Ultrasound and microbubbles to beat barriers in tumors: Improving delivery of nanomedicine

Successful delivery of drugs and nanomedicine to tumors requires a functional vascular network, extravasation across the capillary wall, penetration through the extracellular matrix, and cellular uptake. Nanomedicine has many merits, but penetration deep into the tumor interstitium remains a challenge. Failure of cancer treatment can be caused by insufficient delivery of the therapeutic agents. After intra-venous administration, nanomedicines are often found in off-target organs and the tumor extracellular matrix close to the capillary wall. With circulating microbubbles, ultrasound exposure focused toward the tumor shows great promise in improving the delivery of therapeutic agents. In this review, we address the impact of focused ultrasound and microbubbles to overcome barriers for drug delivery such as perfusion, extravasation, and transport through the extracellular matrix. Furthermore, we discuss the induction of an immune response with ultrasound and delivery of immuno-therapeutics. The review dis-cusses mainly preclinical results and ends with a summary of ongoing clinical trials.publishedVersio

Real-time intravital multiphoton microscopy to visualize focused ultrasound and microbubble treatments to increase blood-brain barrier permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain. Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.publishedVersio

High-Speed Optical Characterization of Protein-and-Nanoparticle–Stabilized Microbubbles for Ultrasound-Triggered Drug Release

Objective: Ultrasound-triggered bubble-mediated local drug delivery has shown potential to increase therapeutic efficacy and reduce systemic side effects, by loading drugs into the microbubble shell and triggering delivery of the payload on demand using ultrasound. Understanding the behavior of the microbubbles in response to ultrasound is crucial for efficient and controlled release. Methods: In this work, the response of microbubbles with a coating consisting of poly(2-ethyl-butyl cyanoacrylate) (PEBCA) nanoparticles and denatured casein was characterized. High-speed recordings were taken of single microbubbles, in both bright field and fluorescence. Results: The nanoparticle-loaded microbubbles show resonance behavior, but with a large variation in response, revealing a substantial interbubble variation in mechanical shell properties. The probability of shell rupture and the probability of nanoparticle release were found to strongly depend on microbubble size, and the most effective size was inversely proportional to the driving frequency. The probabilities of both rupture and release increased with increasing driving pressure amplitude. Rupture of the microbubble shell occurred after fewer cycles of ultrasound as the driving pressure amplitude or driving frequency was increased. Conclusion: The results highlight the importance of careful selection of the driving frequency, driving pressure amplitude and duration of ultrasound to achieve the most efficient ultrasound-triggered shell rupture and nanoparticle release of protein-and-nanoparticle–stabilized microbubbles.</p

Ultrafast Microscopy Imaging of Acoustic Cluster Therapy Bubbles: Activation and Oscillation

Acoustic Cluster Therapy (ACT®) is a platform for improving drug delivery and has had promising pre-clinical results. A clinical trial is ongoing. ACT® is based on microclusters of microbubbles–microdroplets that, when sonicated, form a large ACT® bubble. The aim of this study was to obtain new knowledge on the dynamic formation and oscillations of ACT® bubbles by ultrafast optical imaging in a microchannel. The high-speed recordings revealed the microbubble–microdroplet fusion, and the gas in the microbubble acted as a vaporization seed for the microdroplet. Subsequently, the bubble grew by gas diffusion from the surrounding medium and became a large ACT® bubble with a diameter of 5–50 μm. A second ultrasound exposure at lower frequency caused the ACT® bubble to oscillate. The recorded oscillations were compared with simulations using the modified Rayleigh–Plesset equation. A term accounting for the physical boundary imposed by the microchannel wall was included. The recorded oscillation amplitudes were approximately 1–2 µm, hence similar to oscillations of smaller contrast agent microbubbles. These findings, together with our previously reported promising pre-clinical therapeutic results, suggest that these oscillations covering a large part of the vessel wall because of the large bubble volume can substantially improve therapeutic outcome.publishedVersio

Contrast Enhanced Magnetic Resonance Imaging of Amyloid-β Plaques in a Murine Alzheimer's Disease Model

Background: Early detection of amyloid-β (Aβ) aggregates is a critical step to improve the treatment of Alzheimer’s disease (AD) because neuronal damage by the Aβ aggregates occurs before clinical symptoms are apparent. We have previously shown that luminescent conjugated oligothiophenes (LCOs), which are highly specific towards protein aggregates of Aβ, can be used to fluorescently label amyloid plaque in living rodents. Objective: We hypothesize that the LCO can be used to target gadolinium to the amyloid plaque and hence make the plaque detectable by T1-weighted magnetic resonance imaging (MRI). Methods: A novel LCO-gadolinium construct was synthesized to selectively bind to Aβ plaques and give contrast in conventional T1-weighted MR images after intravenous injection in Tg-APPSwe mice. Results: We found that mice with high plaque-burden could be identified using the LCO-Gd constructs by conventional MRI. Conclusion: Our study shows that MR imaging of amyloid plaques is challenging but feasible, and hence contrast-mediated MR imaging could be a valuable tool for early AD detection.publishedVersio

Quantification and qualitative effects of different PEGylations on Poly(butyl cyanoacrylate) nanoparticles

Protein adsorption on nanoparticles (NPs) used in nanomedicine leads to opsonization and activation of the complement system in blood, which substantially reduces the blood circulation time of NPs. The most commonly used method to avoid protein adsorption, is to coat the NPs with polyethylene glycol, so called PEGylation. Although PEGylation is of utmost importance for designing the in vivo behavior of the NP, there is still a considerable lack of methods for characterization and fundamental understanding related to the PEGylation of NPs. In this work we have studied four different poly(butyl cyanoacrylate) (PBCA) NPs , PEGylated with different types of PEG-based non-ionic surfactants–Jeffamine M-2070, Brij L23, Kolliphor HS 15, Pluronic F68–or combinations thereof. We evaluated the PEGylation, both quantitatively by nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), and qualitatively by studying zeta-potential, protein adsorption, diffusion, cellular interactions and blood circulation half-life. We found that NMR and ToF-SIMS are complementary methods, while TGA is less suitable to quantitate PEG on polymeric NPs. It was found that longer PEG increases both blood circulation time and diffusion of NPs in collagen gels

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly (ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly (ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.Peer reviewe