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The aim of the present study was to prepare and characterize nanocomposite films to improve the treatment of skin wounds by applying the film as a bandage. To modify chitosan (Cs) and to prepare nanocomposites, a mixture between titanium dioxide nanoparticles (TiO 2 NPs) was performed at different concentrations (2, 5, 10 and 15 wt%) and oleic acid (OA). The thin nanocomposite films were prepared by using casting method. The prepared films (Cs, Cs/TiO 2 NPs, Cs/OA and Cs/OA/TiO 2 NPs) were described by water absorption (swelling study) and biological degradation. Physico-chemical characterizations of Cs, Cs/OA, Cs/TiO 2 NPs and Cs/OA/TiO 2 NPs (with only 15 wt% TiO 2 NPs) films were determined by X-ray diffraction, transmission high-resolution electron microscopy, field emission scanning electron microscopy, thermal analysis and Fourier transform infrared spectroscopy as well as their mechanical properties. Antimicrobial activity against microorganisms has been studied to assess activity against bacteria. The prepared nanocomposite films showed good antimicrobial activity for both Gram-positive and Gram-negative bacteria. The therapeutic effects of Cs-TiO 2 NPs-oleic acid nanocomposites on healing excision wounds were studied in rat animal model. The data obtained revealed that groups treated with nanocomposites showed enhancement wound closure and speed up wound healing time. K E Y W O R D S antimicrobial activity, chitosan and oleic acid, chitosan and titanium dioxide nanoparticles, nanocomposites films, wound healing 1 | INTRODUCTION Bionanocomposites are beneficial and have been used in a wide range of applications in many fields such as physical, biological, biomedical and pharmaceutical applications (Díez-Pascual, 2019; Loureiro, Azoia, Gomes, & Cavaco-Paulo, 2016; Nikalje, 2015). Nanoparticles are an important component of many classes of biopolymers and bionanocomposites (Lenart & Hore, 2018). Numerous research has stated that TiO 2 nanoparticles at a low content (1-5%) may improve the physical-thermal properties of biocomposite films (Oleyaei, Almasi, Ghanbarzadeh, & Moayedi, 2016; Zhang et al., 2017). Khan and his colleagues reviewed a detailed overview of the properties, and applications of nanoparticles (NPs) present in various forms (Khan, Saeed, & Khan, 2019). Wound dressings develop according to the cause of different wounds. Various types of wound dressings, for example, xerogels, charcoal, alginate, chitosan, and hydrogel, have been introduced for this purpose (Abbasalipour, Mirjalili, Khajavi, & Majidi, 2014).

Enzyme cost and stability are the main problems facing industrial applications. Consequently, Aspergillus nigerNRC1ami was isolated from rotten orange and recorded a promising pectinase activity (13.8 U/ml). The enzyme was optimized using citrus peel pectin as the sole carbon source and recorded (40 U/ml). It was purified by two steps purifications and recorded 632 purification folds. The pure enzyme showed 14.7% carbohydrate content and consists of 15 amino acids. Glutamic acid was the major (22%) followed by leucine (10.67%) and threonine was the minor (2.70%). A. niger NRC1ami pectinase was conjugated by covalent coupling to sodium periodate (NaIO4) activated polysaccharides. Galactomannan showed the highest recovered activity (85%) and 94.34% reduction in viscosity. The optimum temperature for the pure enzyme shifted from 40 to 45 °C after the conjugation process. Also, the free enzyme showed its optimum activity at pH 5 compared to pH 4, 5 in the conjugated form case. The thermal stability of the free enzyme greatly improved after the modification process. The conjugated process reduced the activation energy to 36%, prolonged the enzyme half-life 5.6-fold at 60 °C, and increase the deactivation energy (Ed) by about 19% in comparison to the free form. The D value of the conjugated enzyme increased to 13.2-fold at 50 °C compared to the free form. Gibbs's free energy (ΔG) of the enzyme increased after the modification process, while the enthalpy (ΔH) and entropy (ΔS) decreased. Na+ and Zn2+ had a stimulating effect on both enzyme forms. Graphical Abstract

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