The relation of Bleomycin Delivery Efficiency to Microbubble Sonodestruction and Cavitation Spectral Characteristics.

The concurrent assessment of principal sonoporation factors has been accomplished in a single systemic study. Microbubble sonodestruction dynamics and cavitation spectral characteristics, ultrasound scattering and attenuation, were examined in relation to the intracellular delivery of anticancer drug, bleomycin. Experiments were conducted on Chinese hamster ovary cells coadministered with Sonovue microbubbles. Detailed analysis of the scattering and attenuation temporal functions culminated in quantification of metrics, inertial cavitation dose and attenuation rate, suitable for cavitation control. The exponents, representing microbubble sonodestruction kinetics were exploited to derive dosimetric, microbubble sonodestruction rate. High intracorrelation between empirically-attained metrics defines the relations which indicate deep physical interdependencies within inherent phenomena. Subsequently each quantified metric was validated to be well-applicable to prognosticate the efficacy of bleomycin delivery and cell viability, as indicated by strong overall correlation (R2 > 0.85). Presented results draw valuable insights in sonoporation dosimetry and contribute towards the development of universal sonoporation dosimetry model. Both bleomycin delivery and cell viability reach their respective plateau levels by the time, required to attain total microbubble sonodestruction, which accord with scattering and attenuation decrease to background levels. This suggests a well-defined criterion, feasible through signal-registration, universally employable to set optimal duration of exposure for efficient sonoporation outcome.

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.

Intracellular Delivery of Bleomycin by Combined Application of Electroporation and Sonoporation in Vitro.

In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can result in an increased intracellular delivery of anticancer drug bleomycin. CHO cells were treated with electric pulses (1 or 8 high voltage pulses of 800 or 1200 V/cm, 100 µs or 1 low voltage pulse of 100 or 250 V/cm, 100 ms) and with 880 kHz US of 320 or 500 kPa peak negative pressure, 100 % duty cycle, applied for 2 s in the presence or absence of exogenously added contrast agent microbubbles. Various sequential or simultaneous combinations of EP and sonoporation were used. The results of the study showed that i) sequential treatment of cells by EP and sonoporation enhanced bleomycin electrosonotransfer at the reduced energy of electric field and US; ii) sequential combination of EP and sonoporation induced a summation effect which at some conditions was more prominent when the cells were treated first by EP and then by sonoporation; iii) the most efficient intracellular delivery of bleomycin was achieved by the simultaneous application of cell EP and sonoporation resulting in percentage of reversibly porated cells above the summation level; and iv) compared with sequential application of EP and sonoporation, simultaneous use of electric pulses and US increased cell viability in the absence of bleomycin.

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.

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

A short review of biophysical mechanisms for electrotransfer of bioactive 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.

Microbubble sonodestruction rate as a metric to evaluate sonoporation efficiency.

OBJECTIVES: 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. METHODS: To evaluate the rate of microbubble sonodestruction as a function of the ultrasound (US) peak negative ultrasound pressure, 12-MHz diagnostic US was used in the B-scan mode. Chinese hamster ovary cells were exposed to therapeutic US at 880 kHz in the absence or presence of microbubbles. The sonoporation efficiency was evaluated by the sonotransfer of bleomycin, a cytotoxic, membrane-impermeable anticancer drug. RESULTS: At a low microbubble sonodestruction rate of 1/τ < 0.5 second(-1) (τ providing the time necessary to decrease the microbubble concentration to 37% of its initial value), cell viability remained basically unaffected, but the percentage of sonoporated cells did not reach 10%. At higher microbubble sonodestruction rates, the efficiencies of irreversible and reversible sonoporation started to increase linearly and reached the plateau at 5 seconds(-1). CONCLUSIONS: These results show that the microbubble sonodestruction rate can be used to predict the percentage of reversible and irreversible sonoporation.

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.

Extent of cell electrofusion in vitro and in vivo is cell line dependent.

BACKGROUND: Electric pulses delivered to cells that are in close contact may induce cell fusion, by a process termed electrofusion. Recently it has been shown that electrofusion in tumours in vivo depends on tumour type. The aim of this work was to examine the differences in electrofusion in various cell lines, both in vivo and in vitro. MATERIALS AND METHODS: LPB, B16F10 and DC-3F cells in vitro and LLC tumours in vivo were exposed to various electric pulses. The number of fused cells was then evaluated. RESULTS: Cell electropermeabilization was confirmed to be a necessary but non-exclusive condition to obtain a high level of cell electrofusion. The extent of electrofusion depends both on the degree of permeabilization and cell type. CONCLUSION: It was observed that metastatic tumour cells easily electrofuse, suggesting that cell type-specific membrane properties and/or secretion of proteases determine the extent of electrofusion.

Enhancement of photodynamic tumor therapy effectiveness by electroporation in vitro.

The aim of our study was to determine if electroporation could improve the efficacy of photodynamic tumor therapy. A disadvantage of photodynamic therapy is a slow and in some cases insufficient accumulation of photosensitizer in tumor tissue, which could restrict the achievement of an efficient dose. Under the action of electric pulses, cells undergo membrane electroporation, which results in an increased permeability to various exogenous molecules. In this study, murine hepatoma MH22A cells were exposed to light in vitro in the presence of a photosensitizer, either chlorin e6 or aluminum phthalocyanine tetrasulfonate, following electroporation. Accumulation of the photosensitizers was registered by fluorescence microscopy. Cell viability was determined by the MTT assay. Our results demonstrate that electroporation improves an access of chlorin e6 and aluminum phthalocyanine tetrasulfonate to MH22A cells. Electroporation in combination with photosensitization significantly reduces viability of the treated cells even at low doses of photosensitizers.

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.

[Histological analysis of electrochemotherapy influence in Lewis lung carcinoma]. / Epidermoidines plauciu karcinomos elektrochemoterapinio poveikio histologinis tyrimas.

Under the influence of strong electric fields the permeability of tumor cell membranes to poor permeating drugs increases and as a result the tumor growth is inhibited. This new tumor treatment method is named electrochemotherapy. We investigated the electrochemotherapy influence of bleomycin upon mice Lewis Lung Carcinoma by using optical histological and electron microscopic tumor analysis. It was shown that due to electrochemotherapy tumor necrosis area is significantly increased, intercellular gaps enlarge, and a big amount of cells is destroyed. Cell and nucleus membranes break as well as hemorrhage are very often. The observable histological tumor change was noted when electrical pulses of 1300 V/cm and 0.1 ms duration were applied. Pulses of 1700 V/cm and 0.1 ms duration induce total tumor destruction.