CN107228848B - Wide fluorescence spectrum and MRI (magnetic resonance imaging) dual-image functional microsphere tracing mesenchymal stem cells and application - Google Patents
Wide fluorescence spectrum and MRI (magnetic resonance imaging) dual-image functional microsphere tracing mesenchymal stem cells and application Download PDFInfo
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Abstract
The invention relates to a wide fluorescence spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cell, wherein the mesenchymal stem cell is internally swallowed with the wide fluorescence spectrum and MRI dual-image functional microsphere, and the wide fluorescence spectrum and MRI dual-image functional microsphere comprises the following components in percentage by mass: 70-99.9% of polymer, 0.05-20% of fluorescein and 0.05-10% of gadolinium agent; the polymer is poly (lactide-co-glycolide-co-polyethylene glycol) or polystyrene. The invention also discloses application of the mesenchymal stem cells in preparation of tracers and/or therapeutic agents for bone defect repair and/or osteoporosis diseases. The microsphere can provide the functions of broad fluorescence spectrum and MRI double images, reduces the interference of self-background fluorescence when applied in animals, co-cultures the prepared microsphere and mesenchymal stem cells to prepare the microsphere with the functions of broad fluorescence spectrum and MRI double images for tracing the mesenchymal stem cells, and is used for repairing and treating bone defects and osteoporosis bone defects.
Description
Technical Field
The invention relates to the technical field of nano fluorescent probes and stem cells, in particular to a microsphere tracing mesenchymal stem cell with wide fluorescence spectrum and MRI (magnetic resonance imaging) double-image function and application thereof.
Background
At present, osteoporosis, osteoporotic fracture and other diseases have received wide attention from global public health problems, and early accurate diagnosis, reasonable treatment and monitoring of treatment effects are of great importance to prevent or reduce osteoporotic brittle fractures. The pathogenesis of osteoporosis is not clear, and it is thought that osteoporosis is ultimately caused by the disruption of the homeostatic balance of bone, imbalance in the number and function of osteoclasts and osteoblasts, and greater bone resorption than bone formation. Osteoporosis is often accompanied by a decrease in the proliferation and osteogenic differentiation capacity of mesenchymal stem cells, and a decrease in the osteogenic capacity of osteoblasts. Thereby causing a decrease in bone mass and localized osteoporotic bone defects.
Human umbilical cord Mesenchymal Stem Cells (MSCs) are isolated and cultured from neonatal umbilical cords, and have rich sources and convenient material acquisition. The in vitro culture and in vivo implantation have the characteristics of multidirectional differentiation potential, strong proliferation and differentiation capacity, low immunogenicity and the like, and are ideal seed cells for repairing bone defects in bone tissue engineering.
In recent years, the search for a labeling and tracing method of human mesenchymal stem cells implanted into a body and the discrimination of the survival condition of the human mesenchymal stem cells in a receptor still remain the hot research problem of the observation and evaluation of the application of the stem cells in bone defect repair at present. A large number of researches show that the human mesenchymal stem cells are implanted into a living body, the osteogenic differentiation of the mesenchymal stem cells is promoted, the bone structure of an osteoporosis part is improved, the bone or cartilage defect caused by diseases and wounds is effectively repaired, and the human mesenchymal stem cells have wide application value in the future treatment of osteoporosis.
At present, the nano-materials with excellent optical characteristics and applied to optical imaging include gold nanoparticles, quantum dots, porous silica nanoparticles, carbon nanotubes, polymer fluorescent microspheres, etc., wherein the fluorescent microspheres have the following advantages: stable morphological structure, concentrated particle size distribution (50-300 nm); stable and high efficiency luminous efficiency; has excellent biodegradability and biocompatibility; the metabolism rate of the small-molecule fluorescein in vivo is greatly reduced. Therefore, fluorescent microsphere labeling has received much attention and great attention in the field of molecular imaging technology.
Fluorescence Resonance Energy Transfer (FRET) refers to the process in which, after excitation of a Donor (Donor) group, dipole-mediated energy is transferred from the Donor to an Acceptor (Acceptor), and a photon has not been involved, and is therefore non-radiative. After the donor molecule is excited, if the acceptor molecule is located at a suitable distance (less than 10nm) from the donor molecule, the donor in the excited state will transfer energy to the acceptor, inducing the acceptor to be excited, and during this transfer, no emission or reabsorption of photons will be involved. The FRET effect can be generated between the commonly used fluorescein such as coumarin, acridine orange, rhodamine and phycoerythrin in turn, and the fluorescein can be widely applied to the fields of fluorescent probes and the like.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a microsphere tracing mesenchymal stem cell with a wide fluorescence spectrum and MRI double-image function and an application thereof. In the present application, the fluorescence spectrum is known as a fluorescence emission spectrum.
The invention provides a wide fluorescence emission spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cell, wherein the mesenchymal stem cell is internally swallowed with the wide fluorescence emission spectrum and MRI dual-image functional microsphere, and the wide fluorescence spectrum and MRI dual-image functional microsphere comprises the following components in percentage by mass: 70-99.9% of polymer, 0.05-20% of fluorescein and 0.05-10% of gadolinium agent; the polymer is poly (lactide-co-glycolide-co-polyethylene glycol) or polystyrene. The particle size of the microspheres is 50-300 nm.
In the microsphere, the polymer is used as a carrier of the fluorescein and the gadolinium agent. Preferably, the molecular weight of the poly (glycolide-co-lactide-co-polyethylene glycol) is 10000-100000g/mol, wherein the molecular weight of the polyethylene glycol is 2000-5000g/mol, and the material has good biocompatibility and is degradable. The polystyrene is a crosslinked polystyrene microsphere, and the surface of the polystyrene microsphere can also have carboxyl or amino. The particle size of the polystyrene microsphere is 50-200nm, and the relative standard deviation of the particle size is less than 10%.
Furthermore, the surface of the microsphere is also coupled with a modified compound, wherein the modified compound is polyarginine, polylysine, polyimide or polyethylene diamine. The polyarginine repeat unit is 9. The molecular weight of the polyimide may be selected to be 25k g/mol. The modified compound on the surface of the microsphere can increase the proportion of the microsphere entering stem cells and improve the fluorescence brightness of the microsphere-labeled stem cells.
Further, the fluorescein is two or three of coumarin, acridine orange, rhodamine, phycoerythrin and indocyanine green. Preferably, the fluorescein is a combination of coumarin and acridine orange, a combination of acridine orange, rhodamine and phycoerythrin, and a combination of phycoerythrin and indocyanine green. Fluorescence Resonance Energy Transfer (FRET) can occur between fluorescein.
Further, the gadolinium agent is gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA). Gadolinium agents have MRI imaging functions.
Further, when the polymer is a poly (lactide-co-ethylene glycol) copolymer, the preparation method of the microsphere with the wide fluorescence spectrum and the MRI dual-image function comprises the following steps:
(1) dissolving poly (lactide-co-glycolide-co-polyethylene glycol) and fluorescein in an organic solvent to serve as an oil phase, dissolving gadolinium in water to serve as an internal water phase, and dissolving polyacrylic acid and polyvinyl alcohol in water to serve as an external water phase;
(2) the microspheres are prepared by using an oil phase, an internal water phase and an external water phase through a multiple emulsion method, organic solvents of the oil phase are removed completely, and the microspheres with the functions of broad fluorescence spectrum and MRI double images are obtained.
Further, in the step (1), the organic solvent is one or more of chloroform, dichloromethane and dichloroethane. In the step (2), the operation steps of the multiple emulsion method are as follows: firstly, preparing oil phase and internal water phase into primary emulsion, adding the primary emulsion into external water phase to obtain water/oil/water multiple emulsion, and volatilizing the organic solvent of the oil phase to obtain the fluorescent and MRI double-image functional microsphere.
Further, when the polymer is polystyrene, the preparation method of the microsphere with wide fluorescence spectrum and MRI dual-image function comprises the following steps:
dissolving fluorescein and gadolinium agent in an organic solvent, and adding polystyrene microspheres into the organic solvent, wherein the particle size range of the polystyrene microspheres is 50-200nm, and the relative standard deviation of the particle size is less than 10%; and after 24h of reaction, removing the organic solvent, and washing to obtain the microsphere with the functions of broad fluorescence spectrum and MRI. The fluorescein is acridine orange, rhodamine or indocyanine green. The organic solvent is a mixture of chloroform and isopropanol, and the volume ratio of the chloroform to the isopropanol is 1: 3. When washing, the microspheres are washed by alcohol and water until the supernatant has no obvious color
Further, the method also comprises the following steps: coupling the microspheres with the amino groups of the modifying compound by a urethanization reaction.
The urethanization reaction can be assisted by a coupling agent EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide). The amino group of the modified compound can also be coupled with the polystyrene microsphere containing the amino group by using glutaraldehyde as a cross-linking agent.
Further, the preparation method of the wide fluorescence spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cells comprises the following steps:
uniformly mixing the wide fluorescence spectrum and MRI dual-image functional microspheres with a cell culture solution to obtain a microsphere solution, incubating at 4 ℃ for 24h, adding the microsphere solution into the adherent mesenchymal stem cells, culturing at 37 ℃ for 1-4 days, washing with a pure culture medium, removing microspheres which do not enter the mesenchymal stem cells, and obtaining the wide fluorescence spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cells.
Further, the concentration of the microsphere solution is 0.2-1.0 mg/mL.
The invention also discloses application of the wide fluorescence spectrum and MRI (magnetic resonance imaging) dual-image functional microsphere tracing mesenchymal stem cells in preparation of a tracer and/or a therapeutic agent for bone defect repair and/or osteoporosis diseases. In the application process, the mesenchymal stem cells have the functions of fluorescence and MRI dual-image imaging.
By the scheme, the invention at least has the following advantages:
the Fluorescence Resonance Energy Transfer (FRET) is used as an optical molecular ruler within the distance range of 1.0-10.0nm, and has the advantages of high resolution, high sensitivity, simplicity, convenience and the like. The fluorescence emission spectrum of the microsphere is widened by adopting the fluorescein combination which can generate FRET effect, the acceptor fluorescein can emit stronger fluorescence by using the optimal excitation wavelength of the donor fluorescein through the FRET effect, and the fluorescein combination designed based on the FRET effect can emit the fluorescence with longer wavelength because the autofluorescence wavelength is closer to the excitation light, thereby weakening the influence of autofluorescence, reducing the background fluorescence interference when in vivo application and improving the in vivo resolution of the microsphere fluorescence signal. Magnetic Resonance Imaging (MRI) has been applied to imaging diagnosis of various systems throughout the body. The best effect is the craniocerebrum, the spinal cord, the great vessels of the heart, the joint bones, the soft tissues and the like. MRI carries out qualitative and semi-quantitative diagnosis, can be used for making a plurality of sectional images, has higher spatial resolution and is superior to other X-ray images, two-dimensional ultrasound, nuclide and CT examination. The microspheres of the present invention combine the advantages of both fluorescence and MRI imaging.
The Mesenchymal Stem Cells (MSCs) used in the invention are phagocytized by the MSCs and uniformly distributed in the cells after being incubated in a culture solution (MSCM) containing the microspheres for 2-4 days, and the cells can be used for in vivo tracing. Compared with the method for directly marking cells by fluorescein and gadolinium agents, after the fluorescein and the gadolinium agents are loaded by polymers, the microspheres have the characteristics of low cytotoxicity, uniform particle size distribution (160-220nm), easiness in phagocytosis, more stability and high efficiency in-vivo circulation, good biodegradability and biocompatibility, improvement of image signal ratio and the like, and are more favorable for exerting the excellent fluorescence penetration characteristic of the broad fluorescence emission spectrum and reducing the damage to biological tissues.
The invention provides an intuitive, simple and dynamic noninvasive scheme for the broad fluorescence emission spectrum element and the gadolinium agent in the aspect of tracing the MSCs, and the method can be further used for deeply observing and confirming the pathogenesis of osteoporosis, evaluating the application of the MSCs in the osteoporosis bone defect repair and having wide application prospect in the osteoporosis molecular image field.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of microsphere solution on cell survival;
FIG. 2 is a fluorescent picture taken under a single photon laser confocal microscope after culturing mesenchymal stem cells by using the culture solution containing the microspheres;
FIG. 3 is a fluorescence image of the control group of example 2 taken under a single photon laser confocal microscope;
fig. 4 illustrates the CT scan results of microsphere-labeled mesenchymal stem cells of the present invention injected into rat skull.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The Mesenchymal Stem Cells (MSCs) used in the invention are human umbilical cord-derived mesenchymal stem cells (HUCMSCs), and the cells can provide strong self-renewal capacity and proliferation and differentiation capacity.
Example 1
The preparation method of the microsphere with wide fluorescence spectrum and MRI double-image function comprises the following steps:
(1) 5mg of acridine orange, 10mg of rhodamine and 25mg of poly (lactide-co-polyethylene glycol) (PLGA-PEG) were weighed and mixed, and dissolved with 1mL of chloroform/isopropanol (volume ratio: 1:3) solution to obtain an oil phase. 100 μ L of 100mg/mL Gd-DTPA contrast agent in water was prepared as the inner aqueous phase.
(2) Ultrasonically mixing the oil phase and the internal water phase obtained in the step (1) for 5 times, 2s (100W, BILON92-II) each time, and obtaining a primary emulsion.
(3) 4mL of an aqueous polyvinyl alcohol (PVA) solution (5 wt%) was added to the product of step (2), and ultrasonically mixed 5 times (2s, 100W) to form a double emulsion.
(4) The product of step (3) was diluted into aqueous polyacrylic acid (PAA) (5 wt%, 40mL), stirred overnight at room temperature, and evaporated in the dark.
(5) And (3) collecting the microsphere-containing solution obtained in the step (4) by using a 50mL high-speed centrifuge tube, firstly washing the solution by using absolute ethyl alcohol once, centrifuging the solution at a high speed (14500rpm for 20min), discarding the supernatant, performing ultrasonic treatment uniformly, then adding pure water for washing, centrifuging the solution at a high speed (12000rpm for 10min), repeating the centrifuging step for 2 to 3 times, and finally obtaining a precipitate, namely the microsphere-traced mesenchymal stem cells with the functions of broad fluorescence spectrum and MRI.
(6) And (5) dissolving the microspheres obtained in the step (5) in pure water to obtain microsphere aqueous solutions with different concentrations.
The prepared microspheres comprise the following components in percentage by weight: 70% of polymer, 20% of fluorescein and 10% of gadolinium agent.
Example 2
(1) And (3) carrying out normal subculture on the HUCMSCs, and paving the creeping pieces with corresponding specifications at the bottom of a common cell culture dish.
(2) Microspheres (prepared as in example 1) were mixed with MSCM medium to prepare a microsphere culture medium (0.2-1mg/mL), which was placed in an incubator (37 ℃ C., 5% CO)2) And (5) incubating for 24 h.
(3) After 24h, the mesenchymal stem cells are attached to the wall, the culture solution is sucked off, the culture solution (0.2-1mg/mL) with the same volume is added, and the mixture is placed in an incubator (37 ℃, 5% CO)2) The culture is continued for 2 to 4 days, preferably 2 days.
The toxic effect of the microspheres on the MSCs is shown in figure 1, the control group is pure poly (glycolide-co-glycolic acid) -polyethylene glycol copolymer microspheres, no obvious toxicity is observed in the control group and the fluorescent and MRI dual-functional microspheres under the concentration of 1.0mg/mL, and the cell death rates of the two groups are obviously increased under the concentration of 2mg/mL, so that the concentration range of the microspheres is preferably below 1mg/mL when the microspheres are used.
(4) After 2-4 days, removing the culture solution, washing with PBS for 2 times, immersing in 5% paraformaldehyde for 30min, washing with PBS for two times, and making slide specimen by climbing the bottom of the dish.
(5) And (3) acquiring the picture by utilizing a Nuance multispectral imaging system and ultraviolet excitation.
(6) The slide specimen is observed under a single photon laser confocal microscope, the excitation light wavelength is 488nm, the emission wavelength receiving ranges are 500-545nm (Green Green) and 550-580nm (Red), the picture is collected, the result is shown in figure 2, figure 2 shows that fluorescence energy resonance transfer occurs among fluorescein of the microspheres, and when 488nm laser is adopted, the cells have extremely strong Red fluorescence. Meanwhile, a stem cell group in which the acridine orange-loaded microspheres are endocytosed by mesenchymal stem cells is taken as a control group, a fluorescence picture (figure 3) is shot according to the method, the fluorescence brightness (red light) is very weak, and compared with the control group, the red fluorescence intensity of the stem cell is 5-10 times that of the stem cell. Combining with fluorescence photograph data, the concentration of the microsphere is preferably more than 0.2mg/mL, and a relatively obvious fluorescence signal can be observed.
Therefore, when the microsphere is used, the concentration of the microsphere is preferably 0.2-1mg/mL by combining the fluorescence intensity and toxicity data of the microsphere tracing MSCs.
(7) And (3) observing the slide specimen under a two-photon laser confocal microscope, wherein the excitation wavelength is 880nm, and observing and shooting the slide.
(8) Injecting a proper amount of microsphere solution into a glass tubule (diameter is 2mm, length is 10cm, liquid height is 2-3cm), and carrying out MRI scanning to compare signal intensity.
(9) If the fluorescence intensity and the signal intensity obtained in the steps (7) and (8) reach the tracing standard, HUCMSCs (5 × 10) marked by wide fluorescence spectrum and MRI double-image functional microspheres5-5×106)Injecting into local bone defect area (skull) of rat, continuously and dynamically observing by two-photon confocal microscope, comparing fluorescence/signal intensity and distribution, and evaluating local bone defect repairing condition by MRI scanning. As a result, as shown in fig. 4, the mesenchymal stem cell bone defect of the present invention heals better and the repair effect is better than the control group (control).
Example 3
The preparation method of the wide fluorescence spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cells comprises the following steps:
(1) 0.1mg of acridine orange, 0.1mg of rhodamine and 25mg of poly (lactide-co-polyethylene glycol) copolymer were weighed and mixed, and dissolved with 1mL of chloroform/isopropanol (volume ratio: 1:3) solution to obtain an oil phase. 10 μ L of 1mg/mL Gd-DTPA contrast agent in water was prepared as the inner aqueous phase.
(2) Ultrasonically mixing the oil phase and the internal water phase obtained in the step (1) for 5 times, 2s (100W, BILON92-II) each time, and obtaining a primary emulsion.
(3) To the product of step (2) was added 4mL of aqueous PVA solution (5 wt%), and mixed 5 times with ultrasound (2s, 100W) to form a double emulsion.
(4) The product of step (3) was diluted into aqueous PAA (5 wt%, 40mL), stirred overnight at room temperature and evaporated in the dark.
(5) And (3) collecting the microsphere-containing solution obtained in the step (4) by using a 50mL high-speed centrifuge tube, firstly washing the solution by using absolute ethyl alcohol once, centrifuging the solution at a high speed (14500rpm for 20min), discarding the supernatant, performing ultrasonic treatment uniformly, then adding pure water for washing, centrifuging the solution at a high speed (12000rpm for 10min), repeating the centrifuging step for 2 to 3 times, and finally obtaining a precipitate, namely the microsphere-traced mesenchymal stem cells with the functions of broad fluorescence emission spectroscopy and MRI.
(6) And (5) dissolving the microspheres obtained in the step (5) in pure water to obtain microsphere aqueous solutions with different concentrations.
The prepared microspheres comprise the following components in percentage by weight: 99.9% of polymer, 0.05% of fluorescein and 0.05% of gadolinium agent.
Example 4
Carboxylated polystyrene microspheres are supplied by Bangs Lab, Inc., with a particle size of 100nm and a relative standard deviation (CV) of about 5%; adding polystyrene into a chloroform/isopropanol (1/3) solution, wherein the chloroform/isopropanol solution contains 2-4mg/mL acridine orange, 2-4mg/mL rhodamine, 2-4mg/mL indocyanine green and 1-10mg/mL Gd-DTPA, reacting for 24h while volatilizing a solvent, and after the solvent is completely volatilized, washing the microspheres with alcohol and water until the supernatant has no obvious color, so as to obtain the microsphere with wide fluorescence emission spectrum and MRI dual-image function.
The prepared microspheres comprise the following components in percentage by weight: 97.5% of polymer, 2% of fluorescein and 0.5% of gadolinium agent.
Under the same condition of using amount of the fluorescein, the polystyrene microsphere can be obviously observed to have higher fluorescence brightness, and the polystyrene has stronger adsorption effect on the fluorescein. Similarly, the toxicity of the microspheres prepared from polystyrene to the MSCs is close to that of the microspheres prepared from PLGA polymer, while the fluorescence signal of the MSCs traced by the microspheres prepared from polystyrene is improved by 1-2 times, but the MRI signal is weaker by 20-50%.
Example 5
The preparation method of the wide fluorescence emission spectrum and MRI (magnetic resonance imaging) dual-image functional microsphere tracing mesenchymal stem cells comprises the following steps of:
(1) 2mg of acridine orange, 2mg of rhodamine, 2mg of indocyanine green were weighed, mixed with 25mg of poly (glycolide-co-lactide), and dissolved in 1mL of chloroform to obtain an oil phase. 100 μ l of Gd-DTPA contrast agent was prepared as the inner aqueous phase.
(2) And (2) ultrasonically mixing the oil phase obtained in the step (1) and the internal water phase for 5 times (2s, 100W, BILON92-II) to obtain a primary emulsion.
(3) To the product of step (2) was added 4mL of aqueous PVA solution (5 wt%), and mixed 5 times with ultrasound (2s, 100W) to form a double emulsion.
(4) The product of step (3) was diluted into aqueous PAA (5 wt%, 40mL), stirred overnight at room temperature and evaporated in the dark.
(5) And (3) collecting the microsphere-containing solution obtained in the step (4) by using a 50mL high-speed centrifuge tube, firstly washing the solution by using absolute ethyl alcohol once, centrifuging the solution at a high speed (14500rpm for 20min), discarding the supernatant, performing ultrasonic treatment uniformly, then adding pure water for washing, centrifuging the solution at a high speed (12000rpm for 10min), repeating the centrifuging at a high speed for 2 to 3 times, and finally dissolving the obtained microsphere precipitate in 1mL pure water.
(6) And (3) uniformly extracting 10 mu l of the solution obtained in the step (5), drying and weighing the solution, and calculating to obtain the concentration of the microsphere solution obtained in the step (5), wherein the concentration of the microsphere with the wide fluorescence emission spectrum and the MRI dual-image function prepared from the poly (glycolide-lactide) is 1-2 mg/mL.
(7) 100 mul of the prepared microsphere solution was extracted, diluted to 2mL with pure water, and the particle size of the microspheres was measured with a nanometer particle size and zeta potential analyzer (Zetasizer Nano ZS90), with the particle size distribution range of the microspheres being 200-300 nm.
Example 6
The method for testing the influence of the microspheres with different concentrations on the cell survival rate comprises the following steps:
(1) the 96-well plate was seeded with 5000 cells per empty.
(2) The microspheres prepared in example 1 were diluted with culture medium to give microsphere solutions of 0.2, 0.4, 0.8, 1, 2mg/mL, wrapped with tinfoil paper, and incubated overnight in a refrigerator at 4 ℃.
(3) Culturing the microsphere culture solution and the culture solution in different concentration experimental groups and blank control groups for 24h and 48h respectively.
(4) And (3) measuring an OD value by using a multifunctional microplate reader (Synergy 2), comparing with a blank control, statistically analyzing the survival rate of cells of each group, and displaying that the cell survival rate of the 2mg/mL microsphere solution is lower than that of the blank control group.
FIG. 1 shows the effect of microsphere solutions with different concentrations on cell survival rate, the control group is pure poly (lactide-co-glycolide-co-polyethylene glycol) blank microspheres (without dye and gadolinium agent), no significant toxicity is observed at the concentration of 1.0mg/mL, and the cell death rate of both groups is significantly increased at the concentration of 2mg/mL, so the dosage of microspheres is preferably below 1.0 mg/mL.
The method for testing the tracing condition of the microsphere comprises the following steps:
(1) 6 non-pregnant female SD rats of 3 months old are subjected to castration surgery to remove ovaries on both sides and are raised for 3 months after the surgery.
(2) After anesthesia of SD rat with 3.6% chloral hydrate, the external skin of femur is incised to expose periosteum, and a round bone defect area with the depth of 1mm is slowly chiseled out at a constant speed on the cortex of femur by using a punch electric drill with the diameter of 3 mm.
(3) Taking 5 × 10 from HUCMSCs marked by wide fluorescence emission spectrum and MRI dual-functional microspheres5-5×106The cells were resuspended in 30. mu.l PBS, drawn out with a 1mL syringe, injected into the round defect area of the femoral cortex, and the skin was sutured.
(4) And (3) scanning the bone repair condition of the bone defect part regularly by a small animal fluorescence image system, Micro-CT and MRI.
By continuously observing the fluorescence change and bone repair conditions of the bone defect area and integrating various imaging means, the repair process of the traced stem cells to the bone defect area can be clarified, and the repair mechanism of the stem cells for repairing the bone defect can be obtained.
Fig. 4 illustrates the CT scanning result of the microsphere-labeled mesenchymal stem cells injected into the skull of the rat, compared with the control group (which is not treated), the microsphere-labeled MSCs group has a denser trabecular structure at the bone defect, a narrowed bone gap and an increased bone density, and the area of the bone defect region after repair is only about 20-40% of that of the control group, which proves that the group has a better effect on bone defect repair.
Example 7
In addition, the surface of the microsphere can be connected with some modified compounds, and the modified compounds are polyarginine, polylysine, polyimide or polyethylene diamine. The modified compound can improve the stem cell endocytosis capacity of the microsphere. The specific modification method is as follows:
polyarginine, polyimide or polyethylene diamine is added to the microsphere solution prepared in example 1 or 4, and through a urethanization reaction, amino groups on the surface of the modified compound react with carboxyl groups on the surface of the microsphere and then are connected to the surface of the microsphere. The urethanization reaction can be assisted by a coupling agent EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide). The amino group of the modified compound can also be coupled with the polystyrene microsphere containing the amino group by using glutaraldehyde as a cross-linking agent. And (3) culturing the finally obtained microspheres and the mesenchymal stem cells by using the method in the embodiment 2 to obtain the microsphere tracing mesenchymal stem cells with the functions of wide fluorescence spectrum and MRI (magnetic resonance imaging). Compared with the microspheres of the unmodified compound, the fluorescence intensity of the mesenchymal stem cells traced by the microspheres of the modified compound can be increased by 50-100%, and the MRI brightness can be increased by 30-100%.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A wide fluorescence spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cell is characterized in that: the mesenchymal stem cells are endocytosed with a broad fluorescence spectrum and MRI dual-image functional microsphere, and the broad fluorescence spectrum and MRI dual-image functional microsphere comprises the following components in parts by mass: 70-99.9% of polymer, 0.05-20% of fluorescein and 0.05-10% of gadolinium agent; the polymer is poly (lactide-co-glycolide-co-polyethylene glycol) or polystyrene; the fluorescein is two or three of coumarin, acridine orange, rhodamine, phycoerythrin and indocyanine green; the particle size of the microsphere is 160-220 nm; the surface of the microsphere is also coupled with a modified compound, wherein the modified compound is polyarginine, polylysine, polyimide or polyethylene diamine; the gadolinium agent is gadolinium-diethylenetriamine pentaacetic acid;
the preparation method of the microsphere tracing mesenchymal stem cells with the functions of wide fluorescence spectrum and MRI (magnetic resonance imaging) double images comprises the following steps:
and uniformly mixing the broad fluorescence spectrum and MRI dual-image functional microspheres with a cell culture solution to obtain a microsphere solution, wherein the concentration of the microsphere solution is 0.2-1.0mg/mL, incubating at 4 ℃ for 24h, adding the microsphere solution into the adherent mesenchymal stem cells, and culturing at 37 ℃ for 1-4 days to obtain the broad fluorescence spectrum and MRI dual-image functional microsphere tracing mesenchymal stem cells.
2. The wide fluorescence spectrum and MRI dual image function microsphere tracking mesenchymal stem cell according to claim 1, wherein when the polymer is poly (lactide-co-ethylene glycol), the preparation method of the wide fluorescence spectrum and MRI dual image function microsphere comprises the following steps:
(1) dissolving the poly (lactide-co-glycolide-co-polyethylene glycol) and fluorescein in an organic solvent to serve as an oil phase, dissolving the gadolinium agent in water to serve as an internal water phase, and dissolving polyacrylic acid and polyvinyl alcohol in water to serve as an external water phase;
(2) and preparing microspheres by using the oil phase, the inner water phase and the outer water phase through a multiple emulsion method, and removing organic solvents of the oil phase to obtain the microspheres with the functions of wide fluorescence spectrum and MRI (magnetic resonance imaging).
3. The wide fluorescence spectrum and MRI dual image function microsphere tracking mesenchymal stem cells according to claim 1, wherein when the polymer is polystyrene, the preparation method of the wide fluorescence spectrum and MRI dual image function microsphere comprises the following steps:
dissolving fluorescein and the gadolinium agent in an organic solvent, and adding polystyrene microspheres into the organic solvent; and after 24h of reaction, removing the organic solvent, and washing to obtain the microsphere with the wide fluorescence spectrum and MRI dual-image function.
4. The broad fluorescence spectrum and MRI dual image functional microsphere tracking mesenchymal stem cells according to claim 2 or 3, characterized by further comprising the steps of: coupling the microspheres with the amino groups of the modified compound by a urethanization reaction.
5. The application of the wide fluorescence spectrum and MRI dual-image functional microsphere labeled mesenchymal stem cells according to claim 1 in preparing tracers and/or therapeutic agents for bone defect repair and/or osteoporosis diseases.
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