폴리다이아세틸렌 리포좀 기반의 생체 내 음이온 감지계 개발

Abstract

Section I. Fluorescent turn-on sensing of apoptotic cellsApoptosis, a programmed cell death, is essential for embryonic development, immune-system function, and the maintenance of tissue homeostasis. The appearance of anionic phosphatidylserine (PS) in the outer monolayer of the plasma membrane is a universal indicator of the early and intermediate stages of cell apoptosis.A liposome-based fluorescence sensing system for apoptotic cells has been developed from stimuli-responsive poly(diacetylene)-liposomes for the first time. A proper combination of the liposome components, phosphatidylserine-binding Zn(II)-dipicolylamine and alcohol-terminated components in a ratio of 2:1, has led to an efficient detection system for apoptotic cells, as demonstrated by confocal fluorescence microscopy and FACS analysis. The liposome shows a color change from blue to red-purple and emits fluorescence in the turn-on mode, upon interaction with the analytes. The present system thus avoids the careful washing steps required for those of “always-on” type sensing systems.

Section II. Colorimetric detection and quantification of heparinHeparin is used as an anticoagulant during cardiovascular surgery or to avoid thrombosis. Overdose of heparin, however, can induce complications such as hemorrhage or thrombocytopenia, making it important to monitor the amount of heparin in an easy and simple manner. We have investigated molecular interactions between an artificial liposome decorated with cationic sites and heparin, a biomacromolecule with dense negative charge, for the first time. The electrostatic interactions between the negatively-charged heparin and the positively-charged liposome led to a ratiometric colour change from blue to red. Sigmoid curves were obtained from the UV-Vis titrations of the liposome with heparin, from which linear calibration curves were extracted. Addition of anionic biopolymers such as chondroitin 4-sulfate or hyaluronic acid to the same liposome system led to small colour changes. The sensing protocol was successfully applied to the determination of heparin in a HEPES buffer and in a buffer containing fetal bovine serum, for the concentration ranges of 0.30–5.35 U/mL and 0.67–4.33 U/mL respectively, which covers most part of therapeutic level. The macromolecular ionic interactions proceeded through a sequential morphology change, showing precipitation around the endpoint of the titration and then dissolution of the precipitates later, which was analyzed by dynamic light scattering and transmission electron microscopy. The aggregation behaviour was supported by zeta potential changes during the titration. Thus, we further correlated the zeta potential change with the colourimetric endpoint during the titration of the liposome with heparin, which provides a new and simple heparin quantification method.

Section III. Colorimetric detection and quantification of fatty acidsFatty acids play vital roles in biological processes such as in the construction of cellular membranes, production of energy sources, and signaling molecules. Especially cis-unsaturated fatty acids act as an energy source and signals for metabolic regulation that act through enzymatic and transcriptional networks to modulate gene expression, growth and survival pathways, and inflammatory and metabolic responses. Traditionally, fatty acid isomers are analyzed by gas chromatography or mass spectroscopy. These methods, however, show limitations such as low efficiency, high cost, and time-consuming, and also require complicated skills including the synthesis of fatty acid methyl ester.We have investigated polydiacetylene liposome systems for simple discrimination and quantification of cis-unsaturated fatty acids. An optimized liposome system showed a colorimetric change from blue to red upon interactions with cis-unsaturated fatty acids, enabling the naked-eye detection. The liposome system does not show any colour changes in the case of saturated or trans-unsaturated fatty acids. Interestingly, the liposome showed stoichiometric binding mode toward cis-unsaturated fatty acids, in a ratio of 1:2, from which we were able to determine the concentration of cis-unsaturated fatty acids. The liposome system was applied to the quantification of cis-unsaturated fatty acids in commercial oil under a cooking condition.

Section IV. Cationic polydiacetylene liposomes as antibacterial agentsThe appearance of antibiotic-resistant bacteria in recent years is threatening our life. Therefore, new antibacterial agents are greatly demanded. We have evaluated cationic liposomes as antibacterial agents based on the fact that cationic substrates can bind and disrupt negatively charged bacterial cellular membranes, which could bring about cell lysis. Negatively charged bacterial outer membrane is stabilized by countercations such as Mg2+ and Ca2+, which neutralize and bridge the anionic phosphate groups. As cationic compounds adsorb bacterial surface, the countercations of bacterial outer membrane are expelled. This ionic disturbance destabilizes the outer membrane, causing bacterial death. Polydiacetylene-based liposomes equipped with cationic sites show antibacterial effects toward some Gram-positive and Gram-negative bacteria, as demonstrated by growth inhibition and bactericidal experiments.