The Assessment of Calcium and Bleomycin Cytotoxic Efficiency in Relation to Cavitation Dosimetry.

Microbubble (MB)- and ultrasound (US)-facilitated intracellular Ca2+ delivery, known as sonoporation (SP), is a promising anticancer treatment modality, since it allows a spatio-temporally controllable and side-effect-free alternative to conventional chemotherapy. The current study provides extensive evidence that a 5 mM concentration of Ca2+ in combination with US alone or US and Sonovue MBs can be an alternative to the conventional 20 nM concentration of the anticancer drug bleomycin (BLM). Ca2+ application together with SP induces a similar level of death in Chinese hamster ovary cells to the combination of BLM and SP but does not cause systemic toxicity, as is inherent to conventional anticancer drugs. In addition, Ca2+ delivery via SP alters three vital characteristics essential for viable cells: membrane permeability, metabolic activity and proliferation ability. Most importantly, Ca2+ delivery via SP elicits sudden cell death-occurring within 15 min-which remains similar during 24-72 h and 6 d periods. The extensive study of US waves side-scattered by MBs led to the quantification of the cavitation dose (CD) separately for subharmonics, ultraharmonics, harmonics and broadband noise (up to 4 MHz). The CD was suitable for the prognostication of the cytotoxic efficiency of both anticancer agents, Ca2+ and BLM, as was indicated by an overall high (R2 ≥ 0.8) correlation (22 pairs in total). These extensive analytical data imply that a broad range of frequencies are applicable for the feedback-loop control of the process of US-mediated Ca2+ or BLM delivery, successively leading to the eventual standardization of the protocols for the sonotransfer of anticancer agents as well as the establishment of a universal cavitation dosimetry model.

Microbial Biofuel Cells: Fundamental Principles, Development and Recent Obstacles.

This review focuses on the development of microbial biofuel cells to demonstrate how similar principles apply to the development of bioelectronic devices. The low specificity of microorganism-based amperometric biosensors can be exploited in designing microbial biofuel cells, enabling them to consume a broader range of chemical fuels. Charge transfer efficiency is among the most challenging and critical issues while developing biofuel cells. Nanomaterials and particular redox mediators are exploited to facilitate charge transfer between biomaterials and biofuel cell electrodes. The application of conductive polymers (CPs) can improve the efficiency of biofuel cells while CPs are well-suitable for the immobilization of enzymes, and in some specific circumstances, CPs can facilitate charge transfer. Moreover, biocompatibility is an important issue during the development of implantable biofuel cells. Therefore, biocompatibility-related aspects of conducting polymers with microorganisms are discussed in this review. Ways to modify cell-wall/membrane and to improve charge transfer efficiency and suitability for biofuel cell design are outlined.

Systemic Optimization of Gene Electrotransfer Protocol Using Hard-to-Transfect UT-7 Cell Line as a Model.

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Sudden Cell Death Induced by Ca2+ Delivery via Microbubble Cavitation.

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.

Polyethylene-Carbon Composite (Velostat®) Based Tactile Sensor.

The progress observed in 'soft robotics' brought some promising research in flexible tactile, pressure and force sensors, which can be based on polymeric composite materials. Therefore, in this paper, we intend to evaluate the characteristics of a force-sensitive material-polyethylene-carbon composite (Velostat®) by implementing this material into the design of the flexible tactile sensor. We have explored several possibilities to measure the electrical signal and assessed the mechanical and time-dependent properties of this tactile sensor. The response of the sensor was evaluated by performing tests in static, long-term load and cyclic modes. Experimental results of loading cycle measurements revealed the hysteresis and nonlinear properties of the sensor. The transverse resolution of the sensor was defined by measuring the response of the sensor at different distances from the loaded point. Obtained dependencies of the sensor's sensitivity, hysteresis, response time, transversal resolution and deformation on applied compressive force promise a practical possibility to use the polyethylene-carbon composite as a sensitive material for sensors with a single electrode pair or its matrix. The results received from experimental research have defined the area of the possible implementation of the sensor based on a composite material-Velostat®.

Correlation between the loss of intracellular molecules and cell viability after cell electroporation.

Control of membrane permeability to exogenous compounds by membrane electroporation can lead to cell death, which is related to permanent membrane damage, oxidation stress, leakage of intracellular molecules. In this study, we show that the predominant cell death modality after the application of high voltage electric pulses is related with inability to reseal of initial pores (first stage irreversible electroporation, FirEP). After moderately strong electric pulses, initial pores reseal, however, some cell still die later on due to electric field induced cell stress which leads to delayed cell death (late-stage irreversible electroporation, LirEP). According to our data, the period in which the majority of cells commit to either pore resealing or complete loss of barrier function depends on the intensity of electric field treatment but did not exceed 35 min. Additionally, we show that after electroporation using electric pulse parameters that induce LirEP, some cells can be rescued by supplementing medium with compounds obtained from irreversibly electroporated cells. We determined that the intracellular molecules that contribute to the increase of cell viability are larger than 30 kDa. This serves to prove that the loss of intracellular compounds plays a significant role in the decrease of cell viability after electroporation.

Calcein Release from Cells In Vitro via Reversible and Irreversible Electroporation.

The aim of this study was to investigate the dependence of calcein extraction and cell viability on the parameters of pulsed electric field (PEF). Two different approaches concerning PEF parameters were investigated: (1) extraction efficiency and cell viability dependence on pulse number, exploiting 1200 V/cm 100 µs duration high voltage (HV) electric pulses and (2) extraction efficiency and cell viability dependence on the pulses with different duration (44-400 µs) and electric field strength (600-1800 V/cm) that result in the same amount of electric field energy delivered to Chinese hamster ovary cells. Extraction efficiency was evaluated as a percentage ratio of calcein fluorescence intensity prior and after PEF treatment. Cell viability was evaluated using PI test and cell clonogenic assay. Moreover, calcein release dynamics from cells after 600 V/cm 400 µs, 1200 V/cm 100 µs, and 1800 V/cm 44 µs was evaluated. Our results show that HV pulses induce instant calcein extraction due to reversible electroporation; however, subsequent calcein leakage over time was only observed when 9 HV pulses of 1800 V/cm 44 µs were used. The increased number of pulses resulted in more efficient total calcein extraction. With the same total energy delivered via electric pulses, the increase of calcein extraction efficiency was more dependent on pulse strength rather than pulse duration. The highest calcein extraction efficiency (84.5 ± 7.4%) was obtained using 9 electric field pulses of 1800 V/cm, 44 µs at 1 Hz. Furthermore, the extraction efficiency can be significantly enhanced if external mechanical stress (pipetting) is applied to cells. Cell viability was determined to be dependent on different PEF exposure parameters. It varied from 96.8 ± 4.8 to 31.2 ± 8.9%, implying the possibility to adjust PEF parameter combinations to maintain high cell viability.

Investigation of Microbubble Cavitation-Induced Calcein Release from Cells In Vitro.

In the present study, microbubble (MB) cavitation signal analysis was performed together with calcein release evaluation in both pressure and exposure duration domains of the acoustic field. A passive cavitation detection system was used to simultaneously measure MB scattering and attenuation signals for subsequent extraction efficiency relative to MB cavitation activity. The results indicate that the decrease in the efficiency of extraction of calcein molecules from Chinese hamster ovary cells, as well as cell viability, is associated with MB cavitation activity and can be accurately predicted using inertial cavitation doses up to 0.18 V × s (R2 > 0.9, p < 0.0001). No decrease in additional calcein release or cell viability was observed after complete MB sonodestruction was achieved. This indicates that the optimal exposure duration within which maximal sono-extraction efficiency is obtained coincides with the time necessary to achieve complete MB destruction. These results illustrate the importance of MB inertial cavitation in the sono-extraction process. To our knowledge, this study is the first to (i) investigate small molecule extraction from cells via sonoporation and (ii) relate the extraction process to the quantitative characteristics of MB cavitation acoustic spectra.

Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro.

The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6-72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2-3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results.