Release of vascular agonists from liposome-microbubble conjugate by ultrasound-mediated microbubble destruction: effect on vascular function.

The endothelium is a single cell layer of the vessel wall and a key regulator of blood flow in vascular beds. Local and systemic pathologies have been associated with alterations in endothelial function. However, targeting the endothelium with vasoconstrictor or vasodilator drugs is often accompanied by systemic effects. Here, we evaluated a liposome-microbubble delivery system as a vascular hydrophilic agonist carrier. Phenylephrine (Phe) or acetylcholine (Ach)-loaded liposomes were conjugated to microbubbles. The drug release was triggered by ultrasound (US), and the vascular response was assessed in rat aortic rings using an isolated organ chamber. Aortic rings incubated with Phe-liposome-microbubble conjugate, exposed to US showed a marked contractile response (0.79 ± 0.04 g) compared to empty liposomes conjugated to microbubbles, aortic rings exposed only to US, and Phe-liposome-microbubble conjugate without US exposure that elicited a minimal or no response. Expressed as %, contractile responses were 85.24 ± 4.31% and 12.62 ± 3.23% for Phe-Chol-liposome-microbubble conjugate and empty Chol-liposome-microbubble conjugate exposed to US, respectively. Addition of 1 × 10-5 M Ach to pre-contracted aortic rings decreased the contraction response from 1 to 0.21 g. The addition of Ach-liposome conjugate and exposure to US decreased the contraction response to 0.32 g. Additionally, the ED50 values for Phe and Ach released by US from liposome-microbubble conjugates were 3.6 × 10-8 M ± 2.8 × 10-9 M for Phe and 2.0 × 10-8 M ± 1.8 × 10-9 M. In conclusion, we evaluated a hybrid delivery system that consisted of loaded liposomes conjugated to microbubbles to deliver and release vascular agonists using UMMD.

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response.

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 108 MBs/mL AP-Cav (8 µM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 µM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.

Functional Activity and Endothelial-Lining Integrity of Ex Vivo Arteries Exposed to Ultrasound-Mediated Microbubble Destruction.

Ultrasound-mediated microbubble destruction (UMMD) is a promising strategy to improve local drug delivery in specific tissues. However, acoustic cavitation can lead to harmful bioeffects in endothelial cells. We investigated the side effects of UMMD treatment on vascular function (contraction and relaxation) and endothelium integrity of ex vivo Wistar rat arteries. We used an isolated organ system to evaluate vascular responses and confocal microscopy to quantify the integrity and viability of endothelial cells. The arteries were exposed for 1-3 min to ultrasound at a 100 Hz pulse-repetition frequency, 0.5 MPa acoustic pressure, 50% duty cycle and 1%-5% v/v microbubbles. The vascular contractile response was not affected. The acetylcholine-dependent maximal relaxation response was reduced from 78% (control) to 60% after 3 min of ultrasound exposure. In arteries treated simultaneously with 1 min of ultrasound exposure and 1%, 2%, 3% or 5% microbubble concentration, vascular relaxation was reduced by 19%, 58%, 80% or 93%, respectively, compared with the control arteries. Fluorescent labeling revealed that apoptotic death, detachment of endothelial cells and reduced nitric oxide synthase phosphorylation are involved in relaxation impairment. We demonstrated that UMMD can be a safe technology if the correct ultrasound and microbubble parameters are applied. Furthermore, we found that tissue-function evaluation combined with cellular analysis can be useful to study ultrasound-microbubble-tissue interactions in the optimization of targeted endothelial drug delivery.