Dataset Information

In Vivo Transfection and Detection of Gene Expression of Stem Cells Preloaded with DNA-carrying Microbubbles.

ABSTRACT:

Purpose

To determine whether (a) stem cells loaded with DNA-carrying 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 ?L phosphate-buffered saline were mixed with 200 ?L 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/cm(2) (n = 3) or 2 W/cm(2) (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 ?m ± 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/cm(2) pulses as compared with 1 W/cm(2) 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/cm(2) 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.

SUBMITTER: Tavri S

PROVIDER: S-EPMC4521611 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Publications

In Vivo Transfection and Detection of Gene Expression of Stem Cells Preloaded with DNA-carrying Microbubbles.

Tavri Sidhartha S Vezeridis Alexander A Cui Wenjin W Mattrey Robert F RF

Radiology 20150326 2

<h4>Purpose</h4>To determine whether (a) stem cells loaded with DNA-carrying 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.<h4>Materials and methods</h4>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 stai ...[more]

PMID: 25811427

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Project description:ObjectivesUltrasound-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.BackgroundContrast 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.MethodsCationic 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.ResultsCharge 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/cm(2)). 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.ConclusionsWe conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Dataset Information

In Vivo Transfection and Detection of Gene Expression of Stem Cells Preloaded with DNA-carrying Microbubbles.

Purpose

Materials and methods

Results

Conclusion

Publications

In Vivo Transfection and Detection of Gene Expression of Stem Cells Preloaded with DNA-carrying Microbubbles.

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