LOGÍSTICA

Clearly, the use of nanoparticles can offer new and potentially exciting avenues in biological research and possibly in medical applications, providing that most of the legitimate concerns about the toxicity of their uses could be allayed by further research.

There are some particularly interesting examples of the proposed nanopar-ticle applications, such as the use of organically modified silica nanoparnanopar-ticles that can be optically tracked and used for non-viral gene delivery (Roy et al, 2005). This should circumvent one of the major objections to genetic modifica-tion, for example the use of viral promoters. Although this appears to be promising even in a possible oral application, the lack of suitable means to

target the particles to particular cells considerably lessens its potential useful-ness. It has also been proposed that three-dimensional networks of multi-walled carbon nanotubes (MWCNTs) as scaffolds could serve as biocompatible matrices in restoring, maintaining or reinforcing weakened or damaged tissues by promoting cell seeding and growth in a biocompatible way (Correa-Duarte et al, 2004). However, the lack of bioresorption on which the suggested increased biocompatibility is based is unlikely to be fulfilled under in vivo conditions because most cells have a capacity to internalize nanoparticles. In some instances these particles capable of emitting fluorescence signals may be used in tracking the trafficking of the nanoparticles in biological systems (Cherukuri et al, 2004).

However, to capitalize on this advantage it is essential to show by more than a simple criterion that this internalization has no toxic consequences for the cell, something that is not always done convincingly. Because of the generally perceived huge and potent biological activity of ultrafine nanoparticles (less than 100nm in size), and because the results of several experimental studies indicate their possible toxicity, influential voices in the scientific community and society are advising caution before the wholesale introduction of nanoparti-cles that can come into contact with humans is allowed (European Commission Workshop, 2004; Royal Society and Royal Academy of Engineering, 2004).

Therefore hazard/risk assessment studies performed before allowing commercia-lization of the products of nanotechnology are of paramount importance.

In some instances, as will be described in this chapter, there is undoubted experimental evidence to show that the nanoparticles are cytotoxic. However, as some of the potent biological responses of cells to nanoparticles or even their cytotoxicity may in part, or in some cases fully, originate from their surface chemistry, it is hoped that by coating them with inert substances the nanoparticles can be rendered more biocompatible. Thus, the possible removal of the cytotoxic properties of semiconductor quantum dots with bright photo-stable fluorescence, which show promise in their use as alternatives to organic dyes for biological labelling, would extend their possible uses in biological systems. Even the highly toxic cadmium selenide quantum dots can be rendered non-toxic by appropriate coating of the cadmium selenide (CdSe) core (Derfus et al, 2004). Moreover, surface modification of nanocrystal quantum dots not only changes their physico-chemical properties but also their cytotoxicity (Hoshino et al, 2004). Surface derivatization by the attach-ment of appropriate peptide-, lectin-, metabolism-specific and other ligands and probes to the surface of soluble and insoluble quantum dots can con-siderably extend their usefulness, particularly as research tools in cell biology (Michalet et al, 2005) and microbiology (Kloepfer et al, 2003). By utilizing their bright, long-lasting and probe-specific near-infrared fluorescence, quantum dots have a great potential in studies to map intracellular processes even at the single molecule level, or to establish cell trafficking pathways and targeting and provide the basis for high-resolution cellular imaging.

With appropriate coatings, quantum nanodots can be made to escape endo-somal clearance in the cell and possibly reach the nucleus or other intracellular The Future of Nanotechnology in Food Science and Nutrition 169

targets. However, their possible use as diagnostic (or other) tools in vivo in human therapeutics requires caution and more research work: there is evidence to show that despite their coating, once the quantum dots are internalized, the cells can extract some of their toxic chemical content. Thus, bacteria could extract Cd or Se from the CdSe quantum dots (Kloepfer et al, 2003). This may also happen with other cells (Derfus et al, 2004). It therefore remains to be seen whether the optimism is justified that it may be possible to find low enough concentrations at which the cytotoxicity of the quantum dots is negli-gible but their advantageous properties are still displayed (Michalet et al, 2005).

Similar to other nanoparticles, the cytoxicity of water-soluble fullerenes (carbon buckyballs, buckminsterfullerenes) is a sensitive function of surface derivatization. In two different human cell lines, the lethal dose of the fuller-enes changed over seven orders of magnitude with relatively minor alterations in their structure. Highly aggregated forms made by generating superoxide anionic radicals were substantially more toxic than soluble derivatives and could damage cell membranes, leading to cell death. It may therefore be possible to establish strategies for enhancing the toxicity of fullerenes in cancer therapeutical or bactericidal uses but it may also be possible to reduce undesirable biological effects by appropriate surface derivatization (Sayes et al, 2004).

In addition to their many non-biological applications, the potential biologi-cally to use carbon nanotubes with a diameter as little as a few nanometres and a length of thousands of nanometres, and which can be filled with pharmaceutical and other desirable materials for delivery to bodily tissues, is regarded by many as a major technological development. However, it appears that these insoluble carbon nanotubes can also present major risks in biological applications. There is in fact a substantial body of experimental evidence to suggest that exposure to nanotubes may be harmful even for some cultured cells. Thus, the formation of free radicals, the accumulation of peroxidative products, antioxidant depletion and the loss of cell viability due to major ultrastructural and morphological changes in cultured human keratinocyte cells resulting from their interaction with unrefined single-walled carbon nanotubes (SWCNTs) indicate that these SWCNTs possess strong cytotoxicity, even though this may in reality be due to embedded iron catalyst particles in the nanotubes (Shedova and Castra-nova, 2003). Furthermore, it was also shown that SWCNTs are able to inhibit the proliferation of cells, such as human HEK293 cells by inducing apoptosis and decreasing their adhesive capacity by secreting small ‘isolation’ proteins (Cui et al, 2005). Although undoubtedly the promised advantages of the use of carbon nanotubes are huge, the results of these in vitro studies should caution us against their premature introduction into human medicine or the food chain.

A major component in some of the reactions and responses to nanoparticles is the activation of the immune system. In some instances this can include the activation of the alternative complement system. Some features of these in vitro reactions may also be relevant in vivo. Thus, it was shown that in serum exposed to nanoparticle carbon black, the alternative complement pathway became activated and chemotactic factors were generated by a mechanism

that involved reactive oxygen species (Barlow et al, 2005). Complement activa-tion and macrophage migraactiva-tion induced by carbon nanoparticle-exposed serum has also been suggested as being involved in lung inflammation in vivo. The generation of chemotactic factors in serum, however, not only occurs with carbon nanoparticles, as ultrafine crystalline silica could similarly generate chemotactic serum factors (Governa et al, 2002).

This short overview of the studies with nanoparticles in in vitro systems with cultured cells demonstrates that their possible cytotoxicity is an obstacle to their potential in vivo uses that would have to be overcome. However, it may yet be possible to use the advantageous properties of some of the nanopar-ticles in biological applications, particularly some carbon-based parnanopar-ticles, if the relation between their surface chemistry and their striking cellular activities were better understood. However, even then chemical purity may not be easily achieved. Moreover, there is an obvious limit to the progress in our understanding of the biological responses to nanoparticles and their possible cytotoxicity and genotoxicity if safety studies are only done with cultured cells in in vitro systems. Indeed, from in vitro studies it is difficult, and in some cases impossible, to predict whether these particles would be safe in vivo and would not display the same and significant toxicity that has been observed in vitro. The results obtained in in vivo studies described below warn us that each single potential application of the nanoparticles in complex biological systems needs to be thoroughly investigated before these highly potent bioactive particles can be safely used, particularly in humans.

In vivo (human and animal) reactivity of nanoparticles