Saftey

Question 1

Briefly describe the different fates that nanoparticles can have once in contact with the human body. (1+6)

Answer 1

Direct elimination > local effect

Absorption, biodistributed, cellular uptake, biotransformtion, accumulate, elimination

Question 2

There is a need for enhanced in vitro models and exposure techniques - describe some of the possible strategies to achieve more relevant in vitro models?

Answer 2

3D –> mimic environment, include other cells, appropriate media: air and not liquid.

Question 3

Explain the term “biological identity of nanoparticles”.

Answer 3

Protein corona – the molecules and peptides adsorbed onto the particle through its exposure path with stuff in biofluids.

Depending on this identity, interactions may differ.

Question 4

Describe the protein coronas characteristics and the process of protein corona formation.

Answer 4

Protein bound to surface of NP dynamically.
1. Soft/transisient with low affinity
2. Replaced by high affinity protein (aka hard corona)
* Some proteins bind to other proteins

Question 5

What endpoints/assays can be used to evaluate toxicity?

Answer 5

Membrane integrity, LDH
Metabolic activity, MTT
ROS production, DCFH
Inflammation, ELISPOT or ELISA
DNA mutations, sequence –> look for mutations

Question 6

What is opsonization of nanoparticles and what can be done to avoid such process?

Answer 6

Immune system that decorate the NP with Abs and complement factors – easily recognizable for clearance

PEGylation (also against phagocytosis)

Question 7

Describe LDH assay for membrane integrity

Answer 7

Membrane integrity can be examined with lactate dehydrogenase assay, LDH
o Lactate dehydrogenase found inside cell, upon membrane damage it is found outside.
o Substrate –> Change in color.

Question 8

Describe MTT assay for metabolic activity

Answer 8

Metabolic activity can be examined through Alamar blue assay, MTT
o Transformation of substrate only in metabolically active cells
o MTT, mitocontrial reductase. Change in color –> activity

Question 9

Briefly describe the process of cellular uptake of nanoparticles.

Answer 9

Endocytosis (w./ wo. receptor), diffusion

Go to endosome/lysosome – die or survive > organelles mitochondria or nuclei, or interfere with protein folding in ER

Question 10

Describe DCFH assay for ROS generation

Answer 10

ROS production, DCFH assay:
o Substrate, goes in to cell
o With ROS in cell, substrate is converted to fluorescent molecules –> detectable

Question 11

What could be the reason(s) why nanoparticles bioaccumulate in the body?

Answer 11

Receptors, targeting

EPR (leaky vessels, cancer)

Too big to handle (not degraded or uptake) > frustrated phagocytosis

Question 12

How can nanoparticles cause high level of reactive oxygen species (ROS)?

Answer 12

Redoc ability is crucial!

Alone due to surface and stuff, or with cellular components, change their activity (mitochondria, certain enzymes)

Question 13

Describe inflammation detection, ELISA/ELISPOT

Answer 13

Inflammation, cytokine levels, ELISA
o Investigate the level of secreted pro-inflammatory cytokines TNFalfa and IL-1beta
o Sandwhich ELISA

ELISPOT - have the actual cells –> see what they produce

Question 14

What is oxidative stress?

Answer 14

Too high levels of ROS that antioxidants cannot counter.
> Cytotoxicity, programmed cell death and inflammation, cell cycle arrest

Question 15

Describe how DNA damage can be detected

Answer 15

Sequence gene –> look for mutation

Question 16

Briefly describe the processes of nanoparticle-mediated DNA damage.

Answer 16

Small enough? Go in themselves through nuclear pore and interact

Otherwise - maybe generate ROS, that go inside and bind to DNA (can alter bases)

Question 17

Chose physicochemical properties that you consider could affect the toxicity of nanoparticles and explain why.

Answer 17

Surface chemistry, e.g. metals that can do redox and cause ROS

Size – smaller can enter the cells easier (make chaos)

Stability – if degrade improperly, dangerous

Question 18

Describe the typical set up/steps of an in vitro toxicity test

Answer 18

1. Choose relevant cell model
2. Exposure, How much, how long, how? – relevant for application
3. Evaluation
   All endpoints:
   - Membrane integrity LDH
   - Metabolic activity MTT
   - Gentoxicity, sequence the gene and look mutations
   - ROS self NP,ROS in cell: DCFH substrate  fluorescent with ROS
   - Inflammation: ELISPOT or ELISA to look for cytokines

Question 19

Mention the advantages and disadvantages of in vitro toxicity studies.
In vitro – subcellular systems (organelles, macromolecules)

Answer 19

(-)
- cell line in culture usually immortalized  affect the normal biology of the cells
- Isolated systems are not reliable for conclusions drawn for the complex reality
- May fail to detect intracellular effects such as cross-talk between inflammatory cells

(+)
- rapid and effective screening and ranking.
- provide important tools to understand the toxic effects on cellular and molecular level
- Provide well defined systems for studying structure-toxicity relationships
- Possibility of high-throughput screening

Question 20

What are the three practical challenges in testing the toxicity of nanomaterials?

Answer 20

In vitro in vivo – translate between systems

High-throughput needed - too much cost and time to do with current assays for everything

Standadrization – need to be able to compare