Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols.

Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (µsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4+ and CD8+ central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.