**4. The characteristics of titanate nanosheets (TiNSs)**

effects on dermal tissue when that barrier is broken. Pulmonary exposure to TiO<sup>2</sup>

pulmonary response to intratracheal instillation of nano-sized ultrafine TiO<sup>2</sup>

particles. Administration with ultrafine TiO<sup>2</sup>

the interstitium, even at day 7 post-exposure, whereas 91% of fine TiO<sup>2</sup>

this stage. Shinohara et al. investigated pulmonary clearance kinetics of TiO2

and dose-dependent manner. van Ravenzwaay also observed that inhaled TiO2

olar macrophages, leading to increased inflammatory responses, and that TiO<sup>2</sup>

have reported alteration of immune functions following administration with TiO2

and inflammatory cytokines compared with fine TiO<sup>2</sup>

the amount was higher for ultrafine TiO<sup>2</sup>

the translocation of administered TiO2

the effect of administration with TiO<sup>2</sup>

tration with TiO2

ing a murine asthma model. It was found that TiO2

acquired immunity. NC/Nga mice treated with TiO2

mice receiving intraperitoneal treatment with TiO2

[22]. Studies using intraperitoneal administration of TiO2

in the lungs. Eight-one percent of ultrafine TiO<sup>2</sup>

. In addition, although both ultrafine and fine TiO<sup>2</sup>

to fine TiO<sup>2</sup>

TiO2

166 Cytotoxicity

TiO2

TiO2

the amounts of TiO2

ticles is expected to result in these toxic effects because a barrier comprising pulmonary tissue is not tough. In addition, alveolar macrophages are always ready to engulf particles in the region, leading to inflammatory responses. Sager et al. used F344 rats to examine the

phonuclear neutrophil (PMN) cell number, lactate dehydrogenase (LDH) activity, albumin

administration and then measured alterations in those tissues during that period. Ultrafine

showed a faster decline of the remaining amount in trachea-bronchial nodes than fine

than fine TiO<sup>2</sup>

in the amounts of lavagable and non-lavagable components between fine and ultrafine TiO<sup>2</sup>

micron particles by intratracheal administration to male F344 rats [24], and results confirmed

reached the lungs and draining lymph nodes [25]. These findings indicate that it is easier for

have a greater potential to influence the function of immune cells. Actually, several studies

the respiratory pathway. Chang et al. demonstrated that intratracheal instillation with TiO2 nanoparticles caused an increase in GATA-3 and decrease in T-bet mRNA levels, which are master genes for Th2 and Th1 cell development, respectively [26]. Mishra et al. examined

responsiveness, lung damage and a mixed Th2/Th1 dependent immune response, associated with increases in Stat3, Socs3, NF-κB, IL-6 and TNF-α [27]. The effect of intradermal adminis-

of IL-4 in the skin, IgE and histamine levels in serum, as well as aggravation of skin lesions

understanding of the immunological influences of these particles. Larsen et al. showed that

challenged with aerosols of OVA responded with high production of IgE and IgG1 antibodies specific for OVA in serum with increases in eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF), which suggests induction of a Th2-dominant immune response [28]. Moreover, Moon et al. demonstrated that intraperitoneal injection with TiO<sup>2</sup> nanoparticles results in the damaged development and proliferation of B and T cells, a decreased cytokine production by LPS-stimulated peritoneal macrophages and a decreased

nanoparticles as mentioned above should also be understood in relation to

nanoparticles to migrate from bronchoalveolar to interstitial tissue or be cleared by alve-

in trachea-bronchial and thymic lymph nodes at days 7 and 42 after

nanopar-

in comparison

caused higher levels of polymor-

[23]. Interestingly, they also examined

were translocated to lymph nodes,

. There was also a striking difference

was still lavagable at

nano- and sub-

nanoparticles

nanoparticles

through

was non-lavagable, suggesting migration to

to the thoracic lymph nodes that increased in a time-

nanoparticles as an adjuvant in an experiment utiliz-

nanoparticles augmented airway hyper-

nanoparticles showed over production

nanoparticles and OVA and subsequently

nanoparticles are valuable for an

Techniques to synthesize nano-scaled 2D materials have been studied recently. The basis of this field is derived from the development of methods for manipulating graphene, a carbon nanosheet with a thickness of one atom, which triggered the subsequent development of various 2D nanomaterials [31–33]. It is against this background that inorganic nanosheets have acquired greater interest because they have an ultrathin structure as well as a diversity of compounds and structures leading to various functions [34–36]. Oxide nanosheets are included in the group comprising inorganic nanosheets, and titanate nanosheets (TiNSs) represent a form of oxide nanosheets. Although TiNSs are composed of a TiO6 octahedron as the particles of TiO2 , TiNSs have the unique crystal structure of lepidocrocite, differing from anatase or rutile, which results in a shape having an ultralow thickness and high aspect ratio [34]. In the 1990s, Sasaki et al. first succeeded in delaminating layered titanate into single titanate nanosheets [37, 38], and TiNSs are now incorporated into useful applications such as photocatalysts, semiconductors and dielectric materials [39–42]. However, the following characteristics suggest possible harmful effects of TiNSs. First, it is noteworthy that TiNSs have a very large surface area per gram due to their ultralow thickness, which is generated from the limited height of one and a half of a sideways TiO6 octahedron together with the repeated linkage of the octahedron horizontally [34]. Such a large surface of TiNSs might enhance the toxic machinery of bulk titanium particles. Second, the large surface of TiNSs is known to be negatively charged due to oxygen atoms existing at the edges of the octahedron, and this suggests the possible influence of TiNSs through a cationic interaction. In addition to TiNSs delaminated from layered titanate, it has been reported that TiNSs with a small diamond shape and crystal structure of lepidocrocite can be synthesized in a bottom-up manner [43, 44], which allows TiNSs to be synthesized at a small scale. Dr. Yoshioka, one of our colleagues, has modified that method to synthesize TiNSs in our group. **Figure 1** shows images of TiNSs taken by transmission electron microscopy (TEM). The TiNSs showed a diamond shape with about 20- and 30-nm diagonals, which is almost the same as that reported in a previous study [43]. Additionally, the TiNSs showed the characteristic peaks of a lepidocrocite structure confirmed by X-ray diffraction analysis. Since it was verified that TiNSs could be synthesized, we therefore started to examine the effect of TiNSs on human immune cells using *in vitro* experiments.

vacuole formation was present in monocytes but not in lymphocytes. Q-VD-OPh, a pan-cas-

increase in apoptotic cells caused by TiNS exposure was also observed in the culture of isolated

potential to cause caspase-dependent apoptosis in immune cells, particularly where monocytes

The results obtained from the cell cultures demonstrated the characteristic toxicity of TiNSs for monocytes, comprising apoptosis associated with the striking formation of vacuoles. Therefore, we investigated the presence of intracellular microstructures in monocytes exposed to TiNSs. Monocytes were isolated from human PBMCs, cultured with TiNSs at 10 μg/ml and then harvested at day 1 or 2 after the culture for subsequent TEM observations. The TEM images showed rapid formation of vacuoles in monocytes even at day 1, and the number and size of vacuoles increased at day 2. It is noteworthy that nano-scaled materials with TiNS-like shapes were found within the vacuoles of the monocytes and that most of the material was located near the inner surface of the vacuolar membrane (**Figure 2**). In order to confirm whether these intra-vacuolar nano-scaled materials were TiNSs, we observed the inner surface of the vacuolar membrane in monocytes using scanning electron microscopy (SEM), followed by energy dispersion X-ray (EDX) analysis for titanium. SEM observations showed that there was a rough area in the inner surface of the vacuolar membrane in monocytes harvested at day 1 after the culture with TiNSs. The rough area of the vacuolar membrane was also seen in other monocytes exposed to TiNSs. Analysis of the rough area by EDX confirmed the presence of titanium, in contrast to results for the cytosolic region in TiNS-exposed or control monocytes. These overall findings indicate that TiNSs were actually engulfed by monocytes and included in the vacuoles.

**Figure 2.** Observation of microstructures in TiNS-exposed monocytes by transmission electron microscope (TEM). The images are taken at day 1 after culture with TiNSs. It can be seen that monocytes have obvious vacuoles even at this early time. Additionally, nano-scaled materials with TiNS-like shapes can be seen inside the vacuoles, and most of the nano-scaled material is located near the vacuolar membrane. Finally, it was confirmed by scanning electron microscopy (SEM) with energy dispersion X-ray (EDX) analysis that these materials included titanium, indicating that the material

contained TiNSs. Scale bars of 5 μm (left), 1 μm (upper right) and 100 nm (bottom right) are shown.

show the formation of large vacuoles prior to apoptosis upon exposure to TiNSs.

**6. Identification of intra-vacuolar TiNSs in monocytes**

cells induced by TiNSs as well as asbestos. The

Toxicity of Titanate Nanosheets on Human Immune Cells

http://dx.doi.org/10.5772/intechopen.72234

169

lymphocytes. These findings indicate that TiNSs have the

pase inhibitor, suppressed the increase in Anx+

monocytes as well as CD4+

CD14+

**Figure 1.** TEM images of TiNSs used in the present study and illustrations of a single TiNS and TiO6 octahedron. (A) TiNSs were synthesized by Dr. Yoshioka and observed by TEM. These images show the uniform diamond shapes of TiNSs in our present study. Some TiNSs appear to be piled up showing darker diamonds. Scale bars of 100 (left) and 50 nm (right) are shown. (B) Illustration of single TiNSs having a diamond shape with almost 20- and 30-nm diagonals, based on TEM images. (C) Illustration of a TiO6 octahedron in the orthostatic position, of which TiNSs are composed. TiNSs have the depth of one and a half of a sideways TiO6 octahedron [34].
