Boosting oral bioavailability of taraxasterol via a nanocrystal-mucoadhesive micropatch platform for potent multi-mechanistic protection against drug-induced liver injury

Abstract

Drug-induced liver injury (DILI) is a serious adverse reaction with limited therapeutic options. Taraxasterol (TAS), a natural triterpenoid with hepatoprotective potential, suffers from poor aqueous solubility and low oral bioavailability, hindering its clinical application. Here, we develop an integrated oral delivery platform comprising TAS nanocrystals (TAS NCs) stabilized by D-α-tocopherol polyethylene glycol succinate (TPGS), embedded within chitosan-carbopol polyelectrolyte complex (EPC) micropatches, and encapsulated in enteric-coated capsules (TAS NCs@EPC/CAP). This design sequentially overcomes multiple absorption barriers through a multi-stage process: nanocrystallization enhances dissolution and permits TPGS-mediated P-glycoprotein inhibition and hepatocyte uptake; the mucoadhesive EPC micropatches prolong intestinal residence and transiently open tight junctions; and enteric coating ensures site-specific release in the small intestine. In mice, TAS NCs@EPC/CAP dramatically improved oral bioavailability (AUC increased 3.1-fold vs. free TAS) and achieved sustained liver enrichment. In both acetaminophen-induced intrinsic and isoniazid/rifampicin-induced idiosyncratic DILI models, TAS NCs@EPC/CAP exerted superior hepatoprotection, as evidenced by normalized liver function markers (ALT, AST, LDH), attenuated oxidative stress (ROS, MDA, GSH, SOD), suppressed inflammation (IL-1β, IL-6, TNF-α), and reduced hepatocyte apoptosis. Integrated network pharmacology and experimental validation revealed that TAS NCs@EPC/CAP potentiates the effects of TAS, leading to enhanced activation of the Nrf2/HO-1 antioxidant signaling, more pronounced inhibition of JNK-mediated stress pathways, and greater modulation of the Bax/Bcl-2/caspase-3 apoptotic cascade. This study establishes a clinically translatable oral delivery strategy for TAS and provides a versatile platform for enhancing the bioavailability of poorly soluble natural products, with potent efficacy against diverse forms of DILI.

Graphical abstract

A TPGS-stabilized TAS nanocrystal-loaded EPC micropatch system encapsulated within an enteric-coated capsule (TAS NCs@EPC/CAP) was developed. This design coordinately addresses multiple absorption barriers that have limited the oral delivery of TAS, achieving a 3.1-fold increase in oral bioavailability, sustained systemic exposure, and marked hepatic accumulation. In both acetaminophen-induced intrinsic DILI and isoniazid/rifampicin-induced idiosyncratic DILI mouse models, TAS NCs@EPC/CAP significantly normalized liver function markers. It also attenuated oxidative stress by reducing ROS and MDA levels while restoring GSH and SOD activities. Furthermore, it suppressed pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α and reduced hepatocyte apoptosis, demonstrating potent and broad hepatoprotection.

Highlights

• •A TPGS-stabilized taraxasterol (TAS) nanocrystal–mucoadhesive micropatch system (TAS NCs@EPC/CAP) was developed to overcome multiple barriers limiting the oral delivery of TAS.
• •The system enables pH-triggered intestinal release, prolonged mucosal retention, transient tight-junction modulation, and TPGS-mediated P-gp inhibition, thereby enhancing hepatic uptake.
• •TAS NCs@EPC/CAP exhibited significantly enhanced hepatoprotective efficacy in both APAP-induced intrinsic and INH/RFP-induced idiosyncratic DILI models.

1. Introduction

Drug-induced liver injury (DILI) represents one of the most serious and unpredictable adverse drug reactions encountered in both clinical practice and drug development, and is a leading cause of acute liver failure and drug withdrawal worldwide [1].Clinically, DILI can be broadly classified into intrinsic DILI, which is dose-dependent and predictable, like acetaminophen (APAP) overdose, and idiosyncratic DILI, which is dose-independent, occurs in susceptible individuals, and is often associated with immune or metabolic factors, such as isoniazid (INH) and rifampicin (RFP) [2,3]. Despite their distinct initiating mechanisms, both forms of DILI converge on shared downstream pathological processes, including excessive oxidative stress, inflammatory amplification, mitochondrial dysfunction, and hepatocyte apoptosis [4]. Currently, N-acetylcysteine remains the only FDA-approved therapy for APAP-induced liver injury, offering little benefit in case of idiosyncratic DILI [5]. Therefore, there is a critical unmet need for broadly effective hepatoprotective agents that can target multiple pathological pathways involved in DILI.

Natural products derived from traditional Chinese medicine have attracted increasing attention as potential hepatoprotective agents due to their multitarget pharmacological profiles and long history of clinical use [6]. Taraxasterol (TAS), a pentacyclic triterpenoid isolated from Taraxacum officinale (dandelion) roots, is a key bioactive constituent of several liver-protective herbal formulations and has been reported to possess antioxidant, anti-inflammatory, and anti-apoptotic activities [7]. Accumulating evidence suggests that TAS can attenuate liver injury by modulating redox homeostasis and inflammatory signaling pathways [8,9]. However, despite its promising pharmacological properties, the clinical translation of TAS is severely hindered by its poor aqueous solubility, low intestinal permeability, and extremely low oral bioavailability [10]. Consequently, these biopharmaceutical limitations result in insufficient systemic exposure and inconsistent therapeutic efficacy following oral administration, substantially restricting its clinical potential.

Nanocrystal technology has emerged as an effective formulation strategy to improve the oral absorption of poorly soluble drugs by increasing apparent solubility and dissolution rate through particle size reduction [11]. However, only enhancing dissolution is frequently insufficient for TAS, as intestinal permeability, efflux transport, and gastrointestinal residence time collectively impose additional absorption barriers. Consequently, nanocrystal stabilization and functional surface modification become critical determinants of their in vivo performance. D-α-tocopherol polyethylene glycol succinate (TPGS), an FDA-approved pharmaceutical excipient, possesses an amphiphilic structure and intrinsic P-glycoprotein (P-gp) inhibitory activity [12]. Numerous studies have demonstrated that TPGS significantly enhances intestinal absorption and bioavailability of P-gp–limited drugs [13]. Importantly, TPGS-modified carriers can promote hepatic uptake, thereby providing a unique opportunity for intestinal–hepatic targeting following oral administration [14]. Thus, the TPGS-stabilized TAS nanocrystals are designed to enhance drug bioavailability by improving intestinal absorption and facilitating targeted delivery to the liver, effectively integrating delivery and therapeutic activities.

However, orally administered nanocrystals face additional challenges, including the harsh gastric environment, enzymatic degradation, rapid clearance by the dynamic intestinal mucus layer, and the formidable barrier imposed by epithelial tight junctions (TJs), all of which can compromise absorption and limit in vivo efficacy [15]. Consequently, there is a growing recognition that successful oral delivery requires integrated strategies that simultaneously enhance dissolution, permeability, and intestinal residence [16]. Mucoadhesive micropatch delivery systems offer an attractive solution by prolonging intestinal residence and increasing local drug concentration at the absorption site [17,18]. Among available mucoadhesive materials, polyelectrolyte complexes (EPC) formed via electrostatic interactions between oppositely charged polymers provide distinct advantages, including tunable swelling behavior, strong mucoadhesion, and the ability to transiently modulate epithelial tight junctions [19]. In particular, EPC constructed from chitosan (CS) and carbopol (CP) have been shown to enhance paracellular transport while maintaining acceptable biocompatibility [20,21]. Their compositional tunability allows precise regulation of swelling behavior and drug release kinetics. Upon hydration, these micropatches rapidly absorb water and swell into gel-like networks that physically confine nanocrystals, forming an initial diffusion barrier. As the polymer matrix gradually erodes, nanocrystals are progressively released, constituting a second level of release control. This dual-barrier mechanism not only moderates drug release but also suppresses nanocrystal aggregation and improves formulation stability. Despite these advantages, direct oral administration of mucoadhesive systems may result in premature adhesion or drug release in the stomach. Therefore, site-specific intestinal delivery is essential. Enteric capsules provide an effective means to shield the micropatches from gastric erosion and the hostile gastric environment, ensuring that the patches disintegrate and are released selectively in the intestine, where prolonged mucosal retention can be achieved.

In this study, we developed a TPGS-stabilized TAS nanocrystal-loaded EPC micropatch system encapsulated within an enteric-coated capsule (TAS NCs@EPC/CAP). This system is designed to overcome the key barriers limiting TAS oral delivery: poor solubility, limited permeability, P-gp mediated efflux, and insufficient intestinal residence time. Following enteric release in the intestine, the micropatches adhere to the epithelial surface, undergo controlled swelling, and gradually release intact nanocrystals. Concurrently, TPGS enhances intestinal permeability, suppresses efflux transport, and promotes hepatocyte uptake. Through the coordinated action of solubility enhancement, mucoadhesive retention, controlled release, efflux inhibition, and liver targeting, this platform aims to achieve efficient oral delivery of TAS. Subsequently, we systematically evaluated its pharmacokinetics, intestinal absorption, tissue distribution, and hepatoprotective efficacy in both intrinsic and idiosyncratic DILI models. Furthermore, we employed network pharmacology analyses to identify TAS-related targets and pathways implicated in DILI, followed by experimental validation at the protein level to establish mechanistic consistency (Scheme 1).

Scheme 1.

Schematic illustration of an enteric-coated capsule encapsulating the EPC micropatch system, which is loaded with TPGS-stabilized TAS nanocrystals (TAS NCs@EPC/CAP). TAS nanocrystals were incorporated into chitosan/carbopol polyelectrolyte complexes and filled into enteric-coated capsules for intestine-specific release. After oral administration, the micropatches swell and adhere to the intestinal mucosa, transiently modulate tight junctions, and enhance epithelial permeation, leading to increased hepatic exposure and improved anti-inflammatory, anti-oxidative, and anti-apoptotic effects against DILI.

Overall, this work aims to provide a solid foundation for the oral application of TAS toward clinical use and offers a generalizable strategy for overcoming oral absorption bottlenecks associated with low solubility and poor permeability natural products.

2. Result

2.1. Biopharmaceutical limitations of TAS and the rationale for nanocrystal-based oral delivery

To evaluate the intestinal transepithelial transport capability of TAS, bidirectional permeability studies were conducted using a Caco-2 cell monolayer model. As shown in Fig. S1A, TAS exhibited an extremely low apparent permeability coefficient (P_app), only 0.79 × 10⁻⁶ cm/s across the Caco-2 monolayer. In addition, at the highest administered dose, TAS was unable to dissolve in 250 mL of aqueous media over a wide pH range from 1.0 to 7.5, indicating its intrinsically poor aqueous solubility and suggesting limited oral absorption potential. To further examine the impact of cellular transporters on TAS, a P-gp inhibition study was performed using verapamil. The transepithelial transport efficiency of TAS increased by approximately 3.6-fold in the presence of verapamil (Fig. S1B). Moreover, free TAS exhibited an efflux ratio greater than 2 (Fig. S1C), which was significantly reduced upon verapamil treatment, further confirming that TAS is a substrate of the P-gp efflux pump. Pharmacokinetic studies revealed that following i.v. administration, free TAS displayed a relatively short elimination half-life (T_1/2) and rapid systemic clearance, achieved a high peak plasma concentration (C_max) and a relatively high AUC. These results indicate that i.v. administered TAS attains substantial systemic exposure within a short period but is eliminated quickly. In contrast, oral administration of TAS resulted in a prolonged T_1/2, suggesting extended residence time in vivo; however, the corresponding AUC was markedly reduced, reflecting limited overall systemic exposure. Notably, the absolute oral bioavailability of TAS was only approximately 6.4%, indicating that only a small fraction of the orally administered dose was effectively absorbed into the systemic circulation (Fig. S2). Collectively, these findings demonstrate that TAS is subject to dual absorption barriers, namely poor aqueous solubility and low intestinal permeability, which together account for its extremely low oral delivery efficiency. Reducing drug particle size to the nanometer scale via nanocrystal technology can markedly increase the specific surface area, thereby significantly enhancing the dissolution rate and apparent solubility of the drug [22]. Thus, TAS was formulated into nanocrystals to facilitate effective improvement of its oral bioavailability.

2.2. Preparation and optimization of TAS NCs

The types and concentrations of stabilizers play a pivotal role in the development of nanocrystals. Insufficient stabilizer concentrations may fail to fully cover particle surfaces, resulting in weak interparticle repulsive forces and subsequent aggregation. Conversely, excessively high stabilizer concentrations can enhance interparticle interactions, promote aggregation, or induce micelle formation, ultimately leading to thermodynamic instability [23]. Therefore, appropriate stabilizers and concentrations must be carefully selected to ensure nanocrystal stability during both preparation and storage.

In this study, three commonly used stabilizers P407, PVA, and TPGS were employed to prepare TAS nanocrystals by top-down approach. A series of TAS nanocrystals were prepared using stabilizer concentrations of 0.25%, 0.5%, 1%, 2.5%, and 5% (w/v), and PS and PDI were determined (Fig. S3A–C). For TAS NCs/P407, increasing the P407 concentration beyond 0.5% did not result in a further reduction in particle size. In contrast, for TAS NCs/PVA and TAS NCs/TPGS, increasing the stabilizer concentration from 0.25% to 1% significantly reduced particle size, whereas no significant differences were observed at higher concentrations. Notably, all TAS nanocrystals prepared with 2.5% and 5% stabilizers exhibited increased PDI values, likely due to aggregation induced by excessive stabilizer content. Consequently, a stabilizer concentration of 1% was selected for subsequent experiments. At this optimized concentration, the average particle sizes of TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS were 145.85 ± 5.51 nm, 188.45 ± 25.44 nm, and 147.53 ± 2.19 nm, respectively, with all formulations exhibiting PDI values below 0.3, indicating narrow size distributions and good dispersion stability. All nanocrystals carried a negative surface charge. TEM images revealed that TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS exhibited near-circular form (Fig. 1A–C). The crystalline states of TAS in its free form and in optimized freeze-dried nanocrystal formulations were analyzed by XRPD. As shown in Fig. 1D, free TAS displayed numerous sharp and intense diffraction peaks within the 10°–20° range, indicative of high crystallinity. Although TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS retained similar crystalline features, they exhibited reduced lattice energy, with TAS NCs/TPGS showing the most pronounced reduction. This observation suggests that TAS NCs/TPGS may substantially enhance the solubility and dissolution rate of TAS, thereby improving its bioavailability. Thermal properties of TAS nanocrystals were further investigated by TGA-DSC (Fig. 1E and F). Free TAS exhibited a distinct exothermic peak at 165.88 °C in the DSC curve without a corresponding mass loss in the TGA curve, suggesting a melting or polymorphic transition. A subsequent exothermic event at 384.52 °C was accompanied by a 98.86% mass loss, indicating near-complete decomposition. For TAS NCs/P407, initial mass loss occurred at 56.49 °C, likely due to evaporation of volatile components, while exothermic peaks were observed at 165.88 °C and 403.84 °C, with a mass loss of 96.56% at the latter temperature. TAS NCs/PVA exhibited endothermic peaks near 165.34 °C and 312.39 °C, with the latter corresponding to a significant mass loss. TAS NCs/TPGS showed two exothermic peaks at approximately 139.92 °C and 410.31 °C, with a sharp mass loss reaching 98.25% at the higher temperature. Notably, TAS NCs/P407 and TAS NCs/TPGS exhibited higher decomposition temperatures compared with free TAS and TAS NCs/PVA, indicating superior thermal stability.

Fig. 1.

Preparation and characterization of TAS NCs. TEM images and particle size characterization of (A) TAS NCs/P407, (B) TAS NCs/PVA, and (C) TAS NCs/TPGS. (D) XRPD, (E) DSC, and (F) TG of free TAS, TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS. (G-I) Changes in PS, PDI and zeta potential of TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS stored at 4 °C for 7 days (n = 3). (J) Dissolution profiles of TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS (n = 3). (K) P_app values of TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS in Caco-2 (n = 3). (L) Cellular uptake of TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS in AML12 (n = 3). Each bar represents the mean ± SD. *p < 0.05, **p < 0.01 vs. TAS NCs/TPGS.

Nanocrystals are thermodynamically unstable colloidal dispersions and inherently prone to aggregation due to Ostwald ripening and positive Gibbs free energy changes [24]. Storage stability studies conducted at 4 °C for 7 days showed that all TAS nanocrystal formulations exhibited increases in particle size and zeta potential. Among them, TAS NCs/PVA showed the most pronounced size increase, whereas TAS NCs/TPGS exhibited the smallest size variation. Changes in PDI followed trends consistent with particle size variations, indicating that TAS NCs/TPGS possessed superior storage stability (Fig. 1G–I). Given that nanocrystalization was primarily intended to enhance TAS solubility, dissolution performance was further evaluated. As shown in Fig. 1J, free TAS exhibited a cumulative dissolution of only 25.61% within 24 h. In contrast, nanocrystal formulations significantly improved dissolution, with TAS NCs/P407, TAS NCs/PVA, and TAS NCs/TPGS achieving dissolution rates of 63.62%, 60.60%, and 88.40%, respectively, which represented 2.48-, 2.37-and 3.45-fold increases relative to free TAS. The enhanced dissolution behavior can be attributed to particle size reduction and increased specific surface area, which collectively promote solvent–drug contact and accelerate dissolution kinetics [25]. Therefore, TAS NCs/TPGS showed a more pronounced effect on improving the dissolution of TAS, which may mainly be attributed to the ability of TAS NCs/TPGS to minimize the lattice energy of TAS. Moreover, TPGS has been reported to inhibit P-gp mediated efflux, thereby enhancing the intestinal absorption and bioavailability of P-gp substrate drugs [12,26]. Consistent with these findings, Caco-2 monolayer transport studies demonstrated that TAS NCs/TPGS significantly suppressed P-gp efflux activity (Fig. S1C), resulting in a 2.6-fold increase in the P_app (AP→BL). Moreover, TAS NCs/TPGS exhibited a higher P_app compared with TAS NCs/P407 and TAS NCs/PVA (Fig. 1K). Importantly, cellular uptake studies further revealed that TAS NCs/TPGS significantly enhanced TAS uptake by hepatocytes (Fig. 1L), which are consistent with previous studies reported [12]. Collectively, these results demonstrate that TAS NCs/TPGS not only markedly improves the solubility and dissolution of TAS but also enhances its intestinal absorption and liver targeting capability, enabling an integrated “delivery–therapy” strategy for efficient hepatoprotective treatment.

2.3. Construction and characterization of mucoadhesive nanocrystal-loaded micropatches (TAS NCs@EPC)

TAS NCs@EPC are double-layer micropatches prepared by coating method, which is composed of ethyl cellulose as waterproof backing layer and EPC formed by CS and CP as adhesive layer (Fig. 2A). CS and CP are known to form EPC through electrostatic interactions [21]. To verify EPC formation, CS, CP, physical mixture CS and CP (CS + CP), and Blank EPC micropatches (@EPC) were characterized by FTIR and TG-DSC. As shown in Fig. 2B the absorption band at approximately 1061 cm⁻¹ in the CS spectrum is attributed to the saccharide ring structure, corresponding to the repeating unit of chitosan, which is structurally analogous to polysaccharides such as chitin and cellulose [27]. The bands observed at 1614–1515 cm⁻¹ are assigned to the C=O stretching vibration of the amide group (amide I band) in the acetylated units of CS. While, in the CP spectrum, the broad band at 3300–2500 cm⁻¹ corresponds to –OH stretching vibrations, the peak at 1698 cm⁻¹ to C=O stretching of carboxyl groups, the band at 1452 cm⁻¹ to CH₂ bending vibrations, and the band at 1166 cm⁻¹ to asymmetric C–O–H stretching [28]. The characteristic absorption peaks of both CS and CP were present in the spectra of CS + CP and @EPC, and no new peaks were observed. However, notable differences in peak intensities were detected. Compared with CS + CP, the characteristic peaks of CP in @EPC were markedly weakened, whereas the CS signal at 1061 cm⁻¹ was almost undetectable in the @EPC spectrum. Given that peak overlap or reduced signal intensity may obscure definitive evidence of @EPC formation in FTIR analysis, synchronous thermal analysis was further employed for confirmation. The DSC and TGA curves of CS, CP, CS + CP, and @EPC are shown in Fig. 2C and D. Two exothermic peaks for CS were found by DSC analysis at roughly 90 °C and 230 °C. At 90.91 °C, only a slight mass change was observed in the TG curve, likely attributable to the evaporation of bound water or volatile components. In contrast, a pronounced mass loss was detected at 237.78 °C, corresponding to thermal decomposition of CS, with a final mass reduction of 62.01%. For CP, an exothermic peak was observed at 235.61 °C, accompanied by a significant mass loss, with a final weight reduction of 78.08%. The physical mixture CS + CP exhibited exothermic degradation peaks corresponding to both CS and CP. Notably, @EPC displayed a new endothermic degradation peak around 420 °C, which may be attributed to the disruption of weak electrostatic interactions between polymer components, indicating the formation of a new complex phase. This observation supports the formation of EPC via electrostatic interactions between CS and CP [29]. Furthermore, the total mass loss of @EPC (62.59%) was lower than that of CS + CP (69.36%), suggesting enhanced thermal stability of EPC [30].

Fig. 2.

Preparation and characterization of TAS nanocrystal–loaded EPC micropatches (TAS NCs@EPC). (A) Schematic illustration of the preparation process of TAS NCs@EPC, showing the formation of CS–CP EPC via electrostatic interactions and subsequent incorporation of TAS nanocrystals. (B) FT-IR, (C) TGA and (D) DSC curves of CS, CP, CS + CP, and @EPC. (E) Appearance and (F) SEM images of @EPC, TAS@EPC and TAS NCs@EPC, scale bar = 100 μm. (G) appearance change and (H) swelling index (%) (n = 3) of different TAS formulation-loaded micropatches. (I) Mucoadhesive strength and (J) adhesion duration of drug-loaded EPC patches on porcine intestinal mucosa. (K) In vitro cumulative release profiles of TAS from different EPC-based patches over 24 h (n = 3). Each bar represents the mean ± SD. ***p < 0.001 vs. @EPC; ^###p < 0.001 vs. TAS NCs@CS; ⁺⁺⁺p < 0.001 vs. TAS NCs@CP.

Blank EPC micropatches (@EPC) with varying CS/CP ratios were prepared to optimize physicochemical and mucoadhesive properties. As shown in Fig. S4A–C, increasing CP content markedly improved micropatches flexibility while moderately increasing patch weight and thickness. Mucoadhesion is primarily achieved through polymer swelling upon water absorption, which also governs drug release behavior [31]. Accordingly, the swelling behavior of the patches was investigated (Fig. S4D and E). The @CS exhibited the slowest swelling, whereas @CP showed rapid and extensive swelling due to ionization of carboxyl groups at pH 6.8 [32]. Consequently, EPC swelling increased with increasing CP content. However, the swelling index of @CS-CP (1:1) was slightly lower than that of @CS-CP (2:1), likely due to the formation of a more compact polymer network arising from strong electrostatic interactions between CS and CP. This compact structure was disrupted upon further CP addition, leading to increased swelling for @CS-CP (1:2) and @CS-CP (1:3). Mucoadhesive evaluation using porcine intestinal mucosa (Fig. S4F and G) demonstrated that @CS exhibited weak adhesion force but prolonged retention, whereas @CP and CP-rich EPC showed stronger adhesion but shorter residence times due to excessive hydration and erosion. Considering micropatches integrity, swelling behavior, and adhesion performance, @CS-CP (1:2) was selected as the optimal formulation for subsequent drug loading.

During intestinal patch fabrication, drugs are typically dissolved or dispersed within the polymer matrix to enhance wettability, apparent solubility, and stability [33]. However, the poor aqueous solubility of TAS presents significant challenges in dose loading, formulation, and administration. Surface morphology analysis demonstrated a marked difference between nanocrystal-loaded and free-drug-loaded patches. As shown in Fig. 2E, TAS NCs@EPC exhibited smooth surfaces without visible drug aggregates, whereas TAS@EPC displayed abundant crystalline deposits, indicating poor drug dispersion. SEM images (Fig. 2F) further confirmed homogeneous distribution of TAS nanocrystals within the EPC matrix, with no evidence of phase separation, while free TAS was primarily localized on the patch surface. As shown in Table S1, TAS content showed a strong linear correlation with patch area, confirming uniform drug distribution and enabling accurate dose control by simple adjustment of patch size. Drug loading did not significantly alter the swelling or mucoadhesive properties of EPC patches. As shown in Fig. 2G–J, drug-loaded patches exhibited swelling indices and mucosal adhesion behavior comparable to those of the corresponding blank formulations, indicating that nanocrystal incorporation preserved the functional characteristics of the EPC matrix. In vitro release studies (Fig. 2K) revealed a substantial enhancement in TAS release from nanocrystal-loaded patches. After 24 h, cumulative release from TAS@EPC was limited to 23.58 ± 1.28%, whereas TAS NCs@CS, TAS NCs@CP, and TAS NCs@EPC achieved 70.74 ± 2.53%, 86.04 ± 3.57%, and 80.75 ± 4.73% release, respectively. Release behavior correlated well with matrix swelling, with CP-rich formulations exhibiting faster release profiles. Therefore, the superior performance of TAS NCs@EPC can be attributed to the synergistic integration of nanocrystal-enabled dissolution enhancement and EPC-mediated matrix control. Nanocrystallization markedly improves drug–polymer compatibility and prevents phase separation, ensuring homogeneous drug distribution, while the electrostatically crosslinked EPC network provides controlled hydration, sustained adhesion, and regulated drug diffusion. Together, these features enable efficient drug release without compromising patch stability or mucoadhesive performance, thereby establishing a robust intestinal delivery platform for subsequent absorption and in vivo efficacy studies.

2.4. Intestinal absorption mechanisms of TAS NCs@EPC

In order to investigate the intestinal permeability of different TAS micropatch formulations, the intestinal permeation was evaluated using excised porcine intestinal mucosa mounted in Franz diffusion cells. After 24 h, TAS NCs@EPC exhibited the highest cumulative permeation, followed by TAS NCs@CS and TAS NCs@CP. These results demonstrate that micropatches fabricated from CS, CP, and EPC all effectively enhanced transepithelial transport of TAS, with EPC-based patches showing the strongest permeation-promoting capability. In contrast, due to the poor aqueous solubility of TAS, TAS@EPC achieved only 21.34% permeation, highlighting the critical role of drug solubility in determining epithelial transport efficiency (Fig. 3A). Thus, these findings indicate that permeation-enhancing polymer matrices alone are insufficient to overcome the absorption barrier of TAS without concomitant improvement in drug dissolution.

Fig. 3.

In vitro absorption mechanism of TAS NCs@EPC. (A) Permeation curves of TAS formulation through small intestine ex vitro. (B) P_app, (C) cellular uptake and (D) Relative TEER of TAS formulation in Caco-2 and Caco-2 + mucus. ***p < 0.001 vs. free TAS; ^##p < 0.01, ^###p < 0.001 vs. TAS NCs; ⁺⁺⁺p < 0.001 vs. TAS NCs@CS; ^ˆp < 0.05, ^ˆˆp < 0.01, ^ˆˆˆp < 0.001 vs. TAS NCs@CP; ^$$$p < 0.001 vs. TAS@EPC. (E) Immunofluorescence staining was used to detect the effects of different TAS preparation on ZO-1 and CLDN-4 proteins in Caco-2 cells, and (F, G) quantitative analysis was performed. Nuclei (DAPI, blue), ZO-1 (Alexa Fluor®488, green), CLDN-4 (Alexa Fluor®594, red). Scale bar = 20 μm ***p < 0.001 vs. Control; ^###p < 0.001 vs. free TAS; ⁺⁺⁺p < 0.001 vs. TAS NCs@CS; ^ˆˆˆp < 0.001 vs. TAS NCs@CP. Each bar represents mean ± SD (n = 3).

To further elucidate the absorption-enhancing capacity and underlying mechanisms of TAS NCs@EPC, an in vitro intestinal transport model was established, consisting of a mucus layer mimicking the intestinal mucus barrier and a Caco-2 cell monolayer representing the epithelial TJ barrier. Prior to transport studies, cytocompatibility was evaluated using a CCK-8 assay (Fig. S6). After 24 h exposure to various concentrations of free TAS or TAS NCs@EPC with different particle sizes, Caco-2 cell viability remained above 85%, indicating negligible cytotoxicity and suitability for subsequent transport experiments. Transepithelial transport efficiency of different TAS micropatch formulations was quantified by P_app. As shown in Fig. 3B, TAS nanocrystallization significantly enhanced TAS permeability, with TAS NCs exhibiting a 2.86-fold increase in P_app compared with free TAS, consistent with the ex vivo porcine intestinal permeation results. Except for TAS@EPC, all micropatch-based formulations significantly increased P_app values, among which TAS NCs@EPC achieved the highest permeability. Although the presence of a mucus layer moderately impeded drug transport, the overall permeation trends remained consistent with those observed in the Caco-2 monolayer model, confirming the robustness of the enhancement effect. Furthermore, cellular uptake studies (Fig. 3C) demonstrated significantly increased intracellular accumulation of TAS in Caco-2 cells treated with TAS NCs–loaded micropatches, suggesting that EPC-based patches may enhance transepithelial transport by promoting transcellular uptake. To assess the contribution of the paracellular pathway, TEER was measured to evaluate monolayer integrity and TJ modulation (Fig. 3D). Upon application of the micropatches to the apical side of the Caco-2 monolayer, TEER values decreased in a time-dependent manner and gradually recovered after formulation removal, indicating reversible TJ opening. Notably, EPC-based micropatches induced the most pronounced TEER reduction, suggesting a stronger ability to transiently modulate TJ permeability. These findings collectively indicate that TAS NCs@EPC enhance epithelial transport through a dual mechanism involving both transcellular and paracellular pathways.

Previous studies have reported that CS can enhance oral drug absorption by opening epithelial TJ, potentially through downregulation of tight junction–associated proteins such as ZO-1 and CLDN-4 [[34], [35], [36]], whereas the effects of CP on TJ remain largely unexplored. Therefore, immunofluorescence staining was employed to investigate the impact of EPC on TJ protein expression. As shown in Fig. S7, both @CS and @CP significantly downregulated ZO-1 expression, with the lowest expression observed following @EPC treatment, indicating a synergistic effect within EPC. Additionally, @CS markedly reduced CLDN-4 expression, whereas @CP exerted no significant effect (Fig. S8), suggesting that CS is the primary contributor to TJ modulation within the EPC system. The effects of TAS-loaded micropatches formulations on TJ protein expression were further evaluated. Micropatches all significantly downregulated ZO-1 expression. In particular, CS- and EPC-based micropatches also significantly reduced CLDN-4 expression, whereas free TAS exerted minimal influence on TJ proteins. These results indicate that fabrication of CS, CP, and EPC into patch formulations preserves their intrinsic TJ-modulating properties, while TAS NCs primarily contribute to enhanced transport through improved dissolution and cellular uptake rather than direct TJ disruption (Fig. 3E–G). Overall, these results demonstrate that TAS NCs@EPC markedly enhance intestinal epithelial transport through a synergistic mechanism involving improved drug dissolution and transcellular uptake mediated by nanocrystallization, together with reversible TJ modulation and paracellular transport induced by EPC-based micropatches. This dual-pathway permeation enhancement provides a mechanistic basis for the superior intestinal absorption observed with TAS NCs@EPC and supports their further development as an efficient oral delivery system.

2.5. Preparation of TAS nanocrystal micropatch capsule (TAS NCs@EPC/CAP) and its gastrointestinal distribution

To prevent premature adhesion or drug release before reaching the intestine and to ensure site-specific intestinal delivery, twenty NR–labeled TAS NCs@EPC micropatches were encapsulated into one mice enteric-coated capsule to generate TAS NCs@EPC/CAP (Fig. 4A). To minimize self-adhesion of the micropatches within the capsule, micropatches without/with MS or SID coating were separately loaded into enteric capsules, and their disintegration and release behaviors were evaluated under simulated gastric (pH 1.2) and intestinal (pH 6.8) conditions. All capsules remained intact for at least 2 h in simulated gastric fluid, indicating excellent acid resistance and effective prevention of premature drug release. In contrast, under simulated intestinal conditions, all capsules rapidly disintegrated within 0.25 h, releasing the encapsulated micropatches. Notably, MS-coated micropatches dispersed uniformly in the medium, whereas uncoated and SID-coated patches exhibited pronounced aggregation due to strong intrinsic adhesiveness (Fig. S9). These results suggest that MS coating effectively reduces inter-patch adhesion and improves dispersion behavior following capsule disintegration, which is critical for uniform intestinal distribution.

Fig. 4.

Preparation and intestinal distribution of TAS nanocrystal micropatch capsule. (A) Schematic diagram of oral administration of 20 NR–labeled TAS NCs@EPC/CAP filled into one enteric capsule using a mice capsule applicator. (B) The image of the patch on the small intestine of the mice 2 h after the administration of NR–labeled TAS NCs@EPC/CAP. (C) In vivo imaging detection of the retention of Free NR, NR NCs and NR NCs@EPC/CAP in the gastrointestinal tract at different time points. The distribution of free NR, NR NCs and NR NCs@EPC/CAP loaded capsule in (D) stomach, (E) small intestine and (F) large intestine. Each bar represents the mean ± SD (n = 4). *p < 0.05, **p < 0.01 vs. Free NR; ^#p < 0.05, ^##p < 0.01 vs. NR NCs.

Subsequently, MS-coated NR NCs@EPC/CAP were orally administered to mice using a capsule delivery device. Two hours post-administration, ex vivo examination of the intestinal tract revealed that the released micropatches adhered to the luminal side of the intestinal mucosa and exhibited pronounced swelling (Fig. 4B). This observation confirms that the enteric capsule successfully delivers the micropatches to the intestine, where they are released in response to the intestinal pH and subsequently adhere to the mucosal surface, thereby prolonging intestinal residence time and facilitating sustained local drug exposure.

To further evaluate the gastrointestinal distribution and retention behavior of nanocrystal micropatch capsule, in vivo fluorescence imaging was performed to compare Free NR, NR NCs, and NR NCs@EPC/CAP following oral administration. As shown in Fig. 4C, Free NR exhibited weak fluorescence signals in the stomach and small intestine, which rapidly diminished over time, indicating poor gastrointestinal retention and rapid clearance. NR NC displayed moderately enhanced fluorescence at early time points; however, the signal gradually migrated distally along the gastrointestinal tract, reflecting limited retention capability. In contrast, NR NCs@EPC/CAP exhibited significantly stronger and more persistent fluorescence signals across multiple time points. Notably, intense fluorescence was retained in the small intestine even at 8 h and 12 h post-administration, demonstrating a clear time-dependent retention advantage. Quantitative analysis further confirmed that the radiant efficiency of NR NCs@EPC/CAP in the stomach and small intestine was significantly higher than that of Free NR and NR NCs, while fluorescence signals in the colon remained comparatively low (Fig. 4D, E, F). These findings indicate that the micropatch-loaded enteric capsules preferentially localize and retain nanocrystals within the small intestine—the primary site for drug absorption.

Thus, the enhanced intestinal retention of TAS NCs@EPC/CAP can be attributed to the synergistic integration of enteric pH-triggered capsule disintegration and mucoadhesive EPC-based micropatches. Enteric coating ensures precise intestinal release, while rapid micropatch swelling and strong mucosal adhesion prolong local residence and maintain high luminal drug concentrations. This prolonged intestinal exposure provides a favorable microenvironment for sustained nanocrystal dissolution and epithelial uptake, thereby establishing a critical prerequisite for improved oral absorption and overall bioavailability.

2.6. In vivo pharmacokinetic and tissue distribution study of TAS Ncs@EPC loaded capsules

The pharmacokinetic behavior and tissue distribution of TAS following oral administration of Free TAS, TAS NCs, and TAS NCs@EPC/CAP were systematically evaluated to assess the oral absorption efficiency and in vivo fate of the micropatch-based delivery system. The plasma concentration–time profiles and corresponding pharmacokinetic parameters are shown in Fig. 5A and B, respectively. Following oral administration, all formulations exhibited a rapid increase in plasma TAS concentration, reaching peak levels at approximately 4 h, followed by a gradual decline. Compared with Free TAS, TAS NCs significantly increased systemic exposure by a 1.67-fold increase in AUC_0–t and a marked elevation in C_max, indicating that nanocrystallization effectively improved TAS dissolution and intestinal absorption. Notably, TAS NCs@EPC/CAP achieved the highest plasma exposure among all groups, with C_max and AUC₀–t values that were remarkably higher than those of Free TAS and TAS NCs. In addition, TAS NCs@EPC/CAP significantly prolonged the T₁/₂ and MRT, while markedly reducing Cl/F, collectively demonstrating enhanced absorption efficiency and sustained systemic exposure.

Fig. 5.

Pharmacokinetics and tissue distribution of TAS NCs@EPC/CAP. (A) Plasma drug time curves and (B) main pharmacokinetic parameters of free TAS, TAS NCs and TAS NCs@EPC/CAP. The distribution of TAS in the (C) liver, (D) kidney and (E) spleen of free TAS, TAS NCs and TAS NCs@EPC/CAP. All data are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Free TAS; ^#p < 0.05, ^###p < 0.001 vs. TAS NCs.

To further elucidate the in vivo distribution characteristics, TAS concentrations in major organs (heart, lung, spleen, liver, and kidney) were quantified at different time points following oral administration (Fig. 5C–E and Fig. S10). Compared with Free TAS, TAS NCs significantly increased TAS accumulation across all examined tissues, confirming that nanocrystallization alone improves in vivo absorption and tissue exposure. Importantly, TAS NCs@EPC/CAP consistently exhibited higher and more sustained TAS levels than both Free TAS and TAS NCs, with the most pronounced enhancement observed in the liver. In the liver, TAS NCs@EPC/CAP achieved substantially higher TAS concentrations at 4–12 h post-administration and maintained elevated levels over time, indicating a clear hepatic enrichment and prolonged retention profile. This liver-targeting tendency is particularly advantageous for the treatment of DILI. In addition, TAS NCs@EPC/CAP also showed time-dependent increases in TAS concentration in the kidney and spleen, with overall exposure levels significantly exceeding those of TAS NCs and Free TAS, reflecting improved systemic distribution persistence. Although accumulation in non-hepatic organs was enhanced, the preferential elevation in hepatic exposure suggests a favorable distribution pattern rather than nonspecific tissue accumulation.

Thus, the superior pharmacokinetic and biodistribution performance of TAS NCs@EPC/CAP stemmed from the interplay of nanocrystal-mediated enhancement of drug dissolution and intestinal permeability, enteric protection ensuring site-specific release in the small intestine, and mucoadhesive EPC micropatches that prolong intestinal residence time and maintain a high local drug concentration gradient for absorption. These results demonstrate that TAS NCs@EPC/CAP markedly enhances the oral bioavailability of TAS and achieves sustained liver enrichment, providing a strong pharmacokinetic basis for its improved hepatoprotective efficacy.

2.7. In vitro hepatoprotective effects of TAS NCs against DILI

Because the EPC-based micropatches are designed to exert their therapeutic effects after intestinal absorption rather than directly acting on hepatic cells, in vitro experiments were performed exclusively to evaluate the hepatoprotective effects of free TAS and TAS NCs. SIL, a clinically established hepatoprotective drug widely used for the treatment of acute and chronic hepatitis as well as fatty liver disease [8], was included as a positive control. The cytotoxicity of free TAS and TAS NCs toward AML12 was evaluated using the CCK-8 assay. As shown in Fig. S11, TAS at concentrations ranging from 0 to 20 μg/mL exhibited negligible cytotoxicity. Although mild growth inhibition was observed at higher concentrations (40 and 80 μg/mL), cell viability remained above 80%, indicating an acceptable safety profile of TAS toward AML12 cells. To assess the therapeutic potential of TAS formulations against DILI, AML12 cells were exposed to APAP or a combination of INH and RFP to establish intrinsic and idiosyncratic DILI models, respectively. ALT and AST activities in the culture supernatant were measured as indicators of hepatocellular injury (Fig. 6A and B and Fig. S12A and B). Both APAP and INH + RFP treatments markedly increased ALT and AST activities, confirming successful induction of hepatocyte injury. Treatment with free TAS or TAS NCs significantly reduced ALT and AST levels to an extent comparable to SIL, indicating that both formulations exert pronounced protective effects against intrinsic and idiosyncratic DILI at the cellular level.

Fig. 6.

Protective effect of TAS NCs on APAP-induced AML12 cells. (A, B) The levels of ALT and AST in the supernatant of AML12 cells. (C-F) The levels of ROS, MDA, GSH and SOD in AML12 cells. (G-I) The levels of IL-1β, IL-6 and TNF-α in the supernatant of AML12 cells. (J) TUNEL staining was used to detect the apoptosis of AML12 cells and (K) fluorescence analysis. Nuclei (DAPI, blue), TUNEL (FITC, green). Scale bar = 20 μm. Each bar represents the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Model; ⁺p < 0.05 vs. free TAS; ^ˆp < 0.05 vs. TAS NCs.

Oxidative stress is a central mechanism underlying hepatocyte injury in DILI [37]. Accordingly, intracellular ROS, MDA, GSH, and SOD levels were quantified (Fig. 6C–F and Fig. S12C–F). Compared with the Control group, Model group markedly increased ROS and MDA levels while significantly decreasing GSH and SOD, reflecting severe oxidative stress. In contrast, treatment with free TAS or TAS NCs significantly attenuated ROS and MDA accumulation and restored intracellular GSH and SOD levels. These results demonstrate that TAS, regardless of formulation, effectively mitigates oxidative stress–mediated hepatocyte injury, consistent with its reported antioxidant activity. Inflammatory signaling plays a pivotal role in the progression of APAP- and INH + RFP–induced liver injury. TAS has been reported to suppress inflammation potentially via modulation of the IκBα/NF-κB p65 pathway [38,39]. Therefore, pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in culture supernatants were quantified by ELISA (Fig. 6G–I and Fig. S12G–I). Both DILI models exhibited significant upregulation of all three cytokines. Treatment with free TAS, TAS NCs, or SIL markedly reduced IL-1β, IL-6, and TNF-α levels, with no statistically significant differences among the treatment groups, indicating that TAS exerts robust anti-inflammatory effects comparable to the clinical reference drug. Hepatocyte apoptosis is a critical contributor to DILI progression. TUNEL staining was performed to assess apoptotic cell death in AML12 cells (Fig. 6J, K and Fig. S12J and K). Minimal apoptosis was observed in control cells, whereas APAP and INH + RFP treatments significantly increased the proportion of TUNEL-positive cells. All drug-treated groups exhibited markedly reduced apoptosis. Notably, both free TAS and TAS NCs demonstrated significantly stronger anti-apoptotic effects than SIL, suggesting superior cytoprotective efficacy at the cellular level.

Collectively, these results demonstrate that TAS effectively protects AML12 hepatocytes from both intrinsic and idiosyncratic DILI by attenuating oxidative stress, suppressing inflammatory responses, and inhibiting apoptosis. Importantly, TAS NCs preserved the intrinsic hepatoprotective activity of TAS while offering formulation advantages that facilitate improved in vivo delivery and bioavailability.

2.8. Protective effect of TAS NCs@EPC/CAP on mice with multiple DILI models

To further evaluate the hepatoprotective efficacy of TAS NCs@EPC/CAP in vivo, APAP and INH + RFP were employed to establish mouse models of intrinsic and idiosyncratic DILI, respectively (Fig. 7A and Fig. S13A). In the APAP-induced intrinsic DILI model, mice were orally administered Free TAS or TAS formulations at an equivalent dose of 10 mg/kg once daily for 7 consecutive days. Control and Model groups received 0.5% CMC-Na. One hour after the final administration, APAP (300 mg/kg) was intraperitoneally injected, and mice were sacrificed 12 h later. As shown in Fig. 7B, the liver index in the Model group was significantly increased compared with the Control group, indicating hepatic edema and inflammatory infiltration. Treatment with TAS and its formulations markedly reduced the liver index, with TAS NCs@EPC/CAP exhibiting the lowest value, suggesting superior hepatoprotective efficacy. Tissue appearance detection further revealed that APAP-treated livers displayed rough surfaces and obvious injury features, consistent with previous reports of acute APAP-induced liver damage [40]. These pathological changes were visibly alleviated in all TAS-treated groups, particularly in the TAS NCs@EPC/CAP group (Fig. 7C). Histopathological analysis by H&E staining (Fig. 7D) corroborated these findings. Control group livers showed well-organized hepatocyte architecture, whereas Model group livers exhibited extensive vacuolization, hepatocyte death, nuclear irregularity, and inflammatory cell infiltration. Free TAS partially ameliorated these lesions, while TAS NCs and TAS NCs@EPC/CAP resulted in more pronounced restoration of normal hepatic morphology. Serum biochemical markers of liver injury, including ALT, AST, and LDH, were significantly elevated in APAP-treated mice (Fig. 7E–G). All TAS-treated groups significantly reduced these enzymes levels compared with the Model group, with TAS NCs@EPC/CAP achieving the greatest reductions, indicating the most effective attenuation of hepatocellular injury. In addition, markers associated with cholestasis and hepatic metabolic dysfunction, like AKP [41], TBA [42,43], and TBIL [44], were markedly increased in the Model group. Treatment with TAS formulations significantly lowered these parameters, with TAS NCs@EPC/CAP again showing the most pronounced normalization (Fig. 7H–J), further confirming its superior protective effect against APAP-induced liver injury. To exclude the potential intrinsic hepatoprotective effects of the excipients, a blank carrier group without TAS (NCs@EPC/CAP) was further evaluated. As shown in Fig. S15A, NCs@EPC/CAP treatment did not significantly reduce liver index or serum biochemical markers, including ALT, AST, LDH, AKP, TBA, and TBIL, compared with the APAP Model group. Consistently, liver morphology and H&E staining showed that NCs@EPC/CAP failed to visibly alleviate APAP-induced hepatic injury. These results indicate that the observed hepatoprotective efficacy was mainly attributable to TAS rather than the carrier excipients.

Fig. 7.

Therapeutic effect of TAS NCs@EPC/CAP on APAP-induced DILI mice model. (A) Schematic diagram of APAP-induced DILI mice treatment and grouping. (B) Liver index of mice. (C) Liver appearance and (D) HE staining of mice. Scale bar = 50 μm. (E-J) The activities of ALT, AST, LDH, AKP, TBA and T-BIL in the serum of mice. Each bar represents mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Model; ⁺p < 0.05, ⁺⁺p < 0.01 vs. Free TAS; ^ˆp < 0.05, ^ˆˆˆp < 0.001 vs. TAS NCs; ^$$$p < 0.001 vs. TAS NCs@EPC/CAP.

Consistent trends were observed in the INH + RFP–induced idiosyncratic DILI model (Fig. S13). Compared with the Control group, the Model group exhibited increased liver index, rough liver surfaces, disordered hepatocyte arrangement, and extensive cellular injury. Treatment with Free TAS, TAS NCs, and TAS NCs@EPC/CAP alleviated these pathological changes, with TAS NCs@EPC/CAP demonstrating hepatoprotective efficacy comparable to SIL. Moreover, TAS NCs@EPC/CAP most effectively reduced serum AST, ALT, LDH, AKP, TBA, and TBIL levels, indicating broad protection against INH + RFP–induced hepatic dysfunction. Similar results were observed in the INH + RFP model, where NCs@EPC/CAP did not significantly improve liver morphology, histopathological injury, liver index, or serum liver function markers compared with the Model group (Fig. S15B), further excluding an obvious intrinsic hepatoprotective contribution from the blank carrier under the present experimental conditions.

To further elucidate the mechanisms underlying the enhanced hepatoprotective effects, oxidative stress, inflammatory responses, and apoptosis were systematically evaluated in liver tissues. In both DILI models, hepatic ROS and MDA levels were significantly elevated in Model groups, accompanied by marked depletion of GSH and SOD (Fig. 8A–D and Fig. S14A–D). Free TAS partially reversed these changes, whereas TAS NCs@EPC/CAP exhibited the most pronounced reduction in ROS and MDA and restoration of GSH and SOD, indicating superior in vivo antioxidant capacity. Pro-inflammatory cytokines were further assessed. As shown in Fig. 8E–G and Fig. S14E–G, IL-1β, IL-6, and TNF-α were markedly upregulated in Model groups. TAS treatment significantly suppressed these cytokines, with TAS NCs@EPC/CAP reducing inflammatory levels to near-normal values, demonstrating robust anti-inflammatory activity in vivo. Hepatocyte apoptosis was evaluated by TUNEL staining (Fig. 8H, I and Fig. S14H and I). DILI induction significantly increased the number of TUNEL-positive cells, whereas TAS treatment markedly reduced apoptotic cell death. Notably, TAS NCs@EPC/CAP resulted in the lowest proportion of TUNEL-positive hepatocytes, consistent with the in vitro findings and highlighting its enhanced anti-apoptotic efficacy. Finally, safety was evaluated by hematological analysis (Fig. S16) and histological examination of major organs (Fig. S17). All hematological parameters, including leukocytes, erythrocytes, platelets, granulocytes, lymphocytes, and monocytes, remained within normal ranges. H&E staining of the heart, lung, spleen, and kidney revealed no detectable pathological alterations. These findings demonstrate that TAS and its formulations, including TAS NCs@EPC/CAP, exhibit favorable in vivo safety profiles.

Fig. 8.

The effects of TAS NCs@EPC/CAP on anti-oxidative stress, anti-inflammation and anti-apoptosis in APAP-induced DILI mice liver tissues. (A-D) The levels of ROS, MDA, GSH and SOD in the liver tissue of mice. (E-G) The levels of cellular inflammatory cytokines IL-1β, IL-6, and TNF-α in the liver tissues of mice. (H-I) TUNEL staining was used to detect the apoptosis of mouse liver tissue cells and quantitative analysis (400 ×, green fluorescence indicates apoptotic cells stained with TUNEL, and blue fluorescence indicates cell nuclei stained with DAPI). Each bar represents mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Model;⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs. free TAS; ^ˆˆˆp < 0.001 vs. TAS NCs; ^$$p < 0.01 vs. TAS NCs@EPC/CAP.

2.9. TAS NCs@EPC/CAP alleviates DILI via multi-target mechanism

Network pharmacology was used to detect the potential therapeutic targets of TAS in the treatment of DILI. Potential targets of TAS, as well as intrinsic and idiosyncratic DILI targets, were retrieved from online databases, yielding 616 TAS-related, 1000 intrinsic DILI-related, and 1000 idiosyncratic DILI-related targets, with 80 intersecting targets identified (Fig. 9A).

Fig. 9.

Exploration of the molecular mechanisms underlying TAS-mediated improvement of DILI in mice. (A) Venn diagram showing the overlap between predicted targets of TAS and genes associated with intrinsic drug-induced liver injury (APAP-induced DILI) and idiosyncratic DILI (INH + RFP–induced). (B) GO and (D) KEGG pathway enrichment analysis of the intersected targets. (C) PPI network of the common targets with node size and color reflecting topological importance. Western blot analysis and quantitative densitometry of Nrf2, HO-1, JNK, p-JNK, caspase-3, Bax, and Bcl-2 in (E) the APAP-induced intrinsic DILI model and (F) INH + RFP–induced idiosyncratic DILI model following treatment with free TAS, TAS NCs, TAS NCs@EPC/CAP, or SIL. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Model;⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs. free TAS; ^ˆp < 0.05 vs. TAS NCs; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 vs. TAS NCs@EPC/CAP. Each bar represents mean ± SD (n = 3).

Integrated GO, KEGG, and PPI analyses consistently indicated that TAS-related targets are tightly linked to the core pathological axes of DILI, namely inflammation, oxidative stress, and apoptosis. GO enrichment highlighted biological processes associated with apoptotic regulation, inflammatory activation, and cellular stress–responsive signaling, together with functional features related to kinase activity and oxidoreductase activity and subcellular localization in compartments relevant to injury signaling, including the cytoplasm, plasma membrane, nucleus, and mitochondria (Fig. 9B). KEGG pathway analysis further pinpointed mechanistically coherent pathways, showing significant enrichment in the TNF signaling and MAPK signaling pathways—supporting the involvement of stress-activated kinase cascades such as JNK/p-JNK—as well as in reactive oxygen species–related pathways and drug metabolism–cytochrome P450, consistent with redox imbalance and xenobiotic metabolism dysfunction in DILI. Importantly, the apoptosis pathway was also prominently enriched, implicating mitochondrial apoptosis as a key downstream effector process (Fig. 9D). Concordantly, the PPI network revealed a densely connected interactome with inflammatory hubs (TNF, IL1B, PTGS2), signaling mediators (EGFR, SRC, MTOR), antioxidant and cytoprotective regulators (NFE2L2/Nrf2, HMOX1/HO-1, SOD2, SIRT1), and apoptotic effectors (BCL2, CASP3) occupying central positions, indicating strong crosstalk among these modules (Fig. 9C). Collectively, these multi-omics and network-level results support a multi-target hepatoprotective mechanism of TAS, whereby suppression of JNK/p-JNK–driven inflammatory signaling, reinforcement of the Nrf2/HO-1 antioxidant defense, and attenuation of Bax/Bcl-2–caspase–dependent apoptosis cooperatively contribute to the amelioration of DILI. Importantly, Western blot analysis in DILI mice liver tissues experimentally validated these computational predictions. In DILI Model groups, antioxidant defense pathways were severely impaired, as evidenced by downregulation of Nrf2 and its downstream target HO-1. Concurrently, stress- and inflammation-related signaling was activated, reflected by increased phosphorylation of JNK, elevated Bax/Bcl-2 ratios, and caspase-3 activation. Treatment with TAS formulations reversed these aberrations to varying extents. Importantly, TAS NCs@EPC/CAP showed the most potent activation of the Nrf2/HO-1 pathway, suppression of p-JNK/JNK signaling, and downregulation of Bax/Bcl-2 ratio and caspase-3 expression, achieving effects comparable to or exceeding those of SIL (Fig. 9D–F, Fig. S18). These results suggest that TAS ameliorates DILI through multi-target modulation of oxidative stress, inflammatory signaling, and apoptotic pathways, involving Nrf2/HO-1, TLR4/NF-κB/JNK, and mitochondrial apoptosis-related signaling [38,39,45]. By markedly improving oral bioavailability and intestinal absorption, TAS NCs@EPC/CAP amplifies these intrinsic pharmacological effects, resulting in superior in vivo hepatoprotection.

3. Discussion

DILI remains a leading cause of acute liver failure and drug attrition during development, yet effective therapeutic options are limited. TAS, a natural pentacyclic triterpenoid, has emerged as a promising hepatoprotective candidate due to its multi-target pharmacological activities. However, its clinical translation is fundamentally constrained by poor aqueous solubility and low oral bioavailability, characteristic of BCS class IV compounds, which result in insufficient systemic exposure and inconsistent therapeutic efficacy. In this study, we developed a sequential oral delivery platform, TAS NCs@EPC/CAP, that integrates nanocrystal technology, TPGS functional stabilizer, mucoadhesive polyelectrolyte complex micropatches, and enteric coating to coordinately overcome these barriers. By transforming this poorly soluble and low-permeability compound into a therapeutically effective candidate, our platform achieved a dramatic 3.1-fold enhancement in oral bioavailability and sustained hepatic enrichment, translating into potent protective efficacy in both intrinsic and idiosyncratic DILI mouse models.

The biopharmaceutical hurdles of TAS were first characterized, revealing negligible aqueous solubility, low apparent permeability (Papp = 0.79 × 10⁻⁶ cm/s) across Caco-2 monolayers, and substantial P-gp-mediated efflux (efflux ratio >2, Fig. S1). These findings confirmed that TAS faces dual solubility-permeability limitations. Nanocrystallization alone (TAS NCs) improved dissolution (Fig. 1J) and moderately increased systemic exposure (Fig. 5A). However, the limited pharmacokinetic gain indicated that dissolution enhancement alone cannot overcome permeability and efflux barriers. This observation aligns with the solubility-permeability interplay concept, which identifies permeability and efflux as rate-limiting steps once dissolution is improved [46]. To address this permeability bottleneck, we selected TPGS as a functional stabilizer. TPGS not only generated small and stable nanocrystals (Fig. 1C–G-I) but also inhibited P-gp efflux (Fig. S1C) and promoted hepatocyte uptake (Fig. 1L). Consistent with established nanocrystal principles [47], particle size reduction increased specific surface area and enhanced dissolution. The resulting TAS NCs/TPGS exhibited superior dissolution (88.4% in 24 h) and significantly higher Papp compared to P407- or PVA-stabilized NCs (Fig. 1J and K). These data underscore the critical role of stabilizer functionality beyond mere particle size reduction.

However, even with enhanced dissolution and efflux inhibition, orally administered nanocrystals are rapidly cleared by intestinal peristalsis and the mucus layer, limiting their residence at the absorption site. To address this limitation, we developed a bilayer mucoadhesive micropatch system based on CS and CP. FTIR and thermal analysis confirmed EPC formation via electrostatic interactions (Fig. 2B–D). Optimized EPC micropatches (CS:CP = 2:1) exhibited balanced swelling, strong mucoadhesion, and prolonged retention on intestinal mucosa (Fig. S4). Loading TAS NCs into the EPC matrix (TAS NCs@EPC) preserved these properties (Fig. 2G–J) and enabled homogeneous drug distribution without aggregation (Fig. 2E and F; Table S1). Beyond physical entrapment, the EPC matrix modulates drug release kinetics. Notably, TAS NCs@EPC exhibited substantially higher cumulative release (80.75% at 24 h) than free TAS-loaded patches (23.58%, Fig. 2K), reflecting the dissolution-enhancing effect of nanocrystallization. Importantly, this enhanced release occurred in a sustained manner due to the modulating effect of the EPC matrix.

The intestinal absorption mechanisms of TAS NCs@EPC were investigated using ex vivo permeation and in vitro Caco-2/mucus models. TAS NCs@EPC achieved the highest cumulative permeation across porcine intestinal mucosa (Fig. 3A) and the highest apparent permeability in both Caco-2 monolayers and Caco-2/mucus co-cultures (Fig. 3B), confirming that the formulation effectively overcomes both the mucus barrier and epithelial tight junctions. Mechanistically, this enhanced transport involves two complementary pathways: transcellular uptake and paracellular diffusion. Cellular uptake studies revealed that TAS NCs@EPC significantly increased intracellular TAS accumulation (Fig. 3C), attributable to TPGS-mediated uptake enhancement and direct nanocrystal internalization. This finding aligns with the established role of TPGS as a P-gp inhibitor that modulates ATPase activity and increases membrane fluidity, thereby preventing efflux of solubilized drug molecules back into the intestinal lumen [12]. Concurrently, TEER measurements showed that EPC-based micropatches reversibly decreased transepithelial electrical resistance (Fig. 3D), indicating transient opening of tight junctions. Immunofluorescence staining further confirmed downregulation of tight junction proteins ZO-1 and claudin-4 following treatment with EPC-based formulations (Fig. 3E–G; Fig. S7 and S8). Notably, CS was primarily responsible for tight junction modulation, while CP contributed to mucoadhesion and matrix swelling. This paracellular enhancement is consistent with previous reports describing the ability of chitosan derivatives to interact with negatively charged sialic acid residues on mucus and tight junction protein components [48]. Collectively, these results demonstrate that TAS NCs@EPC enhance intestinal absorption through a dual mechanism involving nanocrystal-facilitated transcellular uptake and EPC-mediated paracellular transport.

To ensure site-specific intestinal delivery, we encapsulated 20 micropatches into a single enteric-coated capsule (TAS NCs@EPC/CAP). The capsule remained intact in simulated gastric fluid (pH 1.2) for 2 h but rapidly disintegrated upon transfer to intestinal pH (6.8), releasing the micropatches (Fig. S9). Coating the micropatches with magnesium stearate effectively prevented self-aggregation upon release. In vivo imaging using NR-labeled micropatches confirmed that TAS NCs@EPC/CAP adhered to the small intestinal mucosa for up to 12 h post-administration, whereas free dye and nanocrystals were rapidly cleared (Fig. 4C–F). This prolonged mucosal retention creates a localized high-concentration drug depot at the epithelial interface, maximizing the concentration gradient that drives passive diffusion [49] and facilitating sustained drug absorption.

Pharmacokinetic studies confirmed the enhanced oral bioavailability of TAS NCs@EPC/CAP. Compared to free TAS, the formulation achieved a 3.1-fold increase in AUC_0-24 and a 5.8-fold elevation in C_max, along with prolonged half-life and reduced clearance (Fig. 5A and B). Tissue distribution analysis revealed that TAS NCs@EPC/CAP achieved the highest drug levels in the liver, with sustained accumulation over 12 h (Fig. 5C), whereas free TAS showed minimal hepatic exposure. This liver-targeting effect and the prolonged systemic circulation resulting from enhanced absorption. As noted by Thant et al. [50], improving the bioavailability of efflux-limited drugs via surfactant-based strategies often enhances target-organ engagement, ensuring that a therapeutic dose reaches hepatocytes before the onset of necrotic cascades. Such hepatic enrichment is particularly advantageous for DILI therapy, as it ensures that therapeutic concentrations reach the primary site of injury.

The therapeutic efficacy of TAS NCs@EPC/CAP was evaluated in two well-established DILI models: APAP-induced intrinsic injury and INH + RFP-induced idiosyncratic injury. APAP toxicity is primarily driven by mitochondrial oxidative stress and glutathione depletion, whereas the INH + RFP model simulates the complex, metabolically activated injury often encountered in tuberculosis treatment. In both models, TAS NCs@EPC/CAP treatment significantly ameliorated liver injury, as evidenced by reduced liver index, improved histological architecture, and normalized serum markers including ALT, AST, LDH, AKP, TBA, and TBIL (Fig. 7, Fig. S13). These protective effects were superior to those of free TAS and TAS NCs, and comparable or better than silymarin. TAS NCs@EPC/CAP attenuated oxidative stress by reducing ROS and MDA levels while restoring GSH and SOD activities (Fig. 8A–D, Fig. S14A–D), it also suppressed the production of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α (Fig. 8E–G, Fig. S14E–G) and reduced hepatocyte apoptosis, as determined by TUNEL staining (Fig. 8H,I, Fig. S14H and I).

To elucidate the molecular basis of TAS-mediated hepatoprotection, we employed network pharmacology. Overlap analysis of TAS targets with DILI-related genes identified 80 common targets (Fig. 9A). GO and KEGG enrichment revealed involvement in oxidative stress, inflammatory responses, and apoptosis pathways, including Nrf2/HO-1, TNF, MAPK, and apoptosis signaling (Fig. 9B–D). Protein-protein interaction network analysis highlighted hub genes such as NFE2L2 (Nrf2), HMOX1 (HO-1), JUN, MAPK8 (JNK), BCL2, CASP3, TNF, and IL1B (Fig. 9C). These hubs aligned closely with the pathways identified by enrichment analysis. Experimental validation by Western blot and immunohistochemistry confirmed that TAS NCs@EPC/CAP treatment upregulated Nrf2 and HO-1 expression, inhibited JNK phosphorylation, decreased the Bax/Bcl-2 ratio, and reduced cleaved caspase-3 levels in liver tissues from both DILI models (Fig. 9E and F). These findings are consistent with previous studies identifying TAS as a potent inducer of the antioxidant response element [39]. By activating the Nrf2/HO-1 pathway and suppressing JNK-mediated stress signaling, our formulation prevents the progression from initial biochemical insult to widespread hepatocellular necrosis.

Despite the promising results, several limitations of this study should be acknowledged. First, while the TAS NCs@EPC/CAP platform demonstrated superior efficacy in mouse DILI models, the translational relevance to human patients requires further validation, particularly given the species differences in drug metabolism and immune responses. Second, the long-term safety and potential chronic toxicity of repeated administration of this formulation have not been evaluated, which is essential for clinical translation. Third, the current study focused on prophylactic administration; whether the platform exerts therapeutic effects after established liver injury remains to be determined. Finally, the scalability and manufacturing reproducibility of the micropatch system need to be optimized for potential clinical translation.

Future work should address these limitations by evaluating therapeutic efficacy in a post-injury treatment regimen, conducting long-term toxicity studies in multiple animal species, and exploring the feasibility of scale-up manufacturing. Additionally, incorporating emerging technologies such as 3D printing for micropatch fabrication could enhance batch-to-batch consistency and enable personalized dosing strategies. Despite these limitations, the present study establishes a rationally designed oral delivery platform that successfully overcomes the multiple barriers limiting TAS absorption, providing a clinically translatable formulation for DILI therapy and a versatile strategy for oral delivery of poorly soluble natural products with multi-target therapeutic potential.

4. Conclusion

In this study, we developed a rationally designed oral delivery platform, designated as TAS NCs@EPC/CAP, that sequentially overcomes the multiple biopharmaceutical barriers limiting the clinical translation of TAS for DILI. By integrating TPGS-stabilized TAS nanocrystals into chitosan-carbopol polyelectrolyte complex micropatches and encapsulating them in enteric-coated capsules, the formulation achieves site-specific intestinal release, prolonged mucosal retention, transient tight junction modulation, and enhanced hepatocyte uptake. This coordinated strategy dramatically improves the oral bioavailability of TAS, achieving a 3.1-fold increase in AUC, and enables sustained hepatic enrichment. These pharmacokinetic enhancements directly translate into superior therapeutic efficacy in both intrinsic and idiosyncratic DILI models. Mechanistically, TAS NCs@EPC/CAP enhanced systemic exposure, prolonged intestinal retention, and increased liver accumulation, which translated into stronger activation of the Nrf2/HO-1 antioxidant pathway, more effective suppression of JNK-associated stress signaling, and greater inhibition of Bax/Bcl-2/caspase-3-mediated apoptosis. With its excellent safety profile and demonstrated efficacy, this platform not only provides a clinically translatable formulation for TAS but also offers a versatile and generalizable strategy to enhance the oral delivery of poorly soluble natural products with multi-target therapeutic potential.

5. Materials and methods

5.1. Materials

TAS was obtained from Xinkang Pharmaceutical Chemical Co., Ltd. (Hubei, China). Silymarin (SIL) was obtained from Tagetmol Chemicals Inc. (MA, USA) Poloxamer 407 (P407) was purchased from Shanghai Yunhong Chemical Preparation and Excipient Technology Co., Ltd. (Shanghai, China). Polyvinyl alcohol (PVA) purchased from Thermo Fisher Scientific Inc. (PA, USA). Ethyl cellulose (N22, 18-24 mPa) was supplied by Ashland (Philadelphia, USA). TPGS, CS (deacetylation degree 80.0% - 90.0%), CP, castor oil (CO), triethyl citrate (TEC, 98%), sodium dodecyl sulfate (SDS), carboxymethylcellulose sodium (CMC-Na) and dimethylbenzene were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Magnesium stearate (MS) and silicon dioxide (SID) were provided by Anhui Sunhere Pharmaceutical Excipients Co.,Ltd. (Huainan, China).

Cell counting kit-8 (CCK-8) GLPBIO Corporation (CA, USA), reactive oxygen species (ROS) assay kit, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), Alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (AKP), total bile acid (TBA), total bilirubin (‌TBIL) test kits were provided by Nanjing JianCheng Bioengineering Institute (Nanjing, China). Interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kits were supplied by ZCIBIO Technology Co., Ltd. (Shanghai, China). One-step TdT-mediated dUTP nick-end labeling (TUNEL) in situ apoptosis kit were supplied by Wuhan Eliruit Biotechnology Co., Ltd. (Wuhan, China). BCA protein assay kit, RIPA lysis buffer, glycine, hematoxylin and eosin (H&E) stains were supplied by Solarbio Science & Technology Inc. (Beijing, China). Triton-X100 and bovine serum albumin (BSA) were from Aladdin Reagent Inc. (Shanghai, China). Recombinant anti-ZO-1 tight junction protein antibody and goat anti-rabbit IgG H&L (Alexa Fluor®488) secondary antibodies were from Abcam Inc. (Cambridge, UK). Claudin 4 monoclonal antibody Alexa Fluor®594 was obtained from Invitrogen Corporation (CA, USA). DAPI was obtained from Beyotime Biotechnology Inc. (Shanghai, China).

5.2. Cells and animals

Caco-2 human colon cancer cell line was purchased from Procell Life Science & Technology Inc. (Wuhan, China) and were cultured in MEM medium (Procell) with 20% FBS (CellMax, Lanzhou, China) and 1% streptomycin/penicillin (CellMax) as described in S1.5.1. AML12 cells obtained from Shanghai Jinyuan Biotechnology Co., Ltd. (Shanghai, China) were cultured in specialized medium for AML12 cells (Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd., Shanghai, China) as detailed in S1.8. All cells were incubated in 5% CO₂ at 37 °C.

Kunming mice (male, 6-8 weeks) were provided by the Animal Care Welfare Committee (ACWC, Guizhou Medical University, No. 2503816). During the experiment, mice were reared at room temperature 24 ± 2 °C with 60-70% humidity under a 12 h light/dark cycle. All animal procedures strictly complied with protocols approved by the ACWC and were performed in accordance with the guidelines described in S1.9.1.

5.3. Biopharmaceutical characterization of TAS

The biopharmaceutical properties of TAS were evaluated using both in vitro and in vivo approaches. Firstly, the saturated solubility of TAS was measured in deionized water at 25 °C according to the method in S1.1, and its apparent permeability was assessed using the Caco-2 cell monolayer model as described in S1.1. Pharmacokinetic parameters and absolute oral bioavailability of TAS in mice were evaluated as described in 5.10.1 and S1.9.1.

5.4. Preparation and characterization of TAS NCs

TAS nanocrystals were prepared using a top-down ultrasonic milling method with P407, PVA, or TPGS as stabilizers. TAS (20 mg) was dispersed in 2 mL of stabilizer solution (0.25%, 0.5%, 1%, 2.5%, and 5%, w/v) and pre-mixed by vortexing, followed by probe ultrasonication (360 W, 3 s on/3 s off cycles) for 6 min in an ice bath to obtain TAS NCs/P407, TAS NCs/PVA, or TAS NCs/TPGS.

Particle size (PS), polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (DLS). Morphology was observed using transmission electron microscopy (TEM). Crystalline state was analyzed by X-ray powder diffraction (XRPD), while thermal behavior was characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). In vitro dissolution was evaluated using a dialysis method in phosphate buffer. Physical stability was assessed by monitoring PS and PDI during storage at 4 °C. Cellular uptake of AML12 and Caco-2 was quantified by intracellular TAS content. All procedures followed the protocols described in S1.2.

5.5. Preparation and characterization of TAS nanocrystal-loaded micropatches (TAS NCs@EPC)

Ethyl cellulose was used to form the backing layer, while EPC of CS and CP served as the mucoadhesive layer. Formation of EPC was confirmed by Fourier transform infrared spectroscopy (FT-IR) and thermal analysis as described in S1.3. Blank micropatches with different CS/CP ratios were prepared and optimized. Swelling behavior was also evaluated to assess the water absorption capacity. Mucoadhesive strength and mucosal residence time were determined using freshly excised porcine small intestinal mucosa according to the methods in S1.3.

Optimized micropatches were loaded with TAS nanocrystals to obtain TAS NCs@EPC. Surface morphology was examined by scanning electron microscopy (SEM). Swelling behavior, mucoadhesive strength, and in vitro drug release were evaluated using paddle dissolution methods under simulated physiological conditions as detailed in S1.4.

5.6. Intestinal absorption mechanisms of TAS NCs@EPC

Ex vivo permeation studies were conducted using porcine intestinal mucosa mounted on Franz diffusion cells. In vitro transport studies were performed using Caco-2 monoculture and Caco-2/mucus co-culture Transwell models as described in S1.5.1. Cellular uptake was quantified by intracellular TAS content following the protocol in S1.5.2. TJ integrity was assessed by transepithelial electrical resistance (TEER) measurements. Expression of tight junction proteins zonular occludens-1 (ZO-1) and claudin-4 (CLDN-4) was visualized by immunofluorescence staining and confocal laser scanning microscopy as detailed in S1.5.3.

5.7. Preparation and intestinal distribution of TAS NCs@EPC/CAP

To prevent premature adhesion and release, Twenty TAS NCs@EPC micropatches were encapsulated into one enteric-coated capsule to obtain TAS NCs@EPC/CAP (Fig. 4A). Anti-adhesion strategies using MS or SID coating were evaluated as described in S1.6. In vitro release was examined in simulated gastric (pH 1.2) and intestinal (pH 6.8) fluids. In vivo intestinal distribution and mucosal adhesion were assessed in mice using Nile red (NR) labeled micropatches and an in vivo imaging system according to the method in 5.10.2 and S1.9.2.

5.8. Identification of putative targets for TAS in DILI by network analysis

To systematically elucidate the molecular mechanisms underlying the therapeutic effects of TAS against intrinsic and idiosyncratic DILI, an integrated network pharmacology strategy was employed. Detailed methods for target prediction, Protein-Protein Interaction (PPI) network construction, and GO/KEGG enrichment are provided in S1.7.

5.9. In vitro hepatoprotective effects of TAS nanocrystals

AML12 cells were treated with APAP or INH + RFP to establish intrinsic and idiosyncratic DILI models. Cells were subsequently treated with Free TAS, TAS NCs, or positive drug SIL. Cell viability was evaluated using CCK-8 assay. Liver injury markers (ALT, AST), oxidative stress indicators (ROS, MDA, GSH, SOD), inflammatory cytokines (IL-1β, IL-6, TNF-α), and apoptosis were assessed using biochemical assays, ELISA, and TUNEL staining following the protocols in S1.8.

5.10. Animal experiments

5.10.1. Oral absorption and absolute bioavailability of TAS

Male Kunming mice were assigned to intravenous and oral Free TAS groups. TAS was administered at 10 mg/kg, plasma TAS concentrations were quantified by HPLC–MS/MS, and pharmacokinetic parameters and absolute oral bioavailability were calculated by non-compartmental analysis. Detailed sampling procedures are provided in S1.9.1.

5.10.2. Gastrointestinal distribution and mucosal retention

The gastrointestinal distribution and retention of Free NR, NR nanocrystals, and NR-labeled NCs@EPC/CAP were compared after oral administration to male Kunming mice. Fluorescence signals in the stomach, small intestine, and colon were recorded at predefined time points using an in vivo imaging system. The intestinal adhesion and swelling of released micropatches were also examined. Detailed procedures are provided in S1.9.2.

5.10.3. Pharmacokinetic and tissue distribution of TAS NCs@EPC/CAP

Forty-eight Kunming mice were randomly divided into three groups and orally administered free TAS, TAS NCs, or TAS NCs@EPC/CAP at an equivalent TAS dose of 10 mg/kg. Plasma and major organs were collected at predefined time points. TAS concentrations were quantified by LC–MS/MS to determine pharmacokinetic profiles and tissue distribution as detailed in S1.9.3.

5.10.4. In vivo evaluation of TAS NCs@EPC/CAP in DILI mice models

Intrinsic and idiosyncratic DILI models were induced in mice using APAP or INH + RFP, respectively. Mice were treated with Free TAS, TAS NCs, TAS NCs@EPC/CAP, or SIL at an equivalent TAS dose of 10 mg/kg according to the methods in S1.9.4. Liver index, histopathology (H&E), serum biochemical parameters (ALT, AST, LDH, AKP, TBA, TBIL), oxidative stress markers, inflammatory cytokines, apoptosis (TUNEL), and signaling pathways (Nrf2/HO-1, JNK, Bax/Bcl-2, caspase-3) were evaluated. Western blot and immunohistochemistry analysis were performed as described in S1.9.5. Safety was assessed by hematological analysis, urinalysis, and histological examination of major organs according to S1.9.6.

5.11. Statistical analysis

All data were analyzed by one-way ANOVA followed by Tukey's post hoc tests. The results were expressed as the mean ± SD of at least three independent experiments. p < 0.05 was considered significant. The number (n) refers to the number of experiments.

CRediT authorship contribution statement

Zimin Cai: Writing – original draft, Visualization, Software, Methodology, Data curation, Conceptualization. Huan Zhao: Visualization, Software, Project administration, Methodology, Conceptualization. Min Wei: Validation, Resources, Investigation, Formal analysis, Data curation. Zhimei Cheng: Resources, Investigation, Formal analysis, Data curation. Huizhou He: Project administration, Investigation, Formal analysis. Jinzhuan Xu: Investigation, Formal analysis, Data curation. Shan Xu: Validation, Investigation. Jing Huang: Validation, Investigation, Data curation. Tingting Xiao: Validation, Investigation. Jianqing Peng: Writing – review & editing, Project administration, Data curation. Runbin Sun: Writing – review & editing, Visualization, Supervision. Qianming Du: Writing – review & editing, Supervision. Yi Chen: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare no competing financial interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.