CN110231316B - Preparation of bimodal nanoprobe and labeling and imaging of mesenchymal stem cells by bimodal nanoprobe - Google Patents
Preparation of bimodal nanoprobe and labeling and imaging of mesenchymal stem cells by bimodal nanoprobe Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a method for synthesizing a bimodal nanoprobe, which comprises the steps of calcining mesoporous silica into silica fine powder, and Gd (NO)3)3·6H2Dissolving O in deionized water to form Gd (NO)3)3Dissolving Gd (NO) in the solution3)3Dropwise adding the solution into the fine silicon dioxide powder, stirring in the dropwise adding process, and then drying and calcining to obtain Gd @ SiO2A complex; the method for preparing AIE-Gd @ SiO2The nano-composite has good biocompatibility and low toxicity, can be used as a nano probe to mark mesenchymal stem cells, can realize fluorescence and nuclear magnetic bimodal imaging, and is applied to the experimental research of the stem cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing a bimodal nanoprobe AIE-Gd @ SiO2The fluorescent nuclear magnetic double-mode imaging marker is used for marking mesenchymal stem cells and realizing fluorescent and nuclear magnetic double-mode imaging of the stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of pluripotent stem cells derived from early-developing mesoderm, and have biological characteristics such as multidirectional differentiation potential, improvement and repair of damaged tissues, immune regulation and support of hematopoietic function. Under appropriate circumstances, they can be directionally differentiated into osteoblasts, chondrocytes, cardiomyocytes, adipocytes and the like. A large number of experimental studies have shown that stem cells differentiate to a large extent into osteoblasts and adipocytes, which are interconvertible. This indicates that inhibiting differentiation of mesenchymal stem cells into adipocytes and increasing their osteogenic differentiation is an important measure for preventing or treating bone diseases caused by decreased bone content, and has attracted particular attention in bone tissue engineering. The twenty-first century has had numerous scientific discoveries in stem cell research that have expanded our understanding of cell biology. Stem cell based therapies have a major impact on the treatment and cure of a wide range of transmitted diseases, such as diabetes, neurodegeneration or cardiovascular diseases. Meanwhile, The first example of The in vivo transplantation experiment of stem cell tracheal implantation has succeeded (Macchiarini P.; Jungebruth P.; Go T.; Asnaghi M. A.; Rees L. E.; Cogan T. A.; Dodson A.; Martorell J.; Bellini S.; Parnigoto P. Clinical transplantation of a tissue-engineered air way [ J ]. The Lancet, 2008, 372, 2023-2030.), and The stem cells can be tracked in vivo, so that we can monitor their biological distribution and their survival in tissue organs, and understand The interaction and mode of The stem cells and The recipient's cells after transplantation.
Recent studies have shown that the use of fluorescent Nanoparticles in the biomedical field has attracted considerable research interest over the past decades (Michalet, X.; Pinaud, F.; Bentolala, L. A.; Tsay, J. M.; Doose, S.; Li, J.; Sundaresan, G.; Wu, A. M.; Gambrir, S.; Weiss, S. Quantum Dots for Live Cells in video Imaging, and diagnostics. Science, 2005, 307, 538. Wang, Wang., He, X.; Yang, X.; Shi H. Functionalized silicon Nanoparticles: A fluorescent Nanoparticles for fluorescent Imaging and chemical Imaging, and Z.; light J. 3, S. 1367. Zon. J. for fluorescent Nanoparticles in video Imaging, 41, 1323-1349.). Fluorescent nanoparticles such as: quantum dots, upconversion nanoparticles, and dye-based silica nanoparticles can be used to visualize or monitor in vivo organisms in real time by fluorescence imaging. Among them, Silica Nanoparticles based on dyes have high stability, are easy to surface-modify, have good biocompatibility, and have been systematically studied (Montalti, M.; Prodi, L.; Rampazzo, E.; Zacheroni, N. Dye [1] blue Silica Nanoparticles as luminescennt organic Systems for nanomedicine. chem. Soc. Rev. 2014, 43, 4243-. However, conventional dyes tend to undergo photobleaching and aggregation-induced quenching when incorporated into high concentrations of silica nanoparticles, thereby hindering their further use in biological imaging. Due to the discovery of Aggregation-Induced Emission light sources AIE, different AIE were embedded in silica nanoparticles for efficient biological imaging by means of physical encapsulation or covalent coupling (Luo, J.; Xie, Z.; Lam, J.W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B.Z. Aggregation [1] Induced Emission of 1-Methyl-1,2,3,4, 5-pentaphnylsilole. Chem. 2001, 240. 122. Ding, D. Li, K.; Liu, 2458, B.Z. bioprobe used Based on AIE fluorine Chem, Res 3, 46. Hong 3, 46. W. J.41, Lang.W. 3, Z
Emulsion, Phenomenon, Mechanism and applications, chem, Commun, 2009, 4332-4353). Due to Gd3+Is a common MR molecular imaging signal component, is a paramagnetic material, and compared with a superparamagnetic material, Gd3+The fluorescent material has low light absorption, is not easy to cause fluorescence quenching effect, and can be used as an MR imaging signal component of a dual-mode imaging probe.
Based on the research background, the fluorescent magnetic nanoparticles synthesized by the method have the characteristics of quantum dots and magnetic particles, are wrapped by a silicon shell, have good biocompatibility, can efficiently mark the human adipose mesenchymal stem cells, and can dynamically monitor the marked condition of the mesenchymal stem cells in vitro through a fluorescence imaging and magnetic resonance imaging dual mode, so that the fluorescent magnetic nanoparticles can be used as a safe and ideal stem cell tracer and applied to the experimental research of the stem cells.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to prepare a bimodal imaging biological probe, and the probe has better biocompatibility and lower toxicity, can be used as a probe to mark mesenchymal stem cells, and can realize fluorescent and nuclear magnetic bimodal imaging.
The technical scheme adopted by the invention is as follows: the preparation method of the bimodal nanoprobe comprises the following steps
Step one, calcining mesoporous silicon dioxide into silicon dioxide fine powder, and adding Gd (NO)3)3·6H2Dissolving O in deionized water to form Gd (NO)3)3Dissolving Gd (NO) in the solution3)3Dropwise adding the solution into the fine silicon dioxide powder, stirring in the dropwise adding process, and then drying and calcining to obtain Gd @ SiO2A complex;
step two, dissolving aggregation-induced emission AIE fluorescent dye in acetonitrile solution to form AIE solution, and then dropwise adding the AIE solution to Gd @ SiO2Stirring and mixing uniformly in the compound, drying, adding the compound into a mixed solution of polyvinylpyrrolidone and glycol, stirring uniformly, adding ammonia water with the mass percentage concentration of 25%, adding ethanol and ethyl orthosilicate, washing with acetonitrile to remove AIE dye which is not combined on the surface, and drying the precipitate to obtain AIE-Gd @ SiO2The nano-composite is the bimodal nanoprobe.
In the first step, the amount of mesoporous silica is 0.1-1 g, the amount of silica fine powder formed after calcination is 0.06-0.6 g, and Gd (NO)3)3·6H2O0.06-0.6 g, Gd (NO)3)3The concentration of the solution is 0.4-4 mol/l.
In the second step, 0.02-0.2 g of aggregation-induced emission AIE fluorescent dye is dissolved in 2000 microliter of acetonitrile solution 300-2Adding the compound into a mixed solution of 0.25-2.5 g of polyvinylpyrrolidone and 25ml of ethylene glycol, stirring uniformly, adding 2.5-25ml of 25% ammonia water by mass percent, and then adding 1.25-12.5ml of ethanol and 25-250ul of ethyl orthosilicate.
Firstly, deionized water is used for ultrasonic preparation of 1mg/ml AIE-Gd @ SiO2Aqueous nanocomposite solution, then diluting the aqueous nanocomposite solution to different concentrations with a culture medium, followed by different concentrations of AIE-Gd @ SiO2The nano-complexes were incubated with mesenchymal stem cells for 24h in an incubator, respectively, and then buffered with phosphateThe solution is washed for three times to remove the unophagocytized AIE-Gd @ SiO2Adding a culture medium containing a thiazole blue MTT solution into the nano compound, continuously culturing for 4 hours, adding a dimethyl sulfoxide solution, oscillating at a low speed for 10min, and measuring the light absorption value of each culture hole by using an enzyme-labeling instrument at the wavelength of 490 nm; for cell proliferation experiments, cytotoxicity was measured by incubating for 24h and washing and then culturing for 5d, and measuring absorbance as described above.
Different concentrations of AIE-Gd @ SiO2Incubating the nano material and the stem cells for 24 hours, cleaning, fixing, and observing fluorescence imaging under a laser confocal microscope; for nuclear magnetic imaging, after the cells are incubated with the nano-complexes with different concentrations for 24 hours, the cells are suspended in a PCR tube mixed by a phosphate buffer solution and an agarose solution after digestion and centrifugation by recombinant pancreatin, and the strength of a magnetic signal of the stem cells marked by the nano-complexes in the agarose solution is observed by nuclear magnetic imaging.
Different concentrations of AIE-Gd @ SiO2The nano material is 5 micrograms/ml, 10 micrograms/ml, 20 micrograms/ml, 40 micrograms/ml and 80 micrograms/ml, the phosphate buffer solution is 50-500 microliters, the agarose amount is 0.05g, the mass fraction is 1%, and the volume is 50-500 microliters.
The invention has the beneficial effects that: the method for preparing AIE-Gd @ SiO2The nano-composite has good biocompatibility and low toxicity, can be used as a nano probe to mark mesenchymal stem cells, can realize fluorescence and nuclear magnetic bimodal imaging, and is applied to the experimental research of the stem cells.
Drawings
FIG. 1 is AIE-Gd @ SiO2A Transmission Electron Microscope (TEM) image of the composite;
FIG. 2 is AIE-Gd @ SiO2Cellular activity profile of complex labeled mesenchymal stem cells, wherein the abscissa is AIE-Gd @ SiO2Concentration (ul/ml), ordinate is cell activity (%);
FIG. 3 is a graph of cell proliferation with the abscissa of AIE-Gd @ SiO2Concentration (ul/ml), ordinate is cell proliferation (%), time is 5 days;
FIG. 4 is AIE-Gd @ SiO2Marking laser confocal microscope images of the mesenchymal stem cells by the nano-composite;
FIG. 5 is AIE-Gd @ SiO2Nuclear magnetic imaging of the nanocomposite-labeled mesenchymal stem cells.
Detailed Description
Preparation of bimodal nanoprobe and labeling of mesenchymal stem cells by bimodal nanoprobe
0.1-1 g of mesoporous silica is firstly calcined into fine powder in a muffle furnace at high temperature, and then 30% of gadolinium is compounded. The process is carried out by mixing 0.06-0.6 g gadolinium nitrate hexahydrate [ Gd (NO)3）3·6H2O]Dissolving the solid in deionized water to prepare a solution, then dropwise adding the solution on mesoporous silica powder, uniformly mixing, drying and calcining at high temperature to obtain Gd @ SiO2A complex;
step two, taking 0.02-0.2 g of aggregation-induced emission (AIE) fluorescent dye to be ultrasonically dissolved in 300-2000 microliter of acetonitrile solution, and synthesizing 0.08-0.7 g of Gd @ SiO2And (3) dropwise adding the AIE solution into the surface dish, stirring and mixing uniformly, and drying. Then taking 0.05-0.5 g of the dried compound, simultaneously adding 0.25-2.5 g of polyvinylpyrrolidone and 25ml of ethylene glycol, mixing and stirring for 12h, adding 2.5-25ml (25%) of ammonia water, carrying out ultrasonic treatment for 30min, adding 1.25-12.5ml of ethanol and 25-250ul of ethyl orthosilicate, carrying out ultrasonic treatment for 1h, finally carrying out centrifugal cleaning with acetonitrile to ensure that the supernatant is colorless, and drying to obtain the product AIE-Gd @ SiO2A nanocomposite. In order to ensure that the synthesized nano material has good dispersibility, the compounded nano material is firstly dispersed in a solution prepared from polyvinylpyrrolidone and deionized water, 0.01 to 0.15 g of polyethylene glycol is added, and the dispersibility is observed by ultrasonic wave.
By using the prepared AIE-Gd @ SiO2The compound is used as a nano probe to mark stem cells, and is characterized in that: different concentrations of 5. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 40. mu.g/ml and 80. mu.g/ml of AIE-Gd @ SiO2The nanocomposite was incubated with mesenchymal stem cells for 24h, followed by washing three times with 5-15 ml of phosphate buffer solution to remove non-phagocytized nanoparticles, followed by addition of a culture containing 0.25g of thiazole blue solutionContinuously culturing the culture medium for 4h, absorbing the culture medium, adding 150-; for cell proliferation experiments, cytotoxicity was measured by incubating for 24h and washing and then culturing for 5d, and measuring absorbance as described above.
The prepared labeled stem cells are used for fluorescence and nuclear magnetic bimodal imaging, and the method is characterized by comprising the following steps: different concentrations of 5. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 40. mu.g/ml and 80. mu.g/ml of AIE-Gd @ SiO2Incubating the nano material and the stem cells for 24 hours, cleaning, fixing, and observing fluorescence imaging under a laser confocal microscope; for nuclear magnetic imaging, after the cells are incubated with the nano-complexes with different concentrations for 24h, the cells are suspended in a PCR tube mixed with 50-500 microliter of phosphate buffer solution and 0.05g of agarose solution after digestion and centrifugation by recombinant pancreatin, and the magnetic signal intensity of the stem cells marked by the nano-complexes in the agarose solution is observed by nuclear magnetic imaging. The experimental result shows that the stem cells marked by the nanoprobe can be used for fluorescence and nuclear magnetic bimodal imaging.
Claims (7)
1. A method for preparing a bimodal nanoprobe is characterized in that: the method comprises the following steps
Step one, calcining mesoporous silicon dioxide into silicon dioxide fine powder, and adding Gd (NO)3)3·6H2Dissolving O in deionized water to form Gd (NO)3)3Dissolving Gd (NO) in the solution3)3Dropwise adding the solution into the fine silicon dioxide powder, stirring in the dropwise adding process, and then drying and calcining to obtain Gd @ SiO2A complex;
step two, dissolving aggregation-induced emission AIE fluorescent dye in acetonitrile solution to form AIE solution, and then dropwise adding the AIE solution to Gd @ SiO2The compound is stirred and mixed uniformly, then dried, then added into a mixed solution of polyvinylpyrrolidone and glycol, stirred uniformly, added with ammonia water with the mass percentage concentration of 25%, then added with ethanol and ethyl orthosilicate, and finally washed by acetonitrile to remove the surfaceDrying the precipitate to obtain AIE-Gd @ SiO2The nano-composite is the bimodal nanoprobe.
2. The method of bimodal nanoprobe preparation according to claim 1, characterized in that: in the first step, the amount of mesoporous silica is 0.1-1 g, the amount of silica fine powder formed after calcination is 0.06-0.6 g, and Gd (NO)3)3·6H2O0.06-0.6 g, Gd (NO)3)3The concentration of the solution is 0.4-4 mol/l.
3. The method of bimodal nanoprobe preparation according to claim 1, characterized in that: in the second step, 0.02-0.2 g of aggregation-induced emission AIE fluorescent dye is dissolved in 2000 microliter of acetonitrile solution 300-2Adding the compound into a mixed solution of 0.25-2.5 g of polyvinylpyrrolidone and 25ml of ethylene glycol, stirring uniformly, adding 2.5-25ml of 25% ammonia water by mass percent, and then adding 1.25-12.5ml of ethanol and 25-250ul of ethyl orthosilicate.
4. A method of labeling stem cells with the bimodal nanoprobe prepared by the method of preparing the bimodal nanoprobe of claim 1, wherein: firstly, deionized water is used for ultrasonic preparation of 1mg/ml AIE-Gd @ SiO2Aqueous nanocomposite solution, then diluting the aqueous nanocomposite solution to different concentrations with a culture medium, followed by different concentrations of AIE-Gd @ SiO2The nanocomposites were incubated with mesenchymal stem cells for 24h, respectively, followed by three washes with phosphate buffer solution to remove the unsuphagocytosed AIE-Gd @ SiO2And (3) adding the nano compound, then adding a culture medium containing thiazole blue MTT solution, continuing to culture for 4 hours, adding dimethyl sulfoxide solution, oscillating at a low speed for 10min, and measuring the light absorption value of each culture well by using a microplate reader at the wavelength of 490 nm.
5. The method of claim 4A method for labeling stem cells by using a bimodal nanoprobe, which is characterized in that: different concentrations of AIE-Gd @ SiO2The nano-composite is respectively 5 micrograms/ml, 10 micrograms/ml, 20 micrograms/ml, 40 micrograms/ml and 80 micrograms/ml, the amount of the thiazole blue is 0.25g, and the concentration is 5 mg/ml; the amount of the phosphate buffer solution is 5-15 ml, and the pH value is 7.4; the amount of dimethyl sulfoxide was 150-.
6. The method of bimodal nanoprobe labeling stem cells of claim 4, the labeled stem cells are subjected to fluorescence and nuclear magnetic bimodal imaging, characterized in that: different concentrations of AIE-Gd @ SiO2Incubating the nano composite material and stem cells for 24 hours, cleaning, fixing, and observing fluorescence imaging under a laser confocal microscope; after stem cells and AIE-Gd @ SiO nano-composites with different concentrations are incubated for 24h, the stem cells are suspended in a PCR tube mixed with a phosphate buffer solution and an agarose solution after digestion and centrifugation by recombinant pancreatin, and the strength of a magnetic signal of the stem cells marked by the nano-composites in the agarose solution is observed by nuclear magnetic imaging.
7. The method of fluorescence and nuclear magnetic bimodal imaging of labeled stem cells according to claim 6, wherein: different concentrations of AIE-Gd @ SiO2The nano material is 5 micrograms/ml, 10 micrograms/ml, 20 micrograms/ml, 40 micrograms/ml and 80 micrograms/ml, the phosphate buffer solution is 50-500 microliters, the agarose amount is 0.05g, the mass fraction is 1%, and the volume is 50-500 microliters.
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