Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

Abstract

OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin–targeted microbubbles. CONCLUSIONS We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy

“…We reported that the transfection efficiencies that can be reached with mRNA sonoporation are significantly lower than when mRNA is electroporated into DCs, however it should be noted that the importance of mRNA sonoporation as a transfection technique lies within its possible in vivo applicability [42,43]. Commercially available microbubble contrast agents were shown to migrate to the tumor-draining lymph nodes upon intradermal injection around the tumor of breast cancer patients [15].…”

Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy

“…In another study ovarian cancer cells were transfected with wild-type p53 tumour suppressor gene using luteinising hormone-releasing hormone analogue (LHRHa)-tUCA to induce apoptosis (1 MHz, 0.5 W/cm 2 , 30 s treatment) [185]. Two studies [186,187] showed that tUCAs loaded with luciferase plasmid can transfect vasculature in vivo. Xie et al [186] used P-selectin tUCAs in a hind limb ischaemia skeletal muscle model (see Figure 3E; 1.6 MHz, 0.6-1.8 MPa, power Doppler, pulsing interval 5 s, PRF 2.5 kHz for 10 min), whereas Tlaxca et al [187] [190].…”

“…Two studies [186,187] showed that tUCAs loaded with luciferase plasmid can transfect vasculature in vivo. Xie et al [186] used P-selectin tUCAs in a hind limb ischaemia skeletal muscle model (see Figure 3E; 1.6 MHz, 0.6-1.8 MPa, power Doppler, pulsing interval 5 s, PRF 2.5 kHz for 10 min), whereas Tlaxca et al [187] [190]. In vivo, the tUCAs were administered intraperitoneally allowing the tUCAs to adhere to the ovarian cancer cells.…”

“…tUCA vs. non-tUCA for enhanced drug delivery Strikingly, when tUCAs for drug delivery are directly compared with their non-targeted UCA counterparts, tUCAs are more efficient both in vitro [184,185,188,189,192] (up to 7.7-fold higher [189]) and in vivo [186,187,190,191] (up to 5-fold higher [186]), regardless of whether the drug was coadministered with the tUCAs or whether the drug was loaded on/in the tUCAs. Although the reasons for this higher efficiency have not yet been investigated, several could be possible.…”

“…Validation of luciferase reporter plasmid transfection in rats using bioluminescence (left two panels) and imunohistochemistry (right four panels; arrows point to plasmid transfection in endothelial and perivascular cells; reprinted (adapted) from Xie et al [186], ß 2014, with permission from Elsevier).…”