(T1530-06-36) Oral Delivery of Anti-TNF-α Antibody via Pickering Emulsion for Ulcerative Colitis Therapy

Purpose: Anti-TNF-α antibodies are widely used and effective therapies for ulcerative colitis (UC), exerting their effects by neutralizing TNF-α to suppress inflammation and regulate immune responses. While intravenous injection is the primary route of administration, this approach faces challenges such as systemic side effects, poor patient compliance, and high treatment costs. Oral delivery can offer improved compliance, cost-effectiveness, and localized release that limits systemic exposure and adverse effects. However, major challenge in the oral delivery of anti-TNF-α antibodies is protecting the antibody from degradation in the gastrointestinal tract and ensuring its delivery to the colonic lamina propria, where it can effectively neutralize TNF-α produced by immune cells. To address this, we developed a Pickering emulsion-based oral delivery platform with two primary objectives: (1) to shield the antibody from enzymatic and acidic degradation during gastrointestinal transit, and (2) to facilitate its transport to the lamina propria, enabling effective TNF-α neutralization at the site of inflammation. Therefore, casein was employed as the particle stabilizer for the Pickering emulsion. Casein possesses the unique ability to form a gel-like clot under acidic gastric conditions¹, which reduces enzymatic degradation and creates a physical barrier that protects the encapsulated antibody. Additionally, the deformable nature of Pickering emulsions² allows them to penetrate the dense colonic mucus layer, promoting deeper tissue access and enhancing interaction with local TNF-α.

Methods: Casein-stabilized Pickering emulsions loaded with anti-TNF-α antibody (Casein-PE@TNF-Ab) were prepared using a sonication method. To optimize the formulation, various concentrations of casein (2, 4, 6, 8, and 10 mg/mL) and oil-to-water ratios (1:8, 1:10, 1:12, 1:14, and 1:16) were evaluated for their effects on appearance, drug loading capacity, particle size, and zeta potential. The stability of Casein-PE@TNF-Ab was assessed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Cellular uptake by RAW264.7 macrophage cells was quantified via flow cytometry using OVA-647 as a fluorescent model antigen, while cytotoxicity was evaluated using the lactate dehydrogenase (LDH) assay. To assess the in vitro TNF-α neutralization capacity, RAW264.7 cells were stimulated with lipopolysaccharide (LPS) to induce TNF-α production, and the levels of TNF-α in the culture supernatant were measured using an ELISA assay.

Results: Optimization of casein concentration revealed that formulations with 2 and 4 mg/mL exhibited phase separation and low drug loading on day 7, indicating poor stability and encapsulation efficiency. Among the 6, 8, and 10 mg/mL groups, the 8 mg/mL formulation showed the highest drug loading, with stable particle size (304.8 ± 5.6 nm) and zeta potential (−47.41 ± 0.79 mV), and was thus selected for further development. Evaluation of water-to-oil ratios (8:1 to 16:1) showed that the 8:1 group exhibited slight aggregation over time, while other ratios maintained stable physicochemical properties. The 12:1 formulation demonstrated the highest drug loading, exceeding the 8:1 ratio by 1.97-fold, and was selected as the final formulation. In SGF, Casein-PE@TNF-Ab showed an increase in particle size, likely due to casein gelation in the acidic environment. The absolute zeta potential remained around 50 mV over 8 hours, indicating colloidal stability. In SIF, both particle size and zeta potential remained relatively stable, confirming the formulation's gastrointestinal stability. Cellular uptake studies in RAW264.7 macrophages revealed that LPS stimulation enhanced uptake by approximately 121% compared to unstimulated cells, indicating inflammation-responsive behavior. LDH assay showed nearly 100% cell viability, confirming biocompatibility. Furthermore, Casein-PE@TNF-Ab significantly reduced TNF-α production in LPS-stimulated RAW264.7 cells, demonstrating effective TNF-α neutralization in an inflammatory environment.

Conclusion: This study demonstrates the development of Casein-PE@TNF-Ab with a narrow particle size distribution, high stability, and efficient drug loading. The formulation exhibited stability in both simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). It significantly enhanced cellular uptake by RAW264.7 macrophages, particularly under LPS stimulation, and showed excellent biocompatibility. Furthermore, Casein-PE@TNF-Ab effectively neutralized TNF-α produced by LPS-stimulated RAW cells in vitro. These findings suggest that Casein-PE@TNF-Ab holds strong potential as an oral delivery platform for anti-TNF-α therapy in the treatment of ulcerative colitis.

References: 1 Wang, Pengjie, et al. (2018) 2. Tatry, Marie-Charlotte, et al (2021).

Acknowledgements: This work was supported in part by a grant from National Institute of Health (P20GM113117, CoBRE-Chemical Biology of Infectious Disease) (H.K) and University of Kansas Start-up fund (H.K)


Figure 1 Optimization of Casein concentration to fabricate Casein-PE@TNF-Ab. (A–D) Effects of varying Casein concentration on the appearance, encapsulation efficiency, particle size, and zeta potential of Casein-PE@TNF-Ab. n=3, All data are represented as mean ± SD.



Figure 2 Optimization of oil-to-water ratio to fabricate Casein-PE@TNF-Ab. (A–D) Effects of varying oil-to-water ratios on the appearance, encapsulation efficiency, particle size, and zeta potential of Casein-PE@TNF-Ab. n=3, All data are represented as mean ± SD.