Ultrasound-Propelled Nanocups for Drug Delivery

Abstract

Ultrasound-induced bubble activity (cavitation) has been recently shown to actively transport and improve the distribution of therapeutic agents in tumors. However, existing cavitation-promoting agents are micron-sized and cannot sustain cavitation activity over prolonged time periods because they are rapidly destroyed upon ultrasound exposure. A novel ultrasound-responsive single-cavity polymeric nanoparticle (nanocup) capable of trapping and stabilizing gas against dissolution in the bloodstream is reported. Upon ultrasound exposure at frequencies and intensities achievable with existing diagnostic and therapeutic systems, nanocups initiate and sustain readily detectable cavitation activity for at least four times longer than existing microbubble constructs in an in vivo tumor model. As a proof-of-concept of their ability to enhance the delivery of unmodified therapeutics, intravenously injected nanocups are also found to improve the distribution of a freely circulating IgG mouse antibody when the tumor is exposed to ultrasound. Quantification of the delivery distance and concentration of both the nanocups and coadministered model therapeutic in an in vitro flow phantom shows that the ultrasound-propelled nanocups travel further than the model therapeutic, which is itself delivered to hundreds of microns from the vessel wall. Thus nanocups offer considerable potential for enhanced drug delivery and treatment monitoring in oncological and other biomedical applications.

Keywords: cancer therapy; cavitation; drug delivery; nanoparticles; ultrasound.

Figure 1

Schematics of the formation of nanocups and a proposed mechanism for nucleating cavitation are shown. (a) Nanocups are produced by a seeded polymerization technique whereby a template nanoparticle comprised of polystyrene (PS) is coated with MMA with divinylbenzene (DVB) as the cross-linker that induces swelling and deformation of the template. After the formation of the “cup,” the nanoparticle suspension is dried. Upon resuspension, the nanocups trap gas within the cavity. (b) The nanocup with nanobubble construct is activated upon exposure to ultrasound. During the rarefactional pressure phase of the ultrasound wave, the nanobubble grows and detaches from the cavity. Once free, the bubble continues to grow uncontrollably until the compressional phase of the ultrasound, which causes the bubble to collapse.

Figure 2

Physical characterization of nanocups demonstrates their uniform shape and size. a) TEM images of representative nanocups. The scale bar represents 500 nm. The inset is a TEM image of a single nanocup, emphasizing the “cup” shape of the nanoparticle. The scale bar represents 100 nm. b,c) DLS size distribution and corresponding correlation function of the nanocups, respectively. d) Surface charge measurements of nanocups.

Figure 3

Tunability of nanocups is shown alongside its influence on cavity size and cavitation response. TEM images of small (100 nm), medium (300 nm), and large (460 nm) seed particles control the diameter and cavity size of the nanocups (that are categorized based on the seed particle diameter). Furthermore, cavity diameter affects the pressure amplitude required to nucleate from cavitation.

Figure 4

Representative images of inertial cavitation events within the tumor. a) Single-frame stills of a B-mode image with a PAM overlay. PAMs were generated during the ±3° acoustic sweep during the background injection and after each of the three injections. b) Summed PAM overlay of the entire acoustic sweep. In all of the images, the dotted white line represents the edge of the tumor (determined by a blind observer). The color bars represent arbitrary units proportional to power. A white arrow demarcates the direction of the ultrasound. The axial and transverse distances are valid for all images. c) Representative maximum PAM signals from a tumor after intravenous injection of SV or nanocups and exposure to ultrasound (0.5 MHz, 1.5 MPa).

Figure 5

Drug delivery with ultrasound and nanocups (NCs). a,b) Representative fluorescent microscope images of CT-26 tumors treated with IgG antibody (green) and nanocups without (a) and with (b) US exposure (0.5 MHz and 1.5 MPa). Scale bar represents 1 mm. The tumor cells are stained blue and the blood vessels are stained red. The images marked by *, **, and *** show varying amounts of antibody inside the tumor (scale bar is 100 μm for all images) for both (a) and (b). The corresponding location of these images are marked on figures (a) and (b) with the respective number of *'s.

Figure 6

Nanocup penetration in a tissue model. a) Representative fluorescent images of the tissue model sliced radially to the channel after exposure to ultrasound for 5 min at 2.2 and 4 MPa. The white dotted lines indicates the edges of the ultrasound focus, and the dotted blue line in represents the edge of the flow vessel. A white arrow demarcates the direction of ultrasound. In the images, the model drug (TRITCD) is labeled in red and the nanocups are labeled in blue (FNCs). The scale bar represents 1 mm, and is valid for all images. b) Average increase in fluorescence intensity profile plots of no cavitation nuclei, SV, and nanocups at both 2.2 MPa and 4 MPa taken down the center line in the direction of ultrasound are shown for each test condition. The white arrow indicates the direction of ultrasound and the blue box represents the flow channel. TRITCD without cavitation nuclei and without ultrasound exposure was used as the reference value. For clarity, only 20 to 25 points are shown with standard deviations.