Ultrasonic-activated iron-doped barium titanate-based piezoelectric Fenton microspheres and their preparation and application

Abstract

This invention discloses an ultrasound-activated iron-doped barium titanate-based piezoelectric Fenton microsphere, its preparation and application methods, and its applications, belonging to the field of biomedical technology. This invention prepares iron-doped barium titanate nanoparticles via a hydrothermal method, which are then coated with phenylboronic acid lipids and loaded onto microfluidically molded gelatin microspheres via click chemistry to obtain PF@MS. Ultrasound triggers the material to generate a piezoelectric potential, driving a Fenton-like reaction that consumes local protons and moderately upregulates the mitochondrial membrane potential to initiate mitophagy. The reaction exhibits a self-limiting characteristic that weakens with increasing local pH, preventing excessive mitochondrial damage. Boronate modification on the nanoparticle surface can enhance intracellular uptake in energy-deficient degenerated nucleus pulposus cells and facilitate lysosomal release. The iron-doped barium titanate-based piezoelectric Fenton microspheres of this invention can restore mitophagy levels, balance extracellular matrix metabolism in intervertebral discs, and improve the retention rate of degenerated intervertebral disc height, making them suitable for the treatment of degenerative diseases such as intervertebral disc degeneration and mitochondrial dysfunction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials and regenerative medicine technology, specifically relating to an ultrasound-activated iron-doped barium titanate-based piezoelectric Fenton microsphere, its preparation method and application, and its application in the repair of intervertebral disc degenerative diseases related to mitochondrial dysfunction. Background Technology

[0002] Intervertebral disc degeneration (IDD) is a common degenerative disease of the spine, which can induce neck and back pain, limited spinal mobility, and impose a heavy medical burden. Disruption of nucleus pulposus (NP) cell homeostasis is the core pathogenic factor of IDD, with mitochondrial dysfunction being a key pathological feature. Current research only attributes mitochondrial damage to ATP depletion. Clinical single-cell transcriptome data confirm that mitochondria in degenerated NP cells maintain a partial depolarization potential range of -60 to -100 mV. This range neither initiates apoptosis nor activates PINK1-Parkin-mediated mitophagy. The continuous accumulation of damaged mitochondria continuously activates the NF-κB inflammatory pathway, inhibits type II collagen synthesis, and exacerbates the degradation of the intervertebral disc matrix.

[0003] Existing methods for mitochondrial regulation have significant shortcomings: chemical uncoupling agents and optogenetic regulation can easily lead to excessive mitochondrial polarization or apoptosis; in vitro microcurrent stimulation lacks a microenvironmental feedback regulation mechanism, making it impossible to precisely regulate to the appropriate potential range for mitochondrial autophagy; simultaneously, existing delivery vectors are difficult to adapt to the ATP-deficient microenvironment of degenerated cells, resulting in low intracellular delivery efficiency. Currently, no material system can simultaneously meet the four major requirements of potential range regulation, microenvironmental feedback limiting excessive response, efficient targeted delivery to diseased cells, and adaptation to the acidic oxidative microenvironment of the intervertebral disc.

[0004] Existing technologies mostly employ open-loop stimulation, lacking mitochondrial regulatory materials with environmental feedback regulation capabilities. Therefore, developing multi-level microspheres based on single-cell pathological data, possessing targeted delivery, piezoelectric catalysis, and pH-responsive self-regulation characteristics to overcome the shortcomings of existing technologies, has become an urgent technical problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing mitochondrial regulation materials, such as insufficient regulatory precision, lack of environmental feedback mechanisms, low uptake efficiency in degenerated cells, and susceptibility to excessive mitochondrial damage, this invention provides an ultrasound-activated iron-doped barium titanate piezoelectric Fenton microsphere, its preparation method, and its applications. This microsphere can generate a piezoelectric potential under ultrasound triggering, driving a Fenton cycle to consume local protons and adjusting the mitochondrial membrane potential to the physiological range for activating mitophagy. Simultaneously, it relies on a local pH increase to limit the continuous catalytic reaction, avoiding excessive mitochondrial polarization. Surface modification with phenylboronic acid can enhance the intracellular uptake efficiency of ATP-deficient degenerated cells, enabling intralysozyme drug release for the repair of mitochondrial-related degenerative diseases such as intervertebral disc degeneration.

[0006] 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 ultrasonically activated iron-doped barium titanate-based piezoelectric Fenton microspheres, comprising the following steps: (1) Iron-doped barium titanate nanoparticles were synthesized by hydrothermal reaction using a mixed solution of Ba(OH)2・8H2O, ammonium lactate, and FeCl3. (2) Phospholipids, cholesterol, DSPE-PEG2000-PBA and iron-doped barium titanate nanoparticles obtained in step (1) were mixed in a solvent and coated with phenylboronic acid functionalized lipid shell by thin film method to prepare phenylboronic acid functionalized core-shell lipid nanoparticles. (3) Using methacrylamide gelatin hydrogel, tannic acid and photoinitiator as the aqueous phase, and isopropyl myristate and Span 80 as the oil phase, hydrogel microspheres were prepared by microfluidic method. Then, the phenylboronic acid functionalized core-shell lipid nanoparticles obtained in step (2) were embedded in the hydrogel microspheres by click chemistry to obtain ultrasonically activated iron-doped barium titanate piezoelectric Fenton microspheres.

[0007] The inventors constructed ultrasound-activated (US) iron-doped barium titanate (Fe-BTO)-based piezoelectric-Fenton microspheres (PF@MS) using the above method. Ultrasound triggers the generation of a piezoelectric potential in Fe-BTO, overcoming the Fenton catalysis bottleneck (Fe...). 3+ / Fe 2+The process involves a cycle (pH elevation) that upregulates MMPs from pathological to physiological levels (−100 ~ −140 mV) by consuming local protons. This process is self-limiting: pH elevation acts as a feedback switch to prevent excessive mitochondrial polarization. To address impaired endocytosis in energy-depleted cells, we designed a "Trojan horse" delivery strategy based on dynamic borate ester bonds (BEBs) to target sialic acid residues and achieve pH-responsive lysosomal escape. This strategy bypasses energy-dependent pathways, ensuring high intracytoplasmic bioavailability. This deterministic MMP regulation restored mitophagic flux from 8% to 35% in vivo and salvaged 89% of intervertebral disc height. This study establishes a mechanically gated metabolic reprogramming framework, enabling a shift from randomized intervention to deterministic, tissue-level energy homeostasis.

[0008] This invention combines materials science with single-cell pathology to construct piezoelectric-Fenton microspheres (PF@MS) as wireless bioelectric reset switches. A mechanochemical feedback loop is established by embedding iron-doped piezoelectric BTO nanoparticles (Fe-BTO) in a responsive matrix. Upon ultrasonic activation, the piezoelectric potential is transferred through Fe... 2+ / Fe 3+ This invention drives a local Fenton response. Unlike traditional uncoupling agents, this invention is designed to act as a metabolic thermostat: it precisely regulates the proton gradient by consuming local protons, thereby "recharging" and restoring MMPs. The key lies in the self-limiting nature of this process: when MMPs enter the therapeutic window (−100 ~ −140 mV), the altered electrochemical potential naturally forms a barrier to further proton consumption, similar to a charger switching to trickle mode, preventing overcharging and ensuring safety.

[0009] To adapt to the harsh metabolic microenvironment of IDD (Independent Disorders of Development), the inventors equipped PF@MS with a "Trojan horse" surface chemistry. Utilizing dynamic phenylboronic acid ester bonds, the microspheres first adhere to cell membrane glycoproteins in a "Velcro" manner to maximize uptake, and then switch to "Trojan horse" mode through ROS / pH-responsive depolymerization, enabling lysosomal escape and targeting of mitochondria. This study confirms that through the synergy of molecular surface engineering and intracellular bioelectronic regulation, PF@MS can effectively perform precise mitochondrial surgery. This strategy bypasses metabolic barriers, definitively restores mitophagy (8%→35%), inhibits matrix degradation, and preserves intervertebral disc height, providing a generalizable material logic for treating degenerative diseases through organelle-level energy editing.

[0010] Furthermore, the molar ratio of Ba(OH)2・8H2O, ammonium lactate, and FeCl3 in step (1) is 20:20:1.

[0011] Furthermore, the hydrothermal reaction conditions described in step (1) are a hydrothermal reaction at 200 °C for 8 h.

[0012] Furthermore, the product of the hydrothermal reaction in step (1) is collected and precipitated by centrifugation, washed and vacuum dried to obtain iron-doped barium titanate nanoparticles.

[0013] Furthermore, in step (2), the weight ratio of phospholipids, cholesterol, DSPE-PEG2000-PBA and iron-doped barium titanate nanoparticles is 120:40:1:18.

[0014] Furthermore, the solvent mentioned in step (2) is chloroform.

[0015] Furthermore, in step (3), the weight ratio of methacrylamide gelatin hydrogel, tannic acid and photoinitiator is 7:1:0.5.

[0016] Furthermore, the click chemistry operation in step (3) is as follows: the lyophilized methacrylamide gelatin microspheres are reconstituted with borate-buffered saline, and then a lipid-coated iron-doped barium titanate nanoparticle suspension prepared with the same buffer solution is added. After vortexing and standing, borate ester click coupling occurs, and the free nanoparticles are removed by washing with PBS.

[0017] The second objective of this invention is to provide ultrasonically activated iron-doped barium titanate-based piezoelectric Fenton microspheres prepared by the method described above.

[0018] A third objective of this invention is to provide the application of the ultrasound-activated iron-doped barium titanate-based piezoelectric Fenton microspheres, as described above, in the preparation of drugs for treating mitochondrial degenerative diseases.

[0019] The mechanism of the material of the present invention is described as follows: Ultrasonic stimulation of Fe-BTO nanoparticles generates piezoelectric potential, promoting the Fe³⁺ / Fe²⁺ cycle, catalyzing the local Fenton reaction to consume extracellular protons, and upregulating the mitochondrial membrane potential to the −100 ~ −140 mV range. This potential range can effectively activate the PINK1-Parkin mitophagy pathway. As protons are continuously consumed, the local pH increases, the piezoelectric catalytic activity decreases, forming an environmental feedback regulation effect, inhibiting the excessive polarization damage to mitochondria caused by continuous proton consumption.

[0020] This invention modifies the surface of nanoparticles with phenylboronic acid groups, which can specifically bind to sialic acid groups on the surface of nucleus pulposus cells, enhancing the intracellular binding and uptake capacity of ATP-deficient degenerated cells. After the nanoparticles enter lysosomes via endocytosis, the boronic acid ester bonds are hydrolyzed under acidic conditions, resulting in lysosomal release and improving mitochondrial targeted delivery efficiency. In vivo experiments have demonstrated that these microspheres can enhance mitophagy levels, improve intervertebral disc matrix synthesis and metabolism, increase the proportion of intervertebral disc structure preserved, and alleviate degeneration progression.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention successfully constructed a piezoelectric-Fenton microsphere (PF@MS), which is not only a drug carrier, but also a wireless, self-limiting mitochondrial maintenance "smart charger". By establishing an ultrasound-driven mechanochemical feedback loop, PF@MS deterministically resets MMPs to the critical window required for PINK1-Parkin mitochondrial autophagy, severing the upstream link between energy metabolism failure and matrix inflammation, and successfully pioneering a bioelectronic organelle editing paradigm to reverse IDD.

[0022] (2) The material of the present invention has pH-dependent reaction regulation characteristics: the ultrasonic-induced piezoelectric Fenton reaction consumes local protons, and the catalytic effect is spontaneously weakened after the pH rises, which can avoid excessive polarization and oxidative damage to mitochondria and achieve interval controllable regulation of mitochondrial membrane potential. (3) The present invention realizes the integrated construction of piezoelectric catalytic unit, targeted lipid shell and injectable hydrogel microsphere carrier. Existing piezoelectric catalytic materials, simple lipid carriers and ordinary hydrogels cannot achieve this multiple synergistic function. (4) This invention enhances the uptake of nanoparticles by energy-deficient degenerated cells through the interaction of phenylboronic acid and sialic acid, thereby achieving lysosomal release and increasing the concentration of effective drugs in the cytoplasm; (5) At the cellular level, this invention can increase the expression of mitophagy-related markers from 8% to 35%; in a rat intervertebral disc degeneration model, it can significantly increase the proportion of intervertebral disc height retention, inhibit matrix metalloproteinase expression, promote type II collagen deposition, and alleviate intervertebral disc degeneration. (6) The present invention provides an injectable hydrogel microsphere that can be used for local drug delivery. It has good biocompatibility and no obvious short-term organ toxicity. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the self-limiting piezoelectric-Fenton microspheres of the present invention used for ultrasound-tunable mitochondrial membrane potential regulation; (A) Human nucleus pulposus single-cell transcriptome analysis results confirm that mitochondrial membrane potential imbalance is a core pathogenic factor in intervertebral disc degeneration; (B) Complete preparation process of PF@MS microspheres: Fe-BTO nanoparticles are prepared by hydrothermal method, and phenylboronic acid modified LFB@NPs are prepared by thin film method; blank GelMA microspheres are prepared by microfluidic control, and PF@MS is obtained by click chemical loading of nanoparticles; (C) Mechanism of action in vivo after local injection into the intervertebral disc: LFB@NPs are released by microspheres, which bind to nucleus pulposus cells through phenylboronic acid-mediated binding, and are released by lysosomes after endocytosis; ultrasound triggers the piezoelectric Fenton reaction, and mitochondrial membrane potential range regulation is achieved by relying on pH feedback, activating PINK1-Parkin mitophagy, and inhibiting inflammation and matrix degradation.

[0024] Figure 2To verify that mitochondrial autophagy deficiency is a core pathway in intervertebral disc degeneration using single-cell transcriptome and tissue samples; (A) Single-cell sequencing experimental procedure for human nucleus pulposus samples; (B) UMAP clustering diagram of nucleus pulposus cells in healthy individuals and degenerated patients; (C) Statistical analysis of cell proportions in the two groups, showing a significant reduction in functional nucleus pulposus cells in the degenerated group; (D, E) Enrichment of differentially expressed genes GO and KEGG, indicating downregulation of mitochondrial function-related pathways and upregulation of inflammatory pathways in degenerated samples; (F) Correlation analysis of mitochondrial function score with matrix degradation and inflammation score; (G) GSEA enrichment analysis, enrichment of mitochondrial stress and NF-κB inflammation pathways in degenerated samples, and downregulation of collagen synthesis pathway; (H) MRI, histology, and immunofluorescence verification of human nucleus pulposus of different degeneration grades and rat degenerated intervertebral disc tissues; (I) Quantitative statistics of fluorescence related to COL2, MMP13, Park2, and LC3; (J) Fluorescence intensity curves of Parkin and LC3 along cell profiles; (K) Western blot quantification of mitochondrial-related proteins in rat nucleus pulposus tissue; (L) JC-1 staining and TEM observation of mitochondrial morphology to distinguish normal / damaged / mitochondrial autophagy vesicles; (M) Quantification of mitochondrial membrane potential JC-1 red-green ratio in human and rat samples; (N) Correlation between mitochondrial membrane potential level, Parkin recruitment, and apoptosis to determine the safe therapeutic potential range.

[0025] Figure 3 Material characterization of core nanounits and composite microspheres; (A) Schematic diagram of LFB@NPs core-shell structure synthesis; (B, C) TEM morphology of bare Fe-BTO and LFB@NPs; (D, E) Elemental EDS mapping of LFB@NPs; (F–I) PFM characterization of LFB@NPs (morphology, d33 coefficient, butterfly curve, phase hysteresis loop); (J) 48 h stability DLS particle size detection; (K) Schematic diagram of PF@MS microsphere assembly principle; (L) SEM images of blank M@MS and PF@MS microspheres; (M) XRD crystal structure pattern; (N) Elemental EDS spectrum of PF@MS; (O) Statistical distribution of microsphere size.

[0026] Figure 4Characterization of PF@MS cell uptake, response release, and pH regulation; (A) Schematic diagram of intracellular interaction of nanoparticles; (B) Fluorescence map of colocalization of LFB@NPs with mitochondria, lysosomes, and endoplasmic reticulum; (C) Fluorescence intensity distribution curve; (D, E) Quantitative analysis of cell uptake after oligomycin ATP depletion treatment by flow cytometry, verifying that phenylboronic acid modification enhances uptake in low-energy environments; (F) Morphology of microspheres after 25 days of degradation in an acidic enzyme-containing microenvironment; (G) pH change curve during the Fenton reaction, confirming that pH increase brings about reaction regulation effect; (H) Cumulative release curve of nanoparticles with and without ultrasonic stimulation; (I) Comparison of microsphere mass loss under PBS and H2O2 environments.

[0027] Figure 5 PF@MS utilizes pH feedback to regulate mitochondrial membrane potential, activate selective mitophagy, and restore cellular energy metabolism; (A) Schematic diagram of in vitro cellular mechanism of action; (B–D) JC-1 fluorescence imaging, red-green ratio quantification, and flow cytometry detection of mitochondrial membrane potential; (E–G) Parkin / LC3-II co-localization immunofluorescence, Park2 and Map1lc3b mRNA quantification; (H) LC3 lipid autophagy marker fluorescence; (J, K) TEM mitochondrial morphology and mitophagic vesicle statistics; (L) Cellular ATP / ADP ratio; (M) Seahorse mitochondrial oxygen consumption rate OCR detection.

[0028] Figure 6 The transcriptome was used to confirm the metabolic phenotype of degenerated nucleus pulposus cells reprogrammed by PF@MS (ultrasound); (A) RNA-seq PCA analysis of treatment / control groups; (B) Volcano plot of differentially expressed genes; (C) Interaction arc plot of key pathways; (D, E) GO / KEGG and GSEA enrichment analysis; (F, G) Immunofluorescence of MMP13 and COL2; (H, J) qPCR quantification of matrix metabolism-related genes; (K) Transcriptome sequencing experimental design.

[0029] Figure 7 The study aimed to evaluate the in vivo treatment of a rat model of intervertebral disc degeneration using imaging and histology. (A) Timeline of animal modeling, drug administration, and detection; (B) T2-MRI images of the intervertebral disc 8 weeks post-surgery; (C) H&E and Safranin O Fast Green tissue staining; (D) Quantitative analysis of the intervertebral disc height index (DHI); (E) Histological degeneration score; (F) Nucleus pulposus water content detection; and (G) Comparison of treatment effects among different groups using multi-index clustering heatmaps.

[0030] Figure 8To verify in vivo tissue immunofluorescence that PF@MS can activate the mitophagy pathway; (A, C) Parkin / LC3-II colocalization fluorescence of intervertebral disc at 4 and 8 weeks postoperatively; (B, D) Corresponding fluorescence intensity distribution; (E, F) Parkin mean optical density statistics; (G, H) LC3-II mean optical density statistics; (I) Schematic diagram of key genes in the mitophagy KEGG pathway.

[0031] Figure 9 Histological verification of matrix metabolic balance in vivo; (A, B) Immunohistochemistry of MMP13 at 4 and 8 weeks; (C, D) Immunohistochemistry of type II collagen COL2 at 4 and 8 weeks; (E, F) Quantification of average optical density of MMP13 and COL2; (G) Schematic diagram of complete repair mechanism pathway in vivo. Detailed Implementation

[0032] 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.

[0033] Example 1

[0034] (I) Experimental Materials and Methods 1. Experimental materials The following experimental materials were all commercially available: barium hydroxide octahydrate, titanium ammonium lactate, ferric chloride; L-α-phosphatidylcholine, cholesterol, DSPE-PEG2000-PBA, gelatin, methacrylic anhydride, tannic acid, photoinitiator LAP, tert-butyl hydroperoxide (TBHP), hyaluronidase, MMP-13, rat primary nucleus pulposus cells, DMEM / F-12 medium, fetal bovine serum, antibodies: COL2, TOMM20, MMP13, Parkin, LC3B, GAPDH, main reagents and kits: JC-1, DCFH-DA, TMRM, Live / Dead staining, CCK-8, TRIzol, RT-qPCR kit.

[0035] 2. Preparation of Fe-BTO@NPs (hydrothermal method) 1) Dissolve 1 mmol Ba(OH)2・8H2O, 1 mmol titanium ammonium lactate, and 0.05 mmol FeCl3 in 30 mL of deionized water; 2) Transfer to a 50 mL polytetrafluoroethylene reactor and hydrothermally heat at 200 ℃ for 8 h; 3) Allow to cool naturally, then centrifuge at 8000×g for 10 min and collect the precipitate; 4) Wash 3 times with deionized water and 2 times with anhydrous ethanol; 5) Vacuum drying at 60 °C overnight yielded Fe-BTO nanoparticles.

[0036] 3. Preparation of LFB@NPs (thin film method) 1) Dissolve phospholipids, cholesterol, DSPE-PEG2000-PBA, and Fe-BTO NPs in chloroform; 2) Rotary evaporation removes the organic solvent, forming a uniform lipid-nanoparticle film; 3) Hydrate with deionized water for 20 min, then extrude 31 times using a 200 nm polycarbonate membrane; 4) Benzylboronic acid-functionalized core-shell lipid nanoparticles LFB@NPs were obtained.

[0037] 4. Preparation of GelMA 1) Dissolve 10 g of type A gelatin in 100 mL of pH 9.0 carbonate buffer solution and stir at 50 °C to dissolve; 2) Add 8 mL of methacrylic anhydride dropwise and react for 3 h; 3) Dilute with PBS 5 times and dialyze for 7 days (MWCO 12-14 kDa); 4) Freeze-dry to obtain GelMA sponge and store at -20 ℃.

[0038] 5. Preparation of PF@MS microspheres (microfluidics + click chemistry) 1) Preparation of aqueous phase: GelMA (7%), tannic acid (1%), and LAP (0.5%) are dissolved in water; 2) Oil phase: Isopropyl myristate + 10% Span 80; 3) Microfluidic focusing into droplets, followed by UV cross-linking to obtain M@MS microspheres; 4) After rehydration of M@MS, LFB@NPs were added, and borate ester click assembly was performed; the specific operation was as follows: 1 mg of lyophilized methacrylamide gelatin microspheres were reconstituted in 1 mL of pH 8.5, 100 mM borate buffered saline for 5 min, and 200 μL of lipid-coated iron-doped barium titanate nanoparticle suspension prepared in the same buffer was added. After vortexing for 10 s, the mixture was allowed to stand at 25 ℃ for 3 min to achieve borate ester click coupling. 5) Wash with PBS and freeze-dry to obtain PF@MS.

[0039] 6. Cell Culture and Injury Models 1) Rat primary nucleus pulposus cells were cultured in DMEM / F-12 + 10% FBS, and passaged ≤3 times; 2) Treat with 100 μM TBHP for 24 h to establish an oxidative stress mitochondrial damage model.

[0040] 7. Material Characterization Methods l1) TEM / STEM-EDS: morphology and elemental distribution.

[0041] l2) PFM: piezoelectric coefficient d33, butterfly curve, phase hysteresis loop.

[0042] l3) DLS: Hydrated particle size and stability.

[0043] l4) SEM: Microsphere morphology.

[0044] l5) XRD / XPS: Crystal phase and Fe valence state changes.

[0045] l6) ICP-MS: Iron ion leaching amount.

[0046] 8. In vitro functional experiments 1) Cellular uptake and endocytosis mechanisms: 4 ℃ / 37 ℃ comparison, endocytosis inhibitors, and sialic acid competition experiments.

[0047] 2) Lysosomal escape and mitochondrial colocalization: Dio labeling + Tom20 / LAMP1 / Calnexin immunofluorescence colocalization.

[0048] 3) Ultrasound / ROS triggered release: dialysis bag method, ANTS / DPX fluorescence tracer, calculation of cumulative release.

[0049] 4) Self-limiting MMP regulation: JC-1 and TMRM staining, flow cytometry / confocal quantification of MMP.

[0050] 5) Mitochondrial autophagy: Parkin / LC3-II immunofluorescence, Western blot, TEM to observe autophagosomes, and BafA1 blocking experiment to verify autophagic flux.

[0051] 6) ROS clearance: DCFH-DA and MitoSOX were used to detect intracellular / mitochondrial ROS.

[0052] 7) Cell compatibility: Live / Dead and CCK-8 assays were used to detect cell viability at 1 / 3 / 5 days.

[0053] 8) Transcriptomics and qPCR; RNA-seq sequencing, GO / KEGG / GSEA analysis; qPCR detection of Col2a1, Mmp13, Prkn, Agrn, etc.

[0054] 9. Animal experiments (rat IDD model) 1) A 30 G needle was used to puncture the Co5 / 6-Co8 / 9 coccygeal vertebrae to establish an IDD model; 2) Intradiscal injection of 2.5 μL microsphere suspension (5 mg / mL); 3) Ultrasonic activation: 0.5 W / cm², 1 MHz, 2 min each time; 4) Perform X-ray and MRI scans at 4 / 8 pm to calculate DHI and T2 signals; 5) Histology: H&E, Safranin O, Fast Green, Immunohistochemistry / Fluorescence; 6) In vivo safety: serum biochemistry, complete blood count, and HE staining of major organs.

[0055] 10. Statistical methods Data are expressed as mean ± SD; t-tests were used for comparisons between two groups, and one-way ANOVA with Tukey post-hoc test was used for comparisons between multiple groups; n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001.

[0056] (II) Experimental Results and Analysis 1. Damage to mitophagy is a core pathogenic factor in IDD. To elucidate the molecular mechanisms of IDD, we performed scRNA-seq on nucleus pulposus tissue from IDD patients and healthy controls. Unbiased clustering revealed different cell populations, with significant depletion of chondrocytes (the primary functional cells) in the IDD samples, confirming the loss of core cellular components in the tissue.

[0057] Differential gene expression analysis of chondrocyte populations pointed to mitochondrial dysfunction as a key event. GO / KEGG pathway enrichment revealed significant downregulation of mitochondrial function-related genes, such as oxidative phosphorylation and NADH dehydrogenase activity; simultaneously, inflammation-related pathways were upregulated. Key findings: Mitochondrial function score was strongly negatively correlated with the inflammation / extracellular matrix (ECM) degradation composite score (R=−0.80, P<0.001), suggesting that mitochondrial dysfunction is an upstream driver of the degenerative cascade. GSEA further confirmed that anabolic pathways (such as type II collagen) were significantly inhibited in IDD, while mitochondrial stress (HSPD1) and pro-inflammatory NF-κB signaling pathways were activated.

[0058] To validate the transcriptome results, we examined human NP tissues and a rat IDD model at different degeneration levels. Degenerated NP tissues were obtained via unilateral double-channel endoscopy (UBE). Microscopic assessment confirmed significant morphological differences between grade II and grade IV samples. Histological and quantitative analyses showed that grade IV tissues had significantly lower cell viability, concentration, and nucleation rate than the grade II control. Consistent with gene expression results, histological and immunofluorescence findings indicated a shift from anabolism to catabolism during the degeneration process, with protein levels showing a decrease in type II collagen and an increase in matrix metalloproteinase 13 (MMP13).

[0059] Given the central role of mitochondrial dysfunction, we specifically investigated mitophagy, a key quality control mechanism in mitochondria. In healthy rats and Grade II human tissues, the mitophagy initiator Parkin co-localized strongly with the mitochondrial marker LC3-II, indicating active mitophagy. However, in degenerated samples (rat IDD, human Grade IV), this co-localization significantly disappeared, and Parkin diffused into the cytoplasm. This blockade was quantitatively confirmed by a significant decrease in the Parkin-LC3-II Pearson correlation coefficient. Impaired mitophagy leads to severe mitochondrial health consequences. Western blot analysis of rat NP tissue protein levels showed that COL2 and Parkin were significantly downregulated in IDD samples, while MMP13 and the LC3-II / I ratio were significantly increased. These results confirm that the absence of Parkin-mediated mitophagy and catabolism phenotypic shift are core characteristics of NP cell degeneration. JC-1 staining revealed widespread loss of MMPs, and transmission electron microscopy (TEM) confirmed the accumulation of swollen, dysfunctional mitochondria and sparse mitophagosomes in degenerated cells.

[0060] Finally, based on the above findings, we determined the therapeutic window for mitochondrial intervention. Through correlation analysis between MMP recovery levels (JC-1 ratio) and mitophagy reactivation (Parkin recruitment) and apoptosis (Caspase-3 activity), we determined the optimal therapeutic range: a JC-1 ratio of 1.4–1.6 maximizes Parkin recruitment and minimizes apoptosis, providing a clear target for treatment design. Figure 2 ).

[0061] 2. Design and characterization of piezoelectric-Fenton-like microspheres To construct a therapeutic system suitable for the complex intervertebral disc microenvironment, we designed a multi-level delivery system based on self-limiting piezoelectric-Fenton nanocomposites. The design comprises three key steps: piezoelectric core synthesis, functionalized lipid shell encapsulation, and embedding in responsive hydrogel microspheres. First, iron-doped barium titanate nanoparticles (Fe-BTO@NPs) were synthesized as the piezoelectric core. TEM revealed a tetragonal crystal form with an average particle size of approximately 180 nm. Piezoelectric power microscopy (PFM) confirmed the piezoelectric properties, and the piezoelectric coefficient d... 33The potential reached 11.0 pm / V. Theoretical analysis showed that a potential of ~11 mV could be generated under 1 MPa mechanical stress, which was sufficient to drive the Fe²⁺ / Fe³⁺ electron transfer to initiate the Fenton reaction. To improve biocompatibility and colloidal stability, a thin-film method was used to functionalize the naked Fe-BTO@NPs with a phenylboronic acid-coated lipid shell, resulting in LFB@NPs. TEM confirmed a uniform core-shell structure with a final particle size of ~200 nm. EDS verified the success of the lipid encapsulation: the phosphorus (lipid) and the Ba, Ti, and Fe signals of the core were co-localized. Importantly, PFM showed that LFB@NPs retained strong piezoelectric function, exhibiting a characteristic butterfly amplitude curve and a 180° phase shift hysteresis loop. Dynamic light scattering (DLS) confirmed the excellent colloidal stability of LFB@NPs: the hydrated particle size remained stable at ~120 nm within 48 h, while the naked Fe-BTO@NPs significantly aggregated.

[0062] Finally, to achieve long-term local delivery within the intervertebral disc, microfluidic technology was used to embed LFB@NPs into methacrylamide gelatin (GelMA) hydrogel microspheres (PF@MS). Scanning electron microscopy (SEM) showed that PF@MS were spherical with an average diameter of 197±12 μm, comparable to blank M@MS microspheres (202±15 μm). The surface of PF@MS was smoother than that of porous M@MS, indicating successful nanoparticle loading. XRD showed characteristic BTO crystal peaks in PF@MS, and EDS further confirmed the presence of LFB@NPs in the hydrogel matrix. Figure 3 ).

[0063] 3. Intracellular distribution and responsive function of PF@MS To achieve therapeutic effects, PF@MS must successfully enter target cells and reach the mitochondria. Confocal microscopy revealed that Dio-labeled nanoparticles and the mitochondrial marker Tom20 were highly co-localized, further confirmed by overlapping intensity curves. Flow cytometry quantitatively verified that both groups exhibited strong fluorescence under basal conditions, indicating efficient uptake. However, after oligomycin-induced ATP depletion, the fluorescence intensity of the Fe-BTO@NPs group decreased sharply and the peak shifted to the left, suggesting highly energy-dependent endocytosis. In contrast, the BLF@NPs group showed a significantly smaller decrease in fluorescence intensity after oligomycin treatment. Quantitative analysis confirmed that BLF@NPs had a higher uptake and retention rate under energy-limited conditions, suggesting that they may utilize non-energy-dependent pathways or enhance membrane affinity to ensure efficient delivery in energy-deprived pathological microenvironments.

[0064] The long-term efficacy of the system depends on the stability and responsive degradation of the PF@MS microsphere carrier. To simulate the microenvironment of degenerated intervertebral discs, PF@MS was incubated in acidic PBS (pH 6.5) containing MMP13 / hyaluronidase. Optical microscopy showed gradual morphological erosion over 25 days: initially intact microspheres gradually collapsed and decreased in density, eventually degrading into very few fragments after 25 days, confirming responsive biodegradability under pathological conditions. The key safety feature of this design is the self-limiting nature of the Fe-BTO-mediated Fenton-like reaction, preventing damage caused by excessive ROS. In vitro H⁺ consumption experiments confirmed that the Fe-BTO component rapidly stabilized the solution pH after an increase, and the reaction terminated autonomously; while free iron ions (FeCl3) caused a continuous and uncontrollable increase in pH, highlighting the crucial role of the BTO framework in ensuring a safe therapeutic window.

[0065] Furthermore, we investigated the on-demand release of nanoparticles within the microspheres. Accumulated release was significantly accelerated in an oxidizing environment (H2O2, simulating pathology); ultrasound (US) further amplified the release, confirming the system's excellent ultrasound-triggered, on-demand release capability. Quantitative validation through mass loss studies showed that PF@MS degraded significantly faster in H2O2 than in PBS, ensuring precise release of the therapeutic payload at the most needed time and location.

[0066] In vitro systemic evaluation of the biocompatibility of the synthetic materials. Live / dead staining showed no significant abnormalities in cell morphology and survival after 24 h of culture in the M@MS and PF@MS groups, confirming excellent cell compatibility. Long-term CCK-8 quantitative effect: at days 1, 3, and 5, cell viability in the M@MS and PF@MS groups was not significantly different from the control group. Simultaneous evaluation of biocompatibility under treatment-triggered conditions: even with ultrasound application, cell viability in the M@MS and PF@MS groups was unaffected, confirming the safety of the combined treatment. Figure 4 ).

[0067] 4. PF@MS restores mitochondrial homeostasis and cellular energy metabolism by self-limiting regulation of MMPs and promoting selective mitophagy. To investigate the therapeutic mechanism of PF@MS, we constructed a mitochondrial damage model using tert-butyl hydroperoxide (TBHP). Severe mitochondrial damage leads to MMP collapse, which in turn weakens the cell's ability to clear damaged organelles. We hypothesized that PF@MS could partially restore MMPs to functional levels, sufficient to activate selective mitophagy. The JC-1 fluorescent probe confirmed that TBHP treatment led to a significant decrease in MMPs, with the red / green fluorescence ratio dropping to approximately 0.5; after sonication activation, PF@MS treatment successfully restored the ratio to 1.3, suggesting that MMPs can be partially and controllably restored, rather than undergoing complete overpolarization.

[0068] This regulatory MMP restoration was sufficient to activate the classical mitophagy signaling pathway. Following PF@MS(US) treatment, Park2 (encoding Parkin) mRNA expression was significantly upregulated by 4.8-fold. Immunofluorescence showed significant recruitment of Parkin protein to mitochondria (LC3-II marker), with enhanced co-localization of green (Parkin) and red (LC3-II) signals. Simultaneously, the elevated mRNA levels of the post-damage mitochondrial quality marker LC3-II were significantly reduced after treatment, suggesting the clearance of excess / damaged mitochondria.

[0069] Monitoring LC3B lipidation further confirmed the initiation of mitophagy. LC3B spots (LC3B-II on autophagosomes) accumulated in TBHP-damaged cells, disappearing after PF@MS treatment, indicating the completion of autophagic flux. TEM provided direct visual evidence: PF@MS(US) treatment reduced swollen and abnormal mitochondria, increased mitophagic vesicles (mito-AVs), and elevated the proportion of normal, healthy mitochondria.

[0070] Finally, the targeted clearance of damaged mitochondria was assessed to determine whether it translated into improved cellular function. Results showed that PF@MS treatment effectively restored the cellular ATP / ADP ratio and rescued mitochondrial oxygen consumption (OCR, Seahorse assay) damaged by TBHP. Furthermore, DCHF-DA staining was used to assess the level of intracellular reactive oxygen species (ROS), a byproduct of mitochondrial dysfunction. Quantitative analysis showed that TBHP exposure induced significant oxidative stress, and PF@MS treatment under ultrasound irradiation effectively cleared excess ROS, reducing fluorescence intensity to near control levels. Figure 5 ).

[0071] 5. PF@MS treatment reprograms the transcriptome of nucleus pulposus cells, transforming them into an anabolic and anti-catabolic state. To elucidate the molecular mechanism of PF@MS therapeutic effects, whole-transcriptome RNA sequencing (RNA-seq) was performed on rat nucleus pulposus cells after PF@MS treatment under ultrasound (US) stimulation for 24 h. PCA plots showed significant separation between the treatment group and the control group, with the first principal component (PC1) accounting for 89.3% of the total variance, suggesting that treatment induces robust and systematic transcriptional reprogramming.

[0072] Volcano plot analysis of differentially expressed genes (DEGs; |log2FC|>1, p<0.05) revealed a dual effect: key matrix metalloproteinases (Mmp13, Mmp12, Mmp3) were significantly downregulated, while collagen-encoding genes (Col2a1) were upregulated. Heatmap visualization clearly showed a shift: pro-inflammatory / catabolistic genes were downregulated in the treatment group, while pro-anabolistic / repair genes were upregulated. GSEA confirmed a significant downregulation of inflammation and catabolism pathways (e.g., "Inflammatory Response"), and a significant upregulation of ECM remodeling and mitophagy-related pathways (e.g., "Collagen Biosynthesis"). GO and KEGG enrichment analyses showed that differentially expressed genes were mainly enriched in macrophage activity and collagen metabolism pathways, suggesting that PF@MS primarily functions by regulating the inflammation-ECM remodeling network.

[0073] Transcriptome results were validated at protein and gene expression levels. Consistent with RNA-seq, immunofluorescence showed a significant decrease in MMP13 protein levels and a significant increase in COL2 deposition and structure in the PF@MS+US group. RT-qPCR confirmed significant downregulation of Mmp13 and significant upregulation of Col2a1; the expression of other key catabolic enzymes (Mmp10, Adamts4) was suppressed, and the core proteoglycan agglutinin (Agrn) was upregulated, further confirming the decisive shift towards ECM synthesis rather than degradation.

[0074] In summary, the multi-level analysis from transcriptomics to protein validation provides a clear mechanistic basis for the therapeutic effect of PF@MS in alleviating IDD. Figure 6 ).

[0075] 6. In vivo therapeutic effects of PF@MS in a rat IDD model To evaluate the in vivo efficacy of PF@MS, a rat caudal disc degeneration (IDD) model was established using acupuncture. Treatment protocol: local injection of microspheres followed by percutaneous ultrasound activation. The treatment effect was systematically evaluated by imaging and histology at 4 and 8 weeks post-surgery. T2-weighted magnetic resonance imaging (MRI) follow-up was the primary evidence for intervertebral disc structure preservation. At 8 weeks, the PF@MS(US) group maintained high T2 signal intensity, indicating adequate hydration and intact ECM, highly similar to healthy, non-acupunctured intervertebral discs; in contrast, the model control group showed significant signal loss (low signal), indicating severe dehydration and degeneration. Histological examination validated the MRI results at the cellular and tissue levels. H&E staining in the PF@MS(US) group showed well-preserved intervertebral disc structure, healthy NP cell count, and clear boundaries between NPs and the annulus fibrosus (AF); the model group showed severe structural disorder, accompanied by fibrosis and significant NP cell loss. Safranin O-Fixed Green staining showed proteoglycan content: abundant and uniformly distributed in the treatment group, indicating effective matrix synthesis; significantly reduced in the model group, confirming substantial matrix degradation.

[0076] Quantitative analysis enhanced morphological observation results. The key degenerative indicator, the disc height index (DHI), was significantly preserved in the PF@MS(US) group, maintaining an original height of 89.2±3.4% at 8 weeks, significantly better than the model control group (52.1±5.2%) and the non-ultrasound-activated microsphere group. Correspondingly, the histological score of the PF@MS(US) group was significantly lower, indicating a significant reversal of degenerative pathology. Moisture analysis further confirmed that the complete treatment group had significantly higher collagen preservation. Multivariate cluster analysis integrating key indicators showed unique characteristics of the PF@MS(US) group, confirming that the synergistic efficacy of combined treatment is an optimized strategy for reversing IDD. In summary, in vivo results confirm that ultrasound-activated PF@MS effectively alleviates IDD by preserving tissue structure, promoting matrix synthesis, and restoring disc height. This study highlights the key role of ultrasound-triggered therapy and provides crucial data support for clinical translation. Figure 7 ).

[0077] 7. PF@MS activates mitophagy and mitochondrial homeostasis in vivo. To elucidate the molecular mechanisms underlying the therapeutic effect in vivo, we investigated the activation of mitophagy, a key mitochondrial quality control pathway. Immunofluorescence analysis was performed on intervertebral disc tissue to assess the co-localization and expression of key biomarkers.

[0078] Consistent with mitophagy activation, the co-localization of Parkin (green, the key E3 ubiquitin ligase) and the autophagosome marker LC3-II (red) was significantly enhanced in the PF@MS(US) group. At 4 weeks post-surgery, immunofluorescence and quantitative intensity curves showed that the overlap between Parkin and LC3-II signals in the PF@MS(US) group was much higher than that in the model and control groups, indicating successful recruitment of autophagosomes to damaged mitochondria. This enhanced mitophagy trend persisted at 8 weeks, with the PF@MS(US) group maintaining excellent Parkin-LC3-II co-localization, confirming the long-term efficacy of ultrasound-activated therapy.

[0079] Quantitative analysis using mean optical density (AOD) showed that Parkin expression was significantly upregulated in the PF@MS(US) group, significantly higher than in the model group at week 4, and remained at a high level at week 8, indicating a sustained pro-mitochondrial autophagy effect. The increase in the PF@MS group alone was much smaller, confirming that ultrasound activation is crucial for the strong triggering of the pathway in vivo. Simultaneously, total mitochondrial quality was assessed using LC3-II levels. Interestingly, LC3-II expression was also significantly increased in the PF@MS(US) group at weeks 4 and 8. The simultaneous increase in both the mitophagy initiator factor (Parkin) and the autophagosome marker (LC3-II) strongly suggests a comprehensive mitochondrial quality control program coordinated by the treatment: not only clearing damaged organelles through mitophagy but also maintaining mitochondrial homeostasis. Transcriptional analysis of the "Mitophagy-animal" pathway further supports this dual effect: treatment significantly upregulated PINK1 and Parkin expression. Activated Parkin ubiquitinates mitochondrial proteins, which are recognized by the autophagy linker, initiating autophagosome formation and clearance. Figure 8 ).

[0080] 8. PF@MS treatment restores matrix metabolism in the body by inhibiting inflammation and promoting collagen synthesis. After confirming that ultrasound combined with PF@MS restores mitochondrial homeostasis, its downstream effects on ECM metabolism were investigated. Treatment effectively rebalanced matrix dynamics: at 4 and 8 weeks, it significantly inhibited the catabolic enzyme MMP13 and strongly promoted the anabolic protein COL2. Quantitative analysis confirmed that the combined treatment was superior to PF@MS alone in gradually restoring tissue integrity. Mechanistically, this repair is driven by MMP regulation and Pink1-Parkin-LC3-II axis activation, downregulating degradation, upregulating matrix synthesis, and promoting the recovery of intervertebral disc function. Figure 9 ).

[0081] (III) Conclusion Guided by clinical single-cell transcriptome mapping, this invention pioneers a bioelectronic organelle editing paradigm to reverse intracellular disorder (IDD). We successfully constructed piezoelectric-Fenton microspheres (PF@MS), which are not only drug carriers but also wireless, self-limiting mitochondrial maintenance "smart chargers." By establishing an ultrasound-driven mechanochemical feedback loop, PF@MS deterministically resets MMPs to the critical window required for PINK1-Parkin mitophagy, severing the upstream link between energy metabolism failure and matrix inflammation.

[0082] In vivo, this precise mitochondrial surgery effectively coordinates tissue-level repair, restoring the anabolic / catabolistic balance (COL2 / MMP13) and preserving intervertebral disc height in a rat model. This invention bridges the gap between molecular electrophysiology and macroscopic tissue regeneration. Ultimately, this strategy represents a conceptual leap from passive pharmacological intervention to active, controllable bioelectronic regulation, providing a universal blueprint for treating degenerative diseases characterized by mitochondrial energy dysfunction. Figure 1 ).

[0083] This invention establishes a mechanism-driven therapeutic paradigm for the precise repair of damaged mitochondria in degenerated tissues by developing a self-regulating piezoelectric-Fenton hydrogel microsphere system (PF@MS) guided by scRNA-seq and activated by ultrasound. Moving from empirical intervention to mechanism-driven design, single-cell transcriptome analysis helps pinpoint MMP imbalance as a key determinant of cell fate, providing a clear biological target for Fe-BTO-mediated spatiotemporal regulation. Crucially, this platform addresses the long-term risks of mitochondrial overpolarization and secondary oxidative damage through a biophysical-biochemical coupled "kinetic self-locking" mechanism: after ultrasound-triggered responses recover to the physiological threshold (−100 ~ −140 mV), the system autonomously enters a plateau phase guided by intrinsic feedback from H⁺ depletion and pH elevation. This autonomous closed-loop regulation further bypasses the energy-dependent endocytosis pathway by covalently anchoring sialic acid residues. This non-energy-dependent internalization, followed by programmed lysis, enables lysosomal escape, ensuring high-fidelity cytoplasmic delivery even in the ATP-depleted microenvironment of degenerated tissues. In summary, this multi-level intelligent platform provides a robust framework for subcellular intervention, bridging the gap between advanced materials design and the physiological limitations of metabolic diseases.

[0084] In summary, this multi-level, self-regulating platform not only provides a powerful tool for correcting mitochondrial dysfunction but also establishes a conceptual framework for the design of intelligent biomaterials. By coupling external physical stimuli (US) with internal chemical feedback and biological signals (MMP / pH), this method paves the way for advanced therapies for a wide range of metabolic and degenerative diseases.

[0085] (I) This invention has the following significant advantages over existing mitochondrial intervertebral disc repair materials: 1. Different Mechanism Levels: This invention achieves a shift from passive stabilization to deterministic closed-loop bioelectric editing. Existing materials often only passively stabilize MMPs, clear ROS, and indirectly activate autophagy pathways, but cannot quantitatively and precisely anchor MMPs within the -100 to -140 mV physiological range. In contrast, this invention relies on ultrasound activation of Fe-BTO to generate piezoelectric potential, breaking through the Fenton Fe³⁺ / Fe²⁺ cycle bottleneck. By consuming local protons to upregulate MMPs and increase pH to terminate polarization through self-limiting feedback, a complete closed loop of material-electrochemistry-cellular electrophysiology-pH self-feedback is formed, achieving deterministic, quantitative, and self-terminating mitochondrial bioelectric editing. Existing materials cannot achieve these multiple functions.

[0086] 2. In the existing technology, the Fe-BTO piezoelectric-Fenton system is only used for pollutant degradation. Even if the physically mixed piezoelectric materials are forcibly combined with Fenton reagents, it is difficult to easily achieve the synergistic effect of piezoelectric potential-driven ion cycling, proton directional consumption, precise potential locking, and pH self-limiting feedback.

[0087] 3. This invention addresses the inherent challenges of degenerated cells. Existing technologies lack a corresponding design for degenerated intervertebral disc cells, which suffer from inherent problems such as energy deficiency and impaired classical energy-dependent endocytic pathways. Existing microsphere / hydrogel materials have not specifically addressed these issues. This invention uniquely employs a dynamic borate ester bond (BEB) Trojan horse delivery strategy: it can target sialic acid residues in cells, enter cells via non-energy-dependent pathways, and escape via pH-responsive lysosomes, significantly improving cytoplasmic bioavailability and achieving an integrated design of material structure, targeted recognition, and intracellular escape.

[0088] 4. Existing technologies can only slightly improve mitophagy and mildly inhibit apoptosis; however, this invention achieves: mitophagy flux recovery from 8% to 35%, and in vivo salvage of 89% of intervertebral disc height, realizing different levels of repair and improvement, precise MMP regulation, and recovery of autophagy flux.

[0089] (II) The present invention conducted the following comparative exploratory experiments, and the results showed that the corresponding therapeutic effects were generally poor: Comparative Example 1 Referring to the scheme of Example 1, experiments were conducted using only pure barium titanate piezoelectric microspheres without iron doping. The results showed that the obtained material had no Fenton catalytic cycling ability, could not effectively drive Fe³⁺ / Fe²⁺ conversion, could not consume local protons, could not upregulate MMP to the physiological range, had no pH self-limiting feedback, showed only a weak increase in mitochondrial autophagy, and had extremely poor intervertebral disc repair effect.

[0090] Comparative Example 2 Referring to the scheme of Example 1, the scheme of physically mixing ordinary Fenton reagent with hydrogel microspheres was used. The results showed that: since the material has no ultrasonic piezoelectric response, it cannot achieve controllable temporal activation, cannot form a potential regulation closed loop, and is prone to mitochondrial overpolarization and cellular oxidative damage. There is no self-limiting protection mechanism.

[0091] Comparative Example 3 Referring to existing literature (CN 120001428 A), Fe-BTO piezoelectric-Fenton material for pollutant degradation was directly applied to nucleus pulposus cells. The results showed that this approach lacked targeted delivery, lysosomal escape, and MMP quantitative regulation design, resulting in low cell entry efficiency, poor bioavailability, inability to achieve deterministic mitochondrial bioelectric editing, and failure to achieve 89% intervertebral disc height rescue effect.

[0092] The above comparative examples fully demonstrate that only under the material system of this invention can precise potential regulation, self-limiting protection, targeted delivery, and excellent in vivo repair effects be achieved. This invention, through a design scheme integrating specific material composition, microstructure, coupling mechanism, delivery strategy, and feedback regulation, ultimately achieves the effects of significantly increasing mitochondrial autophagy flux and high intervertebral disc height rescue rate.

Claims

1. A method for preparing ultrasonically activated iron-doped barium titanate-based piezoelectric Fenton microspheres, characterized in that, Includes the following steps: (1) Iron-doped barium titanate nanoparticles were synthesized by hydrothermal reaction using a mixed solution of Ba(OH)2・8H2O, ammonium lactate, and FeCl3. (2) Phospholipids, cholesterol, DSPE-PEG2000-PBA and iron-doped barium titanate nanoparticles obtained in step (1) were mixed in a solvent and coated with phenylboronic acid functionalized lipid shell by thin film method to prepare phenylboronic acid functionalized core-shell lipid nanoparticles. (3) Using methacrylamide gelatin hydrogel, tannic acid and photoinitiator as the aqueous phase, and isopropyl myristate and Span80 as the oil phase, hydrogel microspheres were prepared by microfluidic method. Then, the phenylboronic acid functionalized core-shell lipid nanoparticles obtained in step (2) were embedded in the hydrogel microspheres by click chemistry to obtain ultrasonically activated iron-doped barium titanate piezoelectric Fenton microspheres.

2. The method according to claim 1, characterized in that, The molar ratio of Ba(OH)2・8H2O, ammonium lactate, and FeCl3 in step (1) is 20:20:

1.

3. The method according to claim 1, characterized in that, The hydrothermal reaction conditions described in step (1) are a hydrothermal reaction at 200°C for 8 hours.

4. The method according to claim 1, characterized in that, The product of the hydrothermal reaction in step (1) is collected by centrifugation, precipitated, washed, and then vacuum dried to obtain iron-doped barium titanate nanoparticles.

5. The method according to claim 1, characterized in that, In step (2), the weight ratio of phospholipids, cholesterol, DSPE-PEG2000-PBA and iron-doped barium titanate nanoparticles is 120:40:1:

18.

6. The method according to claim 1, characterized in that, The solvent mentioned in step (2) is chloroform.

7. The method according to claim 1, characterized in that, In step (3), the weight ratio of methacrylamide gelatin hydrogel, tannic acid and photoinitiator is 7:1:0.

5.

8. The method according to claim 1, characterized in that, The click chemistry operation in step (3) is as follows: the lyophilized methacrylamide gelatin microspheres are reconstituted with borate-buffered saline, and then a lipid-coated iron-doped barium titanate nanoparticle suspension prepared with the same buffer solution is added. After vortexing and standing, borate ester click coupling occurs, and the free nanoparticles are removed by washing with PBS.

9. Ultrasonically activated iron-doped barium titanate-based piezoelectric Fenton microspheres prepared by the method according to any one of claims 1-8.

10. The use of the ultrasonically activated iron-doped barium titanate-based piezoelectric Fenton microspheres of claim 9 in the preparation of a medicament for treating mitochondrial degenerative diseases.

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