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.

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.

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.