Nanocrystalline chitin thin films

Highlights

• Smooth nanocrystalline chitin (chitin NC) thin films were prepared by spincoating.

• Water contents of porous chitin NC films are twice values for amorphous chitin films.

• Enzymatic hydrolysis of chitin NC films was slower relative to amorphous chitin films.

• Protein adsorption was greater on porous chitin NC films than amorphous chitin films.

Abstract

Elucidating the interactions between crystalline chitin and various biomacromolecules is of fundamental importance for designing and fabricating chitin-based biomaterials. This work highlights a simple method to prepare ultrathin films of chitin nanocrystals (chitin NC) by spincoating chitin NCs from a colloidal suspension onto a gold surface modified by an amine-terminated self-assembled monolayer. Atomic force microscopy confirmed that chitin NC films are reasonably smooth and homogeneous, and quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange experiments demonstrated that chitin NC films have twice as much water as amorphous regenerated chitin (RChitin) films of similar thickness. QCM-D data also showed that chitinase-catalyzed hydrolysis of chitin NC films was much slower than that of RChitin films. Chitinase not only degraded, but also caused the swelling of the chitin nanocrystals. BSA adsorption studies demonstrated that chitin NC films have high protein loading capacity, and thus show potential applications for enzyme immobilization.

Introduction

As the second most abundant biopolymer in nature, chitin is widely distributed in the exoskeletons of crustaceans (e.g. shrimps, crabs and lobsters) and insects, the cell walls of fungi, and the beaks of cephalopods (e.g. octopi and squid) (Rinaudo, 2006). In nature, chitin occurs as ordered crystalline microfibrils which associate with other materials, such as proteins, lipids, polysaccharides, calcium carbonate, and pigments to form natural composites (Goodrich & Winter, 2007). Two polymorphs of chitin (α and β) have been reported, differing with respect to the locations and quantity of hydrogen bonds (Muzzarelli, 2012, Zeng et al., 2012). α-Chitin is the most abundant and stable form, and it is derived from crustacean tendons and shells, yeast and fungal cell walls, as well as insect cuticles (Muzzarelli, 2011, Muzzarelli et al., 2012). β-Chitin is less abundant and stable than α-chitin, and it is found in squid pens and tubeworms (Blackwell et al., 1965, Rudall and Kenchington, 1973). Compared to α-chitin, β-chitin is more susceptible to swelling. β-Chitin can be reversibly swelled by water, alcohols or amines, and irreversibly swelled by strongly acidic or basic solutions (Saito et al., 1997, Saito et al., 2000). β-Chitin could also be converted to thermodynamically stable α-chitin via dissolution or extensive swelling under strongly acidic or basic conditions (Noishiki et al., 2003, Saito et al., 1997). Due to its biocompatibility, biodegradability, affinity for proteins, antimicrobial and gel-forming properties, chitin is widely used in the medical and pharmaceutical area, such as wound-dressing materials (Jayakumar et al., 2011, Kumar et al., 2010, Muzzarelli, 2009), drug carriers (Mi et al., 2003, Rejinold et al., 2011), tissue engineering scaffolds (Freier et al., 2005, Noh et al., 2006), enzyme and cell immobilization supports (Krajewska, 2004, Muzzarelli, 1980), and biosensors (Ohashi and Karube, 1995, Ohashi and Koriyama, 1992).

Fundamental knowledge of the interactions between chitin and proteins, polysaccharides, calcium carbonate, enzymes, drugs, cells and synthetic materials is not only important for elucidating biological processes associated with chitin, but also for designing novel chitin-based biomaterials. Model chitin surfaces and the development of surface characterization techniques provide a convenient way to study and quantify these interactions. Recently, our group reported a simple method to prepare homogeneous, smooth and ultrathin chitin films by spincoating trimethylsilyl chitin (TMSChitin) from a mixture of chloroform and tetrachloroethane onto silica or gold surfaces. These TMSChitin films were subsequently regenerated to amorphous chitin upon exposure to the vapor of a hydrochloric acid solution. The regenerated chitin thin films were used as a model surface to study the interactions between chitin and bovine serum albumin (BSA) via a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM) (Kittle et al., 2012), as well as chitinases (Wang, Kittle, Qian, Roman, & Esker, 2013). However, the regenerated chitin films are only representative of amorphous chitin structures, differing in crystallinity from native chitin. Crystalline chitin thin films are expected to provide a better model to study the enzymatic degradation of native chitin, chitin–protein interactions, fungal cell wall component interactions and biomineralization.

Various cellulose model thin films (including both amorphous and crystalline) have been prepared via spincoating or the Langmuir–Blodgett techniques (Ahola et al., 2008a, Edgar and Gray, 2003, Eriksson et al., 2005, Fält et al., 2004, Gunnars et al., 2003, Habibi et al., 2007, Kontturi et al., 2003, Kontturi et al., 2006Maren, 2009, Schaub et al., 1993, Yokota et al., 2007). Edgar and Gray (2003) reported the preparation of smooth cellulose nanocrystalline thin films by spincoating colloidal suspensions of cellulose nanocrystals onto mica surfaces. Habibi et al. (2007) prepared monolayers of cellulose nanocrystals via the Langmuir–Blodgett technique.

Similar to cellulose, native chitin is highly crystalline with some disordered or paracrystalline regions that arise from defects. The disordered or paracrystalline regions are preferentially hydrolyzed or oxidized under certain conditions, whereas crystalline regions remain intact. Thus, rod-like chitin nanocrystals (chitin NCs) or whiskers can be produced (Habibi et al., 2010, Zeng et al., 2012). Chitin NCs are normally prepared by hydrolysis of chitin in hydrochloric acid solutions (Goodrich and Winter, 2007, Tzoumaki et al., 2010, Zeng et al., 2012). The sizes of the chitin NCs are greatly affected by the origin of the chitin, concentration of the hydrochloric acid solutions and the hydrolysis time, with the lengths varying over the range of 150–2200 nm, and the widths over the range of 10–50 nm (Zeng et al., 2012). Recently, Fan, Saito, and Isogai (2008) prepared chitin NCs via 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation of α-chitin with NaClO as a co-oxidant. The resulting rod-like nanocrystals had high surface charges because some hydroxyl groups on the surface were oxidized to carboxylate groups, and the average nanocrystal length and width were 340 nm and 8 nm, respectively.

This work presents a simple method to prepare smooth and ultrathin nanocrystalline chitin films. The morphologies, surface roughnesses, thicknesses and water contents of these films were characterized via atomic force microscopy (AFM), ellipsometry and a quartz crystal microbalance with dissipation monitoring (QCM-D). The chitinase-catalyzed hydrolysis of these chitin films was investigated via QCM-D. The utility of these chitin films as potential enzyme immobilization supports was demonstrated through the adsorption of bovine serum albumin (BSA) onto the films in QCM-D studies.