In the fascinating world of cellular biology, electroporation stands out as a technique that applies electric pulses to temporarily permeabilize cell membranes, allowing the introduction of molecules into cells. But what happens when this method intersects with extracellular vesicles (EVs), those tiny membrane-bound particles cells release to communicate and transport cargo? Recent research delves into electroporation induced changes in extracellular vesicle profile, revealing how this process can reshape these vesicles' characteristics. The vesicles extracellular profile, encompassing size, charge, concentration, and protein content, undergoes notable transformations, offering insights into biophysical dynamics. This blog explores these shifts through facts and figures from scientific studies, painting a picture of how electroporation influences EV behavior at a fundamental level.
Buffer Blues: The Pre-Electroporation Shake-Up
Before the electric pulses even hit, the mere act of suspending EVs in electroporation buffer (EB) triggers significant alterations. In a study using EVs from C2C12 murine myoblast cells, suspension in EB dropped EV concentration dramatically from 7.27 × 10^7 particles per milliliter in controls to 1.59 × 10^7 particles per milliliter. This represents a roughly 78% reduction, highlighting how the buffer's composition—often containing salts and sugars—can destabilize vesicle integrity. Particle size also swells; nanoparticle tracking analysis (NTA) showed an increase from 166.30 nanometers in native EVs to 258.70 nanometers in EB-suspended ones, a statistically significant jump (p = 0.0090). Dynamic light scattering corroborated this, measuring 173.30 nanometers for controls versus 265.22 nanometers post-suspension.
Zeta potential (ZP), a measure of surface charge, becomes less negative, shifting from -14.10 millivolts to -10.14 millivolts (p = 0.032), potentially affecting vesicle stability and interactions. Surface protein concentration dips too, from 2879 micrograms per milliliter to 2095 micrograms per milliliter (p = 0.0266). These changes suggest aggregation or fusion events, as the buffer's osmotic pressure might prompt vesicles to clump together. Interestingly, washing EVs after EB exposure doesn't restore the original profile; instead, it exacerbates issues, with size ballooning to 425.70 nanometers (p = 0.0041) and protein levels plummeting by 89.02% to 68.67 micrograms per milliliter (p < 0.0001). Polydispersity index, indicating size uniformity, rises from 0.32 to 0.82 (p = 0.0021), pointing to a more heterogeneous population.
Electric Jolt: Pulsing Through Profile Changes
When the pulses arrive, electroporation parameters like voltage, number of pulses, and pulse width further tweak the extracellular vesicles profile. Voltages of 500, 750, and 1000 volts don't significantly alter size but do impact other traits. At 1000 volts, ZP sees a 68.79% reduction, becoming more neutral (p = 0.0091). Pulse numbers show similar effects: one pulse (p = 0.0049) and three pulses (p = 0.0046) both lessen ZP negativity. Pulse widths of 10, 20, and 30 milliseconds all yield significant ZP shifts (p < 0.0001).
Surface proteins take a hit across parameters. At 500 volts (p = 0.0321), 750 volts (p = 0.0081), and 1000 volts (p = 0.0227), concentrations decrease. One pulse (p = 0.0229) and three pulses (p = 0.0020) follow suit, as do 10 milliseconds (p = 0.0220) and 20 milliseconds (p = 0.0197). Western blots reveal diminished levels of markers like Annexin A2 and CD9 in EB-treated samples. Polydispersity decreases at 500 volts (p = 0.0095), 20 milliseconds (p = 0.0085), and 30 milliseconds (p = 0.0350), suggesting some homogenization under specific conditions.
Clumping Conundrums: Aggregation in the Spotlight
Electroporation doesn't just tweak; it can cause outright aggregation. In research on exosomes from adipose-derived stem cells (ASCs), electroporation in cytomix buffer induced visible clumping, as seen in dynamic light scattering and electron microscopy. This aggregation increases vesicle size and may stem from membrane disruptions allowing fusion. Zeta potential reductions, making surfaces more neutral, facilitate this sticking together. Protein content drops, with western blots showing altered compositions. For instance, electroporation efficiencies for loading molecules remain low, under 5%, partly due to these aggregative effects. Such changes in vesicles extracellular profile could influence how EVs interact in experimental setups.
Cargo Shake-Up: Depletion and Selectivity Insights
Beyond external profiles, internal cargo feels the impact. Comparing electroporation to ultrasonication for cargo depletion, the latter outperforms in efficiency, as per high-throughput sequencing of proteins and small RNAs. Both methods preferentially deplete membrane proteins over cytoplasmic ones, with no strong selectivity for specific molecules. Electroporation induces micro-scale vesicle formation via membrane fusion or aggregation, altering overall profiles. This cargo loss might explain reduced protein concentrations observed externally.
Voltage Variations: Fine-Tuning the Electric Dance
Diving deeper into parameters, voltages from 500 to 1000 millivolts, pulse counts of 1 to 3, and widths of 10 to 30 milliseconds create a spectrum of effects. Higher voltages exacerbate ZP neutralization and protein loss, while pulse numbers amplify these trends. In ASC exosomes, these settings increased size, potentially hindering diffusion in dense environments. Buffers like trehalose-stabilized ones mitigate some damage, preserving structure better.
Fusion or Fiction: Exploring Membrane Dynamics
Electroporation's pores might lead to fusion events, merging EVs into larger entities. Studies note RNA precipitation and membrane morphology shifts post-treatment. Concentration reductions aren't fully recoverable, suggesting permanent profile remodeling. These dynamics enrich our understanding of EV resilience.
Electrifying Horizons: What This Means for Science
Electroporation induced changes in extracellular vesicle profile illuminate how external forces reshape cellular messengers. From buffer-induced size swells to pulse-driven charge neutralizations, facts like 78% concentration drops and 68.79% ZP reductions underscore the technique's potency. As researchers refine buffers and parameters, these insights could enhance EV manipulation in labs worldwide, sparking innovative approaches to studying vesicle biology.
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Reference:
1. Graybill, P., Jana, A., Kapania, R., Nain, A., & Davalos, R. (2020). Single cell forces after electroporation. Acs Nano, 15(2), 2554-2568. https://doi.org/10.1021/acsnano.0c07020
2. Lira, R., Dimova, R., & Riske, K. (2014). Giant unilamellar vesicles formed by hybrid films of agarose and lipids display altered mechanical properties. Biophysical Journal, 107(7), 1609-1619. https://doi.org/10.1016/j.bpj.2014.08.009
Poon, I., Parkes, M., Jiang, L., Atkin‐Smith, G., Tixeira, R., Gregory, C., … & Baxter, A. (2019). Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell‐derived extracellular vesicles in vitro. Journal of Extracellular Vesicles, 8(1). https://doi.org/10.1080/20013078.2019.1608786
