Adeno-Associated Virus-like Particles' Response to pH Changes as Revealed by nES-DMA

Area of Science:

• Biophysical characterization
• Nanoparticle analysis
• Gene therapy vector development

Background:

• Gas-phase electrophoresis using nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) analyzes nanoparticle size and distribution.
• Volatile electrolytes like ammonium acetate are essential for electrospraying in nES GEMMA.
• Virus-like particles (VLPs), particularly adeno-associated virus 8 (AAV8), are promising non-infectious vectors for gene therapy.

Purpose of the Study:

• To investigate the impact of pH variations on the physical properties of AAV8 VLPs using nES GEMMA.
• To determine how pH affects the size, aggregation, and shape of both empty and DNA-cargo-filled AAV8 VLPs.
• To assess the reliability of nES GEMMA for characterizing VLPs under varying electrolyte conditions.

Main Methods:

• Utilized nES GEMMA for gas-phase electrophoretic separation and size determination of AAV8 VLPs.
• Employed atomic force microscopy (AFM) to corroborate aggregation behavior.
• Applied cryogenic transmission electron microscopy (cryo-TEM) to analyze particle shape and morphology.

Main Results:

• Observed significant, pH-dependent differences in the diameters of empty versus DNA-cargo-filled AAV8 VLPs.
• Detected VLP aggregation influenced by the electrolyte's pH, confirmed by AFM.
• Cryo-TEM revealed changes in particle shape related to cargo presence, not overall size.

Conclusions:

• Electrolyte pH is a critical parameter that must be closely monitored during VLP characterization using nES GEMMA.
• pH variations can significantly alter VLP behavior and physical characteristics.
• Extrapolation of VLP behavior from empty to filled particles requires caution due to cargo-dependent responses.