Betaine-Modified Chitosan Nanoparticles Enhance Oral Insulin Delivery via pH-Triggered Charge Switching, Mucus Penetration, and Lysosomal Escape

Oral insulin delivery is limited by the intestinal mucus barrier and intracellular lysosomal degradation within epithelial cells. To address these sequential barriers, we engineered insulin-loaded zwitterionic charge-switching nanoparticles (INS/Bet-CS-SA NPs) through the rational integration of betaine-modified chitosan and sodium alginate. This design leverages the pH-responsive surface charge transition of the nanocomplex, allowing the formulation to remain compact and positively charged under gastric conditions while undergoing swelling and surface transformation in intestinal media. The optimized nanoparticles exhibited a high insulin loading capacity (71.71%) and a sustained release profile (86.03% within 12 h under simulated gastrointestinal conditions). Especially, the surface zeta potential of INS/Bet-CS-SA NPs shifted from +15.73 ± 3.17 mV at pH 1.2 to -24.22 ± 3.47 mV at pH 6.8 and -25.68 ± 3.93 mV at pH 7.4, confirming the intended charge-switching behavior. This zwitterionic functional surface markedly enhanced mucus penetration and transepithelial transport, yielding apparent permeability coefficients 25.4-fold higher than that of free insulin. Cellular uptake and trafficking analyses suggested caveolae-associated endocytosis and reduced lysosomal retention played an essential role, which might be caused by betaine-associated membrane perturbation and chitosan's proton buffering capacity. In a type 2 diabetic rat model, orally administered INS/Bet-CS-SA NPs induced a sustained hypoglycemic response and achieved a relative bioavailability of 25.36%. The prolonged pharmacodynamic effect was additionally supported by a semimechanistic pharmacokinetic/pharmacodynamic model. Overall, this work provides a zwitterionic functional nanoplatform capable of overcoming multiple gastrointestinal barriers as a promising strategy for the oral delivery of insulin.