Tumor-targeted chemotherapy is an area of active research with the promise to overcome serious problems of current cancer therapy, potentially realizing the “magic bullet” concept formulated by Paul Ehrlich a century ago. The promising approach towards achieving this goal is in the development of stimuli-responsive drug carriers that release their payload locally in tumor tissue under the action of internal (e.g. pH) or external stimuli, such as heat, light, or ultrasound [1
]. Local application of external stimuli requires tumor imaging prior to, and during treatment. Imaging can also be used to monitor drug carrier biodistribution. Such information allows optimal timing of external stimulus application.
In the last decade, advances in nanomedicine have allowed the combination of various functionalities (e.g. chemotherapeutic agent, imaging agent, and targeting moiety) in one molecular or supramolecular construct. These constructs may be macromolecules or nanoparticles. The family of nanoparticles of biomedical importance includes polymeric micelles, liposomes, hollow particles, nano-emulsion droplets etc. Properly designed nanoparticles avoid extravasation to normal tissues and recognition by cells of the reticulo-endothelial system (RES); these properties prolong nanoparticles circulation time after systemic injection. This in turn allows passive targeting of cancerous or inflamed tissues. Passive targeting is based on the so called enhanced permeability and retention (EPR) effect [2
] that allows extravasation of drug-loaded nanoparticles through defective tumor microvasculature characterized by large inter-endothelial gaps. Characteristic pore cutoff size range between 380 and 780 nm have been shown in a variety of tumors, though in some tumors the size may increase up to 2 μm [3
]. In addition to enhanced vascular permeability, tumors demonstrate poor lymphatic drainage ensuring prolonged retention of the extravasated particles in tumor tissue. In contrast to tumors, blood vessels in normal tissues have tight inter-endothelial junctions that do not allow extravasation of nanoparticles.
Efficient tumor accumulation of nanoparticles via the EPR effect requires sufficient particle residence time in circulation. To provide for this, nanoparticles are commonly coated with poly(ethylene oxide) chains that suppress blood protein adsorption and particle recognition by RES cells.
Among feasible external stimuli, ultrasound is especially attractive by virtue of its accessibility, cost effectiveness, and the possibility to combine imaging and therapeutic capabilities. Sonication may be performed non-invasively or with minimum invasiveness through intraluminal, laparoscopic or percutaneous means. For extracorporeal sonication, the transducer is placed in contact with a water-based gel or a water layer on the skin, and no insertion or surgery is required. As a therapeutic modality, ultrasound may be delivered with millimeter precision and may be directed toward deeply located body sites. As an imaging modality, ultrasound delivers real time information.
The information produced by ultrasound imaging may be enhanced by application of ultrasound contrast agents, i.e. microbubbles. For many decades, microbubbles have been used in clinical practice only as ultrasound contrast agents. During the last decade, microbubbles have attracted attention as drug carriers and enhancers of drug and gene delivery and are now being widely investigated for this application [5
]. However, currently used ultrasound contrast agents present a number of inherent problems as drug carriers to solid tumors. Their short circulation time (minutes) and relatively large size (two to ten microns) do not allow effective extravasation into tumor tissue, preventing efficient tumor targeting.
One way to solve this problem is to develop nano-sized microbubble precursors
that would effectively accumulate in tumor tissue (via the EPR effect or through active targeting) and then convert into microbubbles in situ
under the action of tumor-directed ultrasound. With this in mind, we have recently developed novel drug-loaded perfluorocarbon nanoemulsions stabilized by biodegradable amphiphilic block copolymers [1
]. Bubbles are produced from these nanodroplets under the action of ultrasound. Block copolymer shells of nanodroplets provide for high in vivo
stability and allow accumulation in the tumor volume via
the EPR effect; active targeting is also possible. We have shown that without ultrasound, chemotherapeutic drug (paclitaxel, PTX) was tightly retained by nanodroplets stabilized with poly(ethylene oxide)-co-polycaprolactone (PEG-PCL) block copolymer; however, drug was effectively released into tumor volume under the action of tumor-directed ultrasound, which resulted in effective tumor regression [5
]. In addition, nanodroplets produced long-lasting ultrasound contrast that was substantially enhanced upon droplet-to-bubble transition [5
Our first generation of perfluorocarbon nanoemulsion drug carrier comprised perfluoropentane (PFP) as a droplet core [1
]. The PFP has a boiling temperature of 29 °C; however, thermally induced droplet-to-bubble conversion is hampered by high Laplace pressure inside nanodroplets; in contrast, nanodroplets easily convert into microbubbles under the action of therapeutic ultrasound [29
]. Ultrasound-induced droplet-to-bubble transition is called acoustic droplet vaporization (ADV) [32
]. Under the action of therapeutic ultrasound, microbubbles formed via
ADV undergo acoustic cavitation. It has been shown that droplet-to-bubble transition and bubble oscillation result in release of encapsulated drug and enhanced intracellular uptake [5
]. Stable cavitation of microbubbles has been implicated as the main mechanism of enhanced gene or drug delivery [5
Systemically injected PTX-loaded PFP nanoemulsions combined with 1-MHz ultrasound resulted in effective tumor regression in mouse models of breast, ovarian, and pancreatic cancer [5
]. However, PFP easily forms foam if improperly handled; in addition, its irreversible droplet-to-bubble transition is hard to control. In order to replace the PFP as a droplet core, we have screened various perfluorocarbon compounds with a higher stability. Among those, perfluoro-15-crown-5-ether (PFCE, boiling temperature of 146 °C) has attracted special attention due to a plethora of useful properties. PFCE contains 20 equivalent 19
F nuclei that generate a single resonance peak in 19
F magnetic resonance imaging [38
]. It is possible to use existing proton nuclear magnetic resonance (NMR) instrumentation with minor adjustments to detect fluorinated species with high sensitivity (~83% relative to 1
H). It is also highly beneficial that in vivo,
the endogenous fluorine is found primarily in bones and teeth as solid fluorides with undetectable NMR signals. This allows 19
F MRI to be used to track the biodistribution of exogenously administered fluorinated tracers in vivo.
In the present study, we tested the feasibility of using 19
F MRI to determine the localisation of fluorine nanodroplets after systemic injection. The combination of 19
F and proton MRI depicted the anatomic location of the injected nanodroplets.
PFCE nanoemulsions have been investigated earlier along with perfluorooctylbromide nanoemulsions as 19
F MRI contrast agents and antiangiogenic drug delivery agents in a series of works by the Lanza and Wickline’s team [41
]. The perfluorocarbon nanoemulsions used in these works were stabilized by phospholipid membranes functionalized for active delivery to fibrin or neo-angiogenic sites of tumor bearing mice. For these systems, a unique mechanism of intracellular drug delivery was postulated, based on a so-called contact facilitated delivery where phospholipid membranes of nanodroplets merged with cell membranes of target cells thus delivering their drug payload directly into the cytoplasm.
The nanodroplets used in our studies have a different structure. They are stabilized with amphiphilic poly(ethylene oxide) (PEG)-containing block copolymers and therefore are coated with PEG chains, which are expected to show lower nanodroplet uptake by the RES cells; this can also decrease or prevent contact facilitated drug delivery. The mechanism of drug delivery by the perfluorocarbon nanodroplets described in the present work is anticipated to be different from that described for phospholipid-stabilized nanodroplets. However the properties that allow combining imaging and ultrasound-mediated therapy are inherent to both types of nanoemulsions.
This paper reports the first results on the acoustic properties, ultrasound imaging, 19F MRS and MRI, and therapeutic properties of block copolymer stabilized PFCE nanoemulsions.