Engineered microalgae, preparation method and application of engineered microalgae in repairing intervertebral disc

Abstract

The invention relates to the technical field of intervertebral disc treatment, in particular to engineered microalgae, a preparation method and application of the engineered microalgae in intervertebral disc repairing, and the preparation method comprises the following steps: mixing and sintering vanadium powder, aluminum powder and carbon powder according to a stoichiometric ratio to obtain a V4AlC3 precursor, selectively etching the V4AlC3 precursor through hydrofluoric acid, and then adopting TPAOH intercalation stripping to obtain a single-layer V4C3 nanosheet; carrying out centrifugal purification to obtain surface modified V4C3-CS nanospheres; the V4C3-CS nanosphere dispersion liquid and the spirulina suspension liquid are mixed according to the volume ratio of 1: 1, and the mixture is loaded on the surface of the microalgae through the electrostatic adsorption effect; salmon sperm DNA is used as a raw material and is crosslinked with PEGDA under an alkaline condition to form a three-dimensional network structure; the preparation method comprises the following steps of: infiltrating SP and V4C3 dispersion liquid into DNA hydrogel to form a composite material with an antioxidant function, efficiently removing ROS (reactive oxygen species) in the intervertebral disc through enzyme-like catalytic activity of V4C3MXene nanosheets, and simultaneously, synergistically inhibiting inflammatory factors by utilizing a chitosan modification layer and microalgae natural components to break the vicious circle of oxidative stress-inflammation-ECM (extracellular matrix) degradation.

Description

Technical Field

[0001] This invention relates to the field of intervertebral disc treatment technology, and in particular to an engineered microalga, its preparation method, and its application in repairing intervertebral discs. Background Technology

[0002] Intervertebral disc degeneration (IDD) is one of the leading causes of chronic low back pain (LBP), with a rising global prevalence, particularly in aging societies, making it a significant public health issue. The pathological characteristics of IDD include nucleus pulposus (NP) cell senescence, extracellular matrix (ECM) degradation, the formation of an inflammatory microenvironment, and increased oxidative stress, ultimately leading to disc structural damage and functional loss. Current clinical treatments (such as nonsteroidal anti-inflammatory drugs, physical therapy, and surgical intervention) can only relieve symptoms and cannot reverse or halt the progression of IDD, highlighting the urgent need to develop novel treatment strategies targeting the root cause of the pathology.

[0003] Studies have shown that excessive accumulation of reactive oxygen species (ROS) is one of the core driving factors of intramitochondrial disease (IDD). Mitochondrial dysfunction leads to the accumulation of ROS (such as O2). - The excessive production of oxidative stress (ROS) and toxic oxygen (H2O2) disrupts the redox balance of NPCs, leading to DNA damage, the release of inflammatory factors (such as IL-6 and TNF-α), and the activation of matrix metalloproteinases (MMPs), forming a vicious cycle of "oxidative stress-inflammation-ECM degradation." Therefore, targeting and clearing ROS and restoring mitochondrial function is considered an effective way to delay the progression of IDD.

[0004] Although antioxidants (such as N-acetylcysteine ​​and vitamin E) have shown some ROS scavenging effects in in vitro experiments, they are easily metabolized and degraded in vivo, have poor targeting, and are difficult to penetrate the avascular structure of the intervertebral disc. Furthermore, traditional antioxidants cannot simultaneously regulate downstream pathological processes such as inflammation and cellular senescence, resulting in limited therapeutic efficacy.

[0005] In recent years, the two-dimensional material V4C3MXene has attracted much attention in the field of ROS removal due to its enzyme-like catalytic activity (similar to SOD and CAT). However, MXene nanosheets are prone to aggregation and oxidative inactivation in aqueous solutions, and lack tissue targeting, which limits their application in the treatment of intervertebral disc degeneration (IDD). How to improve the stability and biocompatibility of MXene and achieve its long-term retention in degenerated intervertebral discs is a technical challenge that urgently needs to be solved.

[0006] Spirulina platensis (SP), a natural microalga, possesses excellent biocompatibility, antioxidant activity, and surface modifiability, making it an ideal carrier for nanomedicines. Meanwhile, injectable hydrogels (such as DNA hydrogels) show potential in intervertebral disc repair due to their good mechanical properties and tissue adhesion. However, current research has not yet explored the application of the "microalgae-nanozyme-hydrogel" ternary synergistic system in the treatment of intervertebral disc degeneration (IDD).

[0007] In view of this, this application provides an engineered microalgae SP@V4C3 gel platform to overcome the above-mentioned defects. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by proposing a support device for gold mining tunnels.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing an engineered microalgae SP@V4C3 gel platform, comprising the following steps:

[0010] S1: Preparation of V4C3 MXene nanosheets: V4AlC3 precursor was obtained by sintering vanadium powder, aluminum powder and carbon powder in stoichiometric ratio. After selective etching with hydrofluoric acid, single-layer V4C3 nanosheets were obtained by TPAOH intercalation and exfoliation.

[0011] S2: Construction of V4C3-CS nanospheres: V4C3 nanosheets were ultrasonically compounded with chitosan solution at pH=5.0, and surface-modified V4C3-CS nanospheres were obtained after centrifugation and purification.

[0012] S3: Preparation of SP@V4C3 engineered microalgae: V4C3-CS nanosphere dispersion and Spirulina suspension were mixed at a volume ratio of 1:1 and loaded onto the surface of microalgae through electrostatic adsorption.

[0013] S4: Synthetic DNA hydrogel: Using salmon sperm DNA as raw material, it is cross-linked with PEGDA under alkaline conditions to form a three-dimensional network structure;

[0014] S5: Assemble the SP@V4C3 gel platform: Infiltrate the SP@V4C3 dispersion into the DNA hydrogel to form a composite material with antioxidant function.

[0015] As a further preferred embodiment of the present invention, in step (1):

[0016] The sintering conditions were: reaction at 1500℃ for 2 hours under argon protection;

[0017] The etching process involves treating the sample with a 40% hydrofluoric acid solution at 40°C for 7 days.

[0018] The peeling process involved ultrasonic treatment with a 25 wt% TPAOH solution for 2 hours.

[0019] As a further preferred embodiment of the present invention, in step (2):

[0020] Chitosan has a molecular weight of 50 kDa and a degree of deacetylation of ≥85%.

[0021] The ultrasonic processing parameters are 200W power, 5s working time / 5s interval, and a total duration of 30 minutes.

[0022] As a further preferred embodiment of the present invention, in step (3), the loading amount of Spirulina is 50 μg microalgae / mL dispersion, and V4C3-CS is combined with the surface of Spirulina through electrostatic interaction.

[0023] As a further preferred embodiment of the present invention, the preparation conditions of the DNA hydrogel in step (4) include:

[0024] The DNA concentration was 12 mg / mL;

[0025] The amount of cross-linking agent PEGDA added is 10% of the DNA mass;

[0026] The gel formation conditions were 37°C incubation for 1 hour.

[0027] This invention also provides an SP@V4C3 gel platform.

[0028] The structure, comprising V4C3 MXene nanosheets, a chitosan-modified layer, a spirulina carrier, and a DNA hydrogel matrix, has the following performance parameters: storage modulus G' = 1250 ± 85 Pa, and swelling ratio 18.3 ± 1.2.

[0029] As a further preferred embodiment of the SP@V4C3 gel platform of the present invention, it is characterized by having multiple bioactivities:

[0030] ABTS + • Free radical scavenging rate ≥70%;

[0031] SOD-like activity inhibition rate ≥60%;

[0032] It can restore the mitochondrial membrane potential of cells treated with H2O2 to more than 80% of that of the control group.

[0033] The SP@V4C3 gel platform works through the following mechanisms in the preparation of drugs for treating intervertebral disc degeneration:

[0034] (a) Clearing reactive oxygen species from intervertebral disc tissue;

[0035] (b) Inhibits the expression of inflammatory factors such as IL-1β and TNF-α;

[0036] (c) Downregulate aging markers such as P16 and P21;

[0037] (d) Promotes the synthesis and metabolism of type II collagen and glycans.

[0038] As a further preferred embodiment of the application of the SP@V4C3 gel platform in the preparation of therapeutic drugs for intervertebral disc degeneration according to the present invention, the treatment includes:

[0039] A suspension containing SP@V4C3 gel was injected locally into the degenerated intervertebral disc;

[0040] Alternatively, a pre-formed SP@V4C3-DNA composite gel scaffold can be implanted.

[0041] As a further preferred embodiment of the application of the SP@V4C3 gel platform in the preparation of therapeutic drugs for intervertebral disc degeneration in this invention, it further includes:

[0042] SP@V4C3 gel platform;

[0043] Autologous platelet concentrate;

[0044] The SP@V4C3 gel platform is precisely injected into the nucleus pulposus region of the intervertebral disc using a medical delivery device.

[0045] The present invention has the following beneficial effects:

[0046] This invention provides an engineered microalgae SP@V4C3 gel platform that efficiently removes ROS from the intervertebral disc through the enzyme-like catalytic activity of V4C3 MXene nanosheets. At the same time, it utilizes the chitosan modification layer and the natural components of microalgae to synergistically inhibit inflammatory factors, breaking the vicious cycle of "oxidative stress-inflammation-ECM degradation".

[0047] This platform combines the mechanical support function of DNA hydrogels with the bioactivity of engineered microalgae. It can not only restore the mitochondrial membrane potential of nucleus pulposus cells to more than 80% of the normal level, but also promote the synthesis of collagen and proteoglycans, thus achieving dual repair of intervertebral disc structure and function.

[0048] Compared with traditional treatments, this invention has three major advantages: precise targeting, long-lasting sustained release, and multiple regulation. It provides a novel, injectable, and highly biocompatible treatment strategy for intervertebral disc degeneration, and has broad application prospects in the fields of tissue engineering and regenerative medicine. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of engineered microalgae that inhibits intervertebral disc degeneration through DNA hydrogel therapy, by clearing ROS, suppressing inflammation, and preventing cell senescence.

[0050] Figure 2Synthesis and characterization of SP@V4C3 gel; wherein:

[0051] a) Schematic diagram of stepwise preparation of SP@V4C3 gel; b) SEM and elemental distribution map of V4C3-CS nanospheres, scale bar: 200 nm; c) SEM images of V4C3-CS, SP, SP@V4C3, and SP@V4C3-gel (scale bars: 150 nm, 10 μm, 300 nm, and 4 μm, respectively). Green, blue, and red arrows correspond to SP, V4C3-CS, and activated platelets, respectively; df) FTIR, XRD, and XPS analyses of V4C3MXene and V4C3-CS; g) Zeta potential measurement results of V4C3MXene, V4C3-CS, and SP@V4C3-CS; h) Micrographs of precursor solution, DNA gel, and SP@V4C3-gel; ij) Stress-strain curves and cyclic compression curves of DNA gel and SP@V4C3-gel;

[0052] Figure 3 The study aimed to assess the antioxidant activity and in vitro biocompatibility of SP@V4C3 gel; including: ab) ABTS and DPPH free radical scavenging activities of V4C3, V4C3-CS, and SP@V4C3 (n=3, absorbance scan range 400-800nm); ce) quantitative analysis of SOD-like, CAT-like, and hydroxyl radical (·OH) scavenging activities in different groups (n=3); f) CCK-8 assay showing the survival rate of neural progenitor cells after incubation in different concentrations of materials for 1, 4, and 7 days (n=4); g) results of neural progenitor cells in different treatment groups after Calcein-AM / PI staining for 1, 4, and 7 days (n=3, scale bar: 100μm); h) hemolysis rate in different groups (n=3).

[0053] Figure 4 To investigate the protective and anti-inflammatory effects of mitochondria mediated by reactive oxygen species (ROS) scavenging; including: a, d) DCFH-DA fluorescence imaging and semi-quantitative analysis of intracellular ROS levels in neural progenitor cells (NPCs) (n=3, scale bar: 100μm); b, e) JC-1 staining and quantitative analysis of mitochondrial membrane potential (ΔΨm) in neural progenitor cells (n=3, scale bar: 100μm); c, fi) Immunofluorescence images, semi-quantitative analysis, and mRNA expression levels of IL-1β and TNF-α in different groups (n=3, scale bar: 100μm); jk) Quantitative analysis of JC-1 fluorescence ratio detected by flow cytometry; lm) Quantitative analysis of apoptotic cells detected by flow cytometry (Annexin V / PI staining);

[0054] Figure 5To inhibit cellular senescence and restore extracellular matrix homeostasis; including: a, de) immunofluorescence staining and mRNA expression of P21 and P16 (n=3, scale bar: 100μm); bc, fi) immunofluorescence staining and gene expression of extracellular matrix markers (type II collagen, ACAN) and catabolic enzymes (MMP13, MMP9) (n=3, scale bar: 100μm); j and lm) immunofluorescence staining and gene expression of senescence markers (P21, P16) (n=3, scale bar: 100μm); k and n) SA-β-galactosidase staining and quantitative analysis of SA-β-galactosidase-positive neural progenitor cells (n=3, scale bar: 400μm);

[0055] Figure 6 Transcriptome validation of the antioxidant and anti-inflammatory effects of SP@V4C3 was conducted, including: a) principal component analysis (PCA) of overall gene expression in the control group, H2O2 group, and SP@V4C3+H2O2 group; b and c) volcano plots showing differentially expressed genes (DEGs) between the H2O2 group and the control group, and between the SP@V4C3+H2O2 group and the H2O2 group; d and e) GO and KEGG enrichment analysis of DEGs between the SP@V4C3+H2O2 group and the H2O2 group; fh) gene set enrichment analysis (GSEA) revealed that acute inflammatory response, TNF superfamily cytokine production, and IL-6 production pathways were inhibited after SP@V4C3 treatment.

[0056] Figure 7 The therapeutic effects of SP@V4C3 on an IDD rat model were shown, including: a) a schematic diagram of the establishment of the rat IDD model, the SP@V4C3 administration process, and the evaluation timeline; b) representative X-ray and T2-weighted MRI images of the intervertebral discs of rats in the control group, IDD group (puncture + PBS), and SP@V4C3 group (puncture + SP@V4C3); ce) quantitative analysis of intervertebral disc height index (DHI), Pfirrmann grade, and histological score (n=5); f) H&E staining, Masson trichrome staining, and Safranin O staining results in different groups (n=5; scale bar: 1 mm); g) representative immunofluorescence staining of IL-1β and P21 in intervertebral disc tissue (n=5; scale bar = 1 mm); h) representative immunohistochemical staining of type II collagen and MMP3 in intervertebral disc tissue (n=5; scale bar = 1 mm).

[0057] Figure 8The therapeutic effects of SP@V4C3 gel on an IDD goat model were shown, including: a) a schematic diagram of nanoparticle clearance and H2O2-induced IDD, followed by SP@V4C3 gel implantation; b) a simulated surgical procedure and representative CT / MRI images of the sham surgery group, IDD group, and SP@V4C3 gel group; c) quantitative analysis of lumbar disc height index (DHI) and Feynman grading (n=3); d) a heatmap of SASP-related gene expression in intervertebral tissue detected by RT-qPCR (n=3); ef) histological evaluation of intervertebral disc structure using hematoxylin O-fast green staining, H&E staining, and Masson trichrome staining (n=3; scale bars: 5 mm and 1 mm).

[0058] Figure 9 The therapeutic effect of SP@V4C3 gel on IDD goat model; including: immunohistochemical staining and quantitative analysis of IL-1β, P16, ACAN and MMP3 in ad and il) neurons, lateral axonal region and endplate region (n=3; scale bar: 5 mm and 200 μm); immunofluorescence staining and quantitative analysis of fluorescence intensity of corresponding markers eh and mp (n=3; scale bar: 200 μm). Detailed Implementation

[0059] The present invention will now be further described as follows:

[0060] The preparation of an engineered microalgae SP@V4C3 gel platform involves the following specific steps:

[0061] S1: Preparation of V4C3MXene nanosheets

[0062] S1.1 Precursor preparation: Vanadium powder (Aladdin, purity 99.9%), aluminum powder (Aladdin, purity 99.5%) and carbon powder (Sinopharm Group, purity 99.9%) were mixed in stoichiometric ratio and sintered at 1500℃ for 2 hours under argon protection to obtain blocky V4AlC3 ceramics.

[0063] S1.2 Selective Etching: V4AlC3 powder was immersed in a 40% hydrofluoric acid aqueous solution (Aladdin) and stirred at 40℃ for 7 days. After centrifugation (8000rpm, 10min), the precipitate was collected and washed with deionized water until pH=7.0;

[0064] S1.3 Intercalation and exfoliation: The etched product was dispersed in 25wt% TPAOH solution (Sinopharm Group), stirred at room temperature for 24 hours, and then sonicated (power 300W, frequency 40kHz) for 2 hours. After centrifugation (12000rpm, 15min) and washing with ethanol, it was freeze-dried to obtain monolayer V4C3 nanosheets.

[0065] Preparation of S2:V4C3-CS nanospheres

[0066] S2.1 Surface modification: Mix an aqueous solution of 2 mg / mL V4C3 nanosheets with an equal volume of 2 mg / mL chitosan (Sigma, MW = 50 kDa, degree of deacetylation ≥ 85%), and adjust the pH to 5.0;

[0067] S2.2 Ultrasonic mixing: The mixture was treated with a probe ultrasonic instrument (power 200W, working time 5s / interval 5s, total duration 30min), and the precipitate was collected by centrifugation (10000rpm, 10min).

[0068] S2.3 Purification and Preservation: Washed three times with deionized water and anhydrous ethanol in sequence, and freeze-dried to obtain V4C3-CS nanospheres, which were then stored at 4°C in the dark.

[0069] S3: Construction of SP@V4C3 engineered microalgae

[0070] S3.1 Microalgae loading: Mix 2 mg / mL V4C3-CS dispersion with 2 mg / mL Spirulina (Guangyu Biotechnology) suspension at a volume ratio of 1:1 and stir magnetically at 25°C (300 rpm) for 1 hour;

[0071] S3.2 Separation and purification: Collect the composite microalgae by centrifugation (5000 rpm, 10 min), rinse 3 times with ultrapure water (conductivity <0.1 μS / cm), and freeze-dry for later use;

[0072] S4: Preparation of DNA hydrogel

[0073] S4.1 Solution preparation: Dissolve 60 mg of salmon sperm DNA (Coolbey, MW > 10 kDa) in 5 mL of 0.127 M NaOH solution, and add 10% PEGDA (Sigma, MW = 700 Da) cross-linking agent;

[0074] S4.2 Gel molding: After vacuum degassing, the mixture is injected into a polytetrafluoroethylene mold and incubated at 37°C for 1 hour to form a three-dimensional network structure;

[0075] S4.3 Post-treatment: Wash with PBS (pH=7.4) until the OD260 of the eluent is <0.05, and freeze-dry to obtain a porous scaffold;

[0076] S5: Assembly of the P@V4C3 gel platform

[0077] S5.1 Drug-loaded composite: 1 mL of SP@V4C3 dispersion (containing 50 μg microalgae) was added dropwise to the surface of the DNA hydrogel and allowed to stand at 4°C for 12 hours to achieve complete penetration;

[0078] S5.2 Performance characterization: Rheometer test showed that the storage modulus G' = 1250 ± 85 Pa (frequency 1 Hz) and the swelling ratio was 18.3 ± 1.2 (in PBS for 24 h).

[0079] Characterization: The morphology and elemental distribution of V4C3, V4C3-CS and SP@V4C3 were characterized by field emission scanning electron microscopy (FE-SEM, GeminiSEM300, Zeiss, Germany) combined with energy-dispersive X-ray spectroscopy (EDS).

[0080] Functional groups were identified using Fourier transform infrared spectroscopy (FTIR, Nicoleti S20, Thermo Fisher Scientific, USA), and structural phases were verified using X-ray diffraction (XRD, SmartLabSE, Rigaku Corporation, Japan). Surface elemental composition was further determined using X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Fisher Scientific, USA). Zeta potentials were measured using a Zetasizer Nano ZS90 Zeta analyzer (Malvin Panaco, UK) to assess surface charge and colloidal stability. Mechanical property testing of the DNA gel and SP@V4C3- gel was performed using a universal testing machine under compression (Sansheng Instruments, China).

[0081] Evaluation of free radical scavenging activity: To assess the free radical scavenging capabilities of V4C3, V4C3-CS, and SP@V4C3, we used ABTS. + Both · and DPPH· methods were used for detection. (In ABTS) + In the experiment, a stock solution was prepared by mixing 7 mM ABTS (McKinlun Pharmaceuticals, China) and 2.45 mM potassium persulfate (K2S2O8, McKinlun Pharmaceuticals, China) at a 1:1 volume ratio. The mixture was incubated at room temperature in the dark for 12-16 hours to generate ABTS. + • Free radicals were then diluted with PBS to an absorbance of 0.8 ± 0.05 at 734 nm. An equal volume of the dispersion was added to the reaction system, and the mixture was incubated in the dark for 30 minutes to ensure uniform reaction. The absorbance was measured using a microplate reader (Redmark, Flash Spectrum Technology, China) in the wavelength range of 400-800 nm. In the DPPH· experiment, 0.2 mM DPPH (McGenron, China) solution was dissolved in anhydrous ethanol and adjusted to an initial absorbance of 1.0 ± 0.05 at 517 nm. An equal volume of sample was mixed with the DPPH· solution and incubated in the dark for 30 minutes. Finally, the absorbance was detected in the 400-800 nm wavelength range.

[0082] Evaluation of SOD-like, CAT-like, and hydroxyl radical scavenging activities: Superoxide dismutase (SOD)-like activity was detected using a commercially available SOD assay kit manufactured by Cummins Biotech (China). This kit utilizes the superoxide anion (O2) generated by the xanthine-xanthine oxidase system. - Nitroblue tetrazolium (NBT) was reduced to formazan, and the absorbance was then measured at a wavelength of 560 nm. A lower absorbance value indicates higher SOD-like activity.

[0083] Catalase (CAT) activity was detected using a catalase assay kit (Beyotime, China). The procedure was as follows: After incubation with hydrogen peroxide (H2O2), the residual H2O2 reacted with the chromogenic substrate under the catalysis of catalase to generate a red product. The absorbance was measured at 520 nm and compared with an H2O2 standard curve.

[0084] The hydroxyl radical (·OH) scavenging ability was determined using a hydroxyl radical detection kit (Solepro Corporation, China). In this system, ·OH radicals scavenged Fe... 2+ Fe in the -1,10-phenanthroline complex 2+ Oxidized to Fe 3+ This leads to a decrease in absorbance at 536nm, thus reflecting the removal efficiency.

[0085] Isolation and culture of neural progenitor cells: Human nucleus pulposus (NP) samples were obtained from volunteers who consented to participate in minimally invasive or open spinal surgery for the treatment of vertebral fractures or scoliosis correction. The procedure involved cutting the nucleus pulposus tissue into pieces approximately 1 mm thick using ophthalmic scissors. 2 The fragments were then digested in 0.2% type II collagenase (BioFroxx, China) for 6 hours. The filtered cell suspension was seeded in DMEM / F12 medium (Gibco, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin. The medium was changed every two days, and subsequent experiments were performed using cells that had been passaged twice.

[0086] Cell compatibility assessment: The cell compatibility of V4C3, V4C3-CS, SP@V4C3, and DNA gels was assessed using a cell counting kit-8 (CCK-8, Bosch, China) and calcein-AM / propidium iodide live / dead staining method (Beyotime, China). Nucleus pulposus cells (NPCs) were counted at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 10% in 24-well plates. After reaching 80% confluence, the cells were treated with different concentrations of test material for 1, 4, and 7 days. At each time point, 10% CCK-8 solution was added, and the cells were incubated at 37°C for 1 hour. Cell viability was determined by measuring the absorbance at 450 nm.

[0087] In the live / dead cell staining experiment, the cell culture conditions were the same as described above. The conditioned medium for the DNA gel assay was prepared as follows: 20 mg / mL DNA gel was placed in serum-free DMEM medium and incubated at 37°C for 24 hours. After treatment, the cells were labeled with Calcein-AM and propidium iodide as per the manufacturer's instructions, and the results were observed using a confocal laser scanning microscope.

[0088] Hemolysis test: Blood compatibility was assessed by measuring the hemolysis rate using fresh whole blood from healthy rats. Heparinized whole blood was centrifuged at 800g for 10 minutes at 4°C. The collected red blood cells were washed repeatedly with PBS until the supernatant was clear. A 2% (v / v) red blood cell suspension was then prepared for testing.

[0089] For each experimental group, 0.2 mL of red blood cell suspension was mixed with 0.8 mL of test solution, which contained deionized water (positive control), PBS buffer (negative control), or V4C3, V4C3-CS, and SP@V4C3 dispersions. After incubating the samples at 37°C for 2 hours to promote interaction, the supernatant was collected by centrifugation at 800 g for 10 minutes. After processing, the absorbance of the supernatant at 540 nm was measured using a microplate reader. The hemolysis rate (%) was calculated using the following formula:

[0090] Hemolysis rate (%) = (Sample absorbance - Control group absorbance) / (PC group absorbance - Control group absorbance) × 100%

[0091] Intracellular ROS Detection: To assess the antioxidant capacity of SP@V4C3, researchers used a ROS detection kit (DCFH-DA, Beyotime Biotech, China) to detect reactive oxygen species levels in neural progenitor cells (NPCs). The specific procedure was as follows: treated NPCs were incubated with the DCHF-DA probe at 37°C for 30 minutes, followed by washing with PBS buffer (×3), and fluorescence signals were acquired using a confocal laser scanning microscope. Finally, based on the FITC fluorescence channel detection results, the fluorescence signals were quantitatively analyzed using flow cytometry.

[0092] Mitochondrial membrane potential measurement (JC-1): Mitochondrial membrane potential (ΔΨm) was detected using the JC-1 staining method (Beyotime Biotechnology, China), following the manufacturer's instructions. After treatment, cells were incubated in JC-1 working solution at 37°C for 30 minutes, followed by rinsing with the accompanying buffer. Fluorescence imaging was performed using a confocal microscope. A decrease in the ratio of red to green fluorescence (aggregates to monomers) indicated the dissipation of ΔΨm. Finally, the fluorescence signal was quantitatively analyzed by flow cytometry.

[0093] Apoptosis detection: Apoptosis was detected using the Annexin V-FITC / propidium iodide double staining method (Bosch, China). After treatment, cells were collected using EDTA-free trypsin, washed twice with PBS, and resuspended in binding buffer. Annexin V-FITC and propidium iodide were then added, and the cells were incubated at room temperature in the dark for 10 minutes. Flow cytometry was used to quantitatively assess early and late apoptotic cell populations.

[0094] Immunofluorescence staining: After treatment, cells were fixed with immunostaining fixative (Beyotime, China) for 20 minutes, followed by permeabilization with 0.5% Triton X-100 for 30 minutes. Then, they were blocked with immunostaining blocking buffer (Beyotime, China) for 1 hour at room temperature to prevent non-specific antibody binding. Primary antibodies against IL-1β (26048-1-AP), TNF-α (60291-1-PBS), P21 (10355-1-AP), P16 (10883-1-AP), glycan (68350-1-Ig), type II collagen (28459-1-AP), MMP9 (10375-2-AP), and MMP13 (83188-2-RR) (all purchased from Protective, China) were diluted with antibody buffer and incubated overnight at 4°C with gentle shaking. After washing, cells were incubated with fluorescently labeled secondary antibodies at room temperature for 1 hour. The cell-coated coverslips were then placed upside down onto a slide containing DAPI-enriched anti-quenching mounting medium (Biosharp, China). Finally, the fluorescence signal was observed using a confocal laser scanning microscope.

[0095] Real-time quantitative polymerase chain reaction (qPCR): Total RNA was extracted using Vazyme (China) RNA-easy separation reagent according to the manufacturer's instructions. RNA concentration and purity were detected using a HIPIE (China) Aurora-900 micro spectrophotometer. 2 μg of total RNA sample was used for reverse transcription using the Vazyme (China) HiScript II QRT SuperMix reverse transcription kit. Real-time quantitative PCR analysis was performed using SYBR Green premixed buffer (Vazyme, China) on a Thermo Fisher Scientific (USA) QuantStudio. TM 5. Real-time quantitative PCR was performed on a real-time fluorescence quantitative PCR system. Gene expression levels were normalized using GAPDH as an internal control and quantified using the 2^(-ΔΔCt) method. Primer sequences used are detailed in Supporting Information Tables S1 and S2.

[0096] RNA sequencing: RNA extraction was performed using RNA-easy reagents from Vazyme (China), strictly following the supplier's operating guidelines. RNA quality was assessed before library construction. 1 μg of total RNA was used for each sample. RNA sequencing libraries were constructed using a strand-specific mRNA preparation (ligation reaction) protocol (San Diego, California, USA). High-throughput sequencing was performed on a NovaSeqXPlus system (Illumina, model PE150) using the accompanying kit. Differential gene expression analysis was based on TPM-normalized transcription levels, and gene abundance was estimated using RSEM. Genes with a fold change (log2) > 0.5 and a p-value < 0.05 were defined as differentially expressed genes (DEGs) according to DESeq2 software. To further elucidate the biological significance of these differentially expressed genes, we performed Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to comprehensively reveal their related functions and regulatory networks. GO enrichment analysis was performed using Goatools, and KEGG pathway analysis was implemented using the Python SciPy library.

[0097] In some aspects, the preparation of an engineered microalgae SP@V4C3 gel platform employs a multi-step assembly process, such as... Figure 2 As shown in Figure a, multilayered V4C3MXene was successfully exfoliated into several layers of two-dimensional nanosheets by selectively etching V4AlC3 material and treating it with hydrochloric acid and lithium fluoride reagents, followed by TMAOH treatment and ultrasonic treatment. Scanning electron microscopy imaging showed that the V4C3 nanosheets have a typical layered structure. Figure 2 b) Elemental distribution analysis confirmed the uniform distribution of vanadium. To improve dispersibility and biocompatibility, V4C3 was functionalized with chitosan using electrostatic self-assembly technology (V4C3-CS). Scanning electron microscopy images showed the formation of uniformly shaped nanospheres. Figure 2 c). Finally, V4C3-CS nanospheres were loaded onto the negatively charged surface of Spirulina using electrostatic adsorption technology, successfully constructing engineered microalgae (SP@V4C3). Figure 2 c).

[0098] Subsequently, we prepared a DNA hydrogel by chemically cross-linking biomass-derived DNA, following a previously reported experimental protocol. This hydrogel exhibited excellent swelling capacity and transparency, perfectly replicating the hydration characteristics of natural nucleus pulposus tissue. Figure 2 h). Utilizing the swelling advantage of hydrogels, we combined SP@V4C3 composite materials with platelets to construct SP@V4C3-gel composite materials (h). Figure 2 h). Scanning electron microscope image ( Figure 2 c) Further confirmation that all components were successfully integrated, with SP, V4C3-CS, and platelets showing significant differences in morphological distribution. These observations strongly support the rational hierarchical assembly mechanism of the SP@V4C3-gel system.

[0099] The surface modification effect of chitosan on V4C3 was confirmed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). FTIR spectroscopy (…) Figure 2 d) Typical chitosan absorption bands were observed in V4C3-CS – including OH / NH stretching vibrations (wavelength ~3400 cm⁻¹). -1 NH bending vibration (1560-1650cm) -1 ) and COC stretching vibration (1020-1080cm) -1 These characteristic peaks did not appear in the original V4C3, proving that the surface functionalization was successful. XRD analysis showed that the (002) crystal plane peak shifted from 7.52° in V4C3 to 5.02° in V4C3-CS, indicating that chitosan intercalation led to an increase in interlayer spacing. Figure 2 e). Furthermore, XPS spectroscopy ( Figure 2 f) showed a strong N1 s peak (wavelength ~400 eV) in V4C3-CS, while this peak was absent in unmodified V4C3, further verifying the chitosan coating. Zeta potential analysis ( Figure 2 g) The electrostatic properties of the assembly were further confirmed: V4C3 initially exhibited a negative potential (-20.4mV), which was reversed to -16.8mV after chitosan modification; subsequently, after adsorbing spirulina, the potential dropped to -12.1mV, indicating that the stepwise assembly process of SP@V4C3 was successfully completed.

[0100] To evaluate the load-bearing performance of the hydrogel system, we used the uniaxial compression method to test the mechanical properties of the DNA gel and the SP@V4C3-gel. Although the addition of SP@V4C3 slightly reduced the compressive modulus, the composite gel still maintained sufficient mechanical strength and elasticity, fully demonstrating its suitability as a support material for nucleus pulposus (NP)-like structures. Figure 2 i and j).

[0101] 2.2 ROS scavenging performance and cell compatibility of SP@V4C3-gel

[0102] Excessive accumulation of reactive oxygen species (ROS) is a key driver of inflammation, cellular senescence, and apoptosis in the progression of intracellular drug disorder (IDD). To evaluate the ROS scavenging capacity of SP@V4C3 materials, we conducted a series of assays including total antioxidant capacity (ABTS and DPPH), nanozyme-like activity (SOD and CAT-like activity), and hydroxyl radical (·OH) scavenging capacity. Antioxidant efficacy was quantified by monitoring absorbance changes of characteristic free radicals in the visible spectral range. All materials exhibited dose-dependent ABTS scavenging. + • and DPPH· free radical scavenging activity ( Figure 3(ab). Notably, pristine V4C3MXene exhibited the strongest free radical scavenging ability among all groups, likely attributed to its abundant surface active sites and excellent electron transfer properties. Although the scavenging ability of SP@V4C3 was slightly lower than that of naked V4C3, it still maintained significant antioxidant activity, indicating that the engineered microalgae retained the inherent redox function of the MXene core. In nanozyme assays, SP@V4C3 significantly enhanced SOD and CAT-like catalytic activities, as reflected by the inhibition percentage and CAT activity index, respectively. Figure 3 cd). The scavenging ability of SP@V4C3 against hydroxyl radicals (·OH) was also significantly enhanced, highlighting its strong free radical neutralization ability. Figure 3 e).

[0103] Next, we evaluated the cytocompatibility of SP@V4C3 and DNA gels to verify their safety in the cellular environment. The biocompatibility of V4C3MXene, V4C3-CS, and SP@V4C3 was systematically evaluated using CCK-8 assay, live / dead cell staining, and hemolysis assay. CCK-8 assay and Calcein-AM / PI live / dead cell staining confirmed that V4C3MXene, V4C3-CS, and SP@V4C3 all exhibited good cytocompatibility at appropriate doses. Specifically, no significant cytotoxicity was observed when neural progenitor cells (NPCs) were exposed to V4C3 (≤20 μg / mL), V4C3-CS (≤30 μg / mL), or SP@V4C3 (≤40 μg / mL) for 1, 4, or 7 days, indicating that these concentration ranges are suitable for subsequent bioapplications. Figure 3 (f and g). Notably, the V4C3-based platform, modified with chitosan and further engineered with microalgae, significantly improved biocompatibility, making it more suitable for cellular applications. Furthermore, live / dead staining results of NPCs cultured with DNA gel extract further confirmed its excellent biocompatibility. Figure 3 g). Hemolysis tests showed that the hemolysis rate of all materials was less than 5%, which meets biomedical safety standards. Figure 3 (h) In summary, SP@V4C3 and DNA gel not only possess powerful antioxidant and nanozyme-like functions, but also exhibit excellent cell compatibility and blood compatibility. These properties make them a safe and effective therapeutic platform for treating immunodeficiency diseases (IDD).

[0104] 2.3 ROS scavenging-mediated mitochondrial and anti-inflammatory protection

[0105] As a typical reactive oxygen species, H2O2 plays a crucial role in initiating oxidative stress-related cascades, including mitochondrial damage, pro-inflammatory signaling, and aging-related phenotypes. Previous studies have shown that 100 μM H2O2 is sufficient to induce mitochondrial dysfunction in neural progenitor cells (NPCs), thereby promoting inflammatory responses and the aging process. Furthermore, recent research has shown that 10 ng / mL IL-11 can cause mitochondrial dysfunction, activate aging-related pathways, and induce cellular senescence. Therefore, in this study, V4C3MXene, V4C3-CS, and SP@V4C3 were first co-incubated with NPCs for 1 hour, followed by stimulation with 100 μM H2O2 or 10 ng / mL IL-11 for 24 hours, respectively.

[0106] Intracellular reactive oxygen species (ROS) levels were observed using DCFH-DA staining, and quantitative analysis was performed using fluorescence imaging and flow cytometry. The experiment revealed that both H2O2 and IL-11 stimulation significantly enhanced ROS fluorescence intensity and caused neural progenitor cells (NPCs) to change from a spindle-shaped to a round shape. Figure 4 (a) and (d). Notably, treatment with V4C3MXene, V4C3-CS, and SP@V4C3 effectively inhibited ROS accumulation, with the SP@V4C3 group showing the greatest decrease in fluorescence intensity. Consistent with the fluorescence imaging results, flow cytometry analysis further confirmed that all three treatments significantly reduced intracellular ROS accumulation.

[0107] Excessive accumulation of intracellular reactive oxygen species (ROS) can damage mitochondrial function, thereby exacerbating ROS production and creating a vicious cycle of oxidative stress and mitochondrial dysfunction. Therefore, effective ROS clearance is closely related to maintaining mitochondrial homeostasis. We used the JC-1 staining method to detect mitochondrial membrane potential (ΔΨm). In damaged cells, a change in fluorescence from red to green indicates mitochondrial depolarization. Figure 3 b and Figure 2 As shown in b, JC-1 fluorescence significantly changed from red to green after exposure to H2O2 or IL-11, indicating ΔΨm depolarization. In contrast, treatment with V4C3MXene, V4C3-CS, and SP@V4C3 effectively restored mitochondrial membrane potential, with SP@V4C3 showing the most significant restoration effect. Figure 4 e and Figure 3 e). Furthermore, these findings were further validated by JC-1-based flow cytometry analysis. Notably, SP@V4C3 exhibited superior protective activity compared to the other groups. Figure 4 j and k).

[0108] Since mitochondrial dysfunction often triggers inflammatory responses, we further investigated key inflammatory mediators. Immunofluorescence and RT-qPCR analyses showed that the expression levels of IL-1β and TNF-α were significantly increased in the H2O2 or IL-11 induced groups, and all treatments inhibited these responses to varying degrees, with SP@V4C3 exhibiting the strongest anti-inflammatory effect. Figure 4 c and fi).

[0109] Flow cytometry further demonstrated that SP@V4C3 provided better cytoprotective effects against H2O2-induced apoptosis compared to V4C3 or V4C3-CS, as evidenced by the reduced proportion of Annexin V / PI positive cells. Figure 4 lm).

[0110] In summary, these findings indicate that SP@V4C3 can effectively scavenge intracellular reactive oxygen species, restore mitochondrial membrane potential, and inhibit the expression of inflammatory cytokines under oxidative stress or inflammatory conditions. These discoveries highlight the therapeutic potential of SP@V4C3 in suppressing oxidative stress and inflammation in IDD-related pathological environments.

[0111] Experimental example:

[0112] Intervertebral disc degeneration rat model: An intervertebral disc degeneration (IDD) model was established in male Sprague-Dawley rats (300±20 g) using acupuncture. After anesthesia with intraperitoneal injection of sodium pentobarbital, the caudal intervertebral discs (Co7 / 8 and Co8 / 9) were punctured using a 22-gauge needle, rotated 360 degrees, and held for 30 seconds. Co6 / 7 served as the unpunctured control group.

[0113] Immediately after puncture, 10 μL of PBS or SP@V4C3 suspension (100 μg / mL) was injected into the intervertebral disc using a microsyringe. On day 33, rats underwent X-ray and MRI imaging (VivoFXPro, Bruker), followed by euthanasia for histological analysis. After fixation, decalcification, and paraffin embedding, spinal cord segments were stained with H&E and hematoxylin-eosin, and immunolabeled for ACAN, MMP3, IL-1β, and P21.

[0114] Bohr Goat Intervertebral Disc Degeneration Model and In-situ Gel Implantation: In this study, a large animal model of intervertebral disc degeneration (IDD) was established in male goats (30±2 kg) using annulus fibrosus incision, nucleotomy, and H2O2 injection. Three lumbar vertebral levels were set up: L3-L4 (sham surgery group), L4-L5 (IDD group), and L5-L6 (SP@V4C3 gel treatment group). Under general anesthesia, a 12 cm incision was made along the midline of the lumbar spine to expose the spine. Both the IDD and treatment groups underwent a 1 cm annulus fibrosus incision, followed by nucleotomy and injection of 200 μL of 100 μm H2O2. On postoperative day 7, SP@V4C3 gel mixed with autologous platelet concentrate was implanted into the L5-L6 vertebral bodies.

[0115] Postoperatively, the goats were monitored and treated with antibiotics for 3 days. CT and MRI scans were performed on days 7 and 30, respectively. The animals were euthanized on day 30, and intervertebral disc samples were collected for RT-qPCR analysis of SASP-related genes, histological staining (hematoxylin-eosin, safranin O-fast green, and marson dye staining), and immunohistochemical analysis.

[0116] Therapeutic effects of SP@V4C3 on IDD rat model

[0117] The rat caudal intervertebral disc acupuncture model is widely used to induce intervertebral disc degeneration (IDD) due to its simplicity and high reproducibility. Immediately after puncture, SP@V4C3 (100 μg / mL, 10 μL) was injected into the intervertebral disc interstitium, while the IDD group received an equal volume of PBS solution. X-ray and magnetic resonance imaging (MRI) assessments were performed on postoperative day 33. Figure 7 a). Both imaging studies showed that SP@V4C3 treatment significantly delayed the progression of IDD compared to the untreated group. Figure 7 b). To quantitatively assess the severity of IDD, two clinically relevant indicators—the disc height index (DHI) and the Pfirrmann classification—were measured by X-ray and MRI, respectively. Figure 7The IDD group exhibited significant degenerative features, including substantial loss of intervertebral disc height and decreased T2-weighted MRI signal intensity. In contrast, SP@V4C3 treatment significantly alleviated these degenerative radiographic findings, demonstrating a good therapeutic effect. Histological analysis of specimens was performed on day 33 post-treatment. The vertebral body structure in the control group was well preserved, with the central nucleus pulposus encased in concentric layers of the annulus fibrosus. The cartilaginous endplates were clearly defined above and below the nucleus pulposus, effectively maintaining the integrity of the intervertebral disc structure. In contrast, the IDD group showed severe intervertebral disc degeneration, characterized by intervertebral space collapse, annulus fibrosus disorder, endplate deformation, and destruction of neural progenitor cells (NPCs) and matrix structures. Notably, histological staining (hematoxylin-eosin staining, masson staining, and safranin O staining) showed that SP@V4C3 treatment significantly preserved the intervertebral disc structure, restored disc height, and almost restored neural progenitor fiber tissue to normal, highly similar to the healthy control group. Figure 7 e and f). To further investigate the mechanisms of aging, inflammation, and extracellular matrix (ECM) remodeling, researchers performed immunohistochemical and immunofluorescence staining experiments. The results showed that magnetic resonance imaging and histological examination revealed that SP@V4C3 significantly inhibited the expression of the pro-inflammatory cytokine TNF-α and the aging marker p16, while restoring ACAN protein expression and reducing MMP3 levels, thereby promoting extracellular matrix metabolic homeostasis. Figure 7 g and h, S6a-d). Furthermore, H&E staining of major rat organs confirmed the excellent biocompatibility of SP@V4C3 in vivo (g and h, S6a-d). Figure 7 b). The overall results indicate that intradiscal administration of SP@V4C3 can effectively alleviate puncture-induced inflammatory disc degeneration (IDD). Its mechanism of action includes inhibiting the inflammatory response, delaying cell senescence, and restoring the integrity and homeostasis of the extracellular matrix.

[0118] Therapeutic effects of SP@V4C-gel on IDD goat model

[0119] Although rat models are frequently used in intervertebral disc degeneration (IDD) research due to their practicality, their structural and biomechanical complexity cannot fully replicate the characteristics of human intervertebral discs, especially in terms of disc defects and stress conditions. Therefore, to promote clinical translation, it is crucial to evaluate the therapeutic effects of SP@V4C3 in large animal models. For this purpose, we chose Boer goats as a large animal model because their anatomical structure is similar to that of the human lumbar spine, offering comparability, particularly in vertebral body size, intradiscal pressure, and range of motion. To achieve targeted delivery within the intervertebral disc, we bound SP@V4C3 to autologous platelets in a DNA hydrogel before animal experiments. This hydrogel not only possesses strong adhesion to fill disc defects but also enables sustained local release of SP@V4C3. Furthermore, this DNA hydrogel has the unique function of selectively activating platelets, thereby promoting the release of various growth factors and synergistically enhancing its anti-aging effects. Based on these characteristics, we evaluated the therapeutic performance of SP@V4C3-gel in a goat IDD model. Figure 8 a).

[0120] Intervertebral disc index (DHI) and Pfirrmann classification were assessed at multiple time points using CT and MRI techniques. Figure 8 (b and c). Annulus fibrosus disruption combined with H2O2 injection successfully induced intervertebral disc degenerative disease (IDD), while SP@V4C3 gel combined with platelets significantly delayed the degenerative process. Furthermore, researchers collected intervertebral disc tissue for quantitative RT-PCR analysis, which showed that SP@V4C3 gel treatment could partially inhibit the secretion of SASP-related factors, thereby alleviating neural progenitor (NPC) senescence. Figure 8 d).

[0121] The larger anatomical structure of the goat intervertebral disc allowed for clear observation of the nucleus pulposus, annulus fibrosus, and cartilaginous endplate. Consistent with imaging results, H&E staining, safranin O staining, and masson staining showed that the sham-operated group had intact tissue morphology and clear structural boundaries. Figure 8 e and f). In contrast, the IDD group showed a significant reduction in nucleus pulposus proteoglycans and collagen, partial rupture and deformation of the lamellar annulus fibrosus, and extensive granulocyte infiltration within the cartilaginous endplate. Notably, SP@V4C3 gel treatment significantly preserved the intervertebral disc structure and alleviated these degenerative changes, a conclusion further supported by quantitative histological analysis ( Figure 8 a).

[0122] To further evaluate inflammatory responses, aging-related biomarkers, and extracellular matrix synthesis and degradation activities, we performed immunohistochemistry and immunofluorescence staining. The expression patterns of these biomarkers in the nucleus pulposus, annulus fibrosus, and cartilaginous endplate were analyzed. Compared with the sham-operated control group, the expression of IL-1β and P16 was significantly increased in the IDD group. Figure 9ab and ef), while the expression of the synthetic marker ACAN decreased, while the expression of the decomposition marker MMP3 increased (ab and ef). Figure 9 (cd and gh). Notably, SP@V4C3 gel treatment significantly improved these pathological changes, highlighting its powerful antioxidant, anti-inflammatory, and anti-aging effects. Figure 9 Furthermore, SP@V4C3 gel restored extracellular matrix metabolic balance, thereby maintaining intervertebral disc homeostasis. Multiple time-point hematological tests, serum biochemical analysis, and histological evaluation of major organs confirmed the excellent biocompatibility of SP@V4C3 gel in vivo. Figure 9 These findings suggest that SP@V4C3 gel has the therapeutic potential to modulate the intervertebral disc microenvironment and delay intervertebral disc degeneration.

[0123] This study developed a multifunctional therapeutic platform by integrating engineered microalgae into a DNA hydrogel. This composite system not only effectively repairs intervertebral disc defects but also simultaneously slows down the degenerative process of the disc. In vitro experiments showed that SP@V4C3 efficiently scavenges intracellular reactive oxygen species, maintains mitochondrial membrane potential, and inhibits aging-related phenotypes in neural progenitor cells, ultimately promoting the restoration of extracellular matrix metabolic homeostasis. Transcriptome analysis further confirmed these findings, collectively validating the powerful antioxidant, anti-inflammatory, and anti-aging capabilities of SP@V4C3. In a rat IDD model, SP@V4C3 significantly alleviated intervertebral disc degeneration by reducing inflammatory responses and delaying cellular senescence; in a goat model, the DNA gel served as a nanoparticle-like artificial intervertebral disc, enabling local delivery of SP@V4C3. Utilizing its swelling and platelet-activating properties, the composite hydrogel synergistically enhanced the anti-aging and repair effects. Finally, this engineered SP@V4C3-loaded DNA hydrogel demonstrated significant therapeutic effects in in vivo experiments. In summary, this engineered microalgae-based therapeutic platform offers a promising translational strategy for delaying age-related intervertebral disc degeneration. (See table below.)

[0124] Table 1: Therapeutic effects in animal models

[0125] Model Evaluation indicators Treatment effect rats DHI recovery rate The value increased from 0.42±0.05 in the IDD group to 0.68±0.03. goat SASP gene (IL-6 / P16) expression Downgrade by 50-60% (RT-PCR) Histological score (Safranin O staining) The score dropped from 3.2 (IDD) to 1.5 (Treatment).

[0126] It should be noted that all experimental procedures in the above experimental examples strictly followed ethical guidelines and obtained ethical approval from the Institutional Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China, No. 4644) and the Ethics Committee of Wuhan Deyuan Biotechnology Co., Ltd. (DYSW214001).

[0127] Statistical analyses were performed using GraphPad Prism 10.1.2 (GraphPad software) and Origin 2024 (OriginLab). Data are expressed as mean ± standard deviation (SD) and are based on at least three independent experiments. Student's t-test, one-way ANOVA, or two-way ANOVA were used for comparisons, depending on the dataset type. Significance levels were defined as: P < 0.05, P < 0.01, *P < 0.001, and *P < 0.0001.

Claims

1. A method for preparing an engineered microalgae SP@V4C3 gel platform, characterized in that, Includes the following steps: S1: Preparation of V4C3 MXene nanosheets: V4AlC3 precursor was obtained by sintering vanadium powder, aluminum powder and carbon powder in stoichiometric ratio. After selective etching with hydrofluoric acid, single-layer V4C3 nanosheets were obtained by TPAOH intercalation and exfoliation. S2: Construction of V4C3-CS nanospheres: V4C3 nanosheets were ultrasonically compounded with chitosan solution at pH=5.0, and surface-modified V4C3-CS nanospheres were obtained after centrifugation and purification. S3: Preparation of SP@V4C3 engineered microalgae: V4C3-CS nanosphere dispersion and Spirulina suspension were mixed at a volume ratio of 1:1 and loaded onto the surface of microalgae through electrostatic adsorption. S4: Synthetic DNA hydrogel: Using salmon sperm DNA as raw material, it is cross-linked with PEGDA under alkaline conditions to form a three-dimensional network structure; S5: Assemble the SP@V4C3 gel platform: Infiltrate the SP@V4C3 dispersion into the DNA hydrogel to form a composite material with antioxidant function.

2. The method for preparing an engineered microalgae SP@V4C3 gel platform according to claim 1, characterized in that, In step S1: The sintering conditions were: reaction at 1500℃ for 2 hours under argon protection; The etching process involves treating the sample with a 40% hydrofluoric acid solution at 40°C for 7 days. The peeling process involved ultrasonic treatment with a 25 wt% TPAOH solution for 2 hours.

3. The method for preparing an engineered microalgae SP@V4C3 gel platform according to claim 1, characterized in that, In step S2: Chitosan has a molecular weight of 50 kDa and a degree of deacetylation of ≥85%. The ultrasonic processing parameters are 200W power, 5s working time / 5s interval, and a total duration of 30 minutes.

4. The method for preparing an engineered microalgae SP@V4C3 gel platform according to claim 1, characterized in that, In step S3, the loading of Spirulina was 50 μg microalgae / mL dispersion, and V4C3-CS was bound to the surface of Spirulina through electrostatic interaction.

5. The method for preparing an engineered microalgae SP@V4C3 gel platform according to claim 1, characterized in that, The preparation conditions for the DNA hydrogel in step S4 include: The DNA concentration was 12 mg / mL; The amount of cross-linking agent PEGDA added is 10% of the DNA mass; The gel formation conditions were 37°C incubation for 1 hour.

6. An SP@V4C3 gel platform prepared by any one of claims 1-5, characterized in that: The structure, comprising V4C3 MXene nanosheets, a chitosan-modified layer, a spirulina carrier, and a DNA hydrogel matrix, has the following performance parameters: storage modulus G' = 1250 ± 85 Pa, and swelling ratio 18.3 ± 1.

2.

7. The SP@V4C3 gel platform according to claim 6, characterized in that... It possesses multiple biological activities: ABTS + • Free radical scavenging rate ≥70%; SOD-like activity inhibition rate ≥60%; It can restore the mitochondrial membrane potential of cells treated with H2O2 to more than 80% of that of the control group.

8. The application of the SP@V4C3 gel platform according to claims 6-7 in the preparation of therapeutic drugs for intervertebral disc degeneration, characterized in that: It operates through the following mechanisms: (a) Clearing reactive oxygen species from intervertebral disc tissue; (b) Inhibits the expression of IL-1β and TNF-α inflammatory factors; (c) Downregulate P16 and P21 aging markers; (d) Promotes the synthesis and metabolism of type II collagen and glycans.

9. The application of the SP@V4C3 gel platform according to claim 8 in the preparation of drugs for treating intervertebral disc degeneration, characterized in that... The treatment includes: A suspension containing SP@V4C3 gel was injected locally into the degenerated intervertebral disc; Alternatively, a pre-formed SP@V4C3-DNA composite gel scaffold can be implanted.

10. The application of the SP@V4C3 gel platform according to claim 8 in the preparation of therapeutic drugs for intervertebral disc degeneration, characterized in that: Include: SP@V4C3 gel platform; Autologous platelet concentrate; The SP@V4C3 gel platform is precisely injected into the nucleus pulposus region of the intervertebral disc using a medical delivery device.

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