Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm

Abstract

INTRODUCTION From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers. OBJECTIVES Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface. METHODS We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry. RESULTS Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least 4-fold in comparison to homogenous liposomes. CONCLUSIONS Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery

“…LNPs containing cholesterol analogs , DLin-MC3-DMA, DSPC, DMG-PEG-2000 (38.5:50:10:1.5 lipid molar ratios, respectively), and FLuc mRNA, were prepared using a standard microfluidic mixing procedure. 10,12 The structures of cholesterol analogs are shown in Figure 1A. Overall, all LNPs showed a narrow size distribution (see Figure 1B) as measured by dynamic light scattering (DLS) and >90% encapsulation efficiency ( Figure S1).…”

“…Recently, we investigated the substitution of cholesterol in LNP formulations with naturally occurring analogs with different chemical structures and the consequent effect on LNP size, mRNA encapsulation, internalization, and transfection. 10 The substitution of cholesterol (Chol) with βsitosterol (Sito) in the formulation showed a substantial improvement in cell transfection. Upon further inspection of Sito LNPs with a cryo-transmission electron microscope (cryo-TEM), we found that Sito LNPs have a faceted surface, as opposed to spherical Chol LNPs with smooth curvature, likely due to phase separation of comprising lipids, 11 and potentially, the formation of two-dimensional lipid crystals in the LNP membrane.…”

Numerical modeling of phase separation on dynamic surfaces

“…Membrane fusion is recognized as a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. The process of lipid membrane phase separation to concentrate binding lipids within distinct regions of the membrane surface has been shown to enhance membrane fusion [30]. In addition, recent developments on targeted lipid vesicles suggest that the formation of reversible phase-separated patterns on the vesicle surface increase target selectivity, cell uptake and overall efficacy [1,32].…”

Barcoding Biological Reactions with DNA‐Functionalized Vesicles

“…[26] In synthetic membranes,d omain formation can be readily engineered by choosing specific compositions of membrane components [27] and has been shown to enhance the efficiency of peptide-mediated fusion. [26,28] Yet, the potential of mem-brane domains to impact fusion events for the purpose of technological or therapeutic applications has to date received limited investigation [29] and merits further exploration.…”