Authors:

Emma Vinokour

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About Paper:

Virus-like particles (VLPs) derived from the MS2 bacteriophage are promising nanocarriers for the delivery of therapeutic molecular "cargo" to diseased cells. The in vitro cargo-loading process first requires disassembly of the VLP protein capsid and removal of its native RNA, followed by spontaneous capsid re-assembly around an exogenous, negatively charged cargo, typically nucleic acids. However, there remains no standardized framework for packaging and quantifying the encapsulated cargo: protocol parameters such as disassembly incubation time and re-assembly conditions are not well defined, and packaging efficiency varies with cargo type and structure. Here, we compared multiple quantification techniques for MS2 VLPs and found that traditional absorbance-based measures of MS2 concentration can be cargo-dependent and therefore unreliable for assessing encapsulation yield, particularly across heterogeneous cargoes. We determined that area-under-the-curve absorbance from chromatographic separations provides a rapid yet more consistent proxy for encapsulation yield when supported by mass-based reporting of the cargo-to-protein ratio (as compared to a molar-based ratio). During this process we also identified parameters for a more reproducible re-assembly protocol, including an optimal capsid disassembly incubation time. With this standardized framework in place, we then explored how cargo type and structural features influence differences in cargo-packaging efficiency during in vitro VLP reassembly. Specifically, we examined VLP assembly with different nucleic acid cargoes and observed distinct yield and stability profiles, indicating that both chemical composition of cargo (i.e. DNA vs. RNA) and cargo structural determinants, such as stem-loop motifs and secondary structures, modulate packaging efficiency and capsid stability. These findings suggest that maximized VLP assembly and cargo loading require both accurate yield quantification and an understanding of the biophysical principles governing nucleic acid-capsid interactions, including sequence and structural features. This work holds great potential to inform the sustainable and scalable design of emerging VLP-based drug delivery platforms.

Source:

Northwestern University

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Co-authors:

Emma Vinokour