Magnetic nanomaterial-guided anti-inflammatory enhanced mesenchymal stem cell as well as preparation method and application thereof

Abstract

The invention provides a magnetic nanomaterial-guided anti-inflammatory enhanced mesenchymal stem cell as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. The magnetic nanoparticle labeled mesenchymal stem cell provided by the invention is a magnetic nanoparticle (PLL-MNPs) modified by poly L-lysine coated in a mesenchymal stem cell (MSC). The PLL-MNPs has no obvious influence on the cell morphology, differentiation potential, proliferation, apoptosis and cell cycle of the MSC, meanwhile, the PLL-MNPs obviously enhances the anti-inflammatory ability of the MSC, and the conversion of reactive astrocytes / macrophages from a proinflammatory phenotype to an anti-inflammatory phenotype can be promoted. In conclusion, the mesenchymal stem cells marked by the magnetic nanoparticles have a relatively strong anti-inflammatory function, and a new means is provided for application of clinical cell transplantation therapy of the MSC.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to anti-inflammatory enhanced mesenchymal stem cells guided by magnetic nanomaterials, and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have been widely used in the treatment of various diseases due to their wide range of sources, easy access, low immunogenicity, strong proliferation ability and role in immunoregulation, anti-inflammation and tissue repair ([1] Blanc, KLet al. ISCT MSC committee statement on the US FDA approval of allogenic bone-marrow mesenchymal stromal cells. Cytotherapy 27, 413-416 (2025). [2] Zhang, X. et al. MSC-Based Cell Therapy in Neurological Diseases: A Concise Review of the Literature in Pre-Clinical and Clinical Research. Biomolecules 14, 538 (2024).). These unique properties make them promising candidate cells for the treatment of autoimmune diseases and central nervous system damage ([3] Gavasso, S. et al. The Therapeutic Mechanisms of Mesenchymal Stem Cells in MS-A Review Focusing on Neuroprotective Properties. Int J Mol Sci 25, 1365 (2024)). However, the clinical application of cell transplantation therapy faces challenges. Currently, it is difficult to non-invasively monitor the migration, homing, engraftment efficiency and functional capacity of transplanted cells in vivo.

[0003] In recent years, the application of magnetic nanomaterials (MNPs) in the biomedical field has been increasing, especially in cell tracking through magnetic resonance imaging (MRI). The unique physicochemical properties of MNPs give them significant advantages in medical imaging ([4]Luo, GF & Zhang, X. Magnetic nanoparticles for use in bioimaging. Biomater Sci 12, 6224-6236 (2024). [5]Chen, Y. & Hou, S. Application of magnetic nanoparticles in cell therapy. Stem Cell Res Ther 13, 135 (2022).). Although studies have shown that cells labeled with MNPs generally exhibit non-cytotoxicity, they may cause cells to lose their differentiation ability ([6]Chen, Y. & Hou, S. Recent progress in the effect of magnetic iron oxide nanoparticles on cells and extracellular vesicles. Cell Death Discov 9, 195 (2023).). In addition, in central nervous system diseases, transplanted MSCs mainly improve the inflammatory microenvironment by secreting inflammatory-related factors ([7]Liu, AMetal.Human adipose tissue-and umbilical cord-derived stem cells:which is a better alternative to treat spinal cord injury? Neural Regen Res 15, 2306-2317 (2020).). It is currently unclear whether MNPs labeling will affect the function of MSCs to secrete inflammatory cytokines. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an anti-inflammatory enhanced mesenchymal stem cell guided by magnetic nanomaterials. By using poly-L-lysine (PLL)-modified MNPs to treat MSCs, not only has no obvious effect on the characteristics of the MSC cells themselves, but also can effectively enhance the anti-inflammatory ability of MSCs, providing a new option for the treatment of clinical inflammatory-related diseases.

[0005] The present invention provides a magnetic nanoparticle-labeled mesenchymal stem cell (MSC-MNPs). The mesenchymal stem cell is coated with poly-L-lysine-modified magnetic nanoparticles (PLL-MNPs).

[0006] Preferably, the magnetic nanoparticles are zinc-doped magnetic nanoparticles;

[0007] The molecular formula of the zinc-doped magnetic nanoparticles is Zn 0.4 Fe 2.6 O4.

[0008] Preferably, the molecular weight of the poly-L-lysine is 1800 to 10000.

[0009] The present invention provides a method for preparing mesenchymal stem cells labeled with magnetic nanoparticles, comprising the following steps:

[0010] Under vibration conditions, a poly-L-lysine solution is added to an ethanol dispersion of magnetic nanoparticles for 80 to 100 minutes, and the magnetic material is separated to obtain poly-L-lysine-modified magnetic nanoparticles;

[0011] The poly-L-lysine-modified magnetic nanoparticles are used to treat mesenchymal stem cells to obtain mesenchymal stem cells labeled with the magnetic nanoparticles.

[0012] Preferably, the mass ratio of poly-L-lysine to magnetic nanoparticles is (9-11):1.

[0013] Preferably, the volume ratio of the poly-L-lysine solution to the magnetic nanoparticle ethanol dispersion is (0.8-1.2): (1.8-2.2);

[0014] The concentration of the poly-L-lysine solution is 8-12 mg / mL;

[0015] The concentration of the magnetic nanoparticle ethanol dispersion is 0.4-0.6 mg / mL.

[0016] Preferably, the treatment concentration of the poly-L-lysine modified magnetic nanoparticles is 0.08-0.12 μg / μL.

[0017] Preferably, the treatment time is 10 to 14 hours; the treatment temperature is 36 to 38°C.

[0018] The present invention provides the use of the mesenchymal stem cells labeled with the magnetic nanoparticles or the mesenchymal stem cells labeled with the magnetic nanoparticles prepared by the preparation method in preparing diseases to be treated by cell transplantation therapy.

[0019] Preferably, the diseases treated by the drug include inflammation-related diseases.

[0020] The present invention provides mesenchymal stem cells labeled with magnetic nanoparticles, wherein the mesenchymal stem cells are coated with poly-L-lysine-modified magnetic nanoparticles. The present invention evaluated the morphology, differentiation potential, proliferation, apoptosis, and cell cycle of the mesenchymal stem cells labeled with the magnetic nanoparticles. The results showed that coating with the poly-L-lysine-modified magnetic nanoparticles had no significant effect on the cell morphology, differentiation potential, proliferation, apoptosis, or cell cycle of the mesenchymal stem cells. Furthermore, coating with the poly-L-lysine-modified magnetic nanoparticles enhanced the anti-inflammatory capacity of the mesenchymal stem cells. Bio-Plex analysis showed that the poly-L-lysine-modified magnetic nanoparticles promoted the expression of anti-inflammatory cytokines and inhibited the secretion of pro-inflammatory factors, which was further supported by proteomic analysis. Furthermore, co-culturing MSC-MNPs with activated astrocytes / macrophages promoted the transition of reactive astrocytes / macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype. In summary, poly-L-lysine-modified magnetic nanoparticles significantly enhanced the anti-inflammatory function of mesenchymal stem cells, providing new possibilities for the application of mesenchymal stem cells in clinical cell transplantation therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Characterization of magnetic nanoparticles (MNPs) and identification of mesenchymal stem cells (MSCs); A is the morphology of MNPs under transmission electron microscopy (TEM); B is the magnetization curve of dried MNPs in a magnetic field; C and D are the hydrodynamic diameters and zeta potentials of MNPs and PLL-MNPs (n=3); E is the morphology of MSCs and MSC-MNPs; F is the staining results using Alizarin Red, Alcian Blue, and Oil Red O; G and H are the expression results of MSC markers identified by flow cytometry, sample size n=3;

[0022] Figure 2 Figure 3 Effects of magnetic nanoparticles (MNPs) on cell proliferation, apoptosis, and cell cycle after phagocytosis of mesenchymal stem cells (MSCs); A shows Prussian blue and eosin staining of MSCs; B shows CCK-8 assay for MSC proliferation after phagocytosis of MNPs; C and D show Annexin V and propidium iodide (PI) staining and flow cytometry (FACS) analysis; E and F show PI staining and flow cytometry analysis; Data are expressed as mean ± standard error of variance (SEM); *P < 0.05, **P < 0.01; Panel B shows two-way repeated measures ANOVA with Bonferroni post hoc correction, and the other data were tested using Student's T-test; n = 3 for panels B, F, I, and J, and n = 5 for panel D;

[0023] Figure 3 Figure 3. Effects of magnetic nanoparticles (MNPs) on cytokine secretion in mesenchymal stem cells (MSCs). A is a heat map of the expression levels of 27 cytokines in the MSC and MSC-MNPs groups analyzed by Bio-plex. B is a quantitative analysis of the levels of inflammatory factors, cytokines, and vascular endothelial growth factor (VEGF) in the MSC and MSC-MNPs groups. *P < 0.05; **P < 0.01. Student's t-test was used, and the sample size for each group was n = 3.

[0024] Figure 4 Figure 5. Magnetic nanoparticles (MNPs) altered the proteomic characteristics of mesenchymal stem cells (MSCs). A is a volcano plot showing the differentially expressed proteins in the MSC group and the MSC-MNPs group; B and C are the results of Western blot analysis of the MSC group and the MSC-MNPs group; *P < 0.05, ***P < 0.001, n = 3 per group, Student's t-test.

[0025] Figure 5 Figure 3 The effects of MSC-MNPs on the polarized phenotype of reactive astrocytes and macrophages, where A is a schematic diagram of the co-culture of MSC-MNPs and astrocytes or macrophages; B is the relative expression levels of C3, Serping1, Clcf1, B3gnt5, Tm4sf1, and Ptx3 in primary astrocytes detected by RT-qPCR; C is the expression levels of iNOS, CD86, TNF-a, Arg-1, CD206, and IL-10 in macrophages detected by RT-qPCR; *P<0.05, **P<0.01; n=4; C was analyzed using Student's t-test. DETAILED DESCRIPTION

[0026] The present invention provides a mesenchymal stem cell labeled with magnetic nanoparticles, wherein the mesenchymal stem cell is coated with magnetic nanoparticles modified with poly-L-lysine.

[0027] In the present invention, the mesenchymal stem cells have strong immunomodulatory and tissue repair capabilities and are widely used in cell transplantation to treat various diseases. In the embodiment of the present invention, the mesenchymal stem cells are preferably derived from human umbilical cord and purchased from Celera Stem Cell Science and Technology Co., Ltd. (Guangzhou, China).

[0028] In the present invention, the magnetic nanoparticles are preferably zinc-doped magnetic nanoparticles. The molecular formula of the zinc-doped magnetic nanoparticles is preferably Zn 0.4 Fe 2.6The zinc-doped magnetic nanoparticles are prepared by a high-temperature pyrolysis method. For details, see the prior art (Noh SH, NaW, Jang JT, Lee JH, Lee EJ, Moon SH, et al. Nanoscale Magnetism Control via Surface and Exchange Anisotropy for Optimized Ferrimagnetic Hysteresis. Nano Lett. 2012; 12: 3716-21.). The magnetic nanomaterials are mainly composed of metals, alloys, and ferrites (MFe2O4, M = Fe, Co, Ni, Mn, Zn). The prepared zinc-doped magnetic nanoparticles are water-soluble, have good magnetic properties, can respond to magnetic field drive, and have good nuclear magnetic resonance imaging capabilities.

[0029] In the present invention, the molecular weight of the poly-L-lysine is preferably 1,800 to 10,000, and can be 4,000. The poly-L-lysine serves to increase the positive charge loading on the surface of the magnetic nanoparticles, thereby improving the biocompatibility of the magnetic nanoparticles. In the present embodiment, the poly-L-lysine was purchased from Maclean (Shanghai) Co., Ltd.

[0030] The present invention provides a method for preparing mesenchymal stem cells labeled with magnetic nanoparticles, comprising the following steps:

[0031] Under vibration conditions, a poly-L-lysine solution is added to an ethanol dispersion of magnetic nanoparticles for 80 to 100 minutes, and the magnetic material is separated to obtain poly-L-lysine-modified magnetic nanoparticles;

[0032] The poly-L-lysine-modified magnetic nanoparticles are used to treat mesenchymal stem cells to obtain mesenchymal stem cells labeled with the magnetic nanoparticles.

[0033] The invention adds a poly-L-lysine solution into a magnetic nanoparticle ethanol dispersion under vibration conditions, maintains the mixture for 80 to 100 minutes, separates the magnetic material, and obtains magnetic nanoparticles modified with poly-L-lysine.

[0034] In the present invention, the vibration condition is preferably an ultrasonic probe with an amplitude of 30%. The vibration condition is conducive to the molecules in the solution being in a moving state, which is conducive to increasing the contact opportunity between two molecules, thereby improving the product yield.

[0035] In the present invention, the mass ratio of poly-L-lysine to magnetic nanoparticles is preferably (9-11):1, and may be 10:1. The volume ratio of the poly-L-lysine solution to the magnetic nanoparticle ethanol dispersion is preferably (0.8-1.2):(1.8-2.2), and may be 1:2. The concentration of the poly-L-lysine solution is preferably 8-12 mg / mL, and may be 9-11 mg / mL, or 10 mg / mL. The concentration of the magnetic nanoparticle ethanol dispersion is preferably 0.4-0.6 mg / mL, and may be 0.5 mg / mL.

[0036] In the present invention, the holding time is preferably 85 to 95 minutes, and may also be 90 minutes. The method for separating the magnetic material adopts magnetic field force separation.

[0037] In the present invention, the morphology and structure of PLL-MNPs were characterized. The morphology of PLL-MNPs was observed by transmission electron microscopy. The particle size of PLL-MNPs was analyzed by Nano Measurer 1.2 software. The zeta potential and hydrodynamic size of PLL-MNPs were evaluated by dynamic light scattering (DLS). The static magnetic properties of dry MNPs were measured by vibrating sample magnetometer. The results showed that the zinc-doped magnetic nanoparticles (Zn 0.4 Fe 2.6 O4) are cubic in shape with an average diameter of about 60nm. PLL-MNPs have a high -1 The saturation magnetization intensity ensures that the prepared magnetic nanoparticles-labeled mesenchymal stem cells respond quickly to external magnetism, facilitating the long-term tracking of transplanted mesenchymal stem cells by magnetic resonance imaging technology. Compared with unmodified magnetic nanoparticles (MNPs), PLL-MNPs exhibit excellent hydrophilicity and dispersibility. The hydrodynamic diameter of MNPs is 490±50nm, and the hydrodynamic diameter of PLL-MNPs is 253±10nm. The surface modification of PLL improves the dispersibility of MNPs, which is achieved through electrostatic repulsion and steric hindrance generated by high-density amino groups. At the same time, the PLL improves the ability of MNPs to interact with MSCs. The surface modification of PLL has no effect on the cell morphology of MNPs.

[0038] After obtaining the poly-L-lysine-modified magnetic nanoparticles, the present invention treats mesenchymal stem cells with the poly-L-lysine-modified magnetic nanoparticles to obtain mesenchymal stem cells labeled with the magnetic nanoparticles.

[0039] In the present invention, the treatment concentration of the poly-L-lysine modified magnetic nanoparticles is preferably 0.08 to 0.12 μg / μL, and can be 0.1 μg / μL. The density of the mesenchymal stem cells is preferably 1×105 cells / dish~10×10 5 cells / dish, which can be 5×10 5 cells / dish. The mesenchymal stem cells are preferably passaged 3-4 times. The treatment time is preferably 10-14 hours, and may be 12 hours. The treatment temperature is preferably 36-38°C, and may be 37°C. The carbon dioxide concentration during the treatment is preferably 5%.

[0040] In the examples of the present invention, mesenchymal stem cells labeled with magnetic nanoparticles are relatively consistent with mesenchymal stem cells in terms of adipogenesis, osteogenesis and chondrogenesis, indicating that PLL-MNPs have no effect on the differentiation potential of MSC. The results of cell surface marker detection showed that MSC coated with PLL-MNPs expressed CD90 and CD105, but did not express CD34, CD11b, CD19, CD45 or HLA-DR, similar to unlabeled MSC. In addition, Prussian blue and eosin staining results showed that MSC engulfed blue nanoparticles containing iron oxide, and CCK-8 experiments showed that there was no significant difference in the number of cells between the MSC-MNPs group and the MSC group at 6h, 12h, 24h, and 48h. This indicates that MSC did not cause cytotoxicity after engulfing PLL-MNPs. Flow cytometry was used to evaluate the effect of PLL-MNPs on MSC apoptosis, and the results showed that MSC engulfment of PLL-MNPs did not affect cell apoptosis. At the same time, flow cytometric analysis of PI-stained cells showed that there was no significant change in the cell cycle between the MSC-MNPs group and the MSC group, indicating that the phagocytosis of PLL-MNPs by MSCs did not affect the cell cycle.

[0041] Since the mesenchymal stem cells labeled with magnetic nanoparticles have good magnetic responsiveness and can be used to track transplanted cells using magnetic resonance imaging technology, the present invention provides the use of the mesenchymal stem cells labeled with magnetic nanoparticles or the mesenchymal stem cells labeled with magnetic nanoparticles prepared by the preparation method in the preparation of drugs for cell transplantation therapy.

[0042] In the present invention, the diseases treated by the drug preferably include inflammation-related diseases, including neuroinflammation.

[0043] In the present invention, compared to unlabeled mesenchymal stem cells, the magnetic nanoparticle-labeled mesenchymal stem cells promoted the expression of anti-inflammatory cytokines and inhibited the secretion of pro-inflammatory factors. Furthermore, co-culturing MSC-MNPs with activated astrocytes / macrophages promoted the transition of reactive astrocytes / macrophages from a pro-inflammatory to an anti-inflammatory phenotype. This suggests that MNPs significantly enhance the anti-inflammatory function of MSCs, providing a new approach for the treatment of inflammatory diseases.

[0044] The following is a detailed description of the magnetic nanomaterial-guided anti-inflammatory enhanced mesenchymal stem cells, preparation method and application thereof provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Synthesis and identification of poly-L-lysine (PLL) modified magnetic nanoparticles (MNPs)

[0047] Zinc acetate hydrate (purity 97%) and oleic acid were purchased from Aladdin. Ferric acetate (purity 97%) and diphenyl ether (purity greater than 98%) were purchased from Sigma. Poly-L-lysine (PLL, molecular weight 4000) was purchased from MacLean (Shanghai). Zinc-doped magnetic nanoparticles (Zn) were synthesized by high-temperature pyrolysis based on the existing technology (Noh SH, Na W, Jang JT, Lee JH, Lee EJ, Moon SH, et al. Nanoscale Magnetism Control via Surface and Exchange Anisotropy for Optimized Ferrimagnetic Hysteresis. Nano Lett. 2012; 12: 3716-21.). 0.4 Fe 2.6 To impart a positive surface charge to the MNPs, they were modified with PLL via a surface double exchange process, enabling interaction with MSCs. The procedure was as follows: 5 mg of MNPs were dispersed in 10 mL of ethanol to obtain an MNP ethanol solution. Then, 50 mg of PLL was dissolved in 5 mL of deionized water to obtain a PLL aqueous solution. The MNP ethanol solution was exposed to an ultrasonic probe at 30% amplitude. The PLL aqueous solution was added dropwise to the MNP ethanol solution and sonicated in an ice bath at 120 W and 20 kHz for 90 minutes. Finally, the resulting product (PLL-MNPs) was washed three times with deionized water and collected by magnetic separation.

[0048] The morphology of the MNPs was observed using a transmission electron microscope (TEM, model JEM-1230, JEOL Ltd.). Particle size was statistically analyzed using Nano Measurer 1.2 software. The zeta potential and hydrodynamic size of the MNPs and PLL-MNPs were assessed using dynamic light scattering (DLS, Mastersizer 2000, Malvern, Worcestershire, UK). The static magnetic properties of the dried MNPs were measured using a vibrating sample magnetometer (VSM, Lake Shore 7407, USA).

[0049] Zinc-doped magnetic nanoparticles (Zn 0.4 Fe 2.6 O4) is in the shape of a cube with an average diameter of about 60nm ( Figure 1 These MNPs were modified with poly-L-lysine (PLL) to enhance their biocompatibility. PLL-modified MNPs (PLL-MNPs) exhibited a high biocompatibility of up to 76 emu g -1 The saturation magnetization ( Figure 1 (B), indicating their rapid response to external magnetic fields. After surface modification, MNPs exhibited excellent hydrophilicity and dispersibility. The hydrodynamic diameters of MNPs and PLL-MNPs were 490 ± 50 nm and 253 ± 10 nm, respectively. Figure 1 The zeta potentials of MNPs and PLL-MNPs were -6.14 ± 0.32 mV and 34.82 ± 0.52 mV, respectively ( Figure 1 (D) Modification with PLL significantly improved the dispersibility of MNPs, which was achieved through electrostatic repulsion and steric hindrance caused by the high density of amino groups. In addition, PLL endowed MNPs with the ability to interact with MSCs.

[0050] Example 2

[0051] Culture and identification of mesenchymal stem cells (MSCs)

[0052] 1. Materials and Methods

[0053] Human umbilical cord-derived mesenchymal stem cells (MSCs) were purchased from Celera Stem Cell Science and Technology Co., Ltd. (Guangzhou, China). MSCs were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (Gibco). Cells were maintained at 37°C and 5% carbon dioxide in a standard incubator.

[0054] 1. To evaluate cell surface markers and directed differentiation potential, surface antigens were analyzed by flow cytometry. Flow cytometry was used to monitor cell surface antigens, cell apoptosis, and cell cycle distribution. Cell surface antigens were analyzed using a human MSC analysis kit (BD Biosciences, USA). The specific procedure was as follows: MSCs (5×10 cells) treated overnight with PLL-MNPs (0.1 μg / μL) were 5 Cells were collected and incubated with the added antibodies for 30 min at room temperature in the dark. After three washes, cells were analyzed by flow cytometry (Beckman Coulter, CA, USA). Cell apoptosis and cell cycle were assessed using apoptosis and cell cycle analysis kits (Invitrogen). For apoptosis detection, 1×10 6 cells and stained with Annexin V-FITC and propidium iodide (PI). For cell cycle analysis, 1×10 6 The cells were fixed with 70% ethanol and stained with PI. The data were analyzed using FlowJo software.

[0055] 2. To evaluate the differentiation potential of MSCs, passage 3-4 MSCs were treated with PLL-MNPs (0.1 μg / μL) overnight and then induced to differentiate into osteoblasts, adipocytes, and chondrocytes using complete induction and differentiation medium (OriCell, USA). Differentiation was confirmed by staining: Alizarin red staining (for osteoblasts), Oil Red O staining (for adipocytes), and Alcian blue staining (for chondrocytes).

[0056] 3. Real-time quantitative polymerase chain reaction (RT-qPCR)

[0057] Total RNA was extracted from cells using TriZol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA using a reverse transcription system (Promega). The reaction system is shown in Table 1. The reaction program was 37°C for 15 min; 85°C for 5 seconds (inactivation of reverse transcriptase), and incubation at 4°C. Subsequently, 1 μL of cDNA was used for the Eco TM Real-time polymerase chain reaction system (Illumina) was used for polymerase chain reaction amplification. The sequences of primers used are shown in Table 2, the reaction system is shown in Table 3, and the reaction procedure is shown in Table 4. -ΔΔCt The relative expression levels of target genes were evaluated by PCR.

[0058] Table 1 Transcription reaction system

[0059] Reagents Dosage 5xPrimeScriptRTMasterMix(PerfectRealTime) 2.0 μl Total RNA 500ng <![CDATA[RNaseFreedH2O]]> Add to 10 μl

[0060] Table 2 Primers for qPCR detection

[0061]

[0062]

[0063] Table 3 qPCR reaction system

[0064] Reagents Dosage TBGreenPremixExTaqII(2×) 12.5 μl PCRForwardPrimer (10 μM) 1.0 μl PCR Reverse Primer (10 μM) 1.0 μl System 1 reaction solution (cDNA solution) 2.0 μl sterile water 8.5 μl total 25 μl

[0065] Table 4 qPCR reaction conditions

[0066]

[0067] 5. Cell staining

[0068] Mesenchymal stem cell (MSC) staining: MSCs treated with MNPs (0.1 μg / μL) overnight were stained using a Prussian blue staining kit (Abcam, ab150674) and eosin (Solarbio, G1100) according to the manufacturer's instructions.

[0069] Astrocyte staining: Astrocytes were incubated with rat anti-glial fibrillary acidic protein (GFAP; 13-0300, Thermo Fisher Scientific) overnight at 4°C. Subsequently, the cells were incubated with the corresponding secondary antibodies for 1 h at room temperature.

[0070] 6. Cytokine Analysis

[0071] The supernatants of MSCs treated with or without PLL-MNPs for 24 h were collected for cytokine analysis. Cytokine levels were determined using the Bio-Plex system (Bio-Rad) and a 27-linked cytokine kit (M500KCAFOY, Bio-Rad, USA). The kit contains antibodies against the following cytokines: basic fibroblast growth factor (FGF) basic), eosinophil chemotactic factor (Eotaxin), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), regulated cytokines expressed and secreted by activated normal T cells (RANTES), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), granulocyte / macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor antagonist (IL-1ra), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3) , interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (p70) (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17A (IL-17A), interferon-γ-induced protein 10 (IP-10), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF). Three samples were analyzed for each experimental group.

[0072] 7. Data-Independent Acquisition (DIA) Proteomic Analysis

[0073] Total protein was extracted from MSCs treated with or without PLL-MNPs using a radioimmunoprecipitation assay (RIPA) lysate. Protein sequencing and library construction were performed by Guangzhou Kidio. Data analysis was performed using OmicShare and Omicsmart tools. Criteria for identifying differentially expressed proteins included a coefficient of variation less than 0.5, a mean ratio change greater than or equal to 1.5 or less than or equal to 0.67, and a P value less than 0.05 as determined by a Student's t-test.

[0074] 8. Protein Blot

[0075] Proteins extracted from MSC-MNPs or MSCs were separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a 0.45 μm polyvinylidene difluoride membrane. The following primary antibodies were used: anti-apolipoprotein A1 (APOA1, 1:1000, Zenbio, 680033), anti-hepatocyte growth factor activator (HGFAC, 1:1000, Zenbio, 670841), anti-chitinase 3-like protein 1 (CHI3L1, 1:1000, Zenbio, 820410), and anti-TNF receptor-associated factor 3 (TRAF3, 1:1000, Zenbio, 160776). Secondary antibodies used were horseradish peroxidase-anti-rabbit IgG (1:10,000, Abcam, ab6721) and horseradish peroxidase-anti-mouse IgG (1:10,000, Vector Laboratories). Immunoreactivity was detected using a chemiluminescence detection kit (Epizyme, SQ202). Image via ChemiDoc TM The images were captured using the Touch imaging system (Vilber, France), and the signals were quantified using ImageJ software. All data were normalized to the mean ratio of the corresponding control.

[0076] 9. Macrophage Activation

[0077] Mouse monocytic macrophage leukemia cell line (RAW 264.7) was treated with lipopolysaccharide (LPS, 200 ng / mL, Thermo Fisher Scientific) for 24 h.

[0078] 10. Culture and activation of primary spinal cord astrocytes

[0079] Astrocytes were extracted from the spinal cords of C57BL / 6 mice 1-3 days after birth. Spinal cord tissue was isolated from mice and then trypsinized (0.15% trypsin, 7 min, 37°C). The isolated spinal cord cells were suspended in astrocyte culture medium, which consisted of DMEM-F12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). When the cells reached 90% confluence, the culture flask was shaken at 200 rpm for 20 hours to remove neurons, microglia, and oligodendrocytes. The purity of astrocytes was assessed by GFAP immunostaining and DAPI nuclear staining, and only astrocytes with a purity greater than 95% were used in subsequent experiments.

[0080] Astrocyte activation was performed using supernatant from LPS-treated macrophages. RAW 264.7 cells were treated with lipopolysaccharide (LPS, 200 ng / mL, Thermo Fisher Scientific) for 24 h. The supernatant was then collected and added to astrocyte cultures to induce activation.

[0081] 11. Identification of astrocyte / macrophage polarization phenotype

[0082] MSCs (5 × 10 3 cells / mL) were seeded into the upper chamber, and the culture medium was 10% fetal bovine serum-DMEM / F12 (Gibco). The lower chamber was: purified astrocytes (5×10 4 cells / well) or RAW264.7 (seeded at a density of 5×10 4 Astrocytes or RAW264.7 cells were seeded in 6-well plates at a density of 10 cells / well. Prior to addition of the upper chamber, astrocytes or RAW264.7 cells were cultured with LPS-treated RAW264.7 supernatant for 12 hours. After 24 hours of co-culture in the upper and lower chambers, astrocytes were harvested for RNA extraction. Polarization phenotypes were characterized by RT-qPCR.

[0083] 2. Results

[0084] 1. Magnetic nanoparticles (PLL-MNPs) do not affect the differentiation potential or expression of intrinsic surface markers of mesenchymal stem cells (MSCs)

[0085] The International Society for Cellular Therapy (ISCT) has set the following minimum criteria for MSCs: (1) MSCs must adhere to uncoated plastic culture dishes under standard culture conditions; (2) MSCs must simultaneously express CD105, CD73, and CD90, but not CD45, CD34, CD14, CD11b, CD79, CD19, or lineage markers such as HLA-DR; (3) MSCs must be able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro. MSCs that phagocytized MNPs were evaluated according to the International Society for Cellular Therapy (ISCT) criteria for MSC identification. The results showed that there was no significant morphological change in MSCs after phagocytizing PLL-MNPs (MSC-MNPs, 0.1 μg / μL, Figure 1 In addition, the adipogenic, osteogenic, and chondrogenic abilities of MSC-MNPs were comparable to those of untreated MSCs ( Figure 1 (F) Cell surface markers were also examined, and the results showed that MSC-MNPs expressed CD90 and CD105, but not CD34, CD11b, CD19, CD45, or HLA-DR, similar to untreated MSCs. These results suggest that MSC phagocytosis of MNPs does not affect their differentiation potential or the expression of intrinsic cell surface markers.

[0086] 2. Magnetic nanoparticles (MNPs) phagocytosis of mesenchymal stem cells (MSCs) does not affect their proliferation, apoptosis and cell cycle.

[0087] The effects of MSC phagocytosis of PLL-MNPs on cell proliferation, apoptosis, and cell cycle were examined. Prussian blue and eosin staining showed that blue nanoparticles containing iron oxide were phagocytosed by MSCs ( Figure 2 We evaluated cell proliferation by CCK-8 assay, and there was no significant difference between the MSC-MNPs group and the MSC group at 6h, 12h, 24h, and 48h ( Figure 2 These results indicate that MSC phagocytosis of PLL-MNPs did not cause cytotoxicity. To investigate whether PLL-MNPs affect cell apoptosis, FACS analysis was performed on MSCs treated with PLL-MNPs for 24 h. There was no significant difference in the PI and Annexin V double-positive cell population between the MSC-MNPs group and the MSC group ( Figure 2 C, D), indicating that the phagocytosis of MNPs did not affect cell apoptosis. The cell cycle distribution of MSCs after 24 h of PLL-MNPs treatment was also examined. Similarly, no significant changes were observed between the MSC-MNPs group and the MSC group ( Figure 2 (E, F).

[0088] 3. MSC phagocytosis of MNPs promotes its anti-inflammatory function

[0089] After 24 h of PLL-MNPs treatment, the supernatant of MSCs was collected and the cytokine levels were detected using the Bio-plex assay ( Figure 3 Compared with the MSC group, the levels of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were significantly decreased in the MSC-MNPs group ( Figure 3 In contrast, the levels of anti-inflammatory cytokines interleukin-10 (IL-10), interleukin-13 (IL-13), regulatory cytokines expressed and secreted by activated normal T cells (RANTES), and vascular endothelial growth factor (VEGF) were significantly increased in the MSC-MNPs group ( Figure 3 These results indicate that MSC phagocytosis of MNPs promoted the secretion of anti-inflammatory factors while reducing the secretion of pro-inflammatory factors.

[0090] 4. MSC phagocytosis of MNPs changes proteomic characteristics

[0091] To further explore the potential molecular mechanism by which PLL-MNPs affect MSC cytokine secretion, data-independent acquisition (DIA) proteomic analysis was performed using proteins extracted from cultured MSCs treated with or without PLL-MNPs. 237 differentially expressed proteins (DEPs; FDR < 0.05, |FoldChange| > 1.5) were identified between the MSC and MSC-MNPs groups, among which anti-inflammatory and anti-oxidative stress-related proteins (such as apolipoprotein A1 (APOA1), interleukin-13 (IL-13), ferritin light chain (FTL), and anti-hepatocyte growth factor activator (HGFAC)) were upregulated ( Figure 4 In contrast, pro-inflammatory proteins such as cartilage glycoprotein 39 (CHI3L1), bromodomain-containing protein 3 (BRD3), and TNF receptor-associated factor 3 (TRAF3) were downregulated ( Figure 4 To verify the results of DIA, we performed Western blotting experiments, and APOA1 and HGFAC were confirmed to be upregulated in the MSC-MNPs group, while CHI3L1 and TRAF3 were downregulated in the MSC-MNPs group ( Figure 4 Middle C).

[0092] 5. The anti-inflammatory function of MSC-MNPs promotes the transformation of reactive astrocytes / macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype

[0093] The above results indicate that MSC-MNPs have anti-inflammatory function. Based on this, this embodiment also designed an experiment involving the co-culture of MSC-MNPs and reactive astrocytes / macrophages. Reactive astrocytes undergo complex morphological, molecular and functional changes in response to injury. In early studies, astrocytes / macrophages were divided into pro-inflammatory and anti-inflammatory phenotypes based on gene expression, secreted cytokines and function. Using a similar classification method, the effects of MSC-MNPs on the response of astrocytes and macrophages in vitro were further studied, focusing on the phenotype. Cultured reactive astrocytes or macrophages were co-cultured with MSC for 24h ( Figure 5 Cells were collected for RT-qPCR analysis. In the MSC-MNPs group, the levels of astrocyte pro-inflammatory phenotype genes C3 and Serping1 were significantly reduced, while the levels of anti-inflammatory phenotype genes Tm4sf1 and Ptx3 were significantly higher than those in the MSC group ( Figure 5 Middle C); These results suggest that MSC-MNPs promote the phenotypic transition of reactive astrocytes, thereby alleviating neuroinflammation.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mesenchymal stem cell labeled with magnetic nanoparticles, characterized in that: The mesenchymal stem cells are coated with magnetic nanoparticles modified with poly-L-lysine.

2. The mesenchymal stem cells labeled with magnetic nanoparticles according to claim 1, characterized in that: The magnetic nanoparticles are zinc-doped magnetic nanoparticles; The molecular formula of the zinc-doped magnetic nanoparticles is Zn 0.4 Fe 2.6 O4.

3. The mesenchymal stem cells labeled with magnetic nanoparticles according to claim 1, characterized in that: The molecular weight of the poly-L-lysine is 1800-10000.

4. The method for preparing mesenchymal stem cells labeled with magnetic nanoparticles according to any one of claims 1 to 3, characterized in that: The following steps are involved: Under vibration conditions, a poly-L-lysine solution is added to an ethanol dispersion of magnetic nanoparticles for 80 to 100 minutes, and the magnetic material is separated to obtain poly-L-lysine-modified magnetic nanoparticles; The poly-L-lysine-modified magnetic nanoparticles are used to treat mesenchymal stem cells to obtain mesenchymal stem cells labeled with the magnetic nanoparticles.

5. The preparation method according to claim 4, characterized in that: The mass ratio of poly-L-lysine to magnetic nanoparticles is (9-11):

1.

6. The preparation method according to claim 4, characterized in that: The volume ratio of the poly-L-lysine solution to the magnetic nanoparticle ethanol dispersion is (0.8-1.2): (1.8-2.2); The concentration of the poly-L-lysine solution is 8-12 mg / mL; The concentration of the magnetic nanoparticle ethanol dispersion is 0.4-0.6 mg / mL.

7. The preparation method according to claim 4, characterized in that: The treatment concentration of the poly-L-lysine modified magnetic nanoparticles is 0.08-0.12 μg / μL.

8. The preparation method according to any one of claims 4 to 7, characterized in that The treatment time is 10 to 14 hours; the treatment temperature is 36 to 38°C.

9. Use of the mesenchymal stem cells labeled with magnetic nanoparticles according to any one of claims 1 to 3 or the mesenchymal stem cells labeled with magnetic nanoparticles prepared by the preparation method according to any one of claims 4 to 8 in the preparation of a drug for cell transplantation therapy.

10. The use according to claim 9, characterized in that: Diseases treated by the drug include inflammation-related diseases.

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