Abstract
The liposome dialyzer is a small-volume equilibrium dialysis device, built from commercially available materials, that is designed for the rapid exchange of small volumes of an extraliposomal reagent pool against a liposome preparation. The dialyzer is prepared by modification of commercially available dialysis cartridges (Slide-A-Lyzer cassettes), and it consists of a reactor with two 300-μl chambers and a 1.56-cm2 dialysis surface area. The dialyzer is prepared in three stages: (i) disassembling the dialysis cartridges to obtain the required parts, (ii) assembling the dialyzer and (iii) sealing the dialyzer with epoxy. Preparation of the dialyzer takes ∼1.5 h, not including overnight epoxy curing. Each round of dialysis takes 1–24 h, depending on the analyte and membrane used. We previously used the dialyzer for small-volume non-enzymatic RNA synthesis reactions inside fatty acid vesicles. In this protocol, we demonstrate other applications, including removal of unencapsulated calcein from vesicles, remote loading and vesicle microscopy.
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Acknowledgements
This work was supported in part by The National Aeronautics and Space Administration (NASA) Exobiology grant NNX07AJ09G to J.W.S. and in part by a grant (290363) from the Simons Foundation to J.W.S. A.E.E. and N.P.K. were supported by appointments to the NASA Postdoctoral Program, administered by Oak Ridge Associated Universities through a contract with NASA. A.E.E. was supported by a Tosteson Fellowship from the Massachusetts General Hospital Executive Committee on Research. J.W.S. is an Investigator of the Howard Hughes Medical Institute.
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K.A., A.E.E., N.P.K. and L.J. performed the experiments. K.A., A.E.E., N.P.K., L.J. and J.W.S. wrote the manuscript. J.W.S. supervised the research.
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Supplementary Figure 1 Disassembled Slide-A-Lyzers, ready for assembly.
Two gaskets from two Slide-A-Lyzer units and both parts of the plastic frame of one of the Slide-A-Lyzers are used. Note that one of the gaskets (right) still has one dialysis membrane attached.
Supplementary Figure 2 PCR film to create clear sample viewing windows
A: Two pieces of PCR film, with the back paper still attached, cut out in the shape and size big enough to cover the openings in the plastic frame. B: The cut piece of PCR film is applied to the inside of one half of the plastic frame, and pressed into grooves of the inside of the frame with a pipette tip. C: The PCR film applied to the inside of one half of the plastic frame. The film’s sticky side points to the outside of the dialyzer, and the film fully covers the opening in the half-frame.
Supplementary Figure 3 Insertion of needle into gasket.
A: silicone gasket is pre-punctured with a beveled needle (green) and the blunt needle (blue) is inserted into the opening made with the beveled needle. B: The blunt needle is positioned so that the blunt end of the needle goes all the way through the gasket, but it does not stick out more than half a millimeter inside the gasket. C: A silicone gasket is aligned to cover the window on one half of the plastic frame. The second silicone gasket is aligned on top of the first one, with the single remaining dialysis membrane between the gaskets. D: Side view of the one half of the plastic frame with both silicone gaskets aligned on it. One of the two blunt needles going through the gaskets is visible.
Supplementary Figure 4 Assembled dialyzer before application of epoxy.
A: Both halves of the plastic frame assembled with two silicone gaskets, two needles, and one dialysis membrane, ready to be glued. B: The dialyzer assembly, placed in the bar clamp for gluing. The bar clamps are wrapped in plastic to avoid contamination from the epoxy (and avoid the dialyzer being permanently glued to the clamp if some epoxy leaks out). C: The dialyzer assembly in the bar clamp, with silicone gaskets properly aligned.
Supplementary Figure 5 Application of epoxy to dialyzer.
Epoxy, applied to all sides of the dialyzer assembly and the outer diameter of the blunt needles.
Supplementary Figure 6 Completed dialyzer.
A: Completed dialyzer assembly, removed from the bar clamp after the epoxy has fully cured (24h) and with needles capped.
B: Close-up of the finished dialyzer assembly, with needles going through the silicone gaskets and into the dialysis chambers.
Supplementary Figure 7 Dissassembled Slide-A-Lyzer G2.
The disassembled G2 dialyzer cassette.
The assembly of the G2 based dialyzer is analogous to the process described for the standard slide-a-lyzer cassette; the few notable differences are shown in Supplementary Figures 8-9.
Supplementary Figure 8 G2 dialyzer in bar clamp with epoxy applied.
A: Side view of the G2 dialyzer in the vice clamp, showing parallel alignment of the two sample port plugs aligned on top of the assembly. B: Alignment of G2 dialyzer in bar clamp.
Supplementary Figure 9 Completed G2 dialyzer.
A: The top view of the assembled G2 dialyzer, showing the sample ports. B: Front view of the fully assembled G2 dialyzer, showing the tip of the sample port plug sticking into the dialysis chamber.
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Adamala, K., Engelhart, A., Kamat, N. et al. Construction of a liposome dialyzer for the preparation of high-value, small-volume liposome formulations. Nat Protoc 10, 927–938 (2015). https://doi.org/10.1038/nprot.2015.054
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DOI: https://doi.org/10.1038/nprot.2015.054
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