Lipid loss and compositional change during preparation of liposomes by common biophysical methods

Abstract

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward method using HPLC-ELSD to quantify the lipid concentration and membrane composition of liposomes, and apply that method to study the preparation of simple POPC/cholesterol liposomes. We examine many common steps in liposome formation, including vortexing during re-suspension, hydration of the lipid film, extrusion, freeze-thaw, sonication, and the percentage of cholesterol in the starting mixture. We found that the resuspension step can play an outsized role in determining the overall lipid loss (up to ∼50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses (∼10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 minutes and increasing cholesterol concentrations up to 50 mole% had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition more than ∼5% under the conditions we tested. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation protocols. We expect our results can be leveraged for improved preparation of model membranes by researchers in many fields.Statement of SignificanceLiposomes are spherical lipid membranes that can be prepared by a variety of biophysical techniques. Researchers use liposomes in a variety of ways, including fundamental biophysical studies of lipid membranes, in drug delivery, drug formulation, and other biotechnology applications. In this report, we study the process to prepare liposomes by several common techniques and validate how reliable each technique is at producing consistent liposome concentrations and lipid compositions. We identify best practices for researchers to produce reliable liposome preparations.

Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios

“…Gentle hydration again incorporates 20% more than expected. This is surprising in light of recent results that find that cholesterol is, on average, slightly underincorporated (about 10% less than expected) into vesicles of POPC (16:0/18:1PC) via a related gentle hydration technique (36). The remaining methods (ITO electroformation and Pt wire electroformation) again agree with the stock solution, within uncertainty.…”

“…In terms of the total percent of cholesterol, the shifts are significant, roughly 20% of cholesterol is lost upon extrusion of vesicles made by gentle hydration. This value falls in the middle of ranges previously reported: no loss of cholesterol was observed from some membranes (ternary mixtures of 2:1:3 PC-lipids/eggPC/cholesterol, binary mixtures of 33% cholesterol with di(18:1)PC or di(14:0)PC, or binary mixtures of 30/70, 40/60, or 50/50 cholesterol with 16:0/18:1PC), whereas a loss of ∼1/3 to ∼1/2 of cholesterol was observed from other membranes (for binary mixtures of 33% cholesterol with 16:0/18:1PC, di(16:0)PC, or di(20:4)PC (19, 36). In other words, no clear correlation seems to exist between lipid unsaturation and a loss of cholesterol from vesicles upon extrusion.…”

“…For example, gentle hydration could have been conducted in salt solutions (rather than in sugar), or from lipid films deposited on surfaces of tracing paper (57) or polymer-coated substrates (58, 59) (rather than on bare glass). Similarly, gentle hydration of lipid films could have been achieved for different lengths of time using lipids in different ratios (36), followed by either shaking or vortexing (1) (rather than in a static solution) using different types of vortexers (36), and the subsequent multilamellar vesicles could have been made smaller through freeze/thaw cycles or sonication (rather than extrusion) (36, 52, 60). Details of each technique vary from laboratory to laboratory, and it is unclear how these details influence the ratio of lipids in membranes.…”

“…2, insets) that were not incorporated in the vesicles. For example, some common methods for gentle hydration of GUVs leave >50% of all lipids behind in the reservoir, and extrusion can incur a further loss of ∼20% of lipids (35, 36). Reservoirs may not contain the same ratio of lipids as the vesicles.…”