Oral Icariin Nanoparticles Ameliorate Nonalcoholic Fatty Liver Disease by Alleviating Oxidative Stress

Abstract

Chronic inflammatory liver conditions, such as nonalcoholic fatty liver disease (NAFLD), are frequently exacerbated by oxidative stress, rendering the neutralization of reactive oxygen species (ROS) crucial for their amelioration. Oral administration of nanoparticles (NPs) presents an optimal strategy for NAFLD management due to its convenience. This research aimed to overcome the poor water solubility and low bioavailability of icariin (ICA) by engineering an oral nanosystem, ICA-loaded poly­(lactic-co-glycolic acid)-poly­(ethylene glycol) (PLGA) NPs with chitosan (CS) and mannose surface modification (ICA-NPs), to augment ICA’s antioxidant potency against NAFLD. Methods involved comprehensive in vitro characterization of ICA-NPs (including dispersibility, biocompatibility, HepG2 targeting, and ROS scavenging capabilities) and in vivo studies in high-fat diet (HFD)-induced NAFLD mice, assessing liver accumulation, ROS scavenging, and antiobesity effects across multiple experimental groups. Results demonstrated that this targeted oral NP system significantly amplified ICA’s therapeutic impact, leading to substantial reductions in body weight, diminished white adipose tissue accumulation, decreased hepatic lipid content, improved hepatic function, and a notable suppression of ROS in the livers of NAFLD-afflicted mice. In conclusion, by harnessing the precision of targeted delivery, this oral nanosystem not only enhances ICA’s therapeutic efficacy but also establishes a safe and effective platform for the oral administration of herbal remedies for liver conditions, suggesting a promising avenue for advancing herbal medicine in liver disease treatment.

1. Introduction

Fatty liver conditions, predominantly nonalcoholic fatty liver disease (NAFLD), have emerged as the leading hepatic disorder, affecting approximately one-quarter of the global population. Initially considered a benign and stable condition, evidence now indicates that NAFLD can progress rapidly to severe and irreversible liver damage. Untreated, NAFLD can evolve into steatohepatitis with fibrosis and, in extreme cases, may progress to cirrhosis or hepatocellular carcinoma. Research has highlighted that sustained overconsumption of energy-rich foods fosters hepatic steatosis, which triggers oxidative stress, lipid peroxidation, and perpetuates hepatic inflammation. Current clinical approaches to NAFLD management include lifestyle interventions, metabolic risk factor modulation, and the aversion of hepatic complications. However, existing therapeutic options have yet to meet the desired clinical efficacy and safety thresholds, with no treatments endorsed by the US Food and Drug Administration for NAFLD. This underscores an urgent need for the discovery of efficacious and secure pharmacological interventions for managing NAFLD.

Oxidative stress plays a central role in the complex interplay of chronic inflammatory liver diseases, including NAFLD. The persistent disruption of lipid homeostasis is closely associated with an imbalance between pro-oxidants and antioxidants, adversely affecting cellular organelles involved in metabolism. This dysregulation can lead to lipotoxicity, oxidative degradation of lipids, sustained endoplasmic reticulum (ER) stress, and impaired mitochondrial function. Furthermore, heightened oxidative stress can trigger hepatocellular stress pathways, fostering inflammatory responses and fibrotic changes, which are instrumental in the evolution of nonalcoholic steatohepatitis (NASH). The body’s antioxidant mechanisms are crucial in counteracting the harmful effects of oxidative stress, aiding in the re-establishment of lipid metabolism’s normalcy. Broadly, the antioxidant defense mechanisms protect hepatocytes from the ravages of reactive oxygen species (ROS), which may originate from the gastrointestinal (GI) tract as metabolic byproducts, the microbiota (both balanced and imbalanced), and dietary components. This protection is facilitated through an enhanced gut–liver axis. Consequently, strategies that focus on modulating the antioxidant response present a promising avenue for thwarting the initiation and advancement of NAFLD.

Icariin (ICA), a principal bioactive flavonoid derived from the Epimedium genus, has been a cornerstone in the research of traditional Chinese medicine. Extensive pharmacological investigations have revealed its potential in treating a wide spectrum of conditions, including neurodegenerative and cardiovascular diseases, osteoporosis, inflammatory responses, oxidative stress, depressive disorders, and cancers.

Despite its promise, the clinical application of ICA is encumbered by challenges such as suboptimal permeability, susceptibility to rapid degradation, diminished bioavailability, and the absence of targeted specificity, leading to limited therapeutic efficacy. Furthermore, the prospect of safety hazards looms with prolonged or excessive ICA administration, with respiratory complications among the potential adverse effects. Consequently, the enhancement of ICA’s targeted delivery and the attenuation of its toxicities are imperative for its successful therapeutic integration.

In recent years, nanotechnology-driven ICA formulations have emerged as a focal point of interest, showcasing the potential to revolutionize ICA’s clinical landscape by addressing its pharmacokinetic and pharmacodynamic limitations. Notably, PLGA–PEG-based nanoparticles (NPs) have been shown to improve the oral bioavailability of hydrophobic compounds like ICA through enhanced GI stability and cellular uptake. NPs have emerged as a formidable drug delivery platform in the therapeutic landscape of NAFLD. The nanoscale dimensions and tunable surface characteristics of NPs endow them with the capacity to enhance drug bioavailability, safeguard drugs from premature degradation, expedite GI absorption, and augment cellular internalization at the intended site of action. Moreover, NPs can be engineered with specificity for particular tissues, such as the liver, which is pivotal for diminishing drug elimination, curtailing nontarget tissue accumulation, and bolstering the selective uptake by hepatic cells.

Oral drug delivery systems are favored for their potential to enhance patient convenience and minimize adverse effects, thereby increasing treatment adherence. However, the journey of oral NPs through the body is fraught with physiological challenges that can impede their therapeutic efficacy. These obstacles include susceptibility to chemical and enzymatic degradation within the GI tract, the dynamic nature of the mucin layer, the difficulty in traversing the mucin barrier, and rapid clearance from systemic circulation. Chitosan (CS), a naturally occurring polysaccharide composed of β-(1–4)-linked anhydro-d-glucosamine and anhydro-N-acetylglucosamine units, has been extensively utilized to address these challenges. When NPs are coated with CS, they gain a protective shield against degradation in the GI tract and experience enhanced intestinal permeability, which can be instrumental in overcoming the aforementioned physiological barriers and optimizing the therapeutic potential of orally administered NPs. Similarly, mannose modification facilitates hepatocyte-specific targeting through receptor-mediated mechanisms, further improving liver accumulation and efficacy. ,

In this study, we developed a nanotechnology-driven delivery platform for ICA by using a straightforward emulsification method. The process began with the fabrication of PLGA–PEG-based NPs designed to encapsulate ICA, which were then coated with chitosan and functionalized with mannose to enhance mucosal absorption (ICA-NPs). In contrast to previous oral nanoformulations for natural products (e.g., polyphenols or flavonoids), which often focus on single functionalization, our ICA-NP system employs a dual-modification strategy using CS and mannose. This design leverages CS to improve mucoadhesion and intestinal permeability, while mannose enables active targeting to hepatocytes via mannose receptor-mediated endocytosis. , This approach not only enhances oral bioavailability but also provides mechanistic insights into liver-specific ROS scavenging and lipid metabolism regulation, representing a significant advancement over conventional nanocarriers, as illustrated in Figure . In a high-fat diet (HFD)-induced NAFLD murine model, ICA-NPs significantly reduced the production of hepatic inflammatory mediators, leading to the normalization of hepatic function. Histological assessments, including Oil Red O staining and hematoxylin and eosin (H&E) staining, further supported these findings.

1.

Schematic process of preparing chitosan-modified PLGA–PEG NPs loaded with icariin (ICA-NPs) and their application in the treatment of NAFLD. (A) Formulation of ICA-loaded PLGA–PEG NPs via the emulsification-evaporation method and the subsequent conjugation of chitosan and mannose onto the NP surface. (B) After oral administration of ICA-NPs, transiting GI in protection of NPs, (C) ICA is delivered to the liver to play an anti-ROS role.

2. Materials and Methods

2.1. Materials

ICA (98%) was purchased from Anhui Zesheng Technology Co., Ltd. (Anqing, China). PLGA (lactide:glycolide 50:50, molecular weight 38,000 g/mol) and 2′,7′-dichlorofuorescein diacetate (DCFH-DA) kits were bought from Sigma (USA). Dichloromethane was bought from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China). CS (deacetylated chitin), with a degree of deacetylation and a molecular weight of 20–60 kDa, was bought from MKBio. Co., Shanghai, China. D-Mannose (purity >98%, molecular weight of 180.16, CAS: 3458-28-4) was obtained from BioDuly Co., Nanjing, China. The MTT kit was obtained from Beyotime Biotech Inc. (Nanjing, China). Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA), fetal bovine serum (FBS), trypsin, and penicillin-streptomycin were purchased from Gibco BRL (USA). Oleic acid (OA): purity ≥ 99%; palmitic acid (PA): purity ≥ 99%; and bovine serum albumin (BSA): fatty acid-free were bought from Sigma (USA). Human TNF-α and IL-6 ELISA kits were purchased from UpingBio, Co., Shanghai, China.

2.2. Cells

HepG2 cells were procured from Fuheng Biology, located in Shanghai, China. These cells were nurtured in DMEM, supplemented with 10% FBS (Gibco), along with a mixture of 100 μg/mL of streptomycin and 100 IU/mL of penicillin. The cell culture was maintained under a controlled atmosphere of 5% CO₂ at a temperature of 37 °C.

2.3. Preparation of ICA-Loaded NPs (ICA-NPs)

The synthesis of ICA-NPs was achieved through a modified emulsification and evaporation technique, as detailed in a previous study. To begin, 60 mg of ICA and 200 mg of PLGA were dissolved in 5 mL of dichloromethane to form the organic phase, which was then homogenized for 90 s under ice-cold conditions. Subsequently, 15 mL of the aqueous phase, consisting of a 1% poly­(vinyl alcohol) (PVA) solution enriched with 0.3% chitosan, 0.3% mannose, and 0.5% glacial acetic acid, was gently incorporated into the organic phase. This mixture was further homogenized for 6 min on ice, yielding an ICA-encapsulated PLGA emulsion. The emulsion was then transferred to a fume hood and stirred for 6 h to facilitate solvent evaporation and NP solidification. The resulting NPs were isolated by centrifugation at 12,000 rpm for 20 min and subsequently rinsed with ultrapure water three times to remove any residual impurities. The purified NPs were either stored at 4 °C for immediate use or subjected to lyophilization for an extended shelf life.

2.4. Characterization of NPs

The NP size distribution and zeta potential were characterized using a Nano Particle Analyzer SZ-100 from Horiba Scientific, USA. The topographical examination of the NPs was conducted with scanning electron microscopy (SEM, ZEISS Supra 55, Germany). The encapsulation efficiency of ICA was quantified using an ultraviolet–visible spectrophotometer (UV-2700) from Shimadzu Corporation, Japan. The loading rate and ICA-NPs concentrations were ascertained employing a microplate reader (Infinite 200 pro, Switzerland) at an absorbance wavelength of 465 nm, with ICA concentrations extrapolated from a pre-established standard curve. The crystallographic structure of the NPs was meticulously scrutinized through differential scanning calorimetry (DSC) spectra, performed on a thermal analyzer (TA Q10, USA), complemented by Tyndall effect observations.

2.5. In Vitro Cytotoxicity of ICA and ICA-NPs

HepG2 cells were plated at a density of 5,000 cells per well in a 96-well plate, each well filled with 100 μL of complete DMEM. The cells were allowed to adhere and proliferate for 24 h. Following the removal of the spent medium, the cells were exposed to 100 μL of fresh medium supplemented with varying concentrations of ICA and ICA-NPs for an additional 24 h incubation period. Subsequently, cell viability was assessed using the CCK-8 assay.

2.6. Cellular Uptake of NPs

The cells were incubated with Coumarin 6 (C6)-labeled NPs for 4 h, after which they were rinsed twice with phosphate-buffered saline (PBS) to eliminate any unbound NPs. After this, the cells were visualized using a Live Cell Imaging System (EVOSFL Auto, Invitrogen, USA).

2.7. Intracellular ROS Production

Intracellular ROS levels were evaluated by using the DCFH-DA assay kit. HepG2 cells, at a concentration of 5 × 10⁵ cells/mL, were initially cultured in 6-well plates and allowed to settle for 12 h. Subsequently, a fresh complete medium containing varying concentrations of the test compounds was introduced to the plates, and the cells were further incubated for 2 h. Following this, all groups, except the negative control, were challenged with 1 μg/mL lipopolysaccharide (LPS) for 6 h. The cells were then collected, rinsed twice with PBS, and incubated with 10 μM DCFH-DA in the medium for 30 min at 37 °C in a dark environment to facilitate ROS detection. Excess DCFH-DA was removed by a final PBS rinse, and the intracellular ROS levels were quantified using flow cytometry, adhering to the manufacturer’s recommended protocol.

2.8. Animal Study

Male C57BL/6J mice, aged 8 to 10 weeks, were sourced from SPF Biotechnology Co., Ltd. (Beijing, China). To induce the NAFLD model, C57BL/6J mice were randomly allocated to either a standard chow diet or an HFD (D12492, Guangdong Medical Animal Center, Guangdong, China) delivering 60% of their caloric intake from fat for 2 months to induce obesity. This dietary intervention led to a significant increase in body weight, with each mouse reaching an average of 35 g. The conduct of all experimental procedures adhered to the ethical standards and regulatory requirements set forth by the Animal Ethics Committee of Guangdong Province, China. The protocols for animal care and the study were reviewed and approved by the Institutional Animal Care and Use Committee at Guangdong Medical University, with reference number GDMU-2023–000030.

2.9. Hemolysis Assay

To evaluate the in vivo biosafety of the compounds, hemolytic assays were performed on red blood cells (RBCs) following the established methodologies. RBCs were extracted from healthy mice by centrifuging anticoagulated whole blood with PBS at 3000 rpm for 10 min to create a 5% RBC suspension in PBS (v/v). This suspension was then combined with different concentrations of the test compounds. The resulting mixtures were incubated at 37 °C for 4 h with 100 μg/mL of the compounds. Postincubation, the samples were centrifuged at 3000 rpm and 4 °C for 5 min to separate the supernatant, which was subsequently collected. The absorbance of the supernatant was measured at 540 nm using a microplate reader (TECAN, Switzerland) to assess hemolysis. RBCs lysed in distilled water constituted the positive control, while those suspended in PBS served as the negative control.

2.10. In Vivo Biodistribution of NPs

After the NAFLD mouse model was incubated, indocyanine green (ICG), a fluorescent marker, was embedded into the NPs to facilitate tracking. The mice were imaged at intervals of 4, 8, 12, and 24 h postoral administration using the IVIS Lumina LT In Vivo Imaging System (PerkinElmer, Waltham, MA, USA) under isoflurane-induced anesthesia. Following the imaging session, the mice were humanely euthanized, and their internal organs, with a focus on the liver, were extracted for detailed examination. The fluorescent signatures of these organs were documented using the IVIS imaging system.

2.11. Antiobesity Effect of ICA or ICA-NPs

Upon reaching a body weight of 35 g, the NAFLD mice received either saline (n = 6), ICA (n = 6), or ICA-NPs (n = 6) via oral gavage at 2-day intervals over 8 weeks. The administered dose of ICA was set at 1 mg/kg, with the free ICA being dissolved in saline that contained 1% dimethyl sulfoxide (DMSO). Mice in the control group (n = 6), maintained on a regular chow diet, were administered saline. Each experimental group consisted of six mice. The body weight and food consumption of the mice were closely monitored throughout the study duration.

2.12. Assay of Serum and Hepatic Biochemical Indices

Serum and hepatic biochemical indicators were quantified utilizing assay kits, strictly adhering to the manufacturer’s protocol as outlined in the accompanying manuals (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China). The levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum triglycerides (TGs), total cholesterol (TC), and LDL-C in the serum were assessed through an enzymatic colorimetric method.

2.13. Histology

Upon humane euthanasia, the mice were subjected to necropsy to excise and weigh key organs, including the liver, inguinal white adipose tissue (iWAT), heart, spleen, lungs, and kidneys. The excised tissues were immersed in a 4% paraformaldehyde solution (Seven Biotech, Beijing, China) for 48 h to ensure proper fixation. Postfixation, the tissues underwent a dehydration process through a graded series of alcohol, were cleared with xylene, and then were embedded in paraffin wax for histological preparation. Using a paraffin microtome (Leica RM 2255, Leica, Germany), the embedded tissues were sectioned into 5–7 μm-thick slices. These sections were subsequently stained with an H&E staining kit (Solarbio Life Sciences, Beijing, China) to visualize cellular structures. Microscopic examination and imaging of the stained sections were performed using a Nikon microscope (DS-Ri2, Tokyo, Japan). The area of adipocytes was quantified using ImageJ software (NIH, Bethesda, Maryland, USA), providing a quantitative assessment of tissue morphology. The following histopathology scores of livers were analyzed: steatosis (6–33%, 1; 33–66%, 2; >66%, 3); ballooning (none, 0; few, 1; many, 2); and inflammation (200x < 1, 0; 1–2, 1; 2–4, 2; >4, 3).

2.14. Adipocyte Differentiation and Oil Red O Staining

Frozen tissue sections were initially fixed with a 4% paraformaldehyde (PFA) solution, followed by a thorough washing process with three changes of water and subsequent air drying. The sections were then briefly immersed in isopropanol for 30 s to eliminate residual moisture. Following this, the specimens were incubated with an Oil Red staining solution for 20 min, after which they were rinsed again with isopropanol for 30 s. The specimens were then washed with distilled water and counterstained with hematoxylin for 2 min, followed by a rinse with running water to remove excess stain. Once the staining process was complete, the specimens were mounted, and the stained sections were examined under a microscope to assess cellular lipid content and morphology.

2.15. Assay of ROS Level in Livers

The assessment of in vivo ROS levels was executed following with our prior publication. Mice were rendered unconscious using anesthesia before tissue collection. The liver was extracted, swiftly rinsed with PBS, and then immersed in DMEM containing 10 μM DCFH-DA for 30 min at 37 °C in a dark environment to allow for ROS detection. Following a dual rinse with DMEM to remove any unbound DCFH-DA, the livers were placed in a Phenol red-free medium to prevent autofluorescence interference. The fluorescent signal indicative of ROS was captured using the IVIS Lumina LT In Vivo Imaging System (PerkinElmer, Waltham, MA, USA).

2.16. In Vitro Anti-Inflammatory Assessment in the HepG2 NAFLD Model

To evaluate the anti-inflammatory effects of ICA-NPs versus free ICA in a HepG2 NAFLD model, the expression levels of inflammatory cytokines (TNF-α and IL-6) in cell culture supernatants were quantified by ELISA. HepG2 cells were seeded at 5 × 10⁵ cells/well in 6-well plates with complete DMEM. After 24 h, the NAFLD model was induced by treating cells for 24 h with a fatty acid mixture (FAFM). FAFM was prepared to contain 0.5 mM total fatty acids (palmitic acid (PA):oleic acid (OA) = 1:2 molar ratio) and 1% (w/v) fatty acid-free BSA in serum-containing DMEM. Briefly, a 10% BSA stock was used. 100 mM PA stock and 100 mM OA stock were prepared by heating at 70 °C.

Following FAFM induction, cells were washed once with PBS and then treated for an additional 24 h. Experimental groups included: basal (complete medium); PBS (FAFM only); ICA+HFD; and ICA-NPs + HDF. Free ICA and ICA-NPs were administered in serum-free DMEM. After treatment, cell culture supernatants were collected and centrifuged at 2000 rpm for 5 min at 4 °C to remove cellular debris. Inflammatory cytokine levels were subsequently quantified using specific ELISA kits according to the manufacturer’s instructions.

2.17. Confocal Immunofluorescence Analysis of Nrf2 Nuclear Translocation

Nrf2 subcellular localization, indicative of its activation, was assessed by using immunofluorescence confocal microscopy to investigate the Nrf2 pathway in response to oxidative stress. HepG2 cells were cultured in confocal dishes and subjected to experimental treatments as described in Section 2.16 (for NAFLD model induction and drug treatment). Following treatment, cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells were then permeabilized with 0.1% Triton X-100 in PBST (PBS containing 0.1% Triton X-100) for 5 min, followed by three 5 min washes with PBST. Nonspecific binding was blocked with 5% BSA in PBST for 1 h. Primary Nrf2 antibody (1:400 dilution) was then added and incubated overnight at 4 °C. After three PBST washes, cells were incubated with AF488-conjugated secondary antibody (1:400 dilution) for 2 h at room temperature in the dark. Following the final PBST washes, nuclei were stained with 600 μM DAPI for 3 min, followed by three PBST washes. Dishes were then mounted for observation using a confocal microscope.

2.18. Mannose Receptor-Mediated NP Uptake Assay

A D-mannose competitive inhibition assay was performed to investigate the mannose receptor-mediated cellular uptake of NPs, thereby validating targeting specificity. A 500 mM D-mannose stock solution (purity ≥ 99%) was prepared, followed by sterile filtration (0.22 μm). HepG2 cells were seeded and allowed to adhere. Experimental groups included (A) cells treated with C6 stock solution; (B) cells treated with C6-labeled NPs stock solution; (C) cells preincubated with D-mannose followed by C6 treatment; and (D) cells preincubated with D-mannose followed by C6-NPs treatment.

For competitive inhibition groups (C and D), cells were preincubated with 100 μL of 50 mM D-mannose (a 1:10 dilution of the 500 mM stock solution) per well for 1 h at 37 °C in a 5% CO₂ incubator. Following preincubation, or for control groups (A and B), the medium was aspirated, and cells were washed once with PBS. Subsequently, the respective C6 or C6-NPs stock solutions were added to the wells as described above, and cells were incubated for an additional 1 h. After incubation, cells were rinsed twice with PBS to remove unbound NPs, and cells were then covered with fresh culture medium for observation using a fluorescence inverted microscope.

2.19. Statistical Analysis

Based on the assumption that the data are normally distributed, we used a parametric test. Data were presented as the mean ± standard deviation (SD). To ascertain the statistical significance across multiple groups, a one-way analysis of variance (ANOVA) was employed.

3. Results

3.1. Synthesis of ICA-Loaded Chitosan-Modified PLGA–PEG NPs

Enhancing the oral bioavailability of drugs with limited solubility and augmenting their intestinal absorption are persistent hurdles in the realm of oral pharmaceutical development. The primary impediments to the gastrointestinal absorption of hydrophobic drugs include their scant aqueous solubility and the impediment presented by the lipid bilayers of the gastrointestinal tract’s epithelial cells. NPs, with dimensions typically below 500 nm, can emulate chylomicrons, thereby promoting their uptake by the epithelial cells lining the GI tract. ICA, despite its robust antioxidant capabilities, faces significant limitations in clinical application due to its poor aqueous solubility and the potential for dose-dependent side effects.

In this investigation, to capitalize on ICA’s potent antioxidant attributes, ICA-encapsulated oral NPs were fabricated using an emulsification-evaporation technique, as detailed in our preceding studies. During the fabrication process of PLGA–PEG NPs, ICA, previously dissolved in DMSO, was integrated into the oil phase, setting the stage for the creation of a delivery system designed to enhance ICA’s therapeutic potential.

However, the mucosal adhesion of these PLGA–PEG NPs is suboptimal, which consequently results in diminished oral bioavailability. To surmount this challenge, we incorporated naturally derived, biologically active componentschitosan and mannoseto functionalize the surface of the PLGA–PEG NPs. In the fabrication process, both mannose and chitosan were integrated into the aqueous phase, as depicted in the synthesis schematic (Figure A). Chitosan, known for its mucoadhesive properties, promotes a more effective mucosal absorption. Post oral administration, the ICA-loaded PLGA–PEG NPs, now adorned with chitosan and mannose (ICA-NPs), are designed to withstand the acidic gastric environment and proceed to the intestinal tract. The encapsulated ICA, once released, is poised to penetrate the liver, mitigate hepatic steatosis, and repair hepatic injury by neutralizing ROS, as illustrated in Figure B–C.

3.2. Characterization of ICA-NPs

Dynamic light scattering (DLS) measurements revealed that the ICA-NPs have an average diameter of approximately 200 nm (Figure A), with a polydispersity index (PDI) below 0.5, signifying a high degree of uniformity and stability within the NP suspension. The zeta potential of around −54 mV (Figure B) suggests that the NPs possess a negative surface charge, which is instrumental in their affinity for the cationic surfaces within the colonic mucosa, as previously reported. SEM confirmed the regular spherical morphology of the NPs (Figure C).

2.

Characterizations of ICA-NPs. (A) Size distribution and (B) zeta potential of ICA-NPs were measured by an NP size analyzer, respectively. (C) Representative morphology image of ICA-NPs was determined by a scanning electron microscope (scale bar = 2 μm). (D) Chitosan, mannose, PLGA–PEG, ICA, and ICA-NPs modified with chitosan were measured by FT-IR spectrogram. DSC thermograms (E) and TGA diagrams (F) of ICA and ICA-NPs, respectively. In vitro biosafety of CS@ICA-NPs. (G) Cell viability of HepG2 cells treated with different concentrations of ICA or ICA-NPs for 24 h. (H,I) Images and quantification analysis of the hemolysis assay of free ICA and ICA-NPs. Data are expressed as mean ± SD (n = 3). ****P < 0.0001 versus the H₂O group (one-way ANOVA with Dunnett’s post hoc test). (J) Morphology of erythrocytes cocultured with ICA or ICA-NPs for 60 min at 37 °C imaged under a live cell imaging system (scale bar = 100 μm).

The physicochemical characteristics of the ICA-NPs were further elucidated by using Fourier-transform infrared spectroscopy (FT-IR). The absence of significant shifts or the loss of functional group peaks in the spectra indicates that there was no substantial chemical interaction between the drug and the polymer matrix during encapsulation. The FT-IR spectrum of ICA-NPs (Figure D) retains the characteristic peak of the asymmetric stretching vibration of hydroxyl and amino groups in chitosan at 3378.08 cm^–1 alongside the characteristic peak at 1382.89 cm^–1 for C–H bond bending vibrations. Concurrently, characteristic peaks are observed for mannose 0 at 3378.08 cm^–1 (stretching vibration of monosaccharide hydroxyl groups) and 2944.35 cm^–1 (asymmetric stretching vibration of saturated C–H bonds). The peak at 1762.69 cm^–1 corresponds to the carbonyl stretching vibration of the PLGA segment in PLGA–PEG. The peak at 1453.06 cm^–1 corresponds to the bending vibration of saturated C–H bonds, while the peaks at 1180.71, 1130.54, and 1090.92 cm^–1 are attributed to C–O vibrations within the PLGA–PEG structure. Icariin is primarily characterized by peaks at 1650.69, 1598.03, and 1510.68 cm^–1, all attributed to the aromatic ring CC stretching vibrations of the flavonoid aglycone moiety within the molecule. The preservation of characteristic peaks for both the polymer and the drug in the FT-IR spectra of ICA-NPs (Figure D) corroborates this finding. The encapsulation efficiency of ICA within the NPs was determined to be approximately 78%, calculated using a standard curve derived from the UV absorption peak of ICA at 270 nm.

To investigate the in vitro release kinetics of ICA, we adopted the oscillating dialysis bag method based on previous reports, with slight modifications. Briefly, using 100 mL of PBS buffer at pH 7.4 and pH 2 as the release media, respectively, an amount of nanodrug equivalent to 10 mg of ICA was dissolved in the corresponding medium and placed into an 8000–14,000D dialysis bag. The system was then incubated in the dark at 37 °C under continuous shaking at 120 rpm. At predetermined time intervals (1, 2, 4, 8, 12, 24, 36, 48, and 72 h), 3 mL of sample was withdrawn from the release medium and replaced with an equal volume of fresh buffer. The collected samples were centrifuged at 10,000 rpm and 25 °C for 10 min, and the concentration of ICA in the supernatant was measured to calculate the cumulative drug release. All experiments were performed in triplicate, and the results are presented as mean values. As shown in Supplementary Figure S2, less than 20% of ICA was released under acidic conditions, indicating the ability of the NPs to resist gastric acid. In contrast, at pH 7.4, the NP formulation exhibited a clear sustained-release profile.

3.3. DSC and TGA Analysis

DSC is a pivotal analytical technique for evaluating the thermal characteristics of complex systems, offering valuable insights into the influence of the PLGA–PEG NP vehicle on the thermal stability of ICA. As depicted in Figure E, the characteristic heat absorption peak of ICA was no longer evident in the ICA-NPs, suggesting that the crystalline form of ICA was altered to an amorphous state upon complexation with PLGA. This transition to an amorphous state within the nanocomplex potentially enhances the solubility and bioavailability of ICA, underscoring the role of the NP carrier in stabilizing the drug.

Thermogravimetric (TGA) analysis profiles of ICA in its pristine state and postencapsulation are presented in Figure F. Pure ICA demonstrated a rapid, near-linear decomposition from ambient temperature to 400 °C. In contrast, the TGA of ICA-NPs revealed three distinct weight loss events. The initial weight loss, occurring between 0 and 200 °C, is predominantly ascribed to the desorption of moisture from the NP surface. The second phase of weight reduction, ranging from 200 to 300 °C, is attributed to the decomposition of non-cross-linked ICA and the evaporation of process-related volatiles. The most pronounced weight loss rate for ICA was detected between 200 and 300 °C, which is mainly due to the thermal decomposition of ICA, the disintegration of PLGA, and the off-gassing of volatile components during NP fabrication. The final weight loss stage, near 400 °C, reflects a complex series of degradation reactions. Comparative analysis with unencapsulated ICA indicates that ICA-NPs experienced a diminished overall weight loss and a decelerated rate of mass reduction, implying a stabilizing interaction between PLGA and ICA. The encapsulation of ICA within a PLGA matrix through noncovalent bonding likely endows a core–shell architecture, which substantially augments ICA’s thermal resilience.

3.4. In Vitro and In Vivo Biosafety of Free ICA or ICA-NPs

The biocompatibility and biosafety profiles of NP systems are paramount for their clinical utility. In this study, we evaluated the cytotoxic effects of both free ICA and ICA-NPs on HepG2 cells. As depicted in Figure G, free ICA modestly diminished the cell viability at concentrations above the therapeutic threshold. Specifically, ICA was nontoxic to HepG2 at concentrations below 25 μg/mL but became significantly detrimental at a concentration of 50 μg/mL. In contrast, ICA-NPs, even at low concentrations (≤25 μg/mL), demonstrated minimal toxicity, highlighting their excellent biocompatibility with HepG2. Most notably, ICA-NPs maintained a nontoxic profile at elevated concentrations across both cell types. Collectively, these observations indicate that ICA-NPs not only mitigate cytotoxicity but also enhance the overall biosafety of ICA, making them a promising candidate for clinical translation.

To ascertain the in vivo biosafety of ICA and ICA-NPs, their hemolytic effects on murine red blood cells were evaluated, following established protocols. Hemolysis induced by water served as the positive control with a hemolysis rate normalized to 1.0. An equivalent volume of erythrocytes was introduced into a 12-well plate, and varying concentrations of ICA (20 μg/mL, based on the ICA content in the NPs) were added and incubated at 37 °C for 30 min. Distilled water or PBS was used as the positive or negative control, respectively. As illustrated in Figure H–J, free ICA induced little hemolysis. Erythrocytes treated with ICA-NPs showed negligible hemolysis. Moreover, the morphological integrity of erythrocytes subjected to different treatments was examined under a light microscope. Figure J reveals that erythrocytes exposed to water were extensively lysed with minimal cellular remnants. Conversely, the PBS-treated group exhibited a plethora of intact erythrocytes, maintaining their characteristic biconcave shape. Notably, erythrocytes incubated with ICA-NPs retained their membrane integrity and healthy morphology, suggesting that the NP platform significantly attenuates hemolytic potential and enhances the in vivo biocompatibility of ICA.

To ascertain the in vivo toxicity profile of ICA, a comprehensive assessment was conducted on additional vital organs, including the kidneys, spleen, lungs, and heart, harvested from NAFLD mice that had received various treatments. These organs were subjected to H&E staining to scrutinize for any pathological changes. The findings, as presented in Figure S1, revealed no discernible differences across all experimental groups, thereby attesting to the biosafety of ICA-NPs in the context of NAFLD treatment. This histological evaluation further reinforces the potential of ICA-NPs as a safe therapeutic strategy for managing NAFLD without inducing adverse effects on major organs.

3.5. In Vitro and In Vivo Targeting Properties of ICA-NPs

Given the subdued fluorescence of ICA, we coencapsulated C6 or ICG within ICA-NPs to serve as luminescent tracers. To evaluate the biocompatibility and hepatic targeting efficacy of the various ICA formulations, the human hepatoma cell line HepG2 was utilized as a cellular model (Figure A). Following a 2 h coincubation period, a marked escalation in cellular internalization of C6/ICA-NPs was observed in HepG2 cells, surpassing that of C6/ICA (Figure B). These findings suggest that the NP formulation substantially augments the cellular uptake of ICA, underscoring the potential of this approach for enhanced drug delivery to liver cells. To further elucidate this enhanced cellular uptake mechanism, particularly the role of mannose receptors, a D-mannose competitive inhibition assay was performed using C6-NPs. Fluorescence microscopy revealed strong intracellular fluorescent signals for C6-NPs, while free C6 showed a relatively low and diffuse distribution (Figure S3). Preincubation with a high concentration of free D-mannose significantly reduced C6-NPs uptake, strongly suggesting mannose receptor-mediated uptake. Conversely, free C6 uptake was largely unaffected by D-mannose preincubation, confirming the specificity of the competitive effect for the NP formulation. These findings validate the design of mannose-functionalized NPs for targeted delivery to hepatic cells, known to express mannose receptors, thereby enhancing ICA-NPs accumulation and therapeutic efficacy against NAFLD.

3.

In vitro and in vivo targeting of ICA-NPs. Due to the weak fluorescence of ICA, indocyanine green (ICG) was coloaded into the NPs as fluorescent markers. (A-B) Cell uptake and MFI of free ICA or ICA-NPs in HepG2 cells after cocultured for 2 h. Scale bars = 400 μm. Data are expressed as mean ± SD (n = 3), *P < 0.05 and ****P < 0.0001 versus C6/ICA groups. (C) Distribution in vivo and liver-targeting profile of ICA-NPs: (D, E) After oral administration of free ICA/ICG and ICA/ICG-NPs, whole mouse body images were observed using an IVIS Spectrum system at 4, 8, 12, and 24 h; (F) MFI of whole mouse in C and D; (G) Fluorescence images of major organs and colons from mice I.G. of ICA-NPs for 12 h. Data are expressed as mean ± SD (n = 3). not significant (ns), **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the ICG/ICA group (one-way ANOVA with Dunnett’s post hoc test).

As depicted in Figure C–F, both ICA/ICG-NPs and free ICA/ICG exhibited robust fluorescence signals in HFD-induced mice at 4, 8, 12, and 24 h postoral administration. Notably, ICA/ICG-NPs demonstrated a significantly higher mean fluorescence intensity (MFI) compared with free ICA/ICG, suggesting that NP encapsulation substantially enhances the accumulation of ICA in vivo. Quantitative analysis using the IVIS system revealed that the liver accumulation of ICA/ICG-NPs was 2-fold more than that of free ICA/ICG, respectively. These findings imply that nanoformulation significantly augments the targeting and accumulation of ICA in the liver. At the 24 h mark, ICA/ICG-NPs still exhibited discernible fluorescence, whereas the signal from free ICA/ICG had dissipated, indicating that NP formulation can prolong the in vivo retention of the drug. Furthermore, after a 12 h oral administration, the hepatic fluorescence in the ICA/ICG-NPs group was more pronounced than that in the free ICA/ICG group, further suggesting that nanoformulation can enhance the in vivo retention of drugs (Figure G). Collectively, these results underscore the pronounced liver-targeting capabilities of ICA-NPs and the superior targeting efficacy conferred by the nanoformulation.

3.6. Antiobesity Effects of ICA or ICA-NPs in HFD-Induced NFLD Mice

Figure A outlines the temporal sequence and procedures for establishing an NAFLD mouse model through an HFD and subsequent ICA intervention. Following a 10 week HFD regimen, the mice exhibited a nearly 70% increase in body weight. However, upon intragastric (I.G.) administration of ICA or ICA-NPs, the body weight of HFD mice was reduced by 20% and 40%, respectively, relative to the PBS-treated HFD control group (Figure B and C). Figure D presents photographs of the mice and their iWAT. The mass of various adipose depots, including subcutaneous adipose tissue (SAT), perirenal adipose tissue, and inguinal WAT, was significantly diminished with treatment using either free ICA or ICA-NPs, with ICA-NPs demonstrating the most pronounced antiobesity impact among the treatment groups (Figure E). Consistent with our hypotheses, the levels of TG in both serum and liver tissues of the murine model were markedly elevated compared to the control cohort, indicative of enhanced lipid accumulation within the hepatic environment characteristic of NAFLD. Interestingly, the therapeutic intervention with ICA-NPs effectively mitigated the HFD-induced escalation of the levels of TG, TC, HDL-C, and LDL-C, with no significant alterations in PBS or free ICA-treated NAFLD mice (Figure F–I).

4.

Antiobesity in the NAFLD mice model. (A) Schematic diagram of the experimental design. Male C57BL/6 mice were fed an HFD for 16 weeks to induce obesity. (B) Monitoring of mouse body weight changes during treatment. (C) Body weight of mice with different treatments at the 12th week. (D) Typical image of NAFLD mice and iWAT before and after treatment with different formulations of ICA. (E) The weights of SAT, perirenal WAT, and inguinal WAT at the end point of the experiment. (F–I) Blood samples were obtained for the quantification of total TC, TG, HDL-C, and LDL-C. (J) Histological analysis (H&E staining) of the iWAT. All data are expressed as mean ± SD (n = 6). **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the PBS-treated group (one-way ANOVA with Dunnett’s post hoc test).

The influence of ICA on adipocyte morphology was further evaluated through H&E staining. Adipocytes from the HFD group displayed hypertrophy and increased lipid deposition, which were effectively alleviated by treatment with both ICA-NPs and free ICA (Figure J). These findings indicate that both free ICA and ICA-NPs exhibit notable antiobesity properties, with the NP formulation further amplifying ICA’s antiobesity efficacy.

3.7. Effect of ICA on Pathological Changes in the Liver of HFD-Fed NAFLD mice

Figure A–C illustrates the hepatic morphology following 8 weeks of oral ICA administration. The livers from the normal diet group appeared bright red with a smooth texture. In stark contrast, livers from the HFD group exhibited fat vacuoles, disarray in the hepatic cord architecture, signs of hepatocyte rupture, darker staining of liver nuclei, inflammatory cell infiltration, and distortions in hepatocyte shape, hepatic cord arrangement, and hepatic lobule structure. Meanwhile, the group treated with free ICA showed no significant deviations in these hepatic parameters. Specifically, the free ICA group had a scattering of minor fat vacuoles and well-defined hepatic cords, which were less pronounced and fewer in number compared to the HFD-induced model group. Most notably, mice administered ICA-NPs orally showed minimal fat vacuoles, preserved liver cell morphology, and orderly hepatic cord arrangement. Furthermore, blood glucose levels in HFD mice were reduced with ICA treatment, with ICA-NPs showing particularly effective glycemic control (Figure D).

5.

Effects of ICA and ICA-NPs on hepatic steatosis and lipid deposition in HFD-induced NAFLD mice. After 8 week treatment (one dose/2 days), mouse livers were isolated for assay. (A) Typical photographs of the whole liver originated from different treated groups. (B) Weights of livers and (C) Enlargement of livers of different treated groups. (D)­Blood glucose levels in HFD mice. (E) Liver sections stained by H&E with (F) pathological score and (G) Oil Red O dye, respectively. One bar: μm. The levels of (H) AST and (I) ALT in livers in each group, respectively. All data are expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus PBS-treated group (one-way ANOVA with Dunnett’s post hoc test).

H&E and Oil Red O staining similarly exposed substantial lipid accumulation within the livers of the HFD group, characterized by disrupted hepatocyte architecture, variably sized lipid droplets, and overall changes in the model group (Figure E–G). The therapeutic impact of ICA-NPs was particularly pronounced, aligning with our earlier observations. Liver functionality was further assessed through the measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. As depicted in Figure H and I, while ICA showed a trend toward amelioration of liver function impairment caused by HFD, this was not statistically significant. In contrast, ICA-NPs markedly restored the liver function. Collectively, these outcomes suggest that the nanoformulation strategy significantly potentiates the hepatoprotective effects of ICA against HFD-induced hepatic steatosis.

3.8. ICA-NPs Effectively Suppress Inflammatory Cytokine Production in the HepG2 NAFLD Model

To evaluate the anti-inflammatory potential of ICA and ICA-NPs, an in vitro (NAFLD) model was established in HepG2 cells using an FAFM. As anticipated, FAFM induction significantly elevated the levels of proinflammatory cytokines, including TNF-α and IL-6, in the cell culture supernatants, showing a significant difference compared to the normal control group (Figure S4). Treatment with free ICA demonstrated a notable reduction in both TNF-α and IL-6 levels, indicating its intrinsic anti-inflammatory properties. More importantly, ICA-NPs exhibited superior anti-inflammatory effects, further decreasing the concentrations of TNF-α and IL-6 in FAFM-treated HepG2 cells. This observation suggests that the NP formulation efficiently delivered ICA into the cells, thereby powerfully mitigating NAFLD-associated inflammation, consistent with previous findings on the anti-inflammatory roles of icariin and its nanocarrier systems in liver diseases. , The enhanced therapeutic efficacy of ICA-NPs can be attributed to their improved cellular uptake and sustained-release characteristics, leading to a more effective suppression of the inflammatory cascade.

3.9. In Vitro and In Vivo ROS Scavenging Activity

Oxidative stress plays a pivotal role in NAFLD pathogenesis. To comprehensively investigate the antioxidant mechanisms of ICA-NPs, we first evaluated their impact on Nrf2 activation and then assessed direct ROS scavenging and antioxidant enzyme activity.

Confocal immunofluorescence microscopy revealed that in normal HepG2 cells, Nrf2 was primarily localized in the cytoplasm. In the FAFM-induced NAFLD model, while Nrf2 protein was detectable with cytoplasmic fluorescence, its nuclear translocation was significantly limited compared to the normal control and activated states (Figure S5). This suggests an overwhelmed or suppressed Nrf2-mediated antioxidant response under chronic oxidative stress. Treatment with free ICA induced a clear increase in nuclear Nrf2 signaling. More strikingly, ICA-NPs treatment led to a significant and robust increase in Nrf2 nuclear translocation, surpassing both the FAFM model and free ICA groups, indicating strong activation of the Nrf2 pathway.

Consistent with Nrf2 activation, we further evaluated the ROS-neutralizing capacity of the ICA formulations. DCFH-DA staining showed that both free ICA and ICA-NPs markedly suppressed ROS generation in H2O2-challenged HepG2 cells, with ICA-NPs achieving a more pronounced reduction (Figure A and B). Furthermore, ICA-NPs treatment led to a significant decrease in hepatic ROS levels in NAFLD mice, outperforming free ICA in terms of in vivo ROS scavenging activity (Figure C-E). Cellular superoxide dismutase (SOD) activity was also increased, and malondialdehyde (MDA) levels were decreased by ICA-NPs, consistent with their ROS scavenging effects (Figure F and G). Quantitative analyses corroborated the superior ROS-quenching capabilities of both ICA formulations, with ICA-NPs demonstrating particularly potent effects.

6.

In vitro and in vivo assay of ROS scavenging activity of free ICA or CS @ICA-NPs. HepG2 cells and livers with different treatments were stained with DCFH-DA kits and determined by flow cytometry or IVIS imaging system, respectively. (A-B) Flow cytometry (FCM) analysis of ROS level in HepG2 cells challenged with H₂O₂. Totally, 0.2 × 10⁶ HepG2 cells were pretreated with different formulations of ICA (1 mg/mL) for 12 h before being challenged with 400 nM of H₂O₂ for 2 h, the MFI of 20,000 cells was measured using FCM at the FITC channel. Data are expressed as mean ± SD (n = 3) ***P < 0.001 and ****P < 0.0001 versus the PBS-treated group (one-way ANOVA with Dunnett’s post hoc test). (C-E) Fluorescent pictures of livers originated from differently treated mice stained with DCFH-DA kits imaged by an IVIS imaging system, and the relative MFI of DCFH-DA in liver tissues was measured with Living Image 4.5 software. (F-G) Levels of MDA and SOD in different groups of livers. Data are expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus the PBS-treated group (one-way ANOVA with Dunnett’s post hoc test).

These results collectively provide strong evidence that ICA-NPs effectively activate the Nrf2 pathway and contribute to ROS scavenging, leading to a substantial amplification of ICA’s antioxidant potential and therapeutic efficacy against NAFLD-associated oxidative damage.

4. Discussion

Preemptive measures to counteract hepatic lipid accumulation are paramount to arresting the progression of NAFLD to cirrhosis and other severe hepatopathies. ICA, a botanically derived bioactive compound, has garnered substantial interest for its efficacy in alleviating obesity-related metabolic disorders and hepatic steatosis, thereby underscoring its therapeutic potential for NAFLD management. , Nevertheless, its widespread clinical translation has been impeded by formidable challenges, including pronounced hydrophobicity, limited bioavailability, and the risk of off-target adverse effects.

Encouragingly, the development of various ICA nanoformulations offers substantial promise for advancing the clinical translation of ICA-based therapeutics. Previous studies have reported that high doses of ICA (80–100 mg/kg body weight) are required to exert effective ameliorative effects on NAFLD , whereas the present research demonstrates that nanoformulated ICA (ICA-NPs) confers potent ameliorative efficacy in NAFLD at a substantially lower dose of only 1 mg/kg. Despite this progress, a comprehensive understanding of NAFLD pathophysiology and the precise role of ICA within this context remains elusive, with ongoing investigations aimed at clarifying these critical aspects. , Consequently, the integration of nanotechnology into ICA delivery systems represents a pivotal step toward enhancing its therapeutic index and unlocking its full clinical potential for NAFLD treatment.

The overarching objective of the present study was to develop a biocompatible, liver-targeted drug delivery system and evaluate its capacity to improve ICA’s efficacy in regulating hepatic lipid accumulationa core pathological feature of NAFLD. To this end, ICA was encapsulated in CS-mannose-coated PLGA–PEG NPs, yielding ICA-NPs specifically tailored for precise hepatocyte and hepatic targeting. Orally administered NPs typically reach the liver preferentially following ingestion, a process governed by three core mechanisms: − (i) most orally ingested substances are absorbed through the intestinal tract, enter intestinal capillaries, and subsequently converge into the portal veinthis vasculature directly transports them to the liver, and this pathway serves a crucial function in the metabolism and clearance of drugs and nutrients; (ii) smaller NPs penetrate cell membranes more readily and may be recognized and internalized by hepatic phagocytic cells (e.g., Kupffer cells); and (iii) the liver’s unique featuresas a primary detoxification organ with abundant blood supply and high metabolic capacityenable the efficient sequestration and metabolism of NPs upon their entry into the systemic circulation.

To ensure the delivery system’s performance aligns with its liver-targeted design, optimization of the ICA-NP fabrication process was conducted. PLGA NPs were chosen as the delivery vehicle owing to their advantageous characteristics, , including nontoxicity, nonimmunogenicity, biodegradability, and well-established biocompatibilityproperties that render them highly versatile for pharmaceutical use. Traditionally, PLGA NPs are fabricated via the emulsification-evaporation method. , In this study, the PLGA–PEG NPs were first functionalized with chitosan for the enhancement of mucosal adhesion, followed by the fabrication of ICA-NPs via high-pressure homogenization. This technique is well-recognized for efficiently generating uniform and stable NP dispersions. It not only ensures NP stability and bioavailability but also enhances ICA’s targeting efficiency and cellular uptake, thereby potentiating its therapeutic efficacy in NAFLD.

In vitro experiments validated the liver-targeting capacity of ICA-NPs and their internalization into HepG2 cells. Consistently, in vivo pharmacokinetic analyses demonstrated that 24 h following oral administration, the hepatic accumulation of ICA-NPs was approximately twice that of free ICAa finding indicative of superior bioavailability and a sustained-release profile. Compared with free ICA, ICA-NPs displayed substantially augmented hepatic accumulation, highlighting their robust liver-targeting potential. These results demonstrate the enhanced bioavailability and liver-directed distribution of ICA-NPs, which are critical for regulating hepatic lipid accumulation and metabolic dysregulation.

The in vitro and in vivo data of the present study demonstrate that ICA-NPs reduce hepatic ROS levels and increase superoxide dismutase activity. These effects are presumably mediated by the activation of the Nrf2/HO-1 axisthe master regulator of cellular antioxidant defense. Under conditions of oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus, and induces the expression of cytoprotective enzymes (including heme oxygenase-1 [HO-1]); this pathway is well-established to mitigate lipid metabolic dysfunction and inflammation in NAFLD. Although direct activation of Nrf2 by ICA remains undemonstrated, natural flavonoids structurally analogous to ICA (e.g., geniposide) have been shown to exert hepatoprotective effects via this axis, , providing strong support for our hypothesis that ICA-NPs augment Nrf2-mediated antioxidant defense. This mechanism is further supported by the consistency between our phenotypic observations (reduced steatosis and inflammation) and the well-documented outcomes of Nrf2 activation. Considering the pivotal role of ROS in NAFLD progression, antioxidant therapy is considered to be a promising treatment strategy; notably, ICA nanoformulationswith improved hepatic targeting and bioavailabilityconfer superior clinical utility in potentiating ROS inhibition relative to free ICA or nontargeted formulations.

Importantly, the clinical efficacy of antioxidant therapies for NAFLD has been variable in clinical studies, with natural antioxidants exhibiting higher response rates (100% in human trials) than their synthetic counterparts. ICA-NPs integrate the inherent antioxidant activity of a natural compound with nanoformulation-mediated targeting, overcoming the primary limitation of conventional antioxidant therapiesnamely, inadequate hepatic bioavailability. , The synergy of these attributes establishes ICA-NPs as a potential superior therapeutic candidate relative to both free ICA and nontargeted antioxidant formulations.

5. Conclusions

In conclusion, ICA-NPs constitute a groundbreaking category of orally deliverable, biocompatible nanocarriers meticulously engineered for hepatic targeting. These NPs showcase exceptional stability and solubility in aqueous media, facilitating their robust uptake by hepatocytes. Through preclinical animal studies, ICA-NPs have been observed to navigate preferentially to the liver, where they have exerted a considerable influence on lipid metabolism, curtailed lipid deposition, and ameliorated the metabolic anomalies associated with NAFLD. Moreover, these nanocarriers have demonstrated a capacity to bolster liver functionality in NAFLD murine models.

The findings with ICA-NPs highlight their therapeutic relevance in combating obesity and NAFLD, which are burgeoning health concerns with significant implications. Their liver-targeting efficacy and biocompatible nature position ICA-NPs as a promising therapeutic intervention. As such, further investigation of their safety and efficacy in clinical settings is essential to exploiting their full potential against these pervasive metabolic conditions. The advancement of ICA-NPs in research could lead to innovative treatment paradigms, potentially revolutionizing the management and outcomes for patients grappling with obesity and allied liver pathologies.

The data sets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.5c17806.

• In vivo biosafety, in vitro release kinetics, mannose receptor-mediated cellular uptake, in vitro anti-inflammatory effects (TNF-α and IL-6 quantification), and Nrf2 nuclear translocation (PDF)

S.F. and N.M. contributed equally to this paper. S.F.: methodology, visualization, validation, investigation, conceptualization, data curation. Y.Z.: writing-original draft, software, formal analysis. J.L.: methodology, investigation, data curation. B.W.: methodology. Y.Y.: methodology. X.C.: data curation. Z.Z.: funding acquisition, supervision, project administration, writing-review and editing.

This work was supported by the Special Project for Clinical and Basic Sci&Tech Innovation of Guangdong Medical University (GDMULCJC2024115).

Experimental procedures using mice in this study were reviewed and approved by the ethical review board of Guangdong Medical University, and all the experiments were performed in accordance with relevant guidelines and regulations of Animal Ethics Committee of Guangdong Province, China. Animals and protocol were approved by the Ethics Committee of Guangdong Medical University (GDY2002004).

The authors declare no competing financial interest.