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Gene therapy for ocular diseases meditated by ultrasound and microbubbles (Review).

Wan C, Li F, Li H - Mol Med Rep (2015)

Bottom Line: Ultrasound‑targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene‑ and drug delivery.High‑amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane.In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood‑retinal barrier with no significant side effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ultrasound, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China.

ABSTRACT
The eye is an ideal target organ for gene therapy as it is easily accessible and immune‑privileged. With the increasing insight into the underlying molecular mechanisms of ocular diseases, gene therapy has been proposed as an effective approach. Successful gene therapy depends on efficient gene transfer to targeted cells to prove stable and prolonged gene expression with minimal toxicity. At present, the main hindrance regarding the clinical application of gene therapy is not the lack of an ideal gene, but rather the lack of a safe and efficient method to selectively deliver genes to target cells and tissues. Ultrasound‑targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene‑ and drug delivery. When gene‑loaded microbubbles are injected, UTMD is able to enhance the transport of the gene to the targeted cells. High‑amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane. Thereby, the efficiency of gene therapy can be significantly improved. The UTMD‑mediated gene delivery system has been widely used in pre‑clinical studies to enhance gene expression in a site‑specific manner in a variety of organs. With reasonable application, the effects of sonoporation can be spatially and temporally controlled to improve localized tissue deposition of gene complexes for ocular gene therapy applications. In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood‑retinal barrier with no significant side effects. The present review discusses the current status of gene therapy of ocular diseases as well as studies on gene therapy of ocular diseases meditated by UTMD.

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Intracellular fluorescent microscopic images of cells treated with doxorubicin alone or with doxorubicin + US + MB. As early as 1 min after sonoporation, cells treated with doxorubicin + US + MB showed increased intracellular fluorescence compared with cells exposed to doxorubicin alone (35.26 vs. 45.62). This effect increased further at 60 min, where the mean intensity of fluorescence was 37.03 in control cells versus 71.18 in cells treated with doxorubicin + US + MB. Compared with the early fluorescence observed in cells exposed to US + MB, cells treated with doxorubicin alone showed only trace intracellular fluorescence at 60 min. Boxes represent ROIs for measuring levels of fluorescence, and values indicate mean intensity of fluorescence within the ROI. Scale bars, 10 µm. Image taken from Lee et al (71). ROI, region of interest; US, ultrasound; MB, microbubble.
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f6-mmr-12-04-4803: Intracellular fluorescent microscopic images of cells treated with doxorubicin alone or with doxorubicin + US + MB. As early as 1 min after sonoporation, cells treated with doxorubicin + US + MB showed increased intracellular fluorescence compared with cells exposed to doxorubicin alone (35.26 vs. 45.62). This effect increased further at 60 min, where the mean intensity of fluorescence was 37.03 in control cells versus 71.18 in cells treated with doxorubicin + US + MB. Compared with the early fluorescence observed in cells exposed to US + MB, cells treated with doxorubicin alone showed only trace intracellular fluorescence at 60 min. Boxes represent ROIs for measuring levels of fluorescence, and values indicate mean intensity of fluorescence within the ROI. Scale bars, 10 µm. Image taken from Lee et al (71). ROI, region of interest; US, ultrasound; MB, microbubble.

Mentions: Microbubble destruction by ultrasound exposure generates microstreams or microjets that create shear stress on cells and open transient pores in cell membranes, which has the capability of transiently enhancing cell membrane permeability (68). The use of ultrasound with diagnostic microbubbles in cancer treatment to increase the efficiency of chemotherapy through passive, localized delivery has been an emerging area of research. Numerous studies have demonstrated that optimized UTMD-mediated therapy has the potential to improve cancer response to therapy via increased localized drug uptake and targeted therapeutics, which may lead to a lowering of chemotherapeutic drug dosages and systemic toxicity (69,70). Lee et al (71) proved that using low-intensity (0.3 W/cm2) and low-frequency (1 MHz) ultrasound with microbubbles for 10 sec enhanced the chemotherapeutic efficacy of doxorubicin against retinoblastoma Y79 cells in vitro. Cells exposed to ultrasound and microbubbles showed earlier and higher trace intracellular fluorescence than that of cells treated with doxorubicin alone (Fig. 6). There is a significant decrease in cell viability in cells treated with this method compared with cells treated with chemotherapy alone. To investigate the duration and underlying mechanism of increased permeability, the study used scanning electron microscopy to image cells exposed to ultrasound + microbubbles (for 10 or 60 sec). Pores were identified in cells exposed to ultrasound + microbubbles for 60 sec but not in those exposed for 10 sec. However, in vitro fluorescence showed that doxorubicin uptake significantly increased immediately after exposure to ultrasound + microbubbles for 10 sec. These results suggested that the presence of physical pores may not be a pre-requisite for enhanced drug entry into the cells. It is possible that transient electrical changes, endocytosis or other unidentified mechanisms contributed to the enhanced drug uptake. UTMD may become a valuable adjuvant to chemotherapy of RB, whose treatment is often limited by challenges in drug delivery, and may lead to more effective chemotherapy treatments with less damage and side effects to ocular tissues.


Gene therapy for ocular diseases meditated by ultrasound and microbubbles (Review).

Wan C, Li F, Li H - Mol Med Rep (2015)

Intracellular fluorescent microscopic images of cells treated with doxorubicin alone or with doxorubicin + US + MB. As early as 1 min after sonoporation, cells treated with doxorubicin + US + MB showed increased intracellular fluorescence compared with cells exposed to doxorubicin alone (35.26 vs. 45.62). This effect increased further at 60 min, where the mean intensity of fluorescence was 37.03 in control cells versus 71.18 in cells treated with doxorubicin + US + MB. Compared with the early fluorescence observed in cells exposed to US + MB, cells treated with doxorubicin alone showed only trace intracellular fluorescence at 60 min. Boxes represent ROIs for measuring levels of fluorescence, and values indicate mean intensity of fluorescence within the ROI. Scale bars, 10 µm. Image taken from Lee et al (71). ROI, region of interest; US, ultrasound; MB, microbubble.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581786&req=5

f6-mmr-12-04-4803: Intracellular fluorescent microscopic images of cells treated with doxorubicin alone or with doxorubicin + US + MB. As early as 1 min after sonoporation, cells treated with doxorubicin + US + MB showed increased intracellular fluorescence compared with cells exposed to doxorubicin alone (35.26 vs. 45.62). This effect increased further at 60 min, where the mean intensity of fluorescence was 37.03 in control cells versus 71.18 in cells treated with doxorubicin + US + MB. Compared with the early fluorescence observed in cells exposed to US + MB, cells treated with doxorubicin alone showed only trace intracellular fluorescence at 60 min. Boxes represent ROIs for measuring levels of fluorescence, and values indicate mean intensity of fluorescence within the ROI. Scale bars, 10 µm. Image taken from Lee et al (71). ROI, region of interest; US, ultrasound; MB, microbubble.
Mentions: Microbubble destruction by ultrasound exposure generates microstreams or microjets that create shear stress on cells and open transient pores in cell membranes, which has the capability of transiently enhancing cell membrane permeability (68). The use of ultrasound with diagnostic microbubbles in cancer treatment to increase the efficiency of chemotherapy through passive, localized delivery has been an emerging area of research. Numerous studies have demonstrated that optimized UTMD-mediated therapy has the potential to improve cancer response to therapy via increased localized drug uptake and targeted therapeutics, which may lead to a lowering of chemotherapeutic drug dosages and systemic toxicity (69,70). Lee et al (71) proved that using low-intensity (0.3 W/cm2) and low-frequency (1 MHz) ultrasound with microbubbles for 10 sec enhanced the chemotherapeutic efficacy of doxorubicin against retinoblastoma Y79 cells in vitro. Cells exposed to ultrasound and microbubbles showed earlier and higher trace intracellular fluorescence than that of cells treated with doxorubicin alone (Fig. 6). There is a significant decrease in cell viability in cells treated with this method compared with cells treated with chemotherapy alone. To investigate the duration and underlying mechanism of increased permeability, the study used scanning electron microscopy to image cells exposed to ultrasound + microbubbles (for 10 or 60 sec). Pores were identified in cells exposed to ultrasound + microbubbles for 60 sec but not in those exposed for 10 sec. However, in vitro fluorescence showed that doxorubicin uptake significantly increased immediately after exposure to ultrasound + microbubbles for 10 sec. These results suggested that the presence of physical pores may not be a pre-requisite for enhanced drug entry into the cells. It is possible that transient electrical changes, endocytosis or other unidentified mechanisms contributed to the enhanced drug uptake. UTMD may become a valuable adjuvant to chemotherapy of RB, whose treatment is often limited by challenges in drug delivery, and may lead to more effective chemotherapy treatments with less damage and side effects to ocular tissues.

Bottom Line: Ultrasound‑targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene‑ and drug delivery.High‑amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane.In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood‑retinal barrier with no significant side effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ultrasound, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China.

ABSTRACT
The eye is an ideal target organ for gene therapy as it is easily accessible and immune‑privileged. With the increasing insight into the underlying molecular mechanisms of ocular diseases, gene therapy has been proposed as an effective approach. Successful gene therapy depends on efficient gene transfer to targeted cells to prove stable and prolonged gene expression with minimal toxicity. At present, the main hindrance regarding the clinical application of gene therapy is not the lack of an ideal gene, but rather the lack of a safe and efficient method to selectively deliver genes to target cells and tissues. Ultrasound‑targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene‑ and drug delivery. When gene‑loaded microbubbles are injected, UTMD is able to enhance the transport of the gene to the targeted cells. High‑amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane. Thereby, the efficiency of gene therapy can be significantly improved. The UTMD‑mediated gene delivery system has been widely used in pre‑clinical studies to enhance gene expression in a site‑specific manner in a variety of organs. With reasonable application, the effects of sonoporation can be spatially and temporally controlled to improve localized tissue deposition of gene complexes for ocular gene therapy applications. In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood‑retinal barrier with no significant side effects. The present review discusses the current status of gene therapy of ocular diseases as well as studies on gene therapy of ocular diseases meditated by UTMD.

Show MeSH
Related in: MedlinePlus