Genetically engineered hydrated remodeled biomimetic microspheres, and methods of making and using the same

Abstract

This invention provides a genetically engineered biomimetic microsphere for hydration remodeling, its preparation method, and its application, belonging to the field of biomedical technology. This invention develops an injectable composite system, GBA / SRGN@HADA@GelMA, which possesses the ability to regulate hydration and intervene in genes through interface biomimetic design and multi-level functional integration. It uses HADA as a moisturizing outer layer, provides mechanical support through GelMA microspheres, and achieves targeted delivery of SRGN siRNA through G5-GBA loading, thereby achieving synergistic regulation of the degenerative nucleus pulposus microenvironment. GBA / SRGN@HADA@GelMA reduces the expression of inflammatory factors and matrix degradation genes, and significantly promotes the accumulation of HA and lubricating proteins. This enhances the water retention capacity and interfacial lubrication of the nucleus pulposus, thereby improving compressive elasticity and energy absorption capacity. This invention is the first to combine continuous water regulation, lubrication, mechanical buffering, and extracellular matrix homeostasis regulation to synergistically delay intervertebral disc degeneration, providing an excellent treatment strategy for degenerative intervertebral disc diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a genetically engineered hydrated remodeling biomimetic microsphere, its preparation method, and its application. Background Technology

[0002] The hydration microenvironment of biological tissues, composed of a water-rich extracellular matrix (ECM), is a highly self-balancing functional unit. This hydration layer is primarily composed of proteoglycans rich in glycosaminoglycans (GAGs) interwoven with collagen fibers. The densely distributed sulfate and carboxyl groups on the GAGs endow them with a fixed negative charge density, thereby attracting counterions, such as Na⁺, generating the Donnan osmotic effect. This, in turn, drives and stabilizes a large amount of free and bound water into the matrix, forming a gel-like hydration network. This structure can generate expansion pressure through the osmotic effect and play a viscoelastic buffering role, effectively dispersing local contact stress and significantly reducing load impact. However, under the influence of aging, unstable mechanical loads, or fluctuations in the intensity of intense exercise, proteoglycan synthesis is inhibited, and its GAG chains degrade, leading to the overall collapse of the hydration network. As the tissue's water retention capacity declines, its original elastic and viscoelastic responses undergo significant degenerative changes. Matrix degradation and structural instability reinforce each other, forming a vicious cycle that hinders the expansion pressure required to maintain resistance, resulting in the gradual loss of mechanical elasticity and structural support under complex loads.

[0003] To address functional decline caused by imbalances in the tissue hydration microenvironment, current approaches primarily focus on developing elastic filling systems and injectable biomimetic matrices. These methods can effectively improve the pathological state of degenerated tissues, enhance mechanical properties by supplementing the matrix, and restore mechanical support. However, many hydrogel networks lack sufficient fatigue and crack resistance. Under high loads or long-term repeated stress, they are prone to crack propagation and structural relaxation, making it difficult to sustainably and stably resist external loads and maintain osmotic pressure balance. Furthermore, although cartilage matrix-based strategies can provide biochemical signals close to those of natural matrices in the short term, when the hydration structure has severely collapsed or been damaged over a long period, matrix supplementation alone often cannot fundamentally restore the structural hierarchy of the microenvironment.

[0004] A healthy intervertebral disc nucleus pulposus (NP) is a highly hydrated, gelatinous structure, containing 70% to 90% water in young adults. This exceptional hydration capacity provides crucial cushioning and lubrication properties. However, disc degeneration is essentially an inflammation-driven process that disrupts this hydrational balance. Inflammatory factors IL-1β and TNF-α activate the NF-κB signaling pathway, upregulating various matrix metalloproteinases (MMPs) and albumin-degrading enzymes ADAMTS-4 / 5, collectively triggering the oxidative degradation of the nucleus pulposus core proteoglycan network. This degradation directly impairs the nucleus pulposus's ability to attract and retain water molecules, and the resulting oxidative stress also inhibits the expression of the key one-way aquaporin AQP1 on the nucleus pulposus cell membrane. AQP1 expression levels show a strong linear correlation with the T2-weighted magnetic resonance imaging (MRI) signal intensity of the nucleus pulposus. Furthermore, AQP1 is a core factor in maintaining nucleus pulposus hydration and regulating rapid water flow. Therefore, inflammatory matrix degradation and oxidative stress downregulate AQP1 expression and activity, thereby disrupting the water balance within the intervertebral disc. Fernan (SRGN), a proteoglycan secreted by immune cells, is significantly upregulated in severely degenerated intervertebral discs and accelerates extracellular matrix degradation by enhancing the release of inflammatory factors and the infiltration of M1 macrophages. This directly leads to the collapse of the proteoglycan network and rapidly triggers tissue dehydration, becoming a key inflammatory amplifier driving the collapse of intervertebral disc water balance. Therefore, firnan is considered a potential key target for simultaneously intervening in inflammation and restoring water balance.

[0005] In summary, existing technologies cannot simultaneously achieve the synergistic effects of "inhibiting inflammation, protecting aquaporins, maintaining mechanical support, and restoring hydration," making it difficult to fundamentally reverse the multidimensional progression of intervertebral disc degeneration. Therefore, developing a biomimetic treatment system that can target key inflammatory nodes, protect aquaporin function, and provide durable mechanical support has become a crucial technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This invention aims to solve the aforementioned technical problems by providing a genetically engineered biomimetic microsphere for hydration remodeling, its preparation method, and its applications. This invention successfully develops a biomimetic nucleus pulposus system to remodel the hydration microenvironment, achieving a synergistic effect of simultaneously inhibiting inflammation, protecting aquaporins, maintaining mechanical support, and remodeling hydration, thereby fundamentally reversing the multidimensional progression of intervertebral disc degeneration and improving the therapeutic effect on intervertebral disc degeneration.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing genetically engineered hydration-remodeled biomimetic microspheres, comprising the following steps: (1) Dopamine was grafted onto hyaluronic acid through an activation reaction of EDC and NHS to obtain dopamine-modified hyaluronic acid; (2) Methacrylated gelatin microspheres were prepared by microfluidic reaction of methacrylic anhydride and gelatin, and then dispersed in buffer solution and mixed with dopamine-modified hyaluronic acid obtained in step (1) for co-incubation to obtain composite microspheres; (3) After activating 4-guanidinobenzoic acid with DCC and NHS, it was mixed with fifth-generation polyamide-amine dendritic macromolecules to obtain a polymer modified with 4-guanidinobenzoic acid (GBA) (G5-GBA), which was then co-incubated with siRNA to prepare siRNA complex; (4) The composite microspheres obtained in step (2) are mixed with the siRNA complex obtained in step (3), and genetically engineered hydration remodeling biomimetic microspheres are obtained through covalent and electrostatic adsorption reactions.

[0008] This invention prepares a genetically engineered hydration remodeling biomimetic microsphere and develops a biomimetic therapeutic system that can remodel the hydration environment of the nerve root sheath, reduce inflammatory response, and protect the function of aquaporins to slow the multidimensional progression of intervertebral disc degeneration. This invention provides a localized three-dimensional water-retaining scaffold using methacrylamide gelatin (GelMA) microspheres, forming a microporous network capable of storing water and enhancing local retention stability. The scaffold surface is coated with a hyaluronic acid (HA) derivative, specifically dopamine (DA)-modified hyaluronic acid (HADA). The hyaluronic acid matrix possesses strong hydrophilicity and a gel support structure, restoring tissue hydration and lubrication. The DA-branched catechol structure exhibits antioxidant and anti-inflammatory effects, thereby maintaining baseline expression and function of aquaporins. Simultaneously, chemically modified G5-PAMAM is used to construct the cationic polymer G5-GBA. This polymer stably encapsulates SRGN-siRNA and immobilizes it on the microspheres via amide bonds and electrostatic attraction, forming a multifunctional system (GBA / SRGN@HADA@GelMA) that achieves efficient cell penetration and controlled release.

[0009] In vitro experiments showed that the system developed in this invention significantly inhibited SRGN expression, reduced the levels of inflammatory factors (such as TNF-α and IL-1β), and maintained extracellular matrix homeostasis. In vivo studies demonstrated that GBA / SRGN@HADA@GelMA maintained the structural integrity of intervertebral disc tissue, enhanced hydration capacity and elasticity, and slowed the degenerative process. Transcriptome sequencing further showed that GBA / SRGN@HADA@GelMA significantly upregulated the expression of Adcy family members, thereby activating the cAMP-PKA-CREB signaling pathway, enhancing AQP1 transcription and membrane localization, and improving transmembrane water flux, consistent with hydration repair. In summary, this system employs four synergistic mechanisms—targeting SRGN, protecting AQP1, providing mechanical support, and remodeling hydration—providing a novel strategy and theoretical support for the precision treatment of degenerative intervertebral discs.

[0010] Furthermore, the pH of the activation reaction in step (1) is 5-5.5, and the activation reaction time is 15 minutes.

[0011] Furthermore, the grafting reaction conditions described in step (1) are to react at 35°C for 24 hours under light-protected conditions.

[0012] Furthermore, the pH of the buffer solution described in step (2) is 8.5.

[0013] Furthermore, the co-incubation conditions described in step (2) are incubation at 37°C for 4 hours.

[0014] Furthermore, the activation conditions described in step (3) are activation at room temperature for 6 hours.

[0015] Furthermore, the reaction conditions in step (3) are 7 days of continuous stirring and 30 minutes of co-incubation at room temperature.

[0016] Furthermore, in step (4), the N / P ratio of the reaction is controlled to be 12:1.

[0017] The second objective of this invention is to provide genetically engineered hydrated remodeling biomimetic microspheres prepared by the method described above.

[0018] A third objective of this invention is to provide the application of the genetically engineered hydration-remodeling biomimetic microspheres described above in the preparation of drugs for treating intervertebral disc degeneration.

[0019] The beneficial effects of this invention are as follows: The hydration microenvironment relies on proteoglycan matrix and aquaporins (AQPs) to maintain water homeostasis, which is crucial for tissue function. During intervertebral disc degeneration, loss of matrix integrity and accelerated AQP-mediated water transport contribute to tissue failure. Existing hydrogel materials can provide structural support but cannot fully regulate hydration homeostasis, thus failing to prevent degeneration. Here, we designed a GBA / SRGN@HADA@GelMA biomimetic nucleus pulposus system to reshape the hydration microenvironment. This system utilizes GelMA microspheres to construct a water-retaining matrix with enhanced mechanical stability. A dopamine-modified hyaluronic acid (HADA) coating leverages the hydrophilicity of hyaluronic acid to replenish moisture and improve lubrication, while dopamine provides adhesion and antioxidant protection to alleviate stress and support AQP-mediated water flux. Furthermore, the chemically modified GBA / SRGN complex is anchored to the microsphere surface via amide bonds and electrostatic adsorption, achieving nucleic acid protection and controlled release. In in vitro and in vivo experiments, the system suppressed inflammation and protected the extracellular matrix (ECM), enhancing swelling pressure, hydration, and mechanical stability of the nucleus pulposus. Transcriptomic analysis revealed that upregulation of adenylate cyclase (Adcy) and activation of the cAMP-PKA-CREB pathway promoted the transcription and membrane localization of aquaporin 1 (AQP1), thereby enhancing transmembrane water transport. This biomimetic microsphere system for remodeling the hydration microenvironment provides a precise intervention for degenerative intervertebral discs. Attached Figure Description

[0020] Figure 1 Synthesis and characterization of HADA and HADA@GelMA microspheres; (A) Schematic diagram of HADA synthesis; (B) Synthesis and characterization of Gel, GelMA, HA and HADA. 1(C) 1H NMR spectra; (D) XPS full spectra of HA and HADA; (E) UV-Vis absorption spectra of DA, HA, and HADA; (F) Frequency-scan rheological curves of HADA and GelMA hydrogels; (G) Strain-scan rheological curves of HADA hydrogels; (H) Swelling rates of GelMA, HADA, and HADA@GelMA hydrogels over time; (I) In vitro degradation curves of GelMA, HADA, and HADA@GelMA hydrogels; (J) Macroscopic images of HADA@GelMA microspheres, showing their morphology and injectability via syringe; (K) Optical microscope images of HADA@GelMA microspheres; (L) Diameter distribution of HADA@GelMA microspheres; (SEM images of GelMA and HADA@GelMA microspheres at different magnifications. Images; (M) Elemental distribution of carbon (C), nitrogen (N), and oxygen (O) in GelMA and HADA@GelMA microspheres; Quantitative values ​​are expressed as mean ± standard deviation; Significant differences between groups were determined using one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; 1H NMR, 1 H nuclear magnetic resonance; DA, dopamine; HA, hyaluronic acid; HADA, dopamine-modified hyaluronic acid; SEM, scanning electron microscope; XPS, X-ray photoelectron spectroscopy.

[0021] Figure 2Characterization of the GBA / SRGN@HADA@GelMA system; (A) Schematic diagram of the GBA / SRGN complex construction process; (B) Zeta potential and particle size distribution of the GBA / SRGN complex at different N / P ratios; (C) Western blot results of SRGN protein expression in cells treated with the GBA / SRGN complex at different N / P ratios, with β-actin as an internal control; (D) Quantitative analysis of relative SRGN protein expression corresponding to Figure C (n = 3); (E) Cell viability results after treatment with GBA / SRGN complex at different N / P ratios; (F) Flow cytometry histograms and contour plots of cells treated with GBA / SRGN complex at different N / P ratios; (G) Transmission electron microscopy images of GBA / SRGN complex and its corresponding elemental distribution; (H) Zeta potentials of GelMA, HADA@GelMA and GBA / SRGN complex; (I) Schematic diagram of the construction process of GBA / SRGN@HADA@GelMA microspheres; (J) SEM images of GBA / SRGN@HADA@GelMA microspheres at different magnifications and their corresponding elemental distributions; Values ​​are expressed as mean ± standard deviation; Significant differences between groups were determined by one-way ANOVA: ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; DA represents dopamine; HA represents hyaluronic acid; HADA represents dopamine-modified hyaluronic acid; SEM represents scanning electron microscopy; SRGN represents filaggrin.

[0022] Figure 3Lubrication evaluation for the GBA / SRGN@HADA@GelMA system; (A) Representative photographs of the reciprocating tribometer; (B) Schematic diagram of the ball-substrate configuration used for tribological measurements; (CE) Time-dependent coefficient of friction (COF) data recorded under normal loads of 1 N (C), 5 N (D), and 10 N (E), corresponding to GelMA, HADA@GelMA, and GBA / SRGN@HADA@GelMA, respectively; (F) Summary bar charts of average COF at 1, 5, and 10 N; (GI) Scanning electron microscopy of the prepared microspheres ( SEM images: GelMA (G), HADA@GelMA (H), and GBA / SRGN@HADA@GelMA (I), showing the overall morphology and internal porous structure; (J) Elemental analysis of GBA / SRGN@HADA@GelMA microspheres, including P elemental mapping and corresponding EDS spectra; (K) Proposed multi-scale lubrication mechanism in which microsphere-mediated interfacial support and boundary lubrication jointly promote a stable hydration layer to reduce friction; Quantitative values ​​are expressed as mean ± standard deviation; Significant differences between groups were determined using one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0023] Figure 4 To analyze matrix metabolism and inflammation-related indicators in NP cells under LPS treatment; (A) assess the abundance of target proteins in NP cells by Western blotting (internal control: β-tubulin); (B) determine the secretion levels of TNF-α and IL-1β using ELISA; (C) numerically evaluate the protein expression in Figure A (n=3); (D) show the localization of SRGN in NP cells by immunofluorescence staining; (E) detect the distribution of TNF-α protein by immunofluorescence; (F) detect the cellular expression of IL-1β by immunolabeling; (G, H) compare and analyze the transcriptional level of SRGN and its related MFI data (n=3); (I, J) TNF-α mRNA expression and its quantitative fluorescence intensity (n=3); (K, L) assess the mRNA abundance of IL-1β and its corresponding MFI value (n=3); quantitative values ​​are expressed as mean ± standard deviation; significant differences between different groups were determined by one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; IF, immunofluorescence; MFI, mean fluorescence intensity; NP, nucleus pulposus.

[0024] Figure 5To analyze matrix metabolism-related indicators in NP cells under LPS treatment conditions: (A, B) Immunofluorescence was used to detect the localization of COL-II (type II collagen) in NP cells, including related three-dimensional fluorescence distribution maps; (C, D) Immunofluorescence labeling was used to detect MMP-13 expression in NP cells, and corresponding three-dimensional intensity maps were plotted; (E, F) Agrecan (ACAN) protein content was visualized by immunofluorescence staining, and a three-dimensional atlas was plotted; (G, H) The relative mRNA content of type II collagen and the quantitative value based on MFI were detected by immunofluorescence (n = 3); (I, J) The transcriptome and fluorescence intensity of MMP-13 were analyzed (n = 3); (K, L) Agrecan mRNA levels and MFI data were assessed by immunofluorescence (n = 3); Quantitative values ​​are expressed as mean ± standard deviation; Significant differences between different groups were determined by one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; IF, immunofluorescence; MFI, mean fluorescence intensity; NP, nucleus pulposus.

[0025] Figure 6 The study aimed to evaluate the mechanical properties of a rat intervertebral disc degeneration model. The objectives included: (A) conceptual design of a rat tail compression maneuver; (B) conceptual design and calculation algorithm for the disc height index (DHI); (C) statistical evaluation of DHI changes in different groups at weeks 4 and 8; (D) X-ray images of vertebral segments after compression (weeks 4 and 8); (E) MRI scans and related pseudo-color mappings at specified time points for each group; (F) evaluation of the average grayscale values ​​derived from MRI data (n=6); (G) Pfirrmann classification results at weeks 4 and 8 after intervention (n=6); (H) representative settings for axial mechanical testing of intervertebral disc samples; (I) stress-strain trajectories of tissues under compressive load; (J) calculated compressive modulus of intervertebral disc samples (n=6); and (K) numerical calculation of tissue toughness in each group (n=6). Quantitative values ​​are expressed as mean ± standard deviation. Significant differences between groups were determined by one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.05. 0.01, ***p < 0.001, ****p < 0.0001; MRI, magnetic resonance imaging.

[0026] Figure 7Histological analysis of intervertebral disc tissue; (A, B) Histological grading results of HE-stained sections and intervertebral disc samples (n = 6); (C, D) Representative Sudan O-Fast Green (SO-FG) staining results used to assess the morphological integrity of intervertebral tissue (n = 6); (EG) Immunohistochemical (IHC) localization of TGF-β, including statistical quantification of the percentage of positive areas (n = 6); (H, I) Distribution of SRGN in intervertebral disc sections as shown by immunofluorescence technique, and corresponding mean fluorescence intensity (MFI) assessment (n = 6); Quantitative values ​​are expressed as mean ± standard deviation; Significant differences between groups were determined using one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; HE, hematoxylin-eosin.

[0027] Figure 8 Immunofluorescence analysis of matrix-related proteins in intervertebral disc tissue; (AC) Representative immunofluorescence (IF) images of collagen II, agglutinin, and MMP-13 expression in intervertebral disc sections; (DF) The mean fluorescence intensity (MFI) of each marker was statistically quantified accordingly (n = 6); quantified values ​​are expressed as mean ± standard deviation; significant differences between groups were determined by one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0028] Figure 9 Immunofluorescence analysis of molecules related to hydration and lubrication in intervertebral disc tissue; (A) Expression of hyaluronic acid in intervertebral disc tissue as determined by immunofluorescence (IF) staining; (B) Distribution of lubricating proteins in the intervertebral disc as observed by IF staining; (C) MFI analysis of hyaluronic acid signal (n = 6); (D) Quantitative fluorescence assessment of lubricating protein expression (n = 6); Quantitative values ​​are expressed as mean ± standard deviation; Significant differences between groups were determined by one-way ANOVA: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; MFI, mean fluorescence intensity.

[0029] Figure 10(A) Overview of sample clustering using principal component analysis; (B) Volcano plot for identifying gene expression changes; x-axis represents log2FC, y-axis represents -log10 (P-value); (C) Circular heatmap visualization of 20 differentially expressed genes; (D) Connectivity analysis of differentially expressed transcriptomes using PPI mapping; (E) Functional atlas defined by GO and KEGG enrichment categories; (FH) Gene set enrichment analysis (GSEA) atlas for cAMP, salivary secretion, and NF-κB signaling cascades; (I) Assessment of protein expression of AQP1, total CREB, and phosphorylated CREB (p-CREB) in tissues using Western blotting, with β-tubulin as a normalized reference; (J) Schematic diagram of the mechanism by which the cAMP pathway regulates AQP1.

[0030] Figure 11 Characterization of the GBA / SRGN@HADA@GelMA system; Schematic diagram of GBA / SRGN@HADA@GelMA hydrogel microspheres: preparation process, surface anchoring, and mechanism of action within the intervertebral disc; First, GelMA microspheres were prepared using microfluidic technology, while HA was modified into HADA and formed an siRNA polymer complex with G5-GBA; Second, the polymer complex was fixed onto the HADA-coated microspheres through electrostatic interactions and amide bonds, thereby forming a stable hydration and lubrication layer; Finally, after intradiscal injection, the microspheres remodeled the hydration microenvironment by enhancing cAMP-PKA-pCREB signal transduction and AQP membrane localization, improving water transport and promoting matrix homeostasis.

[0031] Figure 12 Here are the Fourier transform infrared spectra of HA and HADA.

[0032] Figure 13 To analyze the results of co-incubating different concentrations of RNase with SRGN-siRNA in the presence or absence of G5-GBA, agarose gel electrophoresis was performed.

[0033] Figure 14 The cumulative release curve of SRGN / GBA over 7 days.

[0034] Figure 15 Live-dead staining images of rat NP cells co-cultured with GBA / SRGN@HADA@GelMA extract for 1, 3, and 5 days.

[0035] Figure 16 Rat NP cells were cultured with GBA / SRGN@HADA@GelMA extract for 1, 3 and 5 days. CCK-8 assay showed that cell viability was comparable to that of the control group.

[0036] Figure 17 Representative macroscopic images of microsphere degradation behavior at different time points from day 1 to day 49 are presented.

[0037] Figure 18 The biochemical analysis of AST, ALT, and creatinine levels was used to evaluate the safety of the GBA / SRGN@HADA@GelMA microsphere therapy system for the whole-body liver and kidneys.

[0038] Figure 19 To visualize the distribution of SRGN within intervertebral disc sections using immunofluorescence, the corresponding mean fluorescence intensity (MFI) was evaluated (sample size n = 6).

[0039] Figure 20 Gene expression heatmaps for the compressed group and the GBA / SRGN@HADA@GelMA group.

[0040] Figure 21 Gene set enrichment analysis (GSEA) map of calcium and extracellular matrix receptor signaling pathways.

[0041] Figure 22 Numerical assessment of AQP1 and p-CREB protein expression. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0043] Example 1

[0044] I. Experimental Methods 1. Synthesis and Characterization of HADA@GelMA (1) An EDC / NHS-mediated amidation strategy was used to graft DA (dopamine) onto the HA (hyaluronic acid) structure to synthesize HADA (dopamine-modified hyaluronic acid). First, HA was dissolved in ultrapure water at 35°C under a nitrogen atmosphere with continuous stirring until a clear solution was obtained. Carboxyl activation was initiated by introducing EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), maintaining the pH in the range of 5.0-5.5 for 15 minutes. Subsequently, DA was added to the container, and the reaction was carried out at 35°C for 24 hours under light-protected conditions. The crude mixture was subjected to extensive dialysis and washed with distilled water to remove residual precursors and byproducts. The final HADA product was obtained by freeze-drying.

[0045] (2) GelMA was prepared by methacrylation of type A gelatin. First, the gelatin was completely hydrated in phosphate-buffered saline (PBS) at 60°C. Then, methacrylic anhydride was added dropwise and the reaction was maintained for 3 hours. After dilution with warm PBS buffer, the liquid was transferred to a semi-permeable membrane and thoroughly dialyzed with deionized water to extract unconverted reactants. After purification, the solution was filtered and freeze-dried to obtain the final polymethacrylamide powder.

[0046] By dissolving the sample in a deuterated solvent, utilizing 1 1H NMR spectroscopy confirmed the chemical structures of GelMA and HADA. XPS was used to further determine the surface elemental composition of HADA, and UV-Vis spectroscopy was used to evaluate its optical absorption properties. The viscoelastic behavior of GelMA and HADA was characterized by frequency and strain scans on a rotational rheometer to obtain the values ​​of G′ and G″. Swelling was assessed by tracking mass changes of the samples in an aqueous medium at predetermined time points, while degradation was determined by the percentage of mass loss during immersion.

[0047] Lyophilized GelMA was dissolved in PBS solution with a photoinitiator added, and GelMA microspheres were prepared using microfluidic emulsification technology, with the GelMA solution serving as the inner aqueous phase and paraffin oil as the continuous oil phase; Span 80 was added as a surfactant to stabilize the droplets. Uniform droplets were formed by adjusting the flow rates of the two phases. These droplets were then cured by UV irradiation, washed, and centrifuged to obtain GelMA microspheres. To prepare HADA@GelMA, pre-prepared GelMA spheres were dispersed in a buffer solution at pH 8.5, and a certain amount of solid HADA was added. This mixture was incubated at 37°C for 4 hours to promote the deposition and binding of HADA on the surface of the GelMA microspheres. After the reaction was complete, the HADA@GelMA microspheres were washed with buffer to remove unbound HADA. The particle size distribution of the HADA@GelMA spheres was determined using dynamic light scattering (DLS), and the samples were dispersed in deionized water to test their hydrodynamic diameter.

[0048] siRNA polymer complexes were prepared by mixing G5-GBA with DEPC-treated siRNA in different ratios and incubating at room temperature for 30 minutes. The N / P ratio represents the molar balance between the amino groups carried by the dendritic polymer and the phosphate groups of the siRNA. The electrophoretic mobility and effective size of these complexes were measured at 25 °C using dynamic light scattering, and structural analysis was performed by transmission electron microscopy. Finally, the composition and spatial arrangement of calcium, oxygen, and phosphorus were determined by energy-dispersive X-ray spectroscopy (EDC).

[0049] 2. Synthesis and Characterization of GBA / SRGN First, GBA (4-guanidinobenzoic acid) was dissolved in dry dimethylformamide (DMF) and activated at room temperature for 6 hours using a DCC / NHS (N,N'-dicyclohexylcarboimide / N-hydroxysuccinimide) system. Then, a G5-PAMAM (fifth-generation polyamidoamine) dendritic polymer in dimethyl sulfoxide (DMSO) was added dropwise to the activated GBA solution, and the reaction was carried out for 7 days with continuous stirring. Purification involved multiple dialysis with DMSO and deionized water to remove unreacted substances, followed by freeze-drying to obtain purified G5-GBA. siRNA polymer complexes were assembled by mixing G5-GBA with DEPC (diethyl pyrocarbonate)-treated siRNA, followed by environmental incubation at room temperature for 30 minutes. These complexes were characterized by their N / P ratio and subjected to dynamic light scattering (DLS) analysis at 25 °C to quantify effective size and zeta potential. Structural insights were provided by transmission electron microscopy (TEM), while the elemental composition, particularly the spatial arrangement of calcium, oxygen, and phosphorus, was depicted by energy-dispersive X-ray spectroscopy (EDS).

[0050] 3. Cell Culture Primitive neural progenitor cells were extracted from the intervertebral tissue of Sprague-Daverid rats. After cell isolation, the cells were amplified in DMEM / F12 medium containing a mixture of 10% fetal bovine serum and 1% antibiotics (100 units / mL penicillin and 100 μg / mL streptomycin). The culture environment was a 37°C incubator under a 5% carbon dioxide atmosphere. To ensure experimental consistency, subsequent bioassays used only second-generation (P2) cells.

[0051] 4. Evaluation of GBA / SRGN knockout efficiency, transfection efficiency, and cytotoxicity. To evaluate the gene transfection efficiency of the GBA / SRGN formulation, we employed Western blotting and flow cytometry. Neural progenitor cell populations were treated with the GBA / SRGN complex at different N / P ratios. After incubation, total cellular protein was collected from the lysate. Homogeneous protein loads were separated using SDS-PAGE and subsequently electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were sequentially incubated with specific primary antibodies and corresponding secondary antibodies. Protein abundance was visualized using an enhanced chemiluminescence system. To quantify transfection efficiency, GFP-tagged siRNA was used as a tracking fluorophore. Neural progenitor cells were distributed in 24-well plates and brought to approximately 50% confluence before being introduced with GFP-siRNA / GBA units at a series of N / P ratios. After 6 hours of exposure, the transfection medium was replaced with the original medium. After an additional 24-hour culture period, cells were collected for flow cytometry evaluation. The biocompatibility of these complexes was validated using a CCK-8 assay. In 96-well plates, neural progenitor cells at 70%-80% confluence were treated for 6 hours, followed by culture medium replenishment. After a 24-hour recovery period, CCK-8 solution was administered, and the optical density at 450 nm was recorded to quantify cell viability in different groups.

[0052] 5. Preparation and characterization of GBA / SRGN@HADA@GelMA microspheres For chemical coupling and loading operations, HADA@GelMA microspheres were suspended in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH=6.0). EDC and NHS were then added to activate the carboxyl groups of HADA under gentle stirring at room temperature. After activation, the microspheres were collected by centrifugation and washed three times with deionized water to remove residual reagents. The GBA / SRGN complex prepared at an optimized N / P ratio was then introduced into the activated microsphere suspension and gently mixed at room temperature, achieving loading through covalent and electrostatic adsorption to obtain GBA / SRGN@HADA@GelMA microspheres.

[0053] Scanning electron microscopy (SEM) was used to characterize the topographic features and internal structure of the microspheres. Before visualization, the samples were washed and lyophilized, fixed onto a conductive substrate, and metallized by sputtering gold plating. Elemental distribution and composition maps were obtained using energy dispersive spectroscopy (EDS). The spatial arrangement of key markers (carbon, nitrogen, oxygen, and phosphorus) was analyzed to verify the successful integration of GBA / SRGN into HADA@GelMA.

[0054] Preparation of extracts from GBA / SRGN@HADA@GelMA: To obtain material extracts, GBA / SRGN@HADA@GelMA microspheres, washed with sterile PBS, were placed in serum-free culture medium and incubated at 37°C for 72 hours. This time arrangement facilitates the complete release of soluble components. After incubation, the culture medium was collected and processed through a filtration membrane to ensure sterility and remove particulate matter, thus obtaining the filtrate for subsequent experiments.

[0055] 6. Biocompatibility of GBA / SRGN@HADA@GelMA Biocompatibility was assessed using CCK-8 assay and live / dead staining. Neural progenitor cells were seeded in 96-well plates and incubated with extracted material on days 1, 3, and 5, while cells not cultured in serum-containing medium served as controls. At each time point, CCK-8 reagent was added and incubated for 2 hours, and the optical density at 450 nm was measured to quantify cell viability. Simultaneously, cells were stained using the Live / Dead kit according to the manufacturer's instructions, and live (green) and dead (red) cells were observed using fluorescence microscopy.

[0056] 7. Lubrication performance evaluation of GBA / SRGN@HADA@GelMA Linear reciprocating friction tests were performed using a CETR UMT-2 ternary friction tester in a ball-to-plane setup. Microsphere samples (GelMA, HADA@GelMA, and GBA / SRGN@HADA@GelMA) were uniformly distributed at the contact interface. Tests were conducted at a frequency of 1 Hz at room temperature, with normal loads of 1, 5, and 10 Newtons, for 600 seconds. Frictional and normal forces were recorded simultaneously to calculate the friction coefficient-time curve. Each group was tested three times (n ≥ 3). After the tests, the microspheres were collected for scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS).

[0057] 8. Western blotting Western blot experiments were performed to quantitatively detect the expression of matrix-related and inflammation-related biomarkers. The proteins detected included agglutinin, COL2, MMP13, SRGN, IL-1β, and TNF-α, all normalized using β-tubulin as an internal reference. Neural progenitor cells were divided into a control group, an LPS group, a HADA@GelMA group, and a GBA / SRGN@HADA@GelMA group. After stimulation with 200 ng / mL LPS, the cultures were washed with PBS and replenished with fresh culture medium for 24 hours. Extracted protein samples were separated by SDS-PAGE electrophoresis and then transferred to membranes. After incubation with specific primary and secondary antibodies, protein signals were identified using an enhanced chemiluminescence system.

[0058] 9. ELISA analysis After treatment with LPS and extraction buffer, the cell cultures were washed three times with PBS. The samples were then stored in fresh culture medium for 24 hours. To assess cytokine secretion, the collected supernatant was subjected to ELISA using specific reagents provided by Aire Biotechnology Co., Ltd., with optical density measured at the specified wavelength using a microplate reader according to the manufacturer's instructions.

[0059] 10. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence (IF) analysis NP cells were first incubated with the extract and then stimulated with lipopolysaccharide (LPS). Cultures were washed three times with PBS and then reintroduced into fresh culture medium for 24 hours. RNA was extracted from the cells, and real-time PCR analysis was used to quantify the mRNA abundance of genes associated with extracellular matrix turnover and inflammation (aggregate, type II collagen, matrix metalloproteinase 13, serine protease inhibitor N, interleukin-1β, and tumor necrosis factor-α). To assess protein levels, a set of parallel samples were immunofluorescence stained, and fluorescence imaging was used to assess intracellular protein distribution and subcellular localization. The primer sequences used are shown in Table 1.

[0060] Table 1 qRT-PCR primer sequences

[0061] 11. Animal Model Construction The animal experimental protocol used was reviewed and approved by the Ethics Committee of Tongji University Yangpu Hospital (Approval No.: LL-2025-SCI-004). Healthy 10-week-old Sprague-Dawley rats were obtained from a qualified laboratory animal supplier. Before modeling, rats were anesthetized with isoflurane inhalation, and a custom-designed compression device was installed at the designated caudal vertebral segment. After device installation, 10 μL of the corresponding material extract was injected into the intervertebral disc segment of each treatment group using a 1 mL syringe, while the control group received an equal volume of sterile phosphate-buffered saline (PBS). Subsequently, the rats were subjected to continuous mechanical compression loading, maintaining the set loading parameters for two weeks to induce intervertebral disc degeneration. The sham-operated group only had the compression device installed, without effective mechanical compression. Through this process, a mechanically induced intervertebral disc degeneration model was established for subsequent in vivo efficacy and mechanism studies.

[0062] 12. X-ray and magnetic resonance imaging assessment At weeks 4 and 8 post-surgery, we performed imaging evaluations using X-rays and magnetic resonance imaging (MRI). Mice were stabilized under inhaled isoflurane anesthesia to allow for examination of the tail segment. First, X-ray images were taken to monitor changes in the vertical dimensions of the intervertebral disc space. The disc height index (DHI) was determined using the three-line centering method with ImageJ software. Subsequently, sagittal T1- and T2-weighted sequences of the tail segment were recorded using a dedicated laboratory MRI scanner. The pathological condition of the intervertebral disc was classified using a modified Pfirrmann grading system (Grade IV), where Grade I represents a healthy structural state and Grade V indicates severe tissue degeneration.

[0063] 13. Histological and immunological analysis After imaging evaluation, samples were removed from the rat tail discs and fixed in 4% paraformaldehyde. Following decalcification and routine processing, the specimens were embedded in paraffin and cut into 5-micrometer-thick sections. HE and Safranin O-Fast Green staining were used to assess tissue structure and extracellular matrix arrangement. Furthermore, we characterized intratissue molecular changes using immunohistochemistry and immunofluorescence. Immunohistochemical analysis was used to detect TGF-β, while immunofluorescence assays targeted markers including MMP13, COL2, SRGN, lubricin, and hyaluronic acid.

[0064] 14. RNA sequencing analysis Eight weeks after modeling, nucleus pulposus tissue samples were obtained from mice that underwent mechanical disc degeneration induction. These samples were divided into simple compression group and combined treatment group, with each group containing six independent samples. Immediately after dissection, the tissues were rapidly frozen in liquid nitrogen and preserved at low temperature until RNA isolation was performed, followed by transcriptome sequencing and subsequent bioinformatics analysis.

[0065] 15. Statistical Analysis Data were processed using GraphPad Prism software. We assessed the normality of each dataset to guide the selection of appropriate test methods. Results conforming to a normal distribution are reported as mean ± standard deviation. In vitro experiments were performed independently in triplicate, while in vivo study groups consisted of six animals per group. For comparisons involving more than two groups, one-way ANOVA combined with Tukey's multiple comparison test was applied. Comparisons between two groups were performed using a two-tailed unpaired Student's t-test, with statistical significance set at p < 0.05.

[0066] II. Experimental Results and Analysis 1. Design and synthesis of HADA@GelMA composite hydrogel microspheres with mechanical and water-retention functions The synergistic effect of microsphere scaffolds and surface hydration coatings was used to overcome three main challenges: moisture retention, in-situ retention, and cellular stress protection. As a supporting matrix, polymethacrylamide (GelMA) microspheres, through their interconnected micropores formed by stacking, create compressible "reservoirs," significantly enhancing the material's in-situ retention within tissues while maintaining dynamic moisture exchange. On the microsphere surface, a dopamine-modified hyaluronic acid (HADA) coating utilizes highly hydrophilic fragments of hyaluronic acid (HA) to construct a stable interfacial hydration layer, providing high water absorption and mitigating frictional wear by improving boundary lubrication. Furthermore, the HADA molecular chains, modified with dopamine (DA), introduce highly active catechol groups, whose powerful reducing ability precisely eliminates localized excess reactive oxygen species (ROS), restoring the function of aquaporins (AQPs) deactivated by oxidative stress. This functionalized hyaluronic acid derivative blocks inflammatory responses through its antioxidant activity and actively modulates the phenotypic transformation of immune cells, thereby synergistically optimizing the repair environment of damaged tissues at the molecular and cellular levels.

[0067] Under EDC / NHS conditions (pH 5.5, for 24 hours), the amino group of DA undergoes an amidation reaction with the carboxyl group of HA to generate HADA ( Figure 1 (A). Proton nuclear magnetic resonance (¹H NMR) spectrum ( Figure 1(B) confirms that DA has been successfully grafted onto HA, as there is a chemical shift between 6.5 and 7.5 ppm, which is the characteristic proton peak of the DA benzene ring. X-ray photoelectron spectroscopy (XPS) shows that HADA exhibits an enhanced N 1s peak at approximately 400 eV, along with a new signal in the nitrogen-related binding energy range, confirming the successful chemical covalent bonding of DA to HA. Figure 1 (C). Furthermore, HADA exhibits a characteristic UV absorption peak at approximately 280 nm derived from the DA catechol structure, further confirming that DA has been successfully incorporated into the HA backbone. Figure 1 (D). FTIR analysis supports the catechol functionalization structure of HADA relative to HA ( Figure 12 Rheological frequency scanning results showed that, across the entire test range of 1 to 10 Hz, the storage modulus (G′) and loss modulus (G″) of HADA hydrogel were significantly higher than those of GelMA hydrogel, indicating a more stable network structure and stronger viscoelasticity. With increasing frequency, both G′ and G″ of HADA showed a slight upward trend, while the increase in GelMA was smaller, suggesting that the introduction of HADA enhanced the material's mechanical response to dynamic loads. Figure 1 (E in Figure 1). This tensile test further confirms the structural stability of HADA. Within the amplitude range of 0.1% to 100%, HADA's G′ remains relatively stable in most strain ranges, only decreasing under high strain conditions, indicating that it has a wide linear viscoelastic range and excellent structural resistance to failure (F in Figure 1).

[0068] To evaluate the ability of these hydrogels to restore the hydration microenvironment of the intervertebral disc, their swelling behavior and degradation characteristics were analyzed. Swelling results showed that HADA had the strongest water absorption capacity, followed by HADA@GelMA, while GelMA had the weakest. Both HADA and HADA@GelMA rapidly absorbed water and maintained a high swelling rate for a short period, indicating their suitability for rapid hydration in water-scarce environments. Figure 1 Degradation tests further showed that GelMA degraded the fastest, HADA degraded the slowest, while HADA@GelMA exhibited a moderate and stable degradation rate, maintaining the material structure and continuously regulating moisture content for a relatively long period. Figure 1 (H). Overall, the introduction of HADA significantly enhanced the water absorption and retention capacity of the hydrogel, thus providing a basis for reconstructing the hydration microenvironment of the intervertebral disc.

[0069] To confirm the successful preparation of the microspheres, they were characterized in three aspects: injectability, particle size distribution, and microstructure. First, the microspheres exhibited a stable dispersion in centrifuge tubes and could be smoothly injected using a syringe, indicating that the system possesses effective injectability and operability. Figure 1 (I). Microscopic observation showed that the microspheres were uniformly spherical and well dispersed. Figure 1 (J). Particle size statistics further show that its size is mainly concentrated at about 150 micrometers, with a narrow distribution and good uniformity. Figure 1 (Middle K). Scanning electron microscopy (SEM) showed that both GelMA and HADA@GelMA microspheres have typical three-dimensional porous network structures, while HADA@GelMA has more continuous pore walls, a denser pore structure, and a more complete overall morphology. Figure 1 (L). Furthermore, the elemental distribution diagram shows that carbon, nitrogen, and oxygen are uniformly distributed within the microspheres, indicating good integration of the material composition and structural stability. Figure 1 (M). In summary, the obtained microspheres possess good injectability, controllable and uniform particle size, and stable porous microstructure, providing a reliable material basis for subsequent functional studies.

[0070] 2. Design and synthesis of GBA / SRGN@HADA@GelMA composite hydrogel microspheres: a novel material for restoring the hydration microenvironment. The advantage of G5 crosslinked polymer modified with GBA for siRNA delivery lies in the synergistic effect of guanidine and phenyl groups. The amino groups form salt bridges and hydrogen bonds with the nucleic acid phosphate backbone / cell membrane phospholipids, thereby enhancing the aggregation stability of siRNA, cell membrane affinity, and moderate hydrophobic interactions. This further improves cellular uptake and promotes endosome membrane rupture and escape, thus overcoming the rate-limiting step in the delivery process. Based on this, this study synthesized the cationic polymer G5-GBA via a chemical route and combined it with negatively charged siRNA through electrostatically driven self-assembly. Figure 2 China A and Figure 13 These favorable biophysical properties of the G5-GBA / siRNA complex greatly facilitate efficient gene delivery.

[0071] To select a suitable delivery formulation, we compared various properties of the GBA / SRGN complex with different N / P ratios. At all tested N / P ratios, G5-GBA induced siRNA aggregation to form nanoscale complexes of approximately 200–300 nm. When the N / P ratio exceeded 4:1, the surface charge of the complex significantly changed from negative to positive. Figure 2(Figure 2, B). With increasing N / P ratio, SRGN protein expression gradually decreased, with the most significant inhibition observed at N / P = 12. Although N / P = 16 still showed some silencing effect, it was generally weaker than that at N / P = 12. Quantitative results were consistent, showing a stepwise decrease in cell viability with increasing N / P ratio, indicating that N / P = 12 was the optimal ratio (Figure 2, C, D). Cell viability assays showed that cell viability was above 90% in most treatment groups, with only a slight decrease in cell viability at N / P = 16, indicating good biocompatibility and effective silencing (Figure 2, E). Furthermore, flow cytometry results showed that intracellular si-SRGN fluorescence signal increased with increasing N / P ratio. At N / P = 12, the fluorescence peak shifted significantly to the right and showed a more stable distribution, indicating optimal cellular uptake efficiency (Figure 2, F).

[0072] Considering the balance between transfection efficiency and toxicity, an N / P ratio of 12:1 was chosen for subsequent experiments. Transmission electron microscopy (TEM) images showed that GBA / SRGN formed a clear spherical structure, while energy-dispersive X-ray spectroscopy (EDS) results confirmed the successful loading of siRNA via phosphorus (P) distribution. Figure 2 (G in Figure 2). Characteristic peaks of phosphorus were observed in the EDS spectrum, further supporting the effective incorporation of phosphorus. Zeta potential measurements showed that the potential of the strongly positively charged G5-GBA dropped to +13.6 mV after binding with siRNA, but a significant potential difference still existed between it and the negatively charged HADA@GelMA (H in Figure 2). This complementary charge indicates that electrostatic adsorption is the core force driving the stable anchoring of the complex on the microsphere surface. In summary, these results demonstrate the successful construction of the G5-GBA nanocomposite and its stable chemical composition.

[0073] In constructing the biomimetic system, HADA@GelMA microspheres were used as the substrate, and multidimensional loading of GBA / SRGN was achieved through interfacial chemical modification. Based on the classic carbodiimide chemistry principle, the addition of EDC / NHS promoted the formation of covalent amide bonds between the spherical matrix and the carrier complex, giving the system excellent structural stability. Furthermore, this chemical coupling and electrostatic adsorption effect not only enhanced the composite microspheres' ability to capture siRNA loads but also ensured subsequent controllable release in complex physiological environments (Figure 2, I and...). Figure 14The morphology, structure, and chemical composition of the integrated GBA / SRGN@HADA@GelMA composite beads were systematically characterized. Scanning electron microscopy (SEM) results showed that these microspheres possess a uniform spherical structure with a clear porous network and uniform elemental distribution, which is beneficial for the loading and release of nucleic acid molecules. Figure 2 China J and Figure 17 Subsequently, biocompatibility assessment results showed that rat neural progenitor cells and NP cells maintained high survival rates and proliferative activity after being co-cultured with GBA / SRGN@HADA@GelMA microsphere extract for 1, 3, and 5 days. Figure 15 and Figure 16 These results demonstrate that the microsphere system constructed in this study is stable and biocompatible, and provides a reliable material basis for subsequent delivery and microenvironment regulation.

[0074] 3. Evaluation of the lubrication performance of hydrated lubricating gel microspheres Linear reciprocating friction tests were conducted using a tribometer with a ball-to-plane configuration to evaluate the lubrication performance of three hydrogel microsphere formulations under shear friction conditions. Figure 3 (A, B). Normal loads of 1, 5, and 10 Newtons were applied at a frequency of 1 Hz to compare the coefficient of friction-time curves and average coefficient of friction values ​​of GelMA, HADA@GelMA, and GBA / SRGN@HADA@GelMA microspheres. Given that the pressure on the intervertebral disc nucleus pulposus—especially in the lumbar region—can rise from approximately 0.3–0.5 MPa under normal posture to 1 MPa under gravitational activity (potentially reaching approximately 2.3 MPa during heavy lifting), 5 and 10 Newtons were chosen as medium to high-intensity load conditions to simulate the increased mechanical stress experienced within the intervertebral disc. The coefficient of friction-time curves show that all three groups maintained a relatively stable low-friction plateau at 1 Newton. GelMA exhibited significant transient changes in the initial stage, subsequently reaching a steady state, indicating a higher sensitivity to early shear disturbances. Conversely, HADA@GelMA showed a lower plateau with less fluctuation. Figure 3(C) This indicates that the hydrophilic coating enhances the retention of interfacial water and stabilizes the hydration layer, thereby improving lubrication stability under low load conditions. The introduction of the GBA / SRGN composite did not affect this lubrication performance, as the curves remained on a similar low plane. The differences became more pronounced when the load increased to 5 N and 10 N. GelMA exhibited greater fluctuations that gradually increased over time, suggesting that lubrication performance gradually deteriorated under higher contact stresses due to dehydration or network fatigue. In contrast, HADA@GelMA and GBA / SRGN@HADA@GelMA maintained a smoother low-friction plateau, indicating that they have stronger resistance to shear-induced lubrication failure (D, E in Figure 3). Quantitative analysis confirmed these observations: at 1 N, the COF values ​​were 0.139 ± 0.005 (GelMA), 0.109 ± 0.004 (HADA@GelMA), and 0.108 ± 0.003 (GBA / SRGN@HADA@GelMA); at 5 N, they were 0.120 ± 0.013, 0.059 ± 0.005, and 0.058 ± 0.002; and at 10 N, they were 0.104 ± 0.011, 0.055 ± 0.001, and 0.055 ± 0.005 (F in Figure 3). These results confirm that the two modified microsphere groups achieved significant friction reduction and improved stability under medium to high intensity loads. Overall, the coefficient of friction remains low throughout the entire load range, indicating that the microsphere system is able to achieve boundary lubrication and the formation of a hydration layer, thereby providing continuous frictional buffering during sustained reciprocating shearing.

[0075] To investigate the underlying mechanism, researchers examined the microspheres after friction using scanning electron microscopy (SEM) and elemental analysis. Following the friction test, the polymethyl methacrylate (GelMA) microspheres exhibited surface roughening, pore wall collapse, or localized damage, indicating that maintaining a continuous lubricating layer under high shear forces is difficult. Figure 3(G in Figure 3). In contrast, HADA@GelMA and GBA / SRGN@HADA@GelMA better preserved their porous structure, which is beneficial for retaining interfacial moisture and maintaining a continuous lubricating medium (H, I in Figure 3). Furthermore, phosphorus (P) – acting as a tracer for the GBA / SRGN composite – remained detectable after friction, indicating that the composite persists at the interface and synergizes with the hydrated HADA network to enhance the shear resistance and continuity of the boundary lubricating layer (J in Figure 3). In summary, the hydrophilic HADA network within the microsphere system firmly anchors bound water, forming a stable hydration layer that promotes low-resistance interfacial shear. As the material absorbs moisture, the expansion pressure under normal loads provides structural support and maintains the interfacial gap. This separation reduces the contact area and prevents lubricant extrusion, thus ensuring the continuity of the boundary film even under moderate to high-intensity loads (K in Figure 3). The phosphorus trace confirms that the GBA / SRGN composite remains at the interface during friction, thereby enhancing the continuity and stability of the hydrated layer. Through this integration of mechanical support, load dispersion, and separation caused by expansion, the system maintains a low coefficient of friction under high stress conditions.

[0076] 4. In vitro evaluation of GBA / SRGN@HADA@GelMA microspheres in restoring hydrated extracellular matrix The nucleus pulposus is primarily composed of charged proteoglycans and type II collagen (COL2), forming a highly hydrated, gel-like structure that attracts water and builds internal pressure using a fixed negative charge. This allows it to distribute pressure loads during daily activities and maintain the mechanical cushioning function of the intervertebral disc. Current research indicates that degeneration typically begins in the nucleus pulposus, with an early decrease in proteoglycan content, subsequently leading to reduced tissue hydration and impaired water retention. This weakens the mechanical integrity of the intervertebral disc and promotes a local microenvironment that is prone to inflammation and catabolism. Based on this, single-cell atlas studies further reveal the existence of a fibrosis-like cell subset in late-stage degenerated nucleus pulposus, with significantly upregulated SRGN genes. SRGN can promote the infiltration of inflammatory factors and macrophages, thereby amplifying the local inflammatory response and accelerating the degenerative process. Therefore, hydration remodeling can enhance the water retention capacity and mechanical support of the matrix, and combined with siSRGN technology, it can inhibit SRGN-mediated inflammation amplification and extracellular matrix degradation stress, thereby exerting its effects through both water retention and inflammation-driven pathways to more effectively stabilize the nucleus pulposus microenvironment and slow down degeneration.

[0077] Protein expression and regulation directly reflect the functional state of cells; therefore, monitoring key protein levels is crucial for understanding the biological response of nucleus pulposus cells to different treatments. Following LPS stimulation, Western blotting revealed that nucleus pulposus cells exhibited typical characteristics of extracellular matrix degradation and increased inflammation; Aggrecan and COL2 expression were significantly decreased, while the expression of MMP13, SRGN, cleaved IL-1β, and TNF-α was significantly upregulated (A and CH in Figure 4), indicating that the cells were in a state of intense catabolic metabolism and inflammation. HADA@GelMA was able to alleviate these changes to some extent, but with limited improvement. In contrast, GBA / SRGN@HADA@GelMA significantly restored Aggrecan and COL2 expression while effectively inhibiting the excessive increase in the expression of MMP13, SRGN, cleaved IL-1β, and TNF-α, demonstrating the significant advantage of this system in preventing excessive extracellular matrix degradation and reducing inflammatory responses.

[0078] Intervertebral disc degeneration exhibits a persistent low-grade inflammatory phenotype. In degenerated discs, long-term increases in TNF-α and IL-1β activate pathways such as NF-κB and MAPK, promoting the expression of degradative enzymes (e.g., MMPs), accelerating the degradation of collagen and proteoglycans, and shifting the homeostasis of the extracellular matrix towards degradation. At the epineurium cell level, inflammation-mediated apoptosis and other types of cell death may increase, leading to decreased cell viability and limited tissue maintenance and repair capabilities. Enzyme-linked immunosorbent assay (ELISA) results showed that LPS stimulation significantly increased the secretion levels of TNF-α and IL-1β in the cell supernatant. In contrast, although HADA@GelMA treatment reduced the release of inflammatory factors to some extent, the inhibition was limited, while the secretion levels of these two inflammatory factors were significantly reduced after GBA / SRGN@HADA@GelMA treatment, approaching baseline levels (Figure 4B). This indicates that the system has a stronger regulatory capacity at the level of inflammatory factor release. Immunofluorescence (IF) results showed that the protein expression of SRGN, TNF-α, and IL-1β was significantly reduced after treatment with siRNA / G5@HADA@GelMA, and qPCR analysis also confirmed that their mRNA transcription levels were significantly inhibited. Figure 4(DI). This result indicates that siRNA-mediated SRGN-targeted intervention inhibits the secretion and expression of inflammatory factors and mechanistically weakens the active inflammatory feedback loop by downregulating key downstream regulators such as TNF-α and IL-1β. Extracellular matrix repair depends on a dynamic balance between synthesis and degradation, with the synthesis phase focusing on the continuous generation and deposition of COL2 and polyglycosides.

[0079] Degradation requires managing the activity pressures of MMPs and polyproteoglycans to avoid sustained net degradation. Therefore, we further evaluated key molecular changes closely related to extracellular matrix homeostasis. LPS stimulation significantly inhibited the expression of COL-II and polyproteoglycans and significantly increased MMP-13 levels, indicating that extracellular matrix synthesis was suppressed while degradation was enhanced. HADA@GelMA alleviated this imbalance to some extent, with the most significant improvement observed in the GBA / SRGN@HADA@GelMA group. Immunofluorescence results showed significantly enhanced COL-II and polyproteoglycan signaling, while MMP-13 expression was significantly reduced (AF in Figure 5). qPCR results were consistent, showing significantly upregulated transcription levels of COL-II and Aggrecan in the GBA / SRGN@HADA@GelMA group, while MMP-13 was strongly inhibited (GL in Figure 5). In summary, this composite system can simultaneously promote the synthesis of extracellular matrix and inhibit excessive degradation, thereby promoting the repair and stability of the ganglion cell microenvironment at multiple levels.

[0080] 5. Evaluation of the in vivo effects of GBA / SRGN@HADA@GelMA microspheres on improving tissue hydration status. To evaluate the protective effect of this system in a mechanically compressed intervertebral disc degeneration model, we established an intervertebral disc degeneration model in rats using a self-made continuous caudal compression device. The device was designed following the method previously described for mature models, with modifications (Figure 6A). After model establishment, X-ray examination of the rat's caudal vertebrae was performed at weeks 4 and 8 postoperatively to assess changes in the disc height index (DHI). DHI was calculated by quantitatively measuring the intervertebral space height on X-ray images according to a standard formula, reflecting the structural integrity of the intervertebral disc (Figure 6B). After modeling, rats received simple compression therapy, HADA@GelMA, or GBA / SRGN@HADA@GelMA. X-ray results showed that the DHI in the simple compression group decreased significantly at weeks 4 and 8, while the intervertebral space collapse in the HADA@GelMA group was slowed to some extent. In contrast, the GBA / SRGN@HADA@GelMA group showed the best DHI retention at both time points (Figure 6, C and D). Further magnetic resonance imaging evaluation showed that the material intervention groups exhibited higher signal intensity on T2-weighted images, indicating an increase in intervertebral disc water content, with the GBA / SRGN@HADA@GelMA group showing the most significant effect (Figure 6, E).

[0081] Quantitative grayscale analysis and Pfirrmann grading further confirmed that, compared with other treatment groups, GBA / SRGN@HADA@GelMA significantly delayed intervertebral disc degeneration and showed a clear advantage in maintaining the integrity of the imaging structure (Figure 6, F, G). Considering that the mechanical properties of healthy intervertebral discs are highly dependent on the water content of the nucleus pulposus and the integrity of the matrix, and that matrix degradation and water loss during degeneration significantly reduce load-bearing capacity, we used a universal testing machine to measure the compressive modulus of the nucleus pulposus to assess the impact of different treatments on mechanical properties (Figure 6, H). Uniaxial compression testing showed that, under the same compressive strain, the stress response of the compression group was significantly reduced, indicating impaired mechanical support capacity, which HADA@GelMA mitigated to some extent. The stress-strain characteristics of the GBA / SRGN@HADA@GelMA group were closer to those of the sham surgery group (Figure 6, I, J). This composite system significantly improved the compressive modulus of the nerve root sheath cyst (NP) and enhanced the tissue's energy absorption capacity, thereby improving toughness (Figure 6, K). In summary, this composite system, with its dual advantages in morphological maintenance and mechanical reinforcement, significantly alleviates structural collapse caused by high loads and achieves deep protection and biorepair of degenerated intervertebral discs at the functional level. Figure 18 ).

[0082] At weeks 4 and 8 post-surgery, a systematic histological and immunological assessment of the intervertebral disc tissue was performed to compare the effects of different treatment methods. Hematoxylin-eosin (HE) staining showed that the overall structure of the intervertebral disc in the sham surgery group was intact, with the nucleus pulposus and annulus fibrosus clearly connected. The compression group showed significant structural disorder at both time points, manifested as nucleus pulposus collapse and tissue-level destruction. In contrast, HADA@GelMA treatment slowed the degenerative process to some extent, while the GBA / SRGN@HADA@GelMA group better maintained the overall shape of the intervertebral disc at weeks 4 and 8, with a more intact nucleus pulposus structure and a relatively clear boundary between the nucleus pulposus and annulus fibrosus (Figure 7, A and B). Safranin O-fast green staining further showed that the matrix component in the nucleus pulposus region of the compression group was significantly reduced, the HADA@GelMA group showed partial recovery, while the GBA / SRGN@HADA@GelMA group had higher matrix content at both time points (Figure 7, C and D). Immunohistochemical and immunofluorescence assays showed that GBA / SRGN@HADA@GelMA treatment effectively reduced the expression of degeneration-related factors (such as SRGN), enhanced the expression of TGF-β, COL-II, and aggrecan, and inhibited the upregulation of MMP-13 expression. Therefore, this is more conducive to maintaining the dynamic balance between extracellular matrix synthesis and degradation in vivo (EI, α, β) in Figure 7. Figure 8 AF and Figure 19 Overall, these results indicate that GBA / SRGN@HADA@GelMA can significantly improve the tissue structure and stromal environment of the intervertebral disc in vivo, thereby effectively delaying intervertebral disc degeneration.

[0083] Hyaluronic acid (HA) and lubricating proteins play crucial roles in the hydration and low-friction microenvironment of intervertebral discs. Due to their strong hydrophilicity and network structure, HA can form a stable hydration layer in the matrix and increase local water content, thereby improving hydration indices in imaging. Furthermore, HA-related hydrogel intervention promotes matrix repair-related performance in animal models of intervertebral discs. Lubricating proteins, typical boundary lubrication-related glycoproteins, are localized in intervertebral disc tissue. They participate in the sliding between collagen bundles within the annulus fibrosus to accommodate torsional and shear loads. The synergistic assembly of lubricating proteins and HA forms a highly hydrated interfacial layer, effectively enhancing the surface's anti-adhesion properties by masking hydrophobic sites and providing strong hydration-mediated repulsion. In this study, GBA / SRGN@HADA@GelMA significantly promoted the expression of HA and lubricating proteins, indicating that this system can reconstruct the hydration and lubrication functions of the nucleus pulposus at the molecular level. Figure 9(AD). In summary, GBA / SRGN@HADA@GelMA restored the mechanical buffering and load-bearing capacity of nanoparticles by regulating the local hydration and lubrication microenvironment, thereby reducing matrix degradation caused by mechanical stress and maintaining the dynamic balance between extracellular matrix synthesis and degradation. This synergistic mechanism of "hydration and lubrication, extracellular matrix homeostasis, and mechanical buffering" may be an important basis for its ability to delay intervertebral disc degeneration.

[0084] 6. Potential ways to restore a moist microenvironment As a key component regulating transmembrane water transport and metabolite flow, the AQP family plays a crucial role in maintaining cellular homeostasis. AQPs mediate osmotic-driven water exchange and synergistically transport key metabolites such as oxygen and carbon dioxide, directly affecting cellular biosynthetic efficiency. Downregulation of AQP expression significantly exacerbates pathological progression during intervertebral disc degeneration. High expression of AQP1 is directly associated with enhanced intervertebral disc hydration, and its central role in delaying matrix degradation and reconstructing the hydration microenvironment is becoming increasingly prominent. To elucidate the molecular regulatory characteristics of GBA / SRGN@HADA@GelMA during mechanically induced intervertebral disc degeneration, transcriptome sequencing analysis was performed on the nucleus pulposus tissue of a compression model rat. Principal component analysis (PCA) results showed significant separation at the transcriptional level between the compression group and the GBA / SRGN@HADA@GelMA group, with good distribution within each sample, indicating that material treatment introduced stable changes in overall gene expression levels. Figure 10 (A). The results of differential expression analysis showed that, compared with the pressurization group alone, 2258 genes were upregulated and 401 genes were downregulated after material treatment, and all of these changes were statistically significant. Figure 10 B, Figure 20 ).

[0085] Among the differentially expressed genes, several molecules associated with water transport, ion homeostasis, and oxidative stress showed significant changes, including aquaporins (Aqp1 and Aqp3) and genes related to ion transport (ATP1A1 and CYBA). Specifically, Aqp1 expression increased in the material-treated group, suggesting that material intervention may affect the hydration state of the neuronal sheath, given its known role in transmembrane water transport. Therefore, changes in Cyba and ATP1A1 expression reflect cellular responses modulating oxidative stress and ion pump function. Cluster analysis of the top 20 differentially expressed genes revealed stable and consistent expression differences between the two groups. Genes associated with matrix degradation and inflammatory responses, such as Mmp13 and Nos2, were elevated in the pressurized group and significantly decreased after material treatment (Figure 10, C). To further analyze the potential functional connections between the differentially expressed genes, a protein-protein interaction network was constructed. A regulatory network centered on Prkaca links genes such as Adcy2, Adcy3, Aqp1, Aqp3, and Atp1a1, forming functional modules related to cAMP signaling, water channel regulation, and ion transport. Furthermore, it is associated with extracellular matrix remodeling molecules (such as Mmp3, Mmp13, and Dcn), reflecting a synergistic relationship between hydration regulation and matrix homeostasis (D in Figure 10). GO enrichment analysis showed that differentially expressed genes are mainly involved in osmolar responses, hyaluronic acid and glycosaminoglycan metabolism, and processes related to mechanostimulation.

[0086] KEGG pathway analysis further revealed that the cAMP signaling pathway, salivary secretion, calcium signaling pathway, extracellular matrix-receptor interaction, and NF-κB signaling pathway were significantly enriched in the material-treated group (E and κB in Figure 10). Figure 21 Gene set enrichment analysis showed that the cAMP signaling pathway and salivary secretion pathway were positively enriched in the GBA / SRGN@HADA@GelMA group, indicating that the molecular functions related to water secretion, transmembrane transport, and signal regulation were generally enhanced (FH in Figure 10). G protein-mediated activation of adenylate cyclase increases intracellular cAMP levels, promotes the release and activation catalyzed by the PKA conversion subunit, and subsequently phosphorylates the downstream transcription factor CREB, thus forming the cAMP-PKA-CREB signaling axis. This signaling axis can affect the expression of aquaporins through transcriptional regulation.

[0087] To further validate the transcriptome sequencing results at the protein level, Western blotting was used to detect key nodes in the signaling pathway (I in Figure 10). Figure 22The AQP1 protein expression loss caused by stress loading was significantly restored after treatment with GBA / SRGN@HADA@GelMA. Material intervention significantly increased CREB phosphorylation levels (p-CREB), while CREB protein remained relatively stable across groups. This is highly consistent with the positive enrichment of the cAMP pathway in transcriptomic analysis, confirming the activation state of this signaling axis. In summary, this study elucidates the molecular mechanism by which the biomimetic microsphere system increases intracellular cAMP concentration by activating adenylate cyclase, thereby activating PKA-mediated CREB phosphorylation, driving AQP1 transcriptional expression and membrane localization, and ultimately achieving efficient remodeling of transmembrane water transport function and water homeostasis in degenerated nerve root membranes (J in Figure 10).

[0088] III. Conclusion This invention develops an injectable composite system—GBA / SRGN@HADA@GelMA—which, through biomimetic interface design and multi-level functional integration, possesses the ability to regulate hydration and intervene in genes. The system uses HADA as a moisturizing outer layer, provides mechanical support through GelMA microspheres, and achieves targeted delivery of SRGN siRNA via G5-GBA loading, thereby realizing the synergistic regulation of the degenerative nucleus pulposus microenvironment. Figure 11 This design aims to suppress inflammatory and matrix degradation signals at the genetic level and physically remodel the hydration and lubrication microenvironment of the nucleus pulposus, thereby restoring mechanical buffering capacity and maintaining extracellular matrix homeostasis. GBA / SRGN@HADA@GelMA reduced the expression of inflammatory factors and matrix degradation genes and significantly promoted the accumulation of HA and lubricating proteins. This enhanced the nucleus pulposus's water retention capacity and interfacial lubricity, thereby improving compressive elasticity and energy uptake. To our knowledge, this is the first time that sustained water regulation, lubrication, mechanical buffering, and extracellular matrix homeostasis regulation have been combined synergistically to delay intervertebral disc degeneration. This study reveals a novel mechanism that combines hydrogel-based biomimetic microenvironment remodeling with therapeutic gene delivery, providing a potentially transformative therapeutic strategy for degenerative disc diseases.

Claims

1. A method for preparing genetically engineered hydration-remodeling biomimetic microspheres, characterized in that, Includes the following steps: (1) Dopamine was grafted onto hyaluronic acid through an activation reaction of EDC and NHS to obtain dopamine-modified hyaluronic acid; (2) Methacrylated gelatin microspheres were prepared by microfluidic reaction of methacrylic anhydride and gelatin, and then dispersed in buffer solution and mixed with dopamine-modified hyaluronic acid obtained in step (1) for co-incubation to obtain composite microspheres; (3) After activating 4-guanidinobenzoic acid with DCC and NHS, it was mixed with fifth-generation polyamide-amine dendritic macromolecules to obtain a polymer modified with 4-guanidinobenzoic acid. Then, it was co-incubated with siRNA to prepare an siRNA complex. (4) The composite microspheres obtained in step (2) are mixed with the siRNA complex obtained in step (3), and genetically engineered hydration remodeling biomimetic microspheres are obtained through covalent and electrostatic adsorption reactions.

2. The preparation method according to claim 1, characterized in that, The pH of the activation reaction in step (1) is 5-5.5, and the activation reaction time is 15 minutes.

3. The preparation method according to claim 1, characterized in that, The grafting reaction conditions described in step (1) are to react at 35°C for 24 hours under light-protected conditions.

4. The preparation method according to claim 1, characterized in that, The pH of the buffer solution in step (2) is 8.

5.

5. The preparation method according to claim 4, characterized in that, The co-incubation conditions described in step (2) are incubation at 37°C for 4 hours.

6. The preparation method according to claim 1, characterized in that, The activation conditions described in step (3) are to activate at room temperature for 6 hours.

7. The preparation method according to claim 1, characterized in that, The reaction conditions described in step (3) are to react for 7 days with continuous stirring, and to co-incubate for 30 minutes at room temperature.

8. The preparation method according to claim 1, characterized in that, In step (4), the N / P ratio of the reaction is controlled to be 12:

1.

9. Genetically engineered hydrated remodeling biomimetic microspheres prepared by the method according to any one of claims 1-8.

10. The use of the genetically engineered hydrated remodeling biomimetic microspheres of claim 9 in the preparation of a drug for treating intervertebral disc degeneration.

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