartilage tissue block of about ^4mm3 in a mixed solution of DMEM and mixed antibiotics (1%).

2) Wash the cartilage tissue block once with DMEM and discard the washing solution.

3) In the control group, human cartilage tissue blocks were cultured in six-well plates using 10% FBS medium (10% FBS formula: 90 ml DMEM/F12 added to 10 ml FBS).

4) The m-CQDs group used 0.025 μg/m lm-CQDs culture medium to culture human cartilage tissue blocks in six-well plates (0.025 μg/m lm-CQDs formula: 2 ml 10% FBS with 5 ul 10 μg/ml m-CQDs added)

5) Culture for 48 hours without changing the medium. After 48 hours, cartilage tissue blocks from each group will be collected for paraffin embedding and mechanical property testing.

2. Paraffin embedding, sectioning and fluorescence microscopy of human cartilage tissue blocks

1) After 48 hours of culture, human cartilage tissue blocks were fixed in 10% formalin for 72 hours.

2) After fixation, use Richman-Gelfand-Hill decalcification solution to decalcify the tissue in a decalcification machine for 2 months.

3) The tissue is then embedded in a separate embedding box and an embedding machine is used to embed the tissue.

4) Using a paraffin microtome, collect 10 adjacent sections at intervals of 0 μm, 100 μm and 200 μm. Two consecutive 6 μm thick sections at each interval are used for safranin-O staining and DAPI nuclear staining respectively.

Observation results under a fluorescence microscope showed that m-CQDs could successfully pass through the dense structure of the surface layer of cartilage tissue and enter deep cartilage cells (Figure 12).

3. Biological nanoindentation instrument (Piuma) to detect the biomechanical properties of tissue blocks

1) Sample preparation: The sample is fixed on the glass petri dish with biological glue to ensure that the sample does not move during indentation. The thickness of the sample should not be too thin, at least 5um, and the indentation depth should not exceed 10% of the thickness of the sample itself.

2) Probe selection: Select an appropriate probe according to the elastic range of the sample. The probe stiffness used in this study is 5.17N/m, and the probe diameter is 25 µm.

3) Install the probe, turn on the interferometer, controller and detection software required for detection, and calibrate the optical signal and probe.

4) Set the indentation parameters (displacement 10 µm, speed 18 µm/s, loading force 80% of the maximum loading force), use single-point testing mode to detect the mechanical properties of the tissue, and the results are expressed as Young's modulus Expressed in the form of value.

The results of the biological nanoindentation test are shown in Figure 13. The results show that compared with the control group, the Young's modulus of cartilage tissue with low concentration m-CQDs (0.025 μg/ml) has no significant change, indicating that the Young's modulus of cartilage tissue with low concentration m-CQDs (0.025 μg/ml) has no obvious toxic effects on cartilage tissue.

Example 5: In vivo experimental study of m-CQDs: Select adult male SD rats of about 200g, inject 40ul m-CQDs (10 μg/ml) into the joint cavity, and use the small animal fluorescence in vivo imaging system (FMT), frozen section technology, and Red-fast green staining and liver and kidney function tests were used to observe the distribution, metabolism and biocompatibility of m-CQDs in cartilage tissue in vivo.

1. Rat joint injection: Select adult SD rats (180-220g), anesthetize them by intraperitoneal injection of 0.3% sodium pentobarbital at a dose of 1ml/100g; conventional 2% iodine, 75% alcohol (deiodination) three times Disinfect the injection site; take 40ul (10μg/ml) m-CQDs and inject it into the joint cavity of the right hind limb of anesthetized rats. The control group is injected with sterile 1×PBS in the same way. During the injection, identify the puncture point and insert the needle ( Avoid major blood vessels and nerves); after injection, press the needle port with sterile cotton, remove the needle and cover it with sterile gauze or hemostatic patch; move the knee joint several times after injection to evenly distribute the drug in the joint cavity.

2. The small animal fluorescence in vivo imaging system (FMT) detects m-CQDs fluorescence signals in the joint cavity in vivo: power on the system; start the FMT machine power supply and computer; about 30 seconds after the computer starts, double-click the TrueQuant software to start the program (Reconstruction Queue background The reconstruction program automatically starts with the TrueQuant software, and its icon is hidden in the lower right corner of the Windows system desktop. If it does not start automatically, it needs to be added manually when rebuilding data in the background).

Animal preparation: Anesthetize the rat in advance, the anesthesia method is the same as above, and remove the hair in the detection area for detection; take the animal imaging box, rotate the knobs on both sides with both hands to open the imaging box, place the rat in the imaging box, and scan and shoot according to the Reasonably adjust the rat's position in the area; rotate the knobs on both sides with both hands simultaneously until the rat is just fixed. The thickness of the rat can be obtained from the degree at the knob. Slowly push the imaging box in, and close the internal imaging box door and the outer door of the machine. .

Photography parameter settings: Create a Database under the Experiment function label, then create a new Study, and create a new Group under the Study path; when establishing the Group, enter the Subjects value according to the number of experimental rats, and select the Agents channel and fluorescent probe. type.

Scanning and image acquisition: After establishing the name and photographing parameters in the Experiment tab, click to enter the Scan tab; select Group and Subject in Select Subjects; perform a fluorescence three-dimensional imaging scan. During fluorescence scanning, the fluorescence signal and the purpose of the experiment are reasonable. Select the scanning area. The scanning area contains at least 35-75 scanning points and no more than 120 scanning points. Click Advanced to adjust the density of the scanning points to ensure that the value of Cassette Depth is consistent with the value of the knobs on both sides of the rat imaging box. Consistent, check the option Add toReconstruction Queue, click Scan, and the FMT system will immediately start fluorescence scanning; after the scan is completed, the Reconstruction Queue software part will automatically calculate and three-dimensionally reconstruct the scanned data in the background.

Data analysis: Enter the Analysis tab, select the scan data to be analyzed in the Data selection, and double-click to open; display the three-dimensional imaging results through 3D Subject; adjust the thresholds in the Analysis tab to display the transparency of the three-dimensional structure, probe concentration, Adjust the fluorescence volume display, projection of each section, and color scale; use the ROI selection function in the upper left corner of the Analysis tab to select the fluorescence signal that needs to be quantified, and display the volume of the corresponding signal in the quantitative data window at the bottom of the page, etc. Precise quantitative information.

3. Frozen tissue sections: 24 hours and 48 hours after intra-articular injection, the rats were killed by cervical dislocation, the complete knee joint was separated, and the distal femur, proximal tibia and soft tissue around the joint were retained; the specimens were immersed in 50% sucrose. After settling to the bottom, transfer to the mouth of the liquid nitrogen tank for quick freezing; use OCT frozen section embedding agent to embed the tissue, and transfer the specimen to the cold chamber of the freezing microtome. The temperature in the cold chamber and the blade temperature are both pre-cooled to -25 ~ -20℃. ; After the knee joint sample is completely embedded in the embedding agent, place it in the cold room for 30 minutes to allow it to cool down to the temperature in the cold room; transfer the embedded sample to the sample fixture, and adjust the position so that the long axis of the knee joint is longer than the length of the blade With the axes parallel, tighten the screws to securely fix the sample to avoid loosening during slicing; use a disposable blade to slice slowly to ensure consistent speed during slicing. Slowly move the blade forward while lifting the film until the tissue is cut. Completely lift the film and place it on the glass slide. Slice and transfer to room temperature before observation. Melt the embedding agent. Soak the slices in 100% ethanol for 10 minutes before observation. To remove air bubbles; place the sections on a glass slide, cover them with a coverslip, and observe the fluorescence signal of the sections under a fluorescence microscope.

4. Safranin-fast green staining:

The rats were sacrificed by cervical dislocation 48 hours after intra-articular injection, and the complete knee joint was separated, retaining the distal femur, proximal tibia and soft tissue around the joint. After the tissue was fixed with 10% formalin for 72 hours, Richman-Gelfand- Hill decalcifying solution, the tissue is decalcified in a decalcifying machine for 2 months, and then the tissue is embedded in a separate embedding box and the tissue is embedded using the embedding machine. Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm using a paraffin microtome, and two consecutive 6 μm thick sections from each interval were used for safranin-fast green staining.

1) 6μm thick paraffin sections, place them on a baking machine and bake at 60°C for 30-60 minutes.

2) Dewax to water: Place the paraffin sections in xylene I, xylene II and 100% ethanol I for 10 minutes, then place the paraffin sections in 100% ethanol II, 95% ethanol, and 80% ethanol in order. , 70% ethanol and dH ₂ O and let stand for 5 minutes.

3) Pipette 50-100 μl of 0.02% Fast Green staining solution and drop it to the location of the paraffin sectioned cartilage tissue. Discard the Fast Green staining solution after 1 minute.

4) Draw 50-100 μl of 1% acetic acid solution and drop it to the location of the paraffin sectioned cartilage tissue, separate the color for 10-15 seconds, and discard the acetic acid solution.

5) Pipette 50-100 μl of 0.2% Safranin-O staining solution and drop it to the location of the paraffin sectioned cartilage tissue. Discard the Safranin-O staining solution after 2 minutes.

6) Dehydration: Place paraffin sections in 95% ethanol I, 95% ethanol II, 100% ethanol I, 100% ethanol II, xylene I and xylene II in sequence, and let stand for 5 minutes for dehydration.

7) Seal the slides with oily mounting medium for observation under a light microscope.

5. Mankin’s scoring criteria: as shown in Table 1.

Table 1: Mankin’s scoring criteria

There are four Mankin’s scores, each of which is scored separately and then summed up to form the final score.

6. Liver and kidney function test: 48 hours after injection into the joint cavity, take about 2 ml of venous blood, centrifuge at room temperature 1200 rpm for 10 minutes, absorb the upper serum, and use a fully automatic biochemical detector to detect the biology of liver function in the serum. Indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), aspartate aminotransferase alanine aminotransferase 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 Figure 14. The results show that 24 hours after injection into the joint cavity, an obvious fluorescence signal can be detected. 48 hours after injection, the fluorescence signal is significantly weaker than that at 24 hours.

Articular cartilage tissue was taken 24 hours and 48 hours after injection into the articular cavity, and the m-CQDs fluorescence signal in the articular cartilage tissue was observed under a fluorescence microscope after frozen sectioning. The results are shown in Figure 15. The results show that m-CQDs can penetrate The dense tissue on the cartilage tissue surface entered the chondrocytes, and the fluorescence signal was obvious 24 hours after injection, and the fluorescence signal was significantly weakened 48 hours after injection.

As shown in Figure 16, the safranin-fast green staining results show that low concentration of m-CQDs (0.025 μg/ml) has no obvious toxic effect on cartilage tissue.

As shown in Figure 17, the liver and kidney function test results showed that low concentration m-CQDs (0.025 μg/ml) had no obvious systemic toxic effects on rats.

Nitrogen-doped carbon quantum dots have a simple in vitro transfection process, fast transfection speed (30 minutes), high transfection efficiency (nearly 100%), high biocompatibility, can be targeted into the cell nucleus, and the fluorescence signal is clear and stable for easy display. Since the carbon quantum dots are small in size (4-5nm) and can smoothly enter the chondrocytes through the dense structure of the cartilage tissue surface, the carbon quantum dots make up for the shortcomings of the existing delivery system and are useful in the research on the mechanisms of cartilage diseases and clinical diagnosis and treatment. There is great application value in it.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. scope.

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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.