nanoparticles and production of reactive oxygen species

Name: Adwait Suratkar; UID: 1213672

Department of Earth, Environmental Science and Geography

University of Birmingham

Nanoparticles and production of reactive oxygen species (ROS): for how long can we deny

this truth?

“Oxygen: In one form its gives life... in another it takes” – Adwait P.Suratkar

Section. 1 Abstract:

Nanotechnology has certainly been one of the most promising fields of research and development. If you

look at it from one angle, nanotechnology has myriads of applications, ranging from health care and

cosmetic products, medical diagnostics; as coating on surfaces; nanoparticles being used in ceramics and

paints which are also a major part of textile industry; they are also being used as anti-microbial agents.

The dark side of nanotechnology is its toxic effects not only on humans, but on other organisms living in

the environment. In this article we will look at how these nanoparticles end up in cells and how they play

a critical role in formation of reactive oxygen species, which eventually decide the fate of the cell.

Oxygen the destroyer:

Oxygen is biologically one of the most important molecule, but also a molecule that is capable of creating

havoc inside a cell in its nascent state or reactive state. Molecular oxygen has two unpaired electrons in its

outer most shell, with same spin in same direction; when these electrons are excited by a reacting species,

they can produce a molecule called singlet oxygen (1O2) which is a powerful oxidant (Klaus Apel 2004)

that is capable of breaking strong chemical bonds. This reaction usually occurs in the presence of ultra-

violet (UV) light, and can also be initiated in the presence of ionizing radiation. Collectively the many

different forms of this reactive oxygen are called as reactive oxygen species (ROS). In this section we will

be looking at the different forms of reactive oxygen that is produced and released inside a cell.

But before that it is important to understand why reactive oxygen species is produced inside a cell or a

body, there are two important reasons for this:




Two combat with invading micro-organisms, pathogens and viruses. In human body, macrophages and

other lymphocytes are known to use ROS as a mechanism to destroy pathogens.

Reactive oxygen species is also produced during the terminal stages of electron transport chain and

oxidative phosphorylation.

Reactive oxygen species are also encountered at the cell membrane (in the form of an incoming signal).

There are also external factors because of which ROS is produced. This article will enlighten you about

the possible ROS production by nanoparticles.

Reactive oxygen species (ROS): This is a highly reactive species of molecular oxygen produced by the

cell during the metabolism of oxygen. They come in various forms (superoxide, hydrogen peroxide,

singlet oxygen, Hypochlorous acid); Hydroxyl radicals (OH- and OH

+) are the most reactive form of

oxygen in the biological system, they have a very short half life but can cause serious damage to

biological material, they are produced as a result of Fenton reaction. Superoxide anion (O2-) is a

negatively charged but highly destructive species of oxygen when it reduced by electrons, production of

this species occurs at the Complex I (NADH: ubiquinone oxidoreductase) of mitochondria (Turrens, J. F.

2003) Hydrogen peroxide is a by product of superoxide reduction. Hydrogen peroxide is the last of the

ROS; on further reduction water molecules are produced.

What happens to all the ROS produced? Every cell has enzymes and antioxidants present in it to

combat and maintain a balance (in terms of concentration) of the reactive oxygen species. Some of the

antioxidant enzyme include; i. Superoxide dismutase (SOD), ii. Catalase; (ii) Glutathione peroxidase

(GPx); (iii) Cu-Zn Dismutase; there are also certain antioxidant that scavenge on the ROS; they include,

(i) Vitamin C; (ii) Vitamin E; (iii) melatonin, etc. The ROS produced in the cells oxidizes mainly the

genetic material, causes lipid peroxidation, oxidative stress; overall a lot of cell damage.




Nanoparticles: These tiny particle are have amazing applications and but some of them are relatively

new to science and very little is known about its supramolecular chemistry. According to the Scientific

Committee on Emerging and Newly Identified Health Risks (SCENIHR) and the Joint Research Centre

(JRC), a nanoparticle is defined as "a natural, incidental or manufactured material containing particles, in

an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in

the number size distribution, one or more external dimensions is in the size range 1 nm – 100 nm."

Nature of nanoparticle plays a critical role:

Nanoparticles come in various shapes (triangles, rods, cones, wires), sizes and can be synthesized from

various elements. Usually the properties of nanoparticles are tailored to suit the application. There are

also intrinsic properties of nanoparticles (novel to a particular class of nanoparticles); they play a critical

role while evaluating the toxicity and the behavior of nanoparticles a point of bio-contact (Andre Nel

2009). Semiconductor nanoparticles (Quantum dots) are highly toxic especially cadmium nanoparticles

(Jasmina Lovri 2005), as they are soluble in nature and release toxic ions that can be produce reactive

oxygen species. The size, shape and the capping agent and the coating on the surface of the nanoparticle

(polymer coating, or a biocompatible coating) play a critical role. Semiconductor nanoparticles produce

ROS by the production of electron hole pairs that are highly reactive in nature, thereby oxidizing or

reducing biomolecules (proteins, enzymes) to produce ROS. Also it is important to note if the

nanoparticle is hydrophobic or hydrophilic in nature, certain nanocrystals do not have any coating at there

surface, this can make them highly toxic, Rutile and anatase (TiO2)13

nanoparticles are a good example

for this, even uncapped silver nanoparticles (1-10nm) that expose the <111> crystal face are possible

ROS generators (mélanie auffan 2009). Carbon nanotubes7 are a class of carbon nanoparticles that are

toxic because they are insoluble in nature. They get accumulated in the body (especially lungs), they are




not engulfed by the phagocytes (indirect mechanism of ROS production) because of their size and ―tube‖

like shape.

Generation of reactive oxygen species by nanoparticles

Fenton chemistry: This occurs in case of very fine iron nanoparticles1 (due to a very large surface area).

The already formed hydrogen peroxide inside the cell is converted into hydroxyl radical (OH-) and (OH

+),

these are as mentioned earlier the most reactive intermediates of ROS. This would further lead to

oxidative stress and cell damage. There need a further investigation though if this effect is enhanced by

the ―nano‖ of iron particles, (more ions are released in a ―nano‖ then in the bulk.). This phenomenon is

also shown by TiO2 nanoparticles (Auffam 2009) (rutile and anatase), but the difference in the case of

TiO2 nanoparticles is that they require UV excitation or absorption of light.

Figure 1: Reference:

Mélanie Auffan, Nature

nanotechnology Vol 4

October 2009 The figure

explains the relation of

size and the novel

―nano‖ properties

associated with it. There

are also many reactions

occurring at the surface

of a nanoparticle, ROS

production being one of

them.




Release of toxic ions can produce ROS: ZnO2 (Anat Lipovsky 2011); chromium (S. J. Stohs 1995) and

AgNPs (silver nanoparticles) release toxic ions. This release occurs by reduction or oxidation of

nanoparticles inside the body or a cell in individual.

Interaction with Mitochondria: The structure, shape and dimensions of mitochondria play a critical in

nanoparticle interaction, accumulation and generation of ROS. Referring to figure 2 it is important to note

that any nanoparticles with diameter < 14 nm will easily cross the outer membrane of mitochondria and

accumulate in the mitochondria. But nanoparticles within the diameter range of 16 to 30 nm will puncture

into the mitochondria and can block important pathways like oxidative phosphorylation, electron

transport chain and lipid metabolism (due to their large size in comparison to the dimensions of

mitochondria), this can stress the mitochondria, disrupt mitochondrial function, a collective effect is

oxidative damage, and production and accumulation of ROS. Certain mitochondria have a high

percentage of cristae (liver mitochondria);

Figure 2: Terrence G. Frey, Carmen A. Mannella; Elsevier Science TIBS 25 – JULY 2000, PII:

S0968-0004(00)01609-1. This is a 3D tomogram of mitochondria, showing the dimensions of the

organelle, a delicate organelle like mitochondria is an easy target of nanoparticles. Also seen in this figure

is Endoplasmic reticulum ( ), responsible for lipid shuttling from & to mitochondria.




Certain mitochondria have a high percentage of cristae (liver mitochondria); Nanoparticles interact with

mitochondria by either disrupting the mitochondrial membrane potential, or accumulation in the

mitochondria. This is another possible route to production of peroxide species. There is evidence in the

literature about the presence of gold (Pan Y 2009; Liming Wang 2011), silver (Martin Kruszewski

Chapter 5 Advances in Molecular toxicology Vol.5 2011) and Zinc oxide nanoparticles in mitochondria

and production of ROS.

Characterizing ROS damage: The damage caused by nanoparticles can be assessed trough following

ways:

MTT essay to assess the mitochondrial damage caused by the nanoparticles and ROS.

Signs of apoptosis and inflammation can be checked using LDH assay.

Also using transcriptomic and genomic methods can be adapted to analyze the cellular response to

incoming nanoparticles, and post ROS response.

Electron Microscopy to visually analyze the damage (swelling of organelles or cell(s))

Conclusion: There is overwhelming evidence that nanoparticles indeed produce (directly or indirectly)

reactive oxygen species. These are early days, and we are still in dark waters until we understand and

confirm the mechanism of ROS production by nanoparticles.




References:

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nanoparticles and production of reactive oxygen species

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