Ultrasonically targeted delivery into endothelial and smooth muscle cells in ex vivo arteries
Introduction
Cardiovascular disease is one of the leading causes of death worldwide, resulting in nearly 17 million deaths annually [1]. Targeted drug and gene delivery to vascular endothelial cells (ECs) and smooth muscle cells (SMCs) has increasingly become a focus for treating cardiovascular diseases and dysfunctions, such as coronary artery disease, hypercholesterolemia, hypertension, and restenosis, because of the pivotal role of these cells in controlling and maintaining vascular functions [2], [3], [4]. In addition, targeted drug and gene delivery to the endothelium is being used to treat cancerous tumors by anti-vascular therapy [5] and myocardial and peripheral ischemia by promoting angiogenesis [6]. Many drug and gene delivery systems are being developed to increase targeting to vascular cells, such as drug-eluting stents [7], catheter-based systems [8], viral vectors [9], [10], and targeted liposomes [11] and microbubbles [12]. However, most current techniques lack either the effectiveness or specificity to adequately treat these disorders while safely administering the therapeutic agent and avoiding toxic systemic effects or require significantly invasive intervention. A safe, effective, and non-invasive method to target delivery of drugs or genes to the specific disease site in the vasculature would greatly benefit cardiovascular treatments.
A novel approach to targeting drug and gene administration is the method of ultrasound-enhanced delivery [13], [14], [15]. Ultrasound-enhanced delivery often exploits cavitation bubble activity, which can be produced by the pressure oscillations of ultrasound [16]. Furthermore, ultrasound pressures above a certain threshold can cause oscillating bubbles to undergo violent collapse known as inertial cavitation [17]. Inertial cavitation is believed to cause transient disruptions in cell membranes, enabling transport of extracellular molecules (e.g., drugs or genes) into viable cells [18], [19], [20]. Cavitation-mediated cellular disruptions can allow uptake of small molecules, macromolecules (e.g., proteins), and genetic material (e.g., plasmid DNA or siRNA). Furthermore, ultrasound-enhanced delivery has been studied in a variety of in vitro and in vivo scenarios and has demonstrated promising therapeutic results after intracellular uptake of drugs and gene expression [14], [16], [21]. Most importantly, ultrasound can be targeted in the body by non-invasive extracorporeal focused ultrasound [22] or by minimally invasive catheter-based transducers [23]. Greater efficacy and reduced side effects could be realized by this targeted therapy.
The vascular endothelium is an attractive target for ultrasound-enhanced delivery because cavitation can be readily produced in the vasculature, which currently can occur to a mild extent during diagnostic imaging [24]. Moreover, cavitation is expected to have limited effects beyond cell layers directly experiencing cavitation activity, and the endothelium is the first point of contact to cavitation activity [25]. A number of researchers are currently studying ultrasound-enhanced gene therapy for cardiovascular disorders in order to control intimal hyperplasia, restore vascular function, or promote angiogenesis [21]. These studies have shown expression of reporter plasmids as well as plasmids with a therapeutic purpose. Clinical potential of this method has been shown by causing protein expression by plasmid DNA [26] or blocking specific proteins by oligonucleotides [27].
A recognized limitation of ultrasound-enhanced delivery is the need for more cells with drug uptake or gene expression [21]. Many studies have demonstrated gene transfection and tissue response in ex vivo and in vivo systems; however, few studies have been directed at quantifying the intracellular uptake of molecules and imaging bioeffects (i.e., intracellular uptake and loss of viability) caused by ultrasound-enhanced delivery in viable tissue [16], [28]. It is important to know the uptake efficiency at different ultrasound energies in order to design and apply this technique for drug or gene delivery applications. In this study, we sought to determine whether ultrasound-enhanced delivery of a model drug can be targeted into ECs and SMCs in ex vivo arteries. This work represents the first known in-depth study to quantify and image the intracellular uptake of molecules and loss of viability to vascular cells by ultrasound-enhanced delivery. We hypothesize that ultrasound can target intracellular uptake of drugs into the vascular endothelium at low to intermediate energy and into SMCs at high energy. To assess this hypothesis, our goals in this study were to image the localization of (i) intracellular uptake of a model drug and (ii) loss of viability to vascular cells and (iii) to specifically quantify endothelial bioeffects in the targeted region.
Section snippets
Porcine carotid artery isolation and preparation
Porcine carotid arteries were chosen for this study because they can be delicately excised without tissue damage, preserving an intact and viable endothelium. Furthermore, carotid arteries are relatively straight vessels without much branching, simplifying handling and imaging of the artery. Porcine carotid arteries were harvested from freshly killed female swine at a local abattoir (Holifield Farms, Convington, GA). Excised arteries were immediately rinsed with sterile phosphate-buffered
Endothelial bioeffects
To test the hypothesis that ultrasound-mediated cavitation can cause intracellular uptake into ECs, we exposed ex vivo arterial segments to ultrasound at three different acoustic energies – termed low, intermediate, and high – and compared these tissues to control artery segments without ultrasound exposure (i.e., sham exposure). Fig. 2 displays representative en face images of the endothelium of a control tissue and a tissue exposed to ultrasound at the intermediate energy. ECs are recognized
Discussion
Previous studies utilizing ultrasound-enhanced gene therapy have shown promising results of positive gene transfection in ex vivo [36] and in vivo [26] cardiovascular tissues. However, few studies have shown intracellular uptake of small molecules or macromolecules (e.g., proteins) for drug delivery applications in ex vivo or in vivo cardiovascular tissues [16], [21]. Moreover, there is a lack of knowledge on the efficiency of this technique in terms of the number of cells affected and the
Acknowledgments
We thank Holifield Farms for their donation of porcine tissue and laboratory members Mangesh Despande, Vladimir Zarnitsyn, Robyn Schlicher, Joshua Hutcheson, Christina Rostad, and Prerona Chakravarty for their helpful discussions. This work was supported in part by the U.S. National Institutes of Health, and a fellowship to DMH from the U.S. Department of Education GAANN program. DMH and MRP are members of the Center for Drug Design, Development and Delivery and the Institute of Bioengineering
References (40)
- et al.
Endothelial cells in physiology and in the pathophysiology of vascular disorders
Blood
(1998) - et al.
Local drug and gene delivery through microbubbles
Prog. Cardiovasc. Dis.
(2001) - et al.
Encapsulated ultrasound microbubbles: therapeutic application in drug/gene delivery
J. Control. Release
(2006) - et al.
An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes
Biophys. J.
(2003) - et al.
Mechanism of intracellular delivery by acoustic cavitation
Ultrasound Med. Biol.
(2006) - et al.
Plasma membrane poration induced by ultrasound exposure: implication for drug delivery
J. Control. Release
(2005) - et al.
Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine
J. Am. Coll. Cardiol.
(2005) - et al.
High-efficiency endovascular gene delivery via therapeutic ultrasound
J. Am. Coll. Cardiol.
(2001) - et al.
Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo
Ultrasound Med. Biol.
(2005) - et al.
Local delivery of E2F decoy oligodeoxynucleotides using ultrasound with microbubble agent (Optison) inhibits intimal hyperplasia after balloon injury in rat carotid artery model
Biochem. Biophys. Res. Commun.
(2004)
Ten-fold augmentation of endothelial uptake of vascular endothelial growth factor with ultrasound after systemic administration
J. Am. Coll. Cardiol.
Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging
Adv. Drug Deliv. Rev.
Measurement and correlation of acoustic cavitation with cellular bioeffects
Ultrasound Med. Biol.
Erosion of artificial endothelia in vitro by pulsed ultrasound: acoustic pressure, frequency, membrane orientation and microbubble contrast agent dependence
Ultrasound Med. Biol.
Detachment and sonoporation of adherent HeLa-cells by shock wave-induced cavitation
Biochim. Biophys. Acta
The role of inflammation in vascular injury and repair
J. Thromb. Haemost.
Bioeffects caused by changes in acoustic cavitation bubble density and cell concentration: a unified explanation based on cell-to-bubble ratio and blast radius
Ultrasound Med. Biol.
Principles for national and regional guidelines on cardiovascular disease prevention: a scientific statement from the World Heart and Stroke Forum
Circulation
The endothelium: a new target for therapy
Vasc. Med.
Endothelium-targeted gene and cell-based therapies for cardiovascular disease
Arterioscler. Thromb. Vasc. Biol.
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