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 (<1 nm) molecules has been shown at acoustic pressures below 1MPa both in vitro and in vivo, the delivery efficiency of larger (>100 nm) 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 6.9 MPa. 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 6.9 MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9 MPa, 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.

Histotripsy produces tissue fractionation through dense energetic bubble clouds generated by short, high-pressure, ultrasound pulses. Conventional histotripsy treatments have used longer pulses from 3 to 10 cycles wherein the lesion-producing bubble cloud generation depends on the pressure-release scattering of very high peak positive shock fronts from previously initiated, sparsely distributed bubbles (the “shock-scattering” mechanism). In our recent work, the peak negative pressure (P−) for generation of dense bubble clouds directly by a single negative half cycle, the “intrinsic threshold,” was measured. In this paper, the dense bubble clouds and resulting lesions (in RBC phantoms and canine tissues) generated by these supra-intrinsic threshold pulses were studied. A 32-element, PZT-8, 500 kHz therapy transducer was used to generate very short (< 2 cycles) histotripsy pulses at a pulse repetition frequency (PRF) of 1 Hz and P− from 24.5 to 80.7 MPa. The results showed that the spatial extent of the histotripsy-induced lesions increased as the applied P− increased, and the sizes of these lesions corresponded well to the estimates of the focal regions above the intrinsic cavitation threshold, at least in the lower pressure regime (P− = 26–35 MPa). The average sizes for the smallest reproducible lesions were approximately 0.9 × 1.7 mm (lateral × axial), significantly smaller than the −6dB beamwidth of the transducer (1.8 × 4.0 mm). These results suggest that, using the intrinsic threshold mechanism, well-confined and microscopic lesions can be precisely generated and their spatial extent can be estimated based on the fraction of the focal region exceeding the intrinsic cavitation threshold. Since the supra-threshold portion of the negative half cycle can be precisely controlled, lesions considerably less than a wavelength are easily produced, hence the term “microtripsy.”

Ultrasound mediated drug delivery using microbubbles is a safe and noninvasive approach for spatially localized drug administration. This approach can create temporary and reversible openings on cellular membranes and vessel walls (a process called “sonoporation”), allowing for enhanced transport of therapeutic agents across these natural barriers. It is generally believed that the sonoporation process is highly associated with the energetic cavitation activities (volumetric expansion, contraction, fragmentation, and collapse) of the microbubble. However, a thorough understanding of the process was unavailable until recently. Important progress on the mechanistic understanding of sonoporation and the corresponding physiological responses in vitro and in vivo has been made. Specifically, recent research shed light on the cavitation process of microbubbles and fluid motion during insonation of ultrasound, on the spatio-temporal interactions between microbubbles and cells or vessel walls, as well as on the temporal course of the subsequent biological effects. These findings have significant clinical implications on the development of optimal treatment strategies for effective drug delivery. In this article, current progress in the mechanistic understanding of ultrasound and microbubble mediated drug delivery and its implications for clinical translation is discussed.

Treatment options for patients with hepatocellular carcinoma (HCC) are limited, in particular in advanced and drug resistant HCC. MicroRNAs (miRNA) are non-coding small RNAs that are emerging as novel drugs for the treatment of cancer. The aim of this study was to assess treatment effects of two complementary miRNAs (sense miRNA-122, and antisense antimiR-21) encapsulated in biodegradable poly (lactic-co-glycolic acid) nanoparticles (PLGA-NP), administered by an ultrasound-guided and microbubble-enhanced delivery approach in doxorubicin-resistant and non-resistant human HCC xenografts. Proliferation and invasiveness of human HCC cells after miRNA-122/antimiR-21 and doxorubicin treatment were assessed in vitro. Confocal microscopy and qRT-PCR were used to visualize and quantitate successful intracellular miRNA-loaded PLGA-NP delivery. Up and down-regulation of miRNA downstream targets and multidrug resistance proteins and extent of apoptosis were assessed in vivo in treated human HCC xenografts in mice. Compared to single miRNA therapy, combination therapy with the two complementary miRNAs resulted in significantly (P < 0.05) stronger decrease in cell proliferation, invasion, and migration of HCC cells as well as higher resensitization to doxorubicin. Ultrasound-guided delivery significantly increased in vivo miRNA-loaded PLGA-NP delivery in human HCC xenografts compared to control conditions by 5–9 fold (P < 0.001). miRNA-loaded PLGA-NP were internalized in HCC cells and anti-apoptotic proteins were down regulated with apoptosis in ~27% of the tumor volume of doxorubicin-resistant human HCC after a single treatment with complementary miRNAs and doxorubicin. Thus, ultrasound-guided delivery of complementary miRNAs is highly efficient in the treatment of doxorubicin- resistant and non-resistant HCC. Further development of this new treatment approach could aid in better treatment of patients with HCC.

Conventional histotripsy uses pulses with ≥3 cycles wherein the bubble cloud formation relies on the pressure-release scattering of the positive shock fronts from sparsely distributed cavitation bubbles. In a recent work, the peak negative pressure (P(-)) threshold for the generation of dense bubble clouds directly by a negative half cycle were measured, and this threshold has been called the “intrinsic threshold.” In this work, characteristics of lesions generated with this intrinsic threshold mechanism were investigated using RBC phantoms and excised canine tissues. A 32-element, PZT-8, 500 kHz therapy transducer was used to generate short (&lt;2 cycles) histotripsy pulses at PRF = 1Hz and P(-) = 24.5–80.7 MPa. The results showed that the spatial extent of the histotripsy-induced lesions increased as the applied P(-) increased, and the lesion sizes corresponded well to the estimates of the focal regions above the intrinsic threshold. The sizes for the smallest reproducible lesions averaged 0.9 × 1.7mm (lateral × axial), significantly smaller than −6 dB beamwidth of the transducer (1.8 × 4.0 mm). These results suggest that predictable, well-confined and microscopic lesions can be precisely generated using the intrinsic threshold mechanism. Since the supra-threshold portion of the negative half cycle can be precisely controlled, lesions considerably less than a wavelength are easily produced (“microtripsy”).