| The cellular uptake of macromolecules such as oligonucleotides, genes, and (glyco)proteins occurs via endocytosis, following which the endosomal contents are routed to lysosomes for enzymatic degradation. In most cases, access to the cytosol is a prerequisite for the macromolecular drug to exert its pharmacological effect. This study is focused on chemical strategies involving acid-sensitive detergents that exploit the acidic endosomal pH in promoting the cytosolic delivery of therapeutic macromolecules. Three weakly basic amphiphiles with ionization constants (pKa) in the endosomal pH range (∼6.0) were synthesized. The detergents displayed a pH-dependent ability to disrupt red blood cell membranes and potential for enhancing the liposome-mediated cytosolic delivery of macromolecules. Mechanistic studies suggested that liposomal collapse into micelles is a prerequisite for drug release and endosomal permeabilization. Efficient bilayer-to-micelle transition was shown to occur only upon accumulation of detergent concentrations as high as 75 mol% within liposomal bilayers. However, from a formulation standpoint, such conditions are unattainably high due to the low limit of incorporation (∼20 mol%) of single-tailed detergents within intact liposomes.; To address this problem, two acid-sensitive detergent dimers, obtained by crosslinking the head groups of single-tailed detergents via acid-labile acetal or acid-stable oxyethylene spacers were synthesized. The so-called 'Bis-Detergents' (BD) form stable vesicles under neutral conditions, but rapidly undergo lamellar-to-micellar transition under acidic conditions. The latter presumably occurs due to acid-catalyzed hydrolysis of the acetal crosslinker, which generates two single-tailed detergents in situ and electrostatic repulsion between protonated tertiary amine head groups (average pKa ∼6.4). Further, BD-containing liposomes were capable of entrapping macromolecules with moderate efficiency (∼10 to 40%) and promote efficient cytosolic delivery of antisense oligonucleotides as demonstrated by fluorescence microscopy and a functional assay based on luciferase up-regulation. The acid-sensitive detergents developed in this study display potential for in vitro delivery applications and for improving macromolecular therapeutics in general. |
