Protein corona formation and structural changes of lipid nanoparticles investigated using QCM-D and waveguide microscopy
Typ
Examensarbete för masterexamen
Program
Publicerad
2021
Författare
Mohammadi, Sara
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Lipid nanoparticles (LNPs) are being increasingly considered for use in drug delivery.
However, understanding the interactions of LNPs with a biological environment is
crucial for successful deliver of their cargo, such as for example mRNA, across cellular
membranes. Upon exposure to a biological fluid it is believed that the surface of
LNPs is spontaneously covered by a protein corona, a layer of adhered biomolecules,
prior to being taken up by the targeted cells. A protein of interest in this context is
Apolipoprotein-E (ApoE), both because it is known to be part of the protein corona
and because it may control endocytic uptake via specific interactions with receptors
in the cell membrane. Furthermore, ApoE-coated LNPs are expected to undergo a
structural change in the acidic environment of the endosome during cargo release to
the cytosol.
In this master thesis, we have studied protein-corona formation and the reaction
of the LNPs covered/not covered with ApoE in an acid environment that mimics
the endosomal environment using label-free surface-based bioanalytical tools such
as quartz crystal microbalance with dissipation monitoring (QCM-D) and waveguide
microscopy. QCM-D provides information based on ensemble averaging of the
biological interactions taking place on the surface of the sensor, while waveguide
microscopy provides both ensemble-averaged data and information based on inspection
of individual LNPs. Further, waveguide microscopy is capable of detecting both
light scattering and fluorescence signals, which provides the possibility to observe
both the labeled interior of the LNPs and binding of unlabeled protein.
We found that ApoE bind to PEG-modified LNPs with a lag-time of tens of minutes
unless mixed with bovine serum albumin (BSA). This is attributed to BSA-induced
PEG shedding which promotes ApoE binding. Inspections using combined labelfree
and fluorescence based waveguide microscopy releveled a weaker than expected
dependence between cargo fluorescence and scattering intensity, suggesting that the
self-assembly process utilized for LNP fabrication may vary with LNP size. It is also
concluded that further liquid handling must be improved to follow ApoE binding to
LNPs using waveguide microscopy.
Beskrivning
Ämne/nyckelord
Waveguide microscopy , Quartz crystal microbalance with dissipation monitoring , label-free , apolipoprotein-E , corona formation , protein corona , lipid nanoparticle , MC3 , drug delivery , mRNA therapy , serum protein , endosomal membrane