Prof. Elena Ryabchikova and her colleagues are the first in the world to show the details of cell interactions and nanoparticles. Their results raise very serious questions as to the fundamental science, as well as to the responsibilities of the developers of drugs, food supplements, cosmetics and other products that use titanium oxide and other nanomaterials.
Many researchers and companies are looking to create nanoforms of different materials for a variety of applications from health care products to energy production and distribution, to industrial chemical production and to drug delivery systems. Is it reasonable to use nanoparticles before understanding their interactions with cells? Will nanoparticles damage cells or will they be a useful carrier for the delivery of drugs into cells? The importance of this research, as well as answers to many questions, can not be overstated.

Structural Features Of TiO2 Nanoparticles Interaction With A Cell

Elena I. Ryabchikova1, Natalia A. Mazurkova2, Nadejda V. Shikina3, Zinfer R. Ismagilov3,
Stanislav N. Zagrebel'niy2

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Lavrent'eva pr. 8, Novosibirsk, 630090 Russia
2Novosibirsk State University, Pirogova str. 2, Novosibirsk, 630090 Russia
3Institute of Catalysis, Siberian Branch of Russian Academy of Science. Lavrent'eva pr.5, Novosibirsk, 630090 Russia

Abstract

We examined the interaction of amorphous, anatase, brookite and rutile titanium dioxide (TiO2) nanoparticles (4--5 nm) with MDCK cells. The cells were incubated in presence of 100 µg/mL TiO2 nanoparticles during 1, 3 and 5 h, and then fixed for electron microscopy. The number of living MDCK cells was counted, using trypan blue assay, after 5 and 15 h treatment with 100 µg/mL TiO2 nanoparticles at 37oC. All of the TiO2 nanoparticles were toxic to the MDCK cells. Different modes of cell-nanoparticle interaction were observed with the four types of TiO2 . The interaction of amorphous, anatase and brookite TiO2 nanoparticles involve intimate cellular mechanisms. This calls for careful examination of nanoparticle-cellular interaction before introducing such particles into living tissues or the environment.

Introduction

Nanotechnologies are increasingly being introduced into modern life, which has caused some concern about the effects and safety of nanomaterials for humans and other living organisms. It is well known that nanomaterials have different chemical and physical properties than their macro-materials. The extremely small sizes of nanomaterials (less than 100 nm at least in one dimension) provide very close contact with a substrate (a cell), and, involve complicated physico-chemical interactions, which are not possible for larger particles. These new interactions result in new properties for the nanoform of well-known bulk materials, i.e., larger particles, including changes in toxicity. Titanium dioxide (TiO2 ) is a well known and widely used, generally non-toxic substance used in applications such as paints, detergents, cosmetics, and bonding teeth and bones. Presently TiO2 nanoparticles are commercially produced and used in products, such as sunscreens, additives in pharmaceuticals and food colorants. However, the nanoforms of TiO2 have different properties and may not be as safe as bulk TiO2 . Recent publications present data with evidence for toxic effects of TiO2 nanoparticles on mammalian and fish cell cultures. The papers described cellular damage after 24 — 48 h incubation of cells and TiO2 and accumulations of the nanoparticles in the phagosomes [Cheng-Yu, 2008; L'Azou, 2008; Vevers, Awadhesh, 2008]. The goal of our work was to examine the early steps of interaction of four crystal forms of TiO2 nanoparticles with a cell and the influence of the particles on cell viability.

Methods

The TiO2 nanoparticles used in this work had identical sizes, about 5 nm, and were different crystal forms (amorphous, anatase, rutile, and brookite). The synthesis of nanoparticles was described earlier [Ismagilov, 2009]. MDCK cells were propagated at 37oC on 6-well plastic dish to 80% confluence and treated by nanoparticles in concentration 100 µg/mL during 1, 3 and 5 h at 37oC. Cells were centrifuged at 3000 run/min. The pellets were fixed by 4% paraformaldehyde and routinely processed for electron microscopy, and embedded in epon-araldite. Ultrathin sections were examined in JEM-1400 (JEOL, Japan) transmission electron microscope.

The effects of amorphous, anatase, rutile, and brookite TiO2 nanoparticles on the MDCK cells were studied using visual observation and trypan blue stain of MDCK cells that were incubated at 37oC during 5 and 15 h in presence of 100 µg/mL of the preparations.

Results

The forms of four types of TiO2 nanoparticles in the ultrathin sections were distinctly different in the electron microscope as presented in figure 1. Amorphous TiO2 nanoparticles were visible as small crumbly aggregations composed of spherical particles having size 4 — 5 nm. Anatase and brookite TiO2 nanoparticles were needle-like shapes and formed various figures like stars and branches, as a whole looking like delicate lace. Fine needles of anatase and brookite were separated from each other and did not agglutinate as the amorphous form did. Rutile TiO2 nanoparticles formed long needles with 4 — 5 nm in diameter, whose composite resemble palm-leave or fan-like aggregations. The size of these rutile aggregations reached 5 — 6 micrometer (µm or mkm).

Figures 1 and 2

Figure 1. Different forms of TiO2 nanoparticles in ultrathin sections: amorphous (upper row, left); anatase (upper row, right); brookite (under row, left) rutile (under row, right). Transmission electron microscopy.

Figure 2. Ultrathin sections of MDCK cells treated with amorphous (left) and rutile (right) TiO2 nanoparticles during 1 h in concentration 100 µg/mL. Penetration of electron dense amorphous TiO2 nanoparticles into cytoplasm is clearly visible. Large coarse rutile conglomerates are located in cell vacuoles. Transmission electron microscopy.

Examination of ultrathin sections of MDCK cell culture incubated with TiO2 nanoparticles for one hour revealed large areas occupied by the particles between the cells and in areas related to cell surface. Amorphous, anatase and brookite TiO2 nanoparticles were spread on the apical surfaces of the MDCK cells , while rutile particles formed large coarse aggregations, mostly located far from a cell. Amorphous and anatase TiO2 nanoparticles were observed in all the foldings and cavities of cell plasma membranes and had deeply penetrated into the cells, as shown in figure 2. Amorphous and anatase TiO2 nanoparticles were found in small vesicles in cytoplasm and in few endosomes. We also observed the interaction of anatase TiO2 nanoparticles with some clathrin-coated pits, indicating involvement of receptor-mediated endocytosis mechanisms for internalization of the anatase. Brookite particles, after one hour incubation, mostly remained on the MDCK flat cell surface; we noted very few pictures of nanoparticles in cell foldings. So, while anatase and amorphous TiO2 nanoparticles readily interacted with MDCK cells and entered the cells during one hour of incubation, brookite particles remained outside of cells. Heavy coarse deposits of rutile TiO2 nanoparticles were seen in cells, inside large vacuoles (figure 2).

Incubation of MDCK cells with amorphous and anatase TiO2 nanoparticles for three hours resulted in accumulation of the particles inside the cells. Nanoparticles were observed in endosomes and phagosomes, and remained in the surface foldings and cavities or invaginations, as shown in figure 3. Brookite TiO2 nanoparticles mostly remained on the cell surface, however some material was found in the plasma membrane folding and cavities. Anatase and amorphous nanoparticles, located between and near the cells, showed a tendency for random agglutination, while brookite agglutinated in close proximity to the cells. It should be noted that not all cells contained TiO2 nanoparticles inside. The main mass of these three forms of TiO2 nanoparticles remained outside the cells, and only very small amounts of the nanoparticles reached inside the cells. The distribution of the rutile TiO2 nanoparticles, after three hours incubation with MDCK cells, was the same as was observed after one hour incubation: MDCK cells contained large vacuoles with coarse rutile aggregations. No signs of active phagocytosis were found in cells with rutile deposits inside. Many cells showed swelling of cytoplasm and mitochondria, vacuolization of cytoplasmic structures.

Figure 3

Figure 3. Ultrathin section of MDCK cell treated by anatase TiO2 nanoparticles during 3 h in concentration 100 µg/ml. The nanoparticles are seen outside of the cell and in cell foldings and invaginations. Asterisk show late endosomes containing nanoparticles. Transmission electron microscopy.

Examination of MDCK cells incubated with TiO2 nanoparticles for five hours revealed anatase and amorphous forms in the phagosomes and endosomes, as well as on the cell surface. The main mass of these nanoparticles remained outside of the cells and appeared agglutinated. Brookite TiO2 nanoparticles, after five hours incubation, mostly remained outside the cells and were rarely found inside MDCK cells in late endosomes. It is interesting that many cells without brookite nanoparticles inside developed a large number of late endosomes (multivesicular bodies) and numerous “coated” vesicles, which are evidence of active membrane exchange between cellular compartments. The agglutination of brookite nanoparticles in the vicinity of the cell surface increased when the cells were incubated for five hours, and the agglutination had lost its “delicate lace” appearance. Incubation of MDCK cells with amorphous, anatase and brookite TiO2 nanoparticles for five hours also lead to swelling of some cells and the development of pathological changes of cytoplasm structures. The conglomerates of rutile TiO2 nanoparticles, after five hours incubation, remained in large vacuoles without visible signs of interaction with cytoplasmic structures.

The study of TiO2 nanoparticles's influence on cell viability showed obvious toxic effects for all the types of the particles on MDCK cells. The number of living cells surviving five hours of incubation was 53%, 76%, 67% and 40%, using 100 µg/mL amorphous, anatase, rutile and brookite, correspondingly, in comparison to those without nanoparticles. The incubation of MDCK cells for 15 hours with amorphous, anatase, and rutile nanoparticles, at the same concentrations, resulted in decreases of living cells to 34%, 57% and 41%, while the viability of brookite treated cells remained similar to the five hour incubation (43%). Amorphous, anatase and rutile TiO2 nanoparticles decreased cell viability with increasing incubation time. Brookite TiO2 nanoparticles altered a large number of MDCK cells and only 40% of cells remained living after five and 15 hours incubation.

Our study showed the MDCK cells interact differently with different forms of TiO2 nanoparticles. The crystal organization of the nanoparticles is very important for their interaction with a cell and determines which mode of cell reaction to influence. The MDCK cells internalized the anatase and amorphous forms of TiO2 nanoparticles (4 — 5 nm) using endocytosis. However,, in contrast with anatase and amorphous TiO2 nanoparticles, brookite was unable to fill cell foldings and reach the cytoplasm. Rutile TiO2 nanoparticles formed large coarse conglomerates, which interact with the MDCK cells without switching on endocytosis. Perhaps the weight of the rutile conglomerates collapses the plasma membrane and thereby rutile appears inside the cell. Rutile's effects on cell structures develop via mechanisms different from those operating in the case of other TiO2 nanoparticles.

Conclusions

Toxic effects of TiO2 nanoparticles on different cultured cells (after 24-48 h incubation) were reported recently [Cheng-Yu, 2008 ; L'Azou, 2008; Vevers, Awadhesh, 2008]. However, these studies did not examine the early stages of nanoparticles interaction with cells and did not examine the dependence of these interactions on different crystal forms of TiO2 nanoparticles. We found close interaction of the nanoparticles with fine cellular mechanisms which are responsible for the internalization of various compounds. It is evident that nanosizes are critical for this interaction. Taken together our data demonstrate the need for caution before introducing nanomaterials into commercial preparations destined for living organisms nanomaterials. Nanomaterials are being further studied as possible delivery systems in chemotherapy, especially for cancers that are difficult to reach, for example brain tumors. A critical step in the development of the carrier is the interaction of the nanomaterial with cells and understanding the nature of how the materials are incorporated into cells.

Acknowledgment

This work was supported by the Russian Ministry of Science and Education, Program “Basic Studies in Natural Sciences. Development of the Scientific Potential of the Higher School” Project # 2.1.1/5642

References

1. Chen-Yu J. et al. (2008), Cytotoxicity of Titanium Dioxide Nanoparticles in Mouse Fibroblast Cells, Chem. Res. Toxicol. 21, 1871-1877 (doi:10.1021/tx800179f)
2. Ismagilov Z.R. et al. (2009), Synthesis and stabilization of nano-sized titanium dioxide, Progress of Chemistry,78(9), 942-955.(English version : Russian Chemical Reviews (2009), 78 (9): 873, doi: 10.1070/RC2009v078n09ABEH004082)
3. L'Azou B. et al. (2008) In vitro effects of nanoparticles on renal cells, Particle&Fibre Toxicol., 5:22 doi:10.1186/1743-8977-5-22.
4. Vevers W., Awadhesh N. (2008), Genotoxic and cytotoxic potential of titanium dioxide (TiO2 ) nanoparticles on fish cells in vitro, Ecotoxicology, 17:410-420 (doi:10.1007/s10646-008-0226-9)

Editors Note: Whether the nanomaterial is part of a drug delivery system to enter cells and tissues or a part of a protection system that should remain outside of the body, the first steps in determining if a nanomaterial is a viable product includes documenting the size and shapes of the particles and if these particles can enter live cells. Prof. Ryabchikova and her team can determine these to ASTM quality standards.

ASA’s Dr. Barbara Price works with Prof. Ryabchikova, as she has for many years, and her team and laboratory to assist all interested parties in the coordination and arranging for their independent product testing and characterization to ASTM quality standards. For further information - contact Dr. Price at tel: 1-808-235-8010 or e-mail: cbmts@asanltr.com



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