The ultrasound contrast imaging properties of lipid microbubbles loaded with urokinase in dog livers and their thrombolytic effects when combined with low-frequency ultrasound in vitro.

A new microbubble loaded with urokinase (uPA-MB) was explored in a previous study. However, its zeta potential and ultrasound contrast imaging properties and its thrombolytic effects when combined with low-frequency ultrasound (LFUS) were unclear. The zeta potential and ultrasound contrast imaging property of 5 uPA-MBs loading with 50,000 IU uPA was respectively detected using a Malvern laser particle analyzer and a Logiq 9 digital premium ultrasound system. Its ultrasound contrast imaging property was performed on the livers of two healthy dogs to compare with SonoVue. And the clot mass loss rate, D-dimer concentration and surface morphology of the clot residues were measured to evaluate the thrombolytic effect after treatment with three doses of 5 uPA-MBs combined with LFUS in vitro. The zeta potential of 5 uPA-MBs (-27.0 ± 2.40 mV) was higher than that of normal microbubbles (-36.95 ± 1.77 mV). Contrast-enhanced imaging of the hepatic vessels using 5 uPA-MBs was similar to SonoVue, while the imaging duration of 5 uPA-MBs (10 min) was longer than SonoVue (6 min). The thrombolytic effect of three doses of uPA-MBs combined with LFUS was significantly better than that of the control group and showed dose dependence. The 5 uPA-MBs have a negative charge on their surface and good echogenicity as ultrasound contrast agents. The 5 uPA-MBs combined with LFUS can promote thrombolysis in a dose-dependent manner.

The antitumor effect of a new docetaxel-loaded microbubble combined with low-frequency ultrasound in vitro: preparation and parameter analysis.

PURPOSE: To develop a novel docetaxel (DOC)-loaded lipid microbubbles (MBs) for achieving target therapy and overcoming the poor water-solubility drawback of DOC. METHODS: A novel DOC-loaded microbubble (DOC + MB) was prepared by lyophilization and the physicochemical properties including ultrasound contrast imaging of the liver were measured. The anti-tumor effect of the DOC + MBs combined with low-frequency ultrasound (LFUS; 0.8 Hz, 2.56 W/cm², 50% cycle duty) on the DLD-1 cancer cell line was examined using an MTT assay. RESULTS: The physicochemical properties of the two tested formats of DOC + MBs (1.0 mg and 1.6 mg) was shown: concentration, (6.74 ± 0.02) × 108 bubbles/mL and (8.27 ± 0.15) × 108 bubbles/mL; mean size, 3.296 ± 0.004 µm and 3.387 ± 0.005 µm; pH value, 6.67 ± 0.11 and 6.56 ± 0.05; release rate, 3.41% and 12.50%; Zeta potential, -37.95 ± 7.84 mV and -44.35 ± 8.70 mV; and encapsulation efficiency, 54.9 ± 6.21% and 46.3 ± 5.69%, respectively. Compared with SonoVue, the DOC + MBs similarly enhanced the echo signal of the liver imaging. The anti-tumor effect of the DOC + MBs/LFUS group was significantly better than that of DOC alone and that of the normal MBs/LFUS groups. CONCLUSIONS: The self-made DOC + MBs have potential as a new ultrasound contrast agent and drug-loaded microbubble, and can obviously enhance the antitumor effect of DOC under LFUS exposure.

The Effect of Docetaxel-Loaded Micro-Bubbles Combined with Low-Frequency Ultrasound in H22 Hepatocellular Carcinoma-Bearing Mice.

A novel lipid micro-bubble (MB) loaded with docetaxel (DOC-MB) was investigated in a previous study. However, its anti-tumor effects and mechanism of action in combination with low-frequency ultrasound (LFUS) in vivo are still unclear. DOC-MBs containing 5.0 mg of DOC were prepared by lyophilization with modification via ultrasonic emulsification. Then, the effects of DOC-MBs combined with LFUS on tumor growth, proliferating cell nuclear antigen (PCNA) expression and cell apoptosis, as well as local DOC delivery, were investigated in H22 hepatocellular carcinoma (HCC)-bearing mice. Compared with the previously prepared DOC-MBs (1.6 mg of DOC loaded), the encapsulation efficiency (81.2% ± 3.89%) and concentration ([7.94 ± 0.04] × 10(9) bubbles/mL) of the DOC-MBs containing 5.0 mg of DOC were higher, but the bubble size (1.368 ± 0.004 µm) was smaller. After treatment with the DOC-MBs and LFUS, the H22 HCC growth inhibition rate was significantly increased, PCNA expression in tumor tissue was significantly inhibited and local release of DOC was induced. In conclusion, new DOC-MBs containing 5.0 mg of DOC were successfully prepared with a high encapsulation efficiency and superior bubble size and concentration, and their combination with LFUS significantly enhanced the anti-tumor effect of DOC in H22 HCC-bearing mice by inhibiting tumor cell proliferation and increasing local drug delivery.

A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation.

INTRODUCTION: Z4A5 is a novel peptide that inhibits platelet aggregation and formation of platelet thrombi, but the mechanism of its anti-platelet effects remains unknown. This study explores the anti-platelet effect and mechanism of Z4A5. METHODS: We investigated the anti-platelet activity of Z4A5 on platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin (TH) in human platelet-rich plasma (PRP). Fibrinogen and PAC-1 binding to glycoproteinIIb/IIIa (GPIIb/IIIa) were measured by flow cytometry. In addition, we investigated the integrin specificity of Z4A5 in attachment and detachment assays using human umbilical vein endothelial cells (HUVEC) and assessed the relative cell number using the MTT assay. RESULTS: In vitro, Z4A5 inhibited ADP-, AA- and TH-induced human platelet aggregation with IC(50) values of 0.46 ± 0.05 µM (n = 10), 0.23 ± 0.0 5 µM (n = 10) and 0.21 ± 0.02 µM (n = 10), respectively. Z4A5 inhibited fibrinogen, and PAC-1 bound to platelet GPIIb/IIIa with IC(50) values of 0.48 ± 0.07 µM (n = 8) and 0.63 ± 0.12 µM (n = 6), respectively. Z4A5 failed to inhibit α(V)ß(3) integrin-mediated HUVEC attachment to vitronectin and did not cause any significant detachment of HUVEC monolayer when compared with the controls. CONCLUSIONS: Z4A5 is a potential anti-platelet drug that inhibits fibrinogen binding to GPIIb/IIIa, but does not affect the structurally similar integrin α(V)ß(3).

The preparation of a new self-made microbubble-loading urokinase and its thrombolysis combined with low-frequency ultrasound in vitro.

Urokinase (uPA) is used widely for thrombosis therapy in the clinic. However, ways to minimize its adverse effect of hemorrhage are still being studied. As a new technique for the local delivery of genes and drugs, ultrasound (US) contrast agents, microbubbles (MBs), have been mentioned. The purpose of this study is to explore a more efficacious and safer thrombolytic method by preparing three groups of self-made microbubble-loading uPA (uPA-MBs) (1 uPA-MBs, 5 uPA-MBs and 10 uPA-MBs) using freeze-drying methods and measuring their thrombolysis when combined in vitro with low-frequency US. The results showed the mean concentration, mean diameter, pH value and encapsulation efficiency of uPA of the three groups of uPA-MBs were approximately 2.08-2.82 × 10(8)/mL, ∼3.13 µm, 6.89-6.99 and from (78.08% ± 0.57%) to (57.23% ± 0.94%), respectively. Under US exposure, the loaded uPA demonstrated bioactivity by agarose fibrin plate and in vitro thrombolysis of the three uPA-MBs also showed higher effects than in the group of those who received uPA-MBs alone, the control group or the US group. In conclusion, the physiochemical properties of these self-made uPA-MBs are suitable for intravenous administration but 1 uPA-MB and 5 uPA-MBs are better than 10 uPA-MBs. uPA-MBs combined with US can decrease the in vitro dosage of uPA for thrombolysis.