Construction method of magnetic dendritic cell micro-robot delivery model for spinal cord injury research

Abstract

The invention belongs to the field of biomedical engineering and neural restoration, and particularly relates to a construction method of a magnetic dendritic cell micro-robot delivery model for spinal cord injury research. The method comprises the following steps: (1) preparing a magnetic dendritic cell micro-robot; (2) delivering the magnetic dendritic cell micro-robot into an experimental animal body through caudal vein injection; (3) targeting the magnetic dendritic cell micro-robot to a spinal cord injury part under the driving of an external rotating magnetic field; and (4) evaluating the pathological mechanism or the drug intervention effect of the spinal cord injury. Through the regulation and control effect of an external rotating magnetic field, active targeted positioning of the magnetic dendritic micro-robot in a spinal cord injury area can be achieved, and a precise delivery path is provided for mechanism research of central nervous system injury.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical engineering and neural repair, and more specifically to a method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research. Background Art

[0002] Spinal cord injury is a severe injury to the central nervous system, often leading to neuronal loss and axonal severance, resulting in lifelong paralysis. Current clinical treatments primarily target secondary spinal cord injury, using methods such as controlling inflammation and administering neurotrophic drugs. However, these treatments lack targeted therapy, leading to systemic side effects and limited localized drug concentrations, resulting in limited efficacy. Studies have shown that immune cells such as Treg cells and M2 macrophages play an important role in neuroprotection and tissue repair.

[0003] Dendritic cells are professional antigen-presenting cells that can promote the repair of spinal cord injuries by regulating local immune responses and building a protective immune microenvironment. Studies have shown that dendritic cells can play an important role in injury repair by secreting neurotrophic factors (such as BDNF and NT-3) and regulating the transformation of T cell subtypes to neuroprotective types (such as Th2 and Treg cells). In recent years, magnetically controlled microrobots have shown great potential in precision drug delivery. Magnetic microrobots have the characteristics of remote control and efficient penetration, and can achieve precise navigation in complex biological environments. Therefore, combining dendritic cells with magnetically controlled microrobots to achieve efficient targeted delivery of neurotrophic factors and local immune regulation is an innovative strategy. Summary of the Invention

[0004] To overcome the shortcomings of the existing technology, the present invention provides a method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research. The beneficial effect is that through the regulation of an external rotating magnetic field, the magnetic dendritic microrobot can be actively targeted in the spinal cord injury area, providing a precise delivery path for the study of the mechanism of central nervous system injury.

[0005] A method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research, the method comprising:

[0006] (1) Preparation of magnetic dendritic cell microrobots;

[0007] (2) delivering the magnetic dendritic cell microrobot into the body of an experimental animal via tail vein injection;

[0008] (3) Driven by an external rotating magnetic field, the magnetic dendritic cell microrobot is targeted to the spinal cord injury site;

[0009] (4) Evaluate the pathological mechanism of spinal cord injury or the effect of drug intervention.

[0010] The step (1) comprises:

[0011] A. Preparation of antigen-loaded magnetic nanoparticles (A91@NPs);

[0012] B. A91@NPs were coated with Escherichia coli membrane (EM) to form EM@NPs;

[0013] C. Co-culture EM@NPs with dendritic cells to form magnetic dendritic cell microrobots;

[0014] D. After preparation, the magnetic dendritic cell microrobot is tested.

[0015] The step (4) comprises:

[0016] A. Regulate the ratio of T cell subtypes and inhibit inflammatory response;

[0017] B. Provide local high concentrations of neurotrophic factors to promote axon regeneration and myelin repair.

[0018] The magnetic nanoparticles comprise the following components:

[0019] A. The core is composed of poly(lactic-co-glycolic acid) (PLGA)-coated Fe3O4 superparamagnetic nanoparticles, which provide magnetic responsiveness;

[0020] B. Surface coating with polyethyleneimine (PEI) for loading allosteric peptide ligand MBP 87-99 A 91 (A91), constituting A91@NPs;

[0021] C. After A91@NPs are coated on the EM surface, it can serve as an immune adjuvant and increase the A91 loading rate and encapsulation efficiency to obtain EM@NPs.

[0022] The external rotating magnetic field has an intensity range of 5-15 mT and a frequency of 1-10 Hz, and is used to precisely control the movement of the magnetic dendritic cell microrobot.

[0023] The dendritic cells enhance their antigen presentation ability by phagocytosing EM@NPs and are able to secrete neurotrophic factors BDNF and NT-3.

[0024] Dendritic cell microrobots, consisting of the following parts:

[0025] A. dendritic cells;

[0026] B. Magnetic nanoparticles containing antigen (A91) and E. coli membrane (EM).

[0027] A method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research. The dendritic cell microrobot is used for analyzing immune regulatory mechanisms and evaluating the efficiency of neurotrophic factor delivery in basic research on spinal cord injury.

[0028] Method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research:

[0029] The dendritic cell microrobot is used in the following experimental scenarios:

[0030] Luminex and single-cell analysis were used to detect the expression of inflammatory factors and the distribution characteristics of cell subtypes in the damaged area;

[0031] Immunofluorescence and transmission electron microscopy were used to observe structural changes during tissue repair;

[0032] The BMS mouse scoring system was used to quantitatively evaluate the correlation between the motor function of experimental animals and the delivery effect of the microrobot.

[0033] The present invention provides a method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research, with the following main features:

[0034] 1. Combination of dendritic cells and microrobots: Dendritic cells form dendritic cell microrobots (DC robots) by engulfing magnetic nanoparticles modified with the allosteric peptide ligand A91 (EM@NPs), giving them the ability to move under magnetic control.

[0035] 2. Magnetic controlled precise delivery: Through an external rotating magnetic field, the magnetic dendritic microrobot can be efficiently controlled to actively target the spinal cord injury area.

[0036] 3. Dual-function design: The magnetic dendritic microrobot has the dual functions of immune regulation and delivery of neurotrophic factors.

[0037] 4. Biocompatibility and safety: The magnetic dendritic microrobot material uses PLGA, PEI and Fe3O4 nanoparticles to ensure biodegradability and low toxicity in the body.

[0038] 5. Remodeling of the local immune microenvironment: by inducing CD4 + The T cell subtype shifts toward Th2 and Treg cells, regulates the conversion of local macrophage and microglia subtypes toward protective ones, and reduces inflammatory cytokine levels. This invention can establish a neuroprotective immune microenvironment in the local area of ​​spinal cord injury, promoting axonal regeneration and neurological function recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0040] Figure 1 Schematic diagram of the EM@NPs structure;

[0041] Figure 2 Prepare a flow chart for DC robots;

[0042] Figure 3 Surface charge detection, magnetic testing, and fluorescence labeling technology were used to verify the endocytic effect diagram;

[0043] Figure 4 This is the DC robots motion trajectory diagram;

[0044] Figure 5 This is the result of the biocompatibility experiment of cck-8;

[0045] Figure 6 is the target localization map (fluorescent staining);

[0046] Figure 7 This is a Luminex technology and single-cell RNA sequencing diagram;

[0047] Figure 8 Immunofluorescence was used to evaluate scar formation and nerve fiber remodeling;

[0048] Figure 9 This is a transmission electron microscopy image of the myelin regeneration effect;

[0049] Figure 10 A diagram for evaluating motor function recovery effects for BMS scoring and behavioral analysis;

[0050] Figure 11 This is a safety assessment effect diagram. DETAILED DESCRIPTION

[0051] Example 1:

[0052] Figure 2 Preparation process of DC robots: The preparation and detection of the present invention includes the following steps:

[0053] 1. Preparation of A91@NPs: Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared using an emulsification method. Fe3O4 nanoparticles were then encapsulated to impart magnetic properties. Polyethylenimine (PEI) was then added to the PLGA NPs to impart a positive charge using a self-assembly method. The A91 peptide (A91@NPs) were synthesized by electrostatic adsorption.

[0054] 2. Preparation of EM@NPs: A91@NPs were coated with Escherichia coli (E. coli) membranes to form magnetic nanoparticles with a core-shell structure.

[0055] Transmission electron microscopy (TEM) was used to observe the spherical structure and coating thickness of EM@NPs, and the particle size was approximately 191-197 nm;

[0056] 3. Loading of dendritic cells: EM@NPs were co-cultured with dendritic cells (DCs) for 6 hours to allow them to enter the DC cells through endocytosis;

[0057] Use fluorescence labeling technology to verify the endocytosis effect: for example, FITC-labeled EM@NPs were co-cultured with DCs, and the localization of nanoparticles inside DCs was observed using confocal laser scanning microscopy (CLSM);

[0058] Figure 6 For targeted positioning (fluorescence staining), use fluorescent labeling technology to verify the endocytic effect:

[0059] Six hours after injection, fluorescence was present almost throughout the body in the Free-DiR group. In contrast, fluorescence intensity at the lesion site was very low at one and six hours after injection. In the DC group, fluorescence intensity at the lesion site increased slightly compared to the Free-DiR group. Similar to the DC group, fluorescence in the DC Robot (M-) group showed a higher fluorescence signal at the lesion site compared to the Free-Bronchial group.

[0060] 4. After preparation, the magnetic dendritic cell microrobots (DC robots) were tested:

[0061] Surface charge detection: The surface charge of the DC robot was measured using a Zeta potential meter;

[0062] Magnetic test: saturation magnetization was measured by vibrating sample magnetometer (VSM);

[0063] Use fluorescence labeling technology to verify the endocytosis effect: for example, FITC-labeled EM@NPs were co-cultured with DCs, and the localization of nanoparticles inside DCs was observed using confocal laser scanning microscopy (CLSM);

[0064] Biocompatibility assessment: The effect of the DC robot on cell viability was tested using a cell counting kit (CCK-8) to ensure its non-cytotoxicity;

[0065] Figure 3 Surface charge detection, magnetic testing, and fluorescent labeling technology are used to verify the endocytosis effect:

[0066] Before and after charging, the surface charge of the nanoparticles changed from −8.717 ± 3.298 mV (A91@NPs) to −14.42 ± 4.232 mV (EM@NPs), which was close to the surface charge of pure EM vesicles (−15.12 ± 2.882 mV);

[0067] According to the magnetization curve, EM@NPs exhibited typical superparamagnetic behavior, with a saturation magnetization value of 31.69emu·g -1 ;

[0068] EM@NPs were first localized in lysosomes after being taken up by DCs.

[0069] Figure 5 The results of CCK-8 biocompatibility test are as follows:

[0070] The effect of DC robots on cell viability was detected by a cell technology kit (CCK-8), and the results showed that there was no significant difference in DC activity between the DC robot group and the pure DC group.

[0071] The present invention utilizes a magnetic control system, a navigation system, and the chemotactic properties of DC robots to control DC robots:

[0072] The magnetic control system is a Helmholtz coil system consisting of three pairs of orthogonal coils, which can generate a uniform rotating magnetic field (RMF) with a magnetic field strength range of 5-15mT and a frequency range of 1-25Hz.

[0073] The navigation system first uses an optical microscope to observe the DC robot's trajectory in real time, completing visual tracking tasks. Simultaneously, a closed-loop feedback system combines real-time position tracking with autonomous path planning, enabling high-precision control of the DC robot's navigation path.

[0074] Figure 4 This is the DC robots motion trajectory diagram:

[0075] The DC robots completed five-pointed star trajectory motion, right-angle turning motion and simulated blood vessel channel experiments (DC robots can pass through microchannels smoothly), indicating that DC robots can perform precise motion control.

[0076] The uniform rotating magnetic field generated by the Helmholtz coil system is used to achieve multi-mode motion, including linear motion, curved motion and complex trajectories (such as pentagram trajectory).

[0077] The chemotactic characteristics of DC robots are controlled as follows: after SCI damage, the main chemotactic factor for DC migration is CCL2. DC robots express the CCL2 receptor (CCR2), so DC robots can actively move to areas with high CCL2 concentrations (SCI damaged areas). Combined with the above-mentioned navigation system, DC robots can more accurately target the SCI damaged areas.

[0078] The present invention established a mouse spinal cord injury model: under general anesthesia and aseptic conditions, laminectomy was performed to expose the T10 spinal cord segment. A heavy impact device was used to induce spinal cord contusion: a 5.0g rod was dropped from a height of 5.0cm to hit the exposed spinal cord. Then, the muscle layer and skin layer were carefully sutured in sequence. Two hours after spinal cord injury, mice were injected intravenously with 1×10 6 DCs or DC robots (dissolved in 100 μL PBS).

[0079] The present invention performs targeted delivery of DC robots: a self-made rotating magnetic field device is used to guide the DC robots to be delivered to the injured area, and the magnetic field is continuously applied for 30 minutes.

[0080] Fluorescence imaging and magnetic resonance imaging (MRI) were used to evaluate the distribution and targeting of DC robots in vivo.

[0081] Biological evaluation effect:

[0082] Luminex technology was used to detect the levels of inflammatory factors in the spinal cord injury area:

[0083] Upregulate the concentration of pro-inflammatory factors (IFN-γ, TNF-α) and upregulate the concentration of anti-inflammatory factors (such as IL-4, IL-10).

[0084] Using single-cell RNA sequencing (scRNA-seq) technology, we constructed scRNA-seq libraries using microplates to analyze the proportions and subtypes of local cells in the spinal cord:

[0085] The proportions of T cells, oligodendrocytes, and mononuclear DCs increased, while the proportions of microglia, macrophages, and astrocytes decreased. Expression of genes encoding Th2 and Treg cell subtypes was upregulated (Foxp3 and Ikzf2), while expression of genes encoding Th1 and CD8+ T cell subtypes was downregulated (Cd8a, Ifng, and Gata3). Expression of genes encoding pro-inflammatory macrophages and microglia (Cxcl2, Il1a, Nfkbiz, and Tnf) was downregulated, while expression of genes encoding anti-inflammatory macrophages and microglia (Arg1) was upregulated.

[0086] Figure 7 For Luminex technology, single-cell RNA sequencing:

[0087] Protein level analysis using Luminex was performed to assess cytokine levels in spinal cord samples from four orthogonal groups at two time points: 3 and 7 days post-injury (dpi). At 3 dpi, IFN-γ and GM-CSF levels were significantly downregulated in the DC robot (M+) group compared with the PBS group. Furthermore, IL-4 and IL-10 expression increased in the DC and DC robot (M-) groups compared with the PBS group, with a significant increase in IL-4 and IL-10 expression in the DC robot (M+) group. At 7 dpi, IL-1β, IL-12p70, IFN-γ, and GM-CSF levels were significantly downregulated in the DC robot (M+) group compared with the PBS group. TNF-α levels were significantly downregulated in the DC robot (M+) group compared with the DC group. IL-4 and IL-10 levels were significantly increased in the DC robot (M+) group compared with the PBS group. Seven days after immunization, the levels of IL-12p40, IL-6, and IL-17 in the DC robots (M+) group were also downregulated compared with the PBS group, but there was no significant difference.

[0088] Subpopulations were extracted from the total dataset to further investigate the heterogeneity of T cells, microglia, macrophages, OPCs, and astrocytes between the PBS and DC robot (M+) groups. In the T cell subsets, the DC robot group showed lower expression levels of Cd8a, Ifng, and Gata3 than in the PBS group. The DC robot group showed higher expression levels of Foxp3 and Ikzf2 than in the PBS group. In the macrophage and microglia subsets, the DC robot group showed lower expression levels of the pro-inflammatory markers Cxcl2, Illa, Nfkbiz, and Tnf than in the PBS group. The DC robot group showed higher expression levels of the anti-inflammatory marker Argl than in the PBS group. Also in the microglia subset, the DC robot group showed lower expression levels of the demyelination markers Apoe and Gpnmb than in the PBS group. In the OPC subset, the DC robot group showed higher levels of expression of the myelin regeneration marker Tnr than in the PBS group ( Figure 6 Among astrocyte subpopulations, the DC robot group showed lower expression levels of the scarring marker Aqp4 than the PBS group. These data suggest that DC robots alleviate neuroinflammation and modulate the microenvironment to protect against SCI.

[0089] BDNF and NT-3 expression levels were detected by ELISA:

[0090] The levels of BDNF and NT-3 in the DC robot group were significantly higher than those in the control group at 3, 7, and 14 days after injury.

[0091] Immunofluorescence was used to evaluate scar formation and nerve fiber remodeling; transmission electron microscopy was used to evaluate myelin regeneration:

[0092] Figure 9 For transmission electron microscopy assessment of remyelination effects:

[0093] Representative images of regenerating myelinated axons captured by TEM at 35 dpi.

[0094] Figure 8 For immunofluorescence assessment of scar and nerve fiber remodeling:

[0095] The nerve fibers in the control group and the DC robot (M+) group were tightly connected and regular, while the fibers in the other treatment groups were only partially connected.

[0096] The number of nerve fiber trajectories and the average length of trajectories in the DC robot (M+) group were higher than those in the other treatment groups and were close to those in the healthy control group.

[0097] After 35 days, the number of nerve fibers and myelin thickness in the DC robot group were significantly higher than those in the other groups.

[0098]

[0099]

[0100] Diffusion tensor imaging (DTI), a magnetic resonance imaging (MRI) technique, was used to quantitatively analyze the trajectories of nerve bundles at the damaged site.

[0101] Motor function recovery was assessed using BMS score and gait analysis:

[0102] Figure 10 Figure 2. Evaluation of motor function recovery effect using BMS score and gait analysis.

[0103] Behavioral data of SCI mice were collected at 35 dpi using the SMART 3.0 video tracking system. In the open field test, 2D and 3D activity maps showed that the DC Robot (M+) group had significantly longer total distance traveled, distance traveled within the open field, average speed, and number of entries into the open field compared to the other groups. In the elevated plus maze test, the DC Robot (M+) group had significantly longer total distance traveled, distance traveled within the open arms, duration of stay in the open arms, and number of entries than the other groups. In both tests, the DC Robot (M+) group displayed lower immobility time.

[0104] The average BMS score of the DC robot group reached 5.20 after 35 days, which was significantly higher than that of the control group.

[0105] Open field and elevated plus maze tests were used to verify the recovery of motor function.

[0106] Safety assessment: Histopathological analysis (HE staining) of major organs (heart, liver, spleen, lung, and kidney) was performed.

[0107] Detect liver function indicators (such as LDH, ALT) to verify biosafety.

[0108] Figure 11 Safety assessment effect diagram:

[0109] In vivo histopathological examination of the heart, liver, spleen, lungs, and kidneys was performed using HE staining at 35 dpi. The results showed that the morphology and structure of the main organs in the DC robot (M+) treatment group were normal, without any obvious signs of damage. The activity levels of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were evaluated to determine liver function. These findings indicate that the liver function of mice treated with DC robot (M+) was comparable to that of the other groups. These results indicate that DC robot has no obvious biotoxicity to mice.

[0110] Result analysis:

[0111] Experimental results showed that the SCI mice treated with the DC robot experienced a shift in the local immune microenvironment of the injured area toward a protective state, with a significant decrease in inflammatory factors and a significant increase in nerve growth factor concentrations. Transmission electron microscopy revealed significantly better axonal regeneration and myelination in the treated group compared to the control group.

[0112] in conclusion:

[0113] This study combines dendritic cells with magnetically controlled microrobot technology to construct a targeted delivery model for studying the pathology of acute spinal cord injury. This model enables precise localization of magnetic dendritic cell microrobots in the early stages of injury. By delivering antigens and neurotrophic factors, it is used to study the cellular mechanisms of axonal regeneration after spinal cord injury and assess its relevance to neurological function. This technology provides a novel experimental tool for basic research on traumatic central nervous system diseases, exploring potential targets for inflammation regulation and neural repair, and has important biomedical research value.

Claims

1. A method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research, characterized in that: The method includes: (1) Preparation of magnetic dendritic cell microrobots; (2) delivering the magnetic dendritic cell microrobot into the body of an experimental animal via tail vein injection; (3) Driven by an external rotating magnetic field, the magnetic dendritic cell microrobot is targeted to the spinal cord injury site; (4) Evaluate the pathological mechanism of spinal cord injury or the effect of drug intervention.

2. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 1, characterized in that: The step (1) comprises: A. Preparation of antigen-loaded magnetic nanoparticles (A91@NPs); B. A91@NPs were coated with Escherichia coli membrane (EM) to form EM@NPs; C. Co-culture EM@NPs with dendritic cells to form magnetic dendritic cell microrobots; D. After preparation, the magnetic dendritic cell microrobot is tested.

3. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 1, characterized in that: The step (4) comprises: A. Regulate the ratio of T cell subtypes and inhibit inflammatory response; B. Provide local high concentrations of neurotrophic factors to promote axon regeneration and myelin repair.

4. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 2, characterized in that: The magnetic nanoparticles comprise the following components: A. The core is composed of poly(lactic-co-glycolic acid) (PLGA)-coated Fe3O4 superparamagnetic nanoparticles, which provide magnetic responsiveness; B. Surface coating with polyethyleneimine (PEI) for loading allosteric peptide ligand MBP 87-99 A 91 (A91), constituting A91@NPs; C. After A91@NPs are coated on the EM surface, it can serve as an immune adjuvant and increase the A91 loading rate and encapsulation efficiency to obtain EM@NPs.

5. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 1, characterized in that: The external rotating magnetic field has an intensity range of 5-15 mT and a frequency of 1-10 Hz, and is used to precisely control the movement of the magnetic dendritic cell microrobot.

6. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 2, characterized in that: The dendritic cells enhance their antigen presentation ability by phagocytosing EM@NPs and are able to secrete neurotrophic factors BDNF and NT-3.

7. A dendritic cell microrobot, characterized in that It consists of the following parts: A. dendritic cells; B. Magnetic nanoparticles containing antigen (A91) and E. coli membrane (EM).

8. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 7, characterized in that: The dendritic cell microrobot is used for analyzing immune regulatory mechanisms and evaluating the efficiency of neurotrophic factor delivery in basic research on spinal cord injury.

9. The method for constructing a magnetic dendritic cell microrobot delivery model for spinal cord injury research according to claim 8, characterized in that: The dendritic cell microrobot is used in the following experimental scenarios: Luminex and single-cell analysis were used to detect the expression of inflammatory factors and the distribution characteristics of cell subtypes in the damaged area; Immunofluorescence and transmission electron microscopy were used to observe structural changes during tissue repair; The BMS mouse scoring system was used to quantitatively evaluate the correlation between the motor function of experimental animals and the delivery effect of the microrobot.

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