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Nanotoxikologie (Subject) / Teil 2 (Lesson)
Klausurfragen
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- Can you give an example for an interaction of NP with proteins, which causes a stabilization of the protein? Chymotrypsin was stabilized against denaturation by binding Au-NP => Substrate specifity altered due to binding to Au NP (after binding to Au NP cationic substrates were favored)
- a) However an interaction of proteins with NP can also cause a destabilization of the proteins. Which example is typically for it? b) How can you confirm a loss of protein activity (denaturation)? a) •denaturation (loose activity) due to binding to NP surfaces was observed for lysozyme incubated with MWCNT => Lysozyme contains hydrophobic Trp residues who strongly interact with the MWCNT (hydrophobic pi-pi stacking) b) •if Trp binds to MWCNT the Trp will be lost in UV-VIS spectrum•it can be also observed in CD-spectrum, that the molar ellipticity is decreasing when the Trp disappears
- a) What can result as proteins interacting with NP? b) Can it be dangerous, if the protein properties are changing due to an interaction with NP? a) •influencing the protein folding (often irreversible) => decrease of the protein stability, the function of proteins (e.g. enzymatic activity) and protein - protein interaction (e.g. protein fibrillation) b) •yes, this can also influence the interaction with proteins and the immune systeme (e.g. previously covered cryptic epitopes can get exposed and can then be recognized by the immune system)
- a) What is called as „protein corona“? b) What is called as „protein corona evolution“? a) •total bound protein by the NP often referred to as “protein corona” b) •the protein corona is changing its composition over time (slower proteins with higher stability replacing faster binding proteins with lower stability) = protein corona evolution
- a) What is called as „hard corona“? b) What is called as „soft corona“? a) • proteins bind very stable (but need high energy input = high ΔG) => are likely to stay associated with the NP b) •proteins bind not stable (but need low energy input = small ΔG) => are likely to desorb
- Can you explain why smaller particles have a higher toxicity than bigger particles at the same dosage rate? because to have the same dosage rate, you need more particles => toxicity↑ but the toxicity is on the same level as we look at the surface/mL ratio (surface per volume)
- a) What can decreased protein binding cause an impact to the NP? b) What might be the difference in consideration of the uptake between NP and Protein-covered NP? c) Does a protein-binding increase or decrease an agglomeration of NP? a) •Plasma retention time ↑=> enhanced circulation time in blood•protein binding should be decreased to prevent a long retention time in the body b) •protein-covered NP might be incorporated better => stealth mode (no opsonization) c) •depends on the proteins => often stabilization by electrostatic repulsion
- What kind of interaction of NP in the pulmonary reuptake way can be expected? pulmonary surfactant contains 90% lipids (80% of them are phospholipids) => the negatively charged phosphate group causes an interaction with positively charged NP
- What can happen to NP in the different digestion fluids? NP agglomerate in saliva compared to water → „disappear“ in gastric juice (dissolution) → „reform“ when being transferred in intestinal fluid („new“ NP contained silver, chlorine and sulfur)
- a) SiO2 have been dispersed in 3 media (H2O, PBS and BSA with PBS). What kind of dispersion state would you exspect due to the media? b) Can you explain the effect of the serum proteins? c) What can be exspected if you compare the NP size between water dispersed and BSA dispersed NP? a) •in PBS they are not so well dispersed (agglomeration => particles > 200nm)•in BSA the agglomerates are getting smaller (redispersion/disagglomeration) => stronger effect in higher concentrations of BSA•only in water monodispersed b) •serum proteins are dispersing the SiO2 NP in a polydispersed form => they’re breaking the agglomerization in PBS due to their coating function c) •small increase in size => NP are binding to serum proteins = coated with them to protein corona (usually about 20nm, protein size is usually 5-7nm)
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- Silver Nanoparticles have been incubated with serum proteins. Bound proteins have been eluted and analyzed. However, the released amount of proteins is very low and much less than expected. What could be the reason? strong binding of silver NP with sulfur molecules of cystein (in the proteins)
- Please find examples/applications for the interaction of NP with a) carbohydrates b) nucleic acids a) •Cu NP => Glucose sensor•Ag and Au NP are used for DLS to show protein-carbo hydrate interactions b) •interaction of Ag NP with DNA and RNA (anionic phosphate)•Gold nanobeacons (gold NP functionalized with a fluorophore-labelled hairpin-DNA => cancer therapy)