Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Summary

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Abstract

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

Introduction

The cell membrane, composed primarily of phospholipids, cholesterol, and proteins, is a crucial component of all living cells¹. With organization driven by lipid amphiphilicity, the cell membrane functions as a protective barrier and regulates how the cell interacts with its surrounding environment². Several cellular processes are dependent on the lipid and protein composition of the membrane^1,2. For example, cell membrane interactions are important for effective drug delivery³. Pharmaceuticals, biologics, nanomaterials, biological toxins,....

Protocol

1. Developing uni-lipid vesicles

1. Thin-film hydration method
   1. Preparation and storage of lipid stock solutions
      NOTE: All steps using chloroform need to be performed in a chemical fume hood. Chloroform should always be pipetted using solvent safe carbon fiber pipette tips. Solutions containing chloroform should always be stored in glass vials.
      1. Prepare a 10 mg/mL lipid stock solution by adding the appropriate volume of chloroform into the vial containing the lipid powder and mix well. For example, add 20 mL of chloroform to 200 mg of L-α-phosphatidylcholine (egg, chicken) (eggPC). The stock solution may be made at a different co....

Results

This protocol details methods for forming supported and suspended lipid bilayers (Figure 1). The first step to forming a supported lipid bilayer is to develop lipid vesicles. The mini extruder allows for small volumes of lipid vesicles to be prepared (1 mL or less), while the large extruder allows for 5-50 mL of lipid vesicles to be prepared in one batch. Size distributions of uni-lipid vesicles formed by either the mini or large extruder are shown in Figure 2A........

Discussion

This protocol allows for the formation of lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. Here, critical steps are presented to form each of these structures. When forming lipid vesicles, it is important to extrude above the transition temperature of the lipid³⁹. When below the transition temperature, the lipid is physically present in its ordered gel phase³⁹. In this ordered phase the hydrocarbon lipid tails are fully extended allowing for close pac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine  (POPC, 16:0-18:1 PC)	Avanti Polar Lipids	850457
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS, 16:0-18:1 PS)	Avanti Polar Lipids	840034
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE)	Avanti Polar Lipids	850757
1,2-dioleoyl-sn-glycero-2-phospho-L-serine (DOPS, 18:1 PS)	Avanti Polar Lipids	840035
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 18:1 (Δ9-Cis) PC)	Avanti Polar Lipids	850375
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 18:1 (Δ9-Cis) PE)	Avanti Polar Lipids	850725
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:0 EPC (Cl Salt))	Avanti Polar Lipids	890703
3 mL Luer-Loc syringes	BD	309657
40 mL sample vial, amber with polytetrafluoroethylene (PTFE)/rubber liner	Duran Wheaton Kimble	W224605
Acetonitrile	Sigma-Aldrich	271004
Alconox	Fisher Scientific	50-821-781
Ammonium formate	Millipore Sigma	LSAC70221
C18, 3.5 um x 50 mm column, SunFire	Waters	186002551
Chloroform	Millipore Sigma	LSAC288306
Cuvette UV Micro LCH 8.5 mm, 50 um, RPK	Sarstedt	67.758.001
Di(2-ethylhexyl) phthalate (DEHP)	Millipore Sigma	36735
Dimethyl sulfoxide (DMSO)	Millipore Sigma	LSAC472301
Ethanol	Pharmco	111000200
Filter supports, 10 mm	Avanti Polar Lipids	610014	Size for mini extruder
Folded capillary zeta cell	Malvern Panalytical	DTS1070
Isopropanol	Sigma-Aldrich	190764-4L
Kimwipes	Kimberly Clark	34256
L-α-phosphatidylinositol (soy) (Soy PI)	Avanti Polar Lipids	840044
L-α-phosphitidylcholine (Egg, Chicken)	Avanti Polar Lipids	840051
LiposoFast ® LF-50	Avestin, Inc.
Methanol	Sigma-Aldrich	179337 - 4L
Mini-extruder set with holder/heating block	Avanti Polar Lipids	610000
MultiScreen-IP Filter Plate, 0.45 µm, clear, sterile	Millipore Sigma	MAIPS4510	for PAMPA studies
Nitrogen gas, ultrapure	TechAir	NI T5.0
Nuclepore hydrophilic membranes, polycarbonate, 19 mm, 0.1 um	Whatman	800309	Size for mini extruder
Nuclepore hydrophilic membranes, polycarbonate, 25 mm, 0.1 um	Whatman	110605	Size for large extruder
Parafilm	Bemis	PM999
Phosphate buffer saline (PBS), 10x	Genesee Scienfitic	25-507X	Dilute to 1x
Qsoft 401 software	Biolin Scientific
Quartz Crystal Microbalance with Dissipation Q-Sense Analyzer	Biolin Scientific
Scintillation vials, borosilicate glass vials, 20 mL	Duran Wheaton Kimble	986561
Silicon Dioxide, thin QSensors	Biolin Scientific	QSX 303
Sodium chloride (NaCl)	Millipore Sigma	LSACS5886
Sodium dodecyl sulfate (SDS)	Fisher Scientific	BP166-100
Solvent Safe pipette tips	Sigma-Aldrich	S8064
Sphingomyelin (Egg, Chicken)	Avanti Polar Lipids	860061
Trizma base	Millipore Sigma	LSACT1503
Trypsin-ethylenediaminetretaacetic acid	Caisson Labs	TRL01-6X100ML
Whatman drain disc, 25 mm	Whatman	230600	Size for large extruder
Zetasizer ZS90	Malvern Panalytical
Zetasizer 7.01 software	Malvern Panalytical