Mri traceable cell microcarriers and methods of making same

Abstract

The application relates to the technical field of biology, and particularly discloses a cell microcarrier capable of MRI tracing and a preparation method thereof. The preparation method of the cell microcarrier capable of MRI tracing comprises the following steps: in step one, gamma-Fe2O3 nanoparticles and water are uniformly mixed through ultrasonic mixing, silk fibroin is added and uniformly stirred, then a baicalin solution and castor oil are added and uniformly mixed and stirred, ultrasonic emulsification is carried out at 3-5 DEG C to obtain a mixed emulsion; in step two, the mixed emulsion prepared in step one is added drop by drop into castor oil, stirred for 20-35 min, then cooled, anhydrous ethanol is added, extraction is carried out, centrifugal separation is carried out, and drying is carried out to obtain the cell microcarrier. The cell microcarrier prepared in the application can be used for supporting stem cells to exert better therapeutic effects in vivo.

Description

Technical Field

[0001] This invention relates to specific biotechnology fields, and in particular to an MRI-trackable cell microcarrier and its preparation method. Background Technology

[0002] Cerebrovascular diseases have become one of the leading causes of death and disability in humans, with ischemic stroke being the most common. Ischemic neuronal cell damage is most prominently characterized by neuronal death. Traditionally, it was believed that neurons in the adult central nervous system cannot regenerate, which is one of the fundamental causes of disability due to cerebral ischemia. Since the 1990s, the discovery of neural stem cells (NSCs) with self-renewal and pluripotent differentiation capabilities has provided new treatment ideas and approaches for many previously difficult-to-treat neurological diseases.

[0003] Microcarriers are tiny spherical particles that allow adherent cells to grow in a monolayer on the surface of the particle in a suspended state during three-dimensional cell culture. This provides a larger contact area than conventional planar culture, facilitating large-scale cell culture and collection. Microcarriers can also serve as substrates for carrying stem cells. In vitro, three-dimensional culture using microcarriers can rapidly expand a large number of cells; in vivo, microspheres carrying stem cells can be injected into tissue defects for regenerative therapy.

[0004] Currently, in neurodegenerative diseases such as stroke, some studies have used microcarriers as carriers for stem cell repair. Microcarriers act as a matrix to support stem cell survival in vivo and enhance therapeutic efficacy. However, most traceable microcarriers are labeled with bioluminescent substances such as fluorescent agents, and the fluorescence signal can be captured using small animal in vivo imaging or stereofluorescence microscopy. Biofluorescent labels typically suffer from drawbacks such as low fluorescence intensity and easy quenching. Summary of the Invention

[0005] To enable microcarriers to support stem cells and achieve better therapeutic effects in vivo, this application provides an MRI-traceable cell microcarrier and its preparation method.

[0006] This application provides a method for preparing MRI-traceable cell microcarriers, which employs the following technical solution:

[0007] A method for preparing MRI-traceable cell microcarriers includes the following preparation steps:

[0008] Step 1: Ultrasonically mix γ-Fe2O3 nanoparticles and water until homogeneous, add silk fibroin and stir until homogeneous, then add baicalin solution and castor oil, mix and stir until homogeneous, and ultrasonically emulsify at 3-5℃ to obtain a mixed emulsion.

[0009] Step 2: Add the mixed emulsion prepared in Step 1 dropwise to castor oil, stir for 20-35 minutes, then cool, add anhydrous ethanol, extract, centrifuge, and dry to obtain cell microcarriers.

[0010] By adopting the above technical solution, due to the superparamagnetism of γ-Fe2O3 nanoparticles, a stronger relaxation effect is achieved under the same magnetic field conditions. Using them as cell microcarriers for stem cell loading can achieve good invisibility. γ-Fe2O3 nanoparticles and silk fibroin aqueous solution are coated with an oil phase and rapidly stirred to form micro-droplets. Silk fibroin is coated on the surface of the magnetic nanoparticles, and then baicalin is added. The silk fibroin forms hydrogen bonds for cross-linking. Silk fibroin has good biocompatibility, toughness, and mechanical strength. Baicalin is a commonly used drug for treating cerebrovascular diseases. Combining the two can not only achieve the effect of repairing damaged parts of the human body, but also enable the cell carrier to better encapsulate γ-Fe2O3 nanoparticles, improving the cell loading effect.

[0011] Optionally, the weight ratio of silk fibroin to baicalin is (18-22):1.

[0012] By adopting the above technical solution, the combination of silk fibroin and baicalin forms a hydrogen bond cross-linking network that better encapsulates the magnetic nanoparticles, improving the stability of the nanoparticles and enhancing their in vivo imaging effect.

[0013] Optionally, the castor oil accounts for (0.2-0.8):1 of the total weight of silk fibroin and baicalin.

[0014] By adopting the above technical solution, castor oil was used as the oil phase to encapsulate the aqueous phase during the preparation of cell microcarriers, and cell microcarriers with MRI traceability were successfully prepared using the above concentration.

[0015] Optionally, the concentration of baicalin in the baicalin solution is 0.5-1.7 mg / mL.

[0016] By employing the aforementioned technical approach, baicalin, as the main active ingredient of Scutellaria baicalensis, has been demonstrated in experimental studies to exert neuroprotective functions in central nervous system diseases such as stroke by regulating inflammation, inhibiting oxidative stress, and blocking apoptosis pathways. Baicalin can exert its neuroprotective effects through multiple pathways; by loading baicalin onto cellular microcarriers and transferring it to damaged sites, it can exert targeted therapeutic effects.

[0017] Optionally, in step one, baicalin and castor oil are then added and mixed and stirred for 1.5-2.5 hours.

[0018] By employing the above technical solution, baicalin and silk fibroin are fully cross-linked by hydrogen bonds through mixing and stirring, forming a network structure that encapsulates magnetic nanoparticles. This process prepares a product that can carry cells and therapeutic drugs and accurately locate and trace them in vivo, thereby achieving the purpose of assessing prognosis and guiding treatment.

[0019] Optionally, in step two, the mixed emulsion prepared in step one is added dropwise to castor oil at 80-95°C and stirred for 20-35 minutes.

[0020] By adopting the above technical solutions, increasing the temperature increases the cross-linking rate within the droplets. Cross-linking allows the droplets to spherize more quickly, reducing the aggregation between droplets. The increased degree of cross-linking makes the cell microcarriers more stable.

[0021] Optionally, in step one, silk fibroin is added and stirred until homogeneous, and then Tris-HC1 buffer is added.

[0022] By adopting the above technical solution and adding Tris-HC1 buffer, the pH environment of the silk fibroin solution is stabilized, enabling the silk fibroin to form a strong interaction with γ-Fe2O3 nanoparticles.

[0023] Secondly, this application provides an MRI-traceable cell microcarrier, prepared by any of the above-described methods for preparing an MRI-traceable cell microcarrier.

[0024] By adopting the above technical solution, the cell microcarrier prepared in this application can be tracked by MRI, and can carry cells and therapeutic drugs and accurately locate and track them in vivo to guide treatment.

[0025] In summary, this application has the following beneficial effects:

[0026] 1. Since this application uses γ-Fe2O3 nanoparticles, which are superparamagnetic, they result in a stronger relaxation effect under the same magnetic field conditions. Using them as cell microcarriers for stem cell loading can achieve good cloning properties.

[0027] 2. This application preferably uses baicalin. As the main active ingredient of Scutellaria baicalensis, baicalin has been shown in studies to protect neurons in central nervous system diseases such as stroke by regulating inflammation, inhibiting oxidative stress, and blocking apoptosis pathways. Baicalin can protect neurons through multiple pathways. By loading baicalin onto cellular microcarriers and transferring it to damaged sites, it can exert a targeted therapeutic effect.

[0028] 3. This application combines silk fibroin with baicalin. Silk fibroin is coated on the surface of magnetic nanoparticles, and then baicalin is added to form hydrogen bond cross-links with silk fibroin. Silk fibroin has good biocompatibility, toughness and mechanical strength. Baicalin is a commonly used drug for treating cerebrovascular diseases. Combining the two can not only achieve the effect of repairing damaged parts of the human body, but also enable the cell carrier to better coat γ-Fe2O3 nanoparticles and improve the cell carrying effect. Detailed Implementation

[0029] The present application will be further described in detail below with reference to comparative examples and embodiments.

[0030] Example

[0031] Example 1

[0032] A method for preparing MRI-traceable cell microcarriers includes the following preparation steps:

[0033] Step 1: Weigh 70 mg of γ-Fe2O3 nanoparticles and 0.6 ml of deionized water and sonicate for 6 min to mix evenly. Add 160 mg of silk fibroin to the γ-Fe2O3 solution and stir evenly. Then add 10 mol / L Tris-HCl buffer to adjust the pH to 6. Then add 8 ml of 1 mg / ml baicalin solution and 84 mg of castor oil in DMP solvent. Mix and stir mechanically for 2 h. Cool to 4 °C and sonicate for 8 min to emulsify to obtain a mixed emulsion.

[0034] Step 2: Add the mixed emulsion prepared in Step 1 dropwise to 30 mg castor oil at 85 °C, stir for 30 min, then cool to room temperature, add anhydrous ethanol for extraction, centrifuge at 2000 rpm for 10 min, discard the supernatant, repeat 3 times, dry at 40 °C for 4 h to obtain cell microcarriers, and store at 4 °C for later use.

[0035] Example 2

[0036] A method for preparing MRI-traceable cell microcarriers includes the following preparation steps:

[0037] Step 1: Weigh 70 mg of γ-Fe2O3 nanoparticles and 0.6 ml of deionized water, and sonicate for 6 min to mix evenly. Add 126 mg of silk fibroin to the γ-Fe2O3 solution and stir evenly. Then add 10 mol / L Tris-HCl buffer to adjust the pH to 6. Next, add 14 ml of 0.5 mg / ml baicalin solution and 26.6 mg of castor oil (DMP solvent). Mix and stir mechanically for 1.5 h. Cool to 3 °C and sonicate for 8 min to emulsify, obtaining a mixed emulsion.

[0038] Step 2: Add the mixed emulsion prepared in Step 1 dropwise to 30 mg castor oil at 80 °C, stir for 20 min, then cool to room temperature, add anhydrous ethanol for extraction, centrifuge at 2000 rpm for 10 min, discard the supernatant, repeat 3 times, dry at 40 °C for 4 h to obtain cell microcarriers, and store at 4 °C for later use.

[0039] Example 3

[0040] A method for preparing MRI-traceable cell microcarriers includes the following preparation steps:

[0041] Step 1: Weigh 70 mg of γ-Fe2O3 nanoparticles and 0.6 ml of deionized water, and sonicate for 6 min to mix evenly. Add 149.6 mg of silk fibroin to the γ-Fe2O3 solution and stir evenly. Then add 10 mol / L Tris-HCl buffer to adjust the pH to 6. Next, add 4 ml of 1.7 mg / ml baicalin solution and 125.12 mg of castor oil (DMP solvent). Mix and stir mechanically for 2.5 h. Cool to 5 °C and sonicate for 8 min to emulsify, obtaining a mixed emulsion.

[0042] Step 2: Add the mixed emulsion prepared in Step 1 dropwise to 30 mg castor oil at 95 °C, stir for 35 min, then cool to room temperature, add anhydrous ethanol for extraction, centrifuge at 2000 rpm for 10 min, discard the supernatant, repeat 3 times, dry at 40 °C for 4 h to obtain cell microcarriers, and store at 4 °C for later use.

[0043] Example 4

[0044] The method for preparing an MRI-traceable cell microcarrier differs from Example 2 in that the weight ratio of silk fibroin to baicalin is 17:1, the amount of silk fibroin is 102 mg, the amount of baicalin (0.5 mg / ml) is 12 mL, and the amount of castor oil is 21.6 mg.

[0045] Example 5

[0046] The method for preparing an MRI-traceable cell microcarrier differs from Example 3 in that the weight ratio of silk fibroin to baicalin is 23:1, the amount of silk fibroin is 102 mg, the amount of baicalin is 12 mL at 1.7 mg / ml, and the amount of castor oil is 97.92 mg.

[0047] Example 6

[0048] A method for preparing an MRI-traceable cell microcarrier differs from Example 2 in that castor oil accounts for 0.1:1 of the total weight of silk fibroin and baicalin, and the castor oil content is 13.3 mg.

[0049] Example 7

[0050] A method for preparing an MRI-traceable cell microcarrier differs from Example 3 in that castor oil accounts for 0.9:1 of the total weight of silk fibroin and baicalin, and the castor oil content is 140.76 mg.

[0051] Example 8

[0052] A method for preparing MRI-traceable cell microcarriers differs from Example 2 in that the mixing and stirring in step one is performed for 1.4 hours. The specific preparation steps in step one are as follows:

[0053] Weigh 70 mg of γ-Fe2O3 nanoparticles and 0.6 ml of deionized water, and sonicate for 6 min to mix evenly. Add 126 mg of silk fibroin to the γ-Fe2O3 solution and stir evenly. Then add 14 ml of 0.5 mg / ml baicalin solution and 26.6 mg of castor oil (DMP solvent), mix and stir mechanically for 1.4 h, cool to 3 °C and sonicate for 8 min to obtain a mixed emulsion.

[0054] Example 9

[0055] A method for preparing MRI-traceable cell microcarriers differs from Example 3 in that the mixing and stirring in step one is performed for 2.6 hours. The specific preparation steps in step one are as follows:

[0056] Weigh 70 mg of γ-Fe2O3 nanoparticles and 0.6 ml of deionized water, and sonicate for 6 min to mix evenly. Add 149.6 mg of silk fibroin to the γ-Fe2O3 solution and stir evenly. Then add 4 ml of 1.7 mg / ml baicalin solution and 125.12 mg of castor oil (DMP solvent), mix and stir mechanically for 2.6 h, cool to 5 °C and sonicate for 8 min to obtain a mixed emulsion.

[0057] Comparative Example

[0058] Comparative Example 1

[0059] A method for preparing MRI-traced cell microcarriers differs from Example 1 in that the raw materials do not include baicalin, and the specific preparation steps in step one are as follows:

[0060] Step 1: Weigh 70mg of γ-Fe2O3 nanoparticles and 0.6ml of deionized water and sonicate for 6min to mix evenly. Add 160mg of silk fibroin to the γ-Fe2O3 solution and stir evenly. Then add 84mg of castor oil in DMP solvent and mix mechanically for 2h. Cool to 4℃ and sonicate for 8min to obtain a mixed emulsion.

[0061] Comparative Example 2

[0062] A method for preparing MRI-traced cell microcarriers differs from Example 1 in that the raw materials do not include silk fibroin, and the specific preparation steps in step one are as follows:

[0063] Step 1: Weigh 70mg of γ-Fe2O3 nanoparticles and 0.6ml of deionized water, and sonicate for 6min to mix evenly. Then add 8ml of 1mg / ml baicalin solution and 84mg of castor oil (DMP solvent), mix and stir mechanically for 2h, cool to 4℃ and sonicate for 8min to obtain a mixed emulsion.

[0064] Performance testing

[0065] Detection methods

[0066] 1. Cell culture results: 5×10… of the obtained primary neural stem cells… 5 When the cells grew to 1 / 2 the height of the bottom of the dish, 25 μg / mL of the cell microcarriers prepared in Examples 1-9 and Comparative Examples 1-2 were added. The control group was added only with PBS. The cells were incubated together for 24 h in an incubator at 37°C, 5% CO2 and 95% humidity.

[0067] Then, neural stem cells carried by cell microcarriers were injected into an ischemic hypoxic adult mouse model via vein or artery, and the migration and survival of the transplanted neural stem cells in vivo were monitored in real time 7 days after transplantation using 3.0T MRI imaging technology.

[0068] Table 1 Performance Test Results

[0069]

[0070]

[0071] As can be seen from Examples 1-9 and Comparative Examples 1-2, and Table 1, the neural stem cells carried by the cell microcarriers prepared in Examples 1-9 can achieve good survival rates, especially in Examples 1-3, where the survival rate of cells after 7 days is relatively high. Examples 1-3 and Comparative Examples 1-2 demonstrate that baicalin can be used to induce stem cell proliferation and differentiation, significantly improving stem cell survival rates. The combination of this drug and stem cell therapy can effectively promote the recovery of neurological function after ischemic brain injury. Combining baicalin with silk fibroin, which has good biocompatibility, toughness, and mechanical strength, and baicalin being a commonly used drug for treating cerebrovascular diseases, the combination of the two can achieve the effect of repairing damaged areas in the human body and also improve the cell carrying capacity.

[0072] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing MRI-traceable cell microcarriers, characterized by: The preparation steps include the following steps: Step one, uniformly mix the γ-Fe2O3 nanoparticles and water by ultrasonic mixing, add silk fibroin and stir until uniform, then add Tris-HCl buffer solution, and then add baicalin solution and castor oil, mix and stir until uniform, and then ultrasonic emulsify at 3-5℃ to obtain a mixed emulsion; wherein the weight ratio of the silk fibroin to baicalin is (18-22):1, and the castor oil accounts for (0.2-0.8):1 of the total weight of the silk fibroin and baicalin; the mixing and stirring time is 1.5-2.5h; Step two, add the mixed emulsion prepared in step one drop by drop into castor oil, stir for 20-35min, then cool, add anhydrous ethanol, extract, centrifuge, and dry to obtain the cell microcarriers.

2. The method for preparing an MRI-traced cell microcarrier according to claim 1, characterized in that: The concentration of baicalin in the baicalin solution is 0.5-1.7mg / mL.

3. The MRI-traceable cell microcarriers are prepared by the preparation method of the MRI-traceable cell microcarriers according to claim 1 or 2.

Images