Cationic versus Neutral Microbubbles for Ultrasound-mediated Gene Delivery in Cancer

Abstract

Purpose:To test whether plasmid-binding cationic microbubbles (MBs) enhance ultrasound-mediated gene delivery efficiency relative to control neutral MBs in cell culture and in vivo tumors in mice. Materials and Methods:Animal studies were approved by the institutional animal care committee. Cationic and neutral MBs were characterized in terms of size, charge, circulation time, and DNA binding. Click beetle luciferase (CBLuc) reporter plasmids were mixed with cationic or neutral MBs. The ability of cationic MBs to protect bound plasmids from nuclease degradation was tested by means of a deoxyribonuclease (DNase) protection assay. Relative efficiencies of ultrasound-mediated transfection (ultrasound parameters: 1 MHz, 1 W/cm 2 , 20% duty cycle, 1 minute) of CBLuc to endothelial cells by using cationic, neutral, or no MBs were compared in cell culture. Ultrasound-mediated gene delivery to mouse hind limb tumors was performed in vivo (n = 24) with insonation (1 MHz, 2 W/cm 2 , 50% duty cycle, 5 minutes) after intravenous administration of CBLuc with cationic, neutral, or no MBs. Tumor luciferase activity was assessed by means of serial in vivo bioluminescence imaging and ex vivo analysis. Results were compared by using analysis of variance. Results:Cationic

Ultrasound molecular imaging: Moving toward clinical translation

“…In addition to reporting absolute values obtained from calculating the difference in imaging signal, the ratio of imaging signal pre and post destruction can be used as a value which is independent of tissue attenuation and system settings, which becomes important when translating the technique from small to large animal experiments and eventually to patients where tissue attenuation can vary substantially depending on body habitus and location of the imaging area within the body [14]. However, the main disadvantages are the required time-consuming post-processing, hampering the real-time work flow of ultrasound imaging and the fact that microbubbles need to be destroyed, which by itself can have unwarranted biological consequences caused by microbubble-induced cavitation [15, 16]. …”

Nucleic acid delivery with microbubbles and ultrasound

“…So, the plasmid or oligonucleotide should be placed on the microbubble surface, which is easily accomplished by electrostatic binding. Indeed, a direct comparison study has determined that microbubble-plasmid complexes provided significantly better transfection than co-administered plasmid and microbubbles incapable of complex formation [5, 28]. …”

“…DSPC/DSTAP combination has become applicable generally, with a number of published studies from several laboratories [2, 5, 32-34]. In this formulation, microbubbles have to be purified from unincorporated lipid micelles by a short centrifugal flotation in a cell culture centrifuge (∼100-200 g, for several minutes); plasmid can be then added directly to the aqueous microbubble dispersion for electrostatic binding.…”

“…It has been confirmed that plasmid attached onto DSTAP/DSPC microbubbles that carry a grafted PEG brush is partially protected from nuclease degradation [5]. …”

“…The latter goal has not been achieved yet, even for a clinical trial stage. Certain attractive features of ultrasound transfection maintain the interest in this approach; research in this area continues to advance and is rapidly accelerating lately (e.g., [2-5]. In this review we provide examples of the approaches to ultrasound-enhanced nucleic acid delivery.…”