“Bio-Mediated Green Synthesis of Chitosan Nanoparticles Using Egyptian Sargassum latifolium: Box–Behnken Design Optimization and Therapeutic Evaluation”

Marine macroalgae are recognized as rich sources of bioactive compounds with nutritional and pharmacological relevance; however, their clinical translation remains hindered by poor bioavailability and the absence of sustainable nanoformulation strategies. This study reports the development of statistically optimized, bio-mediated chitosan nanoparticles loaded with the methanolic extract of the brown alga Sargassum latifolium (SLE/CsNPs), collected from the Red Sea coast. The formulation was based on a dual-crosslinking approach in which algal polysaccharides interact with chitosan through polyelectrolyte complex formation, while sodium tripolyphosphate (TPP) served as an ionic crosslinker. GC–MS phytochemical profiling of the extract revealed a diverse array of bioactive constituents, notably diterpenoids and sesquiterpenes. A Box–Behnken design was employed to optimize formulation variables affecting particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (EE%). The optimized SLE/CsNPs exhibited favorable physicochemical properties, including a PS of 81.12 ± 2.56 nm, PDI of 0.24 ± 0.001, ZP of +45.77 ± 0.53 mV, and EE% of 77.12 ± 1.45%. Transmission electron microscopy confirmed the spherical morphology and nanoscale dimensions of the optimized nanoparticles. In vitro antioxidant assays showed that SLE/CsNPs improved the antioxidant activity of the free extract by approximately 1.6-fold (IC₅₀ = 70.64 ± 0.74 µg/mL vs . 111.8 ± 1.21 µg/mL for free SLE). The SLE/CsNPs also exhibited enhanced antiproliferative activity against Hep-G2 and Caco-2 cells, with IC₅₀ values of 8.41 ± 0.64 and 4.86 ± 0.17 µg/mL, respectively. Both SLE and SLE/CsNPs exhibited substantially lower cytotoxicity toward WI-38 normal fibroblasts than toward the cancer cell lines, supporting their tumor selectivity. Furthermore, SLE/CsNPs demonstrated enhanced antimicrobial activity relative to free SLE against clinically relevant strains, including Candida albicans , Gram-negative bacteria ( Escherichia coli and Salmonella typhi ), and Gram-positive bacteria ( Bacillus subtilis and Staphylococcus aureus ). These findings support the dual-crosslinking strategy as a sustainable green synthesis approach for developing multifunctional chitosan-based nanocarriers with improved biological activity.