An in vitro comparative study on clot lysis efficiency of urokinase and reteplase with the synergy of ultrasound needle.

Objectives: Ultrasound Needle, which is an improved ultrasonic horn device, has shown great potential for promoting the diffusion of thrombolytic drugs within clots and enhancing clot lysis efficiency. However, the clot lysis efficiency of different thrombolytic drugs with the synergy of Ultrasound Needle remains unknown. In this study, we aimed to compare the lysis efficiency of the non-fibrin-specific drug urokinase and fibrin-specific drug reteplase with the synergy of Ultrasound Needle. Materials and methods: Twenty-five milliliters of human blood was incubated for 1.5 h to form in vitro clots and then received the corresponding treatment protocols: control group (normal saline), US group (10 min of Ultrasound Needle treatment), UK group (30000IU of urokinase), r-PA group (2 mg of reteplase), US + UK group, and US + r-PA group. After treatment, the morphological changes of the clots were analyzed by B-mode ultrasound imaging and hematoxylin and eosin (H&E) staining. Lysis efficiency was evaluated based on the relative end weight (final weight/initial weight). The fibrin density of the different groups after treatment was assessed by immunofluorescence staining. Results: Morphological examination and relative end weight analysis showed that combination therapies induced a more thorough dissolution of clots compared with single therapies, and the US + r-PA group exhibited higher lysis efficiency than the US + UK group. In addition, immunofluorescence staining showed that the US + r-PA group had fewer remaining thrombus fibrins than the US + UK group after treatment. Conclusions: The Ultrasound Needle can significantly improve the clot lysis efficiency of both fibrinolytic drugs, and fibrin-specific reteplase exhibited superior lysis efficiency over non-fibrin-specific urokinase with the synergy of the Ultrasound Needle.

Ultrasound-Induced Tumor Perfusion Changes and Doxorubicin Delivery: A Study on Pulse Length and Pulse Repetition Frequency.

OBJECTIVES: To investigate the appropriate combination of pulse length (PL) and pulse repetition frequency (PRF) when performing ultrasound stimulated microbubble (USMB) to enhance doxorubicin (DOX) delivery to tumors. METHODS: A total of 48 tumor-bearing mice were divided into four groups, namely groups A-D. The mice in groups B-D were treated with chemotherapy and USMB treatment with different combinations of PL and PRF, and group A was control. Contrast-enhanced ultrasound imaging was conducted to analyze tumor blood perfusion. Fluorescence microscopy and high-performance liquid chromatography were used to qualitatively and quantitatively analyse DOX release. The structural changes of tumors were observed under light microscope and transmission electron microscope. Furthermore, another 24 tumor-bearing mice were treated with sonochemotherapy and some related inflammatory factors were measured to explore the underlying mechanism. RESULTS: With PL of three cycles and PRF of 2 kHz, the tumor perfusion area ratio increased by 26.67%, and the DOX concentration was 4.69 times higher than the control (P < .001). With PL of 34.5 cycles and PRF of 200 Hz, the tumor perfusion area ratio decreased by 12.7% and DOX did not exhibit increased extravasation compared with the control. Microvascular rupture and hemorrhage were observed after long PL and low PRF treatment. While vasodilation and higher levels of some vasodilator inflammatory factors were found after treatment with short PL and high PRF. CONCLUSIONS: USMB treatment using short PL and high PRF could enhance tumor blood perfusion and increase DOX delivery, whereas long PL and low PRF could not serve the same purpose.

Optimal treatment occasion for ultrasound stimulated microbubbles in promoting gemcitabine delivery to VX2 tumors.

Ultrasound stimulated microbubbles (USMB) is a widely used technology that can promote chemotherapeutic delivery to tumors yet the best treatment occasion for USMB is unknown or ignored. We aimed to determine the optimal treatment occasion for USMB treatment to enhance tumor chemotherapy to achieve the highest drug concentration in tumors. Experiments were conducted on VX2 tumors implanted in 60 rabbits. Gemcitabine (GEM) was intravenously infused as a chemotherapeutic agent and USMB was administered before, during or after chemotherapy. USMB was conducted with a modified diagnostic ultrasound at 3 MHz employing short bursts (5 cycles and 0.125% duty cycle) at 0.26 MPa in combination with a lipid microbubble. Subsequently, tumor blood perfusion quantitation, drug concentration detection, and fluorescence microscopy were performed. The results showed that the group that received USMB treatment immediately after GEM infusion had the highest drug concentration in tumors, which was 2.83 times that of the control group. Fifteen tumors were then treated repeatedly with the optimal USMB-plus-GEM combination, and along with the GEM and the control groups, were studied for tumor growth, tumor cell proliferation, apoptosis, and related cytokine contents. The combined treatment significantly inhibited tumor growth and promoted apoptosis. The levels of related cytokines, including HIF-1α, decreased after six combination therapies. These results suggest that the optimal treatment occasion for USMB occurs immediately after chemotherapy and tumor hypoxia improves after multiple combination therapies.

A minimally invasive strategy to evacuate hematoma by synergy of an improved ultrasonic horn with urokinase: An in vitro study.

OBJECTIVES: In this study, Ultrasound Needle-an improved minimally invasive ultrasonic horn device was used to explore its potential of synergizing with urokinase in enhancing clots lysis in an in vitro intracranial hematoma model. MATERIALS AND METHODS: Ten milliliter bovine blood was incubated for 3 h at 37℃, and coagulated into clot to mimic intracranial hematoma in vitro. Ultrasound Needle was an improved ultrasonic horn with a fine tip (1.80 mm) and metallic sheath, and had a frequency of 29.62 kHz. The 10,000 IU urokinase was injected through the metallic sheath during the vibration of Ultrasound Needle tip to lyse the clots for 8 min under different working parameter settings (n = 8) to explore the influence of parameters Amplitude (%) and Duty (%) on clot lysis weight (W0 ). The maximum temperatures were measured by an infrared thermometer during the treatment process. The W0 of different treatment groups (US (Ultrasound Needle), US + NS (normal saline), UK (urokinase), US + UK, n = 8) were compared to verify the synergistic lysis effect of Ultrasound Needle combined with urokinase at optimal working parameters (40% Amplitude, 20% Duty; input power 4.20 W; axial tip-vibration amplitude 69.17 µm). Clots samples after treatment were fixed overnight for macroscopic examination. And fluorescent frozen sections and scanning electron microscopy examination were performed to show microscopic changes in clots and evaluate the cavitation effect of Ultrasound Needle on promoting drug diffusion within the clots. RESULTS: The clot lysis weight W0 increased with the parameters Amplitude (%) and Duty (%), reached a peak (2.435 ± 0.137 g) at 40% Amplitude and 20% Duty (input power 4.20 W), and then decreased. Higher Amplitude (%) and Duty (%) led to higher maximum temperature, and W0 was negatively correlated with the maximum temperature after the peak (r = -0.958). At the optimal parameter setting, the maximum temperature was 33.8 ± 0.9℃, and the W0 of the US + UK group was more than four times of UK alone group (2.435 ± 0.137 g vs. 0.607 ± 0.185 g). Fluorescent frozen sections confirmed that the ultrasound energy of Ultrasound Needle could mechanically damage the clot tissues and promote the intra-clots drug diffusion. Macroscopic examination showed that US + UK group caused larger clots lysis area than UK alone group (2.08 cm2 vs. 0.65 cm2 ). In addition, electron microscopy examination exhibited that the fibrin filaments of the clots in US + UK group were lysed more thoroughly compared to single treatment groups. CONCLUSIONS: Ultrasound Needle, an improved ultrasonic horn device, can mechanically damage the clot tissues and exhibit an excellent synergistic lysis effect with thrombolytic drugs. Therefore, Ultrasound Needle has great potential in providing a new minimally invasive strategy for rapid intracranial hematoma evacuation.