**1. Introduction**

Chitin is one of the most abundant polysaccharides and acts as a structural material in biological systems. It has a regular structure consisting of β(1→4)-linked repeating units of *N*-acetyl D-glucosamine [1–3]. It remains an unutilized biomass resource, principally because of its intractable bulk structure and insolubility in water and common organic solvents. An efficient approach to the functional materialization of chitin is nanofibrillation, that is, the fabrication of nanocrystals and nanofibers [4–10] because of the exceptional properties of bio-based nanomaterials, such as their lightweight character, high tensile strength, low thermal expansion coefficient, biocompatibility, and nanosheet formability for sensing and electronic devices [11–17]. We previously developed a facile method for fabricating chitin nanofibers (ChNFs) with widths of approximately 20–60 nm based on a bottom-up approach where self-assembling regeneration from a chitin/ionic liquid (1-allyl-3-methylimidazolium bromide (AMIMBr)) ion gel using methanol was achieved [18,19]. This was based on our finding that AMIMBr efficiently dissolves and swells chitin [20]. Filtration of the resulting ChNF/methanol dispersion resulted in a film with a highly entangled nanofiber morphology.

We also previously reported styrene-in-water Pickering emulsion polymerization using self-assembled ChNFs as stabilizers to fabricate ChNF/polystyrene composite particles [21,22]. The stabilizers were additionally modified by anionic maleyl groups to provide amphiphilicity and simultaneously enhance dispersibility in aqueous media. Pickering emulsions are emulsions of any type, either oil-in-water, water-in-oil, or even multiple types which are stabilized by solid particles or other solid materials. These replace surfactants in general emulsions [23,24]. Pickering emulsion polymerization using polymerizable

**Citation:** Watanabe, R.; Izaki, K.; Yamamoto, K.; Kadokawa, J.-i. Preparation of Nanochitin/ Polystyrene Composite Particles by Pickering Emulsion Polymerization Using Scaled-Down Chitin Nanofibers. *Coatings* **2021**, *11*, 672. https://doi.org/10.3390/ coatings11060672

Academic Editor: Philippe Evon

Received: 20 May 2021 Accepted: 31 May 2021 Published: 1 June 2021

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hydrophobic substrates has proven to be a fascinating method for fabricating composite particles with well-defined morphologies [25]. In our system using maleylated ChNFs as stabilizers, particle sizes were controllable, where the sizes gradually decreased with increasing weight ratios of ChNFs to styrene from 0.02:1 to 0.2:1; however, a further increase in the ChNF ratio could not be obtained to produce smaller particles because of the insufficient dispersibility of the ChNF stabilizer under these higher content conditions in aqueous media.

Nevertheless, we found that the self-assembled ChNFs had a bundle-like structure and were constructed through the assembly of thinner fibrils [26]. Accordingly, the treatment of a ChNF film with aqueous sodium hydroxide for partial generation of amino groups on the chitin chains resulted in disentanglement of the bundles by cationization and electrostatic repulsion in 1.0 mol/L aqueous acetic acid with ultrasonication. This enabled individual thin fibril materials called scaled-down ChNFs (SD-ChNFs) to be obtained [27,28].

In the present study, we use SD-ChNFs as stabilizers for Pickering emulsion polymerization of styrene to fabricate composite particles (Figure 1). Due to their better dispersibility than the aforementioned maleylated ChNFs in aqueous media, nanochitin/polystyrene composite particles with smaller sizes could be obtained with a higher stabilizer content. As previous Pickering emulsion approaches for the fabrication of polystyrene particles have been conducted mostly using inorganic stabilizers such as SiO2 and Fe2O3 substrates [29–32], the present materials have an advantageous potential to be used in biologicalandmedicinalapplicationsowingtothebiocompatibilityofChNFs.

#### **2. Materials and Methods**

## *2.1. Materials*

Chitin powder from crab shells was purchased from Wako Pure Chemicals (Tokyo, Japan). An ionic liquid, AMIMBr, was prepared by the reaction of 1-methylimidazole with 3-bromo-1-propene according to a previously described method [33]. Other reagents and solvents were commercially available and used without further purification.

#### *2.2. Preparation of SD-ChNFs*

A mixture of chitin (0.120 g, 0.590 mmol) with AMIMBr (1.00 g, 4.92 mmol) was allowed to stand at room temperature for 24 h and subsequently heated with stirring at 80 ◦C for 24 h to obtain a chitin ion gel (10 wt%). The gel was then soaked in methanol (30 mL) at room temperature for a week for regeneration, followed by ultrasonication (Branson 1510, 42 kHz, 70 W, Branson Ultrasonics Corporation, Brookfield, CT, USA) for 4 h to yield a self-assembled ChNF dispersion with methanol. The dispersion was subjected to filtration to isolate ChNFs, and the isolated ChNFs were dried to obtain a ChNF film (0.117 g).

A mixture of the resulting film (0.117 g) with 30 wt% aqueous sodium hydroxide (30.0 mL) was heated at 80 ◦C for 5 h. The produced film was separated by filtration, washed with water and methanol, and dried to yield a partially deacetylated ChNF film (0.0824 g). From the integrated ratio of the signals assignable to acetamido protons to the signals assignable to anomeric (H1) protons in the 1H-NMR spectrum of the sample hydrolyzed from the produced partially deacetylated (PDA-)ChNF film in DCl/D2O, the degree of deacetylation (DDA) value was estimated to be 23.0%.

A mixture of the partially deacetylated ChNF film with 1.0 mol/L aqueous acetic acid (10.0 mL) was then ultrasonicated using a homogenizer (Branson Advanced-Digital Sonifier 450 (20 kHz, 400 W), Branson Ultrasonics Corporation, Brookfield, CT, USA) at room temperature for 30 min to yield an aqueous dispersion of SD-ChNFs.

#### *2.3. Preparation of Nanochitin/Polystyrene Composite Particles*

The typical experimental procedure was the following (run 5 in Table 1): Styrene (0.356 g, 3.42 mmol) was added to the aqueous dispersion of SD-ChNFs (0.501 g) and the mixture was ultrasonicated (Branson Advanced-Digital Sonifier 450 (20 kHz, 400 W), Branson Ultrasonics Corporation, Brookfield, CT, USA) for 30 min to produce a Pickering emulsion. After N2 gas was bubbled into the emulsion for 5 min at 0 ◦C, potassium persulfate (2.80 mg, 0.0104 mmol, 0.35 mol% with styrene) was added. The resulting emulsified mixture was then kept at 70 ◦C for 24 h under stirring in a nitrogen atmosphere. The reaction mixture was centrifuged for 45 min at 3500 rpm, and the residual product was lyophilized to yield composite particles (0.0657 g). For scanning electron microscopy (SEM) measurement, a mixture of the product (1.00 mg) and water (10.0 mL) was ultrasonicated for 1 h to yield a re-dispersion.


**Table 1.** Average diameters and yields of composite particles.

aDetermined by SEM images.
