On-Demanand intelligent controlled-release bionic oral targeting delivery system based on yeast microcapsules and application thereof in treatment of intervertebral disc degeneration

Abstract

The invention provides an On-Demand intelligent controlled-release bionic oral targeting delivery system based on yeast microcapsules and an application of the On-Demand intelligent controlled-release bionic oral targeting delivery system in treatment of intervertebral disc degeneration. The loaded nanoparticles are formed by self-assembly of a thioether bond-containing ROS responsive high-molecular polymer PBTEa synthesized based on a Michael addition reaction and a natural anti-inflammatory drug component micromolecule, namely Celasttrol (Cel), through a non-covalent bond. The nanoparticles (Cel-PBTEa) and yeast microcapsules are compounded through electrostatic interaction to form a yeast microcapsule On-Demanand intelligent controlled-release bionic oral targeting delivery system (Cel-PBTEa (at) YC), the size of the nanoparticles is about 350 nm, the nanoparticles are similar to spheres, the dispersity is good, and the nanoparticles are compounded with YC to form a micron-sized oral delivery system. The bionic nano material prepared by the invention can be effectively delivered to a degenerated intervertebral disc in a targeted manner, and the activation of a nucleus pulposus cell inflammatory small body pathway is inhibited, so that the degeneration of the intervertebral disc is inhibited.

Description

Technical field:

[0001] The present invention relates to a targeted drug delivery system in the field of biomedicine, in particular to a smart nanoparticle with intervertebral disc targeting and on-demand controlled release function, as well as a preparation method and application thereof in the treatment of intervertebral disc degeneration. Background technology:

[0002] Over 600 million people worldwide suffer from low back pain, the primary cause of which is intervertebral disc degeneration (IDD). The intervertebral disc, the largest avascular tissue in the human body, is primarily composed of the extracellular matrix (ECM). Degeneration of the intervertebral disc is accompanied by vascular invasion and macrophage infiltration. Inflammatory responses lead to decreased ECM synthesis and increased expression of matrix metalloproteinases (MMPs), ultimately causing IDD. Current clinical treatments primarily rely on nonsteroidal anti-inflammatory drugs (NSAIDs), which only alleviate symptoms, and surgery, which may cause degeneration of adjacent segments. New therapeutic strategies are urgently needed. In recent years, nanomedicine has made significant progress in the treatment of IDD, including delivery systems such as sustained-release hydrogel microspheres, targeted nanofullerenes, and ultrasound-responsive nanobubbles. However, these technologies often rely on invasive local injections. In contrast, oral administration offers a good safety profile but faces the challenge of low bioavailability. Based on this, this study innovatively proposed the use of yeast microcapsules (YC) to achieve non-invasive oral targeted delivery. Through the endocytosis mediated by the macrophage Dectin-1 receptor and the recruitment effect of macrophages in the degenerative intervertebral disc, the precise delivery of drugs was achieved, the inflammatory microenvironment of the degenerative intervertebral disc was perceived in real time, and the on-demand intelligent controlled release of therapeutic drugs was realized, providing a new idea for the treatment of IDD. Summary of the invention:

[0003] (1) Technical problems solved

[0004] This invention addresses the current lack of oral intervertebral disc-targeted drug delivery methods and proposes a noninvasive oral intervertebral disc-targeted drug delivery vehicle, its preparation, and its application. This delivery system exhibits excellent biocompatibility and intervertebral disc-targeted enrichment, significantly improving the bioavailability of oral drug delivery systems and demonstrating excellent inhibitory effects on intervertebral disc degeneration both in vivo and in vitro.

[0005] (2) Technical solution

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An oral targeted delivery system for the intervertebral disc loaded with On-Demand intelligent controlled-release nanoparticles. The nanoparticles (Cel-PBTEa) are formed by self-assembly of Cel and PBTEa, a high molecular weight polymer synthesized by Michael addition polymerization with ROS intelligent response. The oral targeted delivery system is formed by electrostatic interaction between Cel-PBTEa and yeast microcapsules (YC), abbreviated as Cel-PBTEa@YC. This system has excellent therapeutic effects in inhibiting intervertebral disc degeneration in vitro and in vivo.

[0008] The preparation method of Cel-PBTEa@YC comprises the following steps:

[0009] S1: Weigh 20.0 g of yeast powder into a 500 mL beaker, add 200 mL of 1 mol / L NaOH solution, and stir thoroughly with a glass rod (300 rpm, 15 min) until completely dispersed and a uniform suspension is formed. Transfer the mixture to a 500 mL three-necked flask and place it in an 85°C electric constant-temperature oil bath with continuous mechanical stirring (400 rpm) for 1 h. Observe that the color of the system gradually darkens to brown. After the reaction, centrifuge at 3000 rpm for 8 min, discard the supernatant, and wash the pellet three times with 200 mL of double-distilled water. Then, resuspend the cell wall residue in 200 mL of double-distilled water and add concentrated hydrochloric acid (V hydrochloric acid:V water = 1:10000, concentrated hydrochloric acid concentration 12 mol / L) in the appropriate ratio. Adjust the pH of the system to 4.0-5 using precision pH paper. 0, and then placed in a 65°C oil bath with constant temperature stirring (300 rpm) for reaction for 1 hour; the reaction solution was centrifuged at 3000 rpm for 8 minutes, and the precipitate was washed twice with 200 mL of double-distilled water, concentrated by rapid centrifugation (3000 rpm, 3 minutes), and washed four times with isopropanol (40 mL / time, with vortex oscillation for 30 seconds) and twice with acetone (40 mL / time, ultrasonic treatment for 5 minutes / time). After each washing, the precipitate was collected by centrifugation at 3000 rpm for 5 minutes, and finally washed twice with double-distilled water. The resulting precipitate was pre-frozen at -80°C for 24 hours and then freeze-dried in a vacuum freeze dryer (0.1 mbar, -50°C) for 48 hours to obtain light yellow yeast cell wall (YC) powder;

[0010] S2, under ice bath conditions, BDD (1,4-ButaneDiol,Diacrylate), DTT (Dithiothreitol) and AP (5-Amino-1-Pentanol) of different feed ratios were co-dissolved in N, N-dimethylformamide (DMF) (total concentration: 1 mg / mL), reacted for 6 hours, and then the temperature was raised to room temperature for 6 hours. Then, the solution was further reacted at 70°C for 12 hours, and after cooling, the solution was transferred to a dialysis bag (MWCO 2000). It was dialyzed twice with a water / DMF mixture (50:50, volume ratio) at 4°C and dialyzed twice with pure water (the external phase was replaced every 1 hour). Finally, the solution was freeze-dried to obtain the final product PBTEa;

[0011] S3, weigh 10 mg PBTEa and 5 mg Cel, dissolve the above mixture fully in 300 μL DMSO, then use a 1 mL insulin needle to draw up the above solution, slowly drip it into 5 mL PBS, stir it with a magnetic stirrer for 15 minutes, and then transfer it to a dialysis bag (3500 kDa). Change the liquid every 30 minutes for a total of 3 times. Remove the liquid after 1.5 hours and obtain Cel-PBTEa nanoassembly after freeze-drying.

[0012] In step S4, a 10 mg / mL YC suspension was prepared by weighing a certain amount of YC. Vortexing (5 minutes) and ultrasonic dispersion (5 minutes) were performed intermittently to allow for full swelling. Cel-PBTEa nanoparticles were added at varying dosage ratios. The suspension was stirred at room temperature for 8 hours, centrifuged at 2000 g for 10 minutes, washed three times with water, and lyophilized to obtain Cel-PBTEa@YC.

[0013] (3) Beneficial effects

[0014] The yeast microcapsule-based non-invasive oral targeted delivery system for intervertebral discs proposed in this application is loaded with On-Demand intelligent controlled-release nanoparticles. These nanoparticles can intelligently respond to the inflammatory microenvironment of the degenerative intervertebral disc and release therapeutic drugs on demand. They further improve their targeting on the basis of effectively increasing their enrichment in the intervertebral disc. The preparation method is simple, and they can effectively inhibit the activation of the inflammasome pathway of nucleus pulposus cells and inhibit intervertebral disc degeneration, making them suitable for large-scale promotion and application. Description of the drawings:

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments.

[0016] Figure 1 TEM morphology of yeast and yeast microcapsules;

[0017] Figure 2is the nuclear magnetic resonance hydrogen spectrum (1H-NMR) of PBTEa;

[0018] Figure 3 For formulation optimization (a) Cel-PBTEa (b) Cel-PBTEa@YC;

[0019] Figure 4 are the particle size and potential of tripterygium wilfordii-PBTEa / yeast microcapsules;

[0020] Figure 5 To study the stability of yeast microcapsules in macrophages;

[0021] Figure 6 TEM results of PBTEa and Cel-PBTEa before and after H2O2 intervention;

[0022] Figure 7 This is the electron microscopy of tripterygium wilfordii-PBTEa / yeast microencapsulated elements;

[0023] Figure 8 The general appearance of Cel-PBTEa@YC has changed;

[0024] Figure 9 To simulate the gastrointestinal environment release of Cel-PBTEa@YC and its ROS response release;

[0025] Figure 10 In vitro fluorescence imaging of PBTEa@YC loaded with Icg;

[0026] Figure 11 CCK-8 cell activity assay for macrophages and nucleus pulposus cells;

[0027] Figure 12 This is histological staining of the cross-section of the intervertebral disc of SD rats;

[0028] Figure 13 The uptake of intact yeast (IY) by macrophage RAW 264.7 cells;

[0029] Figure 14 Near-infrared fluorescence imaging of small animals in vivo (a) in vivo fluorescence imaging of animals (b) in vitro fluorescence imaging of major organs (c) in vitro fluorescence imaging of major lymph nodes and intervertebral discs (MLN: mesenteric lymph node; ALN: axillary lymph node; ILN: inguinal lymph node; IVD: intervertebral disc);

[0030] Figure 15 MRI evaluation of the in vivo efficacy of the yeast microcapsule oral delivery system (a) MRI imaging results (b) Pfirrmann score;

[0031] Figure 16Histological staining of intervertebral disc (a) H&E, Safranin Fast Green and Alcian Blue staining (b) Histological scoring;

[0032] Figure 17 H&E and Giemsa staining of major organs (heart, liver, spleen, lung, and kidney);

[0033] Figure 18 Transcriptome quality control and enrichment analysis (a) Principal component analysis (PCA) (b) Overview of differentially expressed genes heat map (c) GSEA enrichment analysis;

[0034] Figure 19 GSEA enrichment analysis for the IL-1β+Cel_vs_IL-1β group;

[0035] Figure 20 To verify the molecular mechanism at the protein and animal levels (a) The protein expression level of TLR2 / MyD88 / P65 / Nlrp3 axis after Cel and C29 intervention (b) The inhibitory effect of Cel and C29 on TLR2 / MyD88 / P65 / Nlrp3 at the animal level

[0036] Prevent intervertebral disc degeneration Specific implementation method:

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] 1. Preparation method:

[0040] Weigh 20.0 g of yeast powder into a 500 mL beaker, add 200 mL of 1 mol / L NaOH solution, and stir thoroughly with a glass rod (300 rpm, 15 min) until completely dispersed and a uniform suspension forms. The mixture is then transferred to a 500 mL three-necked flask and placed in an 85°C oil bath with continuous mechanical stirring (400 rpm) for 1 h. The color of the system is observed to gradually darken to brown. After the reaction, centrifuge at 3000 rpm for 8 min, discard the supernatant, and wash the pellet three times with 200 mL of deionized water. Resuspend the cell wall residues in 200 mL of deionized water and add concentrated hydrochloric acid (V hydrochloric acid:V water = 1:10,000, 12 mol / L) as appropriate. Adjust the pH to 4.0-5 using precision pH paper. 0, and then placed in a 65°C oil bath with constant temperature stirring (300 rpm) for reaction for 1 hour; the reaction solution was centrifuged at 3000 rpm for 8 minutes, and the precipitate was washed twice with 200 mL of double-distilled water, concentrated by rapid centrifugation (3000 rpm, 3 minutes), and washed four times with isopropanol (40 mL / time, combined with vortex oscillation for 30 seconds) and twice with acetone (40 mL / time, ultrasonic treatment for 5 minutes / time). After each washing, the precipitate was collected by centrifugation at 3000 rpm for 5 minutes, and finally washed twice with double-distilled water. The resulting precipitate was pre-frozen at -80°C for 24 hours and then freeze-dried in a vacuum freeze dryer (0.1 mbar, -50°C) for 48 hours to obtain light yellow yeast cell wall (YC) powder.

[0041] BDD (1,4-Butanediol, diacrylate), DTT (Dithiothreitol), and AP (5-Amino-1-Pentanol) were dissolved in N,N-dimethylformamide (DMF) at varying feed ratios (total concentration: 1 mg / mL) in an ice bath and reacted for 6 hours. The temperature was then raised to room temperature and allowed to react for 6 hours. The solution was then reacted at 70°C for a further 12 hours. After cooling, the solution was transferred to a dialysis bag (MWCO 2000). The solution was dialyzed twice against a 50:50, volume ratio, water / DMF mixture at 4°C and twice against pure water (external phase was changed every hour). Finally, the solution was lyophilized to obtain the final product, PBTEa.

[0042] Weigh 10 mg PBTEa and 5 mg Cel, fully dissolve the mixture in 300 μL DMSO, then use a 1 mL insulin needle to draw up the solution, slowly drip it into 5 mL PBS, stir it with a magnetic stirrer for 15 minutes, and then transfer it to a dialysis bag (3500 kDa). Change the liquid every 30 minutes for a total of 3 times. Remove the liquid after 1.5 hours and freeze-dry it to obtain Cel-PBTEa nanoassembly.

[0043] A 10 mg / mL YC suspension was prepared by weighing a certain amount of YC. Vortexing (5 minutes) and ultrasonic dispersion (5 minutes) were performed intermittently to allow for full swelling. Cel-PBTEa nanoparticles were then added at varying dosage ratios. The suspension was stirred at room temperature for 8 hours, centrifuged at 2000g for 10 minutes, washed three times with water, and lyophilized to obtain Cel-PBTEa@YC.

[0044] 2. Characterization of Cel-PBTEa@YC

[0045] Figure 1 The morphology of intact yeast and yeast microcapsules. It can be seen in the left picture that the contents of the intact yeast have not been removed, and the electron beam cannot penetrate the yeast, so it appears black; the right picture shows the yeast microcapsules with the contents removed. Through acid, alkali and organic solvent treatment, most of the substances in the yeast are effectively removed to form yeast hollow microcapsules, which provide sufficient space for subsequent drug loading. In order to introduce positively charged groups into the ROS-responsive copolymer, PBTE-PBAE copolymer (denoted as PBTEa) was prepared by Michael addition polymerization of BDD, DTT and AP. Due to the different reaction rates of BDD-DTT and BDD-AP, the reaction was first carried out in an ice bath to avoid the sudden polymerization of BDD-DTT; then the temperature was raised to 70°C to promote the reaction between AP and acrylate to form the final product PBTEa. In the 1H-NMR hydrogen spectrum of PBTEa ( Figure 2 ), the characteristic peaks of BDD (4.12ppm, -OCOCH2), DTT (3.74ppm, -CH2CH(OH)CH) and AP (1.25-1.60ppm, -CCH2CH2CH2OH; 3.58ppm, -CH2CH2CH2OH) units all appeared, confirming the successful synthesis of PBTEa. For this oral delivery system, the formulation of each component of its preparation was optimized, such as Figure 3 As shown, different feed ratios were set (Cel:PBTEa = 1:1, 1:2, 1:5, 1:10), the drug loading capacity was detected and calculated, and a bar graph was drawn with the feed ratio as the horizontal axis and the drug loading capacity (%) as the vertical axis. The formulation of Cel-PBTEa nanoparticles was optimized. The results showed that when the feed ratio of Cel to PBTEa was 1:2, the drug loading capacity was the highest at 23.53% ± 2.16%; subsequently, based on the optimal formulation of Cel-PBTEa, we constructed the Cel-PBTEa@YC nanoparticle / yeast microcapsule delivery system through electrostatic interaction with yeast microcapsules. Similarly, different feed ratios of Cel-PBTEa and YC were set. The results showed that when the feed ratio of Cel-PBTEa to YC was 1:1, the drug loading capacity was the highest at 6.46% ± 0.49%).

[0046] The results of dynamic light scattering (DLS) particle size analysis showed that the particle size of PBTEa and Cel formed nanoparticles was reduced compared with that of single PBTEa nanoparticles (from 485.43±16.91nm to 353.09±75.542nm). Dynamic monitoring of the particle size changes of PBTEa nanoparticles and Cel-PBTEa nanoparticles revealed that the particle size fluctuation did not exceed 10% of its own particle size within 48 hours, indicating that the polymer self-assembled nanoparticles had good stability. The ζ-potential showed that the positive charge value of PBTEa decreased slightly after binding to Cel, but it still showed a positively charged complex overall; the absolute value of the negative charge of yeast decreased after the yeast contents were removed to form yeast microcapsules, and it still showed a negative potential after complexing with positively charged Cel-PBTEa nanoparticles, but its absolute value decreased ( Figure 4 ), further illustrating that Cel-PBTEa and YC mainly form the Cel-PBTEa@YC nanoparticle / yeast microcapsule system through electrostatic interaction.

[0047] In addition, the stability of yeast microcapsules in macrophages was further investigated within 7 days after uptake by RAW 264.7. Figure 5 As shown, DiI fluorescent dye clearly shows the cell membrane boundary of RAW 264.7, and FITC-labeled yeast microcapsules are clearly visible in macrophages with intact morphology; during dynamic observation for up to 7 days, yeast microcapsules can stably exist in macrophages without structural damage or disintegration.

[0048] like Figure 6 TEM results showed that PBTEa was a spherical structure of about 350 nm in size with a relatively uniform morphology; the size of PBTEa was slightly reduced after Cel was wrapped, which may be due to the electrostatic interaction between the positively charged PBTEa and the negatively charged Cel, resulting in a decrease in the particle size of the nanoparticles. After 48 hours of H2O2 (10 mM) intervention, TEM results showed that the nanoparticles disaggregated and it was difficult to observe aggregated nanoparticles. The mechanism of action may be that under the action of H2O2, the sulfide bond of PBTEa is oxidized to sulfoxide or sulfone, and its hydrophilicity is significantly improved, resulting in the hydrolysis of the ester bond in the polymer chain. Figure 7 The transmission electron microscope image of Cel-PBTEa@YC was displayed, showing the nanoparticles loaded in the cavity of the yeast microcapsule. Further elemental analysis detected the presence of elements such as C, N, and O in the system. Compared with ordinary polymers, the raw materials for synthesizing PBTEa contain DTT, in which the S element is a component of a monosulfide bond. The detection of the S element in the elemental analysis indicates that the yeast microcapsule contains Cel-PBTEa, confirming that the yeast microcapsule has successfully loaded the Cel-PBTEa nanocomplex.

[0049] The ROS responsiveness of Cel-PBTEa nanoparticles was further studied. At 2 h, the color difference between the experimental group and the control group was not obvious. At 4 h, the difference gradually appeared. At 48 h, the experimental group was almost clear and transparent, while the color change of the control group was not obvious within 48 h. Figure 8 These results suggest that PBTEa may undergo chemical structural changes under the action of ROS, causing changes in its hydrophilic and hydrophobic properties, which are reflected in the changes in appearance; Cel-PBTEa responsively releases Cel under the action of ROS, and the appearance of PBTEa changes towards clarity after degradation.

[0050] In order to study the in vitro release of Cel-PBTEa@YC, different pH values were set to simulate the process of drug release in the gastrointestinal tract ( Figure 9 ), under pH 1.2 (simulated stomach) conditions, the drug release amounts of Cel-PBTEa@YC and Cel-PBAE@YC in the first 2 h were 20.81% and 11.89%, respectively; under pH 6.8 (simulated intestine) conditions, the cumulative release amounts in the first 6 h were 40.23% and 33.12%, respectively, with no statistically significant differences; at pH 7.4 (simulated intestine) without the addition of H2O2, the cumulative release amounts of Cel-PBTEa@YC and Cel-PBAE@YC within 24 h were 68.30% and 70.77%, respectively; however, after the addition of H2O2, the cumulative release amount of Cel-PBTEa@YC within 24 h was 82.72%, which was 1.3 times that of Cel-PBAE@YC. These results indicate that Cel-PBTEa@YC can achieve ROS-responsive release and has the ability to deliver loaded drugs to sites of oxidative stress.

[0051] The in vitro fluorescence imaging system observed that the fluorescence intensity of the delivery system gradually increased within 24 hours after H2O2 intervention, indicating that the delivery material gradually released the encapsulated fluorescent substance Icg under the action of H2O2; the fluorescence intensity in the control group gradually weakened over time, and the PBTEa@YC-loaded Icg was unable to release Icg in the absence of H2O2, and the fluorescence intensity gradually decreased over time after the fluorescent substance was quenched ( Figure 10 ).

[0052] 3. Application scenarios and functional verification:

[0053] 1. Cytotoxicity: Add 100 μL of cell suspension (add intervention, IL-β or LPS or Cel) to each well of a 96-well plate and incubate in a cell culture incubator for 24 h; add 10 μL of CCK-8 solution to each well, set up cell-free control wells (add only CCK-8 and cell culture solution) and negative control wells (add CCK-8 and cell suspension without other drug intervention); continue to culture in a 37°C cell culture incubator for 1 h; use a microplate reader to measure the absorbance at a wavelength of 450 nm, and calculate the cell viability based on the absorbance of the control and experimental groups, see [see Table 1]. Figure 11

[0054] Results: When the drug concentration was 250 ng / mL, the survival rate of nucleus pulposus cells and macrophages could be maintained above 80% under three different intervention conditions; when the concentration was 50 ng / mL, the cell survival rate was above 90%, indicating the low cytotoxicity and good biocompatibility of the delivery system.

[0055] 2. Tissue toxicity: After 8 weeks of oral administration, the intervertebral discs of SD rats were taken to prepare paraffin sections for morphological and immunofluorescence staining. Figure 12 .

[0056] Results: Compared with the Con group, the intervertebral disc morphology in the Cel, Cel-PBTEa, YC, and Cel-PBTEa@YC groups did not change significantly, and the nucleus pulposus tissue morphology and annulus fibrosus structure remained well, indicating no local tissue toxicity. The histological staining levels of safranin fast green and alcian blue were relatively uniform among the groups, indicating that the delivery system did not have a significant inhibitory effect on ECM components.

[0057] 3. Cellular uptake: Yeast or yeast microcapsules were labeled with FITC, and Cel-PBTEa nanoparticles were labeled with FITC. The labeling method for yeast or yeast microcapsules was as follows: 0.5 mg of FITC was weighed and dissolved in 5 mL of double-distilled water to obtain a 100 ng / mL FITC aqueous solution. 0.5 mg of yeast or yeast microcapsules was ultrasonically dispersed in 1 mL of pure water, 1 mL of FITC aqueous solution was added, and the mixture was made up to 8 mL with water. The mixture was incubated overnight at room temperature on a multi-point magnetic stirrer. The next day, the incubated product was centrifuged at 2000 rpm and washed with water until the supernatant was colorless. The supernatant was then stored in a refrigerator at 4°C protected from light with tin foil. The FITC labeling method for Cel-PBTEa nanoparticles was as follows: After preparing Cel-PBTEa nanoparticles, FITC was added to each of them to a final concentration of 200 ng / mL. The mixture was incubated overnight at room temperature on a multi-point magnetic stirrer. The unlabeled FITC was removed by dialysis (MWCO 2000) the next day. The resulting solution was used for cellular uptake studies.

[0058] RAW 264.7 cells were seeded in a 12-well plate and incubated in a 37°C cell culture incubator for 24 hours. FITC-labeled yeast or yeast microcapsules were added to RAW264.7 cells and incubated with the cells for 0.5 hours, 1 hour, 2 hours, and 4 hours. For nucleus pulposus cells, lysosomes were labeled with 200 nM Lyso Tracker Red, and FITC-labeled Cel-PBTEa nanoparticles or free FITC were added and incubated with the cells for 0.5 hours, 1 hour, 2 hours, and 4 hours. The cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 minutes. After washing three times with PBS, the cell nuclei were stained with DAPI. Finally, the green fluorescence was observed under a fluorescence microscope, and the cell uptake efficiency was detected by flow cytometry. Figure 13 .

[0059] Results: In the initial phase of yeast-macrophage co-incubation (0.5 h), macrophage phagocytosis of yeast was relatively weak, with scattered yeast particles visible within the cells. Over time, the macrophage phagocytosis of yeast significantly increased, with numerous yeast particles visible within the cells (4 h). Macrophage phagocytosis of yeast exhibited a clear, time-dependent, and increasing pattern.

[0060] 4. Biological targeted distribution: Indocyanine green (Icg), a fluorescent dye with a negative charge, was used as a model drug instead of Cel. Referring to the preparation method in 1.2.3 (9) and (10), Icg was loaded into the PBTEa and yeast microcapsule delivery system to obtain Icg-PBTEa and Icg-PBTEa@YC oral delivery systems; the experimental animals were divided into three groups (pure Icg group, Icg-PBTEa group and Icg-PBTEa@YC group), and the fluorescence distribution was observed after oral administration; the experimental animals were placed in the small animal living imaging system ( Trilogy, LI-COR, USA); the near-infrared wavelength was set to 800 nm; the animals were anesthetized with isoflurane inhalation anesthetic at a flow rate of 0.7 L / min; the respiratory rate and heart rate of the animals were observed to avoid excessive anesthesia-induced death; near-infrared in vivo imaging images were acquired at the above time points;

[0061] After collecting in vivo live imaging images at the last time point (48 h), the experimental animals were euthanized, and their major internal organs, gastrointestinal tract, axillary lymph nodes (ALN), inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), and intervertebral discs (IVD) were removed for ex vivo fluorescence imaging of these tissues. Figure 14

[0062] Results: Oral administration of Icg-PBTEa@YC demonstrated excellent targeting within the degenerative disc. Specifically, at eight different time points over a 48-hour period, the free drug (Icg) and drug nanoparticle (Icg-PBTEa) groups exhibited no significant fluorescence distribution within the degenerative disc, indicating low aggregation efficiency within the target region for both drug forms. In contrast, the drug-nanoparticle / yeast microcapsule group (Icg-PBTEa@YC) exhibited significant fluorescence enrichment within the same degenerative disc region. Its fluorescence intensity peaked at 6 hours and then gradually decreased, but at 48 hours, it remained significantly higher than the other two control groups, demonstrating the excellent tissue targeting and sustained-release properties of the yeast microcapsule delivery system.

[0063] After in vivo fluorescence imaging, we removed the animals' major organs, gastrointestinal tract, major lymph nodes, and intervertebral disc tissue for ex vivo fluorescence imaging. Consistent with the in vivo imaging results, the Icg-PBTEa@YC group showed significant fluorescence accumulation in the intervertebral disc. Furthermore, the fluorescence intensity in the gastrointestinal tract and lymph nodes was significantly higher than in the other control groups, suggesting that the in vivo transport pathway of yeast microcapsules is related to intestinal epithelial cells and lymph nodes. In summary, studies in a rat model of intervertebral disc degeneration following acupuncture demonstrated that yeast microcapsules significantly increased local drug accumulation in the intervertebral disc, achieving targeted delivery to the degenerated disc.

[0064] 5. Efficacy and systemic safety of oral targeted delivery system: Experimental SD rats (200-230g) were divided into 6 groups, namely control group (Con), intervertebral disc degeneration group (IDD), yeast microcapsule group (YC), tripterine group (Cel), tripterine nanoparticle group (Cel-PBTEa), and tripterine-PBTEa / yeast microcapsule (Cel-PBTEa@YC), with 5 rats in each group. Cel was orally administered at a concentration of 3 mg / kg 0.5 h after acupuncture modeling, and the YC group was orally administered at a concentration of 70 mg / kg. Oral gavage was performed once every 2 days, and imaging examinations were performed at the 4th and 8th week time points. Venous blood was collected from the rats after the 8th week examination for hematological examination, and the main internal organs, gastrointestinal tract and intervertebral disc were taken for histological staining.

[0065] See Figure 15 , the T2 signal of the intervertebral disc in the IDD group decreased significantly at 4 weeks, and the relative disc height index (%DHI) was significantly lower than that of the control group. The oral empty yeast microcapsule group (YC group) was close to the IDD group in the above two indicators, with no significant difference, indicating that the empty yeast microcapsules had no obvious therapeutic effect on intervertebral disc degeneration; the treatment groups (Cel, Cel-PBTEa, Cel-PBTEa@YC) inhibited the decline of T2 signals to varying degrees. The above imaging results confirmed the good effect of Cel-PBTEa@YC in inhibiting IDD at the animal level, showing better therapeutic effects than free drugs and nanoformulations. Yeast microcapsules played a good targeting role as a delivery medium, while other treatment groups lacked a delivery system and could not achieve drug enrichment in the degenerated intervertebral disc, so the efficacy was limited at the animal experimental level. After completing the imaging evaluation, the intervertebral disc samples of the experimental animals were collected, fixed, decalcified, sliced and stained, as shown in Figure 2. Figure 16 As shown, histological staining (H&E, Safranin Fast Green, and Alcian Blue) clearly visualized the histological structure of the intervertebral disc. Compared with the control group, the IDD group demonstrated a significant decrease in nucleus pulposus cells and disorganized annulus fibrosus laminae at both weeks 2 and 4, with histological scores increasing from 5.0 to 12.0 and 12.6, respectively. Compared with the IDD group, the Cel-PBTEa@YC group showed significant improvements in nucleus pulposus morphology and area, and a more uniform annulus fibrosus structure. After 8 weeks of treatment, the histological scores of the Cel, Cel-PBTEa, and Cel-PBTEa@YC groups decreased by 30% (p < 0.05), 35% (p < 0.05), and 55% (p < 0.05), respectively. The Cel-PBTEa@YC group showed the most significant improvement in disc morphology, consistent with the aforementioned imaging results, demonstrating the significant advantages of this oral delivery system in the treatment of IDD.

[0066] After SD rats were treated with YC, Cel, Cel-PBTEa, and Cel-PBTEa@YC orally for 8 weeks, their major organs, including heart, liver, spleen, lung, kidney, and gastrointestinal tissues, were collected for H&E and Giemsa staining. Figure 3-9 As shown in Figures 3-10, no obvious histological damage or pathological changes were observed in the drug-treated groups, which was consistent with the observation results of the blank control group. Therefore, this oral drug delivery system has good biosafety and does not cause significant systemic adverse reactions.

[0067] 6. Mechanism of Treatment for Intervertebral Disc Degeneration: Experimental animal nucleus pulposus cells were divided into three groups (control group, IL-1β 5 ng / mL group, and IL-1β 5 ng / mL + Cel 50 ng / mL group), with three replicates per group. After 24 hours of cell attachment, the culture medium was replaced and the treatment continued for 48 hours. Total RNA was extracted using RNAisoPlus and, after passing quality control (A260 / A280 = 1.9-2.1), RNA-Seq analysis was performed by IGENEBOOK. RNA integrity was assessed using Qsep400. 1 μg of total RNA was used to construct a library using VAHTS mRNA-seq V8, and 150 bp paired-end sequencing was performed on a Novaseq 6000. Low-quality sequences were filtered using Cutadapt, aligned to the rat genome using Hisat2 (allowing two mismatches), and FPKM-normalized expression was calculated using FeatureCounts. Differentially expressed genes were identified using an FDR < 0.05 and |log2FC| > 1 (EdgeR). Functional enrichment analysis was performed using ClusterProfiler (GO / KEGG / GSEA, q<0.05), and the specific molecular mechanism was verified by Western blotting (WB) and micro-computed tomography (Micro-CT) techniques.

[0068] result: Figure 18 Principal component analysis showed that the biological replicates of the three groups, Con, IL-1β, and IL-1β+Cel, clustered together without significant deviation, indicating strong consistency within the three groups and good biological repeatability. The three groups could be clearly distinguished, with large expression differences between the groups, indicating that different interventions caused changes in the expression patterns of nucleus pulposus cells. Figure 19The heat map in Figure 2 shows differentially expressed genes among the groups. It can be seen that approximately two-fifths of the differentially expressed genes showed decreased expression after IL-1β treatment, while Cel treatment partially restored these decreased gene expression. Another three-fifths of the differentially expressed genes showed significant increases after IL-1β treatment, while Cel treatment suppressed these increases. This differential gene heat map further demonstrates the differences in gene expression patterns among the groups. Compared to the IL-1β group, Cel treatment suppressed activation of the NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways, demonstrating a significant inhibitory effect on IL-1β-induced activation of these inflammatory signaling pathways.

[0069] To confirm that Cel acts through the TLR2 / MyD88 / P65 / Nlrp3 axis, proteins in this pathway were detected by Western blotting. Figure 20 As shown in the data, TLR2 expression was significantly upregulated after IL-1β administration, while p-P65 phosphorylation was enhanced. After C29 or Cel intervention, TLR2 and p-P65 expression decreased significantly, while P65 expression did not change significantly. Nlrp3 and Caspase-1, downstream of the pathway, also showed the same trend as TLR2 and p-P65. MMP13 expression was significantly inhibited after C29 or Cel administration, and Col2a1 expression was significantly decreased under IL-1β induction. After blocking the TLR2 signaling pathway (C29 or Cel intervention), its expression was restored to a certain extent. At the animal level, small animal Micro-CT scanning was performed 8 weeks after local injection of Cel or C29 into the caudal intervertebral disc of rats. The DHI of the IDD group was significantly decreased. Blocking the TLR2 signaling pathway (C29 or Cel intervention) can partially restore the intervertebral disc height, thereby inhibiting intervertebral disc degeneration.

[0070] Finally, it should be noted that the above embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention. In addition, after reading the technical contents of the present invention, those skilled in the art may make various changes, modifications, or variations to the present invention, and all such equivalent forms also fall within the scope of protection defined by the present application.

Claims

1. A delivery system with oral targeted delivery capability to the intervertebral disc and the ability to inhibit intervertebral disc degeneration, characterized in that: The delivery system is loaded with intelligent responsive controlled-release nanoparticles Cel-PBTEa, wherein the nanoparticles Cel-PBTEa are formed by self-assembly of the small molecule compound Celastrol Cel and PBTEa through non-covalent bonds. The yeast microcapsule oral targeted delivery system is abbreviated as Cel-PBTEa@YC in English.

2. The oral targeted delivery system according to claim 1, characterized in that The preparation method of Cel-PBTEa@YC comprises the following steps: S1: Weigh 20.0 g of yeast powder into a 500 mL beaker, add 200 mL of 1 mol / L NaOH solution, and stir thoroughly with a glass rod (300 rpm, 15 min) until completely dispersed and a uniform suspension is formed. Transfer the mixture to a 500 mL three-necked flask and place it in an 85°C electric constant-temperature oil bath with continuous mechanical stirring (400 rpm) for 1 h. Observe that the color of the system gradually darkens to brown. After the reaction, centrifuge at 3000 rpm for 8 min, discard the supernatant, and wash the pellet three times with 200 mL of double-distilled water. Then, resuspend the cell wall residue in 200 mL of double-distilled water and add concentrated hydrochloric acid (V hydrochloric acid:V water = 1:10000, concentrated hydrochloric acid concentration 12 mol / L) in the appropriate ratio. Adjust the pH of the system to 4.0-5 using precision pH paper. 0, and then placed in a 65°C oil bath with constant temperature stirring (300 rpm) for reaction for 1 hour; the reaction solution was centrifuged at 3000 rpm for 8 minutes, and the precipitate was washed twice with 200 mL of double-distilled water, concentrated by rapid centrifugation (3000 rpm, 3 minutes), and washed four times with isopropanol (40 mL / time, with vortex oscillation for 30 seconds) and twice with acetone (40 mL / time, ultrasonic treatment for 5 minutes / time). After each washing, the precipitate was collected by centrifugation at 3000 rpm for 5 minutes, and finally washed twice with double-distilled water. The resulting precipitate was pre-frozen at -80°C for 24 hours and then freeze-dried in a vacuum freeze dryer (0.1 mbar, -50°C) for 48 hours to obtain light yellow yeast cell wall (YC) powder; S2, under ice bath conditions, BDD (1,4-ButaneDiol,Diacrylate), DTT (Dithiothreitol) and AP (5-Amino-1-Pentanol) of different feed ratios were co-dissolved in N, N-dimethylformamide (DMF) (total concentration: 1 mg / mL), reacted for 6 hours, and then the temperature was raised to room temperature for 6 hours. Then, the solution was further reacted at 70°C for 12 hours, and after cooling, the solution was transferred to a dialysis bag (MWCO 2000). It was dialyzed twice with a water / DMF mixture (50:50, volume ratio) at 4°C and dialyzed twice with pure water (the external phase was replaced every 1 hour). Finally, the solution was freeze-dried to obtain the final product PBTEa; S3, weigh 10 mg PBTEa and 5 mg Cel, fully dissolve the above mixture in 300 μL DMSO, then use a 1 mL insulin needle to draw up the above solution, slowly drip it into 5 mL PBS, stir it with a magnetic stirrer for 15 minutes, and then transfer it to a dialysis bag (3500 kDa). Change the liquid every 30 minutes for a total of 3 times. Remove the liquid after 1.5 hours and obtain Cel-PBTEa nanoassembly after freeze-drying. In step S4, a 10 mg / mL YC suspension was prepared by weighing a certain amount of YC. Vortexing (5 minutes) and ultrasonic dispersion (5 minutes) were performed intermittently to allow for full swelling. Cel-PBTEa nanoparticles were added at varying dosage ratios. The suspension was stirred at room temperature for 8 hours, centrifuged at 2000 g for 10 minutes, washed three times with water, and lyophilized to obtain Cel-PBTEa@YC.

3. The preparation method of Cel-PBTEa@YC according to claim 2, characterized in that: The mass ratio of the tripterygium wilfordii Cel and PBTEa is Cel:PBTEa=1:1, 1:2, 1:5, 1:10, and the preferred mass ratio is 1:

2.

4. The preparation method of Cel-PBTEa@YC according to claim 3, characterized in that The mass ratio of Cel-PBTEa to yeast microcapsule YC is Cel-PBTEa:YC=2:1, 2:2, 2:4, 2:10, 2:20, and the preferred mass ratio is 1:

1.

5. Use of the Cel-PBTEa@YC according to any one of claims 1 to 4 in the preparation of a drug for treating intervertebral disc degeneration.

6. The use according to claim 5, characterized in that The intervertebral disc degeneration animal model is acupuncture intervertebral disc degeneration.

7. An oral intervertebral disc targeted delivery system, characterized in that: The yeast microcapsule delivery carrier is a yeast microcapsule synthesized based on the acid-base organic solvent method, and its English name is Yeast Microcapsules, abbreviated as YC. The nanoparticles loaded therein are formed by self-assembly of a high molecular weight polymer poly(β-thioester-β-amino ester) copolymer (abbreviated as PBTEa in English) generated based on Michael addition polymerization reaction and celastrol Cel. The nanoparticles are abbreviated as Cel-PBTEa, and the composite intervertebral disc oral targeted delivery system is abbreviated as Cel-PBTEa@YC.

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