MULTIBUBBLE - Multifunctional microbubbles for improved image-based diagnosis and drug delivery

In the current project, we have developed a novel technology platform based on microbubbles stabilized by biodegradable nanoparticles. Each microbubble is coated by a few thousand nanoparticles and the nanoparticles are carrying a therapeutic drug. The microbubbles are injected into the blood stream and will serve as a contrast agent for ultrasound enabling imaging of small vessels. We have had a special focus on the development and optimization of ultrasound technology enabling imaging of microbubbles at high frequencies which in general is a challenge. Spatial resolution in an ultrasound image increases with imaging frequency, so higher frequencies will give better resolution which is important when imaging small vessels. The transmitted ultrasound intensity can be increased locally at a disease site giving destruction of the microbubbles with a resulting transient increase in the permeability of the vessel wall at the site of bubble destruction. During bubble destruction, the nanoparticles carried by the microbubbles are released and can then proceed to deliver their therapeutic drug load at the disease site by biodegradation. Encapsulation of therapeutic drugs within the nanoparticles has been an important task. The nanoparticles must also be able to stabilize microbubbles after they have been loaded with the desired drug. We have tried to encapsulate various cytotoxic drugs and also hydrocortisone. Several cytotoxic drugs (e.g., Taxanes) were successfully encapsulated within our nanoparticles. Hydrocortisone was not possible to encapsulate within our nanoparticles and this substance is therefore not compatible with our platform. We have chosen to focus on the encapsulation of Taxanes for now since Taxanes generally present difficulties in formulations as medicines due to their poor solubility in water. The nanoparticles have been characterized with respect to size, size distribution, surface charge, aggregation and morphology. Drug loading and PEGylation have been confirmed and quantified. We have developed at system for acoustic characterization of microbubbles where acoustic attenuation and back-scattering can be measured. With this system, we have characterized the microbubbles acoustically and compared them to commercially available microbubbles. Stability and a long blood circulation time is important for the microbubbles, especially in a therapeutic setting. Considerable effort has been put into this task and we have varied parameters such as size of both the nanoparticles and the microbubbles, type of surfactants, type and amount of PEG (polyethylene glycol) and type of gas. We have been able to obtain a microbubble with significantly improved blood circulation time compared to the commercially available microbubble agent SonoVue. A quantitative ultrasound method, based on pulsed Doppler, has been developed for in vivo measurement of microbubble blood circulation time. In vitro toxicity studies of the nanoparticles have been done and preliminary in vivo studies have been carried out in mice. Biodistribution and circulation time in blood for both nanoparticles and microbubbles have been evaluated in mice. High-speed optical observations of microbubble-cell interactions have been studied in vitro in various acoustical fields and compared to observations obtained with commercially available microbubbles. Preliminary tests with a cytotoxic agent encapsulated within the nanoparticles have been carried out on a few different tumor models (breast and prostate) in mice. Based on the results, we have chosen a tumor model that will be used for a larger proof-of-concept study to be carried out in a different project. We have further developed, optimized and tested new ultrasound imaging techniques for improved imaging of microbubbles at high frequencies and for imaging of microbubbles that are not highly resonant (e.g., due to a thick shell). Currently available microbubble imaging techniques have limitations both for imaging at high frequencies in general and for imaging of microbubbles where the encapsulating shell introduces significant stiffness or damping (e.g., due to a thick shell).

The combination of ultrasound and microbubbles is unique for in vivo imaging of the micro-circulation and hence for detecting and visualizing neo-angiogenesis within soft tissue. In the adult body, small microscopic tumours are highly prevalent. The vast majority of these tiny malignant masses will be quiescent and never cause any macroscopic disease. However, some of them will turn into a potentially life-threatening disease. The onset of tumour neo-angiogenesis marks a transition in tumour progression from a dormant state to a rapidly growing and potentially life-threatening state. Similarly for atherosclerosis, it is believed that intraplaque neo-angiogenesis could be a hallmark of vulnerable plaques. However, commercially available thin-shelled microbubbles imaged with conventional ultrasound imaging techniques have several limitations with respect to fragility, blood circulation time, multi-functionality, capacity for drug loading and detectability at high imaging frequencies. To simultaneously utilize the great potential and overcome important limitations found with conventional microbubble-based contrast agents, the project MULTIBUBBLE aims to develop a novel multifunctional microbubble platform that is completely different from available agents. Our platform based on microbubbles stabilized by nanoparticles is unique and potent, combining the strengths of nanoparticles and microbubbles. For robust ultrasound-based imaging and drug delivery with these new and radically different microbubbles, novel ultrasound technology must also be developed. The project will explore knowledge and combine frontiers in nanomaterial science and ultrasound technology to improve diagnosis and therapy of major diseases such as cancer and atherosclerosis. By developing new knowledge within nano material science and ultrasound technology, translating this knowledge into highly relevant clinical applications, this project will have a profound clinical and social impact.