Dataset Information

Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles.

ABSTRACT: Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome, and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmids coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9%±2.2% (n=9), comparable with lipofection (7.5%±0.8%, n=9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection.

SUBMITTER: Fan Z

PROVIDER: S-EPMC3758692 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles.

Fan Z Z Chen D D Deng C X CX

Journal of controlled release : official journal of the Controlled Release Society 20130611 3

Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. In this study, we condu ...[more]

PMID: 23770009

Similar Datasets

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.

Project description:Neurons in the brain can be damaged or lost from neurodegenerative disease, stroke, or traumatic injury. Although neurogenesis occurs in mammalian adult brains, the levels of natural neurogenesis are insufficient to restore function in these cases. Gene therapy has been pursued as a promising strategy to induce differentiation of neural progenitor cells into functional neurons. Non-viral vectors are a preferred method of gene transfer due to potential safety and manufacturing benefits but suffer from lower delivery efficiencies compared to viral vectors. Since the neural stem and progenitor cells reside in the subventricular zone of the brain, intraventricular injection has been used as an administration route for gene transfer to these cells. However, the choroid plexus epithelium remains an obstacle to delivery. Recently, transient disruption of the blood-brain barrier by microbubble-enhanced ultrasound has been used to successfully improve drug delivery to the brain after intravenous injection. In this work, we demonstrate that microbubble-enhanced ultrasound can similarly improve gene transfer to the subventricular zone after intraventricular injection. Microbubbles of different surface charges (neutral, slightly cationic, and cationic) were prepared, characterized by acoustic flow cytometry, and evaluated for their ability to increase the permeability of immortalized choroid plexus epithelium monolayers in vitro. Based on these results, slightly cationic microbubbles were evaluated for microbubble and ultrasound-mediated enhancement of non-viral gene transfer in vivo. When coupled with our previously reported gene delivery vehicles, the slightly cationic microbubbles significantly increased ultrasound-mediated transfection of the murine brain when compared to commercially available Definity® microbubbles. Temporary disruption of the choroid plexus by microbubble-enhanced ultrasound is therefore a viable way of enhancing gene delivery to the brain and merits further research.

Project description:PurposeTo 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 methodsThe 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.ResultsMean 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.ConclusionStem cells loaded with DNA-carrying MBs can be transfected in vivo with ultrasonic pulses and remain alive to demonstrate robust gene expression.

Project description: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.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.Cationic MBs (+15.8 mV; DNA binding capacity, 0.03 pg per MB) partially protected bound DNA from DNase degradation. Mean CBLuc expression of treated endothelial cells in culture was 20-fold higher with cationic than with neutral MBs (219.0 relative light units [RLUs]/µg protein ± 92.5 [standard deviation] vs 10.9 RLUs/µg protein ± 2.7, P = .001) and was significantly higher (P < .001) than that in the no MB and no ultrasound control groups. Serial in vivo bioluminescence of mouse tumors was significantly higher with cationic than with neutral MBs ([5.9 ± 2.2] to [9.3 ± 5.2] vs [2.4 ± 0.8] to [2.9 ± 1.1] × 10(4) photons/sec/cm(2)/steradian, P < .0001) and versus no MB and no ultrasound controls (P < .0001). Results of ex vivo analysis confirmed these results (ρ = 0.88, P < .0001).Plasmid-binding cationic MBs enhance ultrasound-mediated gene delivery efficiency relative to neutral MBs in both cell culture and mouse hind limb tumors.

Dataset Information

Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles.

Publications

Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets