Artificial intelligence-based optimization for chitosan nanoparticles biosynthesis, characterization and in‑vitro assessment of its anti-biofilm potentiality.

Chitosan nanoparticles (CNPs) are promising biopolymeric nanoparticles with excellent physicochemical, antimicrobial, and biological properties. CNPs have a wide range of applications due to their unique characteristics, including plant growth promotion and protection, drug delivery, antimicrobials, and encapsulation. The current study describes an alternative, biologically-based strategy for CNPs biosynthesis using Olea europaea leaves extract. Face centered central composite design (FCCCD), with 50 experiments was used for optimization of CNPs biosynthesis. The artificial neural network (ANN) was employed for analyzing, validating, and predicting CNPs biosynthesis using Olea europaea leaves extract. Using the desirability function, the optimum conditions for maximum CNPs biosynthesis were determined theoretically and verified experimentally. The highest experimental yield of CNPs (21.15 mg CNPs/mL) was obtained using chitosan solution of 1%, leaves extract solution of 100%, initial pH 4.47, and incubation time of 60 min at 53.83°C. The SEM and TEM images revealed that CNPs had a spherical form and varied in size between 6.91 and 11.14 nm. X-ray diffraction demonstrates the crystalline nature of CNPs. The surface of the CNPs is positively charged, having a Zeta potential of 33.1 mV. FTIR analysis revealed various functional groups including C-H, C-O, CONH, NH, C-OH and C-O-C. The thermogravimetric investigation indicated that CNPs are thermally stable. The CNPs were able to suppress biofilm formation by P. aeruginosa, S. aureus and C. albicans at concentrations ranging from 10 to 1500 µg/mL in a dose-dependent manner. Inhibition of biofilm formation was associated with suppression of metabolic activity, protein/exopolysaccharide moieties, and hydrophobicity of biofilm encased cells (r ˃ 0.9, P = 0.00). Due to their small size, in the range of 6.91 to 11.14 nm, CNPs produced using Olea europaea leaves extract are promising for applications in the medical and pharmaceutical industries, in addition to their potential application in controlling multidrug-resistant microorganisms, especially those associated with post COVID-19 pneumonia in immunosuppressed patients.