In Situ Transfection by Controlled Release of Lipoplexes Using Acoustic Droplet Vaporization

Abstract

Localized delivery of nucleic acids to target sites (e.g., diseased tissue) is critical for safe and efficacious gene therapy. An ultrasound-based technique termed acoustic droplet vaporization (ADV) has been used to spatiotemporally control the release of therapeutic small molecules and proteins contained within sonosensitive emulsions. Here, ADV was used to control the release of lipoplex – containing plasmid DNA with an enhanced green fluorescent protein (eGFP) reporter - from a sonosensitive emulsion. Focused ultrasound (3.5 MHz, mechanical index (MI) ≥ 1.5) generated robust release of fluorescein (i.e., surrogate payload) and lipoplex from the emulsion. In situ release of the lipoplex from the emulsion using ADV (MI=1.5, 30 cycles) yielded a 55% release efficiency, resulting in 43% transfection efficiency and 95% viability with C3H/10T1/2 cells. Without exposure to ultrasound, the release and transfection efficiencies were 5% and 7%, respectively, with 99% viability. Lipoplex released by ADV retained its bioactivity while the ADV process did not yield any measureable sonoporative enhancement of transfection. Co-encapsulation of Ficoll PM 400 within the lipoplex loaded emulsion, and its subsequent release using ADV, yielded higher transfection efficiency than the lipoplex alone. The results demonstrate that ADV could have utility in the spatiotemporal control of gene delivery.

In vitro and in vivo assessment of controlled release and degradation of acoustically responsive scaffolds

“…Various acoustic parameters have been shown to affect the ADV threshold (i.e., the lowest acoustic pressure at which ADV begins to occur) and efficiency (i.e., the fraction of droplets that vaporize at a given acoustic pressure) of sonosensitive emulsions and ARSs [34]. For example, ADV thresholds correlate inversely with US pulse duration, insonation frequency, and PRF [33, 34] while ADV efficiency correlates with acoustic pressure [32, 42]. Supplemental Figure 1 and Figure 3 show that payload release – which is directly related to ADV efficiency - correlated with acoustic pressure, PRF, and number of US exposures for both small and large emulsions.…”

Drug Release from Phase-Changeable Nanodroplets Triggered by Low-Intensity Focused Ultrasound

“…For example, it is difficult to achieve efficient drug concentration in tumor sites because of their limited capacity for loading therapeutic agents, short circulation time, and large micrometer size. To address these problems, phase-change perfluorocarbon (PFC) nanodroplets have been developed 18 , 20 , 24 - 27 in which the liquid in the core of nanodroplets can vaporize to gas phase upon activation by ultrasound energy, called acoustic droplet vaporization (ADV) 28 - 30 . The stability of nanodroplets is increased, and they can accumulate in tumor tissues by passive targeting due to their nano-scaled size.…”

“…Ultrasound is a form of the longitudinal mechanical wave that can be transmitted in the human body and is widely used in tumor imaging and therapy 30 - 35 . Different from the diagnostic ultrasound used in the clinic to force bubble elastic compression and expansion, more ultrasound energy is needed to induce conversion of nanodroplets into microbubbles and trigger local drug release 28 - 32 .…”

Colloids, nanoparticles, and materials for imaging, delivery, ablation, and theranostics by focused ultrasound (FUS)

“…While it is possible to load both hydrophobic and hydrophilic molecules to the stabilizing shells of the emulsions as with microbubbles, loading capacities are typically small 163,164. To overcome this limitation, water-in fluorocarbon-in water double emulsions have been prepared in the expense of the significant increase in the particle size compared to regular phase-change droplets 25,165-167.…”