Final Report for Blood Brain Barrier Opening of Rat by Laser-generated Focused Ultrasound Transducer

A laser-generated carbon nanotube (CNT) transducer has been known to generate a shock wave with large amplitude for lasting sub-microseconds without producing heat. This laser-generated CNT transducer has never been applied to transcranial focused ultrasound (tcFUS) applications. Based on preliminary observation of the Evans blue leaked in the brain tissue of a rat after sonication of the shock wave by a CNT transducer, BBB opening of the rat by the shock wave from the CNT transducer was tried to confirm without damage of vessels. However, peak negative pressure was saturated to -9 MPa which is not enough for cavitation even with all trials of different fabrication processes such as coating methods of the PDMS layer, CNT-PDMS composite layers, backing materials, and CNT solutions. These results and discussion and suggestions were reported. As another application of the CNT transducer for lower peak negative pressure, EEG signals before and after sonication of shock waves for 10 minutes were observed in 3 rats. Shock wave by CNT transducer would be a potential for tcFUS neurostimulation and neuromodulation.

Ultrasound-Microbubble Combined Treatment with Vinca Alkaloids in Prostate Cancer Cells

Therapeutic efficacy of chemotherapy is highly dependent on the ability to deliver drug molecules across tissue and cellular barriers. Ultrasound stimulated microbubbles (USMB) have been shown to enhance the delivery and cytotoxicity of various classes of chemotherapeutic agents. Here, the application of USMB in combination with the chemotherapeutic class vinca alkaloids is investigated. Specifically, vinorelbine tartrate (VRL) and vinblastine sulfate (VBL) of the vinca alkaloid class, which to the best of our knowledge have not been reported in combination with USMB, were used in this study. Cell viability analysis demonstrated that USMB does not enhance the cytotoxicity of either drug. VRL+USMB showed to have an additive response in cell death, whereas VBL+USMB resulted in an additive effect at a low peak negative pressure, and antagonistic at higher pressures. This work suggests that the mechanism of uptake is an important factor in determining the effectiveness of a chemotherapy drug with USMB treatment.

Ultrasound-Microbubble Combined Treatment with Vinca Alkaloids in Prostate Cancer Cells

Therapeutic efficacy of chemotherapy is highly dependent on the ability to deliver drug molecules across tissue and cellular barriers. Ultrasound stimulated microbubbles (USMB) have been shown to enhance the delivery and cytotoxicity of various classes of chemotherapeutic agents. Here, the application of USMB in combination with the chemotherapeutic class vinca alkaloids is investigated. Specifically, vinorelbine tartrate (VRL) and vinblastine sulfate (VBL) of the vinca alkaloid class, which to the best of our knowledge have not been reported in combination with USMB, were used in this study. Cell viability analysis demonstrated that USMB does not enhance the cytotoxicity of either drug. VRL+USMB showed to have an additive response in cell death, whereas VBL+USMB resulted in an additive effect at a low peak negative pressure, and antagonistic at higher pressures. This work suggests that the mechanism of uptake is an important factor in determining the effectiveness of a chemotherapy drug with USMB treatment.

On the molecular release induced by ultrasound and microbubbles in cells

Ultrasound and microbubble (USMB) enhances intracellular uptake through membrane disruption and endocytosis. This study investigates USMB effects on the molecular release incells through membrane-disruption and exocytosis. Retinal pigmented epithelial (RPE) cells were loaded with Alexa 647-transferrin (Tfn) to mark recycling endosomes, LAMP-1 antibody was used to mark lysosomes, GFP-transfected RPE cells were used to mark cytoplasm, and 7-AAD was used to assess cell viability. USMB exposure was done at 570kPa peak negative pressure for 1min. The mean fluorescent intensities (MFI) of markers were measured using flow cytometry. USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells respectively. LAMP-1 antibody MFI increased by 50% and 15-folds in viable and non-viable cells indicating USMB induced release from lysosomes. Furthermore, Tfn release from recycling endosomes increased by 22% only in viable cells. In conclusion, USMB enhances the molecular release from cytoplasm, lysosomes, and recycling endosomes

On the molecular release induced by ultrasound and microbubbles in cells

Ultrasound and microbubble (USMB) enhances intracellular uptake through membrane disruption and endocytosis. This study investigates USMB effects on the molecular release incells through membrane-disruption and exocytosis. Retinal pigmented epithelial (RPE) cells were loaded with Alexa 647-transferrin (Tfn) to mark recycling endosomes, LAMP-1 antibody was used to mark lysosomes, GFP-transfected RPE cells were used to mark cytoplasm, and 7-AAD was used to assess cell viability. USMB exposure was done at 570kPa peak negative pressure for 1min. The mean fluorescent intensities (MFI) of markers were measured using flow cytometry. USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells respectively. LAMP-1 antibody MFI increased by 50% and 15-folds in viable and non-viable cells indicating USMB induced release from lysosomes. Furthermore, Tfn release from recycling endosomes increased by 22% only in viable cells. In conclusion, USMB enhances the molecular release from cytoplasm, lysosomes, and recycling endosomes

Sonoporation generates downstream cellular impact after membrane resealing

Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1 MHz frequency, 0.50 MPa peak negative pressure; 10% duty cycle; 30 s exposure period; 29.1 J/cm2 acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24 h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24 h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12 h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3 h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.

Nanodroplet-mediated catheter-directed sonothrombolysis of retracted blood clots

One major challenge in current microbubble (MB) and tissue plasminogen activator (tPA)-mediated sonothrombolysis techniques is effectively treating retracted blood clots, owing to the high density and low porosity of retracted clots. Nanodroplets (NDs) have the potential to enhance retracted clot lysis owing to their small size and ability to penetrate into retracted clots to enhance drug delivery. For the first time, we demonstrate that a sub-megahertz, forward-viewing intravascular (FVI) transducer can be used for ND-mediated sonothrombolysis, in vitro. In this study, we determined the minimum peak negative pressure to induce cavitation with low-boiling point phase change nanodroplets and clot lysis. We then compared nanodroplet mediated sonothrombolysis to MB and tPA mediate techniques. The clot lysis as a percent mass decrease in retracted clots was 9 ± 8%, 9 ± 5%, 16 ± 5%, 14 ± 9%, 17 ± 9%, 30 ± 8%, and 40 ± 9% for the control group, tPA alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US groups, respectively. In retracted blood clots, combined ND- and tPA-mediated sonothrombolysis was able to significantly enhance retracted clot lysis compared with traditional MB and tPA-mediated sonothrombolysis techniques. Combined nanodroplet with tPA-mediated sonothrombolysis may provide a feasible strategy for safely treating retracted clots.

The Hip Suction Seal, Part I: The Role of Acetabular Labral Height on Hip Distractive Stability

Background: The acetabular labrum has been found to provide a significant contribution to the distractive stability of the hip. However, the influence of labral height on hip suction seal biomechanics is not known. Hypothesis: The smaller height of acetabular labrum is associated with decreased distractive stability. Study Design: Descriptive laboratory study. Methods: A total of 23 fresh-frozen cadaveric hemipelvises were used in this study. Hips with acetabular dysplasia or femoroacetabular impingement–related bony morphologic features, intra-articular pathology, or no measurable suction seal were excluded. Before testing, each specimen’s hip capsule was removed, a pressure sensor was placed intra-articularly, and the hip was fixed in a heated saline bath. Labral size was measured by use of a digital caliper. Maximum distraction force, distance to suction seal rupture, and peak negative pressure were recorded while the hip underwent distraction at a rate of 0.5 mm/s. Correlations between factors were analyzed using the Spearman rho, and differences between groups were detected using Mann-Whitney U test. Results: Of 23 hips, 12 satisfied inclusion criteria. The maximum distraction force and peak negative pressure were significantly correlated ( R = −0.83; P = .001). Labral height was largely correlated with all suction seal parameters (maximum distraction force, R = 0.69, P = .013; distance to suction seal rupture, R = 0.55, P = .063; peak negative pressure, R = −0.62, P = .031). Labral height less than 6 mm was observed in 5 hips, with a mean height of 6.48 mm (SD, 2.65 mm; range, 2.62-11.90 mm; 95% CI, 4.80-8.17 mm). Compared with the 7 hips with larger labra (>6 mm), the hips with smaller labra had significantly shorter distance to suction seal rupture (median, 2.3 vs 7.2 mm; P = .010) and significantly decreased peak negative pressure (median, −59.3 vs −66.9 kPa; P = .048). Conclusion: Smaller height (<6 mm) of the acetabular labrum was significantly associated with decreased distance to suction seal rupture and decreased peak negative pressure. A new strategy to increase the size of the labrum, such as labral augmentation, could be justified for patients with smaller labra in order to optimize the hip suction seal. Clinical Relevance: The height of the acetabular labrum is correlated with hip suction seal biomechanics. Further studies are required to identify the clinical effects of labral height on hip stability.

Simulation Study on the Influence of Multifrequency Ultrasound on Transient Cavitation Threshold in Different Media

Through the introduction of multifrequency ultrasound technology, remarkable results have been achieved in tissue ablation and other aspects. By using the nonlinear dynamic equation of spherical bubble, the effects of the combination mode of multifrequency ultrasound, the peak negative pressure and its duration, the phase angle difference, and the polytropic index on the transient cavitation threshold in four different media of water, blood, brain, and liver are simulated and analyzed. The simulation results show that under the same frequency difference and initial bubble radius, the transient cavitation threshold of the high-frequency, triple-frequency combination is higher than that of the low-frequency, triple-frequency combination. When the lowest frequency of triple frequencies is the same, the larger the frequency difference, the higher the transient cavitation threshold. When the initial bubble radius is small, the frequency difference has little effect on the transient cavitation threshold of the triple-frequency combination. With the increase of initial bubble radius, the influence of frequency difference on the transient cavitation threshold of the higher frequency combination of triple frequency is more obvious than that of the lower frequency combination of triple frequency. When the duration of peak negative pressure or peak negative pressure of the multifrequency combined ultrasound is longer than that of the single-frequency ultrasound, the transient cavitation threshold of the multifrequency combined ultrasound is lower than that of the single-frequency ultrasound; on the contrary, the transient cavitation threshold of the multifrequency combined ultrasound is higher than that of the single-frequency ultrasound. When the phase angle difference of multifrequency excitation is zero, the corresponding transient cavitation threshold is the lowest, while the change of the polytropic index has almost no effect on the transient cavitation threshold for the multifrequency combination. The research results can provide a reference for multifrequency ultrasound to reduce the transient cavitation threshold, which is of great significance for the practical application of cavitation.

Acoustic Estimation of Resonance Frequency and Sonodestruction of SonoVue Microbubbles

Acoustic properties of ultrasound (US) contrast agent microbubbles (MB) highly influence sonoporation efficiency and intracellular drug and gene delivery. In this study we propose an acoustic method to monitor passive and excited MBs in a real time. MB monitoring system consisted of two separate transducers. The first transducer delivered over an interval of 1 s US pulses (1 MHz, 1% duty cycle, 100 Hz repetition frequency) with stepwise increased peak negative pressure (PNP), while the second one continuously monitored acoustic response of SonoVue MBs. Pulse echo signals were processed according to the substitution method to calculate attenuation coefficient spectra and loss of amplitude. During US exposure at 50–100 kPa PNP we observed a temporal increase in loss of amplitude which coincided with the US delivery. Transient increase in loss of amplitude vanished at higher PNP values. At higher PNP values loss of amplitude decreased during the US exposure indicating MB sonodestruction. Analysis of transient attenuation spectra revealed that attenuation coefficient was maximal at 1.5 MHz frequency which is consistent with resonance frequency of SonoVue MB. The method allows evaluation of the of resonance frequency of MB, onset and kinetics of MB sonodestruction.