Article Cite This: Langmuir XXXX, XXX, XXX−XXX
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Flow Cytometric Analysis To Evaluate Morphological Changes in Giant Liposomes As Observed in Electrofusion Experiments Takeshi Sunami,*,† Kunihiro Shimada,‡ Gakushi Tsuji,†,‡ and Satoshi Fujii§ †
Institute for Academic initiatives and ‡Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan § Kanagawa Institute of Industrial Science and Technology, KSP EAST303, 3-2-1 Sakado, Takatsu-Ku, Kawasaki, Kanagawa 213-0012, Japan S Supporting Information *
ABSTRACT: Liposome fusion is a way of supplying additional components for in-liposome biochemical reactions. Electrofusion is a method that does not require the addition of fusogens, which often alter the liposome dispersion, and is therefore useful for repetitive liposome fusion. However, the details of electrofusion have not been elucidated because of the limitations surrounding observing liposomes using a microscope. Therefore, we introduced fluorescent markers and high-throughput flow cytometry to analyze the morphological changes that occur in liposome electrofusion. (i) The content mixing was evaluated by a calcein− Co2+−EDTA system, in which green fluorescence from dequenched free calcein is detected when the quenched calcein−Co2+ complex and EDTA are mixed together. (ii) Liposome destruction was evaluated from the decrease in the total membrane volume of giant liposomes. (iii) Liposome fission was evaluated from the increase in the number of giant liposomes. By applying the flow cytometric analysis, we investigated the effect of three parameters (DC pulse, AC field, and lipid composition) on liposome electrofusion. The larger numbers or higher voltages of DC pulses induced liposome fusion and destruction with higher probability. The longer application time of the AC field induced liposome fusion, fission, and destruction with higher probability. Higher content of negatively charged POPG (≥19%) strongly inhibited liposome electrofusion.
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INTRODUCTION In recent years, giant liposomes1 have been used as cell-like microcompartments in which various biochemical reactions can be encapsulated.2−4 However, as the volume of internal solution is small (1−1000 fL, 1.2−12 μm in diameter), materials for biochemical reactions run out in the short term. As a method for solving this problem, α-hemolysin, a poreforming membrane protein, was used to supply nutrients such as amino acids from the outside of giant liposomes for longterm in-liposome protein synthesis.5 However, the application of α-hemolysin has some limitations: (1) macromolecules such as proteins are difficult to be supplied because of the limited pore size, (2) low-molecular-weight components preliminarily encapsulated in giant liposomes continuously leak through the α-hemolysin pores, and (3) specific components are difficult to be selectively supplied because various low-molecular-weight components can pass through the α-hemolysin pores. © XXXX American Chemical Society
Liposome fusion can be employed as another method to supply membrane-impermeable components into giant liposomes.6 The application of liposome fusion for nutrient supply has some advantages: (1) macromolecules such as DNA, RNA, and proteins can be supplied and remain encapsulated, (2) only specific components encapsulated in giant liposomes can be supplied, and (3) membrane components such as lipids and membrane proteins can be supplied. Various kinds of liposome fusion methods for giant liposomes have been reported: metal ion-induced fusion,7,8 peptide-induced fusion,8,9 charged amphiphiles-induced fusion,10,11 and DNA-induced fusion.12 We focused on electrofusion of giant liposomes,13 in which an alternating current (AC) field is applied for liposome alignment Received: September 21, 2017 Revised: December 6, 2017 Published: December 7, 2017 A
DOI: 10.1021/acs.langmuir.7b03317 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. Detection of liposome fusion induced by electric stimulation. (A) Detection method of liposome fusion by flow cytometry (FCM). VT liposomes labeled by β-DPH HPC encapsulate quenched calcein−Co2+ complexes. RD liposomes labeled by ATTO633-DOPE encapsulate EDTA. When the VT liposome and RD liposome are fused with each other, cobalt ions are chelated by EDTA, and then dequenched free calcein emits green fluorescence. (B−E) FCM analysis of giant liposomes before and after electrofusion (100 000 data points). The number of DC pulses was 3, DC voltage was 6 kV/cm, and AC application time was 45 s. The mixing ratio of giant liposomes was VT liposomes:RD liposomes = 1:1 (number ratio), but 1:100 elsewhere. Parts D and E are two-dimensional (2D) dot plots of the giant liposomes in region R1 (parts B and C, respectively). (F, G) Confocal images of giant liposomes before and after electrofusion. Merged images of DIC (gray), green fluorescence, and red fluorescence.
cytometry (FCM)18 to analyze the intensities of scattered light and fluorescence emission from each giant liposome. The application of FCM to analyze giant liposomes has some advantages: (1) high throughput, (2) high sensitivity, (3) multiparameter, (4) wide size range, and (5) high reproducibility.19 A modern flow cytometer enables us to evaluate liposome fusion using fluorescence markers with high speed and at the particle level (>10 000 liposomes/s).11 In this article, we established an experimental system using FCM and the evaluation method to analyze morphological changes in giant liposomes in electrofusion. We analyzed liposome electrofusion in various experimental conditions to understand the effects of different parameters (DC pulse, AC field, and lipid composition).
and subsequent direct current (DC) pulses are applied for liposome fusion and fission. The application of liposome electrofusion has the advantage of enabling repeated electrofusion because the solutions are not contaminated by any chemical fusogens. Long-term biochemical reactions, such as those occurring in living cells, may become possible in giant liposomes by the repetitive nutrient supply induced by liposome electrofusion. However, the details of electrofusion, especially the effect of several parameters such as AC, DC, and lipid composition of giant liposomes on fusion efficiency, are not well understood. An analysis method for liposome electrofusion is important to understand the effects of each parameter. Direct observation using a microscope is one of the most popular methods for analyzing the electrofusion of giant liposomes (approximately 10 μm in diameter).13−15 Microscopic observation has a strong advantage that temporal morphological changes in giant liposomes can be investigated from the time-lapse images. The usage of fast digital imaging allowed for the investigation of the fusion dynamics of giant liposomes with microsecond resolution.16 However, the application of microscopes has some limitations: (1) small (∼1 μm) or vertically moving samples are difficult to observe in detail, (2) the number of samples observed at once is not large (
