**4. Titanium dioxide**

*Renewable and Sustainable Composites*

of skin

**Figure 2.**

its reproduction mechanism [16].

**3. Chitin and chitosan overview**

*Optical image of S. aureus stained using Gram's method.*

possesses inherent antimicrobial properties [19].

• Toxic epidermal necrolysis: infection that causes detachment of large amounts

• Blood flow infection that produces high and persistent fever and shock

bloodstream or can spread through the surrounding tissue

• Osteomyelitis: infection of the bones that can reach the bone through the

UV radiation can inhibit the bacteria growth. This is commonly used in medicine to sterilize surgical instruments. UV radiation kills the bacterium by disrupting

In recent years, the use of biopolymers has increased due to their broader application in cosmetics, pharmaceuticals, food industry, biomedical, agriculture, and environmental products. Chitin was discovered in 1811 by Braconnot and chitosan, in 1859, by Rouget [17]. Chitin is made of N-acetyl-D-glucosamine monomer units linked with β(1,4) glycosidic linkage, whereas chitosan is the deacetylated form of chitin [18]. In addition, chitosan presents a rigid crystalline structure due to interand intramolecular hydrogen bonds as a result of the amine and hydroxyl groups [18]. Chitosan is a long chain of hydrophilic polysaccharide with a chemical formula of β(1–4) 2-asetamide-2-deoxy-D-glucopyranose. Chitosan is also known to be a natural polymer with similar molecular structure as cellulose; however it varies in the C-2 chain where the hydroxyl group in cellulose is replaced by amine (NH2). Chitosan is used to inhibit biofouling, being nontoxic and biocompatible, and also

Chitin can be extracted from the exoskeletons of crustaceans and fungi by a two-step chemical processing: demineralization and deproteinization [20]. A wide variety of chemical processes allow preparing chitosan: solution casting, dipping and spray coating, compression molding, blending, layer-by-layer, and

Chitin along with chitosan is considered the most abundant natural polysaccharides after cellulose. Due to the abundance of waste of shrimps, crabs, and other

**76**

3D printing [21].

Titanium dioxide has many commercially available applications in the food industry as a food additive, in the cosmetic industry as a makeup additive and sunscreen, and in the health industry as a drug delivery system [26]. It has also been used as a treatment of wastewater to remove pollutants [27]. Pure titanium dioxide when exposed to ultraviolet light (wavelength less than 400 nm) becomes a photocatalytic material [28]. This is largely dependent on particle size, shape, surface characteristics, and the dioxide crystal structure [29]. Different TiO2 crystal structures (viz., anatase, rutile, and brookite) have different photocatalytic properties [29]. Sayes et al. demonstrated that the anatase polymorph is more reactive under ultraviolet light (UV) [30], which renders it more cytotoxic. Furthermore, Blinova et al. stated that pure anatase induces apoptosis but does not generate ROS, while rutile does initiate cell death through the formation of ROS [31]. Davis et al. proposed that anatase particles under 100 nm have a strong UV absorption [32].

Nevertheless, TiO2 is a proven photocatalytic material under UV light. When this energy is greater than the band gap, i.e., 3.06 eV for rutile and 3.23 eV for anatase [33], the electrons in the valence band (VB) are excited to the conductive band (CB), producing a corresponding hole in the valence band and highly reactive reactants (e<sup>−</sup> and h+ ) on the TiO2 surface, as shown in **Figure 3**.

The electrons and holes react with water and air to form the highly chemical active ROS. There are four main and common ROS: superoxide radical (O2<sup>−</sup>), hydroxyl radical (OH<sup>−</sup>), hydrogen peroxide (H2O2), and singlet oxygen (O<sup>−</sup>) [34]. They range from less acute (O2− and H2O2) to more acute (O2<sup>−</sup> and O<sup>−</sup>) [34]. Not all

**Figure 3.** *Schematic illustration for the photocatalytic reaction of TiO2.*

NPs can produce ROS, and even if they could, many do not produce them all. For example, calcium oxide (CaO) and magnesium oxide (MgO) NPs can only produce O2<sup>−</sup> [34]. ZnO can generate H2O2 and OH<sup>−</sup> but not O2<sup>−</sup> [34], while TiO2 and copper oxide (CuO) NPs can produce all four types of ROS [34]. For the sake of simplicity, the present work focuses only on TiO2 irradiated with UV light and is used as a ROS source.

Such ROS production induced by the irradiation of TiO2 with UV light is shown in the following equations. Eq. (1) describes the energy absorption and the photocatalytic reaction. Eqs. (2)–(4) depict the photocatalytic redox pathways involved in the generation of an O2<sup>−</sup> and an OH<sup>−</sup> at the reaction between the holes with H2O and the electrons with O2<sup>−</sup> [35]. Eqs. (5), (6) describe the generation of H2O2 by reductive and oxidative pathways, respectively.

$$TiO\_2 \star energy \to e^-\_{CB} \star h^\*\_{VB} \tag{1}$$

$$O\_2 + e^-\_{CB} \to O^{2-} \tag{2}$$

$$H\_2O \star h\_{VB}^\* \Rightarrow OH^- + H^+ \tag{3}$$

$$OH^- + h\_{VB}^+ \to OH^- \tag{4}$$

$$\text{O}\_2^- + 2\text{H}^+ \star e\_{\text{CB}}^- \rightarrow \text{H}\_2\text{O}\_2 \tag{5}$$

$$2h\_{\text{VB}}^{\ast} + 2H\_2O \to H\_2O\_2 + 2H^+ \tag{6}$$

**79**

be tested.

*Biodegradable Chitosan Matrix Composite Reinforced with Titanium Dioxide for Biocidal…*

degree against ROS. However, any overproduction of this species initiates a systematic failure that starts by damaging the cell component and ends with the cell death. Human cells are not exempt from the effect of ROS [36], due to their self-

affecting the interaction between deoxyribonucleic acid (DNA) and cell and

Negatively charged O2<sup>−</sup> and OH<sup>−</sup> can exist on the cell surface and cannot penetrate the intracellular regions of the bacteria because of the negative charge barrier; conversely, hydrogen peroxide [39] can easily pass through. Therefore, NPs that produce all ROS have a higher opportunity as a broad-spectrum biocide. Furthermore, there exists evidence that TiO2 increases the membrane permeability of a bacterial cell, which allows ROS to enter and start the cell death process [40].

For the production of the biocomposite, the following materials were used: chitosan powder (coarse ground flakes, deacetylated chitin, poly(D-glucosamine) CAS number: 9012-76-4), acetic acid (glacial ACS reagent (2.5 L, RABA0010–2.5D1)), and TiO2 (anatase polymorph) purchased from Sigma Aldrich® now Millipore Sigma® (22 nm, nanocrystalline colloidal paste for transparent films, >95% anatase

To test the antibacterial properties of this renewable biocomposite, *E. coli* (ACTC 25922) and *S. aureus* (ATCC25923), provided by the Biology Department at the University of Puerto Rico—Mayagüez (UPRM), were used. The bacterial medium used to grow them was the Miller's Luria Broth (LB, CAS number: 91079–40-2, tryptone 10 g/L, yeasts 5 g/L, and sodium chloride 10 g/L), provided by Research Product International (RPI). An in-house Millipore filter provided the necessary deionized water. To provide aseptic conditions and for disinfection, common Lysol® diluted in distilled water was used. The use of protective gear, i.e., face masks, laboratory coats, gloves, and safety glasses, was mandatory throughout the entire experimental work. Laboratories in the UPRM Biology Building and Stéfani

Following the procedure for polymer solution casting, we measured and mixed

chitosan powder with acetic acid and deionized water following this schedule: a mechanical agitator for 1.5 h, a sonicator for 1 h, and a magnetic agitator for another 1 h. The TiO2 powder (anatase polymorph) was added in increments of 0.5 wt% to reach each target concentration and to prepare four different mixtures of TiO2 and chitosan. These mixtures were left inside an oven at 55°C for 48 hrs to be then utilized and characterized. **Figure 4** shows the as-produced films ready to

, pH <1,

by x-ray diffraction, 60–65% porosity, specific surface area 65–75 m<sup>2</sup>

In bacteria the reactive oxygen species attack the cell membrane and proteins,

increasing the gene expression for oxidative proteins [37]. Several studies found that the expression of two oxidative stress genes (catalase Kat A and alkyl hydroperoxide reductase, Ahp C) and a general stress response gene (chaperon protein, DNA K) rose by 52, 7, and 17 times, respectively, as revealed by real-time polymerase chain

*DOI: http://dx.doi.org/10.5772/intechopen.84397*

production in the mitochondria.

reaction (RT-PCR) [38].

**5. Methodology**

**5.1 Materials**

#798495).

Building hosted the present research.

**5.2 Chitosan/TiO2 film production**

### **4.1 Oxidative stress**

The ROS formation is an oxidative stress mechanism due to the generation of an imbalance between the production of free radicals and the ability of the cell to counteract. Different bacteria have particular ways to protect themselves to some *Biodegradable Chitosan Matrix Composite Reinforced with Titanium Dioxide for Biocidal… DOI: http://dx.doi.org/10.5772/intechopen.84397*

degree against ROS. However, any overproduction of this species initiates a systematic failure that starts by damaging the cell component and ends with the cell death. Human cells are not exempt from the effect of ROS [36], due to their selfproduction in the mitochondria.

In bacteria the reactive oxygen species attack the cell membrane and proteins, affecting the interaction between deoxyribonucleic acid (DNA) and cell and increasing the gene expression for oxidative proteins [37]. Several studies found that the expression of two oxidative stress genes (catalase Kat A and alkyl hydroperoxide reductase, Ahp C) and a general stress response gene (chaperon protein, DNA K) rose by 52, 7, and 17 times, respectively, as revealed by real-time polymerase chain reaction (RT-PCR) [38].

Negatively charged O2<sup>−</sup> and OH<sup>−</sup> can exist on the cell surface and cannot penetrate the intracellular regions of the bacteria because of the negative charge barrier; conversely, hydrogen peroxide [39] can easily pass through. Therefore, NPs that produce all ROS have a higher opportunity as a broad-spectrum biocide. Furthermore, there exists evidence that TiO2 increases the membrane permeability of a bacterial cell, which allows ROS to enter and start the cell death process [40].
