Assessing cell viability and electrotransfer efficiency in the concurrent and separate delivery of proteins, small fluorescent molecules, and nucleic acids using varying high-voltage pulse numbers

Abstract

Introducing proteins into living cells via electroporation (EP) could be used for CRISPR-Cas9 gene editing, cell therapy, and intracellular sensing [1], but theoretical model for their delivery in vitro is yet to be established [2]. To gain more insightinto how protein behaves duringEP, its potential synergies and interferences with other migrating molecules, such as small fluorophores and nucleic acids, could be observed: its co-delivery with small fluorophores, like propidium iodide (PI), may allow to monitor membrane integrity in real time, whilst concurrent electrotransfer with nucleic acids may be applied for more complex experimental designs, involving Cas9 with guide RNA. Due to proteins' size and charge, their migration through small electropores may be impeded [3,4]. Adjusting pulse numbers might improve their uptake by providing more time for their entry, however it should be optimized in terms of cell viability and its ability to maintain the cargoes for longer periods. The aim of this study is to determine how the efficiency of concurrent and separate delivery of proteins, PI, and pDNA into mammalian cells and their viability depends on different high-voltage (HV) pulse numbers. Chinese Hamster Ovary (CHO) cells were suspended in low-conductivity EP medium (0.2 S/m, 270 mOsm, pH 7.2) with bovine serum albumin (BSA) Alexa Fluor 488 and PI or far-red (633 nm excitation, 675/25 nm emission) fluorescent protein mCardinal-encoding plasmid(pRFP), and then received 1,9,or17HVpulses(1Hz,1000V/cm,60μs).Controls were not exposed to HV pulse. BSA+ (488 nm and 530/30 nm, respecitvely) and PI+ (488 nm and 585/40 nm, respectively) cell percentages were measured 30 min post-EP using BD Accuri™ C6 flow cytometer. BSA and pRFP uptake was assessed via flow cytometry and ZEISS Axio Observer 7 microscope followed by cell viability determined by MTT-PMS test 24 h post-EP. Data (mean ± SD) were analyzed with a one-way Bonferroni test (p<0.05, n=3). In accordance with the results (Fig. 1 A-D), BSA and PI uptake showed a dose-response to the number of HV pulses, however at 9-17 HV pulses PI+ cell percentage decreased (p<0.05), indicating that while more HV pulses increase membrane permeability, the BSA limits the PI entry. Comparably, with higher HV pulses BSA and pRFP uptake also increased with more pulses 24 h after procedure (p<0.05), however pRFP uptake was smaller in the BSA-supplemented samples suggesting that cells may prioritize survival over producing new proteins (RFP) during electrotransfection. Based on MTT-PMS assay, cell viability decreased with the increasing number of pulses, with the lowest viability (27.28%) observed when both BSA and pRFP were introduced together at 17 HV pulses (p<0.05). This implies a larger disruption of membrane integrity and increased stress when managing multiple macromolecules at once. [...]