Acid-triggered release via dePEGylation of DOPE liposomes containing acid-labile vinyl ether PEG–lipids

Abstract

Four structurally related, acid-labile polyethylene glycol (PEG) conjugated vinyl ether lipids have been synthesized and used at low molar ratios to stabilize the nonlamellar, highly fusogenic lipid, dioleoylphosphatidyl ethanolamine, as unilamellar liposomes. Acid-catalyzed hydrolysis of the vinyl ether bond destabilized these liposomes by removal of the sterically-stabilizing PEG layer, thereby promoting contents release on the hours timescale at pH<5. Structure–property correlations of these compounds suggested that single vinyl ether linkages between the PEG headgroup and the lipid backbone produce faster leakage rates. These studies also suggested that the presence of a slight negative charge at the membrane surface can accelerate the acid-catalyzed leakage process.

Introduction

The intrinsic low pH within endosomal and ischemic tissue environments has motivated the development of several different triggering approaches employing either cosurfactant-stabilized dioleoylphosphatidyl ethanolamine (DOPE) [1], [2], [3], polymers [4], [5], [6], [7], or viral fusion proteins [8], [9] as an acid-sensitive switch for targeted drug delivery. Cosurfactant-stabilized DOPE systems have been the most widely studied because of their excellent fusogenic potential [10], however, a lack of serum stability greatly limits their potential application in vivo. Incorporation of a sterically-stabilizing polyethylene glycol (PEG) coating improves their serum stability, however, this is achieved at the expense of diminished delivery efficiency and fusogenic capacity [2]. These limitations have stimulated the search for triggering strategies in which the latent DOPE fusogenicity is restored upon dePEGylation.

Efficient targeting of drugs to the cytoplasm of folate-receptor positive cells via receptor-mediated endocytosis has been previously demonstrated by Thompson and co-workers [11], [12] using folic acid-modified, acid-sensitive diplasmalogen (DPPlsC) liposomes. This system operates via acid-catalyzed hydrolysis of the DPPlsC vinyl ether bonds at endosomal pH values, resulting in a lamellar-to-micellar phase transition (L_α–H_I; Fig. 1) that significantly enhances liposomal contents release into the cytoplasm of the target cells. Unlike most pH-sensitive liposome formulations [13], DPPlsC liposomes demonstrate good plasma stability at 37 °C, however, their practical utility has been limited by their slow reaction kinetics and the difficulties encountered in the previously reported syntheses of the (Z)-vinyl ether linkages found in the naturally-occurring forms of diplasmalogen lipids.

DePEGylative triggering [13], [14], [15], [16], [17], [18], using binary mixtures of the H_II phase lipid DOPE and a cleavable PEG–lipid conjugate with an H_I phase preference, is a promising new strategy for liposomal drug delivery. It is well known that sterically stabilized phosphatidylcholine-based liposomes containing low molar ratios of PEG–lipids can possess excellent plasma stability and minimal contents leakage during their long circulation in blood [19], [20], [21]. By substituting DOPE and cleavable PEG–lipids for the phosphatidylcholine lipids and distearoyl phosphatidylethandamine-PEG (DSPE-PEG) DSPE–PEG conjugates, respectively, that are typically used in long circulating liposomes, one can develop triggerable liposome formulations that are activated via the dePEGylation pathway illustrated in Fig. 2. In principle, cleavage of the low molar ratios of PEG–lipid in the DOPE-rich membrane, followed by desorption of the PEG byproduct from the liposome surface, will lead to destabilization of the lamellar phase. If this pathway is activated near or within target cells, the resulting membrane fusion and contents release of the dePEGylated DOPE liposome can significantly enhance intracellular delivery of the liposomal cargo. In order to test the effectiveness of this dePEGylative triggering strategy, we have synthesized four acid-labile PEG conjugated vinyl ether lipids (ST302, ST305, ST352, ST355; Fig. 3). These materials were then analyzed for their ability to promote acid-triggered calcein release from DOPE-rich unilamellar liposomes containing low molar ratios of these conjugates.