Mindaugas S. Venslauskas

Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation

Introduction: Intracellular gene electrotransfer by means of electroporation has been on the increase during the past decade. Significant progress has been achieved both in characterizing mechanisms of gene electrotransfer and in optimizing the protocol in many preclinical trials. Recently this has led to initiation of clinical trials of gene electrotransfer to treat metastatic melanomas. Further progress with the method in various clinical trials requires better understanding of mechanisms of gene electrotransfer. Areas covered: A summary of recent progress in understanding mechanisms of gene electrotransfer, imparting general knowledge of cell electroporation and intracellular molecule electrotransfer. Expert opinion: Gene electrotransfer into cells and tissues is a complex process involving multiple steps that lead to plasmid DNA passage from the extracellular region to the cell nucleus crossing the barriers of the plasma membrane, cytoplasm and nucleus membrane. Electrical parameters of pulses used for gene electrotransfer affect the initial steps of DNA translocation through the plasma membrane and play a crucial role in determining the transfection efficiency. When considering gene electrotransfer into tissues it becomes clear that other nonelectrical conditions are also of primary importance.

Mechanisms of transfer of bioactive molecules through the cell membrane by electroporation

A short review of biophysical mechanisms for electrotransfer of biaoctive molecules through the cell membrane by using electroporation is presented. The concept of transient hydrophilic aqueous pores and membrane electroporation mechanisms of single cells and cells in suspension models are analyzed. Alongside the theoretical approach, some peculiarities of drug and gene electrotransfer into cells and applications in clinical trials are discussed.

Adjustment of ultrasound exposure duration to microbubble sonodestruction kinetics for optimal cell sonoporation in vitro.

Cell sonoporation enables the delivery of various exogenous molecules into the cells. To maximize the percentage of reversibly sonoporated cells and to increase cell viability we propose a model for implicit dosimetry for adjustment of ultrasound (US) exposure duration. The Chinese hamster ovary cell suspension was supplemented with microbubbles (MB) and exposed to US, operating at the frequency of 880 kHz, with a 100% duty cycle and with an output peak negative pressure (PNP) of 500 kPa for durations ranging from 0.5 to 30 s. Using diagnostic B-scan imaging we showed that the majority of the MB at 500 kPa US peak negative pressure undergo sonodestruction in less than a second. During this time maximal number of reversibly sonoporated cells was achieved. Increase of US exposure duration did not increase sonoporated cell number, however it induced additional cell viability decrease. Therefore aiming to achieve the highest level of reversibly sonoporated cells and also to preserve the highest level of cell viability, the duration of US exposure should not exceed the duration needed for complete MB sonodestruction.

Microbubble Sonodestruction Rate as a Metric to Evaluate Sonoporation Efficiency

The efficiency of sonoporation is directly related to microbubble cavitation and can be dependent on the microbubble sonodestruction rate. The objective of this study was to investigate whether the rate of microbubble sonodestruction can be used as a parameter to develop an implicit dosimetric method for sonoporation efficiency evaluation.

Analysis of Metrics for Molecular Sonotransfer in Vitro.

Ultrasound induced microbubble (MB) cavitation is used to significantly enhance cell membrane permeabilization, thereby allowing delivery of various therapeutic agents into cells. In order to monitor and quantitatively control the extent of cavitation the uniform dosimetry model is needed. In present study we have simultaneously performed quantitative evaluation of three main sonoporation factors: (1) MB concentration, (2) MB cavitation extent, and (3) doxorubicin (DOX) sonotransfer into Chinese hamster ovary cells. MB concentration measurement results and passively recorded MB cavitation signals were used for MB sonodestruction rate and spectral root-mean-square (RMS) calculations, respectively. Subsequently, time to maximum value of RMS and inertial cavitation dose (ICD) quantifications were performed for every acoustic pressure value. This comprehensive research has led not only to explanation of relation of ICD and MB sonodestruction rate but also to the development of a new sonoporation metric: the inverse of time to maximum value of RMS (1/time to maximum value of RMS). ICD and MB sonodestruction rate intercorrelation and correlation with DOX sonotransfer suggest inertial cavitation to be the key mechanism for cell sonoporation. All these metrics were successfully used for doxorubicin sonotransfer prediction (R(2) > 0.9, p < 0.01) and therefore shows feasibility to be applied for future dosimetric applications for ultrasound-mediated drug and gene delivery.

The Study of Biorhythms in Marine Colonial Hydroids

Biorhythmic activity of the colony of marine hydroids Podocoryne carnea was investigated. Quantitative characteristics were obtained as a result of the analysis of long-term (10–12 h) monitored stolon oscillations. Sliding window Fourier spectrum dynamics as well as the analysis of the system particular trajectories argue that the main feature of hydroid biorhythmic activity is transient chaos. Long-sustained cyclic activity of stolon pieces separated from the colony and the change in stolon oscillation amplitude and frequency after feeding suggest that stolon cyclic behaviour is determined by the network which consists of pacemakers residing in stolon wall cells and the amount and/or state of food particles inside the stolon lumen. The biorhythmic activity of colony from the point of chaos theory is discussed.

Nonlinear oscillations in marine hydroids

Irregular oscillations in a colony of marine hydroids Podocoryne carnea were investigated. Quantitative characteristics were obtained as a result of long term (10-12 h) monitoring of oscillations at arbitrary sites. The sliding window spectra as well as the pulse-to-pulse dynamics argue the transient chaotic behavior of hydroid colony. The significant change of amplitudes and frequencies in intact colony oscillations after feeding and long sustained oscillations of stolons separated from colony suggest that the irregular activity could be determined by the network of pacemakers residing in stolon wall cells. These are influenced mechanically by the amount of digesting food and/or by chemical action of nutrients inside the stolon lumen. The possible correlation of these oscillations which can evoke Ca(2+) waves in stolon wall cells is discussed.