Unraveling the response of a biomimetic actin cortex to electric pulses in vesicles

Perrier, Dayinta L.; Vahid, Afshin; Kathavi, Vaishnavi; Stam, Lotte; Rems, Lea; Mulla, Yuval; Muralidharan, Aswin; Koenderink, Gijsje H.; Kreutzer, Michiel T.; Boukany, Pouyan E.

doi:10.1101/338566

Cited by 2 publications

(1 citation statement)

References 66 publications

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“…Collectively, these results support that oscillatory mechanical stimulation during MagNEI promoted actin remodeling and accelerated membrane repair, consistent with a recent finding that polymerized actin network could slow down membrane resealing after electroporation. [61] 50/100% of cholesterol from cells was also depleted by adding methyl-β-cyclodextrins (MβCD) for 2/10 min [62] but mechanical stimulation did not influence cholesterol recruitment (Figure 4b) and biosynthesis.…”

Section: Mechanical Stimulation Accelerated Cell Membrane Repair To Reduce Perturbationmentioning

confidence: 99%

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities

Tay

Melosh

2021

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that produces transient gene expression which necessitates repeated, expensive doses for treatment to work. [1] However, transfection using viruses, biochemicals, and bulk electroporation warrant considerable concerns due to high costs, low efficiency, limited cell types, high cell death, and toxic immune reactions. For instance, the size of cargo that can be delivered by most viral vectors is typically restricted to 5-10 kbp, although the use of retroviruses [2] and viral engineering could overcome this limitation. This method also faces safety concerns as immunogenic viral genetic elements can be randomly integrated into host genome, necessitating precautionary and costly long-term patient follow-up. [3] During bulk electroporation, the most popular non-viral method, inhomogeneous electric fields can cause Joule heating and bubble formation that dramatically reduce transfection efficiency and cell viability. [4] The longer time needed for cell manufacturing means delayed and costlier treatments for patients. [5] Advances in flow electroporation has also made this process less biologically perturbative as the cells are only briefly treated with electric fields. Such flow-based electroporation systems can also be scaled up to process billion of cells unlike conventional cuvette-based bulk electroporation platforms which have lower throughput. [6][7][8] The limitations of viral and bulk electroporation have motivated the development of non-viral micro and nano cell transfection technologies. [9] Microfluidic platforms coupled with mechanisms including cell squeezing, [10] micro-injection, [11] shear stresses, [12] deterministic mechanoporation, [13] and vortex shedding [14] have been created for high throughput cell transfection. While excellent performance has been achieved with immune cell lines like Jurkat, few papers have reported success for engineering primary immune cells, except for a handful of publications. [13,[15][16][17] The use of highly concentrated, expensive cargo freely floating in microfluidic channels can also quickly increase operating cost. Clogging of microfluidic channels can too disrupt high throughput cell processing.Nanoparticles with their programmable properties are attractive tools for cargo delivery. Smith et al. made use of nanoparticles to deliver DNA [18] and mRNA [19] for reprogramming T cells. McKinlay et al. also created a library of amphiphilic Transfection is an essential step in genetic engineering and cell therapies. While a number of non-viral micro-and nano-technologies have been developed to deliver DNA plasmids into the cell cytoplasm, one of the most challenging and least efficient steps is DNA transport to and expression in the nucleus. Here, the magnetic nano-electro-injection (MagNEI) platform is described which makes use of oscillatory mechanical stimulation after cytoplasmic delivery with high aspect-ratio nano-structures to achieve stable (>2 weeks) net transfection efficiency (efficiency × viability) of 50% in primary human T cells. This is, to the best of the a...

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Section: Mechanical Stimulation Accelerated Cell Membrane Repair To Reduce Perturbationmentioning

confidence: 99%

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities

Tay

Melosh

2021

Small

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Response of an actin network in vesicles under electric pulses

Perrier

Vahid

Kathavi

et al. 2019

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We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

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Unraveling the response of a biomimetic actin cortex to electric pulses in vesicles

Cited by 2 publications

References 66 publications

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities

Mechanical Stimulation after Centrifuge‐Free Nano‐Electroporative Transfection Is Efficient and Maintains Long‐Term T Cell Functionalities

Response of an actin network in vesicles under electric pulses

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