**1. Introduction**

Polysaccharides, such as 2,3-O-acetylated-1,4-β-D-glucomannan, have been shown to have immunomodulatory properties, including the stimulation of cytokines, interleukin 1-beta (IL–1β), and tumor necrosis alpha (TNF-α) in human cell lines [1,2]. The immune system is a complex network of different cell types and mediators responsible for the defense of an organism against pathogens as well as the maintenance of tolerance to innocuous antigens [3]. Immunomodulation plays a crucial role in physiological processes as well as in the treatment and/or prevention of several diseases, including infectious diseases. Therefore, studies focusing on polysaccharide-based nanomaterials with immunomodulatory activities can have an important impact on development of novel vaccine adjuvants. Adjuvants, in the context of vaccines, are defined as components capable of enhancing and/or shaping antigen-specific immune responses [4]. Advances in particle engineering allow the design of new nanomaterials with desired physico-chemical properties, such as composition, size, shape, surface characteristics, and degradability [5], which ultimately will impact their effect as immunomodulators. It is well known that cationic substances, especially particulate materials, can act as immunomodulators [6,7]. In fact, the immunostimulatory activity of cationic cellulose nanocrystals (CNCs) was first described in our previous work in which we found that they induced the secretion of the inflammatory cytokine, IL-1β, in mouse and human macrophage cells [8,9]. CNCs are elongated crystalline rod-like (or needle-like) nanoparticles derived from the most abundant natural biopolymer on earth, cellulose [10–12]. CNCs are green and biocompatible materials with unique physico-chemical properties, including high aspect ratio, low density, large specific surface area, tunable surface chemistry (presence of abundant hydroxyl groups), non-toxic, colloidal stability, and optical and mechanical properties. These favorable features make CNCs as attractive nanoscale materials for applications in various fields such as biomedical, pharmaceutical, nanocomposites, electronics, among many others [13–15]. CNCs can be manufactured on a large scale and a recent study showed that the current standing of industrially produced CNCs was positive, indicating that the evolution of commercial-scale applications would not be hindered by CNCs production [16]. The emerging interest in CNCs for biomedical purposes led us to synthesize a novel functionalized series of wood-based CNCs possessing surface positive charges with potential immunomodulatory activities that could hopefully be further developed in newly engineered vaccine nanoadjuvants. For applications that required a positively charged CNCs surface, synthetic routes for cationization of CNCs were developed such as nucleophilic ring-opening of epoxide, esterification, copper(I)-catalyzed 1,3-cycloaddition and surface initiated-atom transfer radical polymerization [17–22]. In this work, we reported the design, synthesis, characterization and cytotoxicity of wood-based cationic CNCs as potential immunomodulators. A series of colloidally stable cationic CNCs was prepared via surface-initiated single electron transfer living radical polymerization (SI-SET-LRP) technique with different sizes and surface charges. The choice of [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METAC) and aminoethyl methacrylate hydrochloride (AEM) monomers with pendant cationic groups (+NMe3 and + NH3 respectively) was guided in part due to a recent study highlighting the use of NH2-functionalized aluminum oxyhydroxide nanorods for an enhanced immune adjuvant activity [7]. In addition to an effective synthetic route and sound physico-chemical characterization, another important aspect for the potential application of these cationic CNCs as vaccine nanoadjuvant is safety of these nanomaterials. In general, vaccine adjuvant should not induce an overwhelming immune response, otherwise it would have a toxic effect. Therefore, the cytotoxicity of these cationic CNCs was evaluated using three different cell-based assays (MTT, Neutral Red, and LIVE/DEAD®) and relevant immune cells, including two mouse cell lines (J774A.1 and BV2) as well as human peripheral blood mononuclear cells (PBMCs). Overall, we successfully synthesized and characterized a series of cationic CNCs that showed very low cytotoxicity in all tested experimental conditions indicating that these cationic cellulose-based nanomaterials are suitable to be further investigated as immunomodulators and potential vaccine nanoadjuvants.
