Multimodal Approach of Optical Coherence Tomography and Raman Spectroscopy Can Improve Differentiating Benign and Malignant Skin Tumors in Animal Patients.

As in humans, cancer is one of the leading causes of companion animal mortality. Up to 30% of all canine and feline neoplasms appear on the skin or directly under it. There are only a few available studies that have investigated pet tumors by biophotonics techniques. In this study, we acquired 1115 optical coherence tomography (OCT) images of canine and feline skin, lipomas, soft tissue sarcomas, and mast cell tumors ex vivo, which were subsequently used for automated machine vision analysis. The OCT images were analyzed using a scanning window with a size of 53 × 53 µm. The distributions of the standard deviation, mean, range, and coefficient of variation values were acquired for each image. These distributions were characterized by their mean, standard deviation, and median values, resulting in 12 parameters in total. Additionally, 1002 Raman spectral measurements were made on the same samples, and features were generated by integrating the intensity of the most prominent peaks. Linear discriminant analysis (LDA) was used for sample classification, and sensitivities/specificities were acquired by leave-one-out cross-validation. Three datasets were analyzed-OCT, Raman, and combined. The combined OCT and Raman data enabled the best sample differentiation with the sensitivities of 0.968, 1, and 0.939 and specificities of 0.956, 1, and 0.977 for skin, lipomas, and malignant tumors, respectively. Based on these results, we concluded that the proposed multimodal approach, combining Raman and OCT data, can accurately distinguish between malignant and benign tissues.

Dosimetric assessment of antitumor treatment by enhanced bleomycin delivery via electroporation and sonoporation.

Targeted and controlled techniques of intratumoral delivery of chemotherapeutic agents are under extensive development, since they diminish detrimental side-effects of conventional anticancer drugs. We investigated the effectiveness of chemotherapy using bleomycin (1 mg/ml) and Sonovue microbubbles combined with: electroporation (EP), mainly designed for subcutaneous tumor therapy, and sonoporation (SP) - for deeper localized tumors. Research was performed on hepatoma MH-22A tumors in murine models, exposed to EP or SP and combined (EP + SP) treatment. Animal survival time and the rate/ speed of tumor growth reduction were examined. Study demonstrated that both EP or SP and their combination (EP + SP) were able to induce the reduction of tumor volume from the 3rd day after treatment. The employment of EP before SP allowed to significantly reduce the values of inertial cavitation dose (ICD), necessary to induce complete tumor reduction and prolong animal survival. The analysis of ultrasound (US) side-scattered signals and B-scan imaging indicated the occurrence of inertial cavitation at our experimental conditions. Strong (R2 = 0.88; p < 0.0001) correlation of ICD with the survival time of corresponding unrecovered mice indicates the option for the dosimetric control and standardization of cavitation activities for in vivo practice.

A Setup for Microscopic Studies of Ultrasounds Effects on Microliters Scale Samples: Analytical, Numerical and Experimental Characterization.

Sonoporation is the process of cell membrane permeabilization, due to exposure to ultrasounds. There is a lack of consensus concerning the mechanisms of sonoporation: Understanding the mechanisms of sonoporation refines the choice of the ultrasonic parameters to be applied on the cells. Cells' classical exposure systems to ultrasounds have several drawbacks, like the immersion of the cells in large volumes of liquid, the nonhomogeneous acoustic pressure in the large sample, and thus, the necessity for magnetic stirring to somehow homogenize the exposure of the cells. This article reports the development and characterization of a novel system allowing the exposure to ultrasounds of very small volumes and their observation under the microscope. The observation under a microscope imposes the exposure of cells and Giant Unilamellar Vesicles under an oblique incidence, as well as the very unusual presence of rigid walls limiting the sonicated volume. The advantages of this new setup are not only the use of a very small volume of cells culture medium/microbubbles (MB), but the presence of flat walls near the sonicated region that results in a more homogeneous ultrasonic pressure field, and thus, the control of the focal distance and the real exposure time. The setup presented here comprises the ability to survey the geometrical and dynamical aspects of the exposure of cells and MB to ultrasounds, if an ultrafast camera is used. Indeed, the setup thus fulfills all the requirements to apply ultrasounds conveniently, for accurate mechanistic experiments under an inverted fluorescence microscope, and it could have interesting applications in photoacoustic research.

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.

Autofluorescence imaging for recurrence detection in skin cancer postoperative scars.

This clinical study is a first attempt to use autofluorescence for recurrence diagnosis of skin cancer in postoperative scars. The proposed diagnostic parameter is based on a reduction in scar autofluorescence, evaluated in the green spectral channel. The validity of the method has been tested on 110 postoperative scars from 56 patients suspected of non-melanoma skin cancer, with eight patients (13 scars) available for the repeated examination. The recurrence diagnosis within a scar has been made after two subsequent autofluorescence check-ups, representing the temporal difference between the scar autofluorescence amplitudes as a vector. The recognition of recurrence has been discussed to represent the significant deviations from the value of vector angle θ. This new autofluorescence-based method can be easily integrated into the postoperative monitoring of surgical scars and can help diagnose the recurrence of skin cancer from the early stage of scar development.

Physical Methods for Drug and Gene Delivery Through the Cell Plasma Membrane.

The cell membrane represents a major barrier for efficient delivery of exogenous molecules, either pharmaceuticals or genetic material, under both in vitro and in vivo conditions. The number of methods employed to attempt safe, efficient, and local drug and gene delivery has increased during the recent years. One method for membrane permeabilization, electroporation, has already been translated to clinical practice for localized anticancer drug delivery and is termed "electrochemotherapy". Clinical trials for gene delivery using electroporation as well as drug delivery using another cell permeabilization method, sonoporation, are also underway. This review focuses on these two methods, including their fundamental principles and state-of-the-art applications. Other techniques, such as microinjection, magnetoporation, photoporation, electrospray, and hydrodynamic and ballistic gene delivery, are also discussed.

FRET-based method for evaluation of the efficiency of reversible and irreversible sonoporation.

It is widely known that not all of the treated cells survive after introduction of exogenous molecules via any physical method. Therefore, it is important to develop methods that would allow simultaneous evaluation of both molecular delivery efficiency and cell viability. This study presents Förster resonance energy transfer (FRET)-based method that allows molecular transfer and cell viability evaluation in a single measurement by employing two common fluorescent dyes, namely, ethidium bromide and trypan blue. The method has been validated using cell sonoporation. The FRET-based method allows the efficiency evaluation of both reversible and irreversible sonoporation in a single experiment. Therefore, this method could be used to reduce time, labor, and material cost while improving the accuracy of evaluations.

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.

Noninvasive optical diagnostics of enhanced green fluorescent protein expression in skeletal muscle for comparison of electroporation and sonoporation efficiencies.

We highlight the options available for noninvasive optical diagnostics of reporter gene expression in mouse tibialis cranialis muscle. An in vivo multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) expression, providing information on location and duration of EGFP expression and allowing quantification of EGFP expression levels. For EGFP coding plasmid (pEGFP-Nuc Vector, 10 µg/50 ml 10 µg/50 ml ) transfection, we used electroporation or ultrasound enhanced microbubble cavitation [sonoporation (SP)]. The transcutaneous EGFP fluorescence in live mice was monitored over a period of one year using the described parameters: area of EGFP positive fibers, integral intensity, and mean intensity of EGFP fluorescence. The most efficient transfection of EGFP coding plasmid was achieved, when one high voltage and four low voltage electric pulses were applied. This protocol resulted in the highest short-term and long-term EGFP expression. Other electric pulse protocols as well as SP resulted in lower fluorescence intensities of EGFP in the transfected area. We conclude that noninvasive multispectral imaging technique combined with fluorescence spectroscopy point measurements is a suitable method to estimate the dynamics and efficiency of reporter gene transfection in vivo.

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.

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.

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.

Evaluation of ischemic injury of the cardiac tissue by using the principal component analysis of an epicardial electrogram.

Monitoring and control of the heart tissue viability is of crucial importance during heart surgery operations. In most cases the heart tissue suffers from an ischemic injury that causes a decrease in the velocity of electrical excitation propagation in it and influences the shape of the excitation wave front that spreads over the injured area. It is reflected in a more complex shape of the registered epicardial electrogram as compared to normal. A method for quantitative evaluation of the complexity of the shape of the epicardial electrogram based on the principal component analysis is here proposed for evaluation of the ischemic injury of the cardiac tissue. A minimal, yet sufficient, number of the principal components (the optimal basis functions) for truncated expansion of the epicardial electrogram signals could be used as an estimate of signal complexity. The method for determination of such a minimal, yet sufficient, number of principal components were developed by using epicardial electrograms registered during in situ experiments on dogs in which local ischemia was evoked by ligation of a coronary vessel.

Autofluorescence of transplantable hepatoma A22 (MH-A22): prospects of tumor tissue optical biopsy.

AIM: Autofluorescence of experimental tumor (hepatoma A22 (MH-A22)) was employed to discriminate the optical differences between necrotic and non-necrotic tumor, hemorrhagic tumor and healthy tissue. METHODS: The experiment was performed ex vivo using the transplantable tumor from the right haunch of hybrid mice (C57Bl/CBA). Blue LED light (lambda em=405 nm) was applied for autofluorescence excitation and fibre optics based spectrofluorimeter was used for spectra detection. RESULTS: We observed that necrotic tumor tissue is characterized by the absence of endogenous porphyrins fluorescence, and registered spectra do not possess differences in the red spectral region (600-710 nm) in comparison with normalized autofluorescence spectra of muscle. Moreover, only certain segments of non-necrotic tumor bear the fluorescence of endogenous porphyrins. CONCLUSIONS: Based on the experimental results, we suggest that the absence of long-waved fluorescence differences between necrotic tumor tissue and healthy tissue, e.g. muscle can impede the demarcation between healthy and tumor tissue. The uneven distribution of endogenous porphyrins in non-necrotic tumor tissue as well as the absence of endogenous porphyrins fluorescence in the small experimental tumors complicates the localization of cancerous tissue based on the autofluorescence registration.