Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.
Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).
Liposomes, or vesicles – compartments bounded by bilayer membranes comprised of amphiphilic lipids – have found use in numerous biomedical applications as delivery vehicles for pharmaceuticals, models of cell membranes, and for the development of synthetic cells. We and others have also employed liposomes as models of primitive cell membranes in early life.1,2,3,4 Typically, in such systems, we employ single-chain amphiphiles containing only one lipid hydrocarbon tail (e.g., oleic acid), as these molecules are simpler to synthesize without the benefit of the coded protein enzymes modern cells employ.
Liposomes comprised of single-chain lipids are similar to those formed from diacylphospholipids (e.g., 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or POPC) in that the boundary is composed of bilayer membranes. Liposomes formed from either class of lipids can retain a dissolved payload, and can be downsized and purified by different techniques. Several important differences result from the unique chemical characteristics of single-chain lipids. Vesicles formed by phospholipids are stable over a broad pH range, while fatty acid vesicles are only stable at neutral to mildly basic pH (ca. 7 – 9), which requires certain pH buffer for vesicle preparation. Most of the time, this buffer may also contain specific soluble molecules for vesicle encapsulation, which can be either functional materials (e.g., RNA) for compartmentalized biochemical reactions or simple fluorescent dyes (e.g., calcein) for vesicle characterization.
The presence of only a single hydrocarbon chain produces a membrane that is both more permeable to solutes, as well as more dynamic. Additionally, the carboxylic acid head group present in fatty acids results in vesicles that are more sensitive to the presence of salt and divalent cations (e.g., Mg2+). Magnesium is one of the most important divalent cations for catalyzing biochemical reactions in protocells for origin-of-life studies. In early life, prior to the evolution of sophisticated protein enzymes, RNA may have been the dominant polymer, due to its dual capability to self-replicate and perform catalysis. One representative example of a magnesium-requiring RNA related reaction is non-enzymatic RNA copying, first demonstrated in 1960s.5 When chemically activated RNA nucleotides (i.e. 2-methylimidazolide nucleotides) bind to a preexisting primer-template complex, the 3'-hydroxyl group of the primer attacks the 5'-phosphate of an adjacent activated monomer to displace the leaving group (i.e. 2-methylimidazole), and forms a new phosphodiester bond. This RNA copying chemistry requires a high concentration of Mg2+, which needs to be chelated in order to be compatible with fatty acid protocells.6 Another Mg2+-dependent reaction is that catalyzed by the hammerhead ribozyme, which is perhaps the best-characterized catalytic RNA. This ribozyme, which can be reconstituted from two short oligonucleotides, performs a self-cleavage reaction that is convenient to monitor by a gel shift. As such, it is frequently employed as a model ribozyme in origin-of-life studies.7 Due to a requirement by this ribozyme for unliganded magnesium, liposomes are typically constructed by a mixture of fatty acid and fatty acid glycerol esters, which are more stable to magnesium.8,9 In this protocol, we present techniques we have developed for the preparation, manipulation, characterization of these vesicles and demonstrate application of these vesicles as protocells to host non-enzymatic RNA copying and hammerhead ribozyme catalysis.
1. Vesicle preparation
2. Vesicle purification
3. Use of Vesicles in the Presence of Magnesium
4. Non-enzymatic RNA Copying in Vesicles
5. Hammerhead RNA Self Cleavage in Vesicles
6. Giant Fatty Acid Vesicles for Microscopy
We typically perform liposome purifications on size-exclusion columns. Typical liposome preparations contain a fluorophore of some kind. When liposomes are generated and extruded, the species to be encapsulated are present both inside and outside of the liposomes. By purifying liposomes on a size-exclusion resin (Sepharose 4B), unencapsulated solutes are retained within the pores of the resin, while the larger liposomes are not and elute first (Figure 1A). Collecting fractions and plotting fluorescence vs. fraction number (Figure 1B) typically yields a two-peak trace, with the early-eluting fractions corresponding to the liposomes, which are then collected and used in subsequent applications.
We frequently examine nonenzymatic primer extension reactions, which were a likely means of RNA replication prior to the emergence of ribozyme and protein-based RNA polymerases. These reactions typically employ a fluorescently labeled primer (Figure 2A), which is extended by activated monomers. These reactions can be monitored by gel electrophoresis (Figure 2B) and the resulting electropherograms integrated to obtain rate constants for a given reaction condition (Figure 2C).
To demonstrate that RNA could function inside protocells, we employ hammerhead ribozyme self-cleavage (Figure 3A) as a model RNA catalytic reaction. This reaction requires free Mg2+ to facilitate catalysis, and therefore we used OA/GMO vesicles since they are stable in the presence of 5 mM Mg2+. Similar to primer extension reactions, the hammerhead ribozyme self-cleavage reaction can also be monitored by gel electrophoresis (Figure 3B) and later analyzed to acquire the rate constant under specific conditions (Figure 3C).
We image liposomes employing both fluorescence and transmitted light. Liposomes can be labeled using fluorescent lipids, which give a membrane label (Figure 4A), or using a fluorescent solute within their lumen (Figure 4B). Transmitted light can also be used to observe vesicles (also shown in Figure 4B).
Table 1.1 pure oleic acid in chloroform | |||
Component | Stock | Amount | |
Oleic acid | >99% | 11.7 µL | |
Chloroform | 1 mL | ||
Table 1.2 oleic acid and glycerol monooleate (9:1) in chloroform | |||
Component | Stock | Amount | |
Oleic acid | >99% | 10.5 µL | |
Glycerol monooleate | >99% | 1.4 µL | |
Chloroform | 1 mL | ||
Table 1.3 oleic acid with 0.2mol% Rhodamine-PE in chloroform | |||
Component | Stock | Amount | |
Oleic acid | >99% | 1.6 µL | |
Rhodamine-PE in chloroform | 10 mM | 20 µL | |
Chloroform | 1 mL |
Table 1. Fatty acid chloroform solutions.
Figure 1. Vesicle purification and fluorescence characterization of purification fraction. A. Separation of vesicles containing calcein from free calcein on a Sepharose 4B column. B. Vesicle and free calcein peak detection by plotting the fluorescence in each well vs. well number after fraction collection. Please click here to view a larger version of this figure.
Figure 2. Non-enzymatic RNA replication inside OA vesicles. A. Scheme of non-enzymatic RNA primer extension. B. PAGE image of a primer extension reaction inside pure oleic acid vesicles, with conditions as in section 4. C. Linear fit of the natural logarithm of ratio of amount of primer remaining at given time point to the initial amount of primer vs. time over 30 h. Reaction rate, calculated from the slope of ln(P/P0) vs time, is 0.058 h-1. Please click here to view a larger version of this figure.
Figure 3. Hammerhead ribozyme cleavage in OA/GMO vesicles. A. Scheme of hammerhead ribozyme cleavage of fluorescently labeled substrate strand (top). B. PAGE image of hammerhead ribozyme cleavage inside OA/GMO vesicles with 5 mM Mg2+. C. Ribozyme activity inside vesicles. Linear fit of natural logarithm of ratio of amount of substrate remaining at given time point to the initial amount of substrate vs. time in first 4 h. Reaction rate, calculated from the slope of ln(S/S0) vs time, is 0.36 h-1. Please click here to view a larger version of this figure.
Figure 4. Giant Fatty Acid Vesicles for Microscopy. A. Confocal microscopy image of a Rhodamine PE labeled oleic acid vesicle, scale bar 10 μm. B. Confocal microscopy image of oleic acid vesicle containing Alexa488 labeled RNA with membrane shown in transmitted detector (TD) channel, scale bar 5 μm.
Liposomes formed from fatty acids have been suggested by many as potential models for primitive cells due to their high permeability and dynamic properties. The carboxylic head group of single chain fatty acids only allows self-assembly into membranes in a restricted pH range, and the resulting membranes are quite sensitive to the presence of salts. As a result, fatty acid vesicles require different preparation and handling methods compared with phospholipid vesicles.
In this protocol, although we use oleic acid as an example for liposome formation, other long chain unsaturated fatty acids (C14) and their derivatives (ca. myristoleic acid, palmitoleic acid, and the corresponding alcohols and glycerol esters) also form vesicles following the thin film rehydration method as long as the total lipid concentration is above the cmc and the hydration buffer pH is close to the pKa of the fatty acid within the membrane. Other than tris-HCl buffer used in this protocol, other buffer systems (ca. bicine, phosphate, borate) were reported to support fatty acid vesicle formation, though vesicles formed in phosphate or borate buffer are usually quite leaky13. The resulting fatty acid vesicles after rehydration are polydisperse and multilamellar, but are easily converted into small monodisperse unilamellar vesicles by extrusion as described. Compared with sonication as an alternative method for generating small vesicles, extrusion provides more options for the control of vesicle size by applying different pore size membranes. Vesicles after extrusion are usually slightly bigger than the membrane pore size, but by increasing the number of extrusion cycles, vesicles with a narrower size distribution and an average size close to the membrane pore size can be obtained.
In order to synthesize functional protocells, fatty acid vesicles need to host specific biochemical reactions resulting from the encapsulation of RNA or other building blocks. The thin film rehydration method provides an easy way to form vesicles containing desired encapsulated materials. However, the encapsulation efficiency is relatively low and a large fraction of precious materials such as RNA are typically lost during the purification process. In some cases the encapsulation efficiency can be modestly improved by repeated freeze-thaw cycles before extrusion. Microfluidic methods for the high yield preparation of phospholipid liposomes allow for almost 100% encapsulation efficiency, however analogous methods have not yet been developed for fatty acid vesicles.
When handling protocells with either chelated or free Mg2+, purification after magnesium solution addition and repurification before each time point ensures the removal of leaked encapsulated material that might affect the accuracy of reaction rate measurements inside vesicles. Since each purification takes at least 10 min to achieve good separation and to collect vesicle fractions, the analysis of fast reactions is difficult, and the reaction must be stopped prior to column repurification.
The protocol we present here is well suited for the construction of fatty acid liposomes that host reactions mimicking those that might occur in primitive cells. Our protocols also enable potential applications in the development of biomedical delivery systems and bioreactors for other biochemical reactions.
The authors have nothing to disclose.
J.W.S. is an Investigator of the Howard Hughes Medical Institute. This work was supported in part by a grant (290363) from the Simons Foundation to J.W.S. Both A.E.E. and K.P.A. acknowledge support from University of Minnesota startup funds.
sephorose 4B | SIGMA-ALDRICH INC | 4B200 | |
calcein | SIGMA-ALDRICH INC | C0875-10G | |
tris-HCl pH8.0 1M | LIFE TECHNOLOGIES CORP | AM9851 | |
citric acid | SIGMA-ALDRICH INC | 251275-500G | |
sodium hydroxide | SIGMA-ALDRICH INC | 71690-250G | |
potassium hydroxide | Sigma | 30614 | |
oleic acid | Nu-Chek | U-46-A | |
glycerol monooleate | Nu-Chek | M-239 | |
Liss Rhodamine-PE | LIFE TECHNOLOGIES CORP | L1392 | |
magnesium chloride | Fisher/Thermo Fisher Scientific | AM9530G | |
sequagel concentrate | National Diagnostics | EC-830 | |
sequagel DILUENT | National Diagnostics | EC-840 | |
15% TBE-UREA GEL | Thermo Fisher Scientific | EC68852BOX | |
urea | Sigma Aldrich | U6504-500G | |
titon-100x | SIGMA-ALDRICH INC | T9284-100ML | |
RNA primer | IDT | 5'Cy3-GCG UAG ACU GAC UGG | |
RNA template | IDT | 5'-AAC CCC CCA GUC AGU CUA CGC | |
hammerhead substrate strand | IDT | 5'Cy3-GCG CCG AAA CAC CGU GUC UCG AGC | |
hammerhead ribozyme strand | IDT | 5'GGC UCG ACU GAU GAG GCG CG | |
vesicle extruder set | AVANTI POLAR LIPIDS | 610000 | |
fraction collector | Gilson, Inc. | 171041 | |
96-well plates | Fisher | NC9995941/675 | |
plate reader | Molecular Devices | SpectraMax i3 | |
confocal microscope | Nikon | Nikon A1R MP Confocal | |
gel scanner | GE Healthcare Life Sciences | Typhoon 9410 scanner |