A new safety index based on intrapulse monitoring of ultra-harmonic cavitation during ultrasound-induced blood-brain barrier opening procedures.

Ultrasound-induced blood-brain barrier (BBB) opening using microbubbles is a promising technique for local delivery of therapeutic molecules into the brain. The real-time control of the ultrasound dose delivered through the skull is necessary as the range of pressure for efficient and safe BBB opening is very narrow. Passive cavitation detection (PCD) is a method proposed to monitor the microbubble activity during ultrasound exposure. However, there is still no consensus on a reliable safety indicator able to predict potential damage in the brain. Current approaches for the control of the beam intensity based on PCD employ a full-pulse analysis and may suffer from a lack of sensitivity and poor reaction time. To overcome these limitations, we propose an intra-pulse analysis to monitor the evolution of the frequency content during ultrasound bursts. We hypothesized that the destabilization of microbubbles exposed to a critical level of ultrasound would result in the instantaneous generation of subharmonic and ultra-harmonic components. This specific signature was exploited to define a new sensitive indicator of the safety of the ultrasound protocol. The approach was validated in vivo in rats and non-human primates using a retrospective analysis. Our results demonstrate that intra-pulse monitoring was able to exhibit a sudden appearance of ultra-harmonics during the ultrasound excitation pulse. The repeated detection of such a signature within the excitation pulse was highly correlated with the occurrence of side effects such as hemorrhage and edema. Keeping the acoustic pressure at levels where no such sign of microbubble destabilization occurred resulted in safe BBB openings, as shown by MR images and gross pathology. This new indicator should be more sensitive than conventional full-pulse analysis and can be used to distinguish between potentially harmful and safe ultrasound conditions in the brain with very short reaction time.

Comparative determination of placental perfusion by magnetic resonance imaging and contrast-enhanced ultrasound in a murine model of intrauterine growth restriction.

INTRODUCTION: Exploration of placental perfusion is essential in screening for dysfunctions impairing fetal growth. The objective of this study was to assess the potential value of contrast-enhanced ultrasonography (CEUS) and magnetic resonance imaging (MRI) for examining placental perfusion in a murine model of intrauterine growth restriction (IUGR). We also studied the reproducibility of perfusion quantification by CEUS. METHODS: Pregnant Sprague Dawley rat models of IUGR were studied during the third trimester. Unilateral uterine artery ligation induced IUGR. Placental perfusion was evaluated by CEUS and perfusion MRI with gadolinium for both ligated and control fetoplacental units. The kinetic parameters of the two imaging modalities were then compared. RESULTS: The analysis included 20 rats. The study showed good reproducibility of the CEUS indicators. The CEUS perfusion index approximated the blood flow rate and was halved in the ligation group (27.9 [u.a] (±14.8)) versus 61 [u.a] (±22.3) on the control side (P = 0.0003). MRI with gadolinium injection showed a clear reduction in the blood flow rate to 51.2 mL/min/100 mL (IQR 34.9-54.9) in the ligated horn, compared with 90.9 mL/min/100 mL (IQR 85.1-95.7) for the control side (P < 0.0001). The semiquantitative indicators obtained from the kinetic curves for both CEUS and MRI showed similar trends. Nonetheless, values were more widely dispersed with CEUS than MRI. DISCUSSION: The similar results for the quantification of placental perfusion by MRI and CEUS reinforce the likelihood that CEUS can be used to identify IUGR in a murine model induced by uterine vessel ligation.

Linear and nonlinear characterization of microbubbles and tissue using the Nakagami statistical model.

The goal of this work is to exploit the statistical signatures for discrimination between biological tissues and contrast microbubbles in order to develop new strategies for contrast imaging and tissue characterization. For this purpose, the efficiency of the Nakagami statistical model, for describing the ultrasonic echoes of both contrast microbubbles and tissues, was investigated. Experimental measurements have been performed using a linear array probe connected to an open research platform. A commercially available in vitro phantom was used to mimic biological tissue in which SonoVue contrast microbubbles were flowing. Experimental ultrasound echoes have been filtered around the transmitted frequency (fundamental at 2.5MHz) and around twice the transmitted frequency (at 5MHz) for 2nd harmonic analysis, and a logarithmic compression was applied. The signals have been analyzed in order to evaluate the Nakagami parameter m, the scaling parameter Ω and the probability density function at both frequencies. Parametric images based on the Nakagami parameters map (Nakagami-mode images) were reconstructed and compared to B-mode images. Contrary to the B-mode image which is influenced by the system settings and user operations, the Nakagami parametric image is only based on the backscattered statistics of the ultrasonic signals in a local phantom. Such an imaging principle allows the Nakagami image to quantify the local scatterer concentrations in the phantom and to extract the backscattering information from the regions of the weakest echoes that may be lost in the conventional B-mode image. Results show that the tissue and microbubbles characterization is more sensitive in the 2nd harmonic mode when a logarithmic transform is used. These results would be useful for improving the ultrasound image quality and contrast detection in nonlinear mode.

Monolithic silica columns with covalently attached octaproline chiral selector. Dependence of performance on derivatization degree and comparison with a bead-based analogue.

A monolithic silica gel chromatographic matrix was derivatized repetitively with an octaproline-derived chiral selector (CS). The increasingly derivatized column was tested after each derivatization reaction. The enantioseparation ability, resolution and efficiency were found to depend on the content of CS attained after each reaction. Moreover, enantioselectivity and performance of the column with the highest CS coverage were compared to those of a bead-based chiral stationary phase (CSP) counterpart. The octaproline-derivatized monolithic column demonstrated increased enantioseparation factors, resolution and broader applicability than the particle-based column. Finally, the loading capacity of the CSPs was also examined. The monolithic octaproline-derived column permits the separation of 3-20 times higher molar amounts of the tested analytes (depending on the compound considered) than the particle-based counterpart. The enhanced capabilities of the derivatized monolithic column with respect to that of a bead-based counterpart cannot be explained only on the basis of an increased CS coverage. The involvement of an effect produced by the chromatographic silica support structure in the obtained results is discussed.

Evaluation of chirp reversal power modulation sequence for contrast agent imaging.

Over the last decade, significant research effort has been focused on the use of chirp for contrast agent imaging because chirps are known to significantly increase imaging contrast-to-noise ratio (CNR). New imaging schemes, such as chirp reversal (CR), have been developed to improve contrast detection by increasing non-linear microbubble responses. In this study we evaluated the contrast enhancement efficiency of various chirped imaging sequences in combination with well-established imaging schemes such as power modulation (PM) and pulse inversion (PI). The imaging schemes tested were implemented on a fully programmable open scanner and evaluated by ultrasonically scanning (excitation frequency of 2.5 MHz; amplitude of 350 kPa) a tissue-mimicking flow phantom comprising a 4 mm diameter tube through which aqueous dispersions (dilution fraction of 1/2000) of the commercial ultrasound contrast agent, SonoVue(®) were continuously circulated. The recovery of non-linear microbubble responses after chirp compression requires the development and the optimization of a specific filter. A compression filter was therefore designed and used to compress and extract several non-linear components from the received microbubble responses. The results showed that using chirps increased the image CNR by approximately 10 dB, as compared to conventional Gaussian apodized sine burst excitation but degraded the axial resolution by a factor of 1.4, at -3 dB. We demonstrated that the highest CNR and contrast-to-noise ratio (CTR) were achievable when CR was combined with PM as compared to other imaging schemes such as PI.

Focused ultrasound mediated drug delivery from temperature-sensitive liposomes: in-vitro characterization and validation.

Nanomedicine-based delivery with non-invasive techniques is a promising approach to increase local drug concentration and to reduce systemic side effects. Focused ultrasound (FUS) has become a promising strategy for non-invasive local drug delivery by mild hyperthermia. In this study, traditional temperature-sensitive liposomes (TTSLs) encapsulating doxorubicin (DOX) were evaluated for FUS-mediated drug delivery with an in-vitro FUS setup. In-vitro studies showed quantitative release of the DOX from the lumen of the temperature-sensitive liposomes when heated to 42 °C with FUS using 1 MHz sinusoidal waves at 1.75 MPa for 10 min. No release was observed when heated at 37 °C. Moreover, we showed that DOX released from TTSLs by FUS is as efficiently internalized by glioblastoma cells as free DOX at 37 °C. In-vitro therapeutic evaluation showed that exposure of a cell monolayer to FUS-activated TTSLs induced a 60% and a 50% decrease in cell viability compared to cell medium and to TTSLs preheated at 37 °C, respectively. Using an in-vitro 3D cell culture model, the results showed that after FUS-mediated hyperthermia, preheated liposomes induced a 1.7-fold decrease in U-87 MG spheroid growth in comparison to the preheated liposomes at 37 °C. In conclusion, our results show that in-vitro FUS allows the evaluation of TTSLs and does not modify the cellular uptake of the released DOX nor its cytotoxic activity.

Irinotecan delivery by microbubble-assisted ultrasound: in vitro validation and a pilot preclinical study.

Irinotecan is a powerful anticancer drug with severe systemic side effects that limit its clinical application. Drug-targeted delivery with noninvasive methods is required to enhance the drug concentration locally and to reduce these undesirable events. Microbubble-assisted ultrasound has become a promising method for noninvasive targeted drug delivery. The aim of this study is to evaluate the therapeutic effectiveness of in vitro and in vivo irinotecan delivery based on the combination of ultrasound and microbubbles. In the present study, in vitro results showed that the irinotecan treatment with microbubble-assisted ultrasound induced a significant decrease in cell viability of human glioblastoma cells. Moreover, using subcutaneous glioblastoma xenografts, the in vivo preclinical study in nude mice demonstrated that this therapeutic protocol led to a decrease in tumor growth and perfusion and an increase of tumor necrosis. The conclusions drawn from this study demonstrate the promising potential of this therapeutic approach for the anticancer targeted therapy.

New insights into uteroplacental perfusion: quantitative analysis using Doppler and contrast-enhanced ultrasound imaging.

OBJECTIVE: To monitor and quantify uteroplacental perfusion in rat pregnancies by Doppler ultrasound (DUS) and contrast-enhanced ultrasound (CEUS). METHODS: Fourteen rats were randomized in two groups (the CEUS group and the control group). On days 8, 11, 14, 17, 19 and 20 of gestation, we used DUS to measure the resistance index (RI), pulsatility index and blood velocity in the uterine, arcuate and umbilical arteries in both groups. On days 14, 17 and 20, one group was also examined by CEUS. Quantitative perfusion parameters were calculated in 4 compartments (mesometrial triangle, placenta, umbilical cord and fetus) and compared. RESULTS: The DUS measurement showed that the RI of the uterine and arcuate arteries decreased (p < 0.01) from day 14 to day 17, while velocity increased each of these arteries (p < 0.01 and p < 0.05, respectively). Quantification of uteroplacental perfusion by CEUS in bolus mode revealed that blood volume and local blood flow increased from day 14 to day 20 in the mesometrial triangle (p < 0.01) and the placenta (p < 0.05). In the CEUS destruction-replenishment mode, the perfusion parameters showed trends similar to those observed in bolus mode. No microbubbles were detected in the umbilical vein or fetal compartments. The weights of pups in the two groups did not differ significantly. CONCLUSIONS: CEUS estimates of placental perfusion complement the data provided by DUS.

Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue® or BR14® microbubbles.

BACKGROUND: Cell exposure to ultrasound (US) in the presence of contrast agent microbubbles (MBs) can result in cell sonoporation that can be exploited for drug or gene delivery. Anticancer drug bleomycin (BLM), used in sonoporation, can effectively eliminate tumor cells in vitro and in vivo. Nevertheless, sonoporation mechanism is not known, thus different US parameters and MB types are used. Recently, we proposed that efficiency of cell sonoporation can be related to the efficiency of MB sonodestruction. PURPOSE: We analyzed human tumor cells viability in response to BLM, US and MB treatment. METHODS: Human glioblastoma astrocytoma (U-87 MG) or colon cancer (HCT-116) cells were exposed to US in the presence of BLM and either SonoVue® or BR14® MBs. MB sonodestruction was evaluated according to US signal attenuation. RESULTS: Both HCT-116 and U-87 MG cell viability following US exposure decreased up to 30%. Decrease in cell viability followed similar tendency as MB sonodestruction, which suggests direct relationship between MB sonodestruction and BLM intracellular delivery. CONCLUSION: Sonoporation is a feasible method to deliver BLM in to several types of human cancer cell lines. Efficiency of cell sonoporation correlated well with MB sonodestruction, providing a possibility to optimize US parameters by measuring MB sonodestruction.

Doxorubicin delivery into tumor cells with ultrasound and microbubbles.

Doxorubicin is a potent chemotherapeutic whose severe side effects limit its application. Drug-targeted delivery with noninvasive techniques is required to increase the drug concentration locally and to reduce systemic side effects. Microbubble-assisted ultrasound has become a promising strategy for noninvasive local drug delivery. The aim of this study is to evaluate the applicability and the effectiveness of administration of doxorubicin combined with microbubble-assisted ultrasound in human U-87MG glioblastoma and MDA-MB-231 breast cancer cells. In the present study, the doxorubicin delivery aided by microbubble-assisted ultrasound enhanced the death of breast cancer and glioblastoma cells, including the induction of apoptosis. Various microbubbles were evaluated including Vevo Micromarker, BR14, SonoVue and experimental polymer shelled microbubbles. The results showed that Vevo Micromarker microbubble-assisted ultrasound could induce an enhancement of doxorubicin in glioblastoma and breast cancer cell death. Polylactide-Shelled PEG and Vevo Micromarker microbubbles were the best microbubbles for efficient doxorubicin delivery in the U-87 MG and MDA-MB-231 cells, respectively. Moreover, the induction of apoptosis by doxorubicin and Vevo Micromarker microbubble-assisted ultrasound was examined and results showed a positive increment for acoustic pressures above 600 kPa. The conclusions drawn from in vitro study show the potential of this strategy for an in vivo application.

Prognostic value of CA 19.9 levels in colorectal cancer.

Pretreatment serum levels of carbohydrate antigen 19.9 (CA 19.9) and carcinoembryonic antigen were measured in 293 patients with colorectal cancer. Carbohydrate antigen 19.9 was above the cut-off limit of 37 U/mL in 35% of patients. Carbohydrate antigen 19.9 sensitivity was related to tumor stage. Carcinoembryonic antigen was above the cut-off level of 3.5 ng/mL in 61% of patients, and the simultaneous use of two markers increased sensitivity to 66%. The main use of pretreatment levels of CA 19.9 in locoregional cancer is in prognosis. Carbohydrate antigen 19.9 provided more prognostic information than that obtained by conventional staging methods. In patients with Dukes' C tumors, additional information was obtained for allocation of these patients into groups at low or high risk of recurrence. Prognostic significance of carcinoembryonic antigen was not independent of Dukes' classification.