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Microbubble (MB)-assisted ultrasound (US) is an innovative modality for the non-invasive, targeted, and efficient delivery of therapeutic molecules into the brain. Previously, we reported the first metabolomic signature of blood-brain barrier opening (BBBO) induced by MB-assisted US. In the present study, the neurometabolic consequences of acoustically-mediated BBBO on cerebral tissue were investigated using multimodal metabolomics approaches. Sinusoid US waves (1 MHz, peak negative pressure 0.6 MPa, burst length 10 ms, total treatment time 30 s, MB bolus dose 0.7 × 10 5 MBs/g) were applied on the rats' right striatum (ipsilateral side). Brain was collected and both striata were then dissected 3 h, 2 days, and 1 week after BBBO. After tissue preparation, the samples were analyzed using nuclear magnetic resonance spectrometry (NMRS) and high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Our findings showed a slight disruption of metabolic pathways in contralateral striata of animals. Analyses of metabolic pathways indicated changes in amino acid metabolisms. In addition, tryptophan derivate dosages revealed the perturbation of a central metabolite of the kynurenine pathway (i.e., 3-hydroxy-kynurenine). In conclusion, the acoustically-mediated BBBO of the ipsilateral cerebral hemisphere induced significant change in metabolism of contralateral one., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Presset, Bodard, Lefèvre, Millet, Oujagir, Dupuy, Iazourène, Bouakaz, Emond, Escoffre and Nadal-Desbarats.)

The blood-brain barrier (BBB) maintains brain homeostasis, regulates influx and efflux transport, and provides protection to the brain tissue. Ultrasound (US) and microbubble (MB)-mediated blood-brain barrier opening is an effective and safe technique for drug delivery in-vitro and in-vivo. However, the exact mechanism underlying this technique is still not fully elucidated. The aim of the study is to explore the contribution of transcytosis in the BBB transient opening using an in-vitro model of BBB. Utilizing a diverse set of techniques, including Ca 2+ imaging, electron microscopy, and electrophysiological recordings, our results showed that the combined use of US and MBs triggers membrane deformation within the endothelial cell membrane, a phenomenon primarily observed in the US + MBs group. This deformation facilitates the vesicles transportation of 500 kDa fluorescent Dextran via dynamin-/caveolae-/clathrin- mediated transcytosis pathway. Simultaneously, we observed increase of cytosolic Ca 2+ concentration, which is related with increased permeability of the 500 kDa fluorescent Dextran in-vitro. This was found to be associated with the Ca 2+ -protein kinase C (PKC) signaling pathway. The insights provided by the acoustically-mediated interaction between the microbubbles and the cells delineate potential mechanisms for macromolecular substance permeability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Tumor spheroids as well as multicellular tumor spheroids (MCTSs) are promising 3D in vitro tumor models for drug screening, drug design, drug targeting, drug toxicity, and validation of drug delivery methods. These models partly reflect the tridimensional architecture of tumors, their heterogeneity and their microenvironment, which can alter the intratumoral biodistribution, pharmacokinetics, and pharmacodynamics of drugs. The present review first focuses on current spheroid formation methods and then on in vitro investigations exploiting spheroids and MCTS for designing and validating acoustically mediated drug therapies. We discuss the limitations of the current studies and future perspectives. Various spheroid formation methods enable the easy and reproducible generation of spheroids and MCTSs. The development and assessment of acoustically mediated drug therapies have been mainly demonstrated in spheroids made up of tumor cells only. Despite the promising results obtained with these spheroids, the successful evaluation of these therapies will need to be addressed in more relevant 3D vascular MCTS models using MCTS-on-chip platforms. These MTCSs will be generated from patient-derived cancer cells and nontumor cells, such as fibroblasts, adipocytes, and immune cells.

Sonoporation using microbubble-assisted ultrasound increases the permeability of a biological barrier to therapeutic molecules. Application of this method to the round window membrane could improve the delivery of therapeutics to the inner ear. The aim of this study was to assess the safety of sonoporation of the round window membrane in a sheep model. To achieve this objective, we assessed auditory function and cochlear heating, and analysed the metabolomics profiles of perilymph collected after sonoporation, comparing them with those of the control ear in the same animal. Six normal-hearing ewes were studied, with one sonoporation ear and one control ear for each. A mastoidectomy was performed on both ears. On the sonoporation side, Vevo MicroMarker ® microbubbles (MBs; VisualSonics-Fujifilm, Amsterdam, The Netherlands) at a concentration of 2 × 10 8 MB/mL were locally injected into the middle ear and exposed to 1.1 MHz sinusoidal ultrasonic waves at 0.3 MPa negative peak pressure with 40% duty cycle and 100 μs interpulse period for 1 min; this was repeated three times with 1 min between applications. The sonoporation protocol did not induce any hearing impairment or toxic overheating compared with the control condition. The metabolomic analysis did not reveal any significant metabolic difference between perilymph samples from the sonoporation and control ears. The results suggest that sonoporation of the round window membrane does not cause damage to the inner ear in a sheep model.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that still lacks an efficient therapy. The barriers between the central nervous system (CNS) and the blood represent a major limiting factor to the development of drugs for CNS diseases, including ALS. Alterations of the blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) have been reported in this disease but still require further investigations. Interestingly, these alterations might be involved in the complex etiology and pathogenesis of ALS. Moreover, they can have potential consequences on the diffusion of candidate drugs across the brain. The development of techniques to bypass these barriers is continuously evolving and might open the door for personalized medical approaches. Therefore, identifying robust and non-invasive markers of BBB and BSCB alterations can help distinguish different subgroups of patients, such as those in whom barrier disruption can negatively affect the delivery of drugs to their CNS targets. The restoration of CNS barriers using innovative therapies could consequently present the advantage of both alleviating the disease progression and optimizing the safety and efficiency of ALS-specific therapies.

Microbubble (MB)-assisted ultrasound (US) is a promising physical method to increase non-invasively, transiently, and precisely the permeability of the blood-brain barrier (BBB) to therapeutic molecules. Previous preclinical studies established the innocuity of this procedure using complementary analytical strategies including transcriptomics, histology, brain imaging, and behavioral tests. This cross-sectional study using rats aimed to investigate the metabolic processes following acoustically-mediated BBB opening in vivo using multimodal and multimatrices metabolomics approaches. After intravenous injection of MBs, the right striata were exposed to 1-MHz sinusoidal US waves at 0.6 MPa peak negative pressure with a burst length of 10 ms, for 30 s. Then, the striata, cerebrospinal fluid (CSF), blood serum, and urine were collected during sacrifice in three experimental groups at 3 h, 2 days, and 1 week after BBB opening (BBBO) and were compared to a control group where no US was applied. A well-established analytical workflow using nuclear magnetic resonance spectrometry and non-targeted and targeted high-performance liquid chromatography coupled to mass spectrometry were performed on biological tissues and fluids. In our experimental conditions, a reversible BBBO was observed in the striatum without physical damage or a change in rodent weight and behavior. Cerebral, peri-cerebral, and peripheral metabolomes displayed specific and sequential metabolic kinetics. The blood serum metabolome was more impacted in terms of the number of perturbated metabolisms than in the CSF, the striatum, and the urine. In addition, perturbations of arginine and arginine-related metabolisms were detected in all matrices after BBBO, suggesting activation of vasomotor processes and bioenergetic supply. The exploration of the tryptophan metabolism revealed a transient vascular inflammation and a perturbation of serotoninergic neurotransmission in the striatum. For the first time, we characterized the metabolic signature following the acoustically-mediated BBBO within the striatum and its surrounding biological compartments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Presset, Bodard, Lefèvre, Millet, Oujagir, Dupuy, Iazourène, Bouakaz, Emond, Escoffre and Nadal-Desbarats.)

Ultrasound neurostimulation (USNS) is being investigated as a treatment approach for neuropsychiatric and neurodegenerative disorders. Indeed, unlike the existing methods that use electric or magnetic stimulation, it offers the possibility to modulate brain activity in a noninvasive way, with good spatial specificity and a high penetration capacity. However, there is no consensus yet on ultrasound parameters and beam properties required for efficient neurostimulation. In this context, this preclinical study aimed to elucidate the effect of frequency, peak negative pressure (PNP), pulse duration (PD), and focal spot diameter, on the USNS efficiency. This was done by targeting the motor cortex (M1) of 70 healthy mice and analyzing the elicited motor responses (visually and with electromyography). Also, a further investigation was performed by assessing the corresponding neuronal activity, using c-Fos immunostaining. The results showed that the success rate, a metric that depicts USNS efficacy, increased with PNP in a sigmoidal way, reaching up to 100%. This was verified at different frequencies (0.5, 1, 1.5, and 2.25 MHz) and PDs (53.3, 160, and 320 ms, at 1.5 MHz fixed frequency). Moreover, it was shown that higher PNP values were required to achieve a constant USNS efficacy not only when frequency increased, but also when the focal spot diameter decreased, emphasizing a close link between these acoustic parameters and USNS efficacy. These findings were confirmed with immunohistochemistry (IHC), which showed a strong relationship between neural activation, the applied PNP, and the focal spot diameter.

Physical activity is increasingly recognized as a strategy able to improve cancer patient outcome, and its potential to enhance treatment response is promising, despite being unclear. In our study we used a preclinical model of prostate cancer to investigate whether voluntary wheel running (VWR) could improve tumor perfusion and enhance radiotherapy (RT) efficiency. Nude athymic mice were injected with PC-3 cancer cells and either remained inactive or were housed with running wheels. Apparent microbubble transport was enhanced with VWR, which we hypothesized could improve the RT response. When repeating the experiments and adding RT, however, we observed that VWR did not influence RT efficiency. These findings contrasted with previous results and prompted us to evaluate if the lack of effects observed on tumor growth could be attributable to the physical activity modality used. Using PC-3 and PPC-1 xenografts, we randomized mice to either inactive controls, VWR, or treadmill running (TR). In both models, TR (but not VWR) slowed down tumor growth, suggesting that the anti-cancer effects of physical activity are dependent on its modalities. Providing a better understanding of which activity type should be recommended to cancer patients thus appears essential to improve treatment outcomes.

Stroke is the second leading cause of death worldwide. Existing therapies present limitations, and other therapeutic alternatives are sought, such as sonothrombolysis with microbubbles (STL). The aim of this study was to evaluate the change induced by STL with or without recombinant tissue-type plasminogen activator (rtPA) on the acoustic and elastic properties of the blood clot by measuring its sound speed (SoS) and shear wave speed (SWS) with high frequency ultrasound and ultrafast imaging, respectively. An in-vitro setup was used and human blood clots were submitted to a combination of microbubbles and rtPA. The results demonstrate that STL induces a raise of SoS in the blood clot, specifically when combined with rtPA ( p < 0.05). Moreover, the combination of rtPA and STL induces a hardening of the clot in comparison to rtPA alone ( p < 0.05). This is the first assessment of acoustoelastic properties of blood clots during STL. The combination of rtPA and STL induce SoS and hardening of the clot, which is known to impair the penetration of thrombolytic drugs and their efficacy.

Reactive oxygen species (ROS) are hypothesized to play a role in the sonoporation mechanisms. Nevertheless, the acoustical phenomenon behind the ROS production as well as the exact mechanisms of ROS action involved in the increased cell membrane permeability are still not fully understood. Therefore, we investigated the key processes occurring at the molecular level in and around microbubbles subjected to ultrasound using computational chemistry methods. To confirm the molecular simulation predictions, we measured the ROS production by exposing SonoVue® microbubbles (MBs) to ultrasound using biological assays. To investigate the role of ROS in cell membrane permeabilization, cells were subjected to ultrasound in presence of MBs and plasmid encoding reporter gene, and the transfection level was assessed using flow cytometry. The molecular simulations showed that under sonoporation conditions, ROS can form inside the MBs. These radicals could easily diffuse through the MB shell toward the surrounding aqueous phase and participate in the permeabilization of nearby cell membranes. Experimental data confirmed that MBs favor spontaneous formation of a host of free radicals where HO was the main ROS species after US exposure. The presence of ROS scavengers/inhibitors during the sonoporation process decreased both the production of ROS and the subsequent transfection level without significant loss of cell viability. In conclusion, the exposure of MBs to ultrasound might be the origin of chemical effects, which play a role in the cell membrane permeabilization and in the in vitro gene delivery when generated in its proximity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The poor diffusion of intravenous antibiotics in lung tissue makes nosocomial pneumonia challenging to treat, notably in critical patients under mechanical ventilation. The combination of ultrasound and microbubbles (USMB) is an emerging method for non-invasive and targeted enhancement of uptake of various drugs in several organs. This study aims to evaluate if USMB may increase amikacin concentration in condensed lung tissues in a mechanically ventilated rabbit model. When applied 60 or 160 min after the beginning of an intravenous amikacin infusion, USMB increased amikacin concentration in the condensed lung tissue by 1.33 ( p = 0.025) or 1.56-fold ( p = 0.028) respectively. When applied 70 min after the beginning of an intravenous amikacin infusion, USMB increased amikacin concentration in the muscle tissue by 2.52 ( p = 0.025). In conclusion, this study demonstrates that USMB is a promising method for the targeted delivery of amikacin in mechanically ventilated condensed lung, thus opening new therapeutic fields against lung infections., (Copyright © 2020 Espitalier, Darrouzain, Escoffre, Ternant, Piver, Bouakaz and Remerand.)

In solid tumors, rapid local intravascular release of anticancer agents, e.g., doxorubicin (DOX), from thermosensitive liposomes (TSLs) can be an option to overcome poor extravasation of drug nanocarriers. The driving force of DOX penetration is the drug concentration gradient between the vascular compartment and the tumor interstitium. In this feasibility study, we used fibered confocal fluorescence microscopy (FCFM) to monitor in real-time DOX penetration in the interstitium of a subcutaneous tumor after its intravascular release from TSLs, Thermodox®. Cell uptake kinetics of the released DOX was quantified, along with an in-depth assessment of released-DOX penetration using an evolution model. A subcutaneous rat R1 rhabdomyosarcoma xenograft was used. The rodent was positioned in a setup including a water bath, and FCFM identification of functional vessels in the tumor tissue was applied based on AngioSense. The tumor-bearing leg was immersed in the 43°C water for preheating, and TSLs were injected intravenously. Real-time monitoring of intratumoral (i.t.) DOX penetration could be performed, and it showed the progressing DOX wave front via its native fluorescence, labeling successively all cell nuclei. Cell uptake rates (1/k) of 3 minutes were found ( n =241  cells), and a released-DOX penetration in the range of 2500  µ m 2 ·s -1 was found in the tumor extravascular space. This study also showed that not all vessels, identified as functional based on AngioSense, gave rise to local DOX penetration.

The axial resolution of an ultrasound imaging system is inversely proportional to the bandwidth of the emitted signal. When conventional pulsing (CP) is used, the impulse response of the transducer and the excitation signal determine together the shape of the emitted pulse and its bandwidth. A way to increase the ultrasound image resolution is to increase the transducer's limited passband. The resolution enhancement compression (REC) is a coding technique that boosts the signal energy in the transition frequency bands, where the energy transduction of the ultrasound probe is less efficient. Consequently, image quality metrics including axial resolution, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) can be improved. In this paper, the objective is to combine REC with coherent plane-wave compounding (CPWC) in order to achieve better image quality at an ultrafast acquisition rate. Promising results are obtained from both wire and cyst phantoms using an excitation signal designed to provide a 54% increase in bandwidth over the one obtained with a broadband pulse excitation at -6 dB. The experimental bandwidth measured from the backscattered echoes was improved by 49% for the wire phantom, when using the CPWC-REC technique compared to CPWC-CP. Furthermore, the axial resolution as derived from the modulation transfer function of the envelope of the wire target was enhanced by 29%. The CNR and SNR were improved up to 9 and up to 4 dB, respectively, in the cyst phantom. These results reveal that CPWC-REC is able to achieve higher spatial resolution, compared to CPWC-CP, with better SNR and CNR. Moreover, experimental results show that an effective implementation on a research scanner of REC using plane-wave imaging is possible. Consistent in vivo acquisition results on rabbit are presented and discussed.

Background: Microbubbles (MBs) combined with ultrasound sonothrombolysis (STL) appears to be an alternative therapeutic strategy for acute ischemic stroke (IS), but clinical results remain controversial., Objective: The aim of this systematic review is to identify the parameters tested; to assess evidence on the safety and efficacy on preclinical data on STL; and to assess the validity and publication bias., Methods: Pubmed® and Web of ScienceTM databases were systematically searched from January 1995 to April 2017 in French and English. We included studies evaluating STL on animal stroke model. This systematic review was conducted in accordance with the PRISMA guidelines. Data were extracted following a pre-defined schedule by two of the authors. The CAMARADES criteria were used for quality assessment. A narrative synthesis was conducted., Results: Sixteen studies met the inclusion criteria. The result showed that ultrasound parameters and types of MBs were heterogeneous among studies. Numerous positive outcomes on efficacy were found, but only four studies demonstrated superiority of STL versus recombinant tissue-type plasminogen activator on clinical criteria. Data available on safety are limited., Limitations: Quality assessment of the studies reviewed revealed a number of biases., Conclusion: Further in vivo studies are needed to demonstrate a better efficacy and safety of STL compared to currently approved therapeutic options., Systematic Review Registration: http://syrf.org.uk/protocols/.

Contrast-enhanced ultrasound (CEUS) is a non-invasive imaging technique extensively used for blood perfusion imaging of various organs. This modality is based on the acoustic detection of gas-filled microbubble contrast agents used as intravascular flow tracers. Recent efforts aim at quantifying parameters related to the enhancement in the vascular compartment using time-intensity curve (TIC), and at using these latter as indicators for several pathological conditions. However, this quantification is mainly hampered by two reasons: first, the quantification intrinsically solely relies on temporal intensity variation, the explicit spatial transport of the contrast agent being left out. Second, the exact relationship between the acquired US-signal and the local microbubble concentration is hardly accessible. This paper introduces the use of a fluid dynamic model for the analysis of dynamic CEUS (DCEUS), in order to circumvent the two above-mentioned limitations. A new kinetic analysis is proposed in order to quantify the velocity amplitude of the bolus arrival. The efficiency of proposed methodology is evaluated both in-vitro, for the quantitative estimation of microbubble flow rates, and in-vivo, for the classification of placental insufficiency (control versus ligature) of pregnant rats from DCEUS. Besides, for the in-vivo experimental setup, we demonstrated that the proposed approach outperforms the performance of existing TIC-based methods.

Thrombosis is a major cause of several diseases, i.e. myocardial infarction, cerebral stroke and pulmonary embolism. Thrombolytic therapies are required to induce fast and efficient recanalization of occluded vessels. To evaluate the in vitro efficacy of these thrombolytic strategies, measuring clot dissolution is essential. This study aimed to evaluate and validate high resolution ultrasound as a tool to assess the exact volume of clots in 3D and in real time during in vitro thrombolytic drug testing. This new method was validated by measuring the effects of concentration range of recombinant tissue type plasminogen activator on a blood clot during complete occlusion or 70% stenosis of a vessel. This study shows that high resolution ultrasound imaging allows for a real-time assessment of the 3D volume of a blood clot with negligible inter- and intra-operator variabilities. The conclusions drawn from this study demonstrate the promising potential of high resolution ultrasound imaging for the in vitro assessment of new thrombolytic drugs.

Purpose: This preclinical study aimed to evaluate placental oxygenation in pregnant rats by real-time photoacoustic (PA) imaging on different days of gestation and to specify variations in placental oxygen saturation under conditions of maternal hypoxia and hyperoxygenation., Material and Methods: Placentas of fifteen Sprague-Dawley rats were examined on days 14, 17, and 20 of pregnancy with a PA imaging system coupled to high-resolution ultrasound imaging. Pregnant rats were successively exposed to hyperoxygenated and hypoxic conditions by changing the oxygen concentration in inhaled gas. Tissue oxygen saturation was quantitatively analyzed by real-time PA imaging in the skin and 3 regions of the placenta. All procedures were performed in accordance with applicable ethical guidelines and approved by the animal care committee., Results: Maternal hypoxia was associated with significantly greater decrease in blood oxygen saturation (ΔO2 Saturation) in the skin (70.74% ±7.65) than in the mesometrial triangle (32.66% ±5.75) or other placental areas (labyrinth: 18.58% ± 6.61; basal zone: 13.13% ±5.72) on different days of pregnancy (P<0.001). ΔO2 Saturation did not differ significantly between the labyrinth, the basal zone, and the decidua. After the period of hypoxia, maternal hyperoxygenation led to a significant rise in oxygen saturation, which returned to its initial values in the different placental regions (P<0.001)., Conclusions: PA imaging enables the variation of blood oxygen saturation to be monitored in the placenta during maternal hypoxia or hyperoxygenation. This first preclinical study suggests that the placenta plays an important role in protecting the fetus against maternal hypoxia., Competing Interests: The authors have declared that no competing interests exist.

The combination of microbubbles and ultrasound has emerged as a promising method for local drug delivery. Microbubbles can be locally activated by a targeted ultrasound beam, which can result in several bio-effects. For drug delivery, microbubble-assisted ultrasound is used to increase vascular- and plasma membrane permeability for facilitating drug extravasation and the cellular uptake of drugs in the treated region, respectively. In the case of drug-loaded microbubbles, these two mechanisms can be combined with local release of the drug following destruction of the microbubble. The use of microbubble-assisted ultrasound to deliver chemotherapeutic agents is also referred to as sonochemotherapy. In this review, the basic principles of sonochemotherapy are discussed, including aspects such as the type of (drug-loaded) microbubbles used, the routes of administration used in vivo, ultrasound devices and parameters, treatment schedules and safety issues. Finally, the clinical translation of sonochemotherapy is discussed, including the first clinical study using sonochemotherapy.

Background: Mild hyperthermia (mHT) increases the tumor perfusion and vascular permeability, and reduces the interstitial fluid pressure, resulting in better intra-tumoral bioavailability of low molecular weight drugs. This approach is potentially also attractive for delivery of therapeutic macromolecules, such as antibodies. Here, we investigated the effects of mHT on the stability, immunological and pharmacological properties of Herceptin(®), a clinically approved antibody, targeting the human epidermal growth factor receptor 2 (HER-2) overexpressed in breast cancer., Results: Herceptin(®) was heated to 37°C (control) and 42°C (mHT) for 1 hour. Formation of Herceptin(®) aggregates was measured using Nile Red assay. mHT did not result in additional Herceptin(®) aggregates compared to 37°C, showing the Herceptin(®) stability is unchanged. Immunological and pharmacological properties of Herceptin(®) were evaluated following mHT using HER-2 positive breast cancer cells (BT-474). Exposure of Herceptin(®) to mHT preserved recognition and binding affinity of Herceptin(®) to HER-2. Western-blot and cell proliferation assays on BT-474 cells showed that mHT left the inhibitory activities of Herceptin(®) unchanged., Conclusions: The stability, and the immunological and pharmacological properties of Herceptin(®) are not negatively affected by mHT. Further in-vivo studies are required to evaluate the influence of mHT on intra-tumoral bioavailability and therapeutic effectiveness of Herceptin(®).

Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery.

This technical note investigates the involvement of microbubble attenuation and destruction in sonoporation mechanisms. First, we evaluate sonoporation efficiency using Vevo Micromarker, and a comparison is made with BR14 and SonoVue microbubbles. Then, the acoustical properties of the microbubbles are measured to gain insight into the sonoporation mechanisms using a green fluorescent protein as a marker. Using glioblastoma cells, an unprecedented transfection rate of 70% is reached with Vevo Micromarker, corresponding to a 1.5-fold increase compared with the rate achieved with the other microbubbles. Moreover, attenuation and destruction were shown to be two key parameters in sonoporation efficiency.

One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy.

The RNA interference-mediated gene silencing approach is promising for therapies based on the targeted inhibition of disease-relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA.

Electrotransfer of molecules is a well established technique which finds extensive use for gene transfer and holds great promise for anticancer treatment. Despite its widespread application, the mechanisms governing the entry of DNA into the cell and its intracellular trafficking are not yet known. The aim of this study is to unravel the role of the actin cytoskeleton during gene electrotransfer in cells. We performed single-cell level approaches to observe the organization of the actin cytoskeleton in Chinese hamster ovary (CHO) cells. In addition, we performed experiments at the multiple-cell level to evaluate the efficiency of DNA transfer after alteration of the actin cytoskeleton using the drug latrunculin B. Actin patches colocalizing with the DNA at the plasma membrane were observed with additional characteristics similar to those of the DNA aggregates in terms of time, number, and size. The disruption of the microfilaments reduces the DNA accumulation at the plasma membrane and the gene expression. This is the first direct experimental evidence of the participation of the actin cytoskeleton in DNA electrotransfer.

Gene electrotransfer can be obtained not just on single cells in diluted suspension. For more than 10 years, this is a quasi routine strategy in tissue on the living animal and a few clinical trials have now been approved. New problems have been brought by the close contacts of cells in tissue both on the local field distribution and on the access of DNA to target cells. They need to be solved to provide a further improvement in the efficacy and safety of protein expression. There is a competition between gene transfer and cell destruction. Nevertheless, present results are indicative that electrotransfer is a promising approach for gene therapy. High level and long-lived expression of proteins can be obtained in muscles. This is used for a successful method of electrovaccination.

Electropulsation is one of the nonviral methods successfully used to deliver genes into living cells in vitro and in vivo. This approach shows promise in the field of gene and cellular therapies. The present review focuses on the processes supporting gene electrotransfer in vitro. In the first part, we will report the events occurring before, during, and after pulse application in the specific field of plasmid DNA electrotransfer at the cell level. A critical discussion of the present theoretical considerations about membrane electropermeabilization and the transient structures involved in the plasmid uptake follows in a second part.

We study the effect of permeabilizing electric fields applied to two different types of giant unilamellar vesicles, the first formed from EggPC lipids and the second formed from DOPC lipids. Experiments on vesicles of both lipid types show a decrease in vesicle radius, which is interpreted as being due to lipid loss during the permeabilization process. We show that the decrease in size can be qualitatively explained as a loss of lipid area, which is proportional to the area of the vesicle that is permeabilized. Three possible modes of membrane loss were directly observed: pore formation, vesicle formation, and tubule formation.