In vivo transfection and detection of gene expression of stem cells preloaded with DNA-carrying microbubbles

Purpose: To determine whether (a) stem cells loaded with DNAcarrying microbubbles (MBs) can be transfected in vivo, (b) the cells remain alive to express the gene, and (c) gene expression is sufficiently robust to be detected in vivo. Materials and Methods: The study was approved by the Institutional Animal Care and Use Committee. Cationic MBs were prepared, characterized, and loaded with pLuciferase green fluorescent protein (GFP) plasmid. Loading was confirmed with SYBR Gold staining (Life Technologies, Carlsbad, Calif). C17.2 cells were loaded with the DNA-carrying MBs. Two hundred thousand cells suspended in 20 μL phosphate-buffered saline were mixed with 200 μL Matrigel (BD Biosciences, San Jose, Calif) and injected in both flanks of eight nude mice. One of the Matrigel (BD Biosciences) injections contained 50 000 cells pretransfected in vitro by using lipofectamine as a positive control. Nine flanks were exposed to 2.25-MHz ultrasonic pulses at 50% duty cycle for 1 minute at 1 W/cm² (n = 3) or 2 W/cm² (n = 6), and six flanks served as the negative control. Two days later, bioluminescent images were acquired in each mouse every 3 minutes for 1 hour after the intraperitoneal injection of D-luciferin (Perkin Elmer, Waltham, Mass). Differences between groups were assessed by using the nonparametric Kruskal-Wallis test with Wilcoxon rank sum tests for follow-up comparisons. Mice were then killed, plugs were explanted, and alternate sections were stained with hematoxylin-eosin or stained for GFP expression. Results: Mean DNA-loaded MB diameter ± standard deviation was 2.87 μm ± 1.69 with the DNA associated with the MB shell. C17.2 cells were associated with 2-4 MBs each, and more than 90% were viable. Peak background subtracted bioluminescent signal was fourfold higher when cells were exposed to 2 W/cm² pulses as compared with 1 W/cm² pulses (P = .02) and negative controls (P = .002). Histologic examination showed cells within the Matrigel (BD Biosciences) with robust GFP expression only after 2 W/cm² ultrasound exposure and lipofectamine transfection. Conclusion: Stem cells loaded with DNA-carrying MBs can be transfected in vivo with ultrasonic pulses and remain alive to demonstrate robust gene expression.