L2 Nanotoxicity Flashcards
Re. W3 Quiz
List TWO barriers in the successful delivery of nanoparticles to its intended destination in vivo (2 marks).
- MPS (mononuclear phagocyte system) - “immune system” - eats the NP before the NP hits the target
- Corona formation, changing NP size, altering eventual interaction
- Opsonization - barrier - related to opsonization and eventual cell membrane internalisation / endosomal escape
- Haemorheological behaviour of NP dependent on shape/size
- Renal filtration - filters out < 5nm in size
- Blood-Brain-Barrier - prevents entry of NP (or any other foreign matter) into the CNS
- Endosome-lysosome pathway - mechanism by which cells internalise and destroy matter. Endosomes are vesicles (made of phospholipid membranes) which eventually contain the foreign matter. When endosomes ‘transform’ into lysosome, the inside of the lysosome becomes highly acidic (hydrolytic enzymes), in order to destroy/degrade foreign matter
Define opsonization
(1 mark)
The process of non-specific adsorption of proteins on material surfaces.
Accumulation of opsonins (i.e. proteins that attract the cells of the MPS, e.g. antibodies, complement proteins, plasma proteins, etc.) on the surface of NP –> protein corona
(The ‘reason’ why opsonization/MPS naturally ‘exists’ is to keep us healthy in that foreign bacteria are targeted (i.e. proteins attached to outside of cells) for destruction by immune cells (i.e. protective mechanism, vital part of our biology)
Briefly state why opsonization is an issue in NP delivery.
(1 mark)
Changes NP size and surface properties (charge, hydrophobicity, chemistry, etc.)
Gets eaten up by phagocytes - undergoes phagocytosis.
Limits effectiveness of NPs - ligands are blocked from interacting with receptors.
Name ONE strategy to minimise opsonization
(1 mark)
- CD47 synthetic marker to prevent phagocytes (cells of the MPS) from recognising as foreign
- PEG coating - creates a ‘hydration layer’ that prevents protein adsorption
- Mimicking the surface of the red and white blood cells
Which of the following nanoparticle diameters would be most suitable (for most NP materials) for in vivo drug delivery and/or diagnostics?
(A) 0.25 nm
(B) 2.5 nm
(C) 25 nm
(D) 250 nm
(C) 25 nm
Regarding (B) 2.5 nm, size of less than 5 nm will be filtered out by the kidney.
Regarding (D) 250 nm, (generally) larger than 200 nm) are easier for opsonization/MPS recognition.
Which of the following pathways is NOT involved in the internalisation of nanoparticles?
(A) Clathrin-mediated endocytosis pathway
(B) Caveolin-dependent endocytosis pathway
(C) Receptor-independent endocytosis pathway i.e. no need for receptor
(D) Internalization of NP captured in extracellular vesicles
(D) Internalization of NP captured in extracellular vesicles
Receptor mediated endocytosis = internalization of the NP after the NP interacts with a particular surface receptor → interaction then triggers either clathrin proteins or caveolin proteins (both proteins intracellular) which then creates an endosome that encapsulates the NP.
Name ONE strategy for a NP with its cargo to escape the endosomal-lysosomal pathway.
- Membrane fusion (fusing with the endosomal membrane) → mimics viruses; use cationic lipid carrier → two membranes fuse, release cargo
- H+ ions pump - proton sponge - explode of lysosome. The “natural” activity of lysosomes: its primary function is to destroy whatever is in the lysosome. In order to do this, the cell makes the inside of the lysosome very acidic. If you have something in the lysosome that prevents the rise of acidity, the lysosome will continue to try make it acidic, more H+, more Cl-, more water (osmosis) - BANG!
- Protein on the NP surface causing a porous endosomal membrane (or creating pores on the endosomal membrane) - peptide pore formation
- Membrane disruption → charged polycationic/anionic polymers that have capacity to damage membranes → release cargo
Define nanotoxicology
nanotoxicology:
The study of adverse effects in living organisms due to interaction with nano-sized agents.
(study of toxicity profile of nanoparticles)
Which of the following factors/parameters can potentially affect the in vitro/vivo toxicity of NP?
(Total 4 marks; -0.5 for every incorrect option circled, -0.5 for every correct option uncircled; total number of correct options unspecified)
- Chemical composition of NP
- Geometry of NP
- Route of administration
- Subject variability
- Long-term accumulation
- Dose duration
- Delivery media
- Surfactant toxicity
- Degradation profile
- Specific density of NP
- Interaction with mono-nuclear phagocytic system
- Surface charge
- In vivo corona formation
- Dose amount and frequency
- Agglomeration
- Time of day when NP is administered
- Chemical composition of NP
- Geometry of NP
- Route of administration
- Subject variability
- Long-term accumulation
- Dose duration
- Delivery media
- Surfactant toxicity
- Degradation profile
- Specific density of NP
- Interaction with mono-nuclear phagocytic system
- Surface charge
- In vivo corona formation
- Dose amount and frequency
- Agglomeration
- Time of day when NP is administered
Name ONE method (experimental technique or otherwise) of examining the toxic effect of NPs in in vitro cell lines.
- Live/dead cell assay
- Proliferation assay (to see if the cells are happy or dying in the presence of NP within their environment)
- Apoptosis (controlled cell death) / Necrosis (uncontrolled cell death) markers (different markers and biomolecule profile exhibited between the two)
- Monitoring gene/protein expression (normal or abnormal) –> a measure of cell function (i.e. if the cell is doing the ‘right thing’ by producing/expressing the right proteins
Name ONE method (experimental technique or otherwise) of examining the toxic effect of NPs in in vivo organisms.
- Weight loss - easy to do and most commonly done in in vivo animal models
- mortality (survival rates of animals)
- morbidity (particular tissue is dead or damaged)
- symptoms (e.g. rash)
- surrogate markers of toxicity (enzyme/marker level in body fluid [blood plasma, CSF, etc.] e.g. serum liver enzyme levels) - if your body or part of your body is struggling (due to toxic effects) that particular cell/tissue/organism will release certain chemicals/biiomlecules that you can detect (hopefully)
Which of the following terms define the concentration of an administered agent (i.e. drug, NP) whereby 50% of the administered population exhibit a pre-specified toxic or adverse effect?
(A) Median effective dose (ED50)
(B) Median toxic dose (TD50)
(C) Median lethal dose (LD50)
(D) Maximum toxic dose (MTD)
(E) Minimum effective dose (MED)
(A) Median effective dose (ED50)❌(effective for 50% of population)
(B) Median toxic dose (TD50) ✔
(C) Median lethal dose (LD50) ❌ (50% dies)
(D) Maximum toxic dose (MTD) ❌
(E) Minimum effective dose (MED)❌ (smallest concentration at which there is some therapeutic effect)
List TWO potential chemical mechanisms through which nanoparticles can induce toxicity.
(2 marks)
Production of ROS - generation of reactive oxygen species due to surface reactivity
- Release of metallic ions* (trace amounts is usually normal in the body - if there is an imbalance, then the biomechanisms act up). Metallic ions also act as catalysts for ROS generation.
- Surfactant toxicity* - almost all surfactants have a degree of toxicity (as they’re made of organic molecules)
- Disruption of election/ion membrane transport* (both physical and chemical)
List TWO potential physical mechanisms through which nanoparticles can induce toxicity.
(2 marks)
Blocking membrane activity and transport processes
Disruption of membrane structure (e.g. physical puncturing - burst the membrane of either endosome/cell)
Protein conformation/folding - the physical interaction between NP and proteins sometimes result in the change of the physical structure of the protein. → protein function is dependent on protein shape
“Fibrillation” - another term for uncontrolled aggregation of proteins. If proteins cluster around and agglomerate - change in shape + and covering of binding sites - change in function
Reactive oxygen species (ROS) are chemical species of oxygen which are highly reactive. These are usually generated during metabolism, whereby antioxidants keep the ROS levels in check. However, NPs can provide an environment where excess ROS is generated, and hence exert a negative effect on cell function.
Name ONE way ROS can exert a negative effect on cell function.
Imbalance = undesired biochemical activities = oxidative stress
- Interfered intracellular signalling (normal cell functions heavily depend on intracellular signalling)
- Lipid peroxidation: ROS ‘steal’ electrons from lipids
- Leads to membrane damage (outer, mitochondrial, vesicles, nucleus, etc.)
Damage to DNA, lipids, other nucleic material, proteins through oxidation (of amino groups & nucleotides)
