While gas-filled micrometer-sized ultrasound contrast agents vastly improve signal-to-noise ratios, microbubbles have short circulation lifetimes and poor extravasation from the blood. Previously reported fluorocarbon-based nanoscale contrast agents are more stable but their contrast is generally lower owing to their size and dispersity. The contrast agents reported here are composed of silica nanoparticles of ≈100 nm diameter that are filled with ≈3 nm columnar mesopores. Functionalization of the silica surface with octyl groups and resuspension with Pluronic F127 create particles with pores that remain filled with air but are stable in buffer and serum. Administration of high intensity focused ultrasound (HIFU) allows sensitive imaging of the silica nanoparticles down to 10 10 particles mL −1, with continuous imaging for at least 20 min. Control experiments with different silica particles supported the hypothesis that entrapped air could be pulled into bubble nuclei, which can then in turn act as acoustic scatterers. This process results in very little hemolysis in whole blood, indicating potential for nontoxic blood pool imaging. Finally, the particles are lyophilized and reconstituted or stored in PBS (phosphate-buffered saline, at least for four months) with no loss in contrast, indicating stability to storage and reformulation.

Ultrasound is widely applied in medical diagnosis and therapy due to its safety, high penetration depth, and low cost. In order to improve the contrast of sonographs and efficiency of the ultrasound therapy, echogenic gas bodies or droplets (with diameters from 200 nm to 10 µm) are often used, which are not very stable in the bloodstream and unable to penetrate into target tissues. Recently, it was demonstrated that nanobubbles stabilized by nanoparticles can nucleate ultrasound responsive microbubbles under reduced acoustic pressures, which is very promising for the development of nanoscale (<100 nm) ultrasound agents. However, there is still very little understanding about the effects of nanoparticle properties on the stabilization of nanobubbles and nucleation of acoustic cavitation by these nanobubbles. Here, a series of mesoporous silica nanoparticles with sizes around 100 nm but with different morphologies were synthesized to understand the effects of nanoparticle porosity, surface roughness, hydrophobicity, and hydrophilic surface modification on acoustic cavitation inception by porous nanoparticles. The chemical analyses of the nanoparticles showed that, while the nanoparticles were prepared using the same silica precursor (TEOS) and surfactant (CTAB), they revealed varying amounts of carbon impurities, hydroxyl content, and degrees of silica crosslinking. Carbon impurities or hydrophobic modification with methyl groups is found to be essential for nanobubble stabilization by mesoporous silica nanoparticles. The acoustic cavitation experiments in the presence of ethanol and/or bovine serum albumin (BSA) demonstrated that acoustic cavitation is predominantly nucleated by the nanobubbles stabilized at the nanoparticle surface not inside the mesopores. Finally, acoustic cavitation experiments with rough and smooth nanoparticles were suggested that a rough nanoparticle surface is needed to largely preserve surface nanobubbles after coating the surface with hydrophilic macromolecules, which is required for in vivo applications of nanoparticles.

The mechanical effects of cavitation can be effective for therapy but difficult to control, thus potentially leading to off-target side effects in patients. While administration of ultrasound active agents such as fluorocarbon microbubbles and nanodroplets can locally enhance the effects of high intensity focused ultrasound (HIFU), it has been challenging to prepare ultrasound active agents that are small and stable enough to accumulate in tumors and internalize into cancer cells. Here, we report the synthesis of 100 nm nanoparticle ultrasound agents based on phospholipid-coated, mesoporous, hydrophobically-functionalized silica nanoparticles that can internalize into cancer cells and remain acoustically active. The ultrasound agents produce bubbles when subjected to short HIFU pulses (~6 μs) with peak negative pressure as low as ~7 MPa and at particle concentrations down to 12.5 μg mL−1 (7×109 particles mL−1). Importantly, ultrasound agents are effectively uptaken by cancer cells without cytotoxic effects, but HIFU insonation causes destruction of the cells by the acoustically generated bubbles, as demonstrated by XTT and LDH assays and flow cytometry. Finally, we showed that the HIFU dose required to effectively eliminate cancer cells in the presence of ultrasound agents caused only a small temperature increase of ~3.5 °C.

While thermal ablation of various solid tumors has been demonstrated using high intensity focused ultrasound (HIFU), the therapeutic outcomes of this technique are still unsatisfactory because of common recurrence of thermally ablated cancers and treatment side effects due to the high ultrasound intensity and acoustic pressure requirements. More precise ablation of tumors can be achieved by generating cavitating bubbles in the tissue using shorter pulses with higher acoustic pressures, which induce mechanical damage rather than thermal. However, it has remained as a challenge to safely deliver the acoustic pressures required for mechanical ablation of solid tumors. Here, we report a method to achieve mechanical ablation at lower acoustic pressures by utilizing phospholipid-stabilized hydrophobic mesoporous silica nanoparticles (PL-hMSN). The PL-hMSNs act as seeds for nucleation of cavitation events and thus significantly reduce the peak negative pressures and spatial-average temporal-average HIFU intensities needed to achieve mechanical ablation. Substantial mechanical damage was observed in the red blood cell or tumor spheroid containing tissue mimicking phantoms at PL-hMSN concentrations as low as 10 μg mL−1, after only 5 s of HIFU treatment with peak negative pressures ~11 MPa and duty cycles ~0.01%. Even the application of HIFU (peak negative pressure of 17.5 MPa and duty cycle of 0.017%) for 1 min in the presence of PL-hMSN (200 μg mL−1) did not cause any detectable temperature increase in tissue-mimicking phantoms. In addition, the mechanical effects of cavitation promoted by PL-hMSNs were observed up to 0.5 mm from the center of the cavitation events. This method may thus also improve delivery of therapeutics or nanoparticles to tumor environments with limited macromolecular transport.

This work reports that when PEG-lipid-shelled microbubbles with fluorocarbon interior (C4F10, C5F12, or C6F14) are subjected to ultrasound pulses, they produce metastable, fluid-filled nanoparticles that can be re-imaged upon administration of HIFU. The nanoparticles produced by destruction of the microbubbles (MBNPs) are of 150 nm average diameter and can be re-imaged for up to an hour after creation for C 4F10, and for at least one day for C5F12. The active species were found to be fluid (gas or liquid) filled nanoparticles rather than lipid debris. The acoustic droplet vaporization threshold of the nanoparticles was found to vary with the vapor pressure of the encapsulated fluorocarbon, and integrated image brightness was found to increase dramatically when the temperature was raised above the normal boiling point of the fluorocarbon. Finally, the vaporization threshold decreases in serum as compared to buffer, and administration of HIFU to the nanoparticles caused breast cancer cells to completely detach from their culture substrate. This work demonstrates a new functionality of microbubbles that could serve as a platform technology for ultrasound-based theranostics.