Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics.

Microbubbles (MBs) hold substantial promise for medical imaging and therapy; nonetheless, knowledge gaps persist between composition, structure, and in vivo performance, especially with respect to pharmacokinetics. Of particular interest is the role of the poly(ethylene glycol) (PEG) layer, which is thought to shield the MB against opsonization and rapid clearance but is also known to cause an antibody response upon multiple injections. The goal of this study was, therefore, to elucidate the role of the PEG layer in circulation persistence of MBs in the naïve animal (prior to an adaptive immune response). Here, we directly observe the number and size of individual MBs obtained from blood samples, unifying size and concentration into the microbubble volume dose (MVD) parameter. This approach enables direct evaluation of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately assessing the lipid or gas components. We examined the in vivo circulation persistence of 3 µm diameter phospholipid-coated MBs with three different mPEG2000 content: 2 mol % (mushroom), 5 mol % (intermediate), and 10 mol % (brush). MB size and concentration in the blood were evaluated by a hemocytometer analysis over 30 min following intravenous injections of 20 and 40 µL/kg MVD in Sprague-Dawley rats. Interestingly, pharmacokinetic analysis demonstrated that increasing PEG concentration on the MB surface resulted in faster clearance. This was evidenced by a 1.6-fold reduction in half-life and area under the curve (AUC) (p < 0.05) in the central compartment. Conversely, the AUC in the peripheral compartment increased with PEG density, suggesting enhanced MB trapping by the mononuclear phagocyte system. This was supported by an in vitro assay, which showed a significant rise in complement C3a activation with a higher PEG content. In conclusion, a minimal PEG concentration on the MB shell (mushroom configuration) was found to prolong circulation and mitigate immunogenicity.

Effect of Anesthetic Carrier Gas on In Vivo Circulation Times of Intravenously Administered Phospholipid Oxygen Microbubbles in Rats.

OBJECTIVE: For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics. METHODS: Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas. RESULTS: Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles. CONCLUSION: Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air.

Nanobubbles are Non-Echogenic for Fundamental-Mode Contrast-Enhanced Ultrasound Imaging.

Microbubbles (1-10 µm diameter) have been used as conventional ultrasound contrast agents (UCAs) for applications in contrast-enhanced ultrasound (CEUS) imaging. Nanobubbles (<1 µm diameter) have recently been proposed as potential extravascular UCAs that can extravasate from the leaky vasculature of tumors or sites of inflammation. However, the echogenicity of nanobubbles for CEUS remains controversial owing to prior studies that have shown very low ultrasound backscatter. We hypothesize that microbubble contamination in nanobubble formulations may explain the discrepancy. To test our hypothesis, we examined the size distributions of lipid-coated nanobubble and microbubble suspensions using multiple sizing techniques, examined their echogenicity in an agar phantom with fundamental-mode CEUS at 7 MHz and 330 kPa peak negative pressure, and interpreted our results with simulations of the modified Rayleigh-Plesset model. We found that nanobubble formulations contained a small contamination of microbubbles. Once the contribution from these microbubbles is removed from the acoustic backscatter, the acoustic contrast of the nanobubbles was shown to be near noise levels. This result indicates that nanobubbles have limited utility as UCAs for CEUS.

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.