Electroporation is an effective method for delivering plasmid DNA molecules into cells. The efficiency of gene electrotransfer depends on several factors. To achieve high transfection efficiency while maintaining cell viability is a tedious task in electroporation. Here, we present a combined study in which the dynamics of both evaluation types of transfection efficiency and the cell viability were evaluated in dependence of plasmid concentration as well as at the different number of high voltage (HV) electric pulses. The results of this study reveal a quantitative sigmoidal (R² > 0.95) dependence of the transfection efficiency and cell viability on the distance between the cell membrane and the nearest plasmid. We propose this distance value as a new, more accurate output parameter that could be used in further optimization studies as a predictor and a measure of electrotransfection efficiency.

Electroporation—a transient electric-field-induced increase in cell membrane permeability—can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca²⁺ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl₂ effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl₂ concentration is increased.

Intracellular calcium ion delivery via sonoporation has been validated to be a substitute for classical chemotherapy. However, the mechanism behind calcium sonoporation remains unclear to this day. To elucidate the role of calcium in the process of sonoporation, we aimed to investigate the influence of different calcium concentration on cell membrane permeabilization and cell viability after sonoporation. In this study, we present experimental evidence that extracellular calcium plays a major role in cell membrane molecular transport after applying ultrasound pulses. Ultrasound-microbubble cavitation in the presence of different calcium concentration affects fundamental cell bio-physio-chemical conditions: cell membrane integrity, metabolic activity, and colony formation. Corresponding vital characteristics were evaluated using three independent viability tests: propidium iodide assay (20 min–3 h), MTT assay (48 h), and cell clonogenic assay (6 d). The results indicate instant cell death, as the level of cell viability was determined to be similar within a 20 min–48 h–6 d period. Inertial cavitation activities have been determined to be directly involved in calcium delivery via sonoporation according to high correlation (R² > 0.85, p < 0.01) of inertial cavitation dose with change in either cell membrane permeabilization, metabolic activity, and colony formation efficiency. In general, calcium delivery via sonoporation induces rapid cell death, occurring within 20 min after treatment, that is the result of ultrasound mediated microbubble cavitation.

Gene electrotransfer (GET) is recognized as a promising technique for the development of an efficient tool for gene therapy. Such a therapy would have applications in the treatment of a variety of genetic diseases, including cancer. However, despite its wide applicability, the technique is limited by the lack of understanding of the fundamental mechanism of electroporation as well as other important factors that directly or indirectly influence its success rate. In the current study, we analyzed the impact of low concentrations (0–1 mM) of Ca²⁺ on the process of DNA electrotransfer using flow cytometry. The results revealed that the presence of a CaCl₂ concentration as low as 0.25 mM decreased the efficiency of GET by ~1.5-fold and cell viability decreased by ~2–3-fold. In addition, we determined that the observed phenomenon of the decrease in pDNA electrotransfer due to the influence of Ca²⁺ was not the consequence of cell death but rather should be attributed to secondary mechanisms. The data presented in this study provide an insight into the importance of Ca²⁺ in the process of gene electrotransfer that may be directly applicable to in vivo settings.

Study advances current diagnostic efficiency of canine/feline (sub-)cutaneous tumors using machine learning and multimodal imaging data. White light (WL), fluorescence (FL) and ultrasound (US) imaging were combined into hybrid approaches to differentiate between malignant mastocytomas, soft tissue sarcomas and benign lipomas. Support Vector Machine and Ensemble classifiers were optimized via sequential feature selection. US radio-frequency signals were quantitatively analyzed to derive the colormaps of six US estimates, corresponding to spectral and temporal domains of the acoustic field. This resulted in the quantification of 72 morphological features for US; as well as 24 and 12 - for WL and FL data, respectively. Resulting classification efficiency for mastocytoma and sarcoma using US data was >75%; US+FL - 75-80%; US+WL - 85-90% and US+OPTICS - 90-95%. ∼100% classification efficiency was achieved for the differentiation between benign and malignant tumors even using single WL feature for Ensemble classifier. US features, resulting in inferior classification efficiency, were competitive to superior optical, as they were selected during optimization to be added to or replace optical counterparts. Additional tissue differentiation was performed on z-stacks of US colormaps, obtained using 3D arrays of US radio-frequency signals. This resulted in ∼70% differentiation efficiency for mastocytoma and sarcoma as well as >95% for benign and malignant tissues. The obtained additional metric of classification efficiency provides complementary diagnostic support, which for Support Vector Machine can be expressed as: 90.3 ± 1.9% (US+WL)×71.2 ± 0.6% (US_{Depth Profile}). This hybrid criterion adds robustness to diagnostic model and may be very beneficial to characterize heterogeneous tissues.