Therapeutic Effect of Baicalin-Loaded Thermosensitive Liposomal Hydrogel in Autoimmune Uveitis

Abstract

Autoimmune uveitis (AU) is a chronic intraocular inflammatory disease that can lead to irreversible vision loss. Current pharmacotherapies are often associated with serious systemic adverse effects, limited ocular bioavailability, and high recurrence rates. Therefore, developing a safe and sustained local delivery system that effectively regulates ocular inflammation remains an urgent clinical challenge. Baicalin (BA) exerts immunomodulatory effects by activating the aryl hydrocarbon receptor (AhR), yet its poor solubility and instability hinder its clinical application. To address these limitations, this study developed BA-loaded liposomes (BA-Lip) and incorporated them into a chitosan/β-glycerophosphate (CS/β-GP) thermosensitive hydrogel (BA-Lip@CG) to enable localized and sustained BA delivery for AU treatment, and further evaluated its therapeutic efficacy and underlying mechanisms. BA-Lip and BA-Lip@CG were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and rheological analysis. The biocompatibility of BA-Lip@CG was evaluated in ARPE-19, HCEC, and RAW264.7 cells using the CCK-8 assay, and its in vitro anti-inflammatory and antioxidative effects were detected by ELISA and fluorescence staining. In vivo, an experimental autoimmune uveitis (EAU) mouse model was established, with assessments including clinical scoring, retinal histopathology, ROS staining, ELISA for retinal cytokines, Western blot analysis of AhR expression, and flow cytometry for Treg/Teff subsets. BA-Lip@CG exhibited uniform particle size, a positive zeta potential, rapid in situ gelation at ocular surface temperature, and sustained BA release over 96 h. In vitro, it demonstrated excellent cytocompatibility and significantly suppressed IL-6, IFN-γ, and IL-17A secretion, as well as ROS accumulation, outperforming free BA. In vivo, BA-Lip@CG markedly attenuated ocular inflammation, restored retinal architecture, and decreased oxidative stress in EAU mice. Mechanistically, BA-Lip@CG upregulated retinal AhR expression, increased CD4⁺ FoxP3⁺ Tregs, and decreased CD4⁺ IFN-γ⁺ Th1 and CD4⁺ IL-17A⁺ Th17 cells, confirming its potent immunoregulatory activity. Overall, the BA-Lip@CG thermosensitive hydrogel enables sustained BA delivery, enhances ocular retention, and delivers potent anti-inflammatory, antioxidative, and immunomodulatory effects, supporting its potential as a promising therapeutic strategy for AU.

1. Introduction

Autoimmune uveitis (AU) is a chronic, sight-threatening intraocular inflammatory disease and remains a major cause of visual disability worldwide, characterized by recurrent or persistent inflammation of the uveal tract. Without timely and effective control, AU can lead to irreversible retinal damage, optic nerve atrophy, and eventual blindness. Its pathogenesis is complex and primarily driven by dysregulated immunity, including aberrant T-cell activation, excessive secretion of pro-inflammatory cytokines, and autoantibodies mediated immune damage. Current treatment regimens primarily rely on corticosteroids and conventional immunosuppressants, which effectively attenuate inflammation yet are frequently limited by severe systemic side effects, emerging drug resistance, and a high rate of relapse upon dose tapering. Although biologic agents targeting specific inflammatory mediators have expanded therapeutic options, their high cost, long-term safety concerns, and incomplete remission rates restrict widespread use. Given these limitations, finding novel therapeutic drugs with definite efficacy, high safety, and effective control of inflammation has become an urgent priority in AU management.

In traditional Chinese medicine (TCM) theory, AU falls under the category of “Tong Shen Jin Xiao”. Classical prescriptions such as Longdan Xiegan Decoction and Xinzhi Chailian Decoction have shown clinical efficacy in managing uveitis, with Scutellaria baicalensis Georgi (Huangqin) being a common constituent in these formulations. Data mining analyses further indicate that Huangqin is the most frequently used herb in TCM formulas for uveitis treatment. Its major flavonoids, including baicalein, baicalin (BA), and wogonin, have been reported to exert broad therapeutic effects on ocular disorders through anti-inflammatory and antioxidative mechanisms, but their poor solubility and permeability substantially hinder clinical translation. Among these compounds, BA possesses notable immunomodulatory properties and has been demonstrated to ameliorate experimental autoimmune uveitis (EAU) by activating the aryl hydrocarbon receptor (AhR), thereby modulating Treg/Teff balance and CD4⁺ T cell proliferation. As a widely accepted model for noninfectious uveitis, EAU provides a valuable preclinical platform for elucidating disease mechanisms and evaluating potential therapeutic agents for AU, and was therefore employed as the in vivo model in the present study. Nevertheless, BA’s inherently poor aqueous solubility and limited physicochemical stability lead to rapid metabolism and insufficient intraocular retention following subconjunctival administration, preventing it from achieving and maintaining effective ocular therapeutic concentrations and thereby constraining its therapeutic potential. Consequently, developing an efficient drug delivery system to overcome these limitations has become essential for optimizing the pharmacological benefits of BA in AU.

Liposomes are among the most extensively investigated nanocarriers for ocular therapeutics due to their excellent biocompatibility and biodegradability. Several liposomal formulations have already progressed to clinical trials for ophthalmic diseases, and their delivery to anterior or posterior ocular segments can be optimized by adjusting surface charge or lipid composition. Thus, encapsulation of BA in liposomes is expected to enhance its aqueous solubility, bioavailability, and tissue penetration, thereby improving its intraocular therapeutic efficacy. However, the short half-life of liposomes limits their capacity for sustained drug release, which is essential in ocular drug delivery to reduce dosing frequency and maintain therapeutic levels. In recent years, injectable hydrogels have attracted considerable attention for local drug delivery. Chitosan (CS), a natural polysaccharide composed of a copolymer of glucosamine and N-acetylglucosamine, has been widely utilized in ocular drug delivery systems owing to its mucoadhesive properties and ability to enhance transcorneal permeability. Moreover, β-glycerophosphate sodium (β-GP) is a biocompatible anionic compound that is commonly combined with CS to form thermosensitive CS/β-GP hydrogels. This thermoresponsive in situ gelling system prolongs drug residence time on the ocular surface or within periocular tissues, enabling sustained and controlled drug release, thereby overcoming the limitations of conventional ocular formulations.

The present study employs BA as a therapeutic candidate for AU and applies nanotechnology to prepare BA-loaded liposomes (BA-Lip), aiming to address its inherent poor aqueous solubility and poor permeability. The liposomes are further incorporated into a CS/β-GP thermosensitive hydrogel to enhance ocular delivery efficiency, prolong intraocular drug retention, and achieve sustained therapeutic effects. This work evaluates the efficacy and underlying mechanisms of the BA-Lip@CG system in AU intervention, while underscoring the integration of TCM resources with advanced drug delivery technologies, thereby providing a novel and promising approach for its clinical management.

2. Experimental Section

2.1. Preparation of BA-Lip and BA-Lip@CG

BA-loaded liposomes (BA-Lip) were fabricated using the thin-film hydration method. Briefly, cholesterol, egg lecithin, BA, and stearylamine were dissolved in 10 mL of a chloroform–methanol mixture (3:1, v/v) at a weight ratio of 10:20:7:2 (w/w/w/w). The organic solution was then evaporated under reduced pressure (65 °C, 120 rpm) via a rotary evaporator, forming a thin lipid film on the inner wall of the round-bottom flask. The resultant lipid film was desiccated for 24 h to ensure complete solvent removal. The dried lipid film was hydrated with 7 mL of phosphate-buffered saline (PBS) containing 0.5% (w/v) hyaluronic acid, 0.2% (w/v) albumin, and 0.25% (w/v) hydroxypropyl methylcellulose (HPMC). Following 5 min of shaking, the mixture was sonicated on ice for 3 min to obtain BA-Lip. Blank liposomes (Lip) were prepared using the same procedure without the addition of BA.

For the preparation of CS/β-GP thermosensitive hydrogel, 1 mL of 80% β-GP solution was slowly added dropwise to 3 mL of 2% CS solution under continuous stirring in an ice bath. After complete addition, stirring was continued for an additional 10 min, and the pH was adjusted to 7.4 using saturated disodium hydrogen phosphate solution to obtain blank CS/β-GP thermosensitive gel solution. Subsequently, BA-Lip or blank Lip was slowly added dropwise into the blank CS/β-GP hydrogel solution under ice bath conditions with continuous stirring. After thorough mixing, the mixture was stirred for an additional 10 min to yield Lip@CG or BA-Lip@CG with a final BA concentration of 3 mg/mL.

2.2. Characterization of BA-Lip and BA-Lip@CG

The particle size distribution, polydispersity index (PDI), and zeta potential of Lip and BA-Lip were determined by dynamic light scattering (DLS), with each sample analyzed in triplicate. For transmission electron microscopy (TEM) observation of BA-Lip, a 5 μL aliquot of the liposome suspension was deposited onto a carbon-coated copper grid. After standing for 2 min, excess liquid was blotted with filter paper, and the grid was air-dried at room temperature. The morphology of BA-Lip was then observed using a TEM (FEI Talos F200S, FEI) operated at an accelerating voltage of 100 kV. To evaluate the colloidal stability of BA-Lip, the liposome suspension was diluted with ultrapure water and Dulbecco’s Modified Eagle Medium (DMEM), respectively. The diluted samples were incubated at 37 °C, and their particle size was measured by DLS every 24 h for 7 consecutive days, with triplicate measurements taken each time.

The thermosensitive behavior of BA-Lip@CG was assessed by placing the hydrogel solution in vials and incubating at 25 and 34 °C, with the sol–gel transition visually observed and recorded. For scanning electron microscopy (SEM) analysis of BA-Lip@CG, the hydrogel was freeze-dried to remove water. The dried sample was mounted on a sample stage and sputter-coated with a thin layer of gold. The micromorphology of BA-Lip@CG was observed using a SEM (Hitachi SU8010, Japan) operated at an accelerating voltage of 5 kV.

2.3. Rheological Evaluation of Gelation Temperature and Time for BA-Lip@CG

The rheological properties of BA-Lip@CG were analyzed using a rheometer equipped with a cone/plate geometry (C 35/1: φ = 35 mm, angle = 1°). A 1 mL hydrogel sample was loaded onto the rheometer platform and subjected to oscillatory measurements at a constant frequency of 1 Hz. To determine the gelation temperature, the sample was heated from 20 to 50 °C at a rate of 1 °C/min, and the variations in storage modulus (G′, Pa) and loss modulus (G″, Pa) were recorded as a function of temperature. Gelation temperature was defined as the point at which G′ surpassed G″. For gelation time measurement, the temperature was maintained at 34 °C, and G′ and G″ were recorded as a function of time until G′ exceeded G″, indicating the onset of gelation.

2.4. In Vitro Drug Release Studies

The in vitro release of BA from different formulations was performed according to previously reported protocols with slight modifications. Equal amounts of BA, BA-Lip, and BA-Lip@CG were loaded into dialysis bags (molecular weight cutoff: 10–12 kDa). After gelation at 37 °C, the bags were immersed in 20 mL of PBS containing 0.1% Tween 80 and maintained in a shaking incubator at 37 °C and 50 rpm. At predetermined time intervals, 1 mL of release medium was withdrawn and replaced with an equal volume of fresh prewarmed medium. Samples were filtered through a 0.22 μm membrane filter and analyzed by high-performance liquid chromatography (HPLC) to determine the cumulative BA release.

2.5. Cell Counting Kit-8 (CCK-8)

ARPE-19, HCEC, and RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and cultured for 24 h to allow adherence. Cells were then treated with free BA or BA-Lip@CG at various concentrations (0–500 μM) for 24 h. Cell viability was evaluated using the CCK-8 assay kit (Beyotime, Shanghai, China), and absorbance at 450 nm was recorded with a microplate reader.

2.6. Enzyme-Linked Immunosorbent Assay (ELISA)

The concentrations of IL-6, IFN-γ, and IL-17A in the culture supernatants of RAW264.7 cells and in the retinal tissue homogenates from mice were determined using commercial ELISA kits (Multi Sciences, Hangzhou, China) according to the manufacturer’s protocols. Absorbance was measured at 450 nm using a microplate reader.

2.7. Fluorescence Detection of ROS Levels

Intracellular ROS levels in RAW264.7 cells and retinal tissues were assessed using a DCFH-DA fluorescent probe (Beyotime, Shanghai, China) according to the manufacturer’s protocol. For in vitro experiments, RAW264.7 cells were incubated with 10 μM DCFH-DA at 37 °C for 30 min in the dark, followed by washing three times with PBS to remove excess probe. For in vivo assessment, freshly isolated retinal tissues from mice were sectioned, incubated with 10 μM DCFH-DA under the same conditions, and rinsed thoroughly. Fluorescence signals were observed using a fluorescence microscope (Olympus, Japan) and quantified with a microplate reader (excitation: 488 nm; emission: 525 nm).

2.8. Animals and Treatment Protocol

Male C57BL/6J mice (6–8 weeks old, 16–24 g) were procured from Sipeifu Biotechnology Co., Ltd. (Beijing, China) for the establishment of the EAU model. The animals were housed in a specific pathogen-free (SPF) environment with a relative humidity of 60–65% and a temperature of 23 ± 2 °C, maintained on a 12-h light/dark cycle, with free access to standard chow and water. All experimental procedures were approved by the Experimental Animal Management and Ethics Committee of Zhejiang Chinese Medical University (Approval number: 202508–0850).

The immunization protocol was performed as previously described. Briefly, 250 μg of human interphotoreceptor retinoid-binding protein peptide was emulsified with complete Freund’s adjuvant containing 3.5 μg Mycobacterium tuberculosis to prepare an emulsion. Each mouse was immunized via subcutaneous injection of 200 μL emulsion, followed by an intraperitoneal injection of 0.5 μg pertussis toxin, with a second injection administered 24 h later. The mice in the normal control group did not receive any treatment.

On days 11, 15, and 19 postimmunizations, mice in each group (n = 5) received subconjunctival injections of different formulations, including saline (Sal), BA, BA-Lip, Lip@CG, and BA-Lip@CG. Injections were performed using a 31G insulin syringe for precise delivery, with a dosing volume of 5 μL per administration. Clinical scoring was conducted every other day from the onset of immunization, following the previously reported protocol. During the treatment period, mice were continuously monitored for potential local and systemic adverse effects.

2.9. Hematoxylin and Eosin (H&E) Staining

Mouse eyeballs were enucleated at the indicated time points and immediately fixed in 4% paraformaldehyde for 24 h at 4 °C. After dehydration through a graded ethanol series and clearing in xylene, samples were embedded in paraffin and sectioned sagittally at a thickness of 5 μm, ensuring that the optic nerve was included. Sections were deparaffinized, rehydrated, and stained with hematoxylin for 5 min, followed by rinsing and counterstaining with eosin for 2 min. After dehydration and mounting, retinal morphology was observed under a light microscope (Leica Microsystems), and pathological changes were evaluated by assessing inflammatory cell infiltration.

2.10. Western Blot

Mouse retinas were homogenized in RIPA lysis buffer containing PMSF, and supernatants were collected after centrifugation (12,000 × g, 15 min, 4 °C). Protein concentration was determined via BCA assay. Equal amounts of protein (30–50 μg) were denatured, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies against AhR (1:1000) and β-actin (1:5000, internal control). After TBST washes, membranes were incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized using ECL, and intensities were quantified via ImageJ. Relative AhR expression was calculated as the ratio of AhR to β-actin band intensity, with experiments performed in triplicate.

2.11. Flow Cytometric Analysis of Treg and Teff Subsets

Mouse lymph nodes were harvested and minced through a 70 μm cell strainer to obtain single-cell suspensions. After red blood cell lysis, cells were washed and resuspended in PBS containing 2% FBS. Cell concentration was adjusted to 1 × 10⁶ cells/100 μL in staining buffer (PBS + 2% FBS). For Treg analysis, cells were first stained with antimouse CD4-FITC, followed by fixation, permeabilization, and intracellular staining with anti-FoxP3-PE according to the manufacturer’s protocol (eBioscience). For Teff subset analysis, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomycin (500 ng/mL) in the presence of brefeldin A for 4 h at 37 °C. Surface staining with anti-CD4-FITC was followed by intracellular staining with anti-IFN-γ-PE (Th1) and anti-IL-17A-APC (Th17) using a fixation/permeabilization kit. Data were acquired on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

2.12. Statistical Analysis

All statistical analyses were performed using GraphPad Prism 9.0 or SPSS 26.0. Data are presented as the mean ± standard deviation (Mean ± SD). For comparisons between multiple groups, one-way analysis of variance (ANOVA) was applied. When significant differences were detected, posthoc tests were used for pairwise comparisons. For comparisons between two groups, the unpaired Student’s t test was employed for normally distributed data, while the Mann–Whitney U test was used for non-normally distributed data. Each experiment was independently repeated at least three times to ensure reproducibility. A P-value <0.05 was considered statistically significant.

3. Results and Discussion

3.1. Physicochemical Characterization of BA-Lip@CG

To characterize the prepared BA-Lip and BA-Lip@CG, DLS first revealed that the average particle size of blank liposomes (Lip) was 115.94 ± 6.18 nm (Figure A), while BA-Lip exhibited an increased size of 141.04 ± 7.77 nm (Figure B). The increase in particle size following BA encapsulation aligns with the incorporation of BA into the liposomal bilayer, a phenomenon typical of liposomal drug delivery wherein drug loading induces structural adjustments in the lipid membrane. The ζ-potential of Lip and BA-Lip possessed positive surface charges of (23.83 ± 0.81) mV and (16.08 ± 0.66) mV, respectively (Figure C). Compared with neutral or anionic liposomes, cationic liposomes can slow scleral clearance, thereby enhancing retention time within the sclera and promoting sustained drug release to posterior ocular tissues such as the retina. TEM further visualized the bilayer structure of BA-Lip, strongly supporting the successful formulation of BA-Lip (Figure D). Moreover, Stability assessment demonstrated that the particle size of BA-Lip remained stable in both water and DMEM over 7 days (Figure E), suggesting good colloidal stability. Such physicochemical stability is critical for preserving liposome integrity and reproducibility in biological environments, particularly in ophthalmic applications where pharmaceutical formulation robustness affects therapeutic outcomes.

1.

Characterization of Lip, BA-Lip, and BA-Lip@CG. (A–B) Size distribution and (C) zeta potential of Lip and BA-Lip; (D) TEM image and (E) seven-day stability of BA-Lip; (F) thermosensitive gelation behavior, (G) SEM image, (H) macroscopic appearance at day 1, day 3, day 5, and day 7 during storage, (I) rheological determination of gelation temperature, and (J) rheological assessment of gelation time of BA-Lip@CG; (K) release curve of BA.

For the BA-Lip@CG thermosensitive hydrogel, visual observation showed it remained flowable at 25 °C but underwent gelation upon reaching 34 °C (Figure F). Additionally, SEM revealed a porous network structure (Figure G), which not only facilitates uniform drug distribution within the gel matrix but also allows for gradual diffusion-based release, contributing to long-term therapeutic efficacy. To further evaluate the long-term stability of the BA-Lip within the hydrogel matrix, a preliminary assessment was conducted during hydrogel preparation and storage. After storage for 7 days, the BA-Lip@CG hydrogel showed no noticeable sedimentation or macroscopic aggregation, and retained good transparency and homogeneity (Figure H). Furthermore, the hydrogel stored for 7 days was freeze-dried, followed by SEM observation of liposome distribution within the matrix. As depicted in Figure S1, the liposomes were evenly distributed in the gel matrix without evident aggregation, indicating that the hydrogel matrix is able to maintain the physical stability of the encapsulated BA-Lip. In addition, rheological measurements identified the gelation temperature and time of BA-Lip@CG, defined by the intersection points of G′ and G″, as 34 °C and 86 s, respectively (Figure I–J), which were below the ocular surface temperature of 35 °C, enabling in situ gelation following ocular administration for enhanced ocular retention and controlled release. This rapid gelation behavior at physiological conditions ensures that the formulation remains fluid during administration and forms a gel immediately upon contact with the ocular surface, minimizing drug loss and improving patient compliance.

In vitro drug release profiling further demonstrated that free BA was nearly completely released within 24 h, while BA-Lip achieved 86.66 ± 1.17% cumulative release at 60 h followed by the stabilization of the release. In contrast, BA-Lip@CG sustained drug release over 96 h, reaching a cumulative release of 85.43 ± 2.72% at 96 h (Figure K), highlighting the ability of BA-Lip@CG to provide prolonged and controlled drug release. The release kinetics of BA-Lip@CG can be attributed to the synergistic combination of liposomal encapsulation and hydrogel entrapment, which together form a dual-barrier system that mitigates burst release and enables extended therapeutic windows. These results collectively support the potential of BA-Lip@CG as a promising ocular delivery platform for treating AU.

3.2. In Vitro Biocompatibility and Anti-Uveitic Activity of BA-Lip@CG

To evaluate the biocompatibility of the BA-Lip@CG system in vitro, CCK-8 assays first assessed the cytotoxicity of free BA and BA-Lip@CG across concentration gradients (0–500 μM) on ARPE-19 human retinal epithelial cells (a key retinal epithelial target in uveitis), HCEC human corneal epithelial cells (the first ocular barrier during topical administration), and RAW264.7 macrophages (central mediators of inflammatory signaling) (Figure A–C). For all three cell types, both BA and BA-Lip@CG exhibited negligible cytotoxicity across a range of concentrations from 0 to 400 μM, with cell viability remaining above 95%. These findings indicate excellent biocompatibility of both free BA and the BA-Lip@CG formulation, a critical prerequisite for ocular delivery systems intended for prolonged intraocular residence. At 500 μM, viability declined to approximately 80%, prompting the selection of 400 μM as the optimal concentration for subsequent experiments to balance efficacy and biocompatibility.

2.

In vitro biocompatibility and antiuveitic activity of BA-Lip@CG. (A–C) CCK-8 assay for viability of ARPE-19, HCEC, and RAW264.7 cells; (D–F) ELISA detection of inflammatory cytokines (IL-6, IFN-γ, IL-17A) in supernatants; (G) intracellular ROS levels assessed by fluorescence staining. **, P < 0.01; ***, P < 0.001.

To assess the anti-inflammatory effects of BA-Lip@CG, ELISA was employed to quantify pro-inflammatory cytokine levels in LPS-stimulated RAW264.7 supernatants. BA-Lip@CG significantly reduced the secretion of IL-6 (Figure D), IFN-γ (Figure E), and IL-17A (Figure F) compared to the LPS-stimulated group (P < 0.001), with efficacy superior to free BA (P < 0.01, P < 0.001). This enhanced anti-inflammatory activity of the hydrogel-encapsulated liposomal system arises from the liposomal formulation enhancing BA’s membrane permeability and solubility, while the thermosensitive hydrogel prolongs ocular retention, ensuring sustained drug exposure to inflamed cells. Moreover, fluorescence-based detection of intracellular ROS demonstrated that LPS treatment significantly increased ROS accumulation, with ROS not only directly damaging ocular tissues but also amplifying cytokine production via NF-κB and MAPK signaling, forming a pro-inflammatory feedback loop. BA-Lip@CG administration effectively attenuated ROS levels, as evidenced by reduced green fluorescence intensity (Figure G), demonstrating a strong antioxidative capacity. These in vitro results suggest the ability of BA-Lip@CG to target both inflammatory and redox pathways and to enhance ocular bioavailability by leveraging the advantages of liposomal drug delivery and in situ gelling, offering a potential therapeutic strategy against uveitis.

3.3. In Vivo Evaluation of the Therapeutic Efficacy of BA-Lip@CG in EAU

To evaluate the in vivo therapeutic efficacy of BA-Lip@CG, an EAU mouse model was established, which recapitulates key pathological features of human AU, including intraocular inflammation and retinal damage. Throughout the in vivo experimental period, no treatment-related adverse effects were observed. Specifically, the injection sites showed no evidence of redness, swelling, induration, or ulceration, and all mice maintained normal body weight, coat condition, food intake, and behavioral activity, indicating that BA-Lip@CG and the control formulations were well tolerated under the experimental conditions used. Gross ocular imaging (Figure A) revealed that the EAU model group exhibited severe hypopyon, conjunctival hyperemia, and ciliary vessel congestion. In contrast, treatment with BA, BA-Lip, and BA-Lip@CG alleviated these symptoms to varying degrees, with BA-Lip@CG exhibiting the most pronounced therapeutic effect, which was evidenced by clearer mouse eyes and a marked reduction in vascular congestion. Clinical scoring over time further confirmed this efficacy, as shown in Figure B, BA-Lip@CG maintained consistently lower inflammation scores compared to the EAU model, free BA, and BA-Lip, highlighting the formulation’s ability to provide prolonged anti-inflammatory effects in the dynamic in vivo microenvironment. ELISA quantification of inflammatory cytokines in retinal tissues (Figure C–E) demonstrated that BA-Lip@CG robustly suppressed IL-6, IFN-γ, and IL-17A secretion (P < 0.001), outperforming both BA and BA-Lip (P < 0.05, P < 0.01, P < 0.001). Notably, BA-Lip and BA-Lip@CG showed comparable anti-inflammatory effects in vitro, which may be due to the static nature of cell culture systems, where drug concentrations remain stable. In contrast, in vivo, the thermosensitive hydrogel enables sustained BA release, compensating for rapid ocular clearance, ensuring continuous drug exposure to inflamed retinal tissues to enhance local bioavailability. This sustained delivery amplifies BA’s anti-inflammatory potency, as evidenced by the more pronounced cytokine reduction in BA-Lip@CG-treated mice. Such efficacy is critical for preserving retinal function in uveitis, where chronic inflammation often culminates in irreversible vision loss.

3.

In vitro anti-inflammatory evaluation of BA-Lip@CG. (A) Stereomicroscopic images of mouse eyes; (B) temporal changes in clinical inflammation scores; (C–E) ELISA assessment of retinal inflammatory cytokines (IL-6, IFN-γ, IL-17A). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Histopathological analysis of retinal tissues via H&E staining (Figure A) revealed that the EAU and Lip@CG groups displayed severe retinal folding, detachment, and inflammatory cell infiltration, whereas treatment with BA, BA-Lip, and BA-Lip@CG mitigated these pathological changes to varying degrees. Notably, BA-Lip@CG most effectively restored retinal architecture; this structural repair was associated with reduced cytokine levels, as diminished inflammation facilitated tissue remodeling and repair, thereby indicating the potent anti-inflammatory and tissue protective capacity of BA-Lip@CG. Additionally, DHE staining was employed to assess ROS accumulation in retinal tissues (Figure B). Intense red fluorescence intensity was exhibited in the EAU and Lip@CG groups, indicative of severe oxidative stress. BA-Lip@CG treatment significantly diminished the DHE fluorescence, indicating that oxidative stress in the retinas of EAU mice was alleviated, reinforcing the notion that BA-Lip@CG mitigates uveitis by concurrently modulating inflammatory pathways and oxidative damage.

4.

Retinal histology and oxidative stress analysis. (A) H&E and (B) ROS staining of retinal sections.

Taken together, these in vivo results validate that the BA-Lip@CG system exerts robust anti-inflammatory and antioxidative effects, effectively ameliorating uveitic pathology in mice. The enhanced performance of BA-Lip@CG compared to free BA and BA-Lip formulations can be attributed to the synergistic effects of liposomal encapsulation and hydrogel-based sustained release, which together overcome ocular pharmacokinetic barriers, provide improved ocular retention, controlled drug delivery, and targeted anti-inflammatory action.

3.4. Immunomodulatory Mechanisms of BA-Lip@CG

To elucidate the immunomodulatory mechanisms of BA-Lip@CG in EAU, we first evaluated the expression of AhR, a key immunoregulatory transcription factor that mediates anti-inflammatory responses. Prior studies have shown that BA alleviates EAU by activating AhR to suppress inflammation. Western blot analysis of retinal tissues (Figure A–B) revealed a marked decrease in AhR expression in the EAU group compared with the Sham group (P < 0.001). While treatment with free BA or BA-Lip partially restored AhR levels, BA-Lip@CG more significantly upregulated AhR expression (P < 0.05, P < 0.001). This suggests that the combination of liposomal encapsulation and hydrogel-based delivery can sustain drug release and effectively maintain BA concentrations at the retinal target site, thereby improving the bioavailability of BA and promoting AhR-mediated immunomodulatory pathways. These findings are consistent with previous studies indicating that BA can activate AhR to suppress inflammation and ameliorate autoimmune disorders.

5.

In vivo mechanistic analysis of BA-Lip@CG via AhR signaling and T cell subsets. (A–B) Western blot analysis of AhR protein expression in retinal tissue; (C–D) flow cytometry assessment of Treg (CD4⁺ FoxP3⁺), (E–F) Th1 (CD4⁺ IFN-γ⁺), and (G–H) Th17 (CD4⁺ IL-17A⁺) populations. *, P < 0.05, ***, P < 0.001.

Flow cytometric analysis showed that EAU induction caused a significant reduction in CD4⁺ FoxP3⁺ Tregs (P < 0.001, Figure C–D), a change correlating with disease onset. Concurrently, proportions of CD4⁺ IFN-γ⁺ Th1 cells (Figure E–F) and CD4⁺ IL-17A⁺ Th17 cells (Figure G–H) were markedly elevated (P < 0.001), as these populations fuel inflammation and align with a pathogenic immune profile. Treatment with BA and BA-Lip partially reversed these alterations, whereas BA-Lip@CG exerted the most profound immunomodulatory effects, significantly increasing Treg percentages while suppressing both Th1 and Th17 cell populations (P < 0.001). This immunomodulatory profile, characterized by boosting immunosuppressive Tregs and dampening pro-inflammatory Teffs, directly targets the core pathogenic immune imbalance underpinning EAU.

This shift in T cell subset distribution toward an anti-inflammatory phenotype may be attributed to BA enhancing AhR activation, which is known to promote Treg differentiation and inhibit pro-inflammatory Th1/Th17 polarization. By enhancing BA’s bioavailability and prolonging its action, BA-Lip@CG mitigates ocular inflammation not only through direct anti-inflammatory effects but also by reprogramming the immune microenvironment via AhR activation and T cell modulation, underscoring its multiple advantages in the treatment of AU.

3.5. The Translational Challenges and Study Limitations

Despite the favorable therapeutic outcomes of BA-Lip@CG demonstrated in this study, several translational considerations remain before this system can be advanced toward clinical application. Scaling up the production of thermosensitive liposomal hydrogels while maintaining batch-to-batch consistency in particle size, gelation temperature, and drug encapsulation efficiency represents an important technical challenge. Nevertheless, advances in polymer standardization and the availability of industrial-grade chitosan have already enabled consistent manufacturing of thermosensitive hydrogel systems, suggesting that large-scale production of BA-Lip@CG is feasible. Sterilization is another critical requirement for ophthalmic applications. While conventional terminal sterilization, such as autoclaving and γ-irradiation, may compromise hydrogel integrity, filtration-based aseptic processing and low-temperature methods have shown good compatibility with chitosan-based systems and can be adapted to maintain liposomal stability. Long-term storage stability is another critical factor, as thermosensitive hydrogels and liposomes may undergo structural changes or drug leakage over time. Techniques such as controlled refrigeration and cryoprotectant-assisted lyophilization have proven effective for similar formulations and may extend the shelf life of BA-Lip@CG.

Regarding the regulatory pathway, BA-Lip@CG would likely be classified as a drug-device combination product. As its therapeutic effect is primarily mediated by the pharmacological activity of BA, with the delivery system serving to enhance ocular bioavailability and retention, our formulation is anticipated to be regulated as a medicinal product requiring approval through the New Drug Application (NDA) pathway.

In addition, from a study design perspective, the absence of a standard-of-care corticosteroid as a positive control represents a limitation of this study. Although the present work systematically compared BA-Lip@CG with free BA, BA-loaded liposomes, and the vehicle (Lip@CG) to delineate drug-specific effects and carrier contributions, a direct comparison with first-line clinical therapy would more definitively contextualize the therapeutic potential of BA-Lip@CG.

Overall, our findings demonstrate that the BA-Lip@CG thermosensitive hydrogel enables sustained BA release, enhances ocular retention, and exerts potent anti-inflammatory, antioxidative, and immunomodulatory effects in AU. Future studies focusing on process optimization, sterilization strategies, and stability enhancement will further facilitate the translation of this system. Moreover, direct efficacy comparisons with conventional corticosteroid therapy will be an important next step to fully assess the clinical relevance and competitive advantage of BA-Lip@CG. These efforts will support the potential of BA-Lip@CG as a practical and clinically applicable ophthalmic drug delivery platform.

Data and Supporting Information for this research will be made available upon request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09822.

• SEM observation of liposome distribution within the hydrogel matrix (Figure S1) (PDF)

Conceptualization ideas, L.Z.; validation and verification, N.L.; formal analysis, R.Z.; data curation, Y.S.; writingoriginal draft preparation, L.Z.; writingreview and editing, L.Z. and J.L.; visualization preparation, N.L., R.Z., and J.L.; funding acquisition, L.Z. All authors have read and agreed to the published version of the manuscript.

This work was supported by Zhejiang Provincial Traditional Chinese Medicine Science and Technology Program (2025ZR165).

The experimental protocol was approved by the Experimental Animal Management and Ethics Committee of Zhejiang Chinese Medical University (approval no: 202508–0850).

The authors declare no competing financial interest.