Individualized 3D-printed exosome-siRNA-loaded hydrogel scaffold system for spinal cord injury repair and uses thereof

Abstract

The application provides an individualized 3D printing exosome-siRNA loaded hydrogel scaffold system for spinal cord injury repair and application thereof, and belongs to the technical field of biological medicine. The application designs an individualized scaffold based on multi-modal imaging, and realizes printing of an exo-siRNA loaded hydrogel scaffold by using a biological 3D printer. By in-situ transplantation, the irregular broken ends are precisely matched, the gaps are bridged, and the exo-siRNA is released to act on PTEN / mTOR key molecules to promote neural network remodeling, reduce the drug dosage, and reduce the side effects of systemic administration, thereby obviously improving the prognosis and having good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a personalized 3D-printed hydrogel scaffold system loaded with exosome-siRNA for spinal cord injury repair and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Spinal cord injury (SCI) is a devastating neurological disorder that can lead to severe nerve conduction disorders, such as quadriplegia and paraplegia. The primary cause of irreversible SCI damage is the inability of newly formed neurons to "connect" with other neurons across the transverse gap. First, the severed ends of the spinal cord lack the mechanical support for neuronal bridging. Second, early inflammatory responses and multicellular interactions within the SCI microenvironment contribute to the extensive deposition of extracellular matrix (ECM) molecules and the formation of dense fibrous scarring, further hindering the remodeling of nerve conduction at the severed ends. Currently, clinical strategies for SCI treatment primarily focus on symptomatic and supportive care, but these cannot achieve the recovery of motor function in paralyzed patients.

[0004] Recent studies have shown that neuroprotective molecules, stem cells, and exosomes may have therapeutic potential for spinal cord injury (SCI). However, the accessibility of these approaches is severely limited by the tight junctions of the blood-spinal barrier (BSCB). Hydrogel scaffolds are considered a promising drug delivery system (DDS) due to their high plasticity, biocompatibility, and biodegradability. Implantation of hydrogels within the transection region can provide spatial signaling to modulate neuronal remodeling. They have played a crucial role in supporting the spinal cord at the transection site, promoting the reconnection of newly generated neurons, and achieving controlled release. However, most current hydrogel research is based on local injection and mold casting, which cannot accurately match the heterogeneity of non-uniform geometry in SCI cases. The unsuitability of hydrogel implants can lead to adverse consequences, including secondary compression injury, DDS dislodgement, and hydrogel leakage.

[0005] The central nervous system has limited regenerative capacity, thus necessitating effective treatments to enhance axonal growth. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been shown to possess specific immunomodulatory effects and play a crucial role in nerve regeneration. Numerous studies have demonstrated that engineered exosomes can promote SCI treatment. Exosome engineering modifications can endow them with potent therapeutic capabilities by loading small molecules or nucleic acids for targeted drug delivery. PTEN (a homologous gene of phosphatase and tensin missing on human chromosome 10) and mTOR (a mammalian target of rapamycin) are key negative regulators in axonal regeneration. Based on this, encapsulating PTEN-interfering siRNA into exosomes can achieve PTEN / mTOR targeting and promote axonal regeneration. Summary of the Invention

[0006] Based on the aforementioned existing technologies, this invention provides a personalized 3D-printed hydrogel scaffold system loaded with exosome-siRNA for spinal cord injury repair and its applications. This invention designs a personalized scaffold based on multimodal imaging and utilizes a bio-3D printer to print the Exo&siRNA-loaded hydrogel scaffold. Through in situ transplantation, it precisely matches irregular fracture ends, bridges gaps, and sustainably releases Exo&siRNA to act on key PTEN / mTOR molecules, promoting neural network remodeling. This reduces the dosage and side effects of systemic administration, significantly improving prognosis and demonstrating good practical application value.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In a first aspect, the present invention provides a personalized 3D-printed hydrogel scaffold system for spinal cord injury repair loaded with exosome-siRNA, the hydrogel scaffold system comprising at least: a hydrogel scaffold, and engineered exosomes loaded on the hydrogel scaffold;

[0009] The hydrogel scaffold is a photocurable GelMA hydrogel, which is 3D printed to form an individualized scaffold for in-situ implantation at the site of spinal cord injury.

[0010] The engineered exosomes consist of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) carrying siRNA that interferes with the PTEN gene.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned hydrogel scaffold system, the method comprising: preparing a hydrogel scaffold and engineered exosomes; and adding the engineered exosomes to the hydrogel scaffold to obtain the system.

[0012] The specific method for preparing the hydrogel scaffold includes: constructing an individualized scaffold digital model by combining multimodal imaging damage area scanning with three-dimensional imaging, introducing it into a photopolymerization 3D printer, and printing the GelMA hydrogel scaffold.

[0013] The specific method for preparing engineered exosomes includes: obtaining BMSC-derived exosomes and introducing siRNA that interferes with the PTEN gene into the exosomes via electroporation.

[0014] A third aspect of the present invention provides the application of the above-described hydrogel scaffold system in the preparation of in situ treatment products for spinal cord injury.

[0015] Specifically, when using the in-situ treatment product for spinal cord injury, the aforementioned hydrogel scaffold system is implanted in situ into the spinal cord injury defect.

[0016] More specifically, the in situ treatment product for spinal cord injury has at least the following uses:

[0017] a) Enhance neuronal axon regeneration;

[0018] b) Promotes the remodeling of neural networks at severed ends;

[0019] c) Improve motor function recovery after spinal cord injury.

[0020] The beneficial technical effects of one or more of the above technical solutions:

[0021] Compared to conventional intravenous and oral drug delivery methods, this invention provides a personalized 3D-printed hydrogel scaffold system loaded with exosome-siRNA for in situ treatment of spinal cord injury. This invention enhances neuronal axon regeneration through the PTEN / mTOR pathway, promotes neural network remodeling at the spinal cord stump, and significantly improves motor function recovery after spinal cord injury. The implantation of the personalized scaffold after multimodal imaging scans reduces adverse consequences caused by the inability to precisely match the non-uniform geometry of the SCI stump. The in situ hydrogel scaffold allows for sustained and slow drug release, reducing the total drug exposure to the whole body. The exosome-loaded drug delivery system provides a possible encapsulation of therapeutic nucleic acid and protein molecules that are easily degraded in vivo and have low bioavailability, offering specific neuronal targeting and sustained-release effects. This provides a new direction for drug therapy after spinal cord injury. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1This is a characterization of the individualized scaffold constructed using multimodal imaging of the damaged area combined with three-dimensional imaging in Embodiment 1 of the present invention. (A) Small animal CT was used for scanning and three-dimensional reconstruction of the injured T10 vertebral segment. (B) 3D slicer software was used to measure the size of the T10 vertebral foramen. (C) 9.4T MRI of the spinal cord segment at the fracture site was used for imaging and three-dimensional reconstruction.

[0024] Figure 2 This is a similarity fitting analysis between the individualized scaffold digital model and the 3D-printed scaffold in Embodiment 1 of the present invention. (A) (a) Individualized scaffold digital model obtained after optimizing multimodal image reconstruction data using Cinema 4D software. (b) 3D-printed scaffold entity. (c) Scanned model of the scaffold entity. (d) Similarity analysis between the digital model and the scanned model. (B) Similarity analysis results of other different surfaces between the digital model and the scanned model.

[0025] Figure 3 This invention demonstrates how siRNA reduces PTEN gene expression levels, thereby increasing phosphorylation of downstream mTOR pathway proteins. (A) RT-qPCR results of PTEN gene silencing efficiency by different siRNAs. (B) Schematic diagram of the PTEN / PI3K / AKT / mTOR / S6K pathway. (C) Western Blot results of different groups in cell experiments. (D) Western Blot results of different groups in animal experiments.

[0026] Figure 4 This document describes the construction and characterization of exosomes and Exo&siRNA in Example 1 of this invention. (A) Light micrograph of bone marrow mesenchymal stem cells. (B) Flowchart of siRNA introduction into exosomes via electroporation. (C) Exoview particle size distribution results of exosomes and Exo&siRNA. (D) Electron micrograph of exosomes. (E) Exoview detection of CD63 / CD81 / CD9 distribution on the surface of exosomes. (F) Exoview detection results of cy3-siRNA distribution within the Exo&siRNA content.

[0027] Figure 5 This invention demonstrates the detection of the effect of Exo&siRNA on neuronal axon regeneration in Example 1. (A) Confocal image of exosome fluorescence staining after neuronal endocytosis. (B) Confocal image of exofluorescence staining of cy3-labeled siRNA after neuronal endocytosis. (C) Live / dead staining to detect neuronal survival in 2D / 3D cell culture. (D) Analysis of the promoting effect of different groups on ganglion axon regeneration using root ganglia (DRG). (E) Quantitative analysis of ganglion axon density and length in different groups.

[0028] Figure 6Characterization of the 3D-printed personalized Exo & siRNA-loaded hydrogel scaffold in Example 1 of this invention. (A) Electron microscopy image of the porous structure inside the GelMA hydrogel. (B) Electron microscopy image of the large number of exosomes loaded inside the GelMA-Exo group hydrogel. (C) Confocal microscopy analysis of the distribution of exosomes within the hydrogel scaffold. (D) Fourier transform infrared spectroscopy of GelMA and GelMA-Exo. (E) Atomic force microscopy analysis of the Young's modulus of the GelMA hydrogel.

[0029] Figure 7 This invention, in Example 1, describes the detection of the effect of GelMA-Exo & siRNA on the recovery of motor function in rats with spinal cord transection injury. (A) Procedure of scaffold transplantation in rats with spinal cord transection. (B) Nissl staining characterizes the synergistic process of cell migration and scaffold degradation at the injury site. (C) Release of PKH67-stained exosomes in spinal cord tissue after 28 days. (D) Release of Cy3-labeled siRNA in spinal cord tissue after 28 days. (E) Recovery of hindlimb function in different groups after 8 weeks of SCI treatment in rats. (F) Footprint test analysis of hindlimb motor function recovery. Forelimb footprints are shown in red, and hindlimb footprints are shown in blue. (G) Statistical analysis of stride length and width. Hindlimb footprints in the SCI and GelMA groups could not be measured and were not included in the statistics.

[0030] Figure 8 This is an example of the detection of the effect of GelMA-Exo & siRNA on neural network remodeling at the spinal cord transection site in rats with spinal cord transection injury in Example 1 of this invention. (A) Macroscopic tissue images of the spinal cord in different groups 8 weeks after SCI, H&E staining, and Nissl staining. (B) Syringomyelia rate in different groups. (C) Nerve fiber infiltration at the lesion site 8 weeks after surgery. Red represents Tuj-1 positive cells, and blue represents DAPI. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0033] The present invention will be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the selling company. Materials and reagents used in the examples, unless otherwise specified, are commercially available.

[0034] In a typical embodiment of the present invention, a personalized 3D-printed hydrogel scaffold system loaded with exosome-siRNA for spinal cord injury repair is provided. The hydrogel scaffold system includes at least: a hydrogel scaffold and engineered exosomes loaded on the hydrogel scaffold.

[0035] The hydrogel scaffold is a photocurable GelMA hydrogel, which is 3D printed to form an individualized scaffold for in-situ implantation at the site of spinal cord injury.

[0036] The engineered exosomes consist of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) carrying siRNA that interferes with the PTEN gene.

[0037] The hydrogel scaffold is fabricated using the following method: multimodal imaging of the damaged area combined with three-dimensional imaging to construct a personalized scaffold digital model, which is then introduced into a photopolymerization 3D printer to print the GelMA hydrogel scaffold; the implantation of the personalized scaffold after multimodal imaging scanning reduces the adverse consequences caused by the inability to accurately match the non-uniform geometry of the SCI fracture ends.

[0038] The siRNA sequence for interfering with the PTEN gene is shown in SEQ ID NO.5-10; preferably, the siRNA sequence is shown in SEQ ID NO.9-10.

[0039] Specifically, the aforementioned siRNA enhances the phosphorylation of downstream mTOR protein by reducing the expression level of the PTEN gene, thereby promoting neuronal axon regeneration, facilitating the remodeling of neural networks at the severed ends, and significantly improving the recovery of motor function after spinal cord injury.

[0040] A second aspect of the present invention provides a method for preparing the above-mentioned hydrogel scaffold system, the method comprising: preparing a hydrogel scaffold and engineered exosomes; and adding the engineered exosomes to the hydrogel scaffold to obtain the system.

[0041] The specific method for preparing the hydrogel scaffold includes: constructing an individualized scaffold digital model by combining multimodal imaging damage area scanning with three-dimensional imaging, introducing it into a photopolymerization 3D printer, and printing the GelMA hydrogel scaffold.

[0042] The specific method for preparing engineered exosomes includes: obtaining BMSC-derived exosomes and introducing siRNA that interferes with the PTEN gene into the exosomes via electroporation.

[0043] A third aspect of the present invention provides the application of the above-described hydrogel scaffold system in the preparation of in situ treatment products for spinal cord injury.

[0044] Specifically, when using the in-situ treatment product for spinal cord injury, the aforementioned hydrogel scaffold system is implanted in situ into the spinal cord injury defect.

[0045] More specifically, the in situ treatment product for spinal cord injury has at least the following uses:

[0046] a) Enhance neuronal axon regeneration;

[0047] b) Promotes the remodeling of neural networks at severed ends;

[0048] c) Improve motor function recovery after spinal cord injury.

[0049] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0050] Example 1

[0051] 1. Multimodal Imaging and 3D Graphical Design of Individualized Scaffolds: Multimodal imaging using a small animal micro-CT imaging system (Quantum GX2, PerkinElmer, USA) and a 9.4T MRI (Bruker Biospec, Ettlingen, Germany) was employed for scanning the bony structures and transected spinal cord in SD rats after spinal cord transection. For signal excitation and reception, a cylindrical volumetric coil (86 mm inner diameter) and a single-channel surface coil (20 mm diameter) were used in the MRI scans. Animals were anesthetized with 0.5–1.5% isoflurane and placed on heated pads, with respiratory changes monitored. Sagittal and axial T2-weighted scan data were obtained using RARE sequences. Micro-CT and 9.4T MRI scan data were imported into 3D slicer software for three-dimensional imaging. Finally, the 3D data from multimodal imaging were imported into Cinema 4D software for comprehensive model optimization to create an individualized scaffold.

[0052] 2. siRNA transfection: Small interfering RNA (siRNA) targeting PTEN was purchased from RIBOBIO (China). The siRNA sequence is listed in the table below. Following the manufacturer's instructions, the siRNA was transfected into the cells using Lipofectamine 3000 reagent (Invitrogen).

[0053] 3. Preparation of hydrogel-Exo & siRNA suspension: Weigh a certain amount of GelMA60 (EFL, Jiangsu, China) using an electronic balance. Add the prepared photoinitiator LAP to the solid GelMA60 to prepare a 5% concentration hydrogel solution. Use a water bath at 50-60℃, shaking several times until completely dissolved. Filter the solution using a bacterial filter in a clean bench. Then, mix Exo & siRNA into the liquid hydrogel at a ratio of 500 μg: 50 μl to form a suspension.

[0054] 4. Preparation of 3D-printed hydrogel scaffolds: The 3D printer (EFL, Jiangsu, China) was pre-irradiated with ultraviolet light for 30 minutes. Then, the hydrogel-Exo & siRNA suspension was placed in the material tank and the individualized scaffold model was introduced. Printing was performed according to the following settings: light intensity 12s, exposure time 26s, substrate exposure time 28s, layer height 50μm, material tank temperature 37℃, and deposition platform temperature 30℃.

[0055] 5. Real-time quantitative PCR: Total RNA was extracted from PC12 cells using the Ultrapure RNA Kit (CWBIO, China). Briefly, cells were thoroughly lysed using TRIzol lysis buffer (Beyotime, China) and mixed with chloroform at a 1:1 ratio, then centrifuged at 12,000 rpm for 10 minutes. The resulting supernatant was mixed with an equal volume of 70% ethanol and washed several times, then centrifuged at 12,000 rpm with the washing buffer from the adsorption column to obtain purified total RNA. Complementary DNA was then synthesized using the ReverTraAce qPCR RT Kit (TOYOBO, Tokyo, Japan). Quantitative real-time PCR was performed using the Synergy Brand (SYBR) Green Mixture (TOYOBO, Tokyo, Japan) and the Bio-rad IQ5 Real-time PCR System (Bio-Rad, USA) according to the manufacturer's instructions. The PCR amplification cycling conditions included initial denaturation at 94°C for 2 minutes, followed by 40 cycles of 94°C for 15 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. Two... -ΔCt The method normalizes the expression of the target gene to β-actin (internal control).

[0056] 6. Western blot: Proteins from cells or spinal cord tissue were extracted using RIPA buffer, separated by 10-15% SDS-PAGE, and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was washed three times with TBST (10 min x 3 times) and soaked in 5% skim milk for 2 hours. The membrane was then incubated overnight at 4°C with the following primary antibodies: polyclonal rabbit anti-PTEN (1:1000), anti-p-PI3K (1:1000), anti-AKT (1:2000), anti-p-AKT (1:1000), anti-mTOR (1:1000), anti-p-mTOR (1:1000), anti-S6K (1:1000), anti-p-S6K (1:1000), and anti-GAP43 (1:1000); polyclonal mouse anti-NF (1:1000) and β-actin (1:1000). The PVDF membrane was washed with TBST (10 min × 3 times) and incubated with secondary antibody (1:1000 dilution) at room temperature for 2 hours. Proteins were visualized using ultrasensitive chemiluminescence (ECL) reagent (Coyobo, Jiangsu, China) and detected using a Tanon-4600 imaging system (Tanon, Shanghai, China). Relative intensity analysis of proteins was performed using ImageJ software.

[0057] 7. Extraction and Culture of Bone Marrow Mesenchymal Stem Cells (BMSCs): Primary bone marrow mesenchymal stem cells were extracted from 8-week-old SD rats. The specific steps are as follows: First, 8-week-old female SPF-grade SD rats purchased from Jinan Pengyue Experimental Animal Company were placed in an animal incubator. Cell extraction-related items were placed in a clean bench and irradiated with ultraviolet light for half an hour. After deep anesthesia and euthanasia, the rats were immersed in 75% alcohol for 15 minutes for disinfection. All instruments were autoclaved. The hind limbs of the rats were severed at the groin, taking care not to sever the femoral artery and vein. The attached skin and muscle tissue were peeled off to expose the femur. The femoral head and distal tibia were severed, and the cells were flushed out of the femur. Repeated flushing was performed to ensure that more cells were flushed out of the femur. The cells were suspended in DMEM / F12 containing 10% fetal bovine serum and cultured for 2-3 days. After the primary mesenchymal stem cells adhered, the culture medium was changed to remove non-adherent cells and dead cells. When primary mesenchymal stem cells grow to 75%-85%, they are passaged. When the cells are passaged to the third generation, they can be used for subsequent experiments.

[0058] 8. Exosome Extraction: All BMSC-derived exosomes used in the experiment were from the same batch of BMSCs. Exosome-free FBS were prepared by ultracentrifuging FBS at 120,000 × g for 12 hours at 4°C using a Beckman Coulter. When cells reached 80% confluence, the regular culture medium was replaced with exosome-free medium. After 48 hours, the cell supernatant was centrifuged sequentially at 300 × g for 10 minutes, 2000 × g for 15 minutes, and 10,000 × g for 30 minutes at 4°C. After each centrifugation, the supernatant was collected and the precipitate was removed to eliminate cells, cell debris, and apoptotic bodies. The supernatant was filtered through a 0.22-micron filter to remove microcapsules. Then, it was ultracentrifuged at 100,000 × g for 70 minutes at 4°C. The precipitate was resuspended in sterile PBS and ultracentrifuged again at 100,000 × g for 70 minutes at 4 °C. Finally, the precipitate was resuspended in 200 μl of sterile PBS. The exosome protein concentration was measured using the bis(octanoic acid) (BCA, CWBIO) assay and stored at -80 °C.

[0059] 9. Characterization of BMSC-Exosomes and Exo&siRNA: Exosome morphology was determined using TEM (transmission electron microscopy). Exosome samples were added to a copper lattice on the vector and incubated for 2 minutes. The surrounding suspension was wiped dry with filter paper, and 1% negative phosphotungstic acid dye was added to the suspension. The copper lattice was then dried under incandescent light, and images were taken using a Hitachi TEM system. A single-particle interferometric reflectance imaging sensor was used to characterize the particle size and molecular distribution on the surfaces of exosomes and Exo&siRNA. Exosomes and Exo&siRNA were diluted with incubation medium (NanoView Biosciences, Brighton, MA, USA). Then, 35 μL of Exosome or Exo&siRNA samples were transferred to a custom silica chip coated with CD9, CD63, CD81, and negative isotype control antibodies (hamster IgG and rat IgG). After overnight incubation in 24-well plates, the chip was incubated at RT with a mixture of fluorescent antibodies, including anti-CD81 / siRNA (green), anti-CD63 (red), and anti-CD9 (blue). An ExoView R100 (NanoView Biosciences, Brighton, MA, USA) and acquisition software (NanoView Biosciences, Brighton, MA, USA) were used for image and data acquisition and further analysis, respectively.

[0060] 10. Live / Dead Cell Viability Assay: PC12 cell viability was determined using a live / dead cytotoxicity kit according to the manufacturer's instructions. First, working solutions of the required concentrations (2 μM calcein AM and 8 μM PI) were prepared. PC12 and 3D-PC12 cells (grown in GelMA hydrogels) were washed with PBS. Sufficient working solution was added, followed by RT incubation for 45 minutes. Cell viability and proliferation were observed under a confocal laser microscope (Leica TCS SP8, Germany). Live PC12 cells were stained with calcein AM (green), while dead cells were stained with PI (red).

[0061] 11. Acquisition and Culture of Dorsal Root Ganglia (DRG): DRG ganglia were extracted from 8-week-old rats under a stereomicroscope. They were cultured in a mixed medium containing neuron culture medium (Gibco), 50 ng mL-1 NGF (Gibco), 1×B27 neuron supplement (Gibco), 1×penicillin / streptomycin (Gibco), and 1×Glutamax (Gibco) in a 37°C, 5% CO2 incubator. Axon length and density were measured using the Simple Neurite Tracer plugin in ImageJ software, which tracks individual branches relative to the cell body.

[0062] 12. Characterization of the spinal cord scaffold: The morphology and mechanical properties of the spinal cord scaffold were characterized using scanning electron microscopy (SEM) (S-4800, Hitachi, Japan) and atomic force microscopy (AFM; Dimension icon, Bruker, Germany). The spinal cord scaffold was examined using Fourier transform infrared spectroscopy (FTIR) (NEXUS 670, Thermo Nicolet). The localization of PKH67-labeled exosomes within the scaffold was observed using fluorescence confocal microscopy (Leica TCS SP8, Germany).

[0063] 13. Experimental Animals: Adult female SPF-grade SD rats weighing 200-250g were selected for this study and purchased from Jinan Pengyue Animal Breeding Co., Ltd. They were housed in the SPF-grade animal housing at the Animal Center of Shandong Qianfoshan Hospital, at a temperature of 20±2℃, using a natural light / dark cycle (≈12h-12h). The animal experimental protocol was approved by the Ethics Committee of Shandong Qianfoshan Hospital based on the principles outlined in the National Institutes of Health's Animal Care and Use Guidelines (Ethics No.: 2022S577). Personnel involved in the research on the animal models received training in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

[0064] 14. Spinal Cord Injury (SCI) Model and Postoperative Care: Spinal cord injury in rats was performed according to a standard protocol. Animals were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 30 mg / kg. An incision was made in the middle of the back (centered on the spinous process of T10), and the spinous process and lamina of T10 were removed to fully expose the spinal canal. A 3 mm section of the spinal cord was carefully removed with ophthalmic scissors. Forceps were inserted to ensure no nerve fiber residue, and a scaffold was implanted after adequate hemostasis. Sham rats underwent the same procedure for T10 layer resection, but without spinal cord injury. Postoperatively, each rat received an intramuscular injection of 200,000 U of penicillin once daily for 3 days.

[0065] 15. Motor Function Recovery in the SCI Model: Footprint analysis was used to assess body weight support and coordination. The forelimbs and hindlimbs of rats were stained with red and blue ink, respectively. The rats were then allowed to walk on a narrow surface covered with paper. The distance between the two sides of the hind paw was determined as stride length (W). Step length (L), the vertical distance between the forelimbs and hindlimbs, was used to assess coordination ability.

[0066] 16. In vivo release of PKH67-labeled exosomes and cy3-labeled Exo & siRNA: Hydrogel spinal cord scaffolds containing PKH67-labeled exosomes or cy3-labeled Exo & siRNA were implanted. The control group received a PBS suspension containing the same volume of exosomes. After 28 days, the retention levels of exosomes or Exo & siRNA at the damaged site were observed using a small animal imaging system (IVIS Spectrum, PerkinElmer, USA).

[0067] 17. Preparation of Paraffin Sections and Hematoxylin and Eosin (HE) Staining: After deep anesthesia, rats were exposed to the thoracic cavity. The right atrium was opened, and 4°C PBS solution was injected through the left ventricle until the liquid became colorless, then fixed with 4% PFA. The spinal cord from T9 to T11 was then completely excised, dehydrated, and embedded in paraffin. The sections were cultured at 60°C for 1 hour, then dewaxed with xylene and graded ethanol. After hematoxylin staining and differentiation with 10% hydrochloric acid, the sections were observed under a microscope to assess tissue morphology and identify cavity locations.

[0068] 18. Nissl staining: Incubate paraffin sections at 55°C for 30 minutes, dewax with xylene, and immerse in an ethanol gradient. Place the sections in Nissl staining solution, then soak in differentiation solution in a 58°C water bath for 40 minutes. Dehydrate with 100% ethanol and photograph under a microscope.

[0069] 19. Immunofluorescence staining: After antigen retrieval, tissue sections were incubated with BSA for 30 minutes. Then, rabbit anti-Fn, anti-CD68, anti-GAP43, anti-Tuj-1 antibodies, and mouse anti-NF antibodies were added to the sections and incubated overnight at 4°C. Following this, the sections were incubated with the corresponding secondary antibodies in the dark at RT for 50 minutes. After rinsing and drying the sections, DAPI was added and incubated at RT for 10 minutes, then sealed with anti-fluorescence quenching sealing sheets. Images were captured using a fluorescence microscope, and positive cells were quantified using ImageJ software.

[0070] 20. Statistical Analysis: Each data set was independently repeated at least three times, and the values ​​are expressed as mean ± standard error. Statistical significance between two sets of data was assessed using the Student t-test. GraphPad Prism 9 was used for statistical analysis and graphing. A p-value less than 0.05 was considered statistically significant (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001).

[0071] 21. Sequence Information:

[0072]

[0073] Experimental results:

[0074] 1. Characterization of individualized scaffolds by combining multimodal imaging of damaged areas with 3D imaging.

[0075] Multimodal image fusion was performed based on MRI scans of the transverse segment region and CT scans of the T10 thoracic vertebrae in SD rats. By combining the image data from MRI (spinal cord) and CT (supporting bone structures) scans, a digital scaffold model was reconstructed using 3D slicer software (CT reconstruction: Figure 1 A, 1B, MRI reconstruction: 1C)

[0076] 2. Similarity Fitting Analysis and Detection between Individualized Scaffold Digital Model and 3D Printed Scaffold

[0077] digital model ( Figure 2 A(a)) is introduced into a photopolymer 3D printer to print a GelMA hydrogel scaffold ( Figure 2 (A(b)) The printed spinal cord scaffold was fully expanded in PBS for 12 hours. A full-view scan was performed to characterize the similarity between the digitally constructed model and the printed scaffold. Figure 2 A(c) in Figure 2 A(d) and Figure 2 As shown in Figure B, the fitting results indicate that the similarity between the two models exceeds 95% in rat data. This demonstrates that the accuracy of the printed scaffold can achieve individualized scaffold customization.

[0078] 3. siRNA reduces PTEN gene expression levels, thereby increasing the detection of downstream mTOR pathway protein phosphorylation.

[0079] First, the interference efficiency of different siRNAs was evaluated, and the most effective siRNA sequence, Si-PTEN3 (knockdown of 80%), was selected for further study. Figure 3 A). PTEN (phosphatase and angiotensin homolog) is considered one of the most important intrinsic factors leading to neurogenesis failure. It is a negative regulator of axonal regeneration. Downregulation of PTEN protein can enhance axonal regeneration. The PI3K / AKT / mTOR / S6K signaling pathway plays an important role in cell proliferation and is closely related to the PTEN gene. Figure 3 B). Therefore, to investigate the potential mechanisms involved by GelMA-Exo & siRNA, we performed Western blot analysis to explore the relative protein expression levels of these genes. For example... Figure 3As shown in C and 3D, PTEN was significantly reduced in the GelMA-Exo&siRNA group compared to other groups, in both cell and tissue samples. Furthermore, although the expression levels of phosphorylated (active) PI3K (p-PI3K), p-AKT, p-mTOR, and p-S6K were significantly upregulated in the GelMA-Exo&siRNA group, the total amounts of these proteins did not differ statistically under different culture conditions. The GelMA-Exo group also showed increased expression levels of p-PI3K, p-AKT, p-mTOR, and p-S6K, while PTEN expression was decreased. In conclusion, GelMA-Exo&siRNA can interfere with PTEN protein expression and activate the downstream PI3K / AKT / mTOR / S6K pathway.

[0080] 4. Construction and characterization of exosomes and Exo & siRNA

[0081] BMSCs were isolated by gradient centrifugation and screening methods. Figure 4 A). Subsequently, exosomes with the potential to promote nerve regeneration were isolated and purified from the BMSC culture supernatant by ultracentrifugation. Then, siRNA specifically interfering with the PTEN gene was transferred into the prepared exosomes via electroporation to construct engineered exosomes (Exo&siRNA). Figure 4 B). To characterize the changes in surface molecules and particle size of exosomes and engineered exosomes, ExoView's single EV (extracellular vesicle) microarray imaging technology was used. The results showed that the average diameter of Exo&siRNA was 78 nm, and the average diameter of Exo was 67 nm, indicating that the particle size of the engineered exosomes increased. Figure 4 C). Transmission electron microscopy (TEM) shows that exosomes have a typical cup-shaped or spherical morphology. Figure 4 D). Specific antibody capture at each ExoView chip spot allows for the immobilization of antibody-capture-based EV samples for further probing using fluorescently labeled antibodies (CD63, CD81 / cy3-siRNA, and CD68 antibodies). Fluorescence imaging results show that exosome surface molecules are primarily composed of CD81 and CD9 transmembrane proteins. Figure 4 E). To ensure siRNA transfer to therapeutic exosomes, we captured exosome samples using CD81 and CD9 antibodies, respectively. High-throughput results showed that the fluorescently labeled siRNA was well encapsulated in CD81. + and CD9 + in exosomes ( Figure 4 F).

[0082] 5. Detection of Exo&siRNA promoting neuronal axon regeneration

[0083] Confocal microscopy was used to determine the phagocytic activity of exosomes in single nerve cells. The results showed that exosomes could be phagocytosed by PC12 cells. Figure 5 A). Following phagocytosis, engineered exosomes release cy3-labeled siRNA within PC12 cells, which can then exert their corresponding effects. Figure 5 B). Live / dead cell staining was used to study the bioactivity of neurons cultured under 2D and 3D conditions. In 3D-PC12 cells, the green fluorophore of live cells was uniformly distributed in GelMA, while the red fluorescence of dead cells was almost undetectable. Figure 5 C). This indicates that GelMA hydrogel is suitable for culturing nerve cells. Furthermore, a peripheral neuronal dorsal root ganglion (DRG) co-culture system was used to determine whether engineered BMSC-Exo could regulate axonal growth. Peripheral neurons possess a strong intrinsic potential for axonal regeneration and are commonly used in axonal regeneration studies. Immunostaining DRGs with NF and fluorescence results showed that GelMA-Exo & siRNA could promote axonal growth (…). Figure 5 D). Quantitative analysis of neuronal growth showed that the axon length of the GelMA-Exo&siRNA group was 1275.48±152.48 μm, which was longer than that of GelMA-Exo (783.42±78.91 μm), GelMA (495.40±47.06 μm), and the control group (469.49±49.5 μm). The axon density of the GelMA-Exo&siRNA group was 1.52±0.14, 2.46±0.22, and 2.69±0.31 times higher than that of the GelMA-Exo group, GelMA group, and control group, respectively. Figure 5 E). This indicates that GelMA-Exo&siRNA achieves sustained release of Exo&siRNA, and that siRNA can stimulate the PTEN / PI3K / AKT / mTOR pathway, promoting axonal growth.

[0084] 6. Characterization of 3D-printed personalized Exo & siRNA-loaded hydrogel scaffolds

[0085] The internal structure of the hydrogel scaffold was analyzed using high-resolution SEM (scanning electron microscopy). The results showed that the scaffold possessed a porous structure, with a large number of exosomes successfully encapsulated within a cross-linked network. Figure 6 A, 6B). Next, fluorescence analysis was performed on PKH67-labeled exosomes (green) and rhodamine B-labeled GelMA (red). The results showed that the green fluorescence of the exosomes was uniformly distributed across the entire red fluorescence range of the GelMA hydrogel scaffold. Figure 6C). To further investigate the composition of the GelMA-Exo hydrogel scaffold, we performed Fourier transform infrared (FTIR) spectroscopy analysis. The results showed three characteristic vibrational bands (NH, 3113 cm⁻¹). -1 C=O, 1673cm -1 NH, 1594cm -1 This confirms that amide bonds are formed between GelMA and exosomes. Figure 6 D). Mechanical properties of the GelMA hydrogel scaffold were analyzed using atomic force microscopy (AFM). The Young's modulus of the GelMA hydrogel scaffold ranged from 315.4 kPa to 439.2 kPa, exhibiting similar physical properties to surrounding tissues, which could facilitate mechanical matching between the GelMA scaffold and normal spinal cord (Young's modulus = 200-600 kPa). Figure 3 E). Furthermore, similar elasticity can better mimic the actual spinal cord, providing a suitable microenvironment for neuronal regeneration and reconstruction.

[0086] 7. Detection of GelMA-Exo & siRNA promoting motor function recovery in rats with spinal cord transection injury

[0087] To study the in vivo therapeutic effect, a GelMA-Exo&siRNA scaffold was implanted at a transverse site for local delivery of Exo&siRNA. Figure 7 A). The process of neuronal cell infiltration and gel degradation after scaffold implantation was examined. Results showed that the GelMA scaffold could remain in the spinal cord for more than 28 days. As the hydrogel scaffold degraded over time, more and more neurons migrated centripetally, filling the cavities left by the GelMA scaffold and participating in the neuroregeneration process. Figure 7 B). High retention and release efficiency of exosomes in vivo are key to damage repair. A 200 μg drug delivery system loaded with PKH67-labeled exosomes and Cy3-labeled siRNA was implanted into SCI rats. In the control group, the same dose of exosomes in PBS suspension was directly injected into the injury site. The retention levels of Exo and Exo&siRNA in the damaged area were observed using IVIS 28 days post-surgery. Results showed that the fluorescence intensity of GelMA-Exo&siRNA and the GelMA-Exo group was significantly higher than that of the directly injected Exo group (…). Figure 7 C). The Cy3-labeled Exo&siRNA group exhibited strong fluorescence intensity in vivo after 28 days. Figure 7 D). These results indicate that GelMA hydrogel is superior in preserving internal exosomes. Regarding changes in neurological function, the recovery of motor function in the hind limbs of rats was examined 8 weeks post-surgery. Figure 7As shown in Figure E, the SCI group rats exhibited an upward-tilted pelvis and completely lost voluntary hindlimb movement ability. After implantation of GelMA and GelMA-Exo, the physiological performance of the hind limbs improved, with these rats able to partially contact the ground with their pelvis. As for the GelMA-Exo & siRNA group, the hind limb condition of the rats was significantly improved, approaching that of the Sham group, although the rats still required forelimb support to maintain balance at rest. The footprint analysis results were consistent with the above findings, and can be summarized as follows: Figure 7 In F, the forelegs were stained with red ink, and the hind legs with blue ink. Blue ink was not recorded because the SCI group rats could not lift their hind legs. In the GelMA treatment group, the rats showed a reduced stride length, indicating coordinated movement of the forelimbs and hindlimbs. Compared to the Sham group, rats treated with GelMA-Exo and GelMA-Exo&siRNA showed significant improvements in both stride length (L) and stride width (W). Figure 7 G). Implantation of GelMA-Exo & siRNA promoted functional recovery in SCI rats.

[0088] 8. Detection of GelMA-Exo & siRNA promoting neural network remodeling at the spinal cord transection ends in rats.

[0089] Eight weeks after SCI, the long-term recovery effect in rats was evaluated. For example... Figure 8 As shown in A(ac), abnormal spinal cord morphology caused by loss of neural tissue at the injury site was observed in HE staining. Meanwhile, the Nissl body loss rate in the GelMA-Exo&siRNA group was 7.89±1.65%, significantly lower than that in the SCI (25.36±0.98%), GelMA (22.91±1.68%), and GelMA-Exo (15.53±1.11%) groups. In longitudinal spinal cord sections, the cavitation rate in the SCI group was 24.2±3.25%, and the cavitation rate in the GelMA group was 20.48±1.87%. In contrast, both GelMA-Exo and GelMA-Exo&siRNA reduced cavitation and cavitation rates. Eight weeks post-surgery, the cavitation rate in the GelMA-Exo&siRNA group (8.35±1.01%) was significantly lower than that in the GelMA-Exo group (15.12±1.81%). Figure 8B). These results confirm that the GelMA-Exo&siRNA hydrogel reduced cavity area and promoted cell infiltration and tissue remodeling. Axonal regeneration of neurons throughout the injury site is crucial for effective SCI repair. Tuj1, representing an essential microtubule protein involved in early neuronal differentiation, is considered a specific marker of neonatal neurons. Therefore, immunostaining analysis of Tuj-1 was used to assess neuronal and axonal regeneration. Compared with those observed in the SCI and GelMA groups, a significant increase in Tuj-1 markers near the injury site was observed in both the GelMA-Exo&siRNA and GelMA-Exo groups. Figure 8 C). The number of Tuj-1 positive neurons in the lesion area was lower in the GelMA-Exo hydrogel group than in the GelMA-Exo & siRNA hydrogel group. Furthermore, the hydrogel scaffold connected nerve fibers to the filamentous network, suggesting that the GelMA-Exo & siRNA scaffold plays a sustained protective role and promotes regeneration in these neurons.

[0090] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A personalized 3D-printed hydrogel scaffold system loaded with exosome-siRNA for spinal cord injury repair, characterized in that, The hydrogel scaffold system includes at least: a hydrogel scaffold, and engineered exosomes loaded on the hydrogel scaffold; The hydrogel scaffold is a photocurable GelMA hydrogel, which is 3D printed to form an individualized scaffold for in-situ implantation at the site of spinal cord injury. The engineered exosomes consist of exosomes derived from bone marrow mesenchymal stem cells carrying siRNA that interferes with the PTEN gene; The hydrogel scaffold was prepared by the following method: multimodal imaging damage area scanning combined with three-dimensional imaging to construct an individualized scaffold digital model, which was then introduced into a photopolymerization 3D printer to print a GelMA hydrogel scaffold. The forward primer sequence of the siRNA interfering with the PTEN gene is shown in SEQ ID NO.9, and the reverse primer sequence of the siRNA interfering with the PTEN gene is shown in SEQ ID NO.

10.

2. The method for preparing the hydrogel scaffold system according to claim 1, characterized in that, The preparation method includes: preparing a hydrogel scaffold and engineered exosomes; and adding the engineered exosomes to the hydrogel scaffold to obtain the final product. The specific method for preparing the hydrogel scaffold includes: constructing an individualized scaffold digital model by scanning the damaged area using multimodal imaging combined with three-dimensional imaging, and then introducing it into a photopolymerization 3D printer to print the GelMA hydrogel scaffold. The specific method for preparing engineered exosomes includes: obtaining BMSC-derived exosomes and introducing siRNA that interferes with the PTEN gene into the exosomes via electroporation; The forward primer sequence of the siRNA interfering with the PTEN gene is shown in SEQ ID NO.9, and the reverse primer sequence of the siRNA interfering with the PTEN gene is shown in SEQ ID NO.

10.

3. The application of the hydrogel scaffold system of claim 1 in the preparation of products for in situ treatment of spinal cord injury.

4. The application as described in claim 3, characterized in that, The in-situ treatment product for spinal cord injury involves implanting the aforementioned hydrogel scaffold system in situ into the spinal cord injury defect.

5. The application as described in claim 3 or 4, characterized in that, The in situ treatment product for spinal cord injury has at least the following uses: a) Enhance neuronal axon regeneration; b) Promotes the remodeling of neural networks with severed ends; c) Improve motor function recovery after spinal cord injury.

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