Nucleic acid gel for mediating spinal cord injury repair by remodeling residual neural circuits and construction method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to nucleic acid gel for mediating spinal cord injury repair by remodeling a residual neural circuit as well as a construction method and application of the nucleic acid gel. The nucleic acid gel disclosed by the invention integrates PTEN targeted inhibition mediated neurite growth and 5-HT mediated neuron excitement recovery, so that the unification of dormant residual neuron reactivation and spinal cord loop reconstruction is realized in spinal cord injury repair. A novel synergetic normal form is provided for spinal cord injury treatment by synergetic structure reconstruction and functional recovery on a drug delivery platform in a single focus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a nucleic acid gel for mediating repair of spinal cord injury through remodeling of residual nerve circuits and a construction method and application thereof. BACKGROUND

[0002] Spinal cord injury is a serious central nervous system trauma, which can lead to permanent paralysis, sensory loss and autonomic nervous dysfunction. Most spinal cord injury patients show incomplete injury at the anatomical level, and residual spinal cord circuits composed of local interneurons are left. The residual spinal cord circuits can serve as a relay network to transmit nerve impulses across the injury area. However, the above functional reintegration of the residual spinal cord circuits is mainly limited by two coexisting mechanisms: first, after spinal cord injury, the residual neurons are in a dormant state in terms of function, making it difficult to achieve transmission of nerve impulses; second, the ability of axon regeneration is very limited after spinal cord injury, making it difficult to form a synaptic network structure reconnected with the host spinal cord. Therefore, an integrated pharmacological strategy that combines “reactivation of dormant residual neurons” with “reconstruction of residual spinal cord circuits” is urgently needed.

[0003] At the clinical level, epidural electrical stimulation combined with rehabilitation training has enabled some paralyzed spinal cord injury patients to restore some degree of autonomous walking ability, which may be due to the fact that the above intervention can reactivate dormant spinal interneurons in the residual circuit, allowing the transmission of instructions from the brain. In this process, the 5-HT system plays a key role. 5-HT can enhance the excitability of motor neurons by enhancing persistent inward currents (PICs), thereby mediating the transmission of brain-derived instructions. After spinal cord injury, 5-HT signaling is significantly reduced, and enhancing the impaired 5-HT signaling can restore PICs through multiple receptor subtypes (including 5-HT 1A , 5-HT 1B , 5-HT 2A receptors), thereby reducing the activation threshold of dormant neurons, and ultimately making the previously dormant spinal cord circuit enter a functional state. Although this approach has therapeutic potential, developing a pharmacological approach that selectively targets the 5-HT system is still an important bottleneck for the clinical translation of this strategy.

[0004] In addition to reactivating the excitability of residual dormant neurons, reconstruction of local neural circuits in the spinal cord is also required to maintain and transmit the recovered brain-derived 'instructions' for a durable and repeatable functional recovery. This process needs to coordinate the complex multi-step procedures of'regeneration of damaged axons' and'remodeling of damaged synapses', and relies on the sustained support of a favorable neuroimmune microenvironment. Specifically, in the early stage after spinal cord injury, the residual neurons exhibit a transient upregulation of regenerative genes, followed by a chronic and progressive state of persistent silencing of regenerative genes. This is an important reason for the insufficient intrinsic regenerative potential of mature neurons, and highlights the central role of activating the intrinsic mechanisms of residual neurons in axon regeneration and synaptic remodeling. Meanwhile, the infiltrating immune cells after injury can rapidly release a large amount of pro-inflammatory cytokines, creating a poor neuroimmune microenvironment at the injury site, driving the differentiation of fibroblasts and astrocytes, and promoting the excessive deposition of extracellular matrix, thus forming a dense scar in the core of the injury, which forms a persistent barrier to hinder axon extension and synaptic remodeling. The above neuroimmune restriction and the weakened regenerative capacity of residual neurons together pose a major challenge to the reconstruction of spinal cord circuits. In this framework, PTEN, as a key node of coordination, has become increasingly important: neuron-specific Pten deletion can activate the intrinsic program of residual neurons to promote circuit reconstruction; and microglia-specific Pten deletion can drive its polarization to a repairative M2 phenotype, creating a favorable neuroimmune microenvironment. This 'complementary effect of injury inside and outside' suggests that PTEN can be used as a single and operable lever to coordinate the 'intrinsic neurite growth program of neurons' and 'extrinsic neuroimmune optimization of injury'. However, due to the highly conserved structure of the active site of PTEN protein and the limited electrostatic environment, PTEN is still an 'undruggable target' for traditional protein-directed inhibitors, which further prompts us to focus on sequence-specific oligonucleotide therapy, which has achieved revolutionary success in several central nervous system diseases, but is still in a blank state in the field of spinal cord injury treatment. The complexity of multiple pathological factors indicates that achieving meaningful functional recovery requires targeting multiple interlocking pathological mechanisms simultaneously, which also promotes the development of combined strategies aimed at complementary mechanisms. For example, Wang et al. achieved the targeted delivery of 'inhibitory neurotransmitter-receptor activity agents' to inhibitory interneurons, thereby reactivating the residual spinal cord neural circuit and improving motor function. However, histology showed that the axon extension was sparse, indicating that structural reconstruction was still insufficient. In contrast, Liu et al. proposed that near-infrared light genetic strategies could be used to achieve precise connection of residual corticospinal tract (CST) and its target, but this approach lacks direct regulation of neuronal excitability, and its efficacy largely depends on the long and precise long-distance axon regeneration and synaptic remodeling.Despite the encouraging progress represented above, the complex mechanisms of post-injury dysfunction and the complex interactions among various therapeutic factors within the lesion site still pose challenges to the effective coupling of "reactivation of residual neuronal excitability after injury" and "reconstruction of neural circuits after injury". Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a nucleic acid gel for spinal cord injury repair mediated by remodeling residual neural circuits, along with its construction method and applications. This invention constructs a DNA / RNA heterologous hybridization double-stranded nucleic acid hydrogel (SeroPTEN-Chemogene, abbreviated as SeroPTEN-CG), integrating "PTEN-targeted inhibition-mediated neurite growth program" with "5-HT-mediated excitability recovery" for spinal cord injury repair. After local administration at the injury site, this hydrogel can be in situ transformed into a nanogel, integrating "PTEN-targeted inhibition-mediated neurite growth" and "5-HT-mediated neural excitability recovery," thereby achieving a unified approach to "reactivation of dormant residual neurons" and "spinal cord circuit reconstruction" in spinal cord injury repair. By synergistically combining structural reconstruction and functional recovery on a single lesion-based drug delivery platform, a new synergistic paradigm for spinal cord injury treatment is provided.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a nucleic acid gel for mediating spinal cord injury repair by remodeling residual neural circuits, comprising: a scaffold unit formed by three scaffold single-stranded nucleic acids in a base-complementary pairing manner, each scaffold single-stranded nucleic acid having a scaffold adhesive end, wherein the complementary pairing portion of the scaffold single-stranded nucleic acid has two thiophosphate groups uniformly distributed thereon, and each thiophosphate group is modified with a 5-hydroxytryptophan derivative; and a crosslinking unit formed by two crosslinked single-stranded nucleic acids in a base-complementary pairing manner, each crosslinked single-stranded nucleic acid having a crosslink adhesive end.

[0007] In some implementations, the scaffold unit and the crosslinking unit are crosslinked through the scaffold adhesive ends and the crosslinking adhesive ends in a base-complementary pairing manner to form a three-dimensional spatial network structure.

[0008] In some embodiments, the length of the adhesive end of the stent or the cross-linked adhesive end is 15-20 nt, preferably 18 nt.

[0009] In some embodiments, the scaffold unit and the crosslinking unit are in a stable crosslinking state in an isotonic buffer solution at pH 6.8 to 7.6, free of nucleases and esterases at room temperature.

[0010] In some embodiments, the 5-hydroxytryptophan derivative is 5-HTP-Br, with the following structure: .

[0011] In some embodiments, the scaffold unit comprises the following DNA sequence: Ya-2PS: TGGATCC*GCATGACATTCGCC*GTAAG TTGACCTGTGAA (SEQ ID NO: 1); Yb-2PS: CTTACGG*CGAATGACCGAATC*AGCCT TTGACCTGTGAA (SEQ ID NO: 2); Yc-2PS: AGGCTGA*TTCGGTTCATGCGG*ATCCA TTGACCTGTGAA (SEQ ID NO: 3); where "*" indicates the site modified by thiophosphate (PS site); "underline" indicates "adhesive end", that is, the adhesive end of the scaffold.

[0012] At the same time, 12 bases are left empty at the end of each DNA strand to facilitate subsequent complementary pairing and cross-linking with PTEN siRNA.

[0013] In some embodiments, the cross-linking unit is a PTEN siRNA with cross-linked sticky ends, the PTEN siRNA consisting of the following sequence: L1 (Sense strand): rGrGrGrUrUrUrGrArUrArArGrUrUrCrUrArGrC r UrUrCrArCrArGrGrUrCrArA (SEQ ID NO: 4); L2 (Antisense strand): rGrCrUrArGrArArCrUrUrArUrCrArArArCrCrC rUrUrCrArCrArGrGrUrCrArA (SEQ ID NO: 5); where the underlined part represents "viscous end", that is, the cross-linked viscous end.

[0014] A second aspect of the present invention provides a method for preparing a nucleic acid gel, comprising the following steps: (a1) 5-HTP-Br was grafted onto the phosphate thioester sites of three scaffold single-stranded nucleic acids to obtain 5-HTP-DNA. The three 5-HTP-DNAs formed scaffold units through complementary base pairing. (a2) Dissolve the stent unit in an aqueous medium to obtain an aqueous medium solution of the stent unit; (a3) Prepare crosslinking units by dissolving the crosslinking units in an aqueous medium to obtain an aqueous medium solution of the crosslinking units; (a4) The aqueous medium solution of the scaffold unit and the aqueous medium solution of the crosslinking unit are mixed to form a three-dimensional spatial network structure between the direct unit and the crosslinking unit, thereby obtaining the nucleic acid gel; The structure of the 5-HTP-Br is as follows: .

[0015] In some embodiments, the reaction conditions for grafting 5-HTP-Br onto the thiophosphate sites of three scaffold single-stranded nucleic acids are as follows: DNA strands modified with thiophosphate sites (PS) (Ya-2PS, Yb-2PS, Yc-2PS) and compound 5-HTP-Br are dissolved separately in dimethyl sulfoxide (DMSO), and reacted with gentle shaking at 50°C for 30 min. After precipitation with ethanol and removal of solvent by vacuum evaporation, 5-HTP-modified DNA (5-HTP-Ya-2PS, 5-HTP-Yb-2PS, 5-HTP-Yc-2PS) is obtained.

[0016] In some embodiments, the concentration of the DNA strand modified with the phosphate thioester site (PS) is 90-110 μM, preferably 100 μM.

[0017] In some embodiments, the concentration of 5-HTP-Br is 5-7 mM, preferably 6 mM.

[0018] In some implementation schemes, the molar ratio of 5-HTP-Br to scaffold single-stranded nucleic acid in the reaction system is 60:1.

[0019] In some embodiments, the mixing temperature of the aqueous medium solution of the stent unit and the aqueous medium solution of the crosslinking unit is 15~37°C, preferably 20~30°C, so that the preparation and use can be completed at room temperature or close to body temperature.

[0020] In some embodiments, the pH of the mixture of the aqueous medium solution of the stent unit and the aqueous medium solution of the crosslinking unit is 6.8 to 7.6.

[0021] In some embodiments, when the aqueous medium solution of the stent unit is mixed with the aqueous medium solution of the crosslinking unit, the molar ratio of the stent unit to the crosslinking unit is 1:(1~2), preferably 1:1.5.

[0022] In this invention, the aqueous medium refers to water or an aqueous solution. Preferably, the aqueous solution is a buffer solution containing buffer salts. The aqueous solution is preferably capable of creating an environment similar to the in vivo microenvironment of stem cells, such as physiological conditions (37°C, pH 6.8–7.4, 0.9 wt% NaCl, isotonic).

[0023] A third aspect of the invention provides a kit comprising the nucleic acid gel described herein.

[0024] A fourth aspect of the present invention provides the use of the nucleic acid gel or kit described herein in the preparation of a medicament for repairing spinal cord injury.

[0025] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) This invention constructs and verifies the modular DNA / RNA heteroduplex hydrogel system SeroPTEN-CG, which can synergistically solve two core problems in the treatment of spinal cord injury: reactivation of dormant neurons and reconstruction of spinal cord neural circuits. Through in-situ dynamic transformation from hydrogel to nanogel, and by adopting a controllable release strategy of 5-HTP and PTEN siRNA without mutual interference, neuronal excitability is restored on the one hand, and axon elongation and synaptic remodeling are promoted on the other hand, thereby promoting the reintegration of residual neural circuits at the structural and functional levels.

[0026] (2) The spinal cord injury treatment strategy proposed in this invention transforms the unavoidable microglial phagocytosis after injury into favorable neuroimmune regulation, creating a neuroimmune microenvironment conducive to the above-mentioned repair process. It demonstrates the feasibility of combining 5-HT-mediated functional reactivation with PTEN-targeted structural reconstruction, and provides a universal design paradigm for the synergistic integration of small molecule pharmacology and oligonucleotide therapy in spinal cord injury.

[0027] (3) The DNA / RNA heteroduplex hydrogel of the present invention has a highly modular architecture, and the platform can be redirected to other neural circuits and gene targets in the central nervous system by replacing the target ligand, small molecule payload or siRNA sequence.

[0028] (4) The dosage and ratio of 5-HTP-Br and PTEN siRNA used in this invention are proof of concept. Subsequent work can be carried out systematically to optimize them, and a more refined match can be achieved between "excitatory support" and "structural reconstruction", thereby further improving the repair effect. Future research will focus on: the depiction of dose profiles within the narrow therapeutic window of 5-HT, large-scale preparation, and extrapolation to more central nervous system targets and pathological types, so as to fully release its clinical translation potential. Attached Figure Description

[0029] Figure 1A The carbon NMR spectrum of compound 5-HTP-Br in the embodiments of this invention; Figure 1B This is the mass spectrum of compound 5-HTP-Br in the embodiments of the present invention; Figure 2This is a schematic diagram illustrating the chemically selective grafting, self-assembly, and physicochemical characterization of SeroPTEN-CG in this embodiment of the invention. A shows the site-selective covalent grafting of 5-hydroxytryptophan derivatives (5-HTP-Br) onto two phosphate thioester modification sites (PS) on single-stranded DNA (ssDNA) to obtain 5-HTP-DNA conjugates; B shows a comparison of denaturing PAGE images of unmodified ssDNA (Ya-2PS, Yb-2PS, Yc-2PS) and their 5-HTP-Br conjugates (5-HTP-Ya-2PS, 5-HTP-Yb-2PS, 5-HTP-Yc-2PS); C to H show mass shift and chemometric results from mass spectrometry, demonstrating site specificity of the grafting and complete occupancy of the two PS modification sites; I illustrates self-assembly: 5-HTP-Y-motif is PTEN-... The siRNA-Linker crosslinks to form a SeroPTEN-CG hydrogel network; J is a 10% non-denaturing PAGE gel electrophoresis confirming the self-assembly of 5-HTP-Y-motif and PTEN siRNA-Linker; the Sero PTEN CG hydrogel hardly penetrates the gel matrix; K is a SEM image of the freeze-dried Sero PTEN-CG hydrogel, showing an interconnected porous network; L is a rapid and reversible thermal response: gel formation upon mixing at room temperature; liquefaction at 90℃ (5 min); and gel formation again upon cooling to room temperature (1 min); M is a frequency scan showing that the storage modulus (G') is consistently higher than the loss modulus (G'') across the entire frequency range, a typical rheological characteristic of a stable hydrogel; N is a temperature scan showing that G' gradually decreases with increasing temperature, and G' / G'' crossover occurs at approximately 55℃; O is a denaturing PAGE gel electrophoresis showing that RNase H triggers the release of PTEN siRNA from the Sero PTEN-CG hydrogel, and the band intensity varies with RNase H (0–100). The release of 5-HTP is enhanced by the increase of U / mL; P represents the cumulative release of 5-HTP under specified conditions: the release is lowest (<20%) within 72 h at pH=7.4; the release is enhanced (approximately 40% / 72 h) at pH=5.0; and it is synergistically accelerated (>70% / 72 h) at pH=5.0 with the addition of esterase (17 U / mL). Figure 3 This is a schematic diagram illustrating the DNase-mediated generation and receptor targeting of SeroPTEN-CG nanogels in an embodiment of the present invention; wherein, A and B are images showing Cy5.5-SeroPTEN-CG (red) and MAP2 at the lesion site using a mouse spinal cord hemisection model and in vivo confocal imaging. + Neuron (green) colocalization; right side shows representative scan line scan; C and D show in vivo confocal and scan data displaying Cy5.5-SeroPTEN-CG (red) and 5-HT. +Neuronal (green) colocalization; E represents the quantitative analysis of Cy5.5 signal in the lesion area, indicating that the local accumulation of SeroPTEN-CG is significantly higher than that of PTEN-CG; Figure 4 This is an analysis of neuronal uptake, PTEN silencing, axonal regeneration, and pathway enrichment after SeroPTEN-CG intervention in this embodiment of the invention; wherein, A is a schematic diagram: after SeroPTEN-CG is internalized by neurons, RNase H mediates the release of PTENsiRNA, which in turn induces axonal regeneration; B is the co-incubation of neurons with FAM-labeled SeroPTEN-CG, and FCM analysis shows that the fluorescence peak gradually shifts to the right; C is the comparison of FCM results of neurons treated with different preparations (SCR-CG, PTEN-CG, SeroPTEN-CG) at 6 h; D is the quantitative analysis of MFI over time; E is the MFI analysis of each group at 6 h, with the SeroPTEN-CG group showing the highest intracellular signal; F is the axon-guided spot experiment: under CSPG barrier conditions (CSPG, red; Tuj1) + Axon (green; DAPI, nucleus) SeroPTEN-CG significantly increased axonal crossing; virtual scan indicates CSPG interface; G and H are quantitative analyses of the number of crossings (G) and average crossing length (H) for each neuron, and SeroPTEN-CG intervention significantly improved both of these indicators; I represents the state under inhibitory conditions (CSPG). + or CSPG - The effects of three formulations (SCR-CG, PTEN-CG, and SeroPTEN-CG) on axonal elongation were compared under different conditions; J represents the quantitative representation of axonal length: CSPG significantly reduced axonal length, while SeroPTEN-CG significantly reduced axonal length under different conditions. - With CSPG + All conditions can promote axonal elongation; K and L are bubble plots of KEGG (K) and GO (L) enrichment of DEGs after SeroPTEN-CG intervention, highlighting pathways / processes such as PI3K-Akt / mTOR signaling, cytoskeleton regulation, axonal guidance / regeneration, and inflammatory response; M is a volcano plot showing transcriptomic changes, with representative genes (such as PTEN, SOCS3, and mTOR) labeled; N to Q are GSEA enrichment line scans showing positive enrichment of PI3K-Akt (N), cytokine-receptor interaction (O), axonogenesis (P), and neurotransmitter secretion (Q) after SeroPTEN-CG intervention; Figure 5In this embodiment of the invention, SeroPTEN-CG significantly inhibits PTEN expression in neurons; wherein, A is the qRT-PCR quantitative analysis of Pten mRNA among primary neurons, with the CTRL group as a reference for relative expression display; B and C are the PTEN expression of primary neurons after co-incubation with different preparations (SCR-CG, PTEN-CG, SeroPTEN-CG), with representative Western blot images (B) and relative expression display with the CTRL group as a reference (C); Figure 6 In this embodiment of the invention, SeroPTEN-CG reprograms microglia to the M2 type; wherein, A is a Scheme diagram: activated M1 type microglia undergo "off-target" uptake of SeroPTEN-CG nanogel, driving them to polarize towards the M2 type; B and C are Western spectral analyses of PTEN silencing after microglia are co-incubated with different formulations (SCR-CG, PTEN-CG, SeroPTEN-CG). Blot images (B) and statistical analysis results (C); D and E are microglial immunofluorescence imaging, marking the expression of iNOS / Iba1 / DAPI (D) and Arg1 / Iba1 / DAPI (E), respectively; F to I quantitative analysis results show that SeroPTEN-CG can significantly reduce iNOS fluorescence intensity (F) and iNOS / Iba1 index (G), while increasing Arg1 fluorescence intensity (H) and Arg1 / Iba1 index (I), suggesting that it can promote the conversion from M1 type to M2 type; J and K are qRT-PCR analysis of iNOS (J) and Arg1 (K) to confirm that PTEN-CG or SeroPTEN-CG intervention can inhibit iNOS expression and promote Arg1 expression. Figure 7In this embodiment of the invention, SeroPTEN-CG achieves neuroimmunomodulation and neuroprotection by reprogramming microglia to the M2 phenotype. A shows the distribution of CD68 (red), CD206 (green), and DAPI (blue) in the spinal cord tissue of different treatment groups (CTRL, SCR-CG, PTEN-CG, SeroPTEN CG) at the second week after spinal cord injury, using immunofluorescence staining. High-power images of the cephalic (R) and caudal (C) regions are indicated by boxes. B and C respectively quantify the CD68 fluorescence intensity (B) and CD206 / CD68 index (C) in the cephalic and caudal regions. D and E show qRT-PCR detection of the injured spinal cord tissue, revealing that SeroPTEN-CG intervention significantly downregulated pro-inflammatory factors TNF-α, IL-1β, and IL-6 (D), and significantly upregulated anti-inflammatory factors IL-10 and TGF-β (E). F shows the peri-lesional region of each group. Representative immunofluorescence images of the region, labeled MAP2 (green), NeuN (red), and DAPI (blue); the right side (A1ROI-A4ROI) shows high-power images of the corresponding regions; G and H represent quantitative measurements of the density of interneurons around the injury (G) and the average distance (H) from the remaining interneurons to the injury center, indicating that SeroPTEN-CG and PTEN-CG have significant neuroprotective effects, with SeroPTEN-CG showing a more significant effect; I and J show high-power imaging indicating preservation of synaptic structures: synaptophysin (SYN, gray) and MAP2. + Neurons (green) colocalize in the cephalic (I) and caudal (J) regions of the injury, suggesting that functional synaptic structures were preserved; Figure 8 In this embodiment of the invention, SeroPTEN-CG remodels the extracellular matrix and promotes the integration of specific neural circuits; A represents the axon-ECM interface, shown as NF200 (green) and laminins, Ln (red); compared to CTRL and SCR-CG, PTEN CG, especially SeroPTEN-CG, significantly reduces Ln and rearranges Ln along the rostral-caudal axis of the spinal cord; simultaneously, NF200 is visible. +Axons follow the rearranged Ln fibers (A1ROI-A4ROI on the right side is the high-magnification area); B shows the detection of NF200 (green) and CSPG (gray) to assess the inhibition of glial scarring; CTRL and SCR-CG groups show a large amount of CSPG deposition; CSPG after PTEN-CG treatment. Significant decrease was observed, with the most significant decrease in the SeroPTEN-CG group. Axons were also visible crossing the CSPG-rich region in the SeroPTEN-CG group (high-magnification region on the right, B1ROI-B4ROI); (C) shows quantitative analysis of CSPG fluorescence intensity in each treatment group; D shows representative spinal cord sections from the CTRL and SeroPTEN-CG groups, displaying glial network GFAP (green), stromal spinal cord CST (AAV9-hSyn-mCherry) (red), and nuclear DAPI (blue); insets (D1R / D1C, D2R / D2C) are high-magnification images of the cephalic / choledal region and single-channel CST signals; E shows quantitative analysis of CST signal intensity in the cephalic and caudal regions of each group; F shows high-magnification confocal images of the cephalic and caudal regions of the SeroPTEN-CG group, displaying 5-HTergic terminals (5-HT, red) and MAP2. + Neurons (green) and synaptophysin SYN (gray); the right / bottom is an orthographic view indicating 5-HT and MAP2. + Spatial colocalization with SYN; G is a high-magnification confocal image of the cephalic and caudal regions of the SeroPTEN-CG group, showing glutamatergic terminals (vGLUT, red) and MAP2. + Neuron (green) and SYN (gray); right / bottom orthographic view highlighting vGLUT and MAP2. + Colocalization with SYN suggests that glutamatergic synapses are incorporated into the reconstructed neural circuits; Figure 9 This is an example of synaptic remodeling and bladder pathology analysis after SeroPTEN-CG intervention in this invention; wherein, A is a high-magnification confocal image of the SeroPTEN-CG group in the head and tail positions of the injury, showing tyrosine hydroxylase (TH, red) and MAP2. + Neuron (MAP2, green) and synaptophysin (SYN, gray) are shown in a mid-lateral / lower orthogonal view highlighting TH and MAP2. + Co-localization with SYN suggests that SeroPTEN CG intervention can promote the reconstruction of synaptic connections; B represents the high-magnification large view of the urine bladder H&E staining in the sham surgery, CTRL, SCR-CG, PTEN-CG and SeroPTEN CG groups, used to show the improvement of bladder wall structure. Figure 10This is a proteomic analysis of the SeroPTEN-CG treatment in this embodiment of the invention. A and B represent proteomic pathway enrichment analyses after SeroPTEN-CG intervention: KEGG enrichment analysis (A) and GO enrichment analysis (B) show that multiple functional pathways related to neural repair, inflammation regulation, and signal transduction are activated. C is a volcano plot of differentially expressed proteins (DEPs) with representative protein molecules labeled. D to F are KEGG-based GSEA analyses showing significant enrichment of the 5-HTergic synaptic pathway (D), NF-κB signaling pathway (E), and JAK-STAT signaling pathway (F). G to I are GO-based GSEA analyses showing enrichment of positive regulation of axonal regeneration (G), serotonin metabolism (H), and inflammatory response (I), further indicating that SeroPTEN-CG can synergistically regulate key biological processes related to neural regeneration and inflammation. Figure 11 In this embodiment of the invention, SeroPTEN-CG promotes the recovery of motor and sensory function after spinal cord injury; wherein, A is a schematic diagram of the experiment: right spinal cord hemisection and local administration of SeroPTEN-CG; B shows the changes in BMS scores over time in the Sham, CTRL, SCR-CG, PTEN-CG and SeroPTEN-CG groups within 6 weeks postoperatively; C is a comparison of BMS scores among the groups at week 6 postoperatively; D is CatWalk Representative footprints obtained from XT show the gait patterns of each group; E to K represent gait quantification parameters, including gait regularity index (E), base of support (F), swing velocity (G), footprint width (H), maximum contact area (I), maximum peak contact intensity (J), and maximum footprint intensity (K); L represents the results of the plantar hot plate test, showing the withdrawal latency of each group; M to N represent motor evoked potentials (MEP): representative waveforms (M) and their amplitude quantification results (N); O to P represent sensory evoked potentials (SEP): representative waveforms (O) and their amplitude quantification results (P); Q to R represent neuronal activation in the thoracic T8 spinal cord: c-Fos (green), NeuN (red), and DAPI (blue) immunofluorescence staining and their high-power insets (Q1ROI-Q5ROI below); c-Fos staining of the dorsal / intermediate and ventral regions... + / NeuN + Neuronal quantification (R); S to T represent neuronal activation in the L2 lumbar spinal cord: c-Fos (green), NeuN (red), and DAPI (blue) immunofluorescence staining and their high-power insets (S1ROI-S5ROI below); c-Fos staining was also performed on the dorsal / intermediate and ventral regions. + / NeuN + Neurons are quantified (T); Figure 12This is a schematic diagram of serum biochemical index detection in an embodiment of the present invention; wherein, A to D are the study endpoints, and the serum ALT (A), AST (B), BUN (C) and CREA (D) levels of each group of CTRL, SCR-CG, PTEN-CG and SeroPTEN-CG were detected respectively. There were no significant differences among the groups, indicating that this treatment did not cause significant liver and kidney function damage. Figure 13 This is a schematic diagram of histopathological results in an embodiment of the present invention; representative H&E staining results of liver, heart, spleen, lung and kidney in the Sham, CTRL, SCR-CG, PTEN-CG and SeroPTEN-CG groups show that the tissue structure of each group is intact, without necrosis and diffuse inflammatory cell infiltration. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. 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 invention pertains.

[0031] 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 of the present invention. 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, and / or combinations thereof.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: Synthesis and Characterization of SeroPTEN-CG 1. Preparation of 5-HTP-Br To achieve covalent coupling between 5-HTP and ssDNA, we modified 5-HTP with acylethyl bromide (5-HTP-Br) via a three-step synthetic route, as follows: Boc-5-hydroxy-DL-tryptophan (4.00 g, 12.4 mmol) was dissolved in a mixed solvent of tetrahydrofuran (THF, 40.0 mL) and dichloromethane (DCM, 40.0 mL). Tert-butyl-2,2,2-trichloroacetimidate was then added dropwise, and the reaction was stirred at 25 °C for 16 h. After the reaction was complete, the solution was diluted with water (100 mL) and extracted with DCM (50.0 mL × 3). The combined organic phases were washed with saturated brine (50.0 mL × 2), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether (PE) / ethyl acetate (EtOAc) gradient elution, from 5:1 to 2:1) to give compound I (yellow oil, 1.70 g, 4.52 mmol, yield 36.1%).

[0034] Compound I (1.70 g, 4.52 mmol) was dissolved in acetonitrile (ACN, 10.0 mL), and N,N-diisopropylethylamine (DIPEA, 1.17 g, 9.03 mmol, 1.57 mL) and bromoacetic anhydride (2-bromoacetic anhydride, 1.17 g, 4.52 mmol) were added sequentially. After stirring at 25 °C for 3 h, the mixture was diluted with water (30 mL) and extracted with DCM (20.0 mL × 3). The organic phase was washed with saturated brine (20.0 mL × 2), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EtOAc gradient, from 5:1 to 2:1) to give compound II (yellow oil, 1.20 g, 2.41 mmol, yield 53.4%).

[0035] Compound II (800 mg, 1.61 mmol) was dissolved in DCM (5.00 mL), and trifluoroacetic acid (TFA, 1.47 g, 12.8 mmol, 955 μL) was added with stirring. After stirring at 25 °C for 30 min, the mixture was concentrated under reduced pressure to obtain the crude product, which was purified by preparative high-performance liquid chromatography (prep-HPLC) to obtain 5-HTP-Br (pale yellow solid, 458 mg, 1.16 mmol, yield 2.3%, purity 96.0%). 1 H NMR, 13 C NMR and mass spectrometry are respectively as follows: Figure 1A and Figure 1B As shown, carbon NMR spectrum ( 13The 5-HTP-Br was successfully synthesized, with the 5-C NMR spectrum matching the expected structure and the mass spectrometry (MS) measurement confirming that the molecular weight (Da) was consistent with the theoretical calculation.

[0036] 2. Preparation of 5-HTP-DNA conjugate.

[0037] 5-HTP-Br was grafted onto three complementary DNA strands (Ya-2PS: TGGATCC*GCATGACATTCGCC*GTAAG) via a reaction of benzyl bromide and PS groups. TTGACCTGTGAA (SEQ ID NO: 1); Yb-2PS: CTTACGG*CGAATGACCGAATC*AGCCT TTGACCTGTGAA (SEQ ID NO: 2); Yc-2PS: AGGCTGA*TTCGGTTCATGCGG*ATCCA TTGACCTGTGAA (SEQ ID NO: 3); where "*" indicates the phosphate thioester modified site (PS site); "underline" indicates "sticky end", i.e. the sticky end of the scaffold), thereby preparing the 5-HTP-DNA conjugate ( Figure 2 A) The specific steps are as follows: DNA strands modified with phosphate thioesters (PS) (100 μM, Ya-2PS, Yb-2PS, Yc-2PS) and compound 5-HTP-Br (6 mM) were dissolved separately in dimethyl sulfoxide (DMSO). In the reaction system, the molar ratio of 5-HTP-Br to the scaffold single-stranded nucleic acid was 60:1. The reaction was carried out with gentle shaking at 50°C for 30 min. After precipitation with ethanol and removal of the solvent by vacuum evaporation, 5-HTP-modified DNA was obtained, which are 5-HTP-DNA conjugates, specifically including 5-HTP-Ya-2PS, 5-HTP-Yb-2PS, and 5-HTP-Yc-2PS.

[0038] Each ssDNA strand is designed with two phosphate thioester (PS) modification sites (Ya-2PS, Yb-2PS, Yc-2PS). A Y-motif composed of three complementary DNA strands is used to construct the backbone. Two phosphate thioester (PS) groups are uniformly dispersed in each DNA strand to enhance the DNA molecule's resistance to enzymatic degradation and help it penetrate into cells (Ya-2PS, Yb-2PS, Yc-2PS). At the same time, 12 base scaffold sticky ends are left at the end of each DNA strand to facilitate complementary pairing and cross-linking with the cross-linking sticky ends of PTEN siRNA.

[0039] 15% denaturing polyacrylamide gel electrophoresis (PAGE) showed that the bands were clear and monodisperse, and that the ssDNA grafted with 5-HTP-Br migrated more slowly than the ungrafted ssDNA. Figure 2 B), indicating successful 5-HTP-Br grafting. Mass spectrometry analysis showed that the molecular weights of ungrafted Ya-2PS, Yb-2PS, and Yc-2PS were 11724.8 Da, 11694.2 Da, and 11755.6 Da, respectively, while those after 5-HTP-Br grafting increased to 12249.6 Da, 12218.2 Da, and 12280.6 Da, respectively, further proving that 5-HTP-Br was successfully grafted onto ssDNA (B). Figure 2 CH).

[0040] 3. Synthesis and Characterization of SeroPTEN-CG Three 5-HTP-DNA conjugates were mixed in equal molars, annealed at 95°C for 5 min, and allowed to cool naturally to room temperature. They then assembled into a Y-motif through complementary pairing. PTEN siRNA (L1 (Sense strand): rGrGrGrUrUrUrGrArUrArArGrUrUrCrUrArGrC) rUrUrCrArCrArGrGrUrCrArA (SEQ ID NO: 4); L2 (Antisense strand): rGrCrUrArGrArArCrUrUrArUrCrArArArCrCrC rUrUrCrArCrArGrGrUrCrArA (SEQ ID NO: 5); where the underlined part represents the "sticky end", i.e. the cross-linked sticky end) complementary bases are introduced on both sides of the double strand to the sticky end of the Y-motif and used as a cross-linking agent (linker) to cross-link with the Y-motif to form an RNA / DNA hybrid. The specific steps are as follows: the 5-HTP modified Y structure unit is mixed with PTEN siRNA-linker at a molar ratio of Y-motif:linker of 1:1.5 and then co-incubated at room temperature for 30 min. Further confirmation using 10% non-denaturing PAGE electrophoresis revealed that the 5-HTP-Y-motif motif self-assembled with the PTEN siRNA-Linker to form SeroPTEN-CG. SeroPTEN-CG appeared as a clear, monodisperse band with slower migration and minimal penetration into the colloidal interior. Figure 2 I, J). Scanning electron microscopy (SEM) revealed its characteristic porous microstructure, suggesting effective cross-linking between the 5-HTP-Y-motif motif and the PTEN siRNA-Linker at the micrometer scale. Figure 2K). SeroPTEN-CG exhibits a reversible thermoresponsive gel-solution transition: it liquefies upon heating to 90 °C and holding for 5 min, and then spontaneously reverts to its original gel state upon being placed at room temperature for 1 min. Figure 2 L), consistent with the pairing and uncoupling process mediated by Watson-Crick base complementary crosslinking. Further characterization using a rotational rheometer: frequency-dependent rheological results showed that the storage modulus (G′) was consistently higher than the loss modulus (G″) across the entire frequency range, consistent with the viscoelastic properties of typical hydrogels. Figure 2 Temperature-dependent rheological results showed that G′ gradually decreased in the range of 25 °C to 70 °C, and crossed with G″ at approximately 55 °C. Figure 2 (N), further suggesting that the integrity of the hydrogel network structure is dominated by molecular recognition and complementary base pairing. When the crosslinking mediated by complementary base pairing is thermally destroyed, the network structure of SeroPTEN-CG hydrogels disintegrates.

[0041] SeroPTEN-CG hydrogels are assembled through cross-linking between 5-HTP-Y-motif and PTEN siRNA-Linker, forming a DNA / RNA heterologous double strand. The RNA strand in this double strand can be selectively cleaved by RNase H, thereby releasing PTEN siRNA. To verify this RNase H-mediated release, we incubated SeroPTEN-CG with gradient concentrations of RNase H and performed denaturing PAGE gel electrophoresis. Clear bands corresponding to PTEN siRNA were observed, and the band intensity was positively correlated with the RNase H concentration, confirming RNase H-mediated PTEN siRNA release. Figure 2 O). 5-HTP was grafted onto the PS site of ssDNA via ester bonds. The release kinetics of 5-HTP were determined under different pH conditions (neutral pH=7.4 and acidic pH=5.0) and with / without esterase (17 U / mL), and quantitative analysis was performed using high-performance liquid chromatography (HPLC). Results showed that under physiological conditions (pH=7.4), the release of 5-HTP within 72 h was <20%; lowering the pH to acidic (pH=5.0) increased the release to approximately 40%; the acidic environment (pH=5.0) combined with esterase conditions (17 U / mL) produced a synergistic effect, with cumulative release exceeding 70%. Figure 2 The above results indicate that the 5-HTP derivative, after grafting, exhibits good extracellular stability as a serotonergic targeting ligand, while also functioning as a neurotransmitter for rapid intracellular release.

[0042] In summary, grafting 5-HTP-Br at the PS site, combined with complementary base pairing-mediated self-assembly, achieves a defined stoichiometric loading and a quantitative 5-HTP:siRNA ratio. Furthermore, RNase H-mediated siRNA release and pH / esterase-mediated 5-HTP release form two independent triggering mechanisms, avoiding interference and enabling differentiated quantification. This facilitates synergistic therapy and supports a combined strategy for restoring neuronal excitability and reconstructing residual neural circuits in spinal cord injury repair.

[0043] Example 2: Serotonin system targeting and local accumulation at the site of injury After local administration of SeroPTEN-CG nucleic acid gels (SeroPTEN-CG hydrogels) to the lesion, via internal... Nuclease-derived cleavage transforms the nucleotides into SeroPTEN-CG nanogels. To verify this process, the nucleotide gels were co-incubated with increasing concentrations of DNase I at 37°C for 12 h. The supernatant was then collected, and particle size distribution was analyzed using dynamic light scattering (DLS). The resulting nanogel particle size decreased progressively with increasing DNase I concentration, suggesting that the SeroPTEN-CG hydrogels can disassemble and reassemble under DNase-mediated conditions, supporting their feasibility for localized nanostructuring in lesions. Further investigation was conducted to determine whether the aforementioned targeting ability remains in the complex in vivo lesion microenvironment. Spinal cord tissue was harvested 24 h after local administration of the SeroPTEN-CG gels for fluorescence co-localization analysis. The red fluorescence of the SeroPTEN-CG gels selectively enriched in the lesion area and was positive for MAP2 (MAP2 positive). + Mature neurons exhibit high colocalization. Figure 3 AB), MAP2 + Mature neurons represent those that survive the primary injury and participate in remodeling. Simultaneously, they are associated with 5-HT-positive neurons (5-HT...). + Colocalization of neurons suggests that it effectively targets a serotonergic population, which is involved in regulating motor network reactivation. Figure 3 CD). In contrast, there was almost no significant enrichment and co-localization in the PTEN-CG (without 5-HTP-Br modification) group; quantitative analysis showed that the fluorescence intensity of SeroPTEN-CG in the damaged area was significantly higher than that of PTEN-CG (CD). Figure 3 E), suggesting 5-HTP-Br-mediated receptor-specific targeting and local retention, thereby reducing clearance and prolonging in situ retention time. In summary, the SeroPTEN-CG hydrogel undergoes DNase-mediated disassembly to form a nanogel, which then crosses the extracellular matrix (ECM) and targets the serotonin system through receptor-dependent recognition.

[0044] Example 3: Neuronal PTEN Silencing and Axonal Growth This embodiment further investigates whether SeroPTEN-CG can be efficiently internalized by neurons and promote axonal elongation. Figure 4 A). Neurons were co-incubated with FAM-labeled SeroPTEN-CG for 0, 1, 2, 4, and 6 h, and then the mean intracellular fluorescence intensity (MFI) was detected by flow cytometry (FCM). The results showed that the intracellular MFI increased in a time-dependent manner. Figure 4 (B, D) This suggests that SeroPTEN CG nanogels can be rapidly taken up by neurons and accumulated in the cytoplasm, laying the foundation for intracellular siRNA delivery. To compare endocytosis efficiency, intracellular MFI was quantified at 6 h for the SCR-CG (Scramble siRNA-linked, without 5-HTP-Br modification), PTEN-CG, and SeroPTEN-CG groups. The results showed that the fluorescence intensity of SeroPTEN CG was significantly higher than that of SCR-CG and PTEN-CG (B, D). Figure 4 (C, E), this difference can be attributed to ligand-mediated target specificity and enhanced binding affinity.

[0045] To determine whether endocytosis mediates PTEN silencing, neurons were treated with PBS, SCR-CG, PTEN-CG, or SeroPTEN-CG (10 μL injected locally at the spinal cord injury site in each mouse), and qRT-PCR and Western blot analyses were performed. Both PTEN-CG and SeroPTEN-CG significantly downregulated PTEN mRNA, with SeroPTEN-CG showing the strongest effect, consistent with its strongest neuronal endocytosis activity. Figure 5 A). Western blot showed that the decrease in PTEN protein was most significant in the SeroPTEN CG group ( Figure 5 (B, C) indicates a stronger silencing effect at both the transcriptional and translational levels. These results collectively demonstrate that SeroPTEN-CG achieves superior PTEN silencing compared to the control, providing a mechanistic basis for spinal cord neural circuit remodeling.

[0046] To examine whether SeroPTEN-CG could activate the intrinsic growth program of neurons to overcome the inhibitory effect of scar components, we evaluated its ability to antagonize the inhibition of axonal regeneration by chondroitin sulfate proteoglycan (CSPG). In the axonal guided spot assay, SeroPTEN-CG significantly improved the ability of axons to cross the CSPG barrier: the average number of crossings exceeded 2 per neuron, and the average crossing length exceeded 4 μm, both significantly higher than other groups. Figure 4 FH). Subsequently, neurons were cultured in CSPG (CSPG)-containing medium. +) or without CSPG (CSPG - The culture medium was prepared by adding PBS, SCR-CG, PTEN-CG, or SeroPTEN-CG to the medium. Figure 4 (I) The results show that, compared to CSPG - In contrast, CSPG exposure significantly shortened axon length; while in CSPG... + With CSPG - Under both conditions, SeroPTEN-CG significantly promoted axonal elongation. Figure 4 In summary, SeroPTEN-CG effectively promotes axonal elongation, providing a mechanistic basis and translational potential for reconstructing functional neural circuits after spinal cord injury.

[0047] Based on this, transcriptome data after SeroPTEN-CG intervention were further compared to identify differentially expressed genes (DEGs) and their related regeneration pathways. DEGs between the two groups are presented using a volcano plot, with upregulated genes marked in red. Figure 4 M). To investigate the biological functions and pathways involved, KEGG / GO enrichment analysis was conducted. Twenty KEGG pathways included PI3K-Akt, MAPK, mTOR signaling pathway, actin cytoskeleton regulation, and inflammatory responses. Figure 4 K); GO analysis showed significant enrichment in processes such as axonal regeneration, neurite development, and cytokine-mediated signaling pathways (K); Figure 4 L).

[0048] Furthermore, gene set enrichment analysis (GSEA) based on the Hallmark gene set further highlights PI3K-Akt ( Figure 4 N), cytokine-receptor interaction ( Figure 4 O), axonal formation ( Figure 4 P), neurotransmitter secretion ( Figure 6 Positive enrichment of Q). These results indicate that SeroPTEN-CG intervention can significantly remodel signaling pathways related to neural regeneration, damage repair, and inflammation.

[0049] In summary, enhanced neuronal uptake, PTEN silencing, improved axonal regeneration, and enrichment of pro-regeneration pathways collectively support a coherent mechanism: SeroPTEN-CG, through receptor-mediated intracellular delivery, activates the PI3K-Akt / mTOR-related growth program within neurons and promotes axonal elongation. Simultaneously, the transcriptomic enrichment of actin cytoskeleton regulation and axonogenesis further provides a mechanistic basis for reconstructing neural circuits.

[0050] Example 4: Neuroimmune regulation and neuroprotection Spinal cord injury triggers a multifactorial pathological cascade: following primary injury, a rapid progression to inflammation-driven secondary injury occurs, dominated by M1 microglia. The continuous release of pro-inflammatory cytokines from these microglia exacerbates neurotoxicity, disrupts residual neural circuits, and inhibits regeneration-related signals. Reprogramming M1 microglia into M2 microglia with reparative properties is considered a key strategy to block the aforementioned pathological cascade and establish a pro-regenerative neuroimmune microenvironment. Accordingly, this embodiment proposes an adaptive rescue design: utilizing the inherent phagocytic behavior of M1 microglia as a starting point, inducing their polarization towards reparative M2 microglia (…). Figure 6 A). Macrophages were co-incubated with 5 μL hydrogel in 6-well plates for 24 h, and changes in their polarity were then detected. Western blot showed that both SeroPTEN-CG and PTEN-CG significantly inhibited PTEN expression in microglia, and had a clear silencing effect compared to CTRL and SCR-CG. Figure 6 B, C). It is important to emphasize that no significant difference was observed between PTEN-CG and SeroPTEN-CG in microglia compared to neurons. A reasonable explanation is that microglia possess a higher phagocytic capacity, which dominates the entry efficiency of SeroPTEN-CG, thus masking the difference brought about by 5-HTP-Br. Subsequently, LPS-pretreated microglia were treated with SCR-CG, PTEN-CG, or SeroPTEN-CG for 24 h, followed by immunofluorescence analysis (…). Figure 6 D, E). Compared with the CTRL and SCR-CG groups, both PTEN-CG and SeroPTEN-CG significantly downregulated iNOS fluorescence intensity and the iNOS / Iba1 index, while upregulating Arg1 intensity and the Arg1 / Iba1 index. Figure 6 FI suggests that microglia are reprogrammed from M1 to M2 morphology. qRT-PCR further corroborates this: both groups significantly inhibited iNOS transcription and induced Arg1 transcription, with no significant differences between them. Figure 7 J, K). Then, at week 2 post-injury in a mouse spinal cord injury model (a key time point for microglial activation and infiltration), microglial activation (CD68 intensity) and polarization (CD206 / CD68 index) were assessed. Figure 7 A). The CTRL and SCR-CG groups showed elevated CD68 intensity and extremely low CD206 co-expression, suggesting a predominantly pro-inflammatory M1 phenotype. In contrast, SeroPTEN-CG significantly decreased CD68 and increased CD206 co-expression. + / CD206 + The proportion of double-positive microglia ( Figure 7(B, C) indicates M2-type polarization towards tissue repair. It is noteworthy that although PTEN-CG and SeroPTEN-CG showed similar polarization effects in in vitro experiments, SeroPTEN-CG demonstrated a superior effect in in vivo experiments ( Figure 7 (B, C) This may be due to its receptor-mediated retention and bioavailability at the lesion site. qRT-PCR further confirmed that the SeroPTEN-CG group showed significantly decreased levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, while significantly upregulated levels of anti-inflammatory cytokines IL-10 and TGF-β, all superior to other groups. Figure 7 D, E).

[0051] An optimized neuroimmune microenvironment can alleviate neurotoxicity and secondary damage, thereby protecting residual neurons and synaptic structures and providing a structural and functional basis for subsequent spinal cord neural circuit reconstruction. For example... Figure 7 As shown in F, both PTEN-CG and SeroPTEN-CG have significant neuroprotective effects, increasing neuronal activity, as evidenced by a significant increase in interneuron density. Figure 7 G). Among them, SeroPTEN-CG showed the most significant effect, presumably due to the synergistic effect of "target specificity" and "neuronal PTEN inhibition," the latter possibly achieved through activation of the neuronal PI3K-Akt / mTOR pathway, which has been considered equally crucial in neuroprotection in recent years. Furthermore, both PTEN-CG and SeroPTEN-CG significantly shortened the average distance from the surviving interneurons to the injury center (G). Figure 7 Protection at this spatial level (H) is particularly important because neurons closer to the injury center are more conducive to the functional integration of reconstructed neural circuits. Morphological analysis further revealed that the protected surviving neurons retained their mature morphology and co-localized with synaptophysins (SYNs, markers of neuronal synapses), suggesting that synaptic structure and signal transmission capabilities were preserved after spinal cord injury. Figure 8 I, J). Current anti-inflammatory strategies for spinal cord injury are limited by systemic exposure and the therapeutic window. This study utilizes the inevitable phagocytosis of microglia to deliver PTEN siRNA via phagocytosis, transforming the original "off-target" phagocytosis into "on-target" immune reprogramming to promote M2 polarization. Furthermore, because this action relies on phagocytic uptake rather than receptor targeting, it has a natural targeting ability for inflamed tissues, conceptually extrapolating to injuries beyond spinal cord injury. Overall, this strategy helps alleviate the challenge of payload loss due to the clearance of nanomedicines by the innate immune system and proposes a rarely explored inflammation control approach: in In this approach, phagocytosis is not merely an obstacle, but can be engineered to achieve sustained immune regulation.

[0052] Example 5: Extracellular matrix (ECM) remodeling and specific spinal cord circuit integration In addition to neuronal damage, the secondary inflammatory cascade can also induce excessive deposition of extracellular matrix components in fibrotic cells (especially laminin, Ln), significantly promoting the formation of fibrotic scars. The CTRL group showed obvious fibrotic scars, characterized by dense Ln accumulation in the injury area; while after PTEN-CG treatment, especially SeroPTEN-CG treatment, Ln deposition was significantly reduced and rearranged along the rostral-caudal axis of the spinal cord. Figure 8 A). Meanwhile, in the CTRL and SCR-CG groups, Ln fibers encapsulate NF200. + Axons, hindering their further extension; in contrast, the SeroPTEN-CG group exhibited large, continuous regenerated axons consistent with the orientation of the remodeled Ln fibers. Figure 8 A) suggests that SeroPTEN-CG-induced ECM remodeling creates a more permeable microenvironment and structural base for axonal elongation. Furthermore, chondroitin sulfate proteoglycans (CSPGs) were used to assess glial scars; CSPGs are the main inhibitory component against axonal regeneration. In CTRL, a large amount of CSPG deposition was observed after spinal cord injury; while PTEN-CG significantly reduced the load of this inhibitory ECM, especially the reduction effect of SeroPTEN-CG (). Figure 8 B, C). More importantly, in the SeroPTEN-CG group, regenerated axons were observed crossing CSPG-enriched regions, while this crossing was almost never observed in the CTRL and SCR-CG groups. Figure 8 B) suggests that SeroPTEN-CG not only lowers the inhibitory barrier of the ECM, but also promotes axonal crossing of the aforementioned barrier.

[0053] One of the most significant advances in spinal cord injury treatment over the past few decades has been the promotion of reconnection of interrupted neural circuits to restore nerve impulse transmission between projection neurons and their downstream targets. However, given the highly hierarchical structure and highly specific function of spinal cord neural circuits, current research is shifting from "simple structural reconnection" to "precise integration of specific spinal cord circuits." Based on this, we further focus on reconstructing specific relay neural circuits in the injury area. After SeroPTEN-CG treatment, the corticospinal tract (CST) labeled with AAV9-hSyn-mCherry showed greater extension on both the cephalic and caudal sides of the injury, suggesting that SeroPTEN-CG intervention can achieve broader reconnection and more efficient spinal cord circuit reconstruction. Figure 8(D, E). This structural remodeling is consistent with the activation of the intraneuronal PI3K-Akt / mTOR pathway, which has been shown to promote CST axon regeneration and collateral sprouting. Notably, in the lesion area, 5-HTergic axons and MAP2... + Neurons form synaptic connections ( Figure 8 This is thanks to the dual-function design of SeroPTEN-CG: on the one hand, 5-HTP is metabolized into 5-HT, thereby enhancing local 5-HT synthesis and release, which in turn widely activates 5-HT receptors in the damaged segment and enhances synaptic excitability; on the other hand, the delivery of PTEN siRNA targeting the 5-HT system can synergistically promote axon elongation and circuit reconstruction, which is beneficial for building a functionally specific synaptic network.

[0054] Further validation of functionally specific neural circuit integration and detection of glutamatergic innervation. The excitatory synaptic marker vesicular glutamate transporter (vGLUT) was observed in the lesion area in conjunction with MAP2. + Neuronal colocalization ( Figure 9 (G) indicates that glutamatergic transmission has been incorporated into the reconstructed synaptic network. Spatial colocalization analysis shows that 5-HT, vGLUT, and SYN are colocalized and synchronously distributed, suggesting that the 5-HTergic and glutamatergic systems have achieved coordinated reconstruction, forming a multisynaptic relay network. Considering the complementary roles of 5-HTergic regulation in locomotor rhythm and glutamatergic regulation in motor control (locomotor excitation), this synergistic integration provides a structural basis for regulating the function of the CPG (median pattern generator) and descending nerve tracts.

[0055] Regarding autonomic neural circuits, tyrosine hydroxylase-positive (TH) enzymes, essential for bladder reflex regulation, are involved. + The axon also formed synaptic contacts after SeroPTEN-CG treatment. Figure 9 A). Correspondingly, PTEN-CG, especially SeroPTEN-CG, can significantly alleviate bladder dysfunction, including improvement in bladder morphology and decrease in bladder capacity. Figure 10 B) indicates that the reflexive urination function has recovered to some extent.

[0056] The above results confirm that SeroPTEN-CG can simultaneously promote the structural and functional repair of spinal cord neural circuits. To elucidate its regulatory network, further proteomic analysis was conducted. KEGG enrichment revealed 20 enriched pathways, including: neurodegeneration pathways, 5-HTergic synapses, p53 signaling pathways, PI3K-Akt signaling pathways, mTOR signaling pathways, and glutamatergic synapses, all of which are closely related to the pathophysiological processes of spinal cord injury treatment. Figure 10A). GO enrichment suggests that processes related to neural repair and inflammation are significantly activated, including neuronal differentiation, neurogenesis, and cytokine-mediated signal transduction. Figure 10 B). Volcano plots are used to visualize changes in protein expression and are labeled with representative differentially expressed proteins (DEPs). Figure 10 C). Further validation and supplementation were performed using GSEA. KEGG-related GSEA showed significant enrichment of 5-HTergic synapses, NF-κB signaling pathways, and JAK-STAT signaling pathways. Figure 10 DF); in GO-related GSEA, it is enriched in promoting axonal regeneration, 5-HT metabolism, and inflammatory responses, etc. Figure 11 (GI). Notably, the significant enrichment of 5-HT synapses and 5-HT metabolic processes confirmed by GSEA suggests that 5-HT signaling is enhanced in the damaged area, which is consistent with the material design of SeroPTEN-CG to increase excitability by enhancing 5-HT modulation.

[0057] Overall, SeroPTEN-CG enables the reintegration of residual neural circuits by synergistically activating neuronal growth programs and optimizing the neuroimmune microenvironment. This not only achieves the recovery of spinal cord neural circuits after spinal cord injury, but also has the potential to be extended to other central nervous system injuries and guide translational strategies.

[0058] Example 6: Functional recovery and reactivation of spinal cord neural circuits after spinal cord injury To systematically evaluate the effect of SeroPTEN-CG in promoting the recovery of motor and sensory function after spinal cord injury, functional assessments were conducted using the Basso Mouse Scale (BMS) score, gait analysis, hot plate test, and electrophysiological testing. All mice showed normal right limb movement and a BMS score of 9 immediately after administration; complete paralysis occurred immediately after right spinal cord resection, with a BMS score of 0. Figure 11 A, B). During the 6-week recovery period, the CTRL group showed only mild spontaneous recovery, with a BMS of 3.25±0.16 (mean±SEM); the SCR-CG group showed further improvement, with more frequent plantar stamping, and a BMS of 3.63±0.18, but both were significantly lower than the PTEN-CG group (4.25±0.25). In contrast, SeroPTEN-CG showed continuous improvement from week 1, reaching a mean BMS of 5.25 at week 6, with phenotypes including: plantar stamping, hindlimb weight-bearing, off-ground movement, and some hindlimb coordination (…). Figure 11 B). Statistical analysis showed that approximately 40% of mice reached BMS=6 by week 6, and approximately 90% of mice reached ≥5, indicating a significant therapeutic benefit from SeroPTEN-CG. Figure 11 C).

[0059] Gait parameter analysis was further performed using the CatWalk XT system. Immediately after hemisection, the right hind limb failed to form footprints, and gait parameters were close to 0. By week 6, the CTRL and SCR-CG groups showed only limited improvement, manifested as irregular foot strikes and infrequent stepping with the right hind limb. Figure 11 D). SeroPTEN-CG mice showed partially restored coordinated gait, with a gait regularity index exceeding 80% ( Figure 11 E), indicating reactivation and remodeling of residual spinal cord neural circuits. Simultaneously, parameters such as support base, swing speed, and footprint width all showed significant improvement, suggesting that SeroPTEN-CG helps restore neuromuscular coordination and postural stability. Figure 11 F, H); simultaneous improvement of indicators such as maximum contact area, maximum peak contact intensity, and maximum footprint intensity (F, H); Figure 11 The IK (Imbalance Kinematics) indicated improvements in muscle involvement, neuromotor control, and limb-ground interaction. In contrast, the CTRL and SCR-CG groups still showed significant deficiencies in these gait parameters, while the PTEN-CG group showed limited improvement. Figure 11 EK).

[0060] Based on the assessment of motor function, sensory function was evaluated using the plantar hot plate test. The SeroPTEN-CG group showed the most significant sensory recovery (Table 7), with a withdrawal latency of approximately 4.1 s, superior to the PTEN-CG (~5.8 s), SCR-CG (~6.0 s), and CTRL (~6.7 s) groups. Figure 11 L). Electrophysiological results were highly consistent with the aforementioned functional improvements: the amplitude of motor evoked potentials (MEPs) was significantly increased in the SeroPTEN-CG group ( Figure 11 M, N), and the amplitude of sensory evoked potentials (SEPs) was also significantly improved. Figure 11 The results (O, P) indicate that both the descending motor pathway and the ascending sensory pathway have been reconstructed and reactivated.

[0061] Further investigation was conducted to determine the segment-specific activation of interneurons in different spinal cord segments following SeroPTEN-CG intervention. Spinal cord samples were collected 1 hour after treadmill walking for immunofluorescence staining, using c-Fos as a marker of neuronal activation, and neurons were labeled with NeuN. Quantitative analysis focused on c-Fos in the dorsal / intermediate and ventral regions of the thoracic (T8) and lumbar (L2) segments. + / NeuN + Double-positive neurons. c-Fos was observed at T8 and L2 in the sham-operated group. + / NeuN + Neurons were widely distributed, while activation was less in both the CTRL and SCR-CG groups in the two segments mentioned above.Figure 11 Q, S). In contrast, after SeroPTEN-CG treatment, the T8 segment c-Fos + / NeuN + Interneurons showed a significant redistribution from the dorsal / intermediate region to the ventral region, and the number of double-positive neurons in the L2 segment also increased significantly. Figure 12 (R, T). Since the ventral region of the spinal cord plays a crucial role in the initiation and maintenance of rhythmic movement, this ventral enrichment indicates that descending commands can re-reach the distal segments from the injury, and previously "dormant" spinal neural circuits are reactivated. This result is also highly consistent with the design logic of SeroPTEN-CG: that is, to simultaneously integrate 5-HT-mediated excitability restoration and PTEN-mediated activation of neuronal growth programs at the lesion site to reconstruct the transmission pathway of descending commands.

[0062] At the mechanistic level, multiple pieces of evidence corroborate this framework: the extension of CST and 5-HT, vGLUT in MAP2 + Synaptic colocalization on neurons suggests the reconstruction of residual neural circuits after spinal cord injury, and their synergistic integration with rhythm regulation and excitatory drive to support the recovery of motor function. Consistent with this, improvements in MEP and SEP demonstrate functional reconnection rather than simply structural contact. Ventral c-Fos + / NeuN + The preferential activation of interneurons indicates that descending nerve impulses are retransmitted to the spinal cord tissue distal to the injury. At the behavioral level, BMS scores and CatWalk gait parameters demonstrate the recovery of coordinated stepping and controllable movement, consistent with the functional division of labor where "5-HT supports rhythm and glutamate supports execution." Overall, excitatory remodeling and neural circuit reconstruction must be simultaneously mobilized for effective recovery, and SeroPTEN-CG represents an integrated pharmacological strategy that can simultaneously coordinate both at the lesion site.

[0063] Example 7 In vivo biocompatibility In vivo safety was assessed using serum biochemical indicators and histopathological examination. Results showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were similar across all experimental groups, with no significant differences between groups. Figure 13 The results indicate that SeroPTEN CG did not induce significant liver or kidney function damage. H&E staining of the heart, liver, spleen, lungs, and kidneys showed intact tissue structure, without necrosis or diffuse inflammatory cell infiltration, and no significant abnormalities compared to the sham-operated group. ​ ).

[0064] In conclusion, SeroPTEN-CG exhibits good biocompatibility in vivo under this dosing regimen, supporting its feasibility for the treatment of spinal cord injury.

[0065] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is 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 them. 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 nucleic acid gel that mediates spinal cord injury repair by remodeling residual neural circuits, characterized in that, Include: The scaffold unit is formed by three scaffold single-stranded nucleic acids in a base-complementary pairing manner, and each scaffold single-stranded nucleic acid has a scaffold sticky end. The complementary pairing portion of the scaffold single-stranded nucleic acid has two thiophosphate groups evenly distributed, and each thiophosphate group is modified with a 5-hydroxytryptophan derivative. The cross-linking unit is formed by two cross-linked single-stranded nucleic acids in a base-complementary pairing manner, and each cross-linked single-stranded nucleic acid has a cross-linked sticky end.

2. The nucleic acid gel as described in claim 1, characterized in that, The scaffold unit and the crosslinking unit are crosslinked through the adhesive ends of the scaffold and the adhesive ends of the crosslinking in a base-complementary pairing manner, thereby forming a three-dimensional spatial network structure.

3. The nucleic acid gel as described in claim 1, characterized in that, The length of the adhesive end of the scaffold or the cross-linked adhesive end is 15-20 nt, preferably 18 nt.

4. The nucleic acid gel as described in claim 1, characterized in that, The scaffold unit and the crosslinking unit are in a stable crosslinking state under conditions of pH 6.8 to 7.

6.

5. The method for preparing nucleic acid gel according to any one of claims 1-4, characterized in that, Includes the following steps: (a1) 5-HTP-Br was grafted onto the phosphate thioester sites of three scaffold single-stranded nucleic acids to obtain 5-HTP-DNA. The three 5-HTP-DNAs formed scaffold units through complementary base pairing. (a2) Dissolve the stent unit in an aqueous medium to obtain an aqueous medium solution of the stent unit; (a3) Prepare crosslinking units by dissolving the crosslinking units in an aqueous medium to obtain an aqueous medium solution of the crosslinking units; (a4) The aqueous medium solution of the scaffold unit and the aqueous medium solution of the crosslinking unit are mixed to form a three-dimensional spatial network structure between the scaffold unit and the crosslinking unit, thereby obtaining the nucleic acid gel; The structure of the 5-HTP-Br is as follows: .

6. The preparation method according to claim 5, characterized in that, The concentration of the single-stranded nucleic acid in the scaffold is 90~110 μM, and the concentration of 5-HTP-Br is 5~7 mM.

7. The preparation method according to claim 5, characterized in that, The pH of the mixture of the aqueous medium solution of the scaffold unit and the aqueous medium solution of the crosslinking unit is 6.8~7.

6.

8. The preparation method according to claim 5, characterized in that, When the aqueous medium solution of the stent unit is mixed with the aqueous medium solution of the crosslinking unit, the molar ratio of the stent unit to the crosslinking unit is 1:(1~2).

9. A reagent kit, characterized in that, It comprises the nucleic acid gel according to any one of claims 1-4.

10. The use of the nucleic acid gel according to any one of claims 1-4 or the kit according to claim 9 in the preparation of a medicament for repairing spinal cord injury.

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