Nanotoxicology studies the adverse health and environmental effects of nanomaterials. The toxicity of nanoparticles depends on their physicochemical properties like size, shape, charge and coating. Smaller nanoparticles are generally more toxic due to their higher surface area to volume ratio. Positively charged nanoparticles are more toxic than negatively charged or neutral ones. In vitro and in vivo methods are used to screen nanoparticle toxicity. In vitro tests use cell lines while in vivo tests use animal models. Both methods examine proliferation, oxidative stress, necrosis and apoptosis. Further research is still needed to fully understand nanoparticle toxicity mechanisms and their long term effects.

1. Guru Ghasidas Vishwavidayala
Bilaspur C.G.
Assignment 2023
Department of Biotechnology
Subject : Nanobiotechnology
Submitted to
Miss. Laxamani Verma
Assistant Professor
Submitted by
Parth Sharma
MSc II semester
Roll no. 22043125

2. Introduction :
• Nanotoxicology is the field of studies determining the adverse effects of
nanomaterials on human health and the environment.
• Nanotoxicology searches for establishing and identifying the harms of
engineered nanomaterials and study of exposure assessment,
pharmacokinetics, and medicine.
• Particle size and surface area are important characteristics from a
toxicological perspective. As the particle size decreases, its surface area
increases and therefore allows more atoms or molecules to be reflected on
the surface than on the inside of the material.
• The change in the physicochemical and structural properties of engineered
nanomaterial with a decrease in size could be responsible for a number of
material interactions that could lead to toxicological effects.

3. Relationship of toxicity with physiochemical
properties of nanoparticles :
• Size of nanoparticles : The size of NPs and their toxicity have an inverse
relationship. Smaller NPs have high ratio of the surface area-to-volume
which might clarify this opposite connection among size and toxicity.
• Shape and structure of nanoparticles : NPs exhibit different levels of
toxicity based on existence of different shapes like spherical, cylinders,
cubes, sheets, and rods. Spherical NPs are highly inclined to endocytosis as
compared to nanotubes and nanofibers.
• Charge assessment of toxicity of nanoparticles : surface charges strongly
govern the interaction of NMs with biological molecules. Positively charged
particles show high toxicity compared to the negatively charged and
neutral particles because positively charged particles can enter in the cell
easily because of the electrostatic force of attraction as cell membrane is
negatively charged

4. • Nature of coating and effect on toxicity : The shell and secondary coating
is an important factor that influences NMs toxicity. It is used to enhance
the solubility and biocompatibility of NPs in water and other bio-based
fluids. The shell decreases the level of toxicity of NMs and allows selective
interaction with different cells and biological molecules.
Figure: Mechanisms and factors for toxicity of NPs.

5. Figure : Schematic illustration depicting NM-induced cytotoxicity

6. Figure : toxicity of nano particles in plant cells

7. Methods of screening of toxicity :
Assessment
of toxicity
In – vitro In - vivo
• The use of bacteria
or cell lines is done
to understand the
toxicity
• Living system like
mouse or the zebra
fish or some other
animal models.

8. In vitro
method
Proliferation
assay
Necrosis
assay
Oxidative
stress assay
Apoptosis
assay

9. In vitro method
• Proliferation assay
• The evaluation of cellular metabolism
can be done using proliferation assay.
MTT is a salt which is used for the in-
vitro proliferation assay.
• Measures the cell suitability by
conversion of tetrazolium compound
into a purple-colored water insoluble
formazan crystal. The insoluble
formazan is then solubilized using
dimethyl sulphoxide (DMSO) or
ethanol and the color obtained is
quantified by a spectrophotometer at
the wavelength between 500 and 600
nm.
• Oxidative stress assay
• ROSs and reactive nitrogen species
(RNSs) are produced by the exposure
of NPs.
• ROSs and RNSs can be detected by the
reaction of 2,2,6,6-
tetramethylpiperidine (TMP) with
stable O2 * radical which are sensed
by a high cost technique like electron
paramagnetic resonance spectroscopy
(EPR) or fluorescent probe molecule.

10. • Necrosis assay
• Neutral red (2-amino-3-methyl-7-dimethyl-amino-
phenazoniumchloride), a dye which is a weakly cationic and produces
deep red color at slightly acidic pH is used for this purpose.
• It accumulates within the lysosomes and binds by with anionic sites
using electrostatic hydrophobic bonds within the lysosomal matrix.
• Modifications of the cell surface due to the NPs interaction result in
lysosomal fragility, which can also end up in lowering of uptake and
binding of neutral red dye that can differentiate between viable and
dead cells.

11. In vivo
test
Oral harmful
test
Intense dermal
test
Biodistribution
examination
Eye irritation
test

12. In vivo method
• For oral harmfulness test, the mice were
orally administered 5000 mg/kg body
weight (LD50) of colloidal NMs.
• All creatures were sacrificed after 14 d
and skin & liver were gathered for routine
histopathological assessment.
• After 1, 7, and 10 d of exposure, biopsies
of the skin are performed for
histopathological assessments and blood
was taken for measuring biochemical
parameters like cholesterol, triglyceride,
blood glucose, glutamic oxaloacetic
transaminase (GOT), glutamic pyruvic
transaminase (GPT) and hematological
examinations.
• For intense eye irritation, the creatures
were treated with 1.5 and 2.5 ppm
colloidal nanoparticles, individually.
• Briefly 0.1 mL of colloidal suspension was
injected within the conjunctival sac of
one eye of the creature and the other eye
was filled in as a control with similar
volume of refined water.
• The creature was observed for harmful
side effects at 1, 12, 24, 48 and 72 h after
treatment.

13. • For intense dermal poisonousness test the creatures were arbitrarily
isolated in three groups (n = 3) as follows: bunch 1 gets refined water
and groups 2 and 3, get 50 and 100 ppm of colloidal solution,
respectively.
• Biodistribution examines the localization of NPs to tissues and
organs. Radiolabels are used to detect the dead or live animals.

14. Conclusion :
• We know that there are immense use of NPs in biomedical applications,
but they may cause change in gene expression and protein/lipid oxidation.
• Methods for screening the toxicity of NPs such as in-vivo and in-vitro
effects of cell lines, routes of exposure to human, and mechanism of
toxicity of NPs are also developed.
• Many scientists are working hard to find solutions to several pressing
environmental concerns like bioaccumulation, environmental impact,
toxicity and long-term issues.
• Further research is still needed to understand the proper mechanism of
nanotoxicity, standardization of the protocols of toxicity and long-term
effect of NPs on persons’ health and environment impact.

15. References :
• Deepshikha GUPTA et al. Pathways of nanotoxicity: Modes of
detection, impact, and challenges; Front. Mater. Sci. 2021, 15(4):
512–542.
• Asati S, Sahu A, Jain A; The Dark Side of Nano-formulations; Current
Nanotoxicity and Prevention, 2021, Vol. 1, No. 1.