Sodium tripolyphosphate crosslinking of chitosan improves double-layer microsphere stability and preserves ATP production capacity of thylakoid membranes during simulated gastrointestinal digestion.

Thylakoid membranes (TMs), the photosynthetic membrane systems in chloroplasts, are multifunctional bioactive systems capable of light-driven ATP production. Their capacity of ATP production has inspired strategies for cross-species bioenergetic support and treatment of diseases. However, their oral application against intestinal diseases requires stabilization throughout the gastrointestinal tract. To address this, we developed an enteric delivery system by fabricating TMs-loaded sodium alginate (SA) microspheres (TMs-SPMs) via microfluidics, followed by coating with a chitosan-sodium tripolyphosphate (CS-STPP) complex. The binding between SA and CS-STPP was driven by enthalpic and entropic components, corresponding to charge-charge interactions, possible hydrogen bonding and counterion-release entropy. The CS-STPP network formed a dense barrier that effectively suppressed microsphere swelling, provided a cationic electrostatic shield in the stomach, and maintained a physical barrier in the intestine. Consequently, in simulated gastrointestinal digestion, the structural stability of the microspheres and the ATP production capacity of the encapsulated TMs were significantly preserved after the simulated digestion in vitro. The TMs-SPMs@CS-STPP (3:1) exhibited the highest cross-linking density, showing the most effective protection. This work establishes a feasible, food-grade protective strategy to preserve the bioactivity of TMs, supporting its future application in functional foods for gut health.