Introduction: Hydrogels have received increasing attention as versatile platforms for bioactive compounds delivery, due to their viscoelasticity, injectability, and to provide sustained release. Among them, systems based on ascorbyl palmitate (ASC16) have exhibited shear-thinning behavior, biocompatibility, and the ability to form stable coagels, although their preparation requires relatively high formulation temperatures. It is known that the incorporation of polyethylene glycol 400 (PEG₄₀₀) significantly lowers the critical micelle temperature, enhancing their potential for biomedical applications.

Objective: This study aimed to develop and characterise an ASC16 coagel (Coa-ASC16) in PEG₄₀₀ (Coa-ASC16_PEG) through a novel formulation procedure, distinct from previously reported methods, as a low-cost and easy-to-prepare matrix for the encapsulation and controlled release of biomolecules, using ophidian venom proteins (OVPs) as a model.

Methods: Coa-ASC16_PEG was prepared at 2.5% (w/v) using a mixture of PEG₄₀₀ and water in a 4:1 ratio. ASC16 was dissolved in PEG₄₀₀ in glass tubes and heated to 64 °C until complete solubilization was achieved (≤ 2 minutes). The mixture was then cooled to 40 °C and allowed to reach thermal equilibrium, after which OVPs dissolved in water were added and mixed for 10 seconds (Coa-ASC16_PEG/OVP). The formulation was characterized using dynamic rotational rheology tests, a three-interval thixotropy test (3ITT), encapsulation efficiency measurements, and in vitro release assays.

Results: Coa-ASC16_PEG/OVP demonstrated high encapsulation efficiency (96.42 ± 1.06%), retained stability under protein overload, and enabled a sustained release of OVP over time (23.41 ± 6.12% at 360 minutes). Rheological analyses confirmed its viscoelastic behavior, while 3ITT revealed a robust capacity to recover the original viscosity following mechanical stress.

Conclusions: Coa-ASC16_PEG represents a simple, low-cost platform with favorable rheological properties and high protein loading capacity. Its ability to withstand mechanical stress while providing sustained release highlights its potential as a controlled-release system for biomolecules, with potential applications in biomedical research.