Efficient Production of Fluorescent Polydiacetylene-Containing Liposomes for Pathogen Detection and Identification
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Efficient Production of Fluorescent Polydiacetylene-Containing Liposomes for Pathogen Detection and Identification Cassandra J. Wright-Walker, Caroline E. Hansen, Michael A. Evans, Emily S. Nyers, and Timothy W. Hanks Department of Chemistry, Furman University, 3300 Poinsett Highway, Greenville, SC 29613, U.S.A.
ABSTRACT Amphiphilic diacetylenes (DAs) can self-assemble into photopolymerizable liposomes that can be used to construct effective pathogen sensors. Here, modified commercial inkjet printers are used to disperse DAs into water, facilitating self-assembly. The liposomes are of similar size, but are significantly less polydisperse than liposomes formed using conventional sonication methods. The process is efficient, readily scalable and tolerant of structural modification. The derivitization of approximately 5% of the DA head groups and the incorporation of fluorophores into the hydrophobic bilayer allows for the preparation of novel multifluorophore PDA sensing systems that can provide enhanced bacterial discrimination in a single experiment by way of a fluorescent fingerprint. INTRODUCTION Liposomes are bilayer assemblies of long chain amphiphiles that were first characterized in the 1960s [1]. Much of the interest in these structures is due to the synthetic ease with which the hydrophobic layer membrane and the surface can be covalently derivatized, as well as the facile trapping of various functional species within the aqueous interior. In particular, we are interested in systems containing highly conjugated polydiacetylene polymer chains in the hydrophobic layer. This not only stabilizes the assembly, but adds a chromatic functionality that is sensitive to mechanical disturbances of the vesicle. Polydiacetylene (PDA) liposomes, once polymerized, exhibit a deep blue color (λmax=650nm). Upon interaction with stimuli such as temperature, pH, mechanical stress, or biological species, the PDAs will change from blue to bright red (λmax=540nm) due to a disruption of the backbone planarity [1]. In combination with other modifications, these liposomes can be made into environmental sensors (pH, temperature, mechanical stress, biological species, etc.) [2] as well as nanocapsules for the delivery and controlled release of drugs, nanoparticles and other species. The result is a “smart” device capable of detecting and responding to its environment. PDA liposomes have previously been produced by a variety of methods, with the most prevalent being via sonication. Sonication, however, often results in a wide distribution of liposome sizes and may degrade sensitive components [3]. Recently, a modification of the “ethanol injection method” was shown to efficiently produce liposomes from simple phospholipids [4]. We have extended this method to DA-containing lipids, optimized preparation conditions and explored the effects of liposome surface derivitization and bilayer incorporation of fluorophores.
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EXPERIMENTAL DETAILS Materials 10,12-pentacosadynoic acid (PCDA) acquired from GFS Chemicals (Powell, OH). Fluoropho
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