Nanoparticles : Characteristics , Mechanisms and Modulation of Biotoxicity †

Particulates of 1 to 100 nanometers size are being increasingly used for a variety of clinical and commercial purposes due to their large surface-to-volume ratio and unique physico-chemical, mechanical and electronic properties. While utilizing them for their beneficial functions, it has become vital to heed recent alerts on their toxic biological effects in order that benign systems might be developed. In this paper we review emerging mechanisms of nanotoxicity and possible means for remediation. Challenges in monitoring, characterizing and remediation of nanoparticles are also presented.


Introduction
A revolution in materials technology that is changing the way we live is shadowed by a toxic storm rising on the horizon.Indeed the use of nanoparticles in industrial, health and personal care areas is increasing exponentially owing to their special and often novel mechanical, electronic and chemical properties.Examples include fluorescent quantum dots as markers in biological imaging, gold and carbon nanoparticles as controlled drug and nutrient delivery vehicles in medicine and agriculture, metal nanoparticles as fuel cell components, oxide particles as UV filters in cosmetic and personal care products and pigments and lubricants in commodities.Furthermore, inorganic functionalized nanoparticles are used to produce UV resistant and flame retardant plastics for automotive and aviation industries and metallic ones are used as viscosity modifiers for lubricants and controlling the dielectric properties of electronic coatings.Alarmingly, metal oxide and magnetic nanoparticles are increasingly used without adequate knowledge and precaution in areas such as cosmetics to produce brighter pigmentation and UV protection and in medicine for chemotherapy as well as NMR contrast enhancers.While developing new consumer applications of nanosized materials, potential longterm adverse effects on living species must be considered.Also, as the safety of nanomaterials marketed during past decades does not appear to be adequately addressed, methods are needed to determine the cytotoxicological safety of current and future nanomaterials under different scenarios, particularly since cell cytoskeletons and genetic material have been definitely shown to be damaged in the presence of nanoparticles 1,3,4) .It should be noted that even when direct effects on humans are not recognized, some recent reports suggest that nanoparticles suspended in wash water can work their way into organisms with potentially serious health effects to humans through the food chain.Clearly, there is a dire need for a full understanding of the mechanisms by which nanoparticles bio-accumulate, intrude the skin barrier, as well as interact with biomacromolecules, such as proteins and DNA.It also suggests caution regarding the exposure of human beings during the manufacture, transport and handling of nanoparticles. of this interface is dependent on properties of the nanopar ticles, the biophase (protein, cell membranes, endocytic vesicles or organelles), the medium and most importantly any changes in them due to mutual effects within the interfacial region.Those nanoparticle characteristics which predominantly govern the surface behavior include the chemical composition and the resultant solubility, sur face charge, semi-conductivity, size, shape, surface curvature, crystallinity as well as porosity, surface heterogeneity, roughness, surface functionalization with charged groups, peptides or polymers [5][6][7] (Fig. 1).Indeed, it is the combined effect of the nanoparticle properties in the suspending medium as well as the interactive biological components that describe the nano-bio interface 2) (Fig. 2).Properties of the biosubstrate vary depending on the biomolecular moieties and their sequence in the molecule, molecular weight, the configuration of the macromolecule and membranes or cells comprising them.Many of these properties are in turn dependent upon medium properties such as ionic strength, pH, polarity and temperature 8) .The above characteristics govern other important interfacial processes such as adsorption of ions, surfactants, polymers and proteins as well as aggregation/dispersion and solubility of particles 9,10) .These processes are controlled by forces at the nano-bio interface that operate at the whole range of length scales.10,11) These forces determine the dispersion of nanoparticles which in turn is controlled, in Fig. 2. Representation of receptor mediated uptake.This is a specific biological mechanism for particles interacting with the surface membrane and undergoing cellular uptake.The intrinsic nanoparticle characteristics that promote surface binding Fig. 2 Representation of receptor mediated uptake.This is a specific biological mechanism for particles interacting with the surface membrane and undergoing cellular uptake.The intrinsic nanoparticle characteristics that promote surface binding (roughness, hydrophobicity, cationic charge) generally lead to nonspecific binding forces (marked by asterisks) that promote cellular uptake. Incontrast, specific receptor-ligand interactions generally lead to endocytic uptake.A combination of nonspecific binding forces on the surface of spiked particles can lead to direct penetration of the surface membrane without the need to involve endocytic compartments.2) addition to the size, by their charge, hydration and surface polymeric groups 6,[12][13][14][15][16][17][18][19][20] .It has been found recently that the nanoparticles with different surface properties and aggregation behavior can cause different responses to the culture medium, which in turn can lead to different degrees of toxicity.21,22) The toxic effects of inorganic nanoparticles on biological cells have attracted increasing attention of researchers in recent times 3,16,[23][24][25][26] Reported negative biological effects include the production of intracellular reactive oxygen species (ROS) and related oxidative stress 6) , DNA lesions, denaturation, and even particle wrapping by the surface membrane.27,28) As in the case of engineered composite nanoparticles, cytotoxicity of polymeric nanogels can also be driven by surface and morphological heterogeneous effects.2) .It is to be noted that the entire nano-bio interface is a dynamic system where many short and long term interactions occur simultaneously as well as sequentially.
The characterization of the nanoparticle/liquid inter face is possibly the primar y challenge when tr ying to understand the nano-bio interface.While characterization often assumes steady-state behavior to assess the overall behavior of the suspension by monitoring such properties as iso-electric point, size and average aggregate size, these assumptions have to be adjusted when considering the nano-bio interface.Moreover, methods used to monitor relevant properties of large particles under static conditions are most often not valid for nanoparticles.Also many of these methods do not report special inhomogeneities that can result from spatial localization of ionic groups, proteins and lipids as well as imperfections and contaminants on the surface of particles and the cell membranes.The nano-bio interface is highly dynamic and undergoes continuous changes as a result of nanoparticles dissolution and resulting cellular response.While it may take some time to develop substantive investigative methods to explore this interface at the usual level of sophistication in colloid chemistr y, it is worthwhile exploring the nano-bio interface through novel imaging techniques and biological approaches.

Fundamental and complex forces at the nanobio interface
The forces between particles are typically classified into long range and short range forces.The former forces arise from usually attractive van der Waals forces and repulsive or attractive electrostatic interactions, whereas, the latter arise from steric, depletion and hydration interactions.Even though the forces that control interactions between cells and nanoparticles are similar to the forces between two larger colloidal particles, the cellular response and the resulting mutual effects on nanoparticles and the cells render traditional surface chemical methods unreliable.One such phenomenon that is important for inorganic and other hydrophilic or "water loving" nanoparticles is hydration.If water is attracted to nanoparticles more strongly than nanoparticles to each other, hydration can interfere with the aggregation of nanoparticles. 15)Another major variable is the dynamic effect due to the morphologically and chemically compliant nature of the membrane 29) .In addition, inter-particle interactions can be affected by the chemical heterogeneity of the nanoparticles. 30)lso, it is important to note that multiple particles could form rafts that may exhibit markedly different properties than single particles.In addition, as in the case of flotation of mineral particles by air bubbles, possible roll over of the particle on the cell surface has to be recognized.On the other hand, nanoparticles are not passive.Upon interaction with the cell components, they can change (dissolve/grow, aggregate/disperse, or even experience phase transformation).This generates a time-dependent and dynamic nano-bio interface. 31,32)Other complexities such as endocytosis of the nanoparticles and specific receptor-ligand interactions make these interactions difficult to predict by theoretical considerations.

Challenges and Solutions to the Monitoring Nanoparticle systems
Nano-toxicology is an emerging scientific discipline aimed towards studying the impact of nanotechnology on human health 33) as a growing number of consumer products contain engineered nano-particles.One need that must be addressed in this regard is the development and selection of accurate methods for monitoring nano-particles of various products, many being dispersions containing mixtures of not only different nano-sized particles, but also particles of much larger size.
Monitoring the presence of nano-par ticles in dispersions with broad par ticle size distributions is difficult because their effect could be masked by the presence of larger particles or the aggregates of the nano-particles themselves.While there are some methods for measuring systems containing only nano-particles, there are no well established techniques for monitoring nano-particles in the presence of larger ones.Development of such methods is imperative for reliable investigation of nano-particles in natural and engineered systems.The first logical step in this regard is an investigation of currently available techniques.To make the study reliable, it is necessar y that the same set of nano-particles is studied.Hence we measured different samples with acoustic spectroscopy and TEM, since these techniques have been reported to be well suited for characterizing nano-particles with rather narrow size distributions [34][35][36][37][38] . Aoustic techniques can handle concentrated systems (>1% vol), which can not usually be dealt with by most other techniques 39) .Suspensions of 5%wt ZnO particles were prepared by ultrasonicating samples from different manufacturers in the presence of hexametaphosphate.The acoustic measurement yielded a median value of 200 nm to 700 nm for the size of these powders. Indpendent TEM photographs confirmed the trend of size variation from sample to sample.Importantly, the acoustic measurement indicated that all of these dispersions contain dif ferent amounts of nano-par ticles, even though they were specified by the manufacturers as nano-powders rather than large particles.Table 1 presents the averaged content of nano-particles for each ZnO sample.Precision of this measurement, defined as the ratio of the "absolute average deviations" to the "average content" , varied significantly from sample to sample.

Toxicity due to the portion of particles on the Nano levels
Although there is some controversy about the toxicity of nanoparticles, there is by and large enough evidence of their adverse effects.As a noteworthy example, Oberdorster et al. have reported that 36 nm carbon nanoparticles can translocate to the brain via the olfactory nerve upon inhalation in rats. 40)In their investigation, rats exposed to the nanoparticles for six hours exhibited increasing accumulation of nanoparticles within the olfactor y bulb even seven days after exposure.Oberdorster has also shown that 36 nm carbon particles have a significant potency to induce inflammatory lung injury. 41)Lam et al. also demonstrated that carbon nanotubes could cause lung granulomas in mice. 42)Other studies showed that titanium dioxide (TiO2) particles smaller than 20 nm can cause inflammatory reactions in both animals and humans. 43)Gutwein 44) et al. have reported a dramatic decrease in the growth rate for cells exposed to alumina particles for only 2 hours.Table 2 summarizes reported works on cytotoxicity of various nanoparticles and the mechanisms of toxicity along with potentially useful design methods.As of today, most of the research on nanotoxicity has been on the eukaryotic cells.
We have recently investigated cytotoxicity of ZnO, CeO2, and anatase TiO2 nanoparticles on Nitrosomonas europaea cultures. 2,21,45,46)Nitrosomonas europaea (N.europaea) is a microbe whose growth relies on the oxidation of ammonia to nitrite. 47)Therefore, it is used in general for waste water treatment.The presence of nanoparticles could possibly damage the activity of the Nitrosomonas europaea cells, leading to their poor performance in the treatment of industrial and sewage waste of oxidizing ammonia to nitrate.Nitrosomonas europaea in chemostat state were cultured in the presence of ZnO, CeO2 and anatase TiO2 nanoparticles at concentrations of 20 ppm and 200 ppm for 4 hours.The cells were collected for further characterization using TEM.Fig. 3 shows the transmission electron microscopic images of the particles In addition to TEM, a Beckman Optima XL-1 analytical ultracentrifuge with scanning optics in an interference system was employed to investigate the complex distribution in solution by monitoring sedimentation velocity. 48)he morphological changes of bacteria are illustrated in Figure 4a-h.The intercellular particle accumulation was found for cells treated with all three types of nanoparticles except for the 200 nm anatase TiO2. 25 nm anatase TiO2 produced changes in the morphology of the elliptical N. Europaea (Fig. 4a) to an irregular and distorted form (Fig. 4b).Most of the cells that remained in their elliptical shapes contained a cavity.Those cells that are distorted still kept their cytosolic compositions.Hardly any dividing cells were obser ved in the cells with particles.Also, it was difficult to visualize the clear multilayered cell membranes under nanoparticles stress, especially when 200 ppm of nanoparticles were added (Fig. 4c).At the same loading concentration smaller particles distorted the cells more than the larger particles.Cells in the media with 200 nm particles remained more spherical in shape with their membrane largely unperturbed (Fig4.d) than the ones in media with 25 nm particles.At similar total surface area, 200 ppm 200 nm anatase TiO2 nanoparticles show less damage to cell shape as compared to 20 ppm 25 nm anatase TiO2 NPs.However, empty vacuoles were observed inside the cells for all the sizes.The size effect has also been studied on eukaryotic cells.It has been found that for the same type of particles, larger size creates less damage in general.The cells show little morphological distortion in shape under the stress of ZnO nanoparticles (Fig. 4e) as compared  [51][52][53][54] Fullerenes 42) Frustrated phagocytosis: cell wall disruption and enzymes release, generation of ROS, and inflammation due to aggregation of CNT Generation of ROS and resulting oxidative stress Surface functionalization with antioxidants, complete purification to eliminate residual metal ions; coating with polymer matrix to prevent release of toxic metal ions; surface functionalization.Cationic Polymer nanospheres and Dendrimers 24,55,56) Endosomal cellular uptake Modulation of cationic charge density and increasing specific c e l l u l a r i n t e r a c t i o n t h r o u g h functionalization with appropriate receptors Metals Gold nanoparticles 57,58) Denaturing of protein Coating of nanopar ticles with amphipathic agents to modulate c h a r g e , s i z e , d i s p e r s i o n a n d hydrophobicity Silver nanoparticles 59) generation of ROS, Alteration of transport through membrane Capping agents to prevent release of Ag + ions Metals Oxides TiO2 27,60,61) ZnO 25,62) Al2O3 63,64) SiO2 [65][66][67] CeO2 25) Oxidative stress due to generation of ROS and photoactivity; cell death and fibrillation due to interference in macrophage cell membrane functions High dissolution of ZnO nanoparticles under physiological condition and release of toxic cations.

Induce proinflammatory response
Protein denaturation; oxidative stress due to ROS generation Inducing protein aggregation C a p p i n g N M s w i t h ; c o a t i n g with anti-oxidants (ascorbate, glutathione, alpha-tocopher ol (vitamin E)), enzymatic scavengers o f a c t i v a t e d o x y g e n s u c h a s superoxide dismutase, surfactants, polymers or complexing ligands; Control of redox properties; Surface passivation Other Nanoparticles Co/Ni ferrite Nanoparticles, magnetic metallic nanoparticles 68) CdSe 69) Low cell viability due to the release of toxic cations High cell mortality due to easy dissolution and release of ions

E n c a p s u l a t i o n w i t h p o l y m e r, Capping with phosphonic and h y d r o x a m i c a c i d s ; p o s i t i v e l y charged tetraheptylammonium (for Ni nanoparticles)
with cells cultured in media with 25 nm anatase TiO2 particles.However, most of the inner leaflet of the membrane wrapped the interior cell structures and separated from the rest of outer membrane bilayer compartments.The outer cell membrane bilayers become indistinguishable too.No broken membrane debris was observed in the fixed cell sample.Neither were empty cavities/vacuoles seen in the cell.On the other hand, a few dark spots can be observed having entered the cells and gathered within the center of the cells.Similar morphological responses were found for CeO2 treated N. Europaea (Fig. 4f).No particles were observed to intrude the cell, although some can be observed to adhere to the outer membrane leaflet along their longitudinal needle axis (Fig. 4g).Much significant damage was obser ved in cells that were close to CeO2 aggregates, which display large cavities and bubbles in them (Fig. 4h).The cell walls/membranes, however, remain undamaged.Considering the fact that during TEM sample preparation heavy particles as well as cells precipitate first due to centrifugation, it can be assumed that the cells near the CeO2 aggregates would be heavier and show more damage with vacuoles than the cells far away from them.The stresses exerted by the same type of nanoparticles on eukaryotic cells have been reported by us recently. 25)It has been concluded that ZnO particles induce cytotoxicity by dissolution of Zn 2+ ions as well as the uptake of ZnO particle remnants.The nano-ZnO exposure on cells could increase the membrane permeability via Na + and K + fluxes through cells and ionic homeostasis disturbance based on rat tests. 49)Since the N. Europaea has a cell wall and multilayered membrane bilayer shell, it would be very unlikely that the uptake is via endosomal ways.Moreover, a close contact between cells and particles is also prevented by the electrostatic interaction since both are negatively charged.As from previous references on eukaryotic cells, TiO2 nanoparticles would be toxic under UV energy in the cells. 23)The CeO2 particles, on the other hand, have been reported to be safe and can protect the cells against secondary oxidative stress stimulus. 25)Our results of stress exerted by the anatase TiO2 and CeO2 nanoparticles on the prokar yotic N. Europaea, however, showed observations of toxicity.From the morphological observation, all particles were observed to have caused damage irrespective of their sizes and charges.The shrinkage of the inner cell membrane near the cell wall and the release of cytosolic compartments indicate possible high osmotic pressure in and out of the cell membrane under the stress of nanoparticles.TiO2 cause cell membrane dissociation even when possible UV-radiation was screened to some degree under dark culturing conditions.It is also seen from the TEM images that there are many fibrous structures surrounding the cells, which can be considered to result from shedding of the cell walls or membrane compartments.Since the charges on the cellcomplex shifted to the positive side and the original zeta potential for the particles is negative, we propose  that only some positive ionic species from the media get adsorbed onto the cell walls.This statement is validated by the results from three separate analytical ultracentrifuge measurements in Fig. 5, which shows in general that when under nanoparticles stress, the cells become heavier than the control and thus sediment faster.Cells in 200 ppm TiO2 (and CeO2) nanopar ticles media have a sedimentation peak position close to that of the cells with lower NP loading, while an apparent right-shift is seen between the 200 ppm and 20 ppm peak positions of cells in ZnO nanoparticles media (Fig. 5b).This is not unexpected considering the lower dissolution of the TiO2 (and CeO2) nanopar ticles as compared with ZnO nanoparticles.These results correlated well with the light scattering and zeta potential data.

Toward the production of environmental benign particles
The above discussion raises the following questions in order to produce safe nanoparticles: • How do nanoparticles enter cells, and how can this phenomenon as well as subsequent interactions with cell components be controlled?
• What is the hierarchy in morphological, structural, and electronic properties of nanoparticles that govern their penetration into cells?
• What are the mechanisms of generation of reactive oxygen species (ROS) upon nanoparticle-cell interaction?
• How can particle size and surface functionalization control each of these processes?
• How does the heterogeneity of the nanoparticles make further complications in the mysterious nano/bio interface regions?In summary, is it possible to tune out nanotoxicity?Tackling the above questions requires us to develop a new paradigm that will enable a full understanding of nanoparticle-cell membrane and nanoparticlemedia interactions while taking account of the environment-dependent acid-base, redox, electronic, aggregation, and wettability properties of nanoparticles.As stated earlier, the entire nano-biointerface is a dynamic system where many simultaneous and sequential short and long term interactions play different roles at different time and place.The effects have been suggested to be controlled by characteristics of nanoparticles such as hydrophobicity, surface roughness/porosity, solubility-release of toxic species, surface species-contaminations/adsorption during synthesis/history, capacity to produce ROS, structure/composition, surface charge, binding sites competitive for receptor, dispersion/aggregation.Based on the current obser vation/understandings gained with regard to bio-nano interfaces, the following strategies are proposed towards the production of greener nanoparticles: 1) Promotion of aggregation of nanoparticles using Nanoparticles for 4 hours, g 20ppm CeO 2 Nanoparticles, showing the particle align themselves longitudinally on the cell wall, h) 20ppm CeO 2 Nanoparticles, the cells close to the particle aggregates shows more cavities in them. 1) preferably natural reagents.Our results with both fibroblast and the N. Europaea research have shown that particles of the same materials but larger sizes cause less damage to cells.Therefore, aggregation of particles particularly by natural biomacromolecules, soils, or even small living creatures should increase the safety of particles (Fig. 6a).
2) Modification of the nanoparticles with biocompatible coatings to prevent their catalysis of various reactions with water and other surrounding organic materials.On the other hand, the large production of free radicals can also react and degrade organics in matrix, especially bio-macromolecules such as DNA.
To solve the problem, Lee et al.59 have modified the titania nanoparticles with multi-component polymer coatings (Fig. 6b).The inner-most layer is antioxidants (Oligomeric Proanthocyanidins) to capture free radicals generated by titania particles under UV irradiation.Then, a layer of anionic polymers is coated onto the particle surface, which can provide negative charges to the surface of titania particles with attendant decreased possibility for contact or penetration of particles into cell membranes.To protect the integrity of whole particles with coating layers, the outermost layer is fully covered with hydrophilically modified polysiloxane.The hydrophilicity of the particles could facilitate the dispersion in the aqueous media, maximizing the efficiency of sequestering hazardous radicals.Such a chemical and physical grafting approach remarkably reduces the photocatalytic degradation and enables highly effective screening against the UV radiation.
3) Mixing with benign particles.Nanoparticles, when mixed with biocompatible particles or species are likely to be less toxic but still effective for enhancing mechanical as well as electronic properties.

Conclusions
Past work shows that nanoparticles can potentially cross the cell membranes and stress biological cells.[テキストを入力] The presence of the par ticles has been shown to be responsible for the abnormal actin filaments and extracellular matrix behaviors in dermal fibroblasts, which in turn decreases cell growth and proliferation.When possible toxicity effects of 25 nm and 200 nm anatase TiO2 nanoparticles on waste water treatment N. Europaea bacteria were probed, cells were found to be distorted with membrane damage.CeO2 and ZnO nanoparticles affect cell morphologies, too.Possible means for mitigating toxicity include aggregation, surface modulation with multiple polymer coatings, biocompatible functionalization and mixing with benign particles.
It is clear from an examination of the work done with nanoparticles thus far that there is dire need to develop methods to monitor, characterize and tame, so to speak, nanoparticles so that we may fully reap the benefits of nanotechnology for producing better technological and medical products for a long happy life and a healthier planet.

Fig. 5
Fig. 5 The analytical ultracentrifuge result on N. Europaea cells grown in media loaded with 0ppm, 20ppm and 200ppm of a) 25nm anatase TiO 2 Nanoparticles.b) ZnO Nanoparticles.Cells under the stress of NPs have larger sedimentation coefficient than the control.1)

Fig. 5 .
Fig.5.The analytical ultracentrifuge result on N. Europaea cells grown in media loaded with 0ppm, 20ppm and 200ppm of a) 25nm anatase TiO2 Nanoparticles.b) ZnO Nanoparticles.Cells under the stress of NPs have larger sedimentation coefficient than the control.1)

Fig. 6 Fig. 6 .
Fig. 6 Schematic drawing of a) the aggregation strategy, and b) the surface modification strategy for greener nanoparticles by coating.

Table 1
50)ian particle size and percentage of the nano particles (<100nm) in various ZnO samples at 5%wt stabilized at pH 10 with hexametaphosphate.(mediansizes of the particles were provided by manufacturers)50)

Table 2
Potential mechanisms/routes of adverse effects of nanoparticles and possible ways of mitigation (adapted from Ref. 2)