Hydrogel-Forming Ability and Biological Characterization of Exopolysaccharide (EPS) from Porphyridium cruentum

Exopolysaccharides (EPSs) are emerging as sustainable polymers for biomedical hydrogels. Here, we report hydrogels from sulfated EPSs produced by Porphyridium cruentum and ionically crosslinked with Ca²⁺, Ce³⁺, or Cu²⁺ to generate tunable networks with bioactive potential. Rheological analysis showed viscoelastic behavior was primarily governed by cation nature and accessible binding site density, with diminishing gains above 2.5 wt% EPS and limited benefit beyond 10 wt% crosslinker. Ce³⁺ produced the most solid-like gel, Ca²⁺ yielded more thixotropic networks, and Cu²⁺ promoted rapid, heterogeneous crosslinking consistent with fast surface complexation. These network signatures showed distinct in vitro performances. Cation selection tuned antibacterial activity against Staphylococcus aureus and Escherichia coli, with Cu²⁺ achieving rapid bactericidal effects and Ce³⁺ enabling an 8-log reduction after 24 h. The ABTS assay showed that Ca²⁺- and Ce³⁺-crosslinked gels had antioxidant potential (≥40 µM Trolox eq.mg^-1); however, antioxidant capacity was assay dependent. Conditioned-medium assays showed ≥75% viability at day 3 for Ca²⁺- and Ce³⁺-crosslinked gels against human dermal fibroblasts (HDFs), while only Ce³⁺-crosslinked gels were cytocompatible against human keratinocytes (HaCaTs). Cu²⁺-crosslinked gels were highly cytotoxic across all tested conditions. Macrophage cytokine readouts (TNF-α and IL-6) indicated formulation-dependent immunobiological response. This work establishes microalgal EPSs as versatile polymers and links crosslinking chemistry to rheological modulation and multifunctional biomedical performance, while direct wound-healing efficacy remains to be demonstrated in future in vivo or wound repair functional models.