Molecular biology research often involves the introduction of nucleic acids into eukaryotic cells through transfection, which requires appropriate methods depending on cell type and research objectives. Each method has varying levels of transfection efficiency and cell toxicity [14]. However, the ideal method should exhibit a high transfection efficiency with minimal toxicity. Optimization is often necessary to determine optimal transfection conditions [15]. The Vero cell line plays a crucial role in vaccine research; however, an optimized transfection protocol for this cell type is currently lacking. Several factors must be addressed to achieve optimal transfection, including intracellular uptake, endosome acidification, autophagy, immune sensing pathways, and nuclear entry [16]. Numerous factors can influence transfection outcomes. Our focus was to introduce an optimal transfection method for the Vero cell line, which involved careful selection and fine-tuning of the transfection reagent as well as the development of an optimized protocol. In addition, factors such as cell density, incubation time, and the reagent-to-DNA ratio should be considered. Systematically evaluating and adjusting these variables can significantly improve the transfection efficiency in Vero cells, resulting in more reliable and reproducible experimental results [17].

The success of non-viral DNA delivery methods relies on various factors, including the ability of DNA to evade lysosomal degradation, efficient nuclear translocation, decreased cytotoxicity, better control of molecular composition, flexibility in gene size, and lower immunogenicity compared to viral analogs [18]. Studies have shown that lysosomal degradation can be overcome through strategies such as the proton sponge effect in certain cationic polymers, such as PEI. However, efficient nuclear targeting remains a significant challenge during the transfection process. Transfection outcomes can be significantly influenced by factors, such as cell membrane composition and pH, as highlighted by Kim and Eberwine [5].

In this study, we investigated the optimal transfection conditions for the Vero cell line using various chemical transfection agents, including Lipofectamine™ 2000, TurboFect™, X-tremeGENE™ 9, and PEI MAX®. We tested different ratios of DNA and chemical transfection reagents to determine the most effective combination. Our results showed that using 1 µg of each plasmid with 4 µL of TurboFect™ in 6 × 104 cells achieved the highest transfection efficiency (46.5%) and cell viability (94%) compared to other reagents, such as Lipofectamine™ 2000 (42% transfection efficiency and 94% cell viability).

By determining the optimal ratio of reagent to DNA, researchers can enhance the success of transfection experiments while minimizing any negative effects on cell viability. TurboFect™ is a cationic polymer that forms complexes with nucleic acids, thereby facilitating cell attachment, internalization, and endosomal escape. It is particularly suitable for transecting primary cells, difficult-to-transfect cells, and other cell types [19]. Lipofectamine is a cation-lipid-based transfection reagent that forms liposomes to deliver nucleic acids to cells. The composition and structure of liposomes can affect the efficiency and effectiveness of transfection experiments [20].

TurboFect™ has a lower toxicity than Lipofectamine, which contributes to its higher transfection efficiency and cell viability. Choosing TurboFect™ allowed researchers to achieve a balance between effective transfection and high cell viability in Vero cells. In a similar study, CHO-K1 and SH-SY5Y cell lines were transfected with chemical transfection reagents including Lipofectamine 2000, TurboFect 8.0, and ExGen 500. The results showed that transfection efficiency with TurboFect reagent was significantly higher in SH-SY5Y cells compared to the other two types of chemical transfection reagents, and was approximately the same in CHO-K1 cells. The results of this study are consistent with the results of our study and may indicate the success of TurboFect in optimal DNA delivery in other cell lines [21]. We observed a lower transfection efficiency with X-tremeGENE™ 9 and PEI MAX®. PEI, a cationic polymer, exhibited higher cytotoxicity. It can induce toxicity by depolarizing mitochondria and stimulating immune responses. High-molecular-weight PEI (HMW PEI) can form stable polyplexes, but its non-cleavable structure increases its cytotoxicity [22]. PEI– DNA complexes can activate genes involved in cellular responses including apoptosis, stress responses, and oncogenesis [23].

To reduce the cytotoxicity of PEI, researchers can use degradable disulfide-containing polymers, which increase the disassembly rate of the complexes and reduce their binding affinity with intracellular membranes [24]. In addition, free cationic PEI chains embedded in the cell membrane can enhance transfection efficiency by destabilizing the endosomal membrane and hindering its fusion with lysosomes [25].

Overall, our study highlights the importance of selecting an appropriate transfection reagent and the ratio of reagent to DNA to optimize transfection efficiency and maintain cell viability in Vero cells.

Electroporation transfection is a physical method used to introduce external substances into the cells. It involves subjecting cell membranes to high-voltage pulses, which creates tiny pores that allow for the insertion of exogenous genes [26]. Several factors influence the transfection efficiency and cell viability during electroporation. These factors include the electric field strength (voltage), pulse interval (capacitance flow rate), temperature, buffer composition, cell state and volume, and the concentration and structure of foreign genes [27]. Among these factors, electric field strength (voltage) and buffer composition are particularly important in determining transfection efficiency.

In this study, we aimed to identify the optimal electroporation conditions by testing three different electric field intensities (200, 300, and 400 V) and three different buffers with varying ingredients on Vero cell lines. The results showed that all three buffers exhibited the highest transfection efficiency at 300 V, with an electric pulse interval of 850 µF in square waves at all voltages. The cells demonstrated varying levels of adaptation to both electric field intensity and pulse interval. If the voltage is below optimal levels or the electroporation conditions are inadequate for a specific cell strain, no changes occur in the cell membranes, hindering the entry of exogenous genes and reducing the transfection efficiency [28]. Conversely, excessive voltage leads to irreversible cell damage, which significantly affects both cell survival and transfection efficiency [29]. In a similar study investigating the optimal conditions for electroporation of skeletal muscle satellite cells, it was demonstrated that increasing voltage intensity resulted in a decrease in electroporation efficiency and cell viability. DNA dosage was also found to have a significant impact on electroporation success. The optimal voltage at which the highest electroporation efficiency and cell viability were observed varied depending on the cell type, highlighting the importance of determining the optimal electroporation conditions for each cell type [30].

In addition, the results demonstrated that Ebuffer 3 exhibited higher transfection efficiency (38.6%) than the other buffers, and this increase was statistically significant. The composition of the buffer significantly influenced the transfection efficiency. Using RPMI-1640 medium as a shock buffer simplifies this process and reduces cell damage and death after transfection. RPMI-1640 without serum and antibiotics showed higher cell survival after shock than other buffers [10]. Transfection and viability remained unaffected when RPMI-1640 was used as the transfection buffer, even for cells cultured in different media [31].

However, under optimal transfection conditions, the cell viability after electroporation was 56%, which was significantly lower than that of the control. One notable limitation of the electroporation transfection technique is its inherent cellular toxicity, which can range from 50 to 90%. In general, electroporation maintains viability within the range of 30–40% and can be further optimized to maximize transfection efficiency.

HIV-1 lentiviral vectors are commonly used for efficient gene delivery and long-term genetic modification in various cell types, including dividing and non-dividing cells [32]. These vectors offer high titers and low risk of generating replication-competent retroviruses, making them a safe and powerful tool for gene transfer [33]. However, our research indicates that lentivirus particles have a low transduction efficiency in Vero cells, even at high titers, with a reported maximum efficiency of 20%. These findings align with those of previous studies, suggesting that the low transduction efficiency in Vero cells may be due to post-entry restrictions against HIV-1. In particular, innate cellular responses in Vero cells can impede the uncrating process of the virion capsid structure, primarily through the activity of TRIM5 protein isoforms, which possess a defense mechanism against the virus known as ubiquitin ligase activity [34]. Based on these findings, HIV-1 lentiviral vectors are not suitable for efficient transduction of Vero cells. Therefore, alternative lentiviruses derived from different viruses or alternative transfection methods should be considered to achieve optimal and efficient gene transfer into Vero cells [35].

Additionally, we compared various transfection methods to identify the optimal conditions for transfection in Vero cells. The results demonstrated that TurboFect™, a chemical transfection reagent, exhibited the highest transfection efficiency in Vero cells. Non-viral delivery methods, such as chemical transfection, offer advantages over viral vector delivery, including reduced immunogenicity and a lower risk of insertional mutagenesis [36]. Statistically, TurboFect™ significantly outperformed other chemical transfection methods such as electroporation and transduction using lentiviral vectors. Electroporation, although effective for gene delivery, resulted in a high rate of cell death, which negatively affected transfection efficiency. In contrast, transduction using lentiviral vector-based HIV-1 showed low efficiency in Vero cells because of the presence of an intracellular inhibitor for HIV-1 integration [37]. It can be presumed that the high efficiency of TurboFect™ in creating optimal conditions in Vero cells is attributed to the formation of liposome particles, their successful transfer through the lipid membrane, evasion from degradation by lysosomes, and efficient nuclear translocation.