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Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

ABSTRACT: RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

SUBMITTER: Kopechek JA

PROVIDER: S-EPMC4966949 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cardiotoxicity is the major dose-limiting factor in the chemotherapeutic use of doxorubicin (Dox). A delivery vehicle that can be triggered to release its payload in the tumoral microvasculature but not in healthy tissue would help improve the therapeutic window of the drug. Delivery strategies combining liposomal encapsulated Dox (LDox), microbubbles (MBs), and ultrasound (US) have been shown to improve therapeutic efficacy of LDox, but much remains to be known about the mechanisms and the US conditions that maximize cytotoxicity using this approach. In this study, we compared different US pulses in terms of drug release and acute toxicity. Drug uptake and proliferation rates using low-intensity US were measured in squamous cell carcinoma cells exposed to LDox conjugated to or coinjected with polymer MBs. The aims of this study were: (1) to compare the effects of low- and high-pressure US on Dox release kinetics; (2) to evaluate whether conjugating the liposome to the MB surface (DoxLPX) is an important factor for drug release and cytotoxicity; and (3) to determine which US parameters most inhibit cell proliferation and whether this inhibition is mediated by drug release or the MB/US interaction with cells. Low-pressure US (170 kPa) at high duty cycle (stable cavitation) released up to ∼ 70% of the encapsulated Dox from the DoxLPX, thus improving Dox bioavailability and cellular uptake and leading to a significant reduction in cell proliferation at 48 h. Flow cytometry showed that US generating stable oscillations of DoxLPX significantly increased cellular Dox uptake at 4 h after US exposure compared to LDox. Drug uptake was correlated with cytotoxicity at 48 h. Our results demonstrate that Dox-containing liposomes conjugated to polymer MBs can be triggered to release ∼ 70% of their payload using noninertial US. Following release, Dox became bioavailable to the cells and induced significantly higher cytotoxicity compared to nonreleased encapsulated drug. Our findings show promise for targeted drug delivery using this theranostic delivery platform at low US intensities.

Project description:Objectives: Early recanalization of large vessels in thromboembolism, such as myocardial infarction and ischemic stroke, is associated with improved clinical outcomes. Nitric oxide (NO), a biological gas signaling molecule, has been proven to protect against ischemia-reperfusion injury (IRI). However, the underlying mechanisms remain to be explored. This study investigated whether NO could mitigate IRI and the role of NO during acoustic cavitation. Methods: In vivo, thrombi in the iliac artery of rats were induced by 5% FeCl3. NO-loaded microbubbles (NO-MBs) and ultrasound (US) were used to treat thrombi. B-mode and Doppler US and histological analyses were utilized to evaluate the thrombolysis effect in rats with thrombi. Immunohistochemistry, immunofluorescence, and western blotting were conducted to investigate the underlying mechanisms of NO during acoustic cavitation. In vitro, hypoxia was used to stimulate cells, and NO-MBs were employed to alleviate oxidative stress and apoptosis. Results: We developed NO-MBs that significantly improve the circulation time of NO in vivo, are visible, and effectively release therapeutic gas under US. US-targeted microbubble destruction (UTMD) and NO-loaded UTMD (NO + UTMD) caused a significant decrease in the thrombus area and an increase in the recanalization rates and blood flow velocities compared to the control and US groups. We discovered that UTMD induced NO generation through activation of endothelial NO synthase (eNOS) in vivo. More importantly, we also observed significantly increased NO content and eNOS expression in the NO + UTMD group compared to the UTMD group. NO + UTMD can mitigate oxidative stress and apoptosis in the hind limb muscle without influencing blood pressure or liver and kidney functions. In vitro, NO-MBs alleviated oxidative stress and apoptosis in cells pretreated with hypoxia. Conclusion: Based on these data, UTMD affects the vascular endothelium by activating eNOS, and NO exerts a protective effect against IRI.

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Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

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