Intracellular delivery of peptides and siRNAs using microbubble enhanced focused ultrasound

摘要

Bioactive substances such as peptides and nucleic acid based agents have attracted great attention for the next generation drug for various diseases. However, the greatest challenge for using these bioactive substances is the development of their delivery system, especially the method for delivering these substances through the cell membrane. With the advancement of ultrasound and ultrasound contrast agent technology, it has become possible to transiently change the permeability of the cell membrane. Moreover, using a focused ultrasound transducer, it is possible to narrow and focus the ultrasound energy within a small target, avoiding damage to the surrounding tissue. In this research we have searched the possibility of delivering the Bak BH3 peptide, the death domain of the Bc1-2 family of proteins, or the short interfering RNA (siRNA) targeting the enhanced green fluorescent protein (EGFP) using microbubble-enhanced focused ultrasound in an in vitro setting. Using a 1.696 MHz focused ultrasound and a microbubble ultrasound contrast agent OPTISON?, we first tested the stability of BH3 peptide under microbubble-enhanced focused ultrasound exposure and proved that the peptide is stable under these circumstances. Next, we have tested the cell-killing effect of the intracellularly delivered Bak BH3 peptide in HeLa and BJAB cell line and observed a statistically enhanced cell death in BJAB cells but not in HeLa cells, leading to the conclusion that intracellularly delivered BH3 peptide by microbubble-enhanced ultrasound can exert its cell killing effect in some cells. We also investigated if we can silence the EGFP expression in the cell by delivering siRNA targeting the EGFP in both transient and stable EGFP expression cell line. Using a 1.653 MHz focused ultrasound and OPTISON?, in both cases, intracellularly delivered siRNA by microbubble-enhanced ultrasound was able to knock down the EGFP expression, which demonstrates the feasibility of using this novel method for siRNA intracellular delivery. In conclusion, our investigation has proved the feasibility of using micro-bubble enhanced focused ultrasound as a method for intracellular delivery of peptides and siRNAs. Although further optimization of various parameters is necessary, we consider that this method could be a power tool for using these bioactive substances in the clinical field.