Cardiac gene delivery using ultrasound: State of the field.

Over the past two decades, there has been tremendous and exciting progress toward extending the use of medical ultrasound beyond a traditional imaging tool. Ultrasound contrast agents, typically used for improved visualization of blood flow, have been explored as novel non-viral gene delivery vectors for cardiovascular therapy. Given this adaptation to ultrasound contrast-enhancing agents, this presents as an image-guided and site-specific gene delivery technique with potential for multi-gene and repeatable delivery protocols-overcoming some of the limitations of alternative gene therapy approaches. In this review, we provide an overview of the studies to date that employ this technique toward cardiac gene therapy using cardiovascular disease animal models and summarize their key findings.

Subharmonic resonance of phospholipid coated ultrasound contrast agent microbubbles.

Phospholipid encapsulated ultrasound contrast agents have proven to be a powerful addition in diagnostic imaging and show emerging applications in targeted therapy due to their resonant and nonlinear scattering. Microbubble response is affected by their intrinsic (e.g. bubble size, encapsulation physics) and extrinsic (e.g. boundaries) factors. One of the major intrinsic factors at play affecting microbubble vibration dynamics is the initial phospholipid packing of the lipid encapsulation. Here, we examine how the initial phospholipid packing affects the subharmonic response of either individual or a system of two closely-placed microbubbles. We employ a finite element model to investigate the change in subharmonic resonance under 'small' and 'large' radial excursions. For microbubbles ranging between 1.5 and 2.5 µm in diameter and in its elastic state (σ0 = 0.01 N/m), we demonstrate up to a 10 % shift towards lower frequencies in the peak subharmonic response as the radial excursion increases. However, for a bubble initially in its buckled state (σ0 = 0 N/m), we observe a maximum shift of 8 % towards higher frequencies as the radial excursion increases over the same range of bubble sizes - the opposite trend. We studied the same scenario for a system of two individual microbubbles for which we saw similar results. For microbubbles that are initially in their elastic state, in both cases of a) two identically sized bubbles and b) a bubble in proximity to a smaller bubble, we observed a 6 % and 9 % shift towards lower frequencies respectively; while in the case of a neighboring larger bubble no change in subharmonic resonance frequency was observed. Microbubbles that are initially in a buckled state exert no change, 5 % and 19 % shift towards higher frequencies, in two-bubble systems consisting of a) same-size, b) smaller, and c) larger neighboring bubble respectively. Furthermore, we examined the effect of two adjacent bubbles with non-equal initial phospholipid states. The results presented here have important implications in ultrasound contrast agent applications.

Fluid flow influences ultrasound-assisted endothelial membrane permeabilization and calcium flux.

The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30 ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1 MHz ultrasound with 20-cycle bursts, 1 ms PRI, and 2 s duration at a peak negative pressure of 305 kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆t = 1s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery.

Stable Cavitation-Mediated Delivery of miR-126 to Endothelial Cells.

In endothelial cells, microRNA-126 (miR-126) promotes angiogenesis, and modulating the intracellular levels of this gene could suggest a method to treat cardiovascular diseases such as ischemia. Novel ultrasound-stimulated microbubbles offer a means to deliver therapeutic payloads to target cells and sites of disease. The purpose of this study was to investigate the feasibility of gene delivery by stimulating miR-126-decorated microbubbles using gentle acoustic conditions (stable cavitation). A cationic DSTAP microbubble was formulated and characterized to carry 6 µg of a miR-126 payload per 109 microbubbles. Human umbilical vein endothelial cells (HUVECs) were treated at 20−40% duty cycle with miR-126-conjugated microbubbles in a custom ultrasound setup coupled with a passive cavitation detection system. Transfection efficiency was assessed by RT-qPCR, Western blotting, and endothelial tube formation assay, while HUVEC viability was monitored by MTT assay. With increasing duty cycle, the trend observed was an increase in intracellular miR-126 levels, up to a 2.3-fold increase, as well as a decrease in SPRED1 (by 33%) and PIK3R2 (by 46%) expression, two salient miR-126 targets. Under these ultrasound parameters, HUVECs maintained >95% viability after 96 h. The present work describes the delivery of a proangiogenic miR-126 using an ultrasound-responsive cationic microbubble with potential to stimulate therapeutic angiogenesis while minimizing endothelial damage.

The influence of inter-bubble spacing on the resonance response of ultrasound contrast agent microbubbles.

Ultrasound-driven microbubbles, typically between 1 and 8 µm in diameter, are resonant scatterers that are employed as diagnostic contrast agents and emerging as potentiators of targeted therapies. Microbubbles are administered in populations whereby their radial dynamics - key to their effectiveness - are greatly affected by intrinsic (e.g. bubble size) and extrinsic (e.g. boundaries) factors. In this work, we aim to understand how two neighbouring microbubbles influence each other. We developed a finite element model of a system of two individual phospholipid-encapsulated microbubbles vibrating in proximity to each other to study the effect of inter-bubble distance on microbubble radial resonance response. For the case of two equal-sized and identical bubbles, each bubble exhibits a decrease between 7 and 10% in the frequency of maximum response (fMR) and an increase in amplitude of maximum response (AMR) by 9-11% as compared to its isolated response in free-space, depending on the bubble size examined. For a system of two unequal-sized microbubbles, the large bubble shows no significant change, however the smaller microbubble shows an increase in fMR by 7-11% and a significant decrease in AMR by 38-52%. Furthermore, in very close proximity the small bubble shows a secondary off-resonance peak at the corresponding fMR of its larger companion microbubble. Our work suggests that frequency-dependent microbubble response is greatly affected by the presence of another bubble, which has implications in both imaging and therapy applications. Furthermore, our work suggests a mechanism by which nanobubbles show significant off-resonance vibrations in the clinical frequency range, a behaviour that has been observed experimentally but heretofore unexplained.

Hypoglycemic, hepatoprotective, and hypolipidemic effects of hydroalcoholic extract of Eryngium billardieri root on nicotinamide/streptozotocin-induced type II diabetic rats.

BACKGROUND AND PURPOSE: Erynginum billardieri has been used to control diabetes in traditional medicine. This research was performed to study the antidiabetic, hepatoprotective, and hypolipidemic effects of E. billardieri root extract (EBRE) on streptozotocin/nicotinamide-induced type 2 diabetic male rats. EXPERIMENTAL APPROACH: Type two diabetic animals were treated by three different doses of EBRE (100, 200, and 400 mg/kg), orally administered for 4 weeks. Ultimately, after anesthesia, the glucose, insulin, lipid profiles, hepatic enzyme levels in the blood and liver, and pancreas tissues of the animals were analyzed. FINDINGS/RESULTS: Induction of diabetes caused a diminution in insulin level, high-density lipoprotein (HDL), and significantly enhanced the level of other lipid profiles, glucose, and liver enzymes (P < 0.05). Administration of the EBRE to diabetic-male rats significantly reduced glucose level, lipid profiles, and liver enzymes, and increased the level of HDL to near normal. CONCLUSION AND IMPLICATIONS: The results of the present study showed that E. billardieri had a positive effect on diminishing the lipid profiles, liver enzymes, and controlling diabetes. The most effective dose was found to be 100 mg/kg.

Short-term effects of massage with olive oil on the severity of uremic restless legs syndrome: A double-blind placebo-controlled trial.

BACKGROUND: Although the efficacy of olive oil massage has been established for different disorders, no studies have yet focused on the effect of olive oil massage on restless legs syndrome (RLS). In this study, we aimed to evaluate the short-term effects of massage with olive oil in reducing the severity of uremic RLS. METHODS: This double-blind placebo-controlled trial was conducted on 60 patients with uremic RLS (mean age: 51.96 ± 10.15), who were randomly divided into olive oil and placebo groups. The olive oil group received massage with olive oil, while the placebo group received massage with liquid paraffin twice a week during hemodialysis sessions for three weeks. For each leg, 10 mL of the olive oil or placebo was applied and then massaged for five minute [corrected] from the plantar surface of the foot to the area below the knee. The severity of RLS was rated on the first day and one week after the final massage therapy session by using the International Restless Legs Syndrome Study Group (IRLSSG) Rating Scale. RESULTS: In terms of different categories of RLS severity, a significant decline was observed only in the olive oil group from the pre- to post-intervention stages (P = 0.003). After the intervention, the decline in the total RLS severity was more significant in the olive oil group (P < 0.001), compared to the placebo group (P = 0.019). Moreover, a significant difference in the total RLS severity (P < 0.001) and different categories of RLS severity (P = 0.002) was observed after the intervention between the groups in favor of olive oil massage. However, no significant difference was found between groups in pre-intervention stage in this regard (P = 0.363 and P = 0.955, respectively). CONCLUSION: Application of short-term massage with olive oil as a complementary method seems to be effective in reducing the severity of uremic RLS. Further studies are suggested to identify the sustainability of the findings.