Characterizations of Viral Nanoparticles
Zhuohong Wu, Department of NanoEngineering, UC San Diego
5/13/2024
Context Statement
Goal: To characterize the structural integrities of viral junctions and their subunits.
The degree of crosslinking of a viral nanoparticles is an important knowledge to understand how the nanoparticles might interact with the polymer networks. MONET relevance: specifically, to obtain a tougher polymer network, higher crosslinked nanoparticles are desired. The combination of denaturing gel and native gel can tell us helpful information about the surface chemistry and virus/CPs integrity to guide the upcoming experiment designs. We will discuss how these new techniques work with practical examples, as well as the limitations and considerations.
Characterization techniques
For analysis of electrophoretic mobility of the VLPs (as a function of size/charge) and coat proteins (as a function of molecular weight)
For analysis of VLP size; the larger the VLP the faster its elution.
Data
Data confirm the integrity and purity of native PhMV particles.
Quantification can be done by gel densitometry (e.g., ImageJ analysis)
Limitations and caveats
The conjugation of small molecules (a few Dalton) is difficult to differentiate as secondary bands from primary bands, which are difficult to quantify.
Lighter: CP
Heavier: CP-PEG5
Some viral nanoparticles tend to disassemble when the gel experiment is conducted at room temperature. Adding ice bags surrounding the gel tank is always helpful. Also, lowering the voltage while extending the running time could prevent generating too much heat in the gel tank.
It is hard to differentiate from the gel.
If particles disassemble, you could try:
Practical considerations
Native gel and denaturing gel:
Where can I use this technique? For virus or protein covalent modification, I can quantify the degree of modification. The native gel technique is suitable for non-covalent encapsulation when the denaturing technique is not.
How many materials: denatured protein CPs (2.5–3.75 μg per lane).
How to prepare and run the experiments: all samples were denatured by boiling in 1:10 reducing agent and 1:4 SDS blue loading dye for 5 min, followed by running under 180 V for 60 min. For native gel electrophoresis, PhMV VLPs (5 μg per lane) were characterized using 0.8% (w/v) agarose in 0.1 M Tris-borate-EDTA (TBE) running buffer (pH 8.3).
How to analyze the results: The gels were then stained in Coomassie blue staining solution, followed by photography under UV light in a FluorChem R system (Bio-techne). The fluorescently labeled VLPs were analyzed under UV light with a red-light filter before Coomassie Blue staining.
Size exclusion chromatography:
Where can I use this technique? Integrity inspection of viral nanoparticles. Fluorophore attachment inspection.