Microbubble-enhanced Ultrasound Research Articles

Microbubble‐enhanced ultrasound exposure promotes uptake of methotrexate into synovial cells and enhanced antiinflammatory effects in the knees of rabbits with antigen‐induced arthritis

To evaluate whether microbubble-enhanced ultrasound (US) treatment promotes the delivery of methotrexate (MTX) into synovial cells and the enhanced antiinflammatory effects of intraarticular MTX therapy in a rabbit arthritis model. Arthritis was induced in both knees of 53 rabbits by immunization with ovalbumin. MTX including a microbubble agent was then injected into the left and right knee joints, and the right knees were exposed to US (MTX+/US+ group), while the left knees were not (MTX+/US- group). The knee joints were evaluated histologically in 7 rabbits at 5 time points up to day 56. Quantitative gene expression of interleukin-1beta (IL-1beta) in synovial tissue was measured on days 7 and 28. Eight rabbits were used for the measurement of MTX concentration in synovial tissue 12 hours after treatment. To evaluate the effect of microbubble-enhanced US treatment in the absence of MTX, only the microbubble agent was injected into the left and right knee joints of 10 rabbits with or without US exposure, and these animals were evaluated histologically on days 7 and 28. The MTX concentration in synovial tissue was significantly higher in the MTX+/US+ group than in the MTX+/US- group. Synovial inflammation was less prominent in the MTX+/US+ group compared with the MTX+/US- group, judging from the results of the histologic evaluation and the gene expression levels of IL-1beta in synovial tissue. It also appeared that microbubble-enhanced US exposure itself did not affect inflammation. Microbubble-enhanced US exposure promoted the uptake of MTX into synovial cells, which resulted in enhancement of the antiinflammatory effects of the intraarticular MTX injection. These results suggest that application of this technique may have clinical benefit.

Intracellular Delivery of Bak BH3 Peptide by Microbubble-Enhanced Ultrasound

To investigate the possibility of intracellular delivery of Bak BH3 peptide using sonoporation effect by microbubble-enhanced ultrasound. HeLa and BJAB cells were exposed to 1.696-Mhz focused ultrasound with 2% microbubble contrast agents (OPTISON). Cell-impermeable calcein was used as an indicator for successful sonoporation, and propidium iodide staining was used for cell viability assessment. Peptides were also exposed to ultrasound with OPTISON and analyzed with mass spectrometry for evaluation of stability under ultrasound exposure. The effect of transduced Bak BH3 peptide was evaluated by the cell viability of successfully sonoporated cells. Bak BH3 peptides did not undergo mechanical degradation with microbubble-enhanced ultrasound exposure. With the increase of acoustic energy exposure, the sonoporation efficiency saturated both in BJAB and HeLa cells, while direct cell death rate by ultrasound exposure tended to increase. When BJAB cells were treated with 100 microM Bak BH3 peptides, and ultrasound exposure with ultrasound contrast agents (OPTISON), an increased 35% cell death was confirmed. On the other hand, although HeLa cells had a similar trend, they failed to exhibit statistical significance. Our results suggest that microbubble-enhanced focused ultrasound peptide transduction is possible. Further optimization of ultrasound exposure conditions may be necessary.

Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound.

Intrauterine injection of naked DNA expressing luciferase, green fluorescent protein (GFP), or beta-galactosidase (beta-gal) and fluorescein isothiocyanate-labeled oligodeoxynucleotide (FITC-ODN), in combination with microbubble-enhanced ultrasound (US), referred to as the "shotgun method" (SGM), produced high-level protein expression in fetal mice. With the SGM, luciferase expression increased approximately 10(3)-fold in comparison with expression after injection of naked DNA alone. Electron microscopic analysis demonstrated transient formation of pores on the skin surface after intraamniotic (i.a.) injection with the SGM. Widespread expression of GFP and beta-gal and delivery of FITC-ODN were observed in multiple fetal tissues adjacent to the injection points. PCR analysis indicated that germline transfection was only transient following intraperitoneal (i.p) injection, and there was no evidence of transfer of the reporter gene to the offspring. Thus, SGM might provide a useful means to clarify the molecular mechanisms of genetic diseases in utero, as well as a tool to develop gene therapies in utero.

904. Intra-Uterine Gene Transfer with Microbubble-Enhanced Ultrasound (Shotgun Method; SGM) Increased Fetal Gene Expression

904. Intra-Uterine Gene Transfer with Microbubble-Enhanced Ultrasound (Shotgun Method; SGM) Increased Fetal Gene Expression