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. 2010 Jun 1;93(3):1004-15.
doi: 10.1002/jbm.a.32549.

In vitro and in vivo platelet targeting by cyclic RGD-modified liposomes

Rekha Srinivasan  1 , Roger E Marchant, Anirban Sen Gupta
Affiliations

In vitro and in vivo platelet targeting by cyclic RGD-modified liposomes

Rekha Srinivasan et al. J Biomed Mater Res A. .

Abstract

Cell-selective delivery using ligand-decorated nanoparticles is a promising modality for treating cancer and vascular diseases. We are developing liposome nanoparticles surface-modified by RGD peptide ligands having targeting specificity to integrin GPIIb-IIIa. This integrin is upregulated and stimulated into a ligand-binding conformation on the surface activated platelets. Activated-platelet adhesion and aggregation are primary events in atherosclerosois, thrombosis, and restenosis. Hence, platelet-targeted nanoparticles hold the promise of vascular site-selective delivery of drugs and imaging probes. Here, we report in vitro and ex vivo microscopy studies of platelet-targeting by liposomes surface-modified with a cyclic RGD peptide. The peptide-modified liposomes were labeled either with a lipophilic fluorophore or with lipid-tethered Nanogold(R). For in vitro tests, coverslip-adhered activated human platelets were incubated with probe-labeled liposomes, followed by analysis with fluorescence microscopy, phase contrast microscopy, and scanning electron microscopy (SEM). For in vivo tests, the liposomes were introduced within a catheter-injured carotid artery restenosis model in rats and post-euthanasia, the artery was imaged ex vivo by fluorescence microscopy and SEM. All microscopy results showed successful platelet-targeting by the peptide-modified liposomes. The in vitro SEM results also enabled visualization of nanoscopic liposomes attached to activated platelets. The results validate our nanoparticle design for site-selective vascular delivery.

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Figures

Figure 1
Figure 1
Schematic diagram of possible cellular interactions of activated platelets at the site of a vascular injury/inflammation.
Figure 2
Figure 2
Schematic diagram of the method of liposome preparation including a molecular model representation of the lipid bilayer membrane of the liposome, and the dynamic light scattering (DLS) analysis of liposome size distribution before and after extrusion through nanoporous membrane.
Figure 3
Figure 3
Representative fluorescence and phase contrast microscopy images from in vitro studies of active platelet targeting by peptide-modified fluorescently labeled liposomes; (A) RGE-liposome-incubated sample showed minimal fluorescence, while (B) cRGD-liposome-incubated sample showed significantly enhanced fluorescence; parallel analysis of cRGD-liposome-incubated sample with (C) fluorescence and (D) phase contrast microscopy provided evidence for one-to-one correspondence of fluorescence spots with active platelets, thereby emphasizing that the liposome binding is platelet-specific; (E) represents the surface-averaged fluorescence intensity analysis of the RGE-liposome incubated and the cRGD-liposome incubated samples; (F) shows representative flow cytometry data of NBD fluorescence intensity histogram overlay for platelets without any liposome incubation, with liposome incubation but without ADP pre-incubation (predominantly inactive platelets) and with liposome incubation after ADP pre-incubation (predominantly active platelets), establishing that the NBD-labeled cRGD-liposomes have specific enhanced binding to activated platelets.
Figure 4
Figure 4
Representative images from SEM studies of active platelet targeting by peptide-modified Nanogold-labeled liposomes; SEM analysis of collagen-coated coverslip exposed to human platelet suspension in presence of ADP showed (A) monolayer of adhered platelets which were in highly activated condition as evidenced by (B) highly folded membrane structure, ‘spread’ morphology and branched out pseudopods; SEM images in secondary electron mode of (C1) RGE-liposome-incubated platelet and (D1) cRGD-liposome-incubated platelet showed similar membrane morphology and vesicular structures, but corresponding images in backscatter mode showed (C2) minimal gold contrast enhancement on the membrane of RGE-liposome-incubated platelet, while (D2) significant contrast enhancement of the membrane of cRGD-liposome-incubated platelet; in addition, multiple bright spherical vesicular structures (arrowheads in D2) were visible on cRGD-liposome-incubated platelet, which are possibly intact liposomes adhered on the membrane and pseudopods.
Figure 5
Figure 5
Representative ex vivo images from studies of active platelet binding of peptide-modified fluorescently labeled liposomes in vivo in a rat carotid injury restenosis model; (A) shows the schematic of the in vivo model used in the study; (B) and (D) show representative fluorescence and SEM images of the carotid luminal surface without injury; (E) is an SEM image of the carotid luminal surface immediately after frictional injury with inflated balloon catheter, showing significant wall damage and endothelial denudation; after 1 hr of blood repurfusion through such injured carotid, SEM image of excised sections showed (F) numerous adhered platelets and upon magnification (G) arrested platelets in dense fibrin mesh were visible, signifying acute thrombotic environment; the presence of adhered platelets was confirmed by (C) fluorescence imaging after staining the injured section with FITC-tagged anti-rat CD42d (stains platelet GPV); introduction of fluorescently labeled RGE-liposomes in the injured section after (H) 1 hr and (J) 2 hr blood repurfusion showed only minimal staining of platelets; in contrast, introduction of fluorescently labeled cRGD-liposomes showed significant platelet staining in both (I) 1 hr and (K) 2 hr repurfusion situations; for 2 hr repurfusion sections, highly fluorescent clumps of aggregated platelets were also visible; (L) statistical analysis of fluorescence intensity from images of liposome-exposed carotid sections for several batches of animals, confirmed the enhanced binding of cRGD-liposomes at the injury site.
Figure 6
Figure 6
Representative ex vivo SEM images from studies of active platelet targeting by peptide-modified Nanogold-labeled liposomes in vivo in a rat carotid injury restenosis model; the secondary electron mode images of both (A1) RGE-liposome-incubated and (B1) cRGD-liposome-incubated samples showed similar acute thrombotic environment with platelets arrested in dense fibrin mesh, but the corresponding backscatter image (A2) of RGE-liposome-incubated sample showed only minimal contrast enhancement while the backscatter image (B2) of cRGD-liposome-incubated sample showed substantial contrast enhancement of cellular structures arrested in the fibrin mesh, suggesting enhanced binding of Nanogold-labeled cRGD-liposomes to platelets.
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