Focused ultrasound-mediated drug delivery to pancreatic cancer in a mouse model

Abstract

Background: Many aspects of the mechanisms involved in ultrasound-mediated therapy remain obscure. In particular, the relative roles of drug and ultrasound, the effect of the time of ultrasound application, and the effect of tissue heating are not yet clear. The current study was undertaken with the goal to clarify these aspects of the ultrasound-mediated drug delivery mechanism.

Methods: Focused ultrasound-mediated drug delivery was performed under magnetic resonance imaging guidance (MRgFUS) in a pancreatic ductal adenocarcinoma (PDA) model grown subcutaneously in nu/nu mice. Paclitaxel (PTX) was used as a chemotherapeutic agent because it manifests high potency in the treatment of gemcitabine-resistant PDA. Poly(ethylene oxide)-co-poly(d,l-lactide) block copolymer stabilized perfluoro-15-crown-5-ether nanoemulsions were used as drug carriers. MRgFUS was applied at sub-ablative pressure levels in both continuous wave and pulsed modes, and only a fraction of the tumor was treated.

Results: Positive treatment effects and even complete tumor resolution were achieved by treating the tumor with MRgFUS after injection of nanodroplet encapsulated drug. The MRgFUS treatment enhanced the action of the drug presumably through enhanced tumor perfusion and blood vessel and cell membrane permeability that increased the drug supply to tumor cells. The effect of the pulsed MRgFUS treatment with PTX-loaded nanodroplets was clearly smaller than that of continuous wave MRgFUS treatment, supposedly due to significantly lower temperature increase as measured with MR thermometry and decreased extravasation. The time of the MRgFUS application after drug injection also proved to be an important factor with the best results observed when ultrasound was applied at least 6 h after the injection of drug-loaded nanodroplets. Some collateral damage was observed with particular ultrasound protocols supposedly associated with enhanced inflammation.

Conclusion: This presented data suggest that there exists an optimal range of ultrasound application parameters and drug injection time. Decreased tumor growth, or complete resolution, was achieved with continuous wave ultrasound pressures below or equal to 3.1 MPa and drug injection times of at least 6 h prior to treatment. Increased acoustic pressure or ultrasound application before or shortly after drug injection gave increased tumor growth when compared to other protocols.

Keywords: MRgFUS; Pancreatic cancer; Perfluorocarbon nanoemulsions; Ultrasound-mediated drug delivery.

Figure 1

A nanoparticle size distribution for 5% PEG-PDLA/1% PFCE formulation. Fifty-nanometer particles are residual micelles; two-hundred sixty-two-nanometer particles are nanodroplets. Nanodroplet size may be decreased by increasing sonication pressure during emulsification. Micelle fraction can be decreased by decreasing copolymer concentration and/or increasing PFCE concentration [46]. PTX loading slightly increases nanodroplet sizes (e.g., from 260 to 280 nm).

Figure 2

Schematic representation and axial image of mouse on small-animal MRgFUS device. (A) Schematic representation of the mouse positioning on the small animal MRgFUS device; (B) An axial image of mouse 59 on the small animal MRgFUS device with labeled transducer and agar holder. The white arrow indicates the tumor (initial size 455 mm³).

Figure 3

Spiral and grid ultrasound beam patterns used in the study. Transducer speed was 1 and 2.5 mm/s in the (A) 5-mm- and (B) 8-mm-diameter spiral patterns, respectively, and 0.1 mm/s in the (C) grid pattern.

Figure 4

Mouse photographs and whole-body fluorescence images before and after combined PTX-loaded nanodroplet and MRgFUS treatment. Photographs (A, C) and whole-body fluorescence images (B, D) of a mouse before (A, B) and after (C, D) combined treatment with PTX-loaded nanodroplets and MRgFUS. The dashed circles in (B,D) indicate the tumor location. Treatment parameters: MRgFUS was applied 8 h after drug injection; spiral beam pattern (5-mm diameter) shown in Figure 3A; FUS at 3.1 MPa; sonication time of 3 min. The tumor did not recur during a 5-month observation. The former location of the tumor is still slightly visible in D, indicated by the dashed white circle.

Figure 5

Images of mouse, maximum temperature projection in time, MRgFUS trajectory, temperature response for voxels, and time curve. (A) Axial image of a mouse on the MRgHIFU device. The transducer and agar holder are shown. (B) Coronal image through the tumor with region of interest defined. (C) Maximum temperature projection in time within region of interest. White rectangle indicates the grid trajectory boundaries. (D) MRgFUS trajectory with plotted voxel locations indicated. One-millimeter spacing between all points. (E) Temperature response as a function of time for voxels indicated in (D). (F) Time curve of the mean temperature rise in the treatment plane for all voxels >5°C. Total MRgFUS time was 350 s at a pressure of 3.4 MPa.

Figure 6

Mouse photographs and whole-body fluorescence images before and after combined PTX-loaded nanodroplet and MRgFUS treatment. Mouse photographs (A, C) and whole-body fluorescence images (B, D) taken before (A, B) and 3 weeks after treatment (C,D) with PTX-loaded nanodroplets and MRgFUS therapy; ultrasound was applied 8 h after drug injection at 2.4 MPa with a grid trajectory. The tumor location is indicated by a dashed circle in all images.

Figure 7

Temperature response for the mouse shown in Figure 6. The temperature rise for three individual voxels indicated in the treatment path is shown.

Figure 8

Tumor growth curves. Control (N = 3, squares); Tumors treated with PTX-loaded nanodroplets without ultrasound (N = 7, triangles); Best results for tumors treated with PTX-loaded nanodroplets and MRgFUS (N = 4, diamonds).

Figure 9

Effect of ultrasound pressure on the tumor growth curves in the presence of PTX-loaded nanodroplets. No MRgFUS (N = 7, diamonds); MRgFUS at 4.2 MPa (N = 3, triangles); MRgFUS at 4.8 MPa (N = 2, squares).

Figure 10

MRI showing gas-filled intestines in the ultrasound far-field. This image is representative for seven mice that died within several days after MRgFUS treatment. The transducer and agar holder are labeled. The long and short arrows identify the tumor and intestines, respectively.