This is the first study, to our knowledge, providing evidence that ELIP conjugated to two functionally different antibodies enhance stem cell delivery to the arterial wall. Our discussion will focus on the benefits of this methodology in directing and enhancing stem cell delivery to target tissues.
These data establish the suitability of BF-ELIP with ultrasound enhancement as stem cell delivery agents. Previous investigations have demonstrated that ELIP are very useful agents that can serve as drug/gene carriers and diagnostic ultrasound imaging contrast agents.12, 23
When conjugated to specific antibodies, ELIP can be used for tissue or organ-specific targeting. The present study has demonstrated the feasibility of BF-ELIP for stem cell delivery from peripheral blood and bone marrow to target tissues.
In atherosclerosis, the vascular tissue is injured by hypercholesterolemia and inflammation. In response to the injury, large numbers of leukocytes but few vascular stem cells enter the arterial lesions. Resident vascular progenitor cells may die by apoptosis or other mechanisms.1, 9
Early atheroma lesions develop in the intima of the arteries. Here, the lumenal endothelial layer serves as the entry point for both leukocytes and stem cells to transmigrate into the lesions. Increased expression of adhesion proteins, such as ICAM-1 and VCAM-1, is considered to be a key event that triggers vascular inflammatory cell infiltration.24
By utilizing this event, this methodology creates a new avenue for delivering CD34+ stem cells to atherosclerotic intimal lesions. Endogenous stem cells adhere to the surface via adhesion proteins before entering the arterial wall. The conjugation of anti-ICAM-1 antibodies to the surface of liposomes allows binding of liposomes to ICAM-1-expressing endothelial cells of atherosclerotic lesions, thereby specifically targeting cells involved in early plaque formation.
In addition, selection of specific types of stem cells can be achieved through liposomal conjugation of antibodies against cell-type specific surface markers. Bone marrow-derived stem cells are characterized by several cell-surface markers, including CD29, CD34, CD44, CD90 and CD133. Double-conjugation of echogenic liposomes to both ICAM-1 and stem-cell-specific surface markers to produce bifunctional echogenic liposomes provides a mechanism to allow binding of stem cells to ICAM-1-expressing endothelial cells. Because pre-incubation of BF-ELIP with stem cells would obscure the liposomal ICAM-1 binding sites, it is necessary to administer the BF-ELIP prior to the stem cells. Subsequent coating of ICAM-1 expressing endothelium with stem cell binding sites has the added advantage of recruiting endogenous stem cells, as well as exogenous cell populations introduced after BF-ELIP administration.
It is generally believed that the stem cell traffic and homing is physiologically regulated by a “homing” receptor and its ligand, such as the CXCR4/stromal derived factor system.25, 26
Relatively few studies have focused on increasing plaque stability as a means of treating atherosclerosis. As stem cells have the ability to differentiate into multiple cell types, introduction of stem cells into atherosclerotic plaques may result in the production of cell types that are able to stabilize the plaque, such as collagen-producing fibroblasts. In this study, we demonstrated that BF-ELIP selectively binds to CD34 and ICAM-1 could recognize both human peripheral CD34+ mononuclear cells and porcine bone marrow cells and subsequently bridge the cells to aortal endothelium. The recognition and binding seem to be specific, since the control IgG-ELIP did not have such capacity.
This bridging function improves the likelihood that BF-ELIP can enable increased binding of CD34+ cells to atheroma, compared to therapeutic interventions not employing such delivery agents, in which tissue binding of stem cells is entirely dependent upon endogenous cellular mechanisms of increased cell adhesion, involving adhesin-integrin-mediated homing to areas of tissue injury. Because BF-ELIP targets stem cells to endothelial cells via the ICAM-1 mediated adhesion, they may competitively inhibit leukocyte adhesion and therefore reduce inflammatory cell infiltration into atheroma. This notion is supported by the observation that in the BF-ELIP-loaded area, the majority of cells adherent to endothelium are CD34+ cells.
Exciting work in the field of gene therapy demonstrates enhanced cell transfection with the use of low-intensity ultrasound 28-31
. It has been suggested that this increased uptake is due to cavitational effects by ultrasound 32
, a theory supported by the finding that inclusion of Albunex® (an imaging contrast agent) as a cavitation nucleation agent enhances ultrasound-induced transfection 33
. Petechial hemorrhage and microvascular leakage in the myocardium have also been demonstrated as a result of myocardial contrast imaging at 1.7 MHz in rats 34
. The interaction of ultrasound with the encapsulated gas within the BF-ELIP potentially enhances the adhesion to vascular endothelium via radiation force or shear stress mechanisms.
Evidence for clinically relevant sonoporation-enhanced BF-ELIP bridging was provided in the ex vivo porcine aortic segments. Oil Red O staining identified the presence of liposomes on the surface of the aorta. Utilizing Oil Red O as an indicator, we confirmed that ultrasound treatment increased the binding of BF-ELIP-CD34+ stem cell complexes to the aortic endothelial surface, relative to IgG-ELIP controls. This suggests that the aortic surface adherence was mediated by ICAM-1 binding. Although this study was performed on ex vivo tissues under static incubation conditions and may not represent the in vivo situation found in the arteries with blood flow, the ability of BF-ELIP to adhere to lumenal surfaces under simulated physiologic flow conditions35
and in vivo 13,14,36
has been well established. Other factors besides blood flow that may influence the outcomes of the BF-ELIP guided stem cell delivery include the ratio of BF-ELIP to stem cells and ultrasound energy. In vivo studies, presently underway, will establish whether enhancement of stem cell homing to endothelium is a consistent effect of this liposomal agent used in conjunction with sonoporation.
As we have previously demonstrated that ELIP can be efficiently loaded with various therapeutic agents, followed by ultrasound-induced controlled release,16, 23, 27
BF-ELIP show additional promise as a platform technology for optimization of experimental therapeutic protocols involving concurrent delivery of drugs and genes to enhance endothelial penetration and survival of stem cells.
The current study reports a novel strategy to use BF-ELIP to deliver CD34 positive stem cells directly to atheroma with active expression of adhesion proteins. Our data clearly demonstrate that under the ultrasound enhancement, BF-ELIP can selectively bind peripheral and bone marrow-derived CD34+ cells and provide targeted delivery to vascular endothelium under controlled in vitro conditions. This novel methodology for stem cell delivery provides a basis for new strategies to improve stem cell therapy for cardiovascular diseases.