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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 mPEG 2000 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.

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., Competing Interests: Conflict of interest P.G.D., M.B., P.A.D. and V.P. are all inventors or co-inventors on patents related to oxygen microbubble formulation and/or use. M.B. is also a founder and the chief scientific officer of Respirogen Inc. (Boulder, CO, USA). The other authors declare no competing interests., (Copyright © 2023 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Microbubbles are 1-10 μm diameter gas-filled acoustically-active particles, typically stabilized by a phospholipid monolayer shell. Microbubbles can be engineered through bioconjugation of a ligand, drug and/or cell. Since their inception a few decades ago, several targeted microbubble (tMB) formulations have been developed as ultrasound imaging probes and ultrasound-responsive carriers to promote the local delivery and uptake of a wide variety of drugs, genes, and cells in different therapeutic applications. The aim of this review is to summarize the state-of-the-art of current tMB formulations and their ultrasound-targeted delivery applications. We provide an overview of different carriers used to increase drug loading capacity and different targeting strategies that can be used to enhance local delivery, potentiate therapeutic efficacy, and minimize side effects. Additionally, future directions are proposed to improve the tMB performance in diagnostic and therapeutic applications.

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force ( F rad ) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on F rad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an α v β 3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm 2 . Targeting was assessed by measuring MB attachment for varying F rad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary F rad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with α v β 3 integrin density and F rad time. MB attachment and, to a lesser extent specificity, also increased when driven by F rad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB-MB interactions and aggregations forming from secondary F rad effects as MB concentration was increased. These secondary F rad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary F rad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼10 6 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD-MB targeting.

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.

Optimization of contrast-enhanced imaging and focused ultrasound therapy requires a comprehensive understanding of in vivo microbubble (MB) pharmacokinetics. Prior studies have focused pharmacokinetic analysis on indirect techniques, such as ultrasound imaging of the blood pool and gas chromatography of exhaled gases. The goal of this work was to measure the MB concentration directly in blood and correlate the pharmacokinetic parameters with the MB size and dose. MB volume dose (MVD) was chosen to combine the size distribution and number into a single-dose parameter. Different MB sizes (2, 3, and 5 μm diameter) at 5-40 μL/kg MVD were intravenously injected. Blood samples were withdrawn at different times (1-10 min) and analyzed by image processing. We found that for an MVD threshold < 40 μL/kg for 2 and 3 μm and <10 μL/kg for 5 μm, MB clearance followed first-order kinetics. When matching MVD, MBs of different sizes had comparable half-lives, indicating that gas dissolution and elimination by the lungs are the primary mechanisms for elimination. Above the MVD threshold, MB clearance followed biexponential kinetics, suggesting a second elimination mechanism mediated by organ retention, possibly in the lung, liver, and spleen. In conclusion, we present the first direct MB pharmacokinetic study, demonstrate the utility of MVD as a unified dose metric, and provide insights into the mechanisms of MB clearance from circulation.

A photoacoustic contrast mechanism is presented based on the photoacoustic fluctuations induced by microbubbles flowing inside a micro-vessel filled with a continuous absorber. It is demonstrated that the standard deviation of a homogeneous absorber mixed with microbubbles increases non-linearly as the microbubble concentration and microbubble size is increased. This effect is then utilized to perform photoacoustic fluctuation imaging with increased visibility and contrast of a blood flow phantom., (© 2022 Author(s).)

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 × 10 8 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., Competing Interests: Conflict of interest disclosure The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Using thermoluminiscent dosimeters the absorbed dose in the bladder, rectum and thyroid have been evaluated when 200 cGy was applied to the prostate. The treatment was applied with a (60)Co unit. A water phantom was built and thermoluminiscent dosimeters were located in the position where the prostate, bladder, rectum and thyroid are located. The therapeutic beam was applied in 4 irradiations at 0, 90, 180 and 270° with the prostate at the isocenter. The TLDs readouts were used to evaluate the absorbed dose in each organ. The absorbed doses were used to estimate the effective doses and the probability of developing secondary malignacies in thyroid, rectum and bladder., (Copyright © 2013 Elsevier Ltd. All rights reserved.)