Natalya Rapoport – natasha.rapoport@utah.edu
Department of Bioengineering
University of Utah
36 S. Wasatch Dr., Room 3100
Salt Lake City, Utah 84112
USA

Popular version of paper 3aBA1, “Ultrasound-mediated drug targeting to tumors: Revision of paradigms through intravital imaging”
Presented Wednesday morning, May 25, 2016, 8:15 AM in Salon H
171^st ASA Meeting, Salt Lake City

More than a century ago, Nobel Prize laureate Paul Ehrlich formulated the idea of a “magic bullet”. This is a virtual drug that hits its target while bypassing healthy tissues. No field of medicine could benefit more from the development of a “magic bullet” than cancer chemotherapy, which is complicated by severe side effects. For decades, the prospects of developing “magic bullets” remained elusive. During the last decade, progress in nanomedicine has enabled tumor-targeted delivery of anticancer drugs via their encapsulation in tiny carriers called nanoparticles. Nanoparticle tumor targeting is based on the “Achilles’ heels” of cancerous tumors – their poorly organized and leaky microvasculature. Due to their size, nanoparticles are not capable to penetrate through a tight healthy tissue vasculature. In contrast, nanoparticles penetrate through a leaky tumor microvasculature thus providing for localized accumulation in tumor tissue.  After tumor accumulation of drug-loaded nanoparticles, a drug should be released from the carrier to allow penetration into a site of action (usually located in a cell cytoplasm or nucleus). A local release of an encapsulated drug may be triggered by tumor-directed ultrasound; application of ultrasound has additional benefits: ultrasound enhances nanoparticle penetration through blood vessel walls (extravasation) as well as drug uptake (internalization) by tumor cells.

For decades, ultrasound has been used only as an imaging modality; the development of microbubbles as ultrasound contrast agents in early 2000s has revolutionized imaging. Recently, microbubbles have attracted attention as drug carriers and enhancers of drug and gene delivery. Microbubbles could have been ideal carriers for the ultrasound-mediated delivery of anticancer drugs.  Unfortunately, their micron-scale size does not allow effective extravasation from the tumor microvasculature into tumor tissue. In Dr. Rapoport’s lab, this problem has been solved by the development of nanoscale microbubble precursors, namely drug-loaded nanodroplets that converted into microbubbles under the action of ultrasound[1-6]. Nanodroplets comprised a liquid core formed by a perfluorocarbon compound and a two-layered drug-containing polymeric shell (Figure 1). An aqueous dispersion of nanodroplets is called nanoemulsion.

Figure 1. Schematic representation of a drug-loaded nanodroplet

A suggested mechanism of therapeutic action of drug-loaded perfluorocarbon nanoemulsions is discussed below [3, 5, 6]. A nanoscale size of droplets (ca. 250 nm) provides for their extravasation into a tumor tissue while bypassing normal tissues, which is a basis of tumor targeting. Upon nanodroplet tumor accumulation, tumor-directed ultrasound triggers nanodroplet conversion into microbubbles, which in turn triggers release of a nanodroplet-encapsulated drug. This is because in the process of the droplet-to-bubble conversion, particle volume increases about a hundred-fold, with a related decrease of a shell thickness. Microbubbles oscillate in the ultrasound field, resulting in a drug “ripping” off a thin microbubble shell (Figure 2). In addition, oscillating microbubbles enhance internalization of released drug by tumor cells.

Figure 2. Schematic representation of the mechanism of drug release from perfluorocarbon nanodroplets triggered by ultrasound-induced droplet-to-bubble conversion; PFC – perfluorocarbon

This tumor treatment modality has been tested in mice bearing breast, ovarian, or pancreatic cancerous tumors and has been proved very effective. Dramatic tumor regression and sometimes complete resolution was observed when optimal nanodroplet composition and ultrasound parameters were applied.

3A.

3B.

3C.

(Figure 3. A – Photographs of a mouse bearing a subcutaneously grown breast cancer tumor xenograft treated by four systemic injections of the nanodroplet-encapsulated anticancer drug paclitaxel (PTX) at a dose of 40 mg/kg as PTX. B – Photographs of a mouse bearing two ovarian carcinoma tumors (a) – immediately before and (b) – three weeks after the end of treatment; mouse was treated by four systemic injections of the nanodroplet-encapsulated PTX at a dose of 20 mg/kg as PTX; only the right tumor was sonicated. C – Photographs (a, c) and fluorescence images (b, d) of a mouse bearing fluorescent pancreatic tumor taken before (a, b) and three weeks after the one-time treatment with PTX-loaded nanodroplets at a dose of 40 mg/kg as PTX (c,d). The tumor was completely resolved and never recurred)[3, 4, 6].

In the current presentation, the proposed mechanism of a therapeutic action of drug-loaded, ultrasound-activated perfluorocarbon nanoemulsions has been tested using intravital laser fluorescence microscopy performed in collaboration with Dr. Brian O’Neill (then with Houston Methodist Research Institute, Houston, Texas) [2]. Fluorescently labeled nanocarrier particles (or a fluorescently labeled drug) were systemically injected though the tail vein to anesthetized live mice bearing subcutaneously grown pancreatic tumors. Nanocarrier and drug arrival and extravasation in the region of interest (i.e. normal or tumor tissue) were quantitatively monitored. Various drug nanocarriers in the following size hierarchy were tested: individual polymeric molecules; tiny micelles formed by a self-assembly of these molecules; nanodroplets formed from micelles. The results obtained confirmed the mechanism discussed above.

• As expected, dramatic differences in the extravasation rates of nanoparticles were observed.
• The extravsation of individual polymer molecules was extremely fast even in the normal (thigh muscle) tissue; In contrast, the extravasation of nanodroplets into the normal tissue was very slow. (Figure 4. A – Bright field image of the adipose and thigh muscle tissue. B,C – extravasation of individual molecules (B – 0 min; C – 10 min after injection); vasculature lost fluorescence while tissue fluorescence increased. D,E – extravasation of nanodroplets; blood vessel fluorescence was retained for an hour of observation (D – 30 min; E – 60 min after injection).
• Nanodroplet extravasation into the tumor tissue was substantially faster than that into the normal tissue thus providing for effective nanodroplet tumor targeting.
• Tumor-directed ultrasound significantly enhanced extravasation and tumor accumulation of both, micelles and nanodroplets (Figure 5). Also, pay attention to a very irregular tumor microvasculature, to be compared with that of a normal tissue shown in Figure 4.
• The ultrasound effect on nanodroplets was 3-fold stronger than that on micelles thus making nanodroplets a better drug carriers for ultrasound-mediated drug delivery.
• On a negative side, some premature drug release into the circulation that preceded tumor accumulation was observed. This proposes directions for a further improvement of nanoemulsion formulations.

Figure 5. Effect of ultrasound on the extravasation of Fluorescence of blood vessels dropped while that of the tumor tissue increased after ultrasound