Saftey Flashcards
Briefly describe the different fates that nanoparticles can have once in contact with the human body. (1+6)
Direct elimination > local effect
Absorption, biodistributed, cellular uptake, biotransformtion, accumulate, elimination
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?
3D –> mimic environment, include other cells, appropriate media: air and not liquid.
Explain the term “biological identity of nanoparticles”.
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.
Describe the protein coronas characteristics and the process of protein corona formation.
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
What endpoints/assays can be used to evaluate toxicity?
Membrane integrity, LDH
Metabolic activity, MTT
ROS production, DCFH
Inflammation, ELISPOT or ELISA
DNA mutations, sequence –> look for mutations
What is opsonization of nanoparticles and what can be done to avoid such process?
Immune system that decorate the NP with Abs and complement factors – easily recognizable for clearance
PEGylation (also against phagocytosis)
Describe LDH assay for membrane integrity
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.
Describe MTT assay for metabolic activity
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
Briefly describe the process of cellular uptake of nanoparticles.
Endocytosis (w./ wo. receptor), diffusion
Go to endosome/lysosome – die or survive > organelles mitochondria or nuclei, or interfere with protein folding in ER
Describe DCFH assay for ROS generation
ROS production, DCFH assay:
o Substrate, goes in to cell
o With ROS in cell, substrate is converted to fluorescent molecules –> detectable
What could be the reason(s) why nanoparticles bioaccumulate in the body?
Receptors, targeting
EPR (leaky vessels, cancer)
Too big to handle (not degraded or uptake) > frustrated phagocytosis
How can nanoparticles cause high level of reactive oxygen species (ROS)?
Redoc ability is crucial!
Alone due to surface and stuff, or with cellular components, change their activity (mitochondria, certain enzymes)
Describe inflammation detection, ELISA/ELISPOT
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
What is oxidative stress?
Too high levels of ROS that antioxidants cannot counter.
> Cytotoxicity, programmed cell death and inflammation, cell cycle arrest
Describe how DNA damage can be detected
Sequence gene –> look for mutation
Briefly describe the processes of nanoparticle-mediated DNA damage.
Small enough? Go in themselves through nuclear pore and interact
Otherwise - maybe generate ROS, that go inside and bind to DNA (can alter bases)
Chose physicochemical properties that you consider could affect the toxicity of nanoparticles and explain why.
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
Describe the typical set up/steps of an in vitro toxicity test
- Choose relevant cell model
- Exposure, How much, how long, how? – relevant for application
- 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
Mention the advantages and disadvantages of in vitro toxicity studies.
In vitro – subcellular systems (organelles, macromolecules)
(-)
- 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
What are the three practical challenges in testing the toxicity of nanomaterials?
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
