Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer

Abstract

Ultrasound induced microbubble cavitation can cause enhanced permeability across natural barriers of tumors such as vessel walls or cellular membranes, allowing for enhanced therapeutic delivery into the target tissues. While enhanced delivery of small (<1nm) molecules has been shown at acoustic pressures below 1MPa both in vitro and in vivo, the delivery efficiency of larger (>100nm) therapeutic carriers into cancer remains unclear and may require a higher pressure for sufficient delivery. Enhanced delivery of larger therapeutic carriers such as FDA approved pegylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NP) has significant clinical value because these nanoparticles have been shown to protect encapsulated drugs from degradation in the blood circulation and allow for slow and prolonged release of encapsulated drugs at the target location. In this study, various acoustic parameters were investigated to facilitate the successful delivery of two nanocarriers, a fluorescent semiconducting polymer model drug nanoparticle as well as PLGA-PEG-NP into human colon cancer xenografts in mice. We first measured the cavitation dose produced by various acoustic parameters (pressure, pulse length, and pulse repetition frequency) and microbubble concentration in a tissue mimicking phantom. Next, in vivo studies were performed to evaluate the penetration depth of nanocarriers using various acoustic pressures, ranging between 1.7 and 6.9MPa. Finally, a therapeutic microRNA, miR-122, was loaded into PLGA-PEG-NP and the amount of delivered miR-122 was assessed using quantitative RT-PCR. Our results show that acoustic pressures had the strongest effect on cavitation. An increase of the pressure from 0.8 to 6.9MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9MPa, the amount of nanoparticles deposited in cancer xenografts was increased from 4- to 14-fold, and the median penetration depth of extravasated nanoparticles was increased from 1.3-fold to 3-fold, compared to control conditions without ultrasound, as examined on 3D confocal microscopy. When delivering miR-122 loaded PLGA-PEG-NP using optimal acoustic settings with minimum tissue damage, miR-122 delivery into tumors with ultrasound and microbubbles was 7.9-fold higher compared to treatment without ultrasound. This study demonstrates that ultrasound induced microbubble cavitation can be a useful tool for delivery of therapeutic miR loaded nanocarriers into cancer in vivo.

Keywords: Cancer; Drug delivery; Image guidance; Nanocarriers; Therapy; Ultrasound.

Figure 1

Experiment setup for phantom studies. The cavitation initiation transducer was operated at 1.8MHz to induce microbubble cavitation. Detection of cavitation was performed both passively using a 10-MHz single element transducer as well as actively by using a 5-MHz ultrasound imaging transducer. The cavitation initiation and detection transducers were placed perpendicular to each other and co-focused at the cavitation chamber containing microbubbles.

Figure 2

In vivo experimental setup and treatment timeline. The focal beam of the cavitation initiation transducer was electronically steered across 6 locations to cover the entire tumor volume. The high frequency imaging transducer was aimed at the target tumors at 45° angle to provide anatomical guidance before the treatment and to monitor the microbubble perfusion/destruction process during the treatment. Microbubble perfusion and ultrasound insonation were alternated in the 10-minute-length treatment windows.

Figure 3

Inertial cavitation dose (ICD) measured in the tissue mimicking phantom by the 10-MHz passive cavitation detection transducer after varying a) pressure, b) pulse length, c) pulse repetition frequency (PRF), and d) microbubble concentration. Error bars = ± standard deviation (n=6 each). *p < 0.001.

Figure 4

a) Representative images of microbubble solution in tissue mimicking phantom after exposure to ultrasound at different acoustic pressures. b) Hyperechoic flickering cluster (in the red dashed circle), likely caused by cavitating microbubbles, was seen at the focus of the transducer during the exposure to ultrasound at 6.9 MPa. The hyperechoic vertical lines (blue arrows) are artifacts from interferences of the cavitation pulses. c) The signal intensity in the focal region, marked as red box in panel (a), was plotted against the pressures. Note that significant acoustic shadowing was caused by the concentrated microbubble solution before ultrasound exposure. As microbubbles were destroyed by ultrasound, the shadow disappeared, and the phantom image below the cavitation chamber became visible. The focal region of the cavitation transducer appeared hypoechoic after ultrasound exposure, indicating destruction of microbubbles. The spatial extent of microbubble destruction was broadened with increasing pressures. Data are mean ± standard deviation (n=4 each). Images are displayed with a 40dB dynamic range. Other treatment parameters were fixed at pulse length = 5 cycles, PRF = 100Hz, and microbubble concentration = 1×10⁸/mL.

Figure 5

Representative contrast-enhanced ultrasound images of a human colon cancer xenograft during a 2-minute treatment cycle. Image signal increased during the perfusion period with microbubbles floating into the tumor, and then substantially decreased during sonoporation. Microbubble destruction occurred quickly after ultrasound application. The discrete drop of image signal intensity (see images at 70 sec, 80 sec, and 90 sec) corresponded to each steering of focal beam.

Figure 6

a) Representative immunofluorescence images of human colon cancer xenografts treated with different ultrasound pressures show increased amount of SPN (red) delivered into the tumor tissue with increasing pressures compared to no ultrasound (control). Note, tumor vessels are stained in green and tumor cytoskeleton was stained in blue. Ratios of the total area of SPN to the total area of CD31 vessel staining (b) and to the total area of F-actin cytoskeleton staining (c) are also shown. Both ratios increased with increasing pressures. Data are mean ± standard deviation (n= 5 each).

Figure 7

a) Representative maximum intensity confocal immunofluorescence image reconstructed from 3D confocal microscopy data sets of human colon cancer xenograft treated with ultrasound and microbubbles (6.9 MPa) shows extravasated SPN (red) compared to tumor vessels (green) in the imaged volume. Orthogonal slices indicated by the gray dashed lines further confirmed presence of SPN in tumor parenchyma (blue) and away from the vessel lumen (contoured in green). b) Box blot shows changing tumor penetration depth with various acoustic pressure levels. The central mark, upper edge, lower edge, and whiskers of each box indicate median, 75^th, 25^th percentile, and the range of 54 measurements made for each acoustic pressure level.

Figure 8

a) Representative H&E images of human colon cancer xenografts treated with different acoustic pressures. Small areas of hemorrhage (as shown in the close-up view of the boxed area) was observed in some of the treated tumors. b) Box plot shows a slightly increasing trend towards higher hemorrhage at higher acoustic pressures. Each dot represents individual data points and the bars label the mean in each group. Note that there was already hemorrhage in non-treated tumors (n = 5 each).

Figure 9

RT-PCR quantification of the fold change in miR-122 in tumors treated with control PLGA-PEG-NP (left, n = 3 each) or miR122 loaded PLGA-PEG-NP (right, n = 7 each). The miR-122 levels were expressed as fold changes relative to the endogenous levels of miR-122 in control animals with neither nanoparticle administration nor ultrasound and microbubble treatments. *p = 0.004. **p = 0.002. ***p = 0.001.