Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery.We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG) coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air/liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NPs/MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue providing improved therapy and imaging contrast compared to freely distributed NPs.

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Ultrasound-mediated delivery of a novel nanoparticle-microbubble platform. A major obstacle in delivery of nanoparticles (NPs) to tumor cells is the low uptake and heterogeneous distribution of the NPs in tumor tissue. Ultrasound (US) may improve the delivery of encapsulated drug in various ways depending on the frequency and intensity applied, by inducing heating, radiation force or cavitation. We have developed a novel multimodal, multifunctional drug delivery system consisting of microbubbles stabilized by polymeric NPs to be used in US-mediated delivery of NPs. Miniemulsion polymerization was used to prepare NPs of the biocompatible and biodegradable polymer poly(butyl-2-cyanoacrylate) (PBCA). The NPs were coated with PEG to improve the circulation time and biodistribution. Microbubbles stabilized by these NPs were prepared by mixing the NP dispersion with proteins and air using an ultra-turrax. The aim of the present work was to study the cellular uptake of the NP in vitro and the microdistribution of the NP in tumor tissue in vivo. Human prostate cancer cells were incubated with fluorescently labeled (Nile red and DiR) NPs and the cellular uptake measured by flow cytometry and confocal laser scanning microscopy (CLSM). Prostate cancer xenografts were grown subcutaneously on the leg of athymic mice, and NP alone or NPs stabilizing microbubbles were injected intravenously. The particles circulated for 5 min or 24 hr, before the tumors were exposed to US, thus the effect of US both on extravasation and penetration through the extracellular matrix could be studied. The tumors were exposed to a focused US beam at low (300 kHz or 1 MHz) or high (5 MHz) frequency, applying various intensities. The blood vessels were visualized by injection of FITC- tomato lectin 5 min before euthanizing the mice. The distribution of NPs was studied by CLSM, imaging frozen tumor sections along a radial track from the periphery of the tumor sections. The biodistribution of NPs comparing the uptake in normal and tumor tissue, was studied by whole animal optical imaging. The cellular uptake of the NPs in vitro depended on the length and type of PEG used. In vivo, ultrasound enhanced the uptake and improved the distribution of the NPs in the extracellular matrix. In untreated tumors only small amounts of NPs were observed and they were located close to the blood vessels. In the US-exposed tumors, the uptake was enhanced and the NP had penetrated further away from the blood vessels compared with unexposed tumors. US administered 5 min after NP-injection was more efficient than US given after 24 h. This demonstrates that the effect of US on extravasation is more important than the effect on penetration of NPs through the extracellular matrix. A prerequisite for successful cancer therapy is that the cytotoxic drugs reach all the cancer cells. The present results demonstrate that US improves the delivery of NPs, and mainly by increasing the permeability of the capillary wall. Citation Format: Catharina De Lange Davies, Siv Eggen, Stein-Martin Fagerland, Mercy Afadzi, Audun Dybvik Bøhn, Håkon Furu, Rune Hansen, Bjørn Angelsen, Per Stenstad, Yrr Mørch. Ultrasound-mediated delivery of a novel nanoparticle-microbubble platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5618. doi:10.1158/1538-7445.AM2013-5618