Acoustically Driven Microbubbles Enable Targeted Delivery of microRNA-Loaded Nanoparticles to Spontaneous Hepatocellular Neoplasia in Canines

Abstract

Spatially localized microbubble cavitation by ultrasound offers an effective means of altering permeability of natural barriers (i.e. blood vessel and cell membrane) in favor of nanomaterials accumulation in the target site. In this study, a clinically relevant, minimally invasive ultrasound guided therapeutic approach is investigated for targeted delivery of anticancer microRNA loaded PLGA-b-PEG nanoparticles to spontaneous hepatocellular neoplasia in a canine model. Quantitative assessment of the delivered microRNAs revealed prominent and consistent increase in miRNAs levels (1.5-to 2.3-fold increase (p<0.001)) in ultrasound treated tumor regions compared to untreated control regions. Immunohistology of ultrasound treated tumor tissue presented a clear evidence for higher amount of nanoparticles extravasation from the blood vessels. A distinct pattern of cytokine expression supporting CD8+ T cells mediated "cold-to-hot" tumor transition was evident in all patients. On the outset, proposed platform can enhance delivery of miRNA-loaded nanoparticles to deep seated tumors in large animals to enhance chemotherapy.

Keywords: dog model; focused ultrasound; microRNA; microRNA delivery; microbubbles; nanoparticles; targeted delivery.

Figure 1.. Overview of study.

Schematic outline of (A) microRNA-loaded PLGA-b-PEG nanoparticle synthesis and characterization, and (B) in vivo delivery by intravenous infusion of microbubbles and PLGA-b-PEG nanoparticles with an ultrasound transducer positioned on the tumor region for targeted microRNA delivery.

Figure 2.. Characterization of microRNA loaded PLGA-b-PEG NPs and cellular uptake.

(A) size distribution and (B) zeta potential of microRNA-loaded PLGA-b-PEG nanoparticles; (C) microRNA-loading efficiency in PLGA-b-PEG nanoparticles; (D) RNase protection assay for microRNA-loaded in PLGA-b-PEG nanoparticles; (E) Concentration of PLGA-b-PEG nanoparticles estimated by Nanoparticle Tracking Analysis (Nanosight); (F) Endotoxin assay standard plot (green dots) with respect to microRNA-loaded PLGA-b-PEG NP ‎test samples (red dots); (G) Concentration and size distribution of SonoVue microbubble contrast agent estimated by AccuSizer SPOS system; (H) Confocal microscopic images of microRNA-loaded PLGA-b-PEG NPs cellular uptake in HepG2 cells stained with CellLight® Early Endosomes-GFP, BacMam 2.0 Marker and Hoechst 33342, under Bright field, Cy5, GFP, and DAPI filters. (I) Intensity line map over the region of interest (A‘----B‘) shows the intracellular microRNA in PLGA-b-PEG nanoparticles colocalized in endosomes. Data are shown as mean ± SD (n=3).

Figure 3.. Timeline of procedure and evaluation of spontaneous hepatic neoplasia in dog patients.

(A) Schematic outline of complete timeline of procedures involved in the in vivo miRNA delivery study in dog model; (B-D) Ultrasound transducer setup and orientation for specific treatment region in the tumor of each dog patient; Contrast enhanced abdominal computed tomography (CT) images of dog 1 (E), and CT images of dog 2 (F), and dog 3 (G) showing the presence liver tumor mass with different sizes (* - tumor mass). (H) Schematic representation of dog liver anatomy and tumor mass for ultrasound guided microRNA delivery; (I-K) Abdominal ultrasound images of dog patients indicating the liver tumor and region of ultrasound treatment; (L) 3D stereo view of liver lobes and tumor topology in each dog patient.

Figure 4.. RT-PCR quantitation of delivered microRNA.

Quantitative RT-PCR analysis of miR-122 (A, C &E) and anti-miR-21 (B, D &F) in different tissues (normal liver, US treated tumor and untreated tumor), blood, and urine samples of dog patients (Dog 1 (A,B), Dog 2 (C,D) and Dog 3 (E,F)) received US-MB mediated delivery of miRNA loaded PLGA-b-PEG NPs (Violin plot with median, upper and lower quartiles); Comparative radar chart for (G) miR-122 and (H) anti-miR-21 and relative extent delivery in normal liver, treated liver tumor and untreated liver tumor in Dog 1, Dog 2 and Dog 3. Data are shown as mean ± SD (n=3).Unpaired, two tailed Student’s t-test was used for comparison between two groups. *p≤ 0.05, **p≤ 0.001 and ***p≤ 0.0001

Figure 5.. Inflammatory markers expression profile and tissue histopathology.

(A) Quantitative RT-PCR analysis of inflammatory cytokine markers in the blood of the dogs before and after ultrasound guided microRNA delivery (B-D) Immunohistology of CD8+ T cell population in normal liver, untreated liver tumor and US treated liver tumor region; H&E stained sections of (E, H) normal liver, (F, I) untreated liver tumor, and (G, J) US treated liver tumor tissue and associated blood vessel morphology. All data are shown as mean ± SD (n = 3).

Figure 6.. US-MB mediated delivery of PLGA-b-PEG NPs.

Confocal microscopic images of (A) untreated and US treated tumor tissues stained with Anti-PEG, anti-CD-31 and phalloidin (F-actin) red imaged under brightfield, Cy5 (633/670), FITC (488/525) and Phalloidin Red (514/609 nm) respectively; Magnified section of blood vessels in (B&C) untreated and (D&E) US treated tumor tissue showing NPs (Yellow color) permeation across the blood vessel (CD31-Green color) into extracellular space of tumor tissue and corresponding line intensity map of all three channels across the cross-section of blood vessel denoted by A----B and A‘-----B‘ segments.