3.4.3: Working with Lipids

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Learning Goals

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Liposome Preparation and Properties

• Compare the three principal methods of liposome formation — mechanical dispersion, organic solvent dispersion, and detergent dialysis — with respect to encapsulation efficiency, lamellarity, and suitability for incorporating membrane proteins or expensive solutes.
• Explain how extrusion, sonication, and freeze-thaw cycling convert multilamellar vesicles (MLVs) into unilamellar vesicles of defined size, and describe how size-exclusion chromatography separates liposomes from unencapsulated solutes and residual detergent.
• Predict how varying lipid composition — including the degree of fatty acid unsaturation, head group identity, cholesterol content, and incorporation of charged phospholipids — affects the transition temperature (T_M), fluidity, charge density, and permeability of a liposome preparation.

Biomedical Applications of Liposomes

• Explain how lipid nanoparticles encapsulate and protect mRNA cargo — such as the SARS-CoV-2 spike protein mRNA — using ionizable cationic lipids, and describe how liposomes can be targeted to specific cell types for drug or nucleic acid delivery via receptor-mediated endocytosis or membrane fusion.

Chemical Analysis of Lipids

• Describe the principles of gas chromatography (GC) as applied to lipid analysis, including why derivatization to fatty acid methyl esters or trimethylsilyl derivatives is required to increase volatility, and explain how retention time and flame ionization detection enable qualitative and quantitative identification of fatty acid composition.
• Explain how mass spectrometry — particularly electrospray ionization coupled with tandem MS (MS/MS) and photochemical methods such as the Paternò-Büchi reaction — enables structural characterization of lipid isomers at the level of fatty acid chain identity, sn-position, and C=C bond location and geometry, and explain why this level of resolution is important for lipidomics and disease biomarker discovery.

Introduction

Lipids, although small compared to large biopolymers like proteins, nucleic acids, and large glycans, are very heterogeneous in structure, given the large array of fatty acid and isoprenoid chain lengths, numbers of double bonds, etc., that appear in different lipid classes. In addition to analyzing lipid structure, lipids are used in the laboratory to create liposomes (vesicles), which model membrane bilayers and can encapsulate chemical species (drugs, vaccines) for medical use and solubilize membrane proteins. This section will focus on creating lipid vesicles (critical for encapsulating RNA vaccines targeting the SARS-CoV-2 spike protein) and on the chemical analysis of biological lipids, whose composition affects health and disease states.

Liposomes

Liposomes produced in the lab can be unilamellar, which consists of a single bilayer surrounding the internal aqueous compartment, or multilamellar, which consists of multiple bilayers surrounding the enclosed aqueous solution. You can imagine multilamellar vesicles as an onion with multiple layers. Cartoons of unilamellar and multilamellar liposomes are shown in Figure \(\PageIndex{1}\), where each concentric circle represents a bilayer.

Figure \(\PageIndex{1}\): Cartoons of unilamellar and multilamellar liposomes (each concentric circle represents a bilayer)

Liposomes vary in diameter. They can be generally categorized into small (S, diameter < 25 nm), intermediate (I, diameter around 100 nm), and large (L, diameter from 250-1000 nm). If these vesicles are unilamellar, they are abbreviated as SUV, IUV, and LUV, respectively. Their various sizes are shown in Table \(\PageIndex{1}\) below, compared to other large biological structures.

Table \(\PageIndex{1}\): Sizes of liposomes/vesicles compared to other biological structures.

The chemical composition of liposomes can vary widely. Most contain neutral phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), or sphingomyelin (SM), supplemented, if desired, with negatively charged phospholipids such as phosphatidylserine (PS) and phosphatidylglycerol (PG). In addition, single-chain amphiphiles such as cholesterol (C) and detergents can be incorporated into the bilayer membrane, modulating its fluidity and transition temperature (Tm). If present in too great a concentration, single-chain amphiphiles like detergents, which form micelles, can disrupt the membrane so completely that the double-chain amphiphiles become incorporated into detergent micelles, now called mixed micelles, in a process that effectively destroys the membrane bilayer.

Given the large degree of unsaturation at C2, what do you expect the transition temperature of a liposome composed only of egg yolk PC to be? (Vesicles made using more saturated PC from mammalian sources have a Tm of around 40 ^oC.) This high degree of unsaturation makes egg yolk PC very susceptible to oxidation, which could dramatically alter the liposome's properties. Synthetic PC made with saturated fatty acids could alleviate that problem.

The properties of liposomes (charge density, membrane fluidity, and permeability) are determined by the lipid composition and size of the vesicle. The desired properties will, in turn, depend on the specific liposome used. The vesicles offer simple, powerful models for studying the biochemistry and biophysics of natural membranes. Membrane proteins can be incorporated into the liposome bilayer using the exact method you will be using. Beyond these uses, liposomes can encapsulate water-soluble molecules such as nucleic acids, proteins, and toxic drugs. These liposomes can be targeted to specific cells if antibodies or other molecules that bind specifically to the target cell are incorporated into the vesicle's bilayer. Intraliposomal material may then be transferred into the cell by fusion of the vesicle with the cell or by endocytosis of the vesicle.

Since phospholipids will spontaneously form a bilayer structure when placed in water, most efforts in liposome production involve producing vesicles with the desired size, lamellar structure, and physical characteristics, which, as previously stated, are controlled by liposome size and chemical composition. Methods must also be developed to entrap the desired molecule within the vesicle cost-effectively and with minimal leakage. All production methods involve drying organic solvent-solubilized lipids, dispersing the lipids in the appropriate aqueous solution, and forming monolamellar (one bilayer) liposomes or vesicles. Finally, the vesicles are characterized (chemical composition, T_m, permeability, size, etc.)

Drying of lipids

Purified lipids of the desired composition (often egg PC:cholesterol:PS in molar ratios of 0.9:1.0:0.1) are dissolved in a purified, water-free organic solvent mixture (often chloroform/methanol, 2:1 v/v) and dried down in a round-bottom flask on a rotary evaporator under reduced pressure (using a water aspirator) and slightly elevated temperature (20-40 ^oC). Rapid rotation of the flask ensures the lipid is dispersed over a large surface area, increasing the evaporation rate. The dried flask is usually placed under a high vacuum overnight to remove the last traces of solvent. If a small volume (< 1 ml) of lipid solution is used, the solvent can be evaporated under a stream of nitrogen. To avoid trapping residual chloroform in the lipid film, dissolve the film in t-butyl-methyl ether or diethyl ether and dry it several times. Alternatively, the residual solvent can be removed under a high vacuum.

Dispersion of the lipids

Three main methods exist for dispersing lipids into an aqueous solution to form liposomes.

a. mechanical dispersion - in this method, lipid dried onto the inside glass surface of a container is hydrated with an aqueous solution, which peels off the lipid to form multilamellar - MLV - (multiple bilayers separated by water) vesicles. Only a small part of the aqueous solution is encapsulated inside the liposome, so this is not the method of choice for the encapsulation of expensive or rather insoluble solutes. Liposomes of different sizes can be prepared depending on the degree of agitation and the lipid used.

b. organic solvent dispersion - In these methods, the lipids, which are dissolved in organic solvents, are injected through a fine needle, at a slow rate, into an aqueous solution in which the organic solvent may be miscible (such as ethanol) or immiscible (such as ether). In each case, the lipids are oriented at the interface between the organic solvent and aqueous solution to form bilayer structures. Injection of ethanol-dissolved lipids provides a simple way to produce SUVs, but because liposome formation can not occur at an ethanol concentration greater than 7.5%, only a fraction of the total aqueous phase can be entrapped in the vesicle; hence, this technique is not cost-effective for the entrapment of an expensive solute. Alternatively, the lipid can be dissolved in ether and slowly injected into an aqueous solution, which is warmed so that the ether evaporates at the same rate as it is injected. Since the ether is volatilized, large amounts of lipid can be introduced, and the encapsulation efficiency of the aqueous solution is high.

c. detergent dispersion and solubilization - In this method, lipids are solubilized in an aqueous solution by adding detergents. The detergents are slowly removed from the solution, leading to the spontaneous formation of liposomes. Detergents are single-chain amphiphiles that spontaneously form micelles in aqueous solution when the concentration of free lipid rises to a minimum critical value, the critical micelle concentration (CMC); at this concentration, detergent self-association forms a stable aggregate, the micelle. This is illustrated in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): [Single chain amphiphile] in solution vs [Single chain amphiphile] added - the CMC

Table \(\PageIndex{2}\) below shows the properties and CMC of various detergents.

Table \(\PageIndex{2}\): Properties and CMC of various detergents (data from Avanti Polar Lipids)

Name	mM	mg/ml	MW
n-hepty glucopyranoside	70	19.5	278
n-octyl glucopyranoside	23.2	6.8	292
n-nonyl glucopyranoside	6.5	2.0	306
n-decyl maltoside	2.19	1.1	499
n-dodecyl maltotrioside	0.2	0.16	825
Triton X-100 (a)	0.24	0.15	625
Nonidet P-40 (b)	0.29	0.02	603
Tween 20 (c)	0.033	0.04	1364
Brij 98 (d)	0.025	0.04	1527
sodium deoxycholate	2-6	1.7	415
sodium taurocholate	10-15	6.7	538
sodium cholate	14	6.0	431
sodium dodecyl sulfate	8.3	2.4	289

Making liposomes by dialysis

Lipids are deposited in a small container. Then add an aqueous solution containing water-soluble molecules for encapsulation. Add detergent at a concentration above the lipid concentration and above its CMC. The lipid molecules are then "emulsified" in the detergent micelle. The solubilized mixture is then placed in a semipermeable dialysis bag and immersed in a large volume of aqueous solution. The free detergent in solution is in equilibrium with the detergent in the micelle. The bag contains microscopic holes large enough for the monomeric detergent molecule to pass through, but small enough that the large micelle cannot. During this process, the lipid becomes embedded in the micelle, forming a detergent-lipid mixed micelle. As dialysis continues, the monomeric detergent partitions throughout the bag volume and surrounds the bag, while the mixed micelle remains in the bag. If you replace the aqueous solution surrounding the bag several times with fresh solution, the equilibrium in the bag shifts toward the monomeric form. Alternatively, detergent-adsorbing beads (such as Bio Bead SM-2 by Bio-Rad) can be placed in the aqueous solution surrounding the bag to accelerate detergent re-equilibration. Eventually, all the detergent is in this form, and during the slow process, the lipid in the mixed micelle self-associates to form a liposome. A detergent of low monomer molecular weight and a high CMC is most desirable for this method of liposome production. Another method for removing free detergent is gel filtration chromatography. In this technique, molecules of disparate molecular weights can be separated. An explanation follows this discussion. This method of forming unilamellar liposomes is the method of choice when membrane proteins must be inserted into the liposome bilayer to target the liposomes. However, it is not the best method for quantitatively encapsulating expensive soluble molecules.

Making Liposomes by Extrusion

Several techniques can further process these multilamellar liposomes to form unilamellar liposomes. These include probe or bath sonication of the MLVs, extrusion of the MLVs at high pressure through membrane filters of defined pore size, or pH-induced vesiculation, in which a transient pH change destabilizes the MLVs in favor of unilamellar liposomes. Another technique involves fusing SUVs by repeated freezing and thawing, or by Ca²⁺-mediated aggregation of SUVs containing acidic phospholipids (such as PS). 

Figure \(\PageIndex{3}\)shows the structure of vesicles as they undergo multiple freeze/thaw cycles.

Figure \(\PageIndex{3}\): Structure of multilamellar vesicles as they undergo multiple freeze/thaw cycles

Figure \(\PageIndex{4}\) shows the final step in making large unilamellar vesicles by extrusion of freeze/thaw intermediates.

Figure \(\PageIndex{4}\): Conversion of multilamellar vesicles to unilamellar vesicles by extrusion

Once the liposomes are formed, they must be separated from free monomeric lipids, detergents, and unencapsulated solutes. This can be done by dialysis or, more easily, by size exclusion chromatography. Macromolecules of different sizes can be separated on a column in which the stationary phase is a polymerized agarose or acrylamide bead containing pores of various sizes. A small molecule (such as a monomeric detergent, free lipid, or small aqueous solute) in the mobile phase (aqueous buffered solution) may enter the pores in the bead, while a larger macromolecule or aggregate (such as a large protein, a micelle, or a liposome) may not, due to size restriction. As a result, a larger fraction of the column's overall volume is available to smaller molecules, which spend more time on the column and are eluted by the mobile solvent after the larger species. Liposomes can be characterized chemically to determine the average lipid and protein composition of the bilayer (if incorporated) and physically to determine size, permeability, lamellarity, and the amount of encapsulated material. Size is usually determined by electron microscopy or indirectly by light scattering from these large species.

Chemical Analysis of Lipids

Because there are so many lipids and their derivatives, the most sensitive techniques are needed for separation and analysis. Lipidomics focuses on analyzing the structure and function of all lipids in the cell. The most valuable techniques are analysis (and separation) by gas chromatography (GC), followed by mass spectrometry (MS). NMR spectroscopy is also essential. For GC analysis, the lipid must be made volatile, which limits its use in some circumstances. MS analysis is the most sensitive. MS requires ion formation, and techniques like electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) are used.

Gas chromatography

(This section is adapted from https://www.intechopen.com/chapters/64008. Creative Commons Attribution 3.0 License). In GC, gas is the mobile phase that carries lipids through the column's stationary phase. Gas chromatography (GC) separates organic compounds from a mixture in the gas phase. For this purpose, the GC uses interactions among the sample components and the stationary/mobile phases. After lipid extraction with chloroform and/or methanol, the samples (lipid mixture) are usually liquids and must be exposed to a high temperature at the gas chromatograph entrance (injector). Vaporized, the samples are carried by a gas, which is usually a light and inert gas (i.e., hydrogen, helium), through a long capillary column containing a high- or low-polarity material (stationary phase)

The gaseous compounds generated from the vaporized sample interact with the stationary phase, allowing each compound to elute (separate) at a different time (retention time). Because GC considers both the chemical and physical properties of the vaporized compounds, those with greater chemical affinity for the stationary phase elute more slowly, and temperature influences the overall process. This explains why the column stays in an oven programmed to operate over different temperature ranges (i.e., temperature programming).  The mobile phase gas carries the compounds, and their boiling points determine their elution times.

At the end of GC analysis, the electronic detector generates a chromatogram based on retention time by intensity. This allows qualitative identification of lipid compounds by comparing their retention times with certified standards using a flame ionization detector (FID) or by deducing spectral information from a mass spectrometer. Lipid quantification can also be performed using external or internal certified standards in GC analysis.

The main points to consider when assessing FAs by GC analysis are carrier gas flow rate, column length, and temperature, as these can influence lipid retention times and must be precisely standardized. Column length influences analyte resolution, as a longer column provides more theoretical plates (a hypothetical zone in which two phases establish equilibrium). Because fats and oils have high boiling points and are not well retained by the stationary phase, a previous derivatization step is required after lipid extraction from the biological sample, in which triacylglycerols and free fatty acids are converted into their respective free fatty esters with lower boiling points (transesterification/esterification reaction). Several methods are available for FA derivatization.

In cholesterol analysis, sample preparation typically involves a derivatization reaction. This increases compound volatility.  It also generates less polar products. Cholesterol derivatization is usually achieved by using trimethylsilyl compounds through silylation reactions. A common method uses N,O-bis(trimethylsilyl-trifluoroacetamide/trimethylchlorosilane).

Mass spectrometry

(This section is derived from https://doi.org/10.1038/s41467-019-14180-4. Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0/).

Mass spectrometry (MS) has become the method of choice for lipid analysis, offering label-free detection at high sensitivity and structural characterization capability. However, large-scale lipid analysis that reveals all levels of structural information remains a significant analytical challenge in lipidomics. General protocols, for instance, include five levels of structural information: lipid class, fatty acyl identities, fatty acyl sn-positions, and C=C location/geometry (viz cis/trans) in the fatty acyl. Researchers have already reported successful attempts to determine C=C locations in fatty acyls or their sn-positions by MS analysis, enabling characterization of detailed structural moieties and identification of lipid structural isomers. A key advantage of lipid isomer analysis is the high-precision relative quantitation without lipid standards, which are not readily available. Remarkably, our recent study demonstrated a close correlation between the lipid C=C location isomer compositions and Type II diabetes, which is owed to tighter regulation of lipid desaturation, allowing efficient elimination of interferences due to variations among samples."

Various methods have been explored to differentiate lipid C=C location and sn-position isomers. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have been used to determine both sn-positions and C=C locations in GPs. By coupling the Paternò-Bǜchi (PB) photochemical reaction with tandem MS (MS/MS), researchers can qualitatively and quantitatively analyze lipids with C=C specificity in complex biological samples. PB reaction converts the C=C to an oxetane, which can be preferentially fragmented by low-energy collision-induced dissociation (CID).

An ideal analytical tool for lipidomics to survey a wide range of lipids in discovery work should provide detailed information at multiple lipid structure isomer levels (e.g., C=C location/geometry and sn-position) and be feasible for large-scale quantitative analysis. UVPD can assign C=C locations and sn-positions of fatty acyls, while OzID may be the only one that has been demonstrated to assign C=C locations in sn-specific fatty acyls well. One problem with OzID is the long reaction time required for ion-trap implementation; however, recent work has shown it can be performed on LC-compatible timescales in the high-pressure regions of the MS system. For the PB reaction method, both shotgun analysis and an HPLC-PB-MS/MS workflow have been developed to identify multiple C=C isomer locations. Figure \(\PageIndex{5}\) describes methods for determining C=C location and sn-isomers in lipids.

Figure \(\PageIndex{5}\): Methods for the determination of C=C location and sn-isomers in lipids. a The chemical structure of PC 18:1(9Z)/16:0. b Schematic of the experimental setup for online derivatization of unsaturated GPs by coupling 254 nm irradiation with nanoESI-MS. c MS spectrum of PC 16:0/18:1(9Z) after 30 s reaction. d MS³ spectrum of PC 16:0/18:1(9Z) without PB derivatization. Sodiated lipid precursors were first fragmented to generate product ions after headgroup loss (−183 Da). Product ion at m/z 599.5 was further fragmented to release sn-1-specific diagnostic ions at m/z 319 (C16:0) and 345 (C18:1). e Comparison of the relative abundance of sn-position and C=C location-specific ions of PC 16:0/18:1(9Z) without and with PB derivatization using different PB reagents (Bza: benzaldehyde, APh: acetophenone, BPh: benzophenone, AP: acetylpyridine). The error bar represents the standard deviation, n = 3. Cao, W., Cheng, S., Yang, J. et al. Large-scale lipid analysis with C=C location and sn-position isomer resolving power. Nat Commun 11, 375 (2020). https://doi.org/10.1038/s41467-019-14180-4. DOI: https://doi.org/10.1038/s41467-019-14180-4. Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0/.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter addresses two complementary practical dimensions of lipid biochemistry: the laboratory production of lipid vesicles for research and biomedical applications, and the analytical methods used to characterize the extraordinary structural diversity of biological lipids.

Liposomes — closed phospholipid bilayer vesicles enclosing an aqueous compartment — are produced in the laboratory by exploiting the intrinsic tendency of double-chain amphiphiles to self-assemble in water. All production protocols share three stages: lipid dissolution in an organic solvent, dispersion into an aqueous solution, and conversion to vesicles of the desired size and lamellarity. Three major dispersion strategies are available, each with distinct trade-offs. Mechanical dispersion (hydration of a dried lipid film) is simple but produces heterogeneous multilamellar vesicles (MLVs) with low encapsulation efficiency. Organic solvent injection — either ethanol into water or ether into a heated aqueous phase — produces smaller vesicles but is constrained by solvent miscibility and the need to remove the solvent. Detergent dialysis exploits the equilibrium between micellar and monomeric detergent: lipids are co-solubilized with detergent above the critical micelle concentration (CMC), and progressive removal of the detergent by dialysis or adsorption onto hydrophobic beads allows lipid self-assembly into unilamellar vesicles. This last method is preferred when membrane proteins must be incorporated into the bilayer. MLVs can subsequently be converted to large unilamellar vesicles (LUVs) by repeated freeze-thaw cycling followed by extrusion through polycarbonate filters of defined pore size. Final liposome preparations are purified by size-exclusion chromatography, which separates intact vesicles from free lipid monomers, detergent, and unencapsulated solute based on differential access to the column's pore volume.

The physical and functional properties of a liposome preparation — T_M, fluidity, permeability, and surface charge — are determined by lipid composition and must be designed to match the intended application. Highly unsaturated lipids, such as egg yolk PC, lower T_M and increase fluidity but are susceptible to oxidative degradation; synthetic saturated PC is more chemically stable but produces more rigid membranes. Incorporation of cholesterol buffers T_M sensitivity, while charged phospholipids such as PS or PG introduce surface charge that affects colloidal stability and cell interactions. For mRNA vaccine delivery — exemplified by the SARS-CoV-2 lipid nanoparticle platform — ionizable cationic lipids are included to electrostatically stabilize the negatively charged RNA cargo and facilitate endosomal escape after cellular uptake.

The second half of the chapter addresses lipidomics — the comprehensive structural and quantitative analysis of all lipids in a biological sample. The sheer structural diversity of lipids (arising from variations in chain length, saturation, double-bond position and geometry, head-group identity, and sn-position of esterified fatty acids) demands highly resolving analytical methods. Gas chromatography (GC) separates vaporized lipid derivatives based on their interactions with a stationary phase and their boiling points. Because intact glycerolipids and cholesterol are insufficiently volatile, derivatization is required: fatty acids are converted to fatty acid methyl esters (FAMEs) by transesterification, and cholesterol is converted to its trimethylsilyl ether. Detection by flame ionization (FID) allows quantification relative to certified standards, while coupling GC to mass spectrometry (GC-MS) provides structural identification from fragmentation patterns.

Mass spectrometry has become the central tool of lipidomics, offering high sensitivity, label-free detection, and the capacity to resolve lipid structural isomers that differ only in the position or geometry of a single double bond or in the sn-position of an acyl chain. Soft ionization methods such as electrospray ionization (ESI) and MALDI preserve intact lipid ions for analysis. Tandem MS (MS/MS) generates diagnostic fragment ions that identify head groups and acyl chains. For the most detailed structural assignments — including C=C location and cis/trans geometry — advanced methods are required, including ozone-induced dissociation (OzID), ultraviolet photodissociation (UVPD), and the Paternò-Büchi (PB) photochemical reaction, which converts C=C bonds to oxetane adducts that fragment predictably under low-energy collision-induced dissociation. These approaches enable the resolution of structural isomers that would be indistinguishable by conventional MS, a capability with direct clinical relevance: the chapter notes that the distribution of C=C location isomers in plasma lipids correlates with Type II diabetes, illustrating how high-resolution lipidomics is beginning to reveal lipid structural signatures of disease.