Surface modification of liposomes for selective cell targeting in cardiovascular drug delivery

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Abstract

Cardiovascular disease processes such as atherosclerosis, restenosis, and inflammation are typically localized to discrete regions of the vasculature, affording great opportunity for targeted pharmacological treatment. Liposomes are potentially advantageous targeted drug carriers for such intravascular applications. To facilitate their use as drug delivery vehicles, we have considered three components of liposome design: (i) identification of candidate cell surface receptors for targeting; (ii) identification of ligands that maintain binding specificity and affinity; and (iii) prevention of rapid nonspecific clearance of liposomes into the reticuloendothelial organs. In this report, we describe our work in developing liposomal surface modifications that address both targeting and clearance. An arginine–glycine–aspartic acid (RGD) containing peptide was used as a model ligand to target liposomes to the integrin GPIIb–IIIa on activated platelets. Additionally, oligodextran surfactants incorporated into liposomes provided insight into the effect of vesicle perturbations on liposome clearance, and the importance of molecular geometry in designing oligosaccharide surface modifications. Together these studies demonstrate the feasibility of using peptides to guide liposomes to desired receptors, and illustrate the influence of vesicle stability on liposome interactions in vivo. Furthermore, they underscore the importance of simultaneously considering both targeting specificity and vesicle longevity in the design of effective targeted drug delivery systems.

Introduction

The prevalence of localized cardiovascular disease among adults in the United States remains a major cause of morbidity and mortality. One example is restenosis, or reocclusion of coronary vessels following percutaneous transluminal coronary angioplasty (PTCA) and intracoronary stent deployment. A number of systemic pharmacological approaches have been used to inhibit restenosis, but have enjoyed limited clinical success [1], [2], [3]. Such observations are typically attributed to dose-limiting toxicity for drugs utilized in animal models [4], [5], or to the short half-lives typically associated with peptide or oligosaccharide therapeutics [6]. Alternatively, local delivery of drugs directed against particular steps in the pathogenesis of restenosis enhances efficacy by increasing drug concentrations to therapeutic levels in the immediate vicinity of vascular injury [4], [7], [8], [9], [10], [11]. To date, most drug delivery systems lack specificity for damaged tissue, and therefore may adversely affect surrounding tissue, continue to circulate downstream of the injury site, or suffer “washout” of the therapeutic component. Optimal drug delivery, therefore, requires the ability to preferentially localize to sites of injury, while maintaining a reservoir of drug that avoids uptake into uninvolved tissues.

The goal of our research is to exploit differential expression of receptor molecules on the surfaces of cells involved in restenosis, in order to deliver liposomes to the site of disease. In particular, our work focuses on three such receptors (Fig. 1): (i) integrin GPIIb–IIIa on activated platelets; (ii) tissue factor (TF) on vascular endothelial cells (EC); and (iii) E- and/or P-selectin on both cell types. We have concentrated on using these cells for liposome localization due to their inducible activation states and accessibility to the lumen.

In this report we focus on liposomes directed to GPIIb–IIIa. Equally important for successful liposome targeting, however, is the ability to avoid rapid clearance into organs of the reticuloendothelial system (RES). Irrespective of targeting ligands, a means for increasing liposome circulation lifetime is essential for successful application of targeted liposomal drug delivery. Consequently, in this report we also consider the second aspect of a successful targeted delivery system – circulation lifetime.

Interactions between liposomes and proteins are governed by attractive and repulsive intermolecular forces. In the early 1990s, poly(ethylene oxide) (PEO)-grafted phospholipids were shown to dramatically increase liposome survival in the circulation [12], [13], [14]. The excluded volume at the liposome surface presumably is enhanced by the introduction of large, hydrated molecules at the lipid headgroup that inhibit subsequent protein–liposome interactions. Despite the significant prolongation observed with PEO-modified liposomes, few other surface modifications (e.g., GM1 ganglioside) have been observed to alter liposome circulation lifetimes similarly. Here we describe our preliminary observations using oligodextran as a model of a known “passivating” biomaterial surface modification, and the impact that oligosaccharide surfactants have on vesicle structure and subsequent behavior in vivo.

At present, we have investigated peptide- and oligosaccharide-modified liposomes separately and in parallel. Together, however, these studies provide insight into the development of liposome surfaces that simultaneously incorporate molecules for both targeting specificity and circulation longevity. Such a liposome design is depicted in Fig. 2; peptides provide targeting functionality, while oligosaccharides form a highly hydrated interface to prevent nonspecific interactions.

Section snippets

Preparation of peptide (RGD) and oligodextran conjugates

The linear RGD peptide GSSSGRGDSPA was selected based on the known affinity of the RGDS sequence contained within the fibrinogen Aα chain for GPIIb–IIIa [15]. The peptide was synthesized by solid-phase synthesis [16], [17], and coupled to the succinimidyl carboxyester terminus of a distearoylphosphatidylethanolamine (DSPE)–PEO–COOH conjugate (Shearwater Polymers, Birmingham, AL, USA) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as catalysts. After

Peptide–lipid amphiphiles for directing liposomes to platelet GPIIb–IIIa

Current liposomal drug delivery systems suffer from the interrelated problems of rapid clearance and lack of cell specificity. Numerous targeting strategies have been developed that utilize native proteins, antibodies or antibody fragments to impart cellular recognition [20], [21], thereby directing liposomes toward a particular anatomical or pathological site. Here we describe the construction of peptide–lipid amphiphiles that target activated vascular cells, using an RGD peptide as a model

Conclusions

Effective targeted liposomal delivery systems require that both targeting specificity and liposome longevity be addressed. Our data indicate that peptides are capable of directing liposomes to receptors expressed on pathologically stimulated vascular cells such as platelets. Liposomes bearing an RGD peptide bound to activated platelets, while apparently avoiding platelet aggregation due to modest affinity of the peptide. With respect to oligosaccharide surface modifications, liposome clearance

Acknowledgements

The authors thank Gurunathan Murugesan, Mark Ruegsegger, Michael Zagorski, Shu Chen, and Rachael Petro. Funding was provided by NIH HL40047, and facilities by the Center for Cardiovascular Biomaterials. B.J.L. was supported by a Graduate Research Fellowship from the Whitaker Foundation.

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