Chitosan-coated gynostemma pentaphyllum-derived nanovesicles, and preparation method and application thereof

Abstract

The application discloses a chitosan-coated gynostemma pentaphyllum-derived nanovesicle and a preparation method and application thereof, and relates to the field of biological medicines.The chitosan-coated gynostemma pentaphyllum-derived nanovesicle has a core-shell structure, and comprises a gynostemma pentaphyllum-derived nanovesicle core and a chitosan shell layer coated on the surface of the core.The preparation method comprises the following steps: extracting gynostemma pentaphyllum nanovesicles by differential centrifugation, ultracentrifugation and PBS washing and purification; and obtaining the core-shell structure nanovesicle by electrostatic self-assembly, room temperature incubation, centrifugation and neutral buffer washing.The application of the chitosan-coated gynostemma pentaphyllum-derived nanovesicle in the preparation of drugs for treating inflammatory bowel disease is disclosed.The application solves the problems of existing plant-derived nanovesicles, such as easy degradation after oral administration and low intestinal target delivery efficiency; and solves the clinical problems of inflammatory bowel disease, such as difficulty in realizing flora-metabolism-immune multi-level synergistic regulation in oral treatment and difficulty in fundamentally reversing inflammation and barrier damage.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a chitosan-coated Gynostemma pentaphyllum-derived nanovesicle, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing disease characterized by persistent mucosal damage and dysregulation of the immune response. Although current therapies primarily rely on anti-inflammatory and immunosuppressive strategies, their long-term efficacy is often limited by incomplete mucosal healing and poor restoration of the gut microenvironment. Increasing evidence suggests that sustained remission of IBD requires not only controlling inflammation but also the synergistic restoration of epithelial barrier function, immune homeostasis, and microbiome-derived signaling.

[0003] Against this backdrop, microbial metabolites have become important mediators connecting the gut microbiota ecosystem and host mucosal immunity. Among these metabolites, purine metabolites, particularly inosine, have attracted increasing attention due to their immunomodulatory effects and role in epithelial metabolic regulation. However, the development of metabolite-based oral therapies remains limited by rapid gastrointestinal degradation, poor local retention capacity, and limited ability to remodel complex host-microbiota ecosystems. Therefore, strategies utilizing natural biomaterials to remodel the gut microbiota and restore the endogenous metabolic environment offer a more promising and physiologically sound approach for IBD treatment.

[0004] Plant-derived nanovesicles (PDNVs) have recently emerged as ideal ecological regulators and biomimetic nanotherapy platforms due to their biocompatibility, scalability, and inherent bioactive cargo. Gynostemma pentaphyllum, a medicinal plant with well-documented intestinal protective and anti-inflammatory properties, has shown promising therapeutic potential in colitis through its derived nanovesicles (GP-PDNVs). However, natural PDNVs typically exhibit limited gastrointestinal stability and short mucosal retention time, which restricts their sustained interaction with intestinal tissue and gut microbiota. Summary of the Invention

[0005] The purpose of this invention is to provide a chitosan-coated Gynostemma pentaphyllum-derived nanovesicle, its preparation method, and its application. By modifying the chitosan surface, the gastrointestinal stability and colonic mucosal adhesion and retention capacity of the nanovesicles are enhanced, solving the problems of easy degradation and low intestinal targeted delivery efficiency of existing plant-derived nanovesicles after oral administration. At the same time, this platform reshapes the gut microbiota and enriches the purine metabolite inosine, thereby activating the host PPARγ pathway, solving the clinical problem of difficulty in achieving multi-level synergistic regulation of microbiota-metabolism-immunity and the difficulty in fundamentally reversing inflammation and barrier damage in oral treatment of inflammatory bowel disease (IBD).

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A chitosan-coated Gynostemma pentaphyllum-derived nanovesicle has a core-shell structure, comprising a Gynostemma pentaphyllum-derived nanovesicle core and a chitosan shell coating the surface of the core.

[0008] Furthermore, the chitosan-coated Gynostemma pentaphyllum-derived nanovesicles have a particle size of 130~175nm and a Zeta potential of +27mV~+37mV.

[0009] Furthermore, the Gynostemma pentaphyllum-derived nanovesicles are exosome-like nanoparticles extracted from the whole Gynostemma pentaphyllum herb.

[0010] Furthermore, the nucleic acid leakage rate of the chitosan-coated Gynostemma pentaphyllum-derived nanovesicles is less than 30%, and the protein leakage rate is less than 35%.

[0011] The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles provided by this invention exhibit excellent physicochemical properties and gastrointestinal stability. The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles possess a clear core-shell structure, with the chitosan shell uniformly coating the core surface of the nanovesicles. The surface charge is completely reversed from negative to positive (+27mV~+37mV), and the particle size is 130~175nm. Chitosan interface engineering effectively protects the intrinsic active components of the vesicles, maintaining a stable particle size distribution and low polydispersity index in both simulated gastric and intestinal fluids. The nucleic acid leakage rate is less than 30%, and the protein leakage rate is less than 35%. Silver staining protein spectroscopy shows a high degree of consistency in protein composition before and after coating, indicating that the intrinsic bioactive components of the vesicles are completely preserved during the interface engineering process.

[0012] After being coated with chitosan, the nanovesicles achieve specific accumulation and long-term retention in colonic tissue through electrostatic interaction between the positively charged chitosan shell and the negatively charged intestinal mucus layer. Their colonic fluorescence intensity and retention time are significantly better than those of unmodified vesicles, effectively solving the technical defects of poor gastrointestinal stability and short mucosal retention time of natural plant-derived nanovesicles.

[0013] Furthermore, the Gynostemma pentaphyllum-derived nanovesicles have a lipid bilayer structure, a hydration diameter of 92~123 nm, and a Zeta potential of -28~-22 mV.

[0014] A method for preparing chitosan-coated Gynostemma pentaphyllum-derived nanovesicles includes the following steps:

[0015] S100. Homogenize the whole herb powder of Gynostemma pentaphyllum in cold PBS (phosphate-buffered saline), centrifuge to remove cell debris, collect the supernatant and perform ultracentrifugation to precipitate exosome-like nanoparticles, wash with PBS and collect to obtain Gynostemma pentaphyllum-derived nanovesicles.

[0016] S200. Dissolve chitosan in acetic acid solution, adjust pH to 5.0~6.0, filter to obtain chitosan solution; mix the Gynostemma pentaphyllum-derived nanovesicle suspension obtained in step S100 with chitosan solution, incubate at room temperature, centrifuge to collect precipitate, resuspend in buffer solution, wash repeatedly to obtain chitosan-coated Gynostemma pentaphyllum-derived nanovesicles.

[0017] This invention is the first to apply chitosan coating technology to the surface functionalization modification of nanovesicles derived from Gynostemma pentaphyllum. While natural Gynostemma pentaphyllum vesicles are rich in bioactive components, they are easily cleared by the gastrointestinal barrier after oral administration, limiting their therapeutic efficacy. Conventional chitosan coating strategies are mostly designed for synthetic nanocarriers and are difficult to directly adapt to the complex surface characteristics of natural vesicles. To address this technological gap, this invention designs a matching extraction and self-assembly process based on the unique surface charge density, particle size distribution, and active cargo composition of Gynostemma pentaphyllum vesicles. Utilizing the electrostatic interaction between the natural negative charge of the vesicles and the positive charge of chitosan in a room-temperature aqueous phase, a composite delivery carrier with a clear core-shell structure is constructed in one step. The entire preparation process is mild, requiring no organic solvents or high-temperature treatment, effectively protecting the active components such as proteins and nucleic acids inside the vesicles from damage. The chitosan shell acts as a protective barrier before reaching the lesion, enhancing gastrointestinal stability and reducing leakage of contents; its positively charged surface endows the vesicles with significant mucus adhesion ability, greatly prolonging colonic retention time. The intrinsic bioactive substances of Gynostemma pentaphyllum vesicles are completely preserved during this process, allowing them to fully exert their microecological regulatory functions upon reaching the colon. Thus, chitosan and Gynostemma pentaphyllum vesicles form a clear functional complementarity in this invention. Chitosan solves the delivery bottleneck of "how to reach," while Gynostemma pentaphyllum vesicles assume the therapeutic responsibility of "how to take effect after arrival." Together, they achieve a simultaneous improvement in the delivery performance and therapeutic effect of oral nanomedicines.

[0018] Further, in step S100, the plant powder is homogenized in cold PBS, then centrifuged at 3-5°C to remove cell debris, and the supernatant is ultracentrifuged at 3-5°C for 50-70 minutes to precipitate exosome nanoparticles; the precipitate is washed with PBS and collected by repeated ultracentrifugation at 3-5°C.

[0019] Further, the suspension of Gynostemma pentaphyllum-derived nanovesicles obtained in step S100 was mixed with chitosan solution, incubated at 20-28°C, centrifuged at 3-5°C for 15-25 min to collect the precipitate, and the precipitate was resuspended in an equal volume of neutral MES buffer (2-morpholino)ethanesulfonic acid, and washed 1-3 times to obtain chitosan-coated Gynostemma pentaphyllum-derived nanovesicles.

[0020] An application of the chitosan-coated Gynostemma pentaphyllum-derived nanovesicles, specifically their use in the preparation of drugs for treating inflammatory bowel disease.

[0021] The mechanism of action of the chitosan-coated Gynostemma pentaphyllum-derived nanovesicles is based on their synergistic therapeutic effect through the microbiota-inosine-PPARγ axis. Specifically, the nanovesicles promote endogenous inosine production by enhancing colonic retention and regulating the gut microbiota. Inosine, as a key signaling metabolite, activates the PPARγ nuclear receptor pathway in intestinal epithelial cells. On the one hand, it inhibits the NF-κB-driven inflammatory response by downregulating p65 nuclear translocation and pro-inflammatory mediators such as iNOS, COX-2, IL-6, and IL-1β. On the other hand, it restores intestinal barrier function by upregulating the expression of tight junction proteins Claudin-1, Occludin, ZO-1, and mucin MUC2. Transcriptomic analysis showed that the nanovesicles reversed the disease-related transcriptomic profile, with the PPAR signaling pathway being one of the most significantly enriched core pathways, and the expression of the key gene Pparg was restored. Furthermore, the anti-inflammatory and barrier-protective effects could be completely eliminated by using the PPARγ-specific antagonist GW9662, confirming that PPARγ activation is the core molecular basis for the therapeutic effect of these nanovesicles.

[0022] Furthermore, the chitosan-coated Gynostemma pentaphyllum-derived nanovesicles are administered orally.

[0023] The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles provided by this invention exhibited significant therapeutic effects in a DSS-induced acute colitis model. Oral administration of the nanovesicles significantly alleviated weight loss in model mice, markedly reduced Disease Activity Index (DAI) scores, effectively inhibited colonic shortening, and histopathological analysis showed a significant reduction in inflammatory infiltration, repair of crypt structures, and a marked recovery in goblet cell density. The therapeutic effect was comparable to that of the positive control drug sulfasalazine, and superior to that of unmodified Gynostemma pentaphyllum-derived nanovesicles.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles of this invention not only enhance the gastrointestinal stability of oral formulations but also achieve effective retention in the colon. In the lesion region, these vesicles can remodel the gut microbiota composition and specifically enrich the purine metabolite inosine. The enriched inosine then acts as an upstream signal to activate the host PPARγ signaling pathway, thereby synergistically inhibiting colonic inflammation and restoring the integrity of the intestinal mucosal barrier. This integrated strategy, combining engineered nanocarriers with regulation of the microbiota-metabolism-immune axis, provides a promising platform for oral treatment of inflammatory bowel disease, and its efficacy has been demonstrated in experimental animal models of colitis. Attached Figure Description

[0026] Figure 1 Figures showing the preparation, characterization, and in vitro evaluation of GP-PDNVs and CS-GP-PDNVs;

[0027] Figure 2 A graph showing the in vivo biodistribution and colon targeting efficiency of GP-PDNVs and CS-GP-PDNVs;

[0028] Figure 3 A graph showing the correlation between CS-GP-PDNVs and DSS-induced colitis in mice;

[0029] Figure 4 A diagram relating CS-GP-PDNVs to the gut microbiota structure of DSS-induced colitis mice;

[0030] Figure 5 A graph showing the correlation between CS-GP-PDNVs and DSS-induced colitis in mice;

[0031] Figure 6 A graph illustrating the therapeutic effects of CS-GP-PDNVs through the PPAR signaling pathway as revealed by transcriptomic analysis;

[0032] Figure 7A diagram illustrating how CS-GP-PDNVs reduce inflammation and protect barrier integrity in vitro via the inosine-mediated PPARγ activation pathway;

[0033] Figure 8 A diagram related to in vivo verification that the inosine-PPARγ axis is the core mechanism of the therapeutic effect of CS-GP-PDNVs;

[0034] Figure 9 Analysis of mouse colon tissue confirmed that CS-GP-PDNVs synergistically regulate inflammation and barrier repair through the inosine-PPARγ axis.

[0035] Figure 10 Figure showing the in vivo biosafety assessment of CS-GP-PDNVs.

[0036] Figure label:

[0037] Figure 1 In the diagram, A is a schematic diagram of the process for isolating GP-PDNVs from Gynostemma pentaphyllum; B is a representative transmission electron microscope (TEM) image of the isolated GP-PDNVs (scale bar: 500 nm); the inset is a high-magnification view of a single vesicle, showing the bilayer membrane structure (scale bar: 50 nm); C shows the particle size distribution of GP-PDNVs (top image, determined by nanoparticle tracking analysis NTA) and the zeta potential distribution (bottom image, determined by laser Doppler electrophoresis); D is a schematic diagram of the preparation process of CS-GP-PDNVs; E is a representative TEM image of CS-GP-PDNVs, showing the core-shell structure with an electron-dense chitosan coating layer; top image scale bar: 500 nm; bottom image shows a high-magnification view, scale bar: 50 nm; F shows the CS-GP- Particle size distribution (top) and zeta potential distribution (bottom) of PDNVs; G is the nucleic acid (OD260) and protein (OD280) leakage curves of GP-PDNVs and CS-GP-PDNVs after 12 hours of incubation in SIF; H is the nucleic acid and protein leakage curves of GP-PDNVs and CS-GP-PDNVs in SGF; I is the silver-stained protein map of GP-PDNVs and CS-GP-PDNVs; J is the fluorescence microscopy image of Caco-2 cells after 12 hours of co-incubation with DiR-labeled GP-PDNVs or CS-GP-PDNVs, with cell nuclei stained with DAPI (blue); Scale bar: 25µm; ***p<0.001; Data are expressed as mean ± standard deviation (SEM).

[0038] Figure 2In the table, A represents representative near-infrared in vivo fluorescence images of mice at different time points after oral administration of DiR-labeled GP-PDNVs; B represents representative near-infrared in vivo fluorescence images of mice at different time points after oral administration of DiR-labeled CS-GP-PDNVs; C represents the gastrointestinal fluorescence intensity curve of the DiR-GP-PDNVs group; D represents the gastrointestinal fluorescence intensity curve of the DiR-CS-GP-PDNVs group; E represents representative in vitro fluorescence images of the main organs (heart, liver, spleen, lung, and kidney) of the GP-PDNVs group; and F represents representative in vitro fluorescence images of the main organs (heart, liver, spleen, lung, and kidney) of the CS-GP-PDNVs group. Figure 3 In the table, A is a schematic diagram of the in vivo colitis model and treatment regimen; B is the percentage change in body weight relative to the initial body weight (day 0) during the 7-day experiment; C is the daily calculated Disease Activity Index (DAI) score, which integrates weight loss, stool consistency, and rectal bleeding; D is a representative gross image of the colon in each group at the experimental endpoint (day 7); E is a quantitative analysis of colon length; F is a representative hematoxylin-eosin (H&E) stained section of distal colon tissue, scale bar: 200µm (panoramic), 100µm (local magnification); G is the histopathological score based on H&E staining, assessing inflammatory cell infiltration, crypt damage, and epithelial integrity; H is a representative section stained with alexandrite blue to identify goblet cells in colonic crypts, scale bar: 100µm (panoramic), 50µm (local magnification); I is a quantitative analysis of the number of goblet cells per crypt. (n=6); **p<0.01, ***p<0.001; data are expressed as mean ± SEM. Figure 4 In the table, A shows the α-diversity of gut microbiota assessed using Shannon, Simpson, and Chao1 indices (n=6 per group); B shows the principal coordinate analysis (PCoA) plot based on Bray-Curtis distance, displaying β-diversity; C shows the Bray-Curtis rank / distance box plot; D shows the relative abundance of gut microbiota at the phylum level (top 10 phyla); E shows the relative abundance and statistical significance of key bacterial genera (top 15 genera with significant changes); F shows the differential abundance bar chart; G shows the LDA score plot for taxonomic indices; H shows the predicted functional pathways of gut microbiota based on PICRUSt2 analysis (top 10 enriched pathways compared to the CS-GP-PDNVs group; ***p<0.001; data are expressed as mean ± SEM).

[0039] Figure 5In the figures, A is the PCA plot of colonic contents metabolomics; B is the cluster heatmap; C is the volcano plot of differentially regulated metabolites between the DSS group and the CS-GP-PDNVs group, with red dots indicating significantly upregulated metabolites and blue dots indicating significantly downregulated metabolites; D is the bubble plot of KEGG pathway enrichment analysis based on differentially regulated metabolites, with bubble size and color representing the number of metabolites and the significance of enrichment, respectively; E is the heatmap of the relative abundance of key metabolites in the purine metabolism pathway; F is the quantitative analysis of inosine levels in colonic contents; G is the quantitative analysis of guanosine levels in colonic contents; H is the quantitative analysis of hypoxanthine levels in Caco-2 cells; I is the quantitative analysis of inosine levels in Caco-2 cells; **p<0.01, ***p<0.001, ns: no significant difference; data are expressed as mean ± SEM.

[0040] Figure 6 In the table, A represents principal component analysis (PCA) of gene expression profiles in colon tissue; B represents a hierarchical clustering heatmap of all differentially expressed genes (DEGs) among the groups; C represents a bar chart showing the number of upregulated and downregulated DEGs in the comparison between the "DSS vs. control group" and the "DSS+CS-GP-PDNVs vs. DSS group"; D represents a Venn diagram showing the overlap of DEGs obtained from the above two comparisons; E represents a heatmap showing the expression patterns of 93 overlapping DEGs in each experimental group; F represents a bubble chart of KEGG pathway enrichment analysis of the 93 overlapping DEGs, with the PPAR signaling pathway highlighted; G represents the mRNA expression levels of Ppara, Pparg, Apoc3, and Olr1 in mouse colon tissue; **p<0.01, ***p<0.001; data are expressed as mean ± SEM.

[0041] Figure 7In the table, A shows the cell viability of Caco-2 cells after different LPS treatment times determined by the CCK-8 assay; B shows the protein expression levels of PPARγ, iNOS, COX-2, IL-6, and IL-1β in cells pretreated with GP-PDNVs or CS-GP-PDNVs and then stimulated with LPS by Western blot analysis; C shows the relative mRNA expression levels of ADRP-1, CD63, and FABP4 determined by qRT-PCR; and D shows the secretory tight junction proteins (Claudin-1, Occludin, ZO2, and Z2O3) in the cell culture supernatant determined by ELISA. -1) concentration; E is the immunoblot band; F is the secretion of tight junction proteins as analyzed by ELISA; G is a representative immunofluorescence image showing the nuclear translocation of PPARγ (green) and C / EBPα (green) and the expression of iNOS (red), with cell nuclei counterstained with DAPI (blue), scale bar: 50µm; H is the ELISA analysis showing that both inosine and CS-GP-PDNVs can increase the secretion of tight junction proteins, and this effect was blocked by GW9662 combined treatment; I is the immunofluorescence analysis of the corresponding PPARγ-C / EBPα nuclear translocation and iNOS expression. Scale bar: 50µm; **p<0.01, ***p<0.001; data are expressed as mean ± SEM.

[0042] Figure 8 In the table, A is a schematic diagram of the experimental design, including seven treatment groups involving the PPARγ antagonist GW9662 and inosine supplementation; B shows the changes in body weight of mice in each group during the 7-day experiment; C shows the DAI score; D and E show the quantitative analysis of colon length at the experimental endpoint; F shows HE-stained sections, with a panoramic scale bar of 200µm and a local magnification scale bar of 100µm; G shows the quantitative analysis of the number of goblet cells per crypt; **p<0.01, ***p<0.001, ns: no significant difference; data are expressed as mean ± SEM.

[0043] Figure 9In the image, A shows a representative multiplex immunofluorescence image of a colon section, with staining revealing nuclear receptors PPARγ (green), p65 (red), and iNOS (yellow). Cell nuclei are counterstained with DAPI (blue). Scale bar: 50µm. B shows the quantitative analysis of the percentages of PPARγ-positive cells, p65-positive cells, and iNOS-positive cells based on the image in A. C shows the detection of PPARγ, p65, Nos2 (iNOS), Zo1, Ocln (Occludin), and Muc2 in colon tissue by qRT-PCR. Relative mRNA expression levels were normalized using Gapdh as an internal control; D is a representative multiplex immunofluorescence image of colon sections, showing tight junction proteins ZO-1 (green) and Occludin (red) and mucin MUC2 (yellow), with cell nuclei counterstained with DAPI (blue), scale bar: 50µm; E is the quantitative analysis of the positive cell rate of ZO-1, Occludin and MUC2 based on the image in D; *p<0.05, **p<0.01, ***p<0.001; data are expressed as mean ± SEM.

[0044] Figure 10 In the diagram, A is a schematic diagram of the acute toxicity experiment design. Healthy mice were orally administered CS-GP-PDNVs or PBS solvent daily for 7 consecutive days as a control; B shows the change in mouse body weight during the 7-day administration period; C shows the organ indices (organ weight / body weight × 100%) of the heart, liver, spleen, lungs, kidneys, and brain at the experimental endpoint; D shows the main serum biochemical indicators reflecting liver and kidney function: ALT, AST, TP, CREA, and UREA; E shows representative hematoxylin-eosin (H&E) stained sections of the major organs (heart, liver, spleen, lungs, kidneys, and brain) of mice in the control and CS-GP-PDNVs treatment groups, scale bar: 50µm; F shows representative hematoxylin-eosin (H&E) stained sections of the control and various segments of the entire gastrointestinal tract (duodenum, jejunum, ileum, cecum, colon, and rectum), scale bar: 100µm; ns: no significant difference; data are expressed as mean ± SEM. Detailed Implementation

[0045] 1. Materials and Methods

[0046] 1.1. Materials and Reagents

[0047] Chitosan (catalog number: 419419) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Its weight-average molecular weight (Mw) was 315 kDa (determined by gel permeation chromatography), and its degree of deacetylation was 75% (determined by nuclear magnetic resonance spectroscopy), corresponding to a calculated average monomer molecular weight of 171.67 g / mol. According to the supplier's specifications, the viscosity of this chitosan is 800–2000 cP (1 wt% dissolved in 1% acetic acid solution, 25°C), consistent with its high molecular weight grade.

[0048] FITC and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The PPARγ antagonist GW9662 was also purchased from Sigma-Aldrich. The near-infrared lipophilic dye DiR was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). All cell culture media and additives were purchased from Gibco (Carlsbad, California, USA).

[0049] 1.2. Plant materials and extraction

[0050] Gynostemma pentaphyllum saponins were extracted from the dried whole herb. The plant powder was homogenized in cold PBS and then centrifuged sequentially at 500×g, 3,000×g, and 10,000×g at 4°C to remove cell debris. The final supernatant was ultracentrifuged at 100,000×g for 60 min at 4°C to precipitate exosome-like nanoparticles. The precipitate was washed with PBS and collected by repeated ultracentrifugation at 4°C.

[0051] 1.3. Chitosan coating

[0052] Preparation of chitosan stock solution (1 mg / mL): Dissolve 10 mg of chitosan powder in 9 mL of pure water, add 500 μL of glacial acetic acid dropwise until a clear solution is formed, adjust the pH to 5.5 with 1 M NaOH, and bring the volume to 10 mL. Filter the solution through a 0.22 μm filter membrane and store at 4°C. For coating, mix 1 mL of GP-PDNV suspension (1 mg / mL) with the chitosan stock solution with gentle stirring, incubate at room temperature for 60 minutes, then centrifuge at 100,000 g for 20 minutes at 4°C, discard the supernatant, resuspend the precipitate in an equal volume of MES buffer (pH 7.0), and wash twice.

[0053] 1.4. Vesicle Characterization

[0054] Transmission electron microscopy (TEM) observation: 10 µL of freshly prepared GP-PDNVs or CS-GP-PDNVs suspension was dropped onto a carbon film copper grid, adsorbed for 5 minutes, and excess liquid was absorbed with filter paper. Then, 10 µL of 2% (w / v) uranyl acetate solution was used for negative staining for 1 minute, excess stain was absorbed, and the mixture was allowed to air dry at room temperature before imaging. Particle size distribution and concentration were analyzed using nanoparticle tracking analysis (NTA) with a Malvern NanoSight NS300 at 25°C. Zeta potential was measured using a Malvern Zetasizer Nano ZS at 25°C. Surface chemical composition was analyzed using X-ray photoelectron spectroscopy (Thermo Scientific K-Alpha).

[0055] 1.5. Non-targeted lipidomics analysis

[0056] To analyze lipidomics characteristics, a modified methyl tert-butyl ether (MTBE) extraction method was used to treat GP-PDNVs. In short, samples containing internal standard lipids were homogenized in a methanol / water mixture, followed by the addition of MTBE. After sonication in an ice bath (15 min), phase separation was achieved by centrifugation at 12,000 × g for 10 min at 4°C. The lipid-rich upper organic phase was carefully recovered, evaporated to dryness under a gentle nitrogen stream, and finally redissolved in isopropanol / acetonitrile solution (9:1, v / v).

[0057] Lipid extracts (2 μL injection) were analyzed using a UHPLC-MS / MS platform. Separation was performed using a Waters ACQUITYUPLC CSH C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of phase A (acetonitrile:water = 60:40, v / v) and phase B (isopropanol:acetonitrile = 90:10, v / v), both containing 0.1 mM ammonium formate and 0.1% formic acid. Linear gradient elution was used (flow rate 0.3 mL / min), with phase B increasing from 30% to 100% within 20 min. Mass spectrometry data were acquired using a Q-Exactive Plus high-resolution mass spectrometer in electrospray ionization (ESI) mode. MS parameters were optimized as follows: scan range m / z 200–1500, spray voltage 3.2 kV, capillary temperature and auxiliary gas heater temperature both set to 320°C.

[0058] 1.6. Stability assessment in simulated gastrointestinal fluids

[0059] Stability of simulated gastric fluid (SGF, pH 1.2): Nanoparticles were incubated in SGF containing pepsin at 37°C for 30 minutes. Stability of simulated intestinal fluid (SIF, pH 6.8): Nanoparticles were incubated in SIF containing trypsin and lipase at 37°C for 2 hours. After incubation and centrifugation, the supernatant was collected to determine the content of leaked protein and nucleic acids. Simultaneously, changes in particle size, polydispersity index (PDI), and zeta potential over time were monitored using a Zetasizer (Zetasizer Pro, Malvern, UK). Leakage was also assessed by measuring the optical density values ​​of the supernatant at OD260 and OD280.

[0060] 1.7. Cell Culture

[0061] Human colorectal adenocarcinoma cells (Caco-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% non-essential amino acids and 1% penicillin-streptomycin at 37°C under a 5% CO2 atmosphere.

[0062] 1.8. Cellular uptake experiment

[0063] Caco-2 cells were seeded in confocal culture dishes. When the cell confluence reached approximately 70%, DiR-labeled GP-PDNVs or CS-GP-PDNVs were added, and the cells were incubated at 37°C for 12 hours. After incubation, the cells were washed, fixed, and their nuclei were counterstained with DAPI. Images were acquired using an Olympus FV3000RS confocal laser scanning microscope (Olympus, Japan).

[0064] 1.9. In vitro inflammation and barrier function model

[0065] Establishing an inflammation model: Cells were treated with 1 µg / mL LPS at 37°C for 6 hours. Protective studies: Cells were pretreated with GP-PDNVs, CS-GP-PDNVs, or inosine (200 µM, Sigma-Aldrich Chemical, Evry, France) at 37°C for 2 hours before LPS stimulation. Inhibition experiments: One hour before the addition of the protective agent, the PPARγ antagonist GW9662 (10 µM) was added at 37°C.

[0066] 1.10. DSS-induced colitis model and treatment

[0067] Four-week-old male C57BL / 6 mice were randomly assigned to groups (n=6). Acute colitis was induced for 7 consecutive days by adding 3% (w / v) DSS to drinking water. Treatment groups received daily oral gavage with PBS (DSS group), GP-PDNVs 10 mg / mL (DSS+GP-PDNVs group), CS-GP-PDNVs 10 mg / mL (DSS+CS-GP-PDNVs group), sulfasalazine 50 mg / kg (DSS+sulfasalazine group), inosine (50 mg / kg), or GW9662 (1 mg / kg). Body weight, fecal consistency, and rectal bleeding were monitored daily, and the disease activity index (DAI) was calculated. All animal experiments were approved by the Animal Ethics Committee of Hubei University of Medicine (Approval No.: 2025-291). Control group mice drank normal water and received PBS via gavage.

[0068] 1.11. Live imaging

[0069] Biodistribution studies: DiR-labeled nanoparticles were administered orally via gavage. Mice were anesthetized at specified time points and imaged using the IVIS Spectrum (PerkinElmer, Waltham, MA, USA) in vivo imaging system. At the end of the experiment, major organs were removed for ex vivo imaging.

[0070] 1.12. Real-time quantitative PCR (qRT-PCR)

[0071] Total RNA was extracted from colon tissue or cells using TRIzol reagent. cDNA was synthesized using the PrimeScript RT kit (Takara, Japan). qRT-PCR was performed using SYBR Green premixed buffer (Thermo Fisher Scientific, USA) on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression levels of Pparγ, Nfkb1 (p65), Nos2 (iNOS), Zo1, Ocln (occludin), Muc2, Adrp1, and Cd63 were detected, with Gapdh as an internal control, and calculated using the 2^(-ΔΔCt) method.

[0072] 1.13. Western blot analysis

[0073] Cell lysates were prepared using RIPA lysis buffer containing a protease inhibitor (R0010, Solarbio, China). Equal volumes of protein were separated by SEMS-PAGE and transferred to a PVDF (0.45 µm) membrane (IPVH00010, Immobilon-P, Merck Millipore). After blocking with skim milk, the membrane was incubated overnight at 4°C with primary antibody. After washing, it was incubated with secondary antibody at room temperature for 2 hours, and the signal was detected using chemiluminescence on a Tanon-5200 system (Tanon, Shanghai, China).

[0074] 1.14. Immunofluorescence and Multiplex Staining

[0075] Colonic tissue was fixed, paraffin-embedded, and sectioned. After dewaxing, antigen retrieval, and blocking, sections were incubated overnight at 4°C with primary antibody. Multiple staining was performed using Opal fluorescent dye for sequential labeling. Cell nuclei were counterstained with DAPI. Images were acquired using an Olympus FV3000RS automated imaging system and analyzed using ImageJ software.

[0076] 1.15. Enzyme-linked immunosorbent assay (ELISA)

[0077] The secretion level of the target protein was quantified by ELISA. Culture supernatants from Caco-2 cells under different treatments were collected, centrifuged at 1,000×g for 10 minutes at 4°C to remove cells and debris, aliquoted, and stored at -80°C for later analysis. For the assay, samples and serially diluted standards were added to 96-well plates pre-coated with capture antibodies and incubated at room temperature (25°C) for 2 hours. After thorough washing, biotinylated detection antibodies were added and incubated for 1 hour, followed by incubation with streptavidin-horseradish peroxidase conjugate for 30 minutes. Tetramethylbenzidine substrate was added, and the plate was incubated at room temperature in the dark for 15 minutes. Immediately after stopping the reaction, the absorbance was measured at 450 nm (reference wavelength 570 nm) using a microplate reader. All samples were replicated, and protein concentrations were calculated based on the standard curve. The experiment was independently repeated three times.

[0078] 1.16.16S rRNA gene sequencing and bioinformatics analysis

[0079] Microbial DNA was extracted from cecal contents using the QIAamp DNA Stool Mini Kit. The V3-V4 hypervariable region of the 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. The raw sequences were processed using QIIME2. Alpha diversity (Shannon, Simpson, and Chao1 indices) and Beta diversity (principal coordinate analysis based on Bray-Curtis distance) were calculated. Differential flora and functional pathways were analyzed using LEfSe and predicted using PICRUSt2.

[0080] 1.17. Non-targeted metabolomics

[0081] Metabolites were extracted from colonic contents using a methanol-water solution. After centrifugation and filtration, the extracts were analyzed by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS). Raw data were used for peak identification, alignment, and annotation using MS-DIAL software. Metabolite identification was performed by comparing the obtained MS / MS spectra and precise masses with a standard spectral library. Differential metabolite screening: variable projection importance (VIP) > 1.0 in multivariate analysis and p-value < 0.05 in univariate analysis.

[0082] 1.18. RNA Sequencing and Transcriptome Analysis

[0083] A library was constructed from total RNA from colon tissue and sequenced on the Illumina NovaSeq platform. Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of |log2FC|>1 and a corrected p-value <0.05. KEGG pathway enrichment analysis was performed on DEGs using the clusterProfiler software package.

[0084] 1.19. Histological analysis

[0085] Colonic tissue was fixed in 10% neutral buffered formalin for 24 hours, dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Serial sections (4 µm thick) were prepared. After dewaxing and rehydration, sections were stained with hematoxylin (5 min) and eosin, or alicin blue, to specifically show acidic mucin. Sections were routinely dehydrated, cleared, and mounted with neutral resin. Positive and negative controls were included for all staining to ensure batch consistency. Histopathological evaluation was performed independently by two pathologists unaware of the grouping. Goblet cell count: Under a 400x optical microscope (Olympus, Tokyo, Japan), the number of alicin blue-positive cells in at least 30 well-oriented, longitudinally cut crypts was counted, and the results are expressed as the mean number of goblet cells per crypt.

[0086] 1.20. Security Assessment

[0087] Acute toxicity study: Healthy mice were orally administered CS-GP-PDNVs daily for 7 consecutive days. Body weight was recorded. Serum biochemical parameters (ALT, AST, TP, CREA, UREA) were measured. Major organs were weighed to calculate organ indices, and H&E staining was performed.

[0088] 1.21. Statistical Analysis

[0089] All statistical analyses were performed using GraphPad Prism (version 10.4.1) and SPSS (version 27.0). Data are expressed as mean ± standard error (SEM). Normality was assessed using the Shapiro-Wilk test. For two-group comparisons: Student's t-test was used for normally distributed data, and Mann-Whitney U test was used for non-normally distributed data. For multiple-group comparisons: one-way ANOVA was used for normally distributed data, and Kruskal-Wallis test was used for non-normally distributed data, followed by Tukey's post-hoc test (for homogeneity of variance) or Dunnett's T3 test (for heterogeneity of variance) depending on the homogeneity of variance. A p-value < 0.05 was considered statistically significant.

[0090] 2. Results

[0091] 2.1 Chitosan Interface Engineering Induces Physicochemical Property Transformation of Gynostemma pentaphyllum Exosome Nanovesicles and Enhances Their Gastrointestinal Stability. Plant-derived nanovesicles (PDNVs) represent a promising oral delivery platform due to their inherent biocompatibility and bioactivity. However, their transformation potential is often limited by poor physicochemical stability and insufficient epithelial interaction under gastrointestinal conditions. To address these challenges, this invention engineered GP-PDNVs through chitosan interface coating to modulate surface properties and improve oral delivery performance.

[0092] GP-PDNVs according to Figure 1 The procedure shown in A was successful in separating the vesicles. Transmission electron microscopy (TEM) revealed a typical goblet vesicle morphology with a clear lipid bilayer structure. Figure 1 B), consistent with previously reported plant-derived nanovesicles. Nanoparticle tracking analysis (NTA) showed a relatively uniform particle size distribution, with an average hydrated diameter of 107.5 ± 15.3 nm (B). Figure 1 C, top), Zeta potential analysis showed that the surface carried a negative charge (−25.1±3.1 mV) ( Figure 1 C, down).

[0093] To overcome the gastrointestinal barrier, GP-PDNVs are coated with chitosan via electrostatic assembly. Figure 1 (D schematic diagram). X-ray photoelectron spectroscopy (XPS) showed a characteristic N1s peak at approximately 399.8 eV in CS-GP-PDNVs, which is absent in natural GP-PDNVs, confirming successful surface coupling of chitosan (Figure S1A). TEM imaging further revealed a clear core-shell structure, characterized by an electron-dense outer layer surrounding the vesicle core. Figure 1 E). Consistently, NTA analysis showed an increase in hydrated diameter to 152.8 ± 20.1 nm (E). Figure 1 F, above), while the surface charge completely reverses from negative to positive (+31.7±4.5mV) ( Figure 1 F, below), together demonstrate effective interface engineering and surface reconstruction.

[0094] Next, the stability of the vesicles was evaluated under simulated gastrointestinal conditions. Natural GP-PDNVs exhibited rapid aggregation in simulated gastric fluid (SGF, pH 2.0), characterized by increased particle size and polydispersity index (PDI), indicating structural instability. In contrast, CS-GP-PDNVs maintained a stable particle size distribution and low PDI value throughout the incubation process (Figs. S1B-C). Similar stability advantages were also observed in simulated intestinal fluid (SIF, pH 6.8), and dynamic zeta potential monitoring confirmed a persistent positive surface charge (Fig. S1D). Functional integrity assays further showed that CS-GP-PDNVs exhibited significantly reduced cargo leakage compared to unmodified vesicles, with nucleic acid and protein leakage remaining below 30% and 35%, respectively. Figure 1 G, H). Silver staining analysis showed a high degree of similarity in protein profiles between coated vesicles and native vesicles. Figure 1 I) indicates that the internal cargo is preserved during the interface engineering process.

[0095] Given that surface charge is a key determinant of biological interfacial interactions, this invention used the Caco-2 intestinal epithelial model to assess cellular uptake. After 12 hours of incubation, DiR-labeled CS-GP-PDNVs exhibited significantly enhanced intracellular fluorescence compared to GP-PDNVs. Figure 1 The improved internalization of the epithelium (J) indicates enhanced epithelial function. This enhancement can be attributed to the electrostatic attraction between the positively charged chitosan shell and the negatively charged cell membrane, as well as improved structural stability that facilitates endocytic uptake. Extracellular vesicles are typically rich in specific lipid components that play a crucial role in their structural integrity and biological function. To further elucidate the compositional characterization of engineered nanovesicles, this invention performed untargeted lipidomics analysis to characterize the lipid composition of GP-PDNVs. The results showed a high proportion of unsaturated fatty acids in the nanovesicles. Specifically, polyunsaturated fatty acids (PUFAs) accounted for 51.29% of the total lipid content, followed by monounsaturated fatty acids (MUFAs) at 18.37%, saturated fatty acids (SFAs) at 15.51%, and diunsaturated fatty acids (DUFAs) at 13.63% (Figure S2). This lipid profile is highly consistent with the recent lipidomics characterization of Gynostemma pentaphyllum-derived vesicles by Wang et al. The abundant presence of PUFAs may significantly contribute to the membrane fluidity, structural stability, and potential intrinsic biological activity of GP-PDNVs.

[0096] These findings demonstrate that chitosan interface engineering induces synergistic physicochemical property transformations, including surface charge reversal, enhanced structural robustness, and improved biological interface interactions, providing a mechanistic basis for the enhanced oral delivery performance and biological effects explored in subsequent chapters.

[0097] 2.2 Engineered CS-GP-PDNVs achieve enhanced colonic retention and superior therapeutic effects in DSS-induced colitis. To determine whether chitosan interface engineering can be translated into improved in vivo delivery behavior and therapeutic effects, this invention evaluated CS-GP-PDNVs in a DSS-induced acute colitis model.

[0098] In vivo animal imaging based on DiR was used to investigate the in vivo fate of engineered nanovesicles. Following oral administration, natural GP-PDNVs exhibited rapid gastrointestinal transit, with only weak and transient fluorescence detected in the colonic region. Figure 2 A, C). In contrast, DiR-CS-GP-PDNVs exhibited significant colon-specific accumulation and a significantly prolonged retention time ( Figure 2 B, D). Ex vivo imaging further confirmed the preferential enrichment of CS-GP-PDNVs in colon tissue, while the fluorescence signals in other organs remained the lowest and comparable in both groups. Figure 2 These findings suggest that chitosan coating fundamentally reprograms the in vivo delivery kinetics of GP-PDNVs by enhancing mucosal adhesion and local retention, thereby increasing exposure at sites of inflammation. The enhanced gastrointestinal stability of CS-GP-PDNVs is primarily attributed to the polycationic properties of the chitosan coating. In the highly acidic gastric environment, the amino groups of chitosan are rapidly protonated, inducing the formation of a robust, hydrated gel network. This physical barrier effectively protects the lipid bilayer of the nanovesicles from acid-induced dissociation and pepsin degradation.

[0099] The disease outcomes in DSS-treated mice were then assessed. Figure 3 A). DSS administration induced progressive weight loss and an increase in the Disease Activity Index (DAI) score, confirming the successful establishment of the model. Figure 3 BC). Compared with natural GP-PDNVs, CS-GP-PDNVs treatment significantly reduced weight loss and lowered DAI scores, achieving therapeutic effects comparable to the positive control sulfasalazine.

[0100] At a macroscopic level, DSS attack results in significant colonic shortening, a marker of inflammation severity. CS-GP-PDNVs treatment effectively restores colonic length to near-normal levels. Figure 3DE). Histopathological analysis showed that DSS-treated mice exhibited severe inflammatory infiltration, crypt destruction, and epithelial erosion, while CS-GP-PDNVs significantly preserved crypt structure and reduced inflammatory infiltration. Figure 3 FG). Histological scores were significantly improved compared to the GP-PDNVs group and comparable to the positive control sulfasalazine group, indicating enhanced mucosal repair capacity.

[0101] Given the crucial role of goblet cells in maintaining the intestinal chemical barrier, alcine blue staining was used to assess the regeneration of mucus-secreting cells. DSS exposure significantly depleted goblet cells, while CS-GP-PDNVs significantly restored goblet cell density compared to native GP-PDNVs. Figure 3 The results indicate that the mucus layer and structural epithelium repair work synergistically. These results suggest that chitosan interface engineering enhances colonic retention and local bioavailability of GP-PDNVs, which mechanistically translates into superior therapeutic effects characterized by reduced inflammation and full restoration of intestinal barrier integrity.

[0102] 2.3CS-GP-PDNVs reshape the gut microbiota ecosystem and restore inosine-associated purine metabolism signaling

[0103] Mounting evidence suggests that gut microbiota dysbiosis plays a crucial role in the pathogenesis of IBD. To determine whether the therapeutic efficacy of the nanoplatform of this invention involves a microbiota-mediated mechanism, this invention investigated how chitosan interface engineering affects gut microbiome ecology and downstream metabolic output. Given that in vivo pharmacodynamic assessments of this invention showed that CS-GP-PDNVs exhibited superior therapeutic efficacy and colonic retention capacity compared to unmodified GP-PDNVs, this invention strategically selected the CS-GP-PDNVs treatment group as the representative model for all subsequent multi-omics analyses. This approach enabled this invention to elucidate in depth and specifically the underlying ecological and molecular mechanisms driving this optimal therapeutic intervention. Accordingly, this invention performed 16S rRNA gene sequencing on fecal samples to assess the impact of CS-GP-PDNVs on gut microbiota composition and explore the association between microbial alterations and IBD-related phenotypes.

[0104] 16S rRNA sequencing showed that DSS treatment induced significant dysbiosis, manifested as reduced microbial diversity and altered community structure. CS-GP-PDNVs treatment significantly restored α-diversity indices, including Shannon, Simpson, and Chao1 indicators. Figure 4 A). Principal coordinate analysis (PCoA) based on Bray-Curtis distance further indicates that the microbial composition of the CS-GP-PDNVs group shifted towards the healthy control group cluster, reflecting ecological reorganization towards a steady state. Figure 4BC).

[0105] Notably, Alistipes and Alloprevotella were significantly increased, while DSS-associated pathogens were suppressed. Figure 4 DE). LEfSe analysis identified Alistipes as a key discriminant taxonomic group enriched in the CS-GP-PDNVs group ( Figure 4 The emergence of this functional systemic type is particularly important because the Alistipes species are associated with anti-inflammatory metabolites, suggesting that engineered nanovesicles are linked to the reconfiguration of microbiota to a metabolically favorable configuration.

[0106] Furthermore, this invention performed PICRUSt2 analysis to compare predicted metabolic pathways between the DSS group and the CS-GP-PDNVs group. Figure 4 As shown in Figure H, compared with the DSS group, CS-GP-PDNVs treatment significantly enriched several functional pathways. In particular, enrichment was observed in metabolic pathways such as arachidonic acid metabolism and the renin-angiotensin system. These predicted functional changes suggest that CS-GP-PDNVs may modulate the metabolic potential of the gut microbiota, prompting this invention to further conduct non-targeted metabolomics analysis of colonic contents.

[0107] Based on the predicted metabolic pathway changes from PICRUSt2 analysis, this invention subsequently performed non-targeted metabolomics analysis of colonic contents to directly assess the functional consequences of microbiome remodeling. Results showed that the metabolomic landscape significantly shifted towards a healthy phenotype after CS-GP-PDNVs treatment. Figure 5 AB). Among the 154 differentially metabolites identified ( Figure 5 C), purine metabolism has become one of the most significantly regulated pathways ( Figure 5 D). In this pathway, CS-GP-PDNVs treatment significantly restored hypoxanthine and inosine levels (D). Figure 5 EG). Notably, in the Caco-2 cell model, CS-GP-PDNVs selectively restored only inosine levels, not hypoxanthine (EG). Figure 5 (HI). This in vitro finding highlights the importance of inosine as a specifically regulated metabolite.

[0108] 2.4. Transcriptomic Analysis Reveals Activation of the PPARγ Signaling Axis After CS-GP-PDNVs Treatment. To explore the host molecular response behind the therapeutic efficacy of CS-GP-PDNVs, this invention employed RNA sequencing (RNA-seq) to perform transcriptomic analysis on colon tissue. Principal component analysis (PCA) showed significant separation between the experimental groups, with the CS-GP-PDNVs treatment group's transcriptomic landscape shifting towards that of healthy controls, moving away from the DSS model cluster (…). Figure 6A) indicates that the expression patterns of disease-related genes have been significantly normalized.

[0109] Differential expression analysis identified 783 genes significantly regulated by CS-GP-PDNVs treatment, exceeding the number of genes altered solely by DSS-induced inflammation (453 DEGs). Figure 6 BC). Integration of the DEGs dataset using Venn diagram intersection and cluster analysis identified a group of 93 genes that were dysregulated during DSS-induced colitis but recovered to near-baseline levels after CS-GP-PDNVs intervention. Figure 6 D, E). These genes represent potential transcriptional features associated with treatment recovery. Functional enrichment analysis of this core gene set revealed that the PPAR signaling pathway was one of the most significantly enriched pathways. Figure 6 F). Notably, this observation is consistent with metabolomics findings (elevated levels of microbial-derived purine metabolites), suggesting a possible link between metabolic remodeling and host transcriptional regulation.

[0110] To validate the transcriptomic results, this invention performed qRT-PCR analysis on key genes in the PPAR signaling axis. DSS exposure significantly reduced the expression of Ppara and Pparg, while increasing the expression of downstream inflammation-related genes (including Apoc3 and Olr1). CS-GP-PDNVs treatment reversed these transcriptional alterations, restored Pparg expression, and inhibited the abnormal upregulation of downstream targets. Figure 6 In summary, these data suggest that CS-GP-PDNVs treatment is associated with coordinated transcriptional remodeling involving the PPAR signaling pathway, providing molecular evidence for the link between microbial-derived metabolic changes and the host's anti-inflammatory response.

[0111] 2.5. CS-GP-PDNVs and their effector metabolite inosine exert anti-inflammatory and barrier-protective effects by activating PPARγ.

[0112] After multi-omics analysis revealed a coordinating axis involving the gut microbiota, the metabolite inosine, and the host PPARγ pathway, the present invention was then validated directly in vitro.

[0113] In an LPS-induced Caco-2 cell inflammation model, CS-GP-PDNV pretreatment most significantly upregulated PPARγ protein expression. Figure 7 AB), and effectively inhibited the production of key pro-inflammatory mediators such as iNOS, COX-2, IL-6 and IL-1β. Figure 7 B). Meanwhile, CS-GP-PDNVs also showed a significant advantage in promoting the expression of tight junction proteins (Claudin-1, Occludin, ZO-1). Figure 7 C). qRT-PCR confirmed that CS-GP-PDNVs upregulated the expression of downstream target genes of PPARγ ( Figure 7 D).

[0114] To further elucidate the dose-dependent effect of PPARγ, verification experiments were conducted in this invention. For example... Figure 7 As shown in E (left), treatment with incremental concentrations (0, 1, 5, 10 μM) of the specific PPARγ antagonist GW9662 resulted in a corresponding decrease in PPARγ expression, while the expression of pro-inflammatory markers iNOS, COX-2, TNF-α, and IL-1β increased accordingly. Figure 7 E, left). This establishes a clear reverse pharmacological relationship, confirming the crucial role of PPARγ in maintaining cellular homeostasis. GW9662 co-treatment completely eliminated all protective effects conferred by CS-GP-PDNVs, including the loss of anti-inflammatory factor inhibition and the cancellation of barrier protein restoration. Figure 7 (FG). This confirms that activation of the PPARγ signaling pathway is a necessary molecular basis for the function of CS-GP-PDNVs at the cellular level.

[0115] To validate the findings of in vivo metabolomics, this invention directly added inosine in vitro. The results showed that exogenous inosine perfectly replicated the protective effects of CS-GP-PDNVs in a dose-dependent manner, also enhancing PPARγ activation and inhibiting key inflammatory cytokines (including TNF-α and IL-1β). Figure 7 E, right), thereby maintaining barrier integrity ( Figure 7 H, I). More importantly, GW9662 also antagonizes all the beneficial effects of inosine (H, I). Figure 7 H, I). This series of experiments completed the closed-loop mechanism demonstration: CS-GP-PDNVs, through their engineered delivery advantages, may directly or indirectly (through microbiome regulation) increase inosine levels in the local microenvironment; inosine then acts as a key signaling metabolite to activate the PPARγ nuclear receptor pathway in intestinal epithelial cells, ultimately achieving anti-inflammatory and barrier repair effects synergistically.

[0116] 2.6. CS-GP-PDNVs exert their therapeutic effect through the inosine-PPARγ axis.

[0117] To validate the proposed in vivo inosine-PPARγ regulatory axis, this invention conducted a mechanistic intervention study using the selective PPARγ antagonist GW9662 in combination with inosine supplementation. Figure 8A). First, this invention evaluated whether PPARγ activation is essential for the macroscopic therapeutic benefits derived from CS-GP-PDNVs. As expected, oral CS-GP-PDNVs significantly improved DSS-induced colitis, manifested in improved weight recovery, reduced Disease Activity Index (DAI) scores, and prevention of colonic shortening. Figure 8 BD). Notably, these therapeutic effects were largely eliminated after pharmacological inhibition of PPARγ via intraperitoneal injection of GW9662, indicating that PPARγ signaling is a key mediator of the in vivo efficacy of CS-GP-PDNVs. Figure 8 BD).

[0118] Next, this invention investigated whether inosine acts as an upstream effector in this pathway. Direct supplementation with exogenous inosine reproduced the therapeutic effects observed in CS-GP-PDNVs treatment, significantly alleviating macroscopic disease manifestations. Figure 8 Importantly, the protective effect of inosine was also weakened when combined with GW9662 treatment (BC). Figure 8 BE) supports the existence of a functional signaling cascade in vivo, in which inosine promotes therapeutic response in a PPARγ-dependent manner. Histological analysis further confirms this model, showing that CS-GP-PDNVs and inosine-induced mucosal structural repair and goblet cell regeneration both depend on intact PPARγ signaling ( Figure 8 F, G).

[0119] At the molecular level, this invention explores how this axis coordinates inflammation suppression and barrier repair in colonic tissue. Immunofluorescence and qRT-PCR analyses showed that DSS exposure reduced tissue PPARγ expression while promoting nuclear translocation of NF-κB (p65) and upregulation of its downstream target iNOS. Figure 9 A–C). CS-GP-PDNVs or inosine treatment can effectively reverse these pro-inflammatory changes, while combination administration of GW9662 eliminates these protective molecular responses ( Figure 9 Consistent with this, the restoration of tight junction protein (ZO-1 and Occludin) and MUC2 expression also critically depends on PPARγ activity ( Figure 9 (C–E). Integrating macroscopic phenotypes, histological findings, and molecular analyses, these findings support a coherent therapeutic model: engineered CS-GP-PDNVs enhance colonic retention and modulate the gut microbiota ecosystem, leading to increased production of the endogenous metabolite inosine; inosine subsequently acts as a signaling mediator to activate epithelial PPARγ, which coordinates transcriptional programs to suppress NF-κB-driven inflammation and promote mucosal barrier repair, thereby exerting therapeutic efficacy in experimental colitis.

[0120] 2.7. CS-GP-PDNVs exhibited good in vivo biocompatibility.

[0121] To assess the translational feasibility of this oral nanotherapy strategy, this invention systematically evaluated the in vivo biosafety of CS-GP-PDNVs using a short-term, repeated-dose regimen. Healthy mice were orally administered CS-GP-PDNVs daily for 7 consecutive days. Figure 10 A), no obvious signs of systemic toxicity or behavioral abnormalities were observed during this period. Throughout the treatment period, the weight change trend in the CS-GP-PDNVs group was comparable to that in the solvent control group ( Figure 10 B) indicates no significant effect on physiological growth or basal metabolic state. Consistent with this, organ indices of major tissues (including heart, liver, spleen, lungs, kidneys, and brain) showed no significant differences between the two groups. Figure 10 C), indicating no significant organ toxicity. Serum biochemical analysis further supports good safety. Key indicators reflecting liver function (ALT and AST), nutritional status (TP), and renal function (CREA and UREA) were all within the normal physiological range and showed no difference from the control group. Figure 10 D). Furthermore, a comprehensive histopathological evaluation (H&E staining) showed that the major organs ( Figure 10 E) and all segments of the gastrointestinal tract (from the duodenum to the rectum) Figure 10 The tissue structures of F) remained intact, with no changes such as inflammatory infiltration, necrosis, or fibrosis observed. Overall, these results indicate that CS-GP-PDNVs exhibit excellent biocompatibility at therapeutic doses, supporting their potential for further preclinical development as an oral nanotherapy platform.

[0122] 3. Discussion

[0123] This invention develops an orally administered chitosan-functionalized plant-derived nanovesicle platform (CS-GP-PDNVs) and verifies its therapeutic efficacy in experimental colitis. The platform, derived from Gynostemma pentaphyllum, undergoes chitosan surface modification, resulting in a positively charged surface and a clearly defined core-shell nanostructure. Chitosan coating significantly enhances the stability of the vesicles in simulated gastrointestinal fluids, reduces content leakage, protects protein loads, and substantially prolongs colonic retention time—attributable to the electrostatic interaction between the positively charged chitosan layer and the negatively charged mucus layer. It is important to emphasize that chitosan serves only as a pharmacologically inert mucosal adhesion excipient; it has no intrinsic therapeutic effect on severe colitis. The therapeutic effect fundamentally originates from the intrinsic bioactive cargo of GP-PDNVs.

[0124] Enhanced colonic retention led to a profound reshaping of the gut microbiota ecosystem by CS-GP-PDNVs. Multi-omics analysis revealed that treatment not only restored microbial diversity but also selectively enriched beneficial bacteria genera with anti-inflammatory properties (such as *Alistipes* and *Alloprevotella*), creating favorable niches for the colonization of beneficial symbiotic bacteria. PICRUSt2 functional prediction indicated enrichment of the purine metabolism pathway, and subsequent non-targeted metabolomics confirmed that purine metabolism is a regulated core pathway, with significant restoration of inosine levels in colonic contents and Caco-2 cells. This result echoes known studies on barley leaves, pointing to an operable "microbiota-inosine" axis.

[0125] To link changes in microbial metabolism with host response, transcriptomic analysis was performed on colonic tissue. RNA sequencing revealed that CS-GP-PDNVs reversed the disease-related transcriptomic profile, and the PPAR signaling pathway was one of the most significantly enriched pathways in the therapeutic recovery genes. PPARγ, a nuclear receptor highly expressed in the colon, is a key factor in regulating the epithelial barrier, immune homeostasis, and inflammation; various natural products exert IBD protective effects through this pathway. In vitro and in vivo experiments confirmed that both CS-GP-PDNVs and exogenous inosine can activate PPARγ, inhibit pro-inflammatory mediators (iNOS, COX-2, IL-6, IL-1β), and restore tight junction proteins (Claudin-1, Occludin, ZO-1). Crucially, the protective effect completely disappeared after use of the PPARγ inhibitor GW9662, confirming that PPARγ activation is a necessary condition for anti-inflammatory and barrier repair functions, rather than just a related factor.

[0126] This strategy integrates materials engineering with the regulation of the "microbiota-metabolite-host" axis, enabling CS-GP-PDNVs to join the emerging "post-biotic" modulator paradigm—not by delivering a single active ingredient, but by reshaping the gut ecosystem to stimulate the production of endogenous, host-targeting metabolites. Safety assessments showed no systemic toxicity with repeated oral administration (normal body weight, organ indices, serum biochemical parameters, and histological structure), consistent with the excellent biocompatibility of chitosan and plant-derived vesicles. In conclusion, CS-GP-PDNVs provide a promising integrated strategy for oral nanotherapy of inflammatory bowel disease.

[0127] 4. Conclusion

[0128] This invention develops a chitosan-functionalized plant-derived nanovesicle platform (CS-GP-PDNVs) derived from Gynostemma pentaphyllum and demonstrates its therapeutic efficacy in experimental colitis. The invention enhances gastrointestinal stability and colonic retention through chitosan coating, and this platform remodels the gut microbiota and enriches the purine metabolite inosine. Inosine then acts as a key signaling molecule to activate the host PPARγ pathway, thereby suppressing inflammation and restoring intestinal barrier function. This integrated strategy combines materials engineering with the modulation of the "microbiota-metabolite-host" axis, providing a promising approach for oral nanotherapy of inflammatory bowel disease.

Claims

1. A chitosan-coated Gynostemma pentaphyllum-derived nanovesicle, characterized in that, It includes a nanovesicle core derived from Gynostemma pentaphyllum and a chitosan shell layer coating the surface of the core.

2. The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles according to claim 1, characterized in that, The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles have a particle size of 130~175nm and a zeta potential of +27mV~+37mV.

3. The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles according to claim 1, characterized in that, The Gynostemma pentaphyllum-derived nanovesicles are exosome-like nanoparticles extracted from the whole Gynostemma pentaphyllum herb.

4. The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles according to claim 1, characterized in that, The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles exhibit a nucleic acid leakage rate of less than 30% and a protein leakage rate of less than 35%.

5. The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles according to claim 1, characterized in that, The Gynostemma pentaphyllum-derived nanovesicles have a lipid bilayer structure, a hydrated diameter of 92~123 nm, and a zeta potential of -28~-22 mV.

6. A method for preparing chitosan-coated Gynostemma pentaphyllum-derived nanovesicles as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S100. Homogenize the whole herb powder of Gynostemma pentaphyllum in cold PBS, centrifuge to remove cell debris, collect the supernatant and perform ultracentrifugation to precipitate exosome nanoparticles, wash with PBS and collect to obtain Gynostemma pentaphyllum-derived nanovesicles. S200. Dissolve chitosan in acetic acid solution, adjust pH to 5.0~6.0, filter to obtain chitosan solution; mix the Gynostemma pentaphyllum-derived nanovesicle suspension obtained in step S100 with chitosan solution, incubate at room temperature, centrifuge to collect precipitate, resuspend in buffer solution, wash repeatedly to obtain chitosan-coated Gynostemma pentaphyllum-derived nanovesicles.

7. The preparation method according to claim 6, characterized in that, In step S100, the plant powder is homogenized in cold PBS, then centrifuged at 3-5°C to remove cell debris. The supernatant is then ultracentrifuged at 3-5°C for 50-70 minutes to precipitate exosome nanoparticles. The precipitate is washed with PBS and collected by repeated ultracentrifugation at 3-5°C.

8. The preparation method according to claim 6, characterized in that, The suspension of Gynostemma pentaphyllum-derived nanovesicles obtained in step S100 was mixed with chitosan solution, incubated at 20-28°C, centrifuged at 3-5°C for 15-25 min to collect the precipitate, and the precipitate was resuspended in an equal volume of neutral MES buffer. The mixture was washed 1-3 times to obtain chitosan-coated Gynostemma pentaphyllum-derived nanovesicles.

9. An application of chitosan-coated Gynostemma pentaphyllum-derived nanovesicles as described in any one of claims 1 to 5, characterized in that, The application of chitosan-coated Gynostemma pentaphyllum-derived nanovesicles in the preparation of drugs for treating inflammatory bowel disease.

10. The application according to claim 9, characterized in that, The chitosan-coated Gynostemma pentaphyllum-derived nanovesicles are administered orally.

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